Mechanisms of multidrug resistance in a human uterine sarcoma cell line following exposure to discodermolide, a new microtubule-stabilizing compound from a marine sponge

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Mechanisms of multidrug resistance in a human uterine sarcoma cell line following exposure to discodermolide, a new microtubule-stabilizing compound from a marine sponge

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Title:
Mechanisms of multidrug resistance in a human uterine sarcoma cell line following exposure to discodermolide, a new microtubule-stabilizing compound from a marine sponge
Creator:
Turkson, Nicole Y.
Place of Publication:
Tampa, Florida
Publisher:
University of South Florida
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English
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vii, 56 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Discodermolide ( lcsh )
Multidrug resistance ( lcsh )
HeLa cells ( lcsh )
Paclitaxel ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

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General Note:
Thesis (M.S.)--University of South Florida, 2000. Includes bibliographical references (leaves 47-52).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
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027858230 ( ALEPH )
47956358 ( OCLC )
F51-00154 ( USFLDC DOI )
f51.154 ( USFLDC Handle )

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MECHANISMS OF MUL TID RUG RESISTANCE IN A HUMAN UTERINE SARCOMA CELL LINE FOLLOWING EXPOSURE TO DISCODERMOLIDE, A NEW MICROTUBULE-STABILIZING COMPOUND FROM A MARINE SPONGE by NICOLE Y. TURKSON A th esis submitted in partia l fu lfillm ent of the requirements for the degree of Master of Science Department of Marine Science College of Arts and Sciences University of South F l orida August 2000 Co-Major Professor: Pamela Hallock-Muller, Ph.D. Co Major Professor : Ross E. Longley Ph.D.

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Office of Graduate Studies University of South florida Tampa, Florida CERTIFICATE OF APPROVAL Master s Thesis This is to certify that the Master s Thesis of NICOLE Y TURKSON with a major in Marine Science has been approved for the thesis requirement on May 18, 2000 for the Master of Science degree. Examining Committee : .... .,., Major Professor: Dr. Pamela Ph.D. Member.;.I)r. EdwardS. Van Vleet Ph n Dr. A. Isbrucker Ph D

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ACKNOWLEDGEMENTS I wish to thank the members of my thesis committee: Dr. Ross E. Longley, Dr. Pamela Hallock-Muller, Dr. EdwardS. Van Vleet, and Dr. Richard Isbrucker for their tireless help and support I would also like to thank the Harbor Branch Oceanographic Institution and all of the people who gave their valuable time to help me. Thank you Pat Linley Tara Pitts Edward Jefford and Shirley Pomponi for all your help My gratitude goes to the Skell y Foundation for their support. Finally I wish to thank my family and friends for their endless encouragement and for always believing in me

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TABLE OF CONTENTS List of Tables lll List of Figures iv Abstract v Introduction 1 What is Cancer? 1 Multidrug Resistance 1 MDR Proteins 2 ABC Superfamily of Transporters 3 P-gp 3 MRP 6 cMOAT 7 LRP 8 Other Forms of Resistance 8 MDR Reversal 9 Paclitaxel 11 Discodermolide 11 Research Focus and Objectives 13 Materials 14 Antibodies and Reagents 14 Cell Lines 15 Method s 1 7 Cell Culture 17 Drug Solution 18 MTT Colorimetric Assay 18

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Immunoblotting Preparation of Cell Lysate Samples Protein Determination Protein Gel Electrophoresis Coomass ie Blue Staining Protein Transfer to Membrane Immunoblotting Results Microscopy P -gp Staining MRP Staining Tubulin Staining Flow Cytometry MTT Cytotoxicity Assays (48 hr.) Immunob lotting P gp MRP Microscopy P-gp MRP Tubulin Flow Cytometry Discussion Summary Conclusions Suggestions for Further Study References Appendices Appendix A: Immunoblotting Solutions Appendix B: Polyacrylamide Gel Protocols 11 19 19 19 20 20 20 21 22 22 23 23 24 25 2 5 28 28 29 30 30 30 33 36 38 43 45 46 47 53 54 56

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LIST OF TABLES Table 1 Multixenobiotic resistance in marine organisms 5 Table 2 Agents that reverse multidrug resistance 9 Table 3 Compounds that inhibit MRPand/or P-gp-mediated calcein-AM efflux 10 Table 4 IC50 values (nM) for discodermolide w i th and without P-gp and MRP inhibitors 26 Table 5 IC5o values (nM) for paclitaxel with and without P-gp and MRP inhibitors 27 Table 6 Presence and absence of P-gp and MRP in each cell line 44 Table 7 MTT -based drug sensitivity in each cell line 45 Table 8 Microscopy-based drug sensitivity in each cell line 45 lll

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L I ST O F FIGURES Figure 1. Structures ofDiscodermolide and Paclitaxel 12 Figure 2 Discodermo l ide Cell Cytoto x icity 27 Figure 3. P-gp Immunoblot 28 Figur e 4 MRP Immunoblot 29 Figure 5 Confocal images of cells stained for P gp 31 Figure 6. Confoca l images o f cells sta i n e d for MRP 32 Figure 7 Confoca l images o f MES-SA and MES-SA/DX5 cells treated with discodermolide and stained for tubulin 34 Figure 8. Confoca l images o f MES-SA/DX5 + D and PANC-1 c ells treated with discodermolide a nd stained for tubulin 35 Figure 9 MES SA Flow Cytometry Images 37 Figure 10. P ANC-1 F l ow Cytometry Ima ge s 37 IV

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MECHANISMS OF MULTIDRUG RESISTANCE IN A HUMAN UTERINE SARCOMA CELL LINE FOLLOWING EXPOSURE TO DISCODERMOLIDE, A NEW MICROTUBULE-STABILIZING COMPOUND FROM A MARINE SPONGE by NICOLE Y. TURKSON An Abstract of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science College of Arts and Sciences University of South Florida August 2000 Co-Major Professor: Pamela Hallock Muller, Ph.D. Co-Major Professor: Ross E Longley, Ph. D. v

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ABSTRACT Discodermolide is an antimitotic agent derived from the marine sponge, Discod e rmia diss o luta It has a mechanism of action similar to that of paclitaxel a potent anticancer agent which hyperstabili z es microtubules thereby disruptin g cell division. One of the most challenging problems associated with ch e moth e rapy i nvolving paclitaxel is the development of the multidrug resistance (MDR) phenotype, which can ultimately lead to chemotherapeutic failure. MDR can occur in tumor cells exposed to an anticancer agent, and is characteri z ed by the development of cross-resistance to a number of structurally unrelated natural products. The MDR phenotype involves the overexpression of membrane glycoproteins of high molecular weight and a reduction of intracellular drug accumulation. P-glycoprotein (P-gp ), an ATP-dependent trans-membrane efflu x pump, is the primary membrane protein associated with MDR. P-gp reduces cellular drug accumulation by pumping cytotoxic agents from within the cell, thereby reducing their cytoplasmic concentrations A second major membrane protein implicated in MDR is the multidrug resistance-associated protein (MRP), which also pumps cytotoxic compounds out of the cell. The presence ofP-gp has been associated with paclitaxel-induced drug resistance in both clinical and in vitro samples. It is known that cells resistant to paclitaxel remain sensitive to discodermolide, ind i cating a difference in the mechanisms of resistance to these two compounds Preliminary studies have shown the potent i al for cultured mammalian cells to become resistant to discodermolide, however the mechanism of this resistance is yet unknown. This study attempted to determine whether the MRP pump is a mechanism by which discodermolide resistance has occurred in a line of cultured uterine sarcoma cells. A combination of cytotoxicity assays, immunoblotting, fluorescence microscopy and flow cytometry were performed using four separate cell lines having varying degrees of resistance to discodermolide and paclitaxel. VI

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Results indicate that MRP is not the primary mechanism of resistance to discodermolide although data from Western blots and fluorescence microscopy indicate that a low level ofMRP is present in the discodermolide-resistant cell line. Evidence suggests that P-gp is involved and may confer resistance to discodermolide at higher levels of expression than those seen in paclitaxel-resistant cells A number of other resistance mechanisms not explored in this study are also possible. Abstract Approved: __________ _______ Co-Major Professor: Pamela Hallock-Muller, Ph .D. Professor Department of Marine Science Date Approved: Abstract Approved: Co -Major .. Ros; Gngley, Ph D. Harbor Branch Oceanographic Institution Date Approved: Vll

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INTRODUCTION What is Cancer? Cancer is a leading cause of death in the United States second only to heart disease. About 1 in 4 deaths can be attributed to cancer, with more than half a million Americans dying from cancer-related illnesses every year (Ries et al. 1999). The incidence of cancer is on the increase with about 1.2 million new cases expected to be diagnosed in the year 2000 (Wingo et al., 1998). The costs of cancer in health care expenses and lost productivity are overwhelming. The U. S. National Institutes of Health estimate that cancer costs the United States around $107 billion dollars each year. Cancer is not a single disease but rather a group of diseases in which abnormal cells grow and spread at an uncontrolled rate (Ries et al., 1996) Cancer can affect any part ofthe body and may lead to the death of the patient if not treated successfully through chemotherapy, radiation, immunotherapy or surgery. Certain chemicals, viruses and radiation are known to initiate cancer through induction of genetic mutations, but internal causal factors have also been identified, such as immune conditions, hormones and expression of inherited mutations. Environmental, socio-economic and life style choices have also been implicated in the risk of developing cancers (Ries et al., 1999). Multidrug Resistance Patients who initially respond well to chemotherapy may develop resistance during successive rounds of treatment. Resistance to anticancer agents can lead to chemotherapeutic failure, or the development of refractory drug resistant tumors. These types of obstacles can ultimately contribute to lowered patient survival (Krishan et al., 1997) Resistance to chemotherapy in cancer patients can occur in two forms Either the cancer is resistant to chemotherapeutic treatment from the beginning (intrinsic resistance), 1

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or resistance develops through the course of successive treatments (acquired resistance; Goldstein, 1995). Multidrug resistance (MDR) usually falls into the latter category. MDR may occur in tumor ce lls exposed to a particular anticancer agent, and is characterized by the development of cross-resistance to a number of structurally unrelated agents Classes of these compounds include anthracyclines, taxanes, epipodophyllotoxins and vinca alkaloids (Goldstein, 1995). Vinblastine and vincristine (vinca alkaloids), actinomycin D, daunorubicin and doxorubicin (anthracyclines), etoposide (an epipodophyllotoxin) and paclitaxel (Taxol) are a few of the drugs which have shown involvement in multidrug resistance (Bellamy, 1996) MDR is most common in ovarian renal, pancreatic and breast cancers, as well as leukemias, myelomas and lymphomas (Krishan et al., 1997) It is a serious and recurring problem, which often leads to chemotherapeutic failure. For example, paclitaxel, an effect i ve natural product-derived anti-cancer agent used for the treatment of ovarian and breast cancers, shows a response rate of less than 50% in patients with acquired resistance as opposed to a response rate of greater than 50% in patients without resistance (Krishan et al., 1997). MDR Proteins When a cell line resistant to paclitaxel was developed from a paclitaxel-sensitive parent cell line through exposure to step wise increasing concentrations of the drug cellular paclitaxel accumulation was reduced by 88% indicating an extremely high level of resistance (Casazza and Fairchild, 1996). The MDR phenotype is frequently associated with the over expression of cell membrane glycoproteins of high molecular weight and a reduction in cellular drug accumulation (Greenberg et al., 1988). The high molecular weight membrane glycoproteins found in tumor cells exhibiting the MDR phenotype have been demonstrated to act as trans-membrane efflux pumps (As z alos and Ross 1998). These proteins bind to a wide range ofxenobiotic compounds and expel them from the cell before they are able to reach therapeutic levels at their targets (Casaz z a and Fairc hild 1996). 2

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ABC Superfamily of Transporters MDR proteins belong to a large family of evolutionarily conserved genes known as the ATP-binding cassette (ABC) (Bellamy, 1996) Over 50 ABC transporters have been identified to date, mainly from prokaryotic organisms (Higgins, 1992). ABC transporters utilize energy from the hydrolysis of ATP to transport a wide variety of substrates across cell membranes Each ABC transporter d emonstrates relatively high specificity for type of substrate although the ABC family as a whole transports a wide variety of compounds including proteins sugars, amino acids, polysaccharides, peptides, oligopeptides, inorganic ions, or nonendogenous compounds. Some of these transport proteins pump substrates out of the cell, while others accumulate substrates within the cell (Higgins, 1992) The typical ABC transporter is comprised of four membrane-associated domains. Two of these domains form the substrate pathway across the cellular membrane, while the other two function in the binding of ATP and the coupling of ATP hydrolysis to the transport process (Higgins, 1992). It is important to note that transport of compounds across cell membranes does not result in an alteration of the compounds, and the transported compounds are therefore not true substrates. Regulation of many ABC transporters occurs at the synthesis level. Expression of the ABC prot ein may occur in spec ific cell types, or in response to certain growth conditions, such as the exposure to cytotoxic agents to favor survival of the cell. MDR has been demonstrated to occur in non-mammalian organisms. ABC transporters have been implicated in a number of microbial drug resistances, including erythromycin resistance in Staphylococcus (Ross et al., 1990), methotrexate and heavy metal resistance in Leishmania (Higgins, 1992), and chloroquine resistance in the malarial parasite Plasmodium (Foote et al., 1989, 1990). The primary membrane protein associated with MDR in cancer chemotherapy is the Pg lycoprotein pump (P gp ). It is a 170-180 kD ATP-dependent trans-membrane efflux pump which reduces cellular drug accumulation by transporting xenobiotic agents 3

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from the cell and effectively reducing their cytoplasmic concentration (Casazza and Fairchild, 1996). In humans, the increased expression of P-gp results from upregulation ofthe MDR1 gene located on chromosome 7 (Goldstein, 1995). Increased expression of the MDR1 gene in clinical material is often predictive of a poor chemotherapeutic response (Baldini, 1997) and the degree ofPgp expression has been found to correlate with the degree of resistance in cancer cell lines (Kurelec et al., 1992). P -gp has been found to occur in relatively high levels in normal cells of the kidney and adrenal tissues, gastrointestinal epithelium, mucosal cells ofthe small and large intestine, biliary hepatocytes and pancreatic ductules. High levels ofP-gp are also found in cells of the placenta, blood brain and blood-testicle barrier endothelium, and in some bone marrow stem cells (Baldini, 1997). Moderate l evels occur in cells of the lung, colon and liver and lower levels in cells of the testis placenta, brain, lung, prostate, stomach and bone marrow stem cells (Kruh et al., 1995) Low or no expression ofP-gp is found in tissues of th e breast bladder, stomach, esophagus and the head and neck (Goldstein, 1995). It is also known that P-gp is expressed in human tissues during early development, and may be related to functional changes as fetal organs develop (Baldini, 1997). The presence ofP-gp in so many normal tissues coupled with its involvement in multi-drug resistance in cancer cells may be an indication that the normal physiological role of this protein is as a cytoplasmic detoxifier aimed at preventing the accumulation of toxic compounds in cells or tissues of the body. It is believed that P -gp and other such efflux proteins may protect the body by functioning as broad-specificity transport systems by exporting exogenous molecul es found in our environment and in our diets, as well as endogenous metabolites from our bodies (Juranka et al., 1989) Cancers from tissu es which naturally contain significant level s ofP-gp are often found to poss ess intrinsic resistance to chemotherapy, whereas those from tissues which do not normally express P -gp often acquire resistance after treatment has begun. This suggests that the introduction of chemotherapeutic drugs to non-P -gp expressing cells leads to the upregulation of the MDR-1 gene. A high degree of intrinsic drug resistance is commonly found in cancers of the liver, kidney pancreas and adrenal g landsall tissues which normally express a high degree of P-gp Breast cancers and sarcomas are derived from tissues with low natural MDRl gene expression and consequently, often r es pond well initially to chemotherapy. 4

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Various MDR genes have been localized in human, mouse and hamster genomes (Bellamy, 1996). However, there is evidence that P-gp may have developed throughout evolution as a member of a class of general defense mechanisms aimed at protectin g organisms from harmful endogenous and exogenous toxins (Smital and Kurelec, 1998). These mechanisms have been termed "multixenobiotic resistance" (Kurelec, 1992) and occur across broad taxonomies, encompassing both prokaryotes and eukaryotes including bacteria protozoa, yeast, mollusks, insects and fish (Juranka et al., 1989 ; Smital and Kurelec, 1998). Some recent investigations of non-human resistance mechanisms have focused on aquatic and marine species (Table 1) (Kurelec, 1992; Kurelec et al., 1992; Smital and Kurelec, 1998). This is of particular importance now that marine invertebrates are being increasingly screened for new bioactive compounds having pharmaceutical potential (Munro et al., 1999). Of particular interest is the identification of a 125 kD glycoprotein in two species of sponge. This protein reacts with a P-gp-specific monoclonal antibody (clone C219) in immunoblots of celllysates prepared from the marine sponges Geodia cydonium and Verongia aerophoba (Kurelec et al., 1992a ; Kurelec, 1992b). Similarly a 140 kD glycoprotein was identified using the same P-gp antibody in the marine worm Urechis caupo on Western blots (Kurelec, 1 992) Table 1 : Multixenobiotic resistance in marine organisms Worm Sponge Mussel Oyster Snail Organism Urechi s caupo Geodia c y donium Verongia a e rophoba Mytilus galloprovincialis Mytilus edulis Crassostrea gigas Monodonta turbinata Reference (Holland-Toomey and Epel,1993) (Kurelec, 1992) (Kurelec, 1992) (Kurelec and Pivcevic, 1991) (Minier eta!., 1993) (Minier et al. 1993) (Kure l ec eta!., 1995) P-gpl ike efflux proteins have been identified in a number of marine organisms (modified from Srnital and Kurelec, 1998) The presence of the 125 kD glycoprotein in sponge cells was confirmed by microscopy in which both viable and fixed cells were labelled using polyclonal P5

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glycoprotein antibodies. This procedure demonstrated that this protein is localized to the cell surface (Kurelec, 1992; Kurelec et al., 1992). This protein has been shown to efflux various xenobiotics, including vincristine and daunomycin, from the sponge cell, a process which is inhibited by the presence of the MDR-reversing agent verapamil (Kurelec et al., 1992). It has been suggested that this defense mechanism in various aquatic organisms serves to protect against the toxic effects of chemical pollutants. In support of this idea, it has been found that exposure to polluted water appears to induce the expression of multixenobiotic resistance (MXR; Smital and Kurelec 1998). A second high molecular weight membrane protein associated with MDR is known as the multidrug resistance-associated protein (MRP). MRP is a 190 kD membrane-bound glycoprotein (Bellamy, 1996) which belongs to the same family of ABC proteins as P-gp (Broxterman et al., 1995), but shares less than 15% homology of amino acids with P gp (Komarov et al., 1998). The MRP gene which encodes the MRP protein is located on chromosome 16 in humans. MRP is found not only in the plasma membrane, but is also expressed in the Golgi region ofthe cell (Bellamy, 1996), and may originate in membranes of the Golgi network or endoplasmic reticulum (Krishan et al., 1997). MRP was first identified in a human lung cancer cell line demonstrating the MDR phenotype, without over expressing P-gp (Cole et al., 1992). Tumor cells which naturally express MRP, and cells into which MRP has been transfected have been shown to possess increased resistance to a number of anticancer agents such as daunorubicin (Bellamy, 1996), suggesting that MRP may also play an important role in multidrug resistance of tumor cells to chemotherapy MRP has been found to occur naturally in a number of normal tissues, including liver, adrenal, testis, and peripheral blood mononuclear cells (Krishan et al. 1997). MRP has also been identified in tissues of the lung, kidney colon, thyroid, urinary bladder stomach spleen (Sugawara 1998) and skeletal muscle (Kruh et al., 1995). High levels of MRP have been implicated in multi drug resistance in cancers of the lung and pancreas (Miller et al., 1996), and in neuroblastomas, leukemias and cancer of the thyroid (Kruh e t al., 1995) as well as bladder ovarian and breast cancers (Barrand et al., 1997) 6

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MRP-mediated MDR involves some of the same classes of compounds as those that are mediated by P-gp, including vinca alkaloids, epipodophyllotoxins, anthracyclins and actinomycin D (Barrand et al., 1997). However, the substrate specificity has been demonstrated to differ from that ofP-gp (Komarov et al., 1998). For example tumor cells possessing a high degre e ofP-gp expression usually demonstrate a high level of resistance to paclitaxel, whereas those with high MRP expression may be only mildly resistant to paclitaxel (Barrand et al., 1997) Conversely, MRP-overexpressing cells have shown resistance to some heavy metal oxyanions, whereas P-gp confers little resistance to these compounds (Cole and Deeley, 1998) MRP, like P-gp appears to function in the body as a cytoplasmic detoxifier and is also suspected to be involved in the regulation of chloride channel activity (Higgins, 1995) and the maintenance of internal pH (Roepe, 1995). Although the MRP protein operates at the cell surface, where it expels xenobiotics, it originates in the Golgi apparatus It is thought that the function ofMRP in normal cells may be to transport compounds into intracellular compartments. When a cell is tumorous and exposed to an anti-cancer drug, MRP may be recruited to the cell membrane in order to expel the cytotoxic compound (Barrand et al., 1997). cMOAT The human canalicular multispecific or ga nic anion transporter ( cMOAT) is also a member of the ABC superfamily of transporter proteins, and is ATP dependent. Also known as MRP2 it shares a 49% amino acid homology with MRP (Borst and Evers, 1997) cMOAT is localized to the apical membrane ofhepatocytes. It may have possible implication in MDR in human cancer cells c-MOAT has been shown to transport vinblastine and has also been found upregulated in some cancer cells, particularly those with resistance to cisplatin (Borst and Evers 1997). High levels of cMOAT have been identified in resistant head and neck, bladder and prostrate cancer cell lines (Narasaki et al. 1997) but no conclusive evidence has yet been found implicating c-MOAT in MDR of human cancer cells. 7

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Another membrane protein which is over expressed in some resistant tumor cell lines is the human major vault protein known as LRP (lung resistance-related protein) (Komarov et al., 1998). LRP is not a member of the ABC transporter family, and the gene which regulates it is located on chromosome 16 in humans (List, 1997) This 110 kD protein was first identified in non-P-gp lung cancer cell lines exhibiting reduced drug accumulation, and was associated with poor chemotherapeutic response in patients with ovarian carcinoma and acute myeloid leukemia (Bellamy, 1996). Vault proteins function as ribonucl eo protein complexes with multi-subunits, and are involved in nuclear-cytoplasmic trafficking (List 1997). They are thought to mediate bidirect io nal nucleocytoplasmic exchange and vesicular transport of drugs (Komarov et al., 199 8). Transfection studies indicate that LRP alone is not a mechanism of resistance but when overexpressed LRP in cancer cells may contribute to drug resistance by rapidly redistributing anthracyclines from the nucleus to the cytoplasm (List 1997). Michieli et al. (1999) reported a correlation between P-gp and LRP expression in acute non lymphocytic l e ukemias Another study found that the remission rate in patients with acute myeloid leukemias expressing LRP was 33% lower than in thos e not expressing LRP (List, 1997). Overall, the presence ofLRP in tumor cells is believed to be an indicator of drug resistance and a poor prognostic factor in the response to chemotherapy of cancer patients Oth er Forms of Resistance Resistance to chemotherapy in cancer cells can occur at a number of level s. The trans-membrane efflux pump s act at the cellular transport level (Bradshaw and Arceci, 1998) Other cellular mechanisms of resi s tance include alterations in cellular metabolism or drug tar ge ts increased detoxification, altered tubulin, enhanced ability of the cell to repair DNA damage and failure to under go apoptosis, or programmed cell death (Bellamy 1996). Increased levels of glutathione, reduced cellular uptake of cytotoxic dru gs and abnormalities in the expression of th e p53 tumor suppressor gene have also been implicated in drug resistance (Casazza and Fairchild, 1996). 8

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MDR Reversal One feature of trans-membrane efflux pumps associated with MDR is their ability to be reversed by certain blocking agents, or inhibitors, causing the sensitivity of the cell to an anti-cancer agent to be restored in vitro (Casazza and Fairchild, 1996). These chemosensitizing compounds include verapamil, cyclosporins cephalosporins, indole alkaloids, bisbenzylisoquinoline alkaloids (Casazza and Fairchild, 1996), and calmodulin inhibitors (Table 2; Goldstein 1995) One clinical strategy to circumvent MDR in cancer patients is to administer chemotherapies in conjunction with an MDR inhibitor in the hopes of reversing the resistance (Goldstein, 1995). Table 2 : Agents that modulate multidrug resistance Class Antibiotics Antiestrogens Antimalarials Calcium Channe l Blockers Calmodulin Inhibitors Cyclosporins Quinolines Steroids and D e rivativ es Surfactants Examples Cepharanthidine, Cefoperazone, Erythromycin Tamoxifen Torernif e ne Chloroquine Quinidine Quinine Veraparnil, Nica rdapin e, Nifedipine Chlorpromazine Thioridazine Cyc lo sporin A, PSC 833 Quinine, Quinidine Chloroquine Progesterone Tamoxifen Tween 80, Solutol H S-15 Multidrug resistance can be reversed by various compounds wh i ch bind to trans membrane efflux pumps and inhibit the efflux of chemothe rapeutic agents from the cell (modified from Bellamy, 1996 ; Volm, 1998) The use of chemosensitizers might at first seem to be the answer to the problem of reversing multidrug resi stance However, the inhibiting compounds themselves often carry toxicities and side effects. Administration of an anticancer drug in conjunction with an MDR inhibitor can lead to synergistic effects which increase toxiciti es to normal cells of the body which naturally ex press the MDR protein being inhibited (Bellamy 1996) The knowledge that certain MDR inhibitors in spe cific concentrations selectively bind to particular MDR efflux proteins aids in the identification of these proteins in v itr o 9

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{Table 3). For example, when used in a concentration of 10 -20 J.!.M, indomethacin, a methylated indole derivative, serves as an MRP-specific inhibitor (Hollo et al., 1996). Indomethacin in itselfhas strong anti-inflammatory and analgesic-antipyretic properties, and is used to treat patients with rheumatoid and various other types of arthritis (Insel, 1990). It is known to carry severe gastrointestinal and CNS side effects which in conjunction with cancer chemotherapy may cause severely detrimental health problems. Traditionally used as an antimalarial, quinine is a natural product alkaloid derived from the bark ofthe South American cinchona tree (Webster, 1990) Used in concentrations of20 -30 J.!.M, quinine serves as an important inhibitor ofP-gp function (Hollo et al., 1996) Like many drugs however, quinine is associated with a myriad of negative side effects, including those to the gastrointestinal tract, CNS and card iovascular system (Webster, 1990). Table 3: Compounds that inhibit MRP-and /o r P-gp-mediated calcein-AM efflux Compound ICso MRP P-gp Cyclosporin A 2-4 0 .51 Genistein 150-200 >1000 Indomethacin 10-20 >800 Quinine 50-100 20-30 Tamoxifen 3-6 2-5 Verapamil 4-8 2-5 Various modulators ofMDR specifically inhibit a particular efflux pump when administered in certa in concentrations (modified from Hollo et a!., 1996) A common laboratory assay uses the non-fluorescent, lipophilic compound, calcein-acetoxymethylester (Calcein-AM) to measure drug efflux (Feller et al., 1995a) Calcein-AM is a substrate for both MRP and P-gp (Essodaigui et al., 1998) Inside the cell, intracellular esterases cleave the ester bonds of calcein-AM, producing calcein, its highly fluorescent hydrolysis product (Feller et al., 1995a) Testing the efflux/accumulation of fluorescence by a drug resistant tumor cell type against MRPand P gp specific inhibitors provides an indication of which MDR efflux protein may be present in the cell type 10

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Paclitaxel (Taxol ) Paclitaxel is a complex diterpene derived from the Pacific yew tree, Taxus brevifolia (Wani et al., 1971). Paclitaxel is used to treat ovarian and breast cancers, but has also shown activity against a number of other cancers, including esophageal cancer, lung cancer and cancers ofthe head and neck (Casazza and Fairchild, 1996). Paclitaxel induces the formation of stable tubulin polymers in the form of microtubule bundles, thereby interrupting normal cell division (Horwitz et al., 1993). Resistance to paclitaxel is known to involve the over-production ofp-glycoprotein and reduced cellular drug accumulation the classic MDR phenotype (Horwitz et al., 1993). Although paclitaxel often shows an initially favorable response in chemotherapy use against certain malignancies, res istan ce to paclitaxel has proven to be a serious obstacle in the treatment of cancer patients. Overall response rates of tumors to paclitaxel are around 50%, and refractory tumors often develop following treatment (Casazza and Fairchild, 1996). Many tumors exhibit intrinsic resistance to paclitaxel, and those that are initially respondent often d evelo p resistance with successive treatments (Casazza and Fairchi ld 1996) Discodermo lide Discodermolide is a potent anti-mitotic agent derived from the marine sponge Discodermia dissoluta (Longley et al., 1991a and b). (+)-Discodermolide is a lactone bearing, polyhydroxylated alkatetraene (ter Haar et al., 1996) with a mechanism of action similar to that ofpaclitaxel. Both agents are stabilizers of the microtubule structure and also disrupt cell division (Horwitz et al., 1993). Originally, discodermolide was found to have potent in vitro immunosuppressive qualities (Longley et al., 1991a and b). While the mechanism of action by which this compound may function as an immunosuppressive agent was being explored, it was discovered that discodermolide is able to prevent cell proliferation (Longley et al. 1993). Discodermolide has been reported to inhibit mitosis in vitro by inducing bundling of microtubules and hyperstabilizing the tubulin polymer (ter Haar et al., 1 996) No apparent structural similarity exists between discodermolide and paclitaxel (Figure 1 ), altho ugh a common pharmacophore has recently been proposed (Ojima eta!., 1999). 11

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Discodermolide is believed to represent a class of compounds which is structurall y distinct from other natural product compounds which stabili z e tubulin polymer (ter Haar et al., 1996). Although their mechanisms of action appear to be similar, discodermolide is considered to be more potent in stabilizing tubulin polymer at low temperatures, and in the presence of Ca2+, conditions in which paclitaxel-induced tubulin polymers are destabilized (Kowalski et al., 1997) There is evidence that discodermolide possesses a greater affinit y for tubulin than that of paclitaxel, and is therefore able to competitively inhibit the binding of paclitaxel to tubulin (Kowalski et al., 1997) Based on current research, discodermolide holds substantial promise as a therapeutic weapon in the fight against certain cancers, including breast cancer P a clita x el Discodermolide Figure 1: Ch e mical s tructure s o fPaclita x el and discodermolide 12

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Research Focus and Objectives P-gp has been identified as a primary mechanism of MDR in paclitaxel-induced drug resistance (Casazza and Fairchild, 1996) Early studies have shown the potential for cultured human uterine sarcoma cells to become resistant to discodermolide, but the mechanism of this resistance is unknown (Longley and Isbrucker, personal communication). It is known, however, that P-gp-expressing cells that are resistant to paclitaxel remain sensitive to discodermolide (Kowalski et al., 1997). Therefore the primary goal of this study is to determine whether MRP is involved in the discodermolide resistance of a uterine sarcoma cell line. Using four cell lines having varying degrees of sensitivity to discodermolide and paclitaxel, a series of laboratory tests was performed in vitro to examine the potential role ofMRP in discodermolide resistance in MES-SNDX5+D cells a cell line which has shown resistance to discodermolide. Discodermolide activity was tested in the presence and absence ofP-gp-and MRP-specific inhibitors and examined for cytotoxicity and the ability to induce microtubule bundling in treated cells. Irnmunoblotting and microscopy were used to confirm the presence ofP-gp and MRP and an efflux assay was attempted to observe the activity of these pumps. 13

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MATERIALS Antibodies and Reagents The following were obtained from Life Technologies, Gaithersburg, Maryland : RPMI 1640 culture medium, fetal bovine serum (FBS), N-2-hydroxyethylpiperazine N-2ethane sulfonic acid buf fer (HEPES), L-glutamine, antibiotic-antimycotic (penicillin streptomyc i n), gentamycin, trypan blue, trypsin/EDT A, Dulbecco's phosphate buffered saline (DPBS) The following were obtained from the Sigma Chemical Corporation St. Louis, Missouri: paclitaxel, daunorubicin (DNR), doxorubicin verapamil, genistein, indomethacin, quinine, murine monoclonal anti-P-glycoprotein lgG1 (clone F4), rabbit ant i mouse lgG alkaline phosphatase conjugate, goat anti-rat lgG alkaline phosphatase conjugate, FITC-conjugated goat anti-mouse IgG FITC-conjugated rabbit anti rat IgG2a, mouse anti-alpha tubulin, aprotinin leupeptin, phenylmethylsulfonylfluoride (PMSF), Trizma [tris(hydroxymeth y l)aminomethane] acid, Trizma [tris(hydroxymethyl)aminomethane] base Triton X 100 RNase A, propidium iodide (PI), b 9 vine serum albumen (BSA) ( octylphenoxy)-polyethoxyethanol (NP-40), sodium deoxycholate (NaDOC), sodium dodecyl sulfate (SDS) tween 20, B-mercaptoethanol, bromophenol blue, ethylenediaminetetraacetic acid (EDTA), 3-4,5-dimethylthia z ol-2 ,5diphenyl tetrazolium bromide (MTT) nitro blue tetrazolium (NBT), 5 bromo-4-chloro-3indolyl phosphate (BCIP) The following were obtained from Signet Laboratories Inc. Dedham Massachusetts : murine monoclonal anti-P-glycoprotein mouse IgG2a (clone C219) rat monoclonal anti-multidrug resistance associated protein rat lgG2 a (clone MRP1). 14

PAGE 25

The following were obtained from Pharmingen International, San Diego California : rat anti keyhole limpet hemocyanin (anti-KLH), murine anti-human TNF alpha The following were obtained from Bio-Rad Laboratories, Hercules, California : 30% acrylamide/Bis solution (29: 1 ratio of acrylamide : N, N -methylenebisacrylamide ), nitrocellulose membranes (NC), polyvinylidene difluoride membranes (PVDF), high range alkaline phosphatase-conjugated biotinylated molecular weight protein for SDSp AGE, avidin conjugates, Coomassie brilliant blue. Slowfade was purchased from Molecular Probes, Inc., Eugene, Oregon. BCA Protein Assay Kit was obtained from Pierce, Rockford Illinois Costar Tissue culture flasks and 6-well plates were purchased from Corning, Inc., Corning, New York. Nalge 96-well microplates were purchased from Nunc International, Denmark. Cell Lines Four separate cell lines were used in this study: MES-SA (American Type Culture Collection, Manasses, VA; ATCC) is a uterine sarcoma cell line developed from a surgical specimen at the time ofhystorectomy (Harker etal., 1983) MES-SA/DX5 (ATCC) was developed from the MES-SA line by culture in increasing conc e ntrations of doxorubicin (Harker and Sikic 1985) The MES-SA/DX5 cell line expresses high levels ofP-gp and exhibits cross resistance to a number of cytotoxic agents including paclitaxel (Harker and Sikic, 1985). This cell line is still sensitive to discodermolide, indicating a difference in the mechanism of resistance to these two drugs A further MES-SA subline, MES-SA/DX5+D, was developed from MES SA/DX5 cells at the Harbor Branch Oceanographic Institution through the intermittent exposure to increasing concentrations of discodermolide over a period of 45 days This cell line shows resistance to both paclitaxel and discodermolide (unpublished data). An MRP+ pancreatic carcinoma cell line, PANC-1 was used as a positive MRP control. 15

PAGE 26

This cell line was developed from cancerous epitheliod pancreatic duct tissue (Lieber et al., 1975) These cells are not known to express P-gp, but show evidence of a drug efflux system consistent with MRP (Miller et al., 1996). 16

PAGE 27

METHODS Cell Culture All cell lines were maintained in RPMI-1640 tissue culture medium (TCM) containing 10% FBS, 18 mM HEPES buffer, 60 j..lg/ ml L-glutamine, antibiotic antimycotic (I 00 U / ml penicillin 100 j..lg/ml streptomycin), and 0.05 mg/ml gentamycin. P ANC-1 cells were grown in TCM which was also supplemented with 4.5 g/1 D-glucose, 1.0 mM sodium pyruvate, and 5 nM daunorubicin (DNR). MES-SNDX5+ D cells were maintained in TCM containing 5 nM discodermolide. Cells were cultured in 75 cm2 flasks and kept incubated at 37 and 5% C02 Cells were subcultured twice a week Cell lines were kept frozen until ready for use in FBS containing 10% dimethyl sulfoxide (DMSO) at -80C. Cells were quickly thawed by immersion in a 37C water bath slowly diluted in TCM and centrifuged at 3000 x g for 5 minutes. The cell pellets were then gently resuspended in TCM and transferred to a sterile 25 cm2 tissue culture flask containing an appropriate volume of fresh TCM. The cells were incubated at 37C, 5% C02 When the cells reached 95% confluency, they were passed into larger flasks for continued culture. TCM was removed from the cells by inversion, and 5 ml of trypsin/EDT A was added to the flask The cells were incubated for 15 minutes at 3 7 C, then transferred to a new flask containing 20 ml of fresh TCM. MES-SA, MES-SNDX5 and MES SNDX5+ D cell lines were passed at a dilution of 1:10 (I 0% of the previous passage was transferred to the new passage) PANC-1 cells were passed every 3-4 days at a dilution of 1 :20 Cell viability was determined by trypan blue exclusion and manual counting using a hemocytometer. Percent viability was also calculated using the following formula: #live cells I #total cells x 100 = %via bility Cells were used within 14 passages of the frozen cell culture. 17

PAGE 28

Drug Solutions Discodermolide was maintained in stock solution at a concentration of 1 mg/ml 95 % ethanol at -20 C. Paclitaxel stock solution was kept at -20C in 1 mg/ml DMSO. Verapamil stock solution was prepared prior to use at 10 mg/ml EtOH Doxorubicin stock solution was stored fro z en at 0.4 mg/ml sterile water Stock solutions of the compounds were diluted further in TCM prior to use. MTT Colorimetric Assay The MTT assay measures the mitochondrial conversion of the yellow tetrazolium salt 3-4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) to its insoluble purple formazan product and has been demonstrated to reflect viability of cell cultures (Mosmann, 1983; Twentyman et al., 1989). Cells were cultured in 96-well microplates at a density of 6,000 cells per well in TCM with a total volume of 200 111 per well. Cells were incubated at 37C, 5% C02 overnight to allow cells to adhere. Test agents at various concentrations were added to the cells the following day, and the cells were incubated for a further 24, 48 and 72 hours before MTT testing was performed Each cell plate was removed from the incubator app r ox i mately 3 hours prior to 24 48, or 72 hours after test agents were added. Seventy-five 111 of a 5 mg/ml MTT solution were added to all wells before returning the plate to the incubator for 3 hours Plates were centrifuged for 10 minutes at 220 x g. The supernatant was then removed from _the cell plate by inversion. Two-hundred 111 of acidified isopropyl alcohol (1 ml HCl/ 499 ml 2-propanol) was then added to all wells The cell plate was then placed on a plate shaker for 15 minutes. Absorption of plates was determined on a TECAN Spectra II plate reader (TECAN U S Research Triangle Park, NC) at 570 nrn with a reference filter of 650 nrn. Each assay was repeated 2-6 times with a sample size of 3 -5 wells per concentration point. The concentrations of each test agent which produces 50% cytotoxicity (IC5 0 values) were determined from the data, using numbers obtained from the wells containing doxorubicin as 100% cytotoxicity values, and numbers from wells containing cells alone as 0% cytotoxicity values. Numbers were accepted or rejected based on the Q-test (Zar, 1984). 18

PAGE 29

Immunoblotting Cells were cultured into 25 cm2 tissue-culture flasks at a density of 1.5 million cells per flask and a total volume of 6 ml. The flasks were incubated at 37 C, 5% C02 overnight to allow the cells to adhere. Cell protein samples were prepared the following morning and used immediately or aliquoted and frozen for later use A Preparation of Cell Lysate Samples Cells were briefly washed twice with 3 ml of ice-cold Dulbecco's phosphate buffered saline (DPBS) Three-hundred )ll of ice-cold Radio Immuno Precipitation Assay buffer / ethylenediamenetetraacetic acid (RIP NEDT A ; see Appendix A) lysis buffer plus protease inhibitors ( aprotinin leupeptin, PMSF) were then added to each flask, and the cells were scraped into the lysis buffer using a sterile c e ll scraper. The contents of each flask were transferred to a clean 1 5 ml microcentrifuge tube and placed on ice for 30 45 minutes The samples were centrifuged at 4 C for 15 minutes at 10 000-12 000 x g to pellet cell debris. The sup e rnatants were then transferred to fresh tubes and placed on ice ; pellets were discarded. B. Protein Determination Total protein cont e nt for each sample was determined usin g the BCA Protein A s say method. Diluent s olution w as pre par e d using RIP A buffer with protease inhibitors The working reagent was prepared accordin g to manufacturer s ins tructions. Two to twenty )ll of each sample or BSA standard were added to a 96 well plate. Two hundred )ll of working reagent w e r e then added to each well and mixed on a plate shaker for 30 seconds. The plate was incubat e d at 37 C for 30 minutes, and ab s orption was then determined on a TECAN Spectra IT plate reader at 570 nm. Protein concentrations of the sample s w e r e d e termined by comparison to a s tandard curve of known BSA concentrations. 19

PAGE 30

C. Protein Gel Electrophoresis The mini-gel electrophoresis apparatus (Bio-Rad) was cleaned and assembled according to manufacturer's instructions. Acrylamide solution (7.0% or 7.5%; see Appendix A) was prepared according to manufacturer's instructions and immediately poured into the gel casts (7 em x 8 em). The gels were allowed to polymerize for 30 60 minutes. A stacking gel (see Appendix A) was layered above the running gel and a comb was inserted to create 10 lanes (5 mm width) for sample addition The stacking gels were allowed to polymerize for approximately 30 minutes Cell samples were prepared by adding 2X sodium dodecyl sulfate (SDS) Laemmli sample buffer to each protein lysis sample in a 1:1 dilution, and heating them for three minutes in boiling water. A molecular weight standard was also prepared in a dilution of 1:20 in sample buffer. Samples were briefly centrifuged at 4C. Approximately 1530 J..Lg of total protein was loaded in each lane. Electrophoresis was conducted in IX running buffer at 200 volts for 45 60 minutes, until the toluene blue dye marker migrated to the bottom ofthe gel. D. Coomassie Blue Staining Coomassie staining was done to verify that all lanes contained equal amounts of protein. Polyacrylamide gels were carefully placed in Coomassie blue staining solution for 20 minutes, occasional stirring. The gel was washed 4 -5 times in destaining solutipn; 15 minutes per wash. The gel was placed in fixing/drying solution for 10 minutes. The gel was then carefully placed on blotting paper pre-wetted with deioni z ed water and dried in a Bio-Rad Model583 aspirating gel dryer for 90 minutes at 80C. E. Protein Transfer to Membrane All membrane transfers wer e performed using a Bio-Rad's mini gel electrophoresis apparatus. The gel to be transferred was carefully removed from the glass-plate cast and placed in transfer buffer (see Appendix A). A polyvinyl-difluoride (PVDF) or nitrocellulose (NC) membrane was pre wet in deionized water for 2 minutes Membrane, gel and filter papers were equilibrated in transfer buffer for 15 minutes at 20

PAGE 31

room temperature. The gel was transferred at 4 C for 1.5 hours at 100 volts (250 rnA) with stirring. F. Imrnunoblotting After transfer, the membrane was washed twice in TBS-T (see Appendix A), 2 minutes per wash. The membrane was blocked for 45 minutes in 10 ml of fresh TBST I milk (see Appendix A) at room temperature. The membrane was then incubated overnight in 10 ml ofTBS-T/milk containing P-gp or MRP antibody (primary antibody) in a 1:500 dilution. The membrane was placed on a plate shaker in a cold cabinet at 4C overnight. The membrane was washed five times in TBST the following morning with 2 minutes per wash at room temperature. The side part of the membrane containing the molecular weight standards was cut from the rest of the membrane containing the cell samples The membrane with cell samples was then incubated for 60 minutes at room temperature in 10 ml of fresh TBS-T/milk containing anti-mouse IgG or anti-rat IgG (secondary antibody) in a 1 : 1000 dilution, and the membrane with the weight standards was incubated separately in antibody buffer (1% bovine serum albumin in TBST) containing avidin conjugate in a 1:2000 dilution. The membrane was then washed five times in TBST 2 minutes per wash at room temperature. The blot was rinsed twice in alkaline phosphatase buffer (AP buffer) 2 minutes per riose at room temperature and developed in 5 ml of AP buffer containing 0.33 mg/ml nitro blue tetrazolium (NBT) and 0 17 mg/ml 5-bromo-4 chloro-3-indolyl phosphate (BCIP) at room temperature. After removal from the developing solution, the blots were placed in deionized water for 10 minutes to halt developing, changing the water once during this time. The blots were then allowed to dry. 21

PAGE 32

Microscopy P-gp Staining Cells were allowed to adhere overnight to coverslips placed in standard 6-well plates containing appropriate TCM (2 ml per well) at densities of 7x 104 to 1 .2x 1 os cells per well After removing TCM, the cells were rinsed briefly in DPBS at room temperature. The cells were fixed in 3.8 % formaldehyde /3% sucrose in DPBS (2 mllw e ll) for 10 minutes at room temperature. The fixative was removed and the cells were permeabili z ed for 5 minutes at room temperature in 2% Triton X-100 (in DPBS2 ml per well). The cells were then rinsed twice in DPBS. The fixed cells were incubated for 45 minutes in 2 ml of murine monoclonal anti P glycoprotein mouse IgG 1 (clone F4 ; primary antibody) in a 1 :500 dilution in DPBS, with gentle agitation every 15 minutes. Purified mouse anti human TNF-alpha was used as a negative control. The primary antibody solution was then removed and replaced with 2 ml ofDPBS containing FITC conjugated goat anti-mouse (secondary antibody) diluted 1:1000 DPBS. The cells were incubated at room temperature in the dark for 45 minutes, gently agitating the plate every 15 minutes. The secondary antibody solution was then removed and the cells were rinsed twice in DPBS. The cells were incubated for 30 minutes in 1 ml of PI solution (0 02 m g/ ml in DPBS) containing RNase A (0 1 mg/ml in DPBS) at 37 C in the dark Following this incubation the cells were rinsed twice in DPBS The coverslips were removed from the 6 w e ll plate drained, and allowed to air dry The cover slips were mounted onto slides with a drop of Slow Fade and sealed with clear nai l polish. Slides were examined immediately by epifluorescence and laser confocal microscopy using an Olympus Fluoview Scanning Laser Biological Microscope BX50 (Olympus America, Melville, NY) 22

PAGE 33

MRP Staining Cells were seeded onto coverslips as described above, then fixed in 0.5% formaldehyde /3% sucrose for 30 minutes at 4 C. The fixative was then removed and the cells were rinsed in DPBS. Two ml of rat monoclonal anti-multidrug resistance associated protein (primary antibody) diluted 1 : 500 in DPBS with 0.1% Triton X-1 00 were added. Purified rat anti-KLH was used as a control. The cells were incubated in the primary antibody solution for 60 minutes at 4 C with gentle agitation every 15 minutes The primary antibody was then removed, and the cells were again rinsed in DPBS Two ml ofFITC-conjugated anti-rat IgG2a (secondary antibody) diluted 1 : 1000 in DPBS were then added to each well. The cells were incubated with secondary antibody at 4C in the dark for 30 minutes with gentle agitation of the plate every 15 minutes. The secondary antibody solution was then removed and the cells were rinsed twice in PBS. The cells were incubated for 30 minutes in 1 ml of PI solution containing RNAse A at 37C in the dark Following this incubation, the cells were again rinsed twice in DPBS, and slides were prepared and examined as above. Tubulin Staining Cells were allowed to adhere to coverslips overnight in 6-well tissue culture plates containing 2 ml appropriate TCM at densities of 7x 104 cells per well. Test factors diluted in TCM were added to the cells the following day and incubated at 37 C for an additional 24 hO!lfS. After 24 hours, the TCM was removed from the wells. The cells were rinsed in DPBS and fixed in 2 ml of3.7% formaldehyd e/3% sucrose for 10 minutes at room temperature The fixative was removed and th e cells permeabili ze d in 2 ml of 2% Triton X-100 in DPBS for 5 minutes at room temperature The cells were then washed twice in DPBS. Mouse anti-alpha tubulin (primary anitbody) was diluted 1 : 1000 in DPBS was added and the cells were incubated for 45 minutes at room temperature, with gentle agitation every 15 minutes. The primary antibody solution was removed and replac ed with FITC conjugated goat anti-mouse IgG (secondary anitbody) diluted 1:1000 in DPBS The cell s wer e 23

PAGE 34

incubated for 45 minutes at room temperature in the dark, with gentle agitation every 15 minutes. Following the removal of the secondary antibody, the cells were rinsed with DPBS and incubated for 30 minutes in 1 ml of PI solution containing RNAse A at 37 C in the dark. The cells were then rinsed twice in DPBS, and the slides were prepared and examined as above. Flow Cvtometry MES-SA and P ANC-1 cells were cultured into 25 cm2 flasks and allowed to adhere overnight. TCM was removed from the flasks and the cells were washed twice in ice-cold DPBS Following these washes, 2 additional ml of ice-cold DPBS were added to each flask, and the flasks were placed flat in a cold cabinet ( 4 C) for 15 minutes. Cells were then scraped into the cold DPBS using a cell scraper. Cells were declumped by p i petting up and down several times in the corner of the flask, then counted using trypan blue exclusion Cells were diluted to 5 x 1 o5 cells/ml in cold DPBS containing 10% FBS and placed in 1.5 ml centrifuge tubes. The tubes were placed in a 37C water bath for 2 minutes before adding genistein in concentrations of350 400 450 and 500 After this addition, the tubes were returned to the warm water bath for 5 minutes. Two DNR was then added to the tubes and a further incubation of 60 minutes in the warm water bath followed with the gentle inversion of each tube every 20 minutes. After removing the tubes from the warm water bath, the samples were placed in ice for 1 minute and centrifuged at 3,000 x g, 4C for 10 minutes to pellet the cells. The supernatant was removed and the cells resuspended in 1 ml of cold DPBS. The centrifugation was repeated, and the cell pellets were resuspended in 0.5 ml of cold DPBS and placed on ice Samples were measured immediately by flow cytometry (Beckman Coulter Epics E lite, Miami, FL), with an excitation of 488 nm and an emmission of 575 nm. The samples were then incubated at 37C for an additional45 minutes, placed on ice and read a second time. 2 4

PAGE 35

RESULTS MTT Cytotoxicity Assays ( 48 hr.) Specific inhibitors ofP-gp (quinine) and MRP (indomethacin) were used to help elucidate the mechanism(s) of drug resistance to discodermolide in the parental MES-SA human uterine sarcoma cell line and the drug resistant MES-SA/DX5 and MES-SA/DX5+D sub lines and the human pancreatic carcinoma P ANC-1 cell line. Each cell line was incubated with various concentrations of discodermolide in the presence or absence of each specific inhibitor. The IC50 values indicated represent the concentration of discodermolide at which 50% inhibition of growth (Kowalski et al., 1997) was achieved for each cell line under the specific conditions. Data are shown for a 48 hour incubation. The results in Table 4 show that both quinine and indomethacin had little effect on the IC5o value of the MES-SA parental cell line, with IC5o values ranging from 77 nM for the control and 79 nM and 69 nM for quinine and indomethacin, respectively In the MES-SA/DX5 line, however, cells incubated with discodermolide alone yvere approximately 2-fold resistant to discodermolide compared to the parental cell line Addition of quinine to these cells resulted in a reversal of their resistance, with an IC5o of 56 nM, a value which approached that ofthe parental cell line (IC50= 77 nM). Indomethacin treatment had little effect on the ICso of discodermolide for these cells For the M E S-SA/DX5 + D cell line, cells incubated with discodermolide alone were again, approximately 2-fold resistant to discodermolide compared to the parental cell line However incubation with either quinine or indomethacin failed to reverse their resistance to discodermolide. P ANC-1 cells incubated with discodermolide were particularly sensitive to the effects of both quinine and indomethacin, as the ICso values approached 70-fold (quinine) to 700-fold (indomethacin) resistant compared to the PANC-1 control. The PANC-1 control showed sensitivity to discodermolide (1Cso=74 nM) which was similar to that of the MES-SA cell line (IC so= 77 nM) 25

PAGE 36

Table 4: ICsovalues for discodermolide (nM) with and without P-gp and MRP inhibitors -------------------------------------------------------------------------------------------------Cell Line Disco (SD) Disco+ 20flM Quin (SD) Disco+ 20flM Indo (SD) ---------................................................................................................................................................................................................................................... .. MES-SA MES-SAIDX5 MES-SAIDX5+D PANC-1 77 (44) 127 (67) 155 (46) 74 (63) 79 (6) 56 (14) 121 (64) 1.0 (1.4) 69 (63) 168 (66) 127 (43) less than 0.10 (0) ----------------------------------------------------------------------------------------------ICso values reported here are averages. Each assay was perform e d 2 6 times with a sample size of 3 5 we lls. Standard deviations (SD) are reported in parentheses. The same inhibitors ofP-gp and MRP were used to help elucidate the mechanism(s) of drug resistance to paclitaxel in these four cell lines. Each cell line was incubated with various concentrations ofpaclitaxel in the presence or absence of each specific inhibitor. Data is shown for a 48 hour incubation The results in Table 5 show that both quinine and indomethacin had little effect on the IC5o values of paclitaxel on the MES-SA parental cell line, with IC50 values ranging from 13 nM for the control and 7 nM and 38 nM for quinine and indomethacin, respectively. In the MES-SAIDX5 drug-resistant cell line, however, cells incubated with paclitaxel alone were approximately 11-fold resistant to paclitaxel compared to the parental cell line. Addition ofboth quinine and indomethacin resulted in a reversal of their resistance, with IC50 values of 63 nM and 45 nM, respectively For theMESSAIDX5 + D cell line, cells incubated with paclitaxel alone were approximately 14-fold resistant to paclitaxel compared to the parental cell line However, incubation with either quinine or indomethacin failed to reverse their resistance to paclitaxel. P ANC-1 cells incubated with paclitaxel had a sensitivity to paclitaxel (ICso= 20 nM) similar to that of the MES SA cell line (IC50= 13 nM). However, incubation with either quinine or indomethacin caused a sharp increase in resistance, as the ICso values approached 5-fold (indomethacin) to 38-fold (quinine) resistant compared to the PANC-1 control. 26

PAGE 37

Table 5: ICso values for paclitaxel (nM) with and without P-gp and MRP inhibitors Cell Line MES-SA MES-SA/DXS MES-SA/DXS + D PANC-1 Paclitaxel (SD) Paclitaxel + 2011M Quin (SD) Paclitaxe l + 401-LM Indo (SD) 13 (9) 146 (242) 176 (91) 20 (18) 7 (4) 63 (40) 189 (60) 755 (286) 38 (47) 45 (63) 198 (127) 100 (141) IC50 values reported here are averages. Each assay was performed 2 -6 times with a sample size of 3 -5 wells. Standard deviations (SD) are reported in parentheses. Figure 2 demonstrates the trends in cytotoxicity of discodermolide in each cell line when treated with varying concentrations of discodermolide. At the 50% cytotoxicity level it can be seen that of the three uterine sarcoma cell lines the MES-SA line shows the lowest concentration of discodermolide, followed by MES-SA/DX5 and MES SA/DX5+D Of these cell lines, MES-SA/DX5+D has the highest IC50 and reflects the highest degree of discodermolide resistance, whereas MES-SA is the most sensitive to discodermolide. % 100 c 90 y 80 t 70 0 60 t 50 0 40 X 30 c 20 10 t 0 y I Discodermolide Cell Cytotoxicity .. ......... ...................... .......................................................... .... .......... ........ : .. 0 200 400 600 800 1000 Conc.(nM) [j] MES-SA (!] MES-SA/DXS MES-SA/DXS+D Figure 2: Discodermolide Cell Cytotoxicity Cytotoxicity of each cell line when exposed to discodermolide. The point on the graph at which 50% cytotoxic ity is reached reflects the IC50 value in each cell line for discodermolide 27

PAGE 38

Immunoblotting P-gp Protein bands corresponding to 175 -186 kD appeared in the lanes containing the MES-SNDX5 and MES-SNDX5+ D cell lines (Figure 3). No signal was detected in the lanes from the MES-SA and P ANC-1 cell lines. Coomassie staining verified that an equal amount of prot ei n was loaded into all lanes. MW Standard DX5+D DX5 MES-SA PANC-1 Figure 3 : Immunoblot using P-gp antibody. T h e darker shaded section on the left s hows a lan e con t aining molecular weig ht standard. The two t o pmo s t bands in the lane s for MES-SAIDX5+D and MES-SAIDX5 correspond to 1 75186 kD. Arrows at left indicate MW standards in kD units. 28

PAGE 39

MRP Very strong protein bands corresponding to 194-196 kD appeared in the lanes containing the P ANC-1 cell line (Figure 4). Faint bands appeared in the same range in the lanes containing the MES-SA, MES-SA/DX5 and MES-SA/DX5+D cell lines, with the strongest of these faint signals occurring in the MES-SA/DX5+D cell line Coomassie staining verified that an equal amount of protein was loaded into all lanes PANC-1 MES-SA DX5 DX5 + D MW Standard 116 +-Figure 4 : Immunoblot u si ng MRP antibody. The darker s haded section on the right shows a lane containing molecular weight s tanda rd. The bands in the lane s for P ANC -1, MESSA, MES SA/DX5, and M E S-SA/DX5+D correspond to 1 94-196 kD. Arrows at right indicate MW s tandard s in kD units 29

PAGE 40

Microscopy P-gp MES-SA cells and P ANC-1 cells stained for P-glycoprotein displayed background fluorescence only (Figure Sa and Sg) MES-SA and PANC-1 control cells treated with purified mouse anti-human TNF-alpha appear e d simi lar (Figure Sb and Sh) MES-SNDXS cells and MES-SNDXS+D cells stained for P-gp displayed distinct bright green rin g s around cell membranes (Figure Sc and Se) These rings were absent in MESSNDXS and MES-SNDXS+D control cells treated with purified mouse anti-human TNF-alpha (Figure Sd and Sf) where only background fluorescence was visible. MRP P ANC-1 cells displayed the highest level of fluorescence of the four cell lines stained for MRP (Figure 6g) Fluorescence in these cells appeared as a bright speckling of the cytoplasm MES-SNDXS+D cells showed the same pattern of fluorescence as P ANC-1 cells but fluorescence in MES-SNDXS+D cells was much less intense than in PANC-1 cells (Figure 6e). No fluorescence appeared in PANC-1 and MES-SNDXS+ D control cells treated with purified rat anti-KLH (Figure 6h and 6f) The MES-SA and MES-SNDXS cell lines both showed a very faint speckling of fluorescence (Figure 6a and 6c) whereas the controls treated with purified rat anti-KLH were completely dark (Figure 6b and 6d) 30

PAGE 41

Fig u re 5 : A MES-SA cells stained for P-gp, B. MES-SA P-gp control C MES-SA/DX5 cells stained for P-gp, D MES-SA/DX5 P-gp control E MES-SA/DX5 + D cells stained for P-gp F. MES-SA/DX5+D P gp control G PANC 1 cells stained for P-gp H PANC 1 P-gp control. 31

PAGE 42

Figure 6 : A. MES-SA cells stained for MRP, B. MES-SA MRP control, C. MES-SA/DX5 cells sta in e d for MRP, D MES-SA/DX5 MRP co ntrol, E MES-SA/DX5+D cells stained for MRP F MES SNDX5+D MRP control, G. PAN C1 cells stained for MRP H PANC-1 MRP control. 32

PAGE 43

Tubulin MES-SA cells treated with 30nM discodermolide and stained for tubulin showed strong bundling ofmicrotubules (Figure 7a). Microtubule bundling remained strong when 40 !lM indomethacin (Figure 7c) or 40 !lM quinine (photo not shown) were added Control cells stained only for tubulin and not treated with discodermolide or inhibitors and those treated only with inhibitors and stained for tubulin showed normal cytoskeletal matrices. The nuclei had normal morphology and fibrular matrices were thin and hair like with no microtubule bundling visible. MES-SA/DX5 cells treated with 30nM discodermolide and stained for tubulin also showed strong microtubule bundling (Figure 7b). The degree ofbundling d i d not change appreciably when 40 !lM indomethacin (Figure 7d) or 40 !lM quinine (Figure 7f) were added. All controls (same as above) showed normal cytoskeletal matrices, with no microtubule bundling visible MES-SA/DX5+D cells treated with 30nM discodermolide and stained for tubulin showed a much lower degree of bundling than either MES-SA and MES-SA/DX5 cells (Figure 8a) When 40 !lM indomethacin was added, cells showed no more microtubule bundling than without indomethacin (Figure 8c) The degree ofbundling in MES SA/DX5+D cells was the highest in those cells treated with discodermolide and 40 !lM quinine (Figure 8e) MES-SAIDX5+D controls (same as above) all appeared normal P ANC-1 cells treated with 30nM discodermolide and stained for tubulin showed a high degree of microtubule bundling (Figure 8b ). The addition of 40 !lM indomethacin caused no more bundling ofmicrotublules than without indomethacin (Figure 8d) The degree of microtubule bundling was very high in P ANC-1 cells treated with discodermolide and 40 !lM quinine (Figure 8f) P ANC-1 controls (same as above) all appeared normal. 33

PAGE 44

Photo not available Figure 7: A. MES-SA cells treated with discodermolide and stained for tubulin B. MES-SA/DX5 cells treated with discodermolide and s tained for tubulin, C. MES-SA cells treated with discodermolide + indomethacin and stained for tubulin D. MES-SA/DX5 cells treated with discodermolide + indomethacin and stained for tubulin E Photo not available, F. MES-SA/DX5 cells treated with discodermolide + quinine and stained for tubulin G MES-SA cells stained for tubulin H. MES-SA/DX5 cells stained for tubulin 34

PAGE 45

Figure 8: A. MES-SAIDX5+D cells treated with discodermolide and stained for tubulin, B. PANC-lcells treated with discodermolide and stained for tubulin, C. MES-SA/DX5+D cells treated with discodermolide +indom ethaci n and s t ained for tubulin, D PANC-1 cells treated with discodermolide +indomethaci n and stained for tubulin, E MES-SA/DX5 + D ce ll s treated with discodermolide + quinine and st ain ed for tubu l in F. PANC-lcells treated with discodermolide +quinin e and stained for tubulin G. MES-SAIDX5 + D cells s tained for tubulin, H. PAN C-lcells stained for tubulin. 35

PAGE 46

Flow Cytometry MES-SA cells treated with DNR demonstrated a fluorescence peak around 101 log units, a value which remained unchanged after the second 45 minute incubation (data not shown). Fluorescence peaks remained around 101log units for MES-SA cells treated with DNR plus 350 JlM and 400 JlM, genistein after one incubation. Peaks obtained after the second incubation were slimmer than following the first incubation. Fluorescence peaks in MES-SA controls fell below 1 oo log units. P ANC-1 cells treated with DNR also demonstrated a fluorescence peak around 101 log units after the initial incubation (data not shown). This peak shifted to approximately 1 oo.s log units following the second 45 minute incubation. Fluorescence peaks occurred around 101 log units for P ANC-1 cells treated with DNR plus 350 JlM and 400JlM genistein. These peaks remained around 101 log units following the second incubation although they became somewhat slimmer. Fluorescence peaks in P ANC-1 controls fell below 1 oo log units. 36

PAGE 47

Iii A 8 IB D Figure 9: Flow cytometry images of MES-SA cells treated with DNR in the presence and absence of genistein A Cells treated with DNR after one incubation B. Cells treated with DNR after a second incubation C. Cells treated with DNR and 350 genistein D Cells treated with DNR and 350 genistein after a second incubation 8 "' D Figure 10 : Flow cytometry images ofPANC-1 cells treated with DNR in the presence and absence of genistein A Cells treated with DNR after one incubation B. Cells treated with DNR after a second incubation C Cells treated with DNR and 400 genistein D Cells treated with DNR and 400 genistein after a second incubation 37

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DISCUSSION MTT assays were performed to test the cytotoxicity of discodermolide and paclitaxel in the presence and absence ofP-gp and MRP-specific inhibitors. Indomethacin has previously been reported to be an MRP-specific inhibitor with an IC5o of 1020 J.!M for MRP and of>800 J.!M for P gp (Hollo et al., 1996) Quinine has previously been reported to be a P-gp-specific inhibitor with an IC50 of2030 J.!M for Pgp and of 50-100 J.!M for MRP (Hollo et al., 1996). Controls (cells treated only with inhibitors and stained for tubulin) were performed to eliminate the possibility of cytotoxicity caused by the inhibitors alone at these concentrations ICso values indicate that the MES-SA cell line has the highest sensitivity to both discodermolide and pacl i taxel of the uterine sarcoma cell lines (Tables 4 and 5). Values for MES-SA cells treated with quinine and indomethacin suggest that no active efflux pumps are present in this cell line. The MES-SNDX5 cell line, which has previously been shown to express P-gp (Harker and Sikic, 1985), appears to be two-fold more resistant to discodermolide and 22fold _more resistant to paclitaxel than its parent cell line (Tables 4 and 5). Quinine increased the sensitivity of these cells to discodermolide to levels similar to that ofMESSA cells (Table 4) and increased th eir sensitivity to paclitaxel nearly 5-fold (Table 5), indicating a synergistic effect between quinine and discodermolide likely via the inhibition ofP-gp. Indomethacin had no significant effect on IC50 values for theMES -SNDX5 cell line when treated with discodermolide (Table 4), but increased sensitivity to paclitaxel more than 6-fold (Table 5). However, paclitaxel is not known to be effluxed by MRP (Szakacs et al., 1998), and results from immunoblotting and microscopy in this study do not indicate a strong expression ofMRP in the MES-SNDX5 cell line 38

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The ICso value obtained for the MES -SNDX5+D cell line indicate a 2.5-fold resistance to discodermolide when compared to the MES-SA cell line (Table 4). Treatment with quinine increased their sensitivity to discodermolide 2.3-fold. Treatment with indomethacin had a similar effect, suggesting that both P-gp and MRP pumps may actively efflux discodermolide in this cell line. Controls performed using inhibitors alone indicated that there is no cytotoxic effect on cells from these compounds. Treatment with either inhibitor did not significantly alter sensitivity ofMES-SNDX5+D cells to paclitaxel (Table 5). However, data from Western blots (Figures 3 and 4) and fluorescence microscopy (Figures 5 and 6) indicate that the level ofP-gp is strong in this cell line and that a lesser level of MRP is also present Due to the high variability in results, cytotoxicity data for P ANC-1 cells from MTT assays is considered to be unreliable in this study. This cell line appears to have a high sensitivity to discodermolide, which was increased several hundred-fold by treatment with quinine (Table 4). No IC50 value for discodermolide could be obtained for this cell line when treated with indomethacin. Sensitivity of P ANC-1 cells to paclitaxel also appears to be high However treatment with both inhibitors appeared to decr ease their sensitivity to paclitaxel (Table 5) Results from imrnunoblots indicate that P-gp is present in the MES-SNDX5 and MES-SNDX5+D cell lines, but is absent in the MES-SA and P ANC-1 cell lines (Figure 3). The protein band which appeared in the lanes containing MES-SNDX5 and MESSNDXS+D samples occurred between 175 and 186 kD, which corresponds well to the molecular weight of 170 180 kD which has been reported for P-gp (Cassaza and Fairchild, 1996). These results correlate well with res ults obtained from microscopy. A ring of fluorescence was seen on the periphery of each MES-SNDX5 and MES SNDXS+D cell stained for P gp whereas only background fluorescence was seen in MES-SA and P ANC-1 cells when stained for P-gp (Figure 5) Based on these results it appears likely that only the MES -SNDX5 and MES-SNDX5+D cell lines express P-gp. A hi g h amount of protein of approximately 194 196 kD was detected in extracts from P ANC-1 cells using the MRP antibody, which corresponds well to the molecular weight of 190 kD reported for MRP (Bellamy, 1996). Faint bands appeared in the same range in the lanes containin g the MES-SA, MES -SNDX5 and MES-SNDXS+ D cell lines, with the strongest of these faint signals occurring in the MES -SNDX5+ D cell line (Figure 4). It appears, therefore, that the MES-SNDX5 and MES-SNDX5+ D cell 39

PAGE 50

extracts may express both P-gp and MRP. Other human cancer cell lines such as etoposide-selected H69 small cell lung cancer cells, have also been reported to coexpress both P-gp and MRP (Brocket al., 1995). The MES-SA/DX5+D cell line may contain higher levels ofMRP than theMES SA and MES-SA/DX5 cell lines, based on the intensity of the faint bands which appeared at 194 196 kD in the lanes containing samples of these three cell lines. Results obtained from microscopy supports the data from immunoblots P ANC-1 cells stained for MRP showed intense fluorescent speckling of the cytoplasm Faint speckling was seen in all three of the other cell lines but MES-SA/DX5+D cells showed more fluorescence than either the MES-SA or MES-SA/DX5 cell lines (Figure 6). In this study, fluorescence in cells stained for MRP appeared as a bright green speckling of the cytoplasm. In a previous study using similar methods, multi-drug resistant H69AR cells and MRP transfected T5 cells displayed a ring of fluorescence on the periphery of each cell membrane when stained for MRP (Hipfner et al., 1994) Despite this difference in results, it is likely that the fluorescence which resulted in cells stained for MRP in this study reflects MRP in these cells rather than background fluorescence because control cells stained with anti-KLH of the same antibody class appeared completely dark. Results from microscopy confirm that the P ANC-1 cell line expresses high levels of MRP, and that each of the other three cell lines used in this study may express low levels of this protein with the MES-SA/DX5+D cell line expressing the highest of these low levels (Figure 6). Bundling of micro tubules in MES-SA cells treated with 30 nM discodermolide and stained for tubulin suggests a high degree of sensitivity in this cell line to discodermolide (Figure 7) Microtubule bundling remained strong when 40 f.J.M indomethacin or 40 f.J.M quinine were added, indicating that these cells do not have active efflux pumps which are blocked by these inhibitors. No difference was observed in controls which tested the effect of each inhibitor alone in these cell lines, indicating that these compounds do not alter the cytoskeletal matrix. Microtubule bundling in MES-SA/DX5 cells treated with 30 nM discodermolide and stained for tubulin suggests that this cell line is also sensitive to discodermolide though less sensitive than its parent cell line (Figure 7) Although this cell line is reported to express P-gp, the addition of quinine, a P-gp inhibitor (Hollo et al., 1996) did 40

PAGE 51

not appear to increase sensitivity to discodermolide in these microscopy experiments (Figure 7). This could either suggest that discodermolide is not effluxed by the P-gp pump or that quinine simply did not inhibit the P-gp pump in this cell line It is also possible that although a slight MRP presence is suggested by MRP-staining microscopy, the protein is not active in this cell line. No difference in degree of bundling was seen in this cell line with the addition of 40 )..lM indomethacin (Figure 7), a result which could either suggest that discodermolide is not effluxed by MRP, or that these cell lines express a degree ofMRP too low to measure through inhibition of the pump It is important to note that microscopy is too subjective to compare small differences among cells Microscopy prov ides a qualitative visual rather than a quantitative assessment of cell structures The lesser degree of microtubule bundling which resulted in MES-SAIDXS+D cells treated with 30 nM discodermolide and stained for tubulin than that obtained for either of the two cell lines discussed above supports previous work indicating the resistance of this cell line to discodermolide (Isbrucker and Longley personal communication) The addition of an MRP inhibitor had no visible effect on the degree of bundling in these cells (Figure 8). This indicates that either discodermolide is not effluxed by MRP, or that the degree of MRP expression in these cells is too low to appreciate through microscopy. It also remains possible that MRP may be present, but not active in this cell line The addition of a P-gp inhibitor, however, caused such an increase in microtubule bundling in these cells, that it appears likely that discodermolide may be effluxed by P-gp despite the results obtained from MES-SA/DX5 cells (Figure 8). P ANC-1 cells treated with 30 nM discodermolide and stained for tubulin appear to have a high sensitivity to discodermolide (Figure 8) The addition of 40 )..lM indomethacin caused no visible increase in bundling of microtubules P ANC-1 cells are reported to have high MRP expression Therefore it appears that discodermolide is not effluxed by MRP The degree of microtubule bundling was very h i gh in P ANC-1 cells treated with discodermolide and 40 )..lM quinine a strange result since this cell line is not known to express P-gp (Figure 8), nor was P-gp detected by microscopy (Figure 5) or immunoblot (Figure 3) Flow cytometry e x periments were performed for the purpose of determining whether efflux pumps a re active in these four cell lines Measurements taken after the first incubation reflect drug a ccumulation within the cell, whereas those taken following a 41

PAGE 52

second incubation should reflect drug retention by the cell. However, due to time constraints and technical difficulties, experiments were only performed using the MES SA and P ANC-1 cell lines to investigate the functionality of the MRP pump. The shift in fluorescence peaks in P ANC-1 cells indicates that DNR was lost from these cells after a second incubation period (Figure 1 Oa and 1 Ob ). This suggests that MRP actively effluxes DNR, a known MRP substrate (Feller et al., 1995b ), from P ANC-1 cells. That this pump is blocked by genistein (Feller et al., 1995b) is supported by the lack of any shift in fluorescence peaks following the second incubation when 350 J.l.M and 400 J.l.M genistein were present. Slimming of peaks after the second incubation period is most likely a result of normal cell leakage Flow cytometry experiments using MES-SA cells indicate that no MRP pump is active in this cell line. No shifting of fluorescence peaks occurred from the first incubation to the second, in the absence or the presence of genistein (Figure 9) This result alone indicates that this cell line does not have an active MRP pump. However, results obtained through Western blotting (Figure 3) and fluorescence microscopy (Figure 5) indicate that a low level of this protein is present in this cell line, though not necessaril y active From the combinat i on of results obtained in this study, the evidence supporting MRP as the mechanism ofMDR in tumor cells resistant to discodermolide is not strong. It appears likely that discodermolide may be effluxed by P-gp, rather than MRP. The MES-SA/DX5 cell line is known to be P-gp positive (Harker and Sikic, 1985) while still sensitive to discodermolide (Isbrucker & Longley, personal communication), but it is possjble that exposure of these cells to discodermolide causes an increase in P-gp expression. Ifthis is the case, the MES / SA/DX5+D cell line may simply express higher levels ofP-gp than its parent cell line It is also po s sible that discodermolide is pumped by P-gp If paclitaxel has a higher affinity for P-gp than discodermolide, this may explain why the MES-SA/DX5 cell line has a higher resistance to discodermolide than its sensitive parent cell line but is still relatively sensitive to discodermolide compared to the MES-SA/DX5+D cell line MES SA/DX5+D cells may simply express P-gp at levels high enough to confer resistance to both drugs. 42

PAGE 53

However, data from Western blots did not indicate a higher level ofP-gp in the MES-SA/DX5+D cell line than in the MES-SA/DX5 cell line, based on the intensity of the bands (Figure 3). Similarly, microscopy did not reveal a difference in the intensity of fluorescence between these two cell lines when stained for P-gp (Figure 5). That the addition of the P-gp inhibitor quinine to MES-SA/DX5+D cells treated with discodermolide and stained for tubulin caused a visible increase in microtubule bundling whereas the addition of the MRP inhibitor indomethacin appeared to have no visible effect also supports P-gp as the mechanism of resistance to discodermolide (Figure 8). Summary Results from the various tests performed in this study correspond fairly well. The MES-SA cell line was shown to be P-gp-negative and slightly MRPpositive through immunoblotting and P-gp-IMRP-staining microscopy {Table 6). Tubulin-staining microscopy and MTT cytotoxicity data indicated that this cell line is sensitive to discodermolide, with no effect seen when a P-gp or MRP inhibitor is present {Tables 7 and 8). The MES-SA/DX5 cell line was shown to be P-gp-positive and slightly MRP positive through immunoblotting and P-gp-IMRP-staining microscopy {Table 6). Tubulin-staining microscopy and MTT cytotoxicity data suggest that this cell line has intermediate sensitivity to discodermolide relative to the MES-SA parental cell line (Tables 7 and 8). Full sensitivity was restored to these cells by the addition of quinine. obtained from the addition of indomethacin, however, are conflicting MTT cytotoxicity data showed that this cell line retains its resistance when the MRP inhibitor is added (Table 7), whereas tubulin staining microscopy suggests that these cells become sensitive when indomethacin is present {Table 8) The MES-SA/DX5 + D cell line was shown to be P-gp-positive and slightly more MRP-positive than the MES-SA and MES-SA/DX5 cell lines through immunoblotting and P-gp-/MRP-staining microscopy (Table 6) Tubulin-staining and MTT cytotoxicity data indicated that this cell line is resistant to discodermolide {Tables 7 and 8). The addition of the P-gp inhibitor, quinine, appeared to reverse this resistance, whereas the MRP inhibitor, indomethacin did not. 43

PAGE 54

The P ANC-1 cell line was shown to be P-gp-negative and strongly MRPpositive through immunoblotting and P-gp-/MRP-staining microscopy (Table 6). Tubulin staining microscopy and MTT cytotoxicity data suggested that this cell line is sensitive to discodermolide (Tables 7 and 8). Results differed for tubulin-staining microscopy and MTT cytotoxicity data when inhibitors were added Tubulin-staining microscopy indicated that these cells remained when either quinine or indomethacin were added (Table 8), whereas MTT data indicated that the cells became resistant, a result which is attributed to error (Table 7). Table 6 : Presence and absence ofPgp and MRP in each cell line Irnrnunoblotting P-gp Microscopy MRP Micro scopy MES-SA P-gp0 MRP+ P-gp0 MRP + MES-SA/DX5 P-gp ++++ MRP + P -gp ++++ MRP+ MES-SAJDX5 + D P-gp ++++ MRP++ P-gp ++++ MRP++ PANC-1 P-gp0 MRP ++++ P-gp0 MRP ++++ Summarizations are based on results from irnrnunoblotting, P-gpand MRP-staining micro scopy + +++ represents the highest le ve l of protein ; + represents the lowest level of protein P-gn0 represents no detectable protein 44

PAGE 55

Table 7: MIT-based drug sensitivity in each cell line MES-SA MES-SA/DX5 MES-SA/DX5+D Discodermolide w / Quinine w / lndomethacin Sensitive Intermediate Sensitive Sensitive Sensitive Resistant Resistant Intermediate Intermediate PANC-1 Sensitive Sensitive Sensitive Summarizations are based on MTT cytotoxicity data using each cell line with discodermo lid e in the presence or absence of20 jlM P-gp and MRP inhibitors. Table 8 : Microscopy-based drug sensitivity of each cell line MES-SA MES-SA/DX5 D iscodermo I ide w / Quinine w / lndomethacin Sensitive Sensitive Sensitive MES-SA/DX5+D PANC-1 Sensitive Resistant Sensitive Sensitive Sensitive Resistant Sensitive Sensitive Sensitive Summarizations are based on tubulin-staining microscopy data using each cell line with discodermolide in the presence or absence of 40 jlM P-gp and MRP inhibitors. Conclusions It is possible that resistance t o discodermolide is conferred by a mechanism other than the P-gp or MRP efflux pumps Because of their similar mechanisms of action, attention should be paid to the mechanisms of paclitaxel resistance, as similar changes may occur in cells resistant to discodermolide There is evidence that cells are able to alter their relative tubulin isotype composition in response to drug exposure, which presents one mechanism ofMDR in paclitaxel resistant cells (Haber, et al., 1995). 45

PAGE 56

The LRP and cMOAT efflux pumps offer other possibilities, as do alterations in cellular metabolism, enhanced ability of the cell to repair DNA damage, failure to undergo apoptosis, increased levels ofGSH, and abnormalities in the expression ofthe p53 tumor suppressor gene, as well as other mechanisms not considered here The possibility also exists that resistance to discodermolide is conferred by some as yet unidentified mechanism It does appear, however that P-gp plays some role in resistance to discodermolide, and that MRP can be rejected as the primary cause. Suggestions for Further Study There are a number of experiments which could give more certainty to the results obtained in this study Flow cytometry or other active efflux experiments involving all four cell lines, pump-specific substrates and various inhibitors need to be performed in order to fully test the functionality of both the P-gp and MRP efflux pumps Tubulin staining microscopy was performed only with cells treated with discodermolide These experiments need to be repeated using paclitaxel for purposes of comparison. Beyond these experiments a wide range of tests are possible which could provide a more complete picture of drug resistance involving discodermolide Western blotting should be performed to test for the presence of other efflux proteins, such as LRP and cMOAT. Mechanisms of resistance involving the p53 tumor suppressor gene should be explored, as well as the other mechanisms of resistance mentioned above Since there is evidence for the expression of a drug-binding P-glycoprotein in marine sponges (Kurelec, 1992), it would be interesting to perform efflux assays using discodermolide paclitaxel and various inhibitors in sponge cells, particularly Discodermia dissoluta The presence of such an efflux protein in this species would first need to be identified Such experiments may provide further evidence supporting P glycoproteins as a broad taxonomic defense mechanism against the toxic effects of harmful chemicals both intrinsic and extrinsic, and offer additional insight into the role played by drug efflux proteins in cancer chemotherapy. 46

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APPENDICES 53

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Appendix A: Immunoblotting Solutions RIPA Lysis Buffer I50 mMNaCl I% NP-40 0.5%NaDOC 0.1% SDS 50 mM Tris, pH 8 0 2 mM EDTA, pH 8 0 2X SDS Laemmli Sample Buffer 67 mM Tris HCl, pH 6 8 2%SDS 0.1% Bromophenol Blue 0 2% B-mercaptoethanol I 0% Glycerol dH20 to 10 ml Coomassie Brilliant Blue O.I% Coomassie 40% Methanol I 0% Acetic acid 50% dH20 54 Protease fuhibitors 3 Aprotinin 10 Leu pep tin 100 PMSF 5X Running Buffer 15. 1 g Tris base 72.0 g Glycine 5.0 g SDS dH20 to 1 liter Destaining Solution 40% Methanol 1 0% Acetic acid 50%dH20

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Fixing and Drying Solution 10% Ethanol 4% Glycerol dH20 to 500 ml TBS-T 50 mM Tris, pH 7.4 200mMNaCl 0.1% Tween-20 dH20 to 500 ml TBS-T / 10% Milk TBS-T 1 0% Nonfat dry milk powder Appendix A: (continued) 55 Transfer Buffer 25 mM Tris 0.7 M Glycine dH20 to 1 liter pH to 7.2-7 3 at 25 oc Alkaline Phosphatase Buffer 100 mM Tris, pH 9.5 100 mM NaCl 5 mMMgCl2 dH20 to 500 ml Developing Solution AP Buffer 0 .33 mg/ml NBT 0.17 mg/m l BCIP

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Appendix B : Polyacrylamide Gel Protocols Composition of7. 50% Running Gel 20% Acryl/0.8% Bis 1 25 ml 1M Tris HCl, pH 8.8 1.88 ml dH20 1 80 ml 10% SDS 50 ).!1 10%APS 25 ).!1 TEMED 2 5 )ll Total volume 5 ml/gel Composition of 4 % Stacking Gel 30% Acryl/0.8% Bis 0 .33 ml 1M Tris HCl pH 6 0 0.31 ml dH20 1.83 m1 10 % SDS 25 ).!1 10%APS 12 5 ).!1 TEMED 1.25 ).!1 Total volume 2 5 ml!gel 56


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