Cytotoxicity Of Vincristine Research Articles

99mTc-sestamibi is a substrate for P-glycoprotein and the multidrug resistance-associated protein.

99mTc-sestamibi (99mTc-MIBI) is a substrate for the P-glycoprotein (P-gp) pump but it is not known whether it is a substrate for the multidrug resistance-associated protein (MRP) pump. Therefore, 99mTc-MIBI was evaluated in the GLC4 cell line and its doxorubicin-resistant MRP-, but not P-gp-, overexpressing GLC4/ADR sublines as well as in the S1 cell line and its MRP-transfected subline S1-MRP. 99mTc-MIBI concentration decreased in the GLC4/ADR sublines with increasing MRP overexpression and was lower in S1-MRP than in S1. 99mTc-MIBI plus vincristine increased 99mTc-MIBI concentration in GLC4 lines compared with 99mTc-MIBI alone. 99mTc-MIBI efflux raised with increasing MRP expression in the GLC4 lines. Glutathione depletion elevated 99mTc-MIBI concentration in GLC4/ADR150x. Cross resistance for 99Tc-MIBI, used to test cytotoxicity of the Tc compound, was observed in GLC4/ADR150x vs GLC4. 99Tc-MIBI induced a synergistic effect on vincristine cytotoxicity in GLC4/ADR150x. These results show that 99mTc-MIBI is involved in MRP-mediated efflux. The fact that 99mTc-MIBI efflux is influenced by MDR1 and MRP expression must be taken into account when this gamma-rays-emitting complex is tested for tumour efflux measurements.

Enhancement of drug accumulation by andrastin A produced by Penicillium sp. FO-3929 in vincristine-resistant KB cells.

In the course of our screening for compounds that reverse multidrug resistance, we found that the cytotoxicity of vincristine was enhanced 1.5-20-fold depending on the concentration of andrastin A in vincristine-resistant KB cells (VJ-300). Andrastin A alone had no effect on the growth of drug sensitive KB cells and VJ-300 cells. On the other hand, andrastin A (25 and 50 micrograms/ml) significantly enhanced accumulation of [3H]vincristine in VJ-300 cells. Andrastin A (50 micrograms/ml) completely inhibited the binding of [3H]azidopine to the P-glycoprotein in VJ-300 cells. The result suggests that andrastin A directly interacts with P-glycoprotein and inhibits the efflux of antitumor agents in drug resistant cells.

Modulating activity of indomethacin to vincristine cytotoxicity in various human carcinoma cells.

The modulating activity of indomethacin to vincristine (VCR) was investigated in thirty human pulmonary carcinoma cell lines (adenocarcinoma 9, large cell carcinoma 9, squamous cell carcinoma 6, small cell carcinoma 6) and five other cell lines (colon carcinoma 2, melanoma 1, teratocarcinoma 1, thymoma 1). The survival of these cell lines treated with VCR with or without indomethacin (2 microg/ml) for 72 hours were examined using MTT assay, and IC50 values were calculated. When the cutoff level of potent combined effect in clinical use was set at 2-fold increase of sensitivity, the positive rate was 100% for adenocarcinomas and large cell carcinomas, 25% for squamous cell carcinomas, 33% for small cell carcinomas. Mean modulating index was 2.91 in adenocarcinomas, 1.92 in squamous cell carcinomas, 3.06 in large cell carcinomas and 1.67 in small cell carcinomas. Of the cell lines of other tumors, three cell lines (colon carcinoma 1, melanoma 1, teratocarcinoma 1) showed the potent combined effect of VCR and indomethacin, while indomethacin was not effective in modulating activity to VCR in a thymoma cell line and fibroblast cells. In conclusion, it is considered that modulating activity of indomethacin for VCR is a general effect for various human cancer cells, and combined use with VCR and indomethacin may be a useful modulation therapy for the advanced lung cancer.

P-5-560 - High concentration of daunorubicin and daunorubicinol in human malignant astrocytomas after systemic administration of liposomal daunorubicin

The role of non-caspase protease activation in drug-induced cell death of glioma cells was examined. Neither calpain inhibitors I or II, phenylmethylsulfonyl fluoride (PMSF), Nα -p-tosyl-l-lysine chloromethyl ketone (TLCK), N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), E64, leupeptin nor pepstatin inhibit the cytotoxicity of vincristine, cisplatin, doxorubicin, cytarabine, camptothecin, BCNU or VM26 in two malignant glioma cell lines, T98G and LN-229. However, DNA fragmentation induced by VM26 is inhibited by calpain inhibitor I, PMSF, TLCK and TPCK, and that induced by camptothecin by calpain inhibitors I and II and TPCK. Moreover, protease inhibitors fail to abrogate CD95 ligand-induced apoptosis even though DNA fragmentation is attenuated by calpain inhibitor II and TPCK. Thus, non-caspase protease activation is not required for drug-induced apoptosis of glioma cells. Protease inhibitor-mediated inhibition of DNA fragmentation operates downstream of the commitment point for cell death.

P-5-559 - Local chemotherapy of cerebral glioma during operation

The role of non-caspase protease activation in drug-induced cell death of glioma cells was examined. Neither calpain inhibitors I or II, phenylmethylsulfonyl fluoride (PMSF), Nα -p-tosyl-l-lysine chloromethyl ketone (TLCK), N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), E64, leupeptin nor pepstatin inhibit the cytotoxicity of vincristine, cisplatin, doxorubicin, cytarabine, camptothecin, BCNU or VM26 in two malignant glioma cell lines, T98G and LN-229. However, DNA fragmentation induced by VM26 is inhibited by calpain inhibitor I, PMSF, TLCK and TPCK, and that induced by camptothecin by calpain inhibitors I and II and TPCK. Moreover, protease inhibitors fail to abrogate CD95 ligand-induced apoptosis even though DNA fragmentation is attenuated by calpain inhibitor II and TPCK. Thus, non-caspase protease activation is not required for drug-induced apoptosis of glioma cells. Protease inhibitor-mediated inhibition of DNA fragmentation operates downstream of the commitment point for cell death.

Augmentation of vincristine cytotoxicity by megestrol acetate.

To determine the effect of a semisynthetic progesterone, megestrol acetate (MA), on the cytotoxicity of various chemotherapeutic agents including vincristine, doxorubicin, actinomycin-D, taxol, vinblastine and colchicine in cell lines with or without P-gp expression. Three cell lines with high P-gp expression (two colon cancer and one leukemia), and a control cell line with no P-gp expression were exposed to chemotherapeutic agents in the presence or absence of MA and drug sensitivity was determined using the MTT colorimetric assay. P-gp-170 expression was detected by flow cytometry using JSB-1 monoclonal antibody and the functionality of MDR expression was tested by rhodamine-123 uptake studies. In vitro drug accumulation studies were performed using [3H]-vincristine. The results were subjected to paired t-test analysis and 95% confidence intervals were determined in cytotoxicity tests. MA augmented the cytotoxicity of vincristine, but not doxorubicin, actinomycin-D, taxol, vinblastine or colchicine in the three P-gp-expressing cell lines, whereas verapamil augmented the cytotoxicity of doxorubicin and vincristine. MA did not augment the cytotoxicity of vincristine in the P-gp-negative HUT-102 cell line. MA augmented vincristine cytotoxicity in P-gp-expressing cell lines. However, this phenomenon did not occur with the other classic MDR drugs. Therefore, the augmentation of vincristine cytotoxicity by MA can be explained either by involvement of a different mechanism that coexists with the mdr-1 phenotype or by the presence of a different affinity or binding site on the P-gp molecule for MA compared to that for the other classic MDR drugs and verapamil.

Indomethacin enhances the cytotoxicity of VCR and ADR in human pulmonary adenocarcinoma cells.

The ability of anti-inflammatory agents to modulate cellular sensitivity to anticancer drugs was investigated for pulmonary carcinoma cells in vitro. We examined the drug sensitivity of two pulmonary adenocarcinoma cell lines (76-2, 77-4) in the presence of two drugs, an anticancer drug and an anti-inflammatory agent, for 72 hr by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with 96 well plates. Anticancer drugs used for screening test were cyclophosphamide (CPM), mitomycin C (MMC), adriamycin (ADR), 5-fluorouracil (5FU), vindesine (VDS), cisplatin (CDDP), cytarabine (Ara C), methotrexate (MTX), etoposide (VP-16), and vincristine (VCR). Anti-inflammatory agents examined as modulators to anticancer drugs were aspirin, mefenamic acid, ibuprofen, sulindac, piroxicam, phenacetin, dicrofenac, ketoprofen, tolmetin and indomethacin. Screening tests showed indomethacin to be the most effective modulator, resulting in more than a 3-fold increase in cytotoxicity of VCR as compared with that produced by VCR alone. Study of each of the ten anticancer drugs in combination with indomethacin showed VCR to be the most effective anticancer drug in this combination. In 76-2 cells, the concentration of VCR producing 50% growth inhibition (IC50) for VCR alone and VCR in combination with 2 micrograms/ml indomethacin were 1.58 +/- 0.16 and 0.52 +/- 0.1 ng/ml respectively, which represents a 3-fold decrease. In 77-4 cells, the IC50 for VCR alone and VCR in combination with 2 micrograms/ml indomethacin were 2.86 +/- 0.2 and 0.52 +/- 0.11 ng/ml respectively, which represents a 3.8-fold decrease. Our studies indicate that clinically achievable concentrations of indomethacin may be useful in modulating VCR resistance in human pulmonary adenocarcinoma cells, so that combined use of VCR and indomethacin may be of potential clinical significance in the treatment of lung cancer.

Circumvention of drug resistance in cancer cells by a new fluoropyrimidine analogue

The potentiation of antitumor drugs by their analogues is one of strategies for overcoming cellular resistance to chemotherapy. In attempts to improve the efficacy of 5-fluorouracil (5-FU) and overcome 5-FU resistance, we synthesized new 5- FU derivatives and evaluated the potentiation effect on 5-FU cytotoxicity. 5-Fluorouracil-1-yl-3-butanone (5-FUBO), a newly synthesized 5-FU derivative, significantly potentiated the cytotoxic effect of 5-FU, and the potentiation of 5-FU by 5-FUBO in 5-FU resistant cells was higher than in their drugsensitive parental cells. Surprisingly, it potentiated the cytotoxicities of vincristine and vinblastine. The potentiation of these drugs by 5-FUBO in mutidrugresistant (MDR) cells was more than in their drugsensitive parental cells. It was also shown that 5-FUBO increased the intracellular accumulation of [3H]vincristine in P388/M cells, and this is related with increased cytotoxicity of vincristine. Northern blot analysis showed that the potentiation effect of MDR-related antitumor drugs by 5-FUBO was caused by inhibition of MDRl gene expression. Also, 5-FUBO cytotoxicity in K562 cells was increased significantly upon transformation with c-Ha-ras.

Enhancement of Vincristine Cytotoxicity in Drug-Resistant Cells by Simultaneous Treatment With Onconase, an Antitumor Ribonuclease

Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Anti-tumor ribonuclease, combined with or conjugated to monoclonal antibody MRK16, overcomes multidrug resistance to vincristine in vitro and in vivo

Onconase, a ribonuclease isolated from Rana pipiens oocytes and early embryos, is a member of the RNase A superfamily. Onconase has anti-neoplastic properties both in vitro and in vivo, and is undergoing clinical evaluation. In the present study, Onconase was combined with or conjugated to MRK16, an anti-P-glycoprotein (Pgp) monoclonal antibody. The interaction of these combinations with vincristine (VCR) against parental and multidrug resistant (MDR), Pgp expressing, human colon carcinoma cells caused increased VCR cytotoxicity in vitro and enhanced survival of athymic nude mice given transplants of drug resistant HT-29(mdr1) cells in vivo. The results suggest that combination treatment with Onconase and other agents that modulate the chemosensitivity of Pgp-expressing human tumor cells has the potential to overcome MDR.

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies.

Expression of Bcl-xL Can Confer a Multidrug Resistance Phenotype

It has been suggested that genes that regulate apoptotic cell death may play an important role in determining the sensitivity of tumor cells to chemotherapy. We have recently cloned a member of the bcl-2 family, bcl-x. To test whether bcl-XL expression affects the sensitivity of tumor cells to chemotherapy, we have created stable cell lines overexpressing bcl-XL and have tested these cells for resistance to cell death induced by metabolic inhibitors and chemotherapeutic agents. Bcl-XL expression dramatically reduces the cytotoxicity of bleomycin, cisplatin, etoposide, vincristine, hygromycin B, and mycophenolic acid for up to 4 days in culture. Bcl-XL does not prevent cells from undergoing cell cycle arrest in response to these drugs, but rather prevents treated cells from undergoing apoptosis. Cell-cycle analysis on cells treated with the chemotherapeutic agents bleomycin, cisplatin, etoposide, and vincristine, show that the drugs cause growth arrest in different positions within the cell cycle. Bcl-XL expressing cells treated with chemotherapeutic drugs retain their proliferative ability after the drugs are removed. Interestingly, vincristine-treated cells expressing bcl-XL become polyploid after drug removal. These data show that bcl-XL protects cells from a wide variety of apoptotic stimuli, acts in multiple positions within the cell cycle, and confers a multidrug resistance phenotype. The ability of bcl-XL to prevent apoptotic cell death in response to chemotherapy-induced DNA damage and cell-cycle arrest may contribute to the accumulation of chromosomal aberrations within tumors. The expression of bcl-XL in tumor cells is likely to be an important indicator of chemotherapeutic efficacy.

Microtubule Changes in Hematologic Malignant Cells Treated with Paclitaxel and Comparison with Vincristine Cytotoxicity

ABSTRACT Using immunofluorescence microscopy with an anti-alpha tubulin monoclonal antibody, we examined the microtubule changes induced by paclitaxel in pathologic cells from 38 hematologic malignancies, and compared the morphologic and cytotoxic changes with those induced by vincristine in vitro. Malignant cells cultured without paclitaxel or vincristine (controls) showed well-developed microtubules radiating from a microtubule organizing center (MTOC). Malignant cells cultured with paclitaxel showed bundling of microtubules and those cultured with vincristine showed crystal formation of the microtubules. In 28 lymphoid malignancies, the difference in the percentage of cells showing microtubular changes with paclitaxel and vincristine was significant (paired t test) after 2h P<0.01, after 4h P<0.05, and after 20h P<0.05. As paclitaxel is not in clinical use for the treatment of hematologic malignancies in Japan, no clinical material was available. In 6 out of 9 patients with lymphoid malignancies with a previous history of combined chemotherapy including vincristine, paclitaxel produced microtubular changes in a higher percentage of cells than did vincristine. In 10 myeloid malignancies, the percentages of the cells with microtubular changes induced by paclitaxel was also higher than those induced by vincristine (P<0.05 in 2 h, P<0.01 in 4h, and P<0.05 in 20h). Although the number of studied cases was small, this suggests that paclitaxel is as effective as vincristine in certain hematologic malignancies. As microtubular changes induced by paclitaxel and vincristine are easy to assess, the study of microtubular changes induced in vitro by antimicrotubular agents may prove useful in predicting the chemotherapeutic effect in vivo of these agents.

Novel nucleoside transport inhibitors of natural origin.

The salvage pathway of nucleotide biosynthesis is one of the attractive targets for cancer chemotherapy. Dipyridamole, a nucleoside transport inhibitor, may be used for blocking the salvage pathways. Studies have demonstrated that dipyridamole can reduce the rescue effect of exogenous nucleosides and potentiated the cytotoxicity of acivicin, an antimetabolite of de novo pathways of nucleotide biosynthesis, in hepatoma cells1, 2. The inhibitory effect of dipyridamole was cell growth phase -dependent. Cultured hepatoma cells in lag and log phase were highly sensitive to dipyridamole; by contrast, cells in stationary phase were insensitive3, 4. The combination of acivicn and dipyridamole administered in rats bearing s.c. transplanted hepatoma yielded a summation effect, decreasing NTP and dNTP pools5. The synergistic effect of dipyridamole and acivicin was also demonstrated in colon cancer cells6. There have been reports that dipyridamole potentiated the cytotoxicity of several antimetabolites including methotrexate, 5-fluorouracil and N-phosphonacetyl-L-aspartate (PALA)7, 8, 9. In addition to potentiating antimetabolites, dipyridamole was found to enhance the cytotoxicity of doxorubicin, etoposide, vincristine and mitoxantrone10, 11, 12. These synergistic effects of dipyridamole and drugs which are not antimetabolites were due to, at least in part, increased intracellular concentrations of anticancer drugs. Drug sensitivity of multidrug resistant cells may be modulated by dipyridamole13. The use of dipyridamole in cancer chemotherapy has drawn much attention and clinical trials have been underway14, 15. The study of dipyridamole indicates that blocking nucleoside salvage pathways by inhibition of nucleoside transport may be one of the effective ways in chemotherapy. By examination of a series of compounds of natural origin, we have found that green tea polyphenols (GP), antibiotics C3368-A (CA) and C3368-B (CB) are highly active in blocking nucleoside transport in cancer cells. Investigations have demonstrated that GP, CA or CB show synergistic effects with anticancer drugs.

N-alkylated 1,4-dihydropyridines: new agents to overcome multidrug resistance.

New N-alkylated 1,4-dihydropyridine derivatives were synthesized and their ability to overcome multidrug resistance was examined in vincristine-resistant P388 cells (P388/VCR cells). Compounds that possessed an arylalkyl substituent on the dihydropyridine ring nitrogen were more potent than verapamil in potentiating the cytotoxicity of vincristine against P388/VCR cells. However, neither drug effectively enhanced the antitumor activity of vincristine in tumor-bearing mice. Introduction of basic nitrogen-containing substituents on the side chain of 1,4-dihydropyridines gave improved activity in vitro and in vivo. The piperazine derivative 12c and 12o were more than 10 times as potent as verapamil in vitro. Four compounds selected for in vivo testing showed superior antitumor activity in P388/VCR-bearing mice in combination with vincristine. The structure-activity relationships of the compounds are discussed.

Sensitizing properties of new N-(2-arylalkyl) propionamides in drug-sensitive and multidrug-resistant tumor cells.

The propionamides 5 and 6 have been synthesized and tested for stimulation of antitumor drug activity. 5 and 6b increase vincristine cytotoxicity in drug-sensitive murine tumor cells; 5 also increases the toxicity in multidrug resistant cells. Dissimilar trends in sensitive and resistant cells have been observed for the stimulating activity of several propionamides of this family and structurally related verapamil with their molar refractivity, suggesting different size requirements for the sensitizers in sensitive and resistant cells.

Dominant Expression of Multiple Drug Resistance After In Vitro X-irradiation Exposure in Intraspecific Chinese Hamster Ovary Hybrid Cells

Exposure of Chinese hamster ovary (CHO) cells to fractionated x irradiation in vitro has led to expression of a distinctive multiple-drug-resistant phenotype. This phenotype is characterized by overexpression of P-glycoprotein without an increase in P-glycoprotein messenger RNA or gene amplification; increased glutathione S-transferase (GST) activity; and resistance to vincristine, colchicine, and etoposide but not to doxorubicin. To investigate whether this phenotype is dominant or recessive, we established intraspecific hybrids by fusion of x-ray-treated, drug-resistant CHO cells (DXR-10I or DXR-10II) with drug-sensitive CHO cells (E29). Drug resistance levels were determined in the wild-type CHO cell line AuxB1, the drug-sensitive E29 line, the x-ray-pretreated lines, and the hybrid lines by colony-forming assay of cells grown in increasing concentrations of colchicine, vincristine, or doxorubicin. The hybrids were characterized by analysis of DNA content, P-glycoprotein expression by Western blotting, GST activity by use of 1-chloro-2,4-dinitrobenzene as substrate, and sensitivity to reversal of resistance to vincristine by exposure to verapamil. These hybrids proved resistant to colchicine (two-fold) and vincristine (five- to seven-fold) but not to doxorubicin. After the hybrids were exposed to verapamil, vincristine cytotoxicity was increased 10- to 12-fold. The hybrid lines exhibited levels of P-glycoprotein comparable to those of the unfused x-ray-treated parent cell line, suggesting that P-glycoprotein overexpression is a dominant trait in these hybrid lines. Interpretation of the role of increased GST activity in these hybrids was inconclusive because of the very high levels of GST in the drug-sensitive cell-fusion partner. The multiple-drug-resistant phenotype following x-ray treatment of CHO cells in vitro was dominantly expressed. Overall, these data are consistent with the hypothesis that this phenotype is a consequence of the dominant genetic alteration resulting from exposure to x irradiation. This work adds weight to our hypothesis that there is a biological basis for the expression of clinical drug resistance in certain patients whose tumors have been previously irradiated.

CHEMOSENSITIZATION OF MURINE FIBROSARCOMA CELLS TO DRUGS AFFECTED BY THE MULTIDRUG RESISTANCE PHENOTYPE BY THE ANTIDEPRESSANT TRAZODONE - AN EXPERIMENTAL-MODEL FOR THE REVERSAL OF INTRINSIC DRUG-RESISTANCE

A variety of resistance phenotypes to cytotoxic agents in bacteria, protozoa parasites and mammalian cells are mediated by evolutionarily conserved proteins of the mdr family. The finding that chloroquine resistance in the malarial parasite, Plasmodium falciparum, that is mediated by an mdr-1 gene product can be circumvented by tricyclic antidepressant drugs has stimulated the present study to assess whether this class of agents might also modulate the multidrug resistance (MDR) phenotype(s) in mammalian tumor cells. The possible chemosensitizing effects of nine antidepressant drugs have been tested against the UV-2237M murine fibrosarcoma line and its MDR variant. At nontoxic concentrations all nine antidepressants markedly enhanced the cytotoxicity of ADR against the parental cells but were much less effective against the MDR cells. The most active antidepressant, trazodone, also enhanced the cytotoxicities of vinblastine and vincristine, but not those of actinomycin D, mitomycin C, or 5-fluorouracil. The parental cells treated with trazodone exhibited an increased accumulation of intracellular ADR, but lacked detectable alterations in the expression and drug-binding activity of plasma membrane P-glycoprotein, and trazodone did not affect the activities of isolated protein kinase C and calmodulin. These data suggest that the antidepressant drug trazodone may be useful in the reversal of the intrinsic drug resistance of tumor cells that express low levels of P-glycoprotein.

Cytotoxicity of adriamycin, idarubicin, and vincristine in acute myeloid leukemia: chemosensitization by verapamil in relation to P-glycoprotein expression.

A 4-day colorimetric tetrazolium dye (MTT) assay was used to assess the cytotoxicity of adriamycin (ADM), vincristine (VCR), and idarubicin (IDA) in blasts isolated from 37 patients with newly diagnosed and pretreated acute myeloid leukemia (AML). The effect of verapamil (VRP) as a chemosensitizer was studied in relation to the expression of the membrane efflux pump P-glycoprotein (PGP) as determined by a semiquantitative flow-cytometric proceder. A slight positive correlation was found between the fraction of cells expressing PGP and the ID50 values for ADM and VCR, but not between cellular PGP content and sensitivity to IDA. The overall data showed no significant sensitization effect of VRP. However, in specimens with more than 10% cells expressing PGP, 2 microM VRP sensitized cells to ADM and VCR significantly. The median of sensitization ratios (SRs), i.e., the ratios of cytotoxic drug ID50 in the absence/presence of VRP, were 1.89 and 2.0, respectively. No sensitizing effect of VRP on the cytotoxicity of IDA was observed. Related to the clinical status, the median fraction of PGP-positive blasts was elevated fourfold in pretreated patients (n = 16) in comparison to patients with de novo AML (n = 19). No differences in ID50 values were observed between newly diagnosed and pretreated patients. However, SRs for ADM and VCR were higher in samples of pretreated patients compared with de novo AML. PGP-mediated cellular drug resistance may thus be circumvented in leukemic blasts by application of chemosensitizers or, potentially, alternative anthracyclines.

Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro.

This study has provided evidence that exposure of the wild-type MCF-7 human breast carcinoma cell line to the mutagen ethyl methane sulphonate (EMS), followed by selection in vincristine (VCR), resulted in a stably-resistant subline, designated VCREMS, which expressed an approximately 14-fold level of resistance to VCR. This VCREMS subline showed cross-resistance (3-fold) to adriamycin (ADR) and to etoposide (3-fold), but not to cisplatin. The addition of a non-toxic concentration of verapamil (6.6 microM) significantly enhanced VCR cytotoxicity only in the resistant subline. This resistance was associated with over-expression of P-glycoprotein (Pgp), but without a concomitant increase in Pgp mRNA or gene amplification. In addition, activities of total glutathione S-transferases (GST) and glutathione peroxidase were elevated in this resistant subline, with over-expression of the GST-pi isozyme and its associated mRNA being identified, without gene amplification. This VCR-selected resistant MCF-7 cell line therefore provides another example of a breast carcinoma subline in which there is co-ordinate over-expression of both Pgp and GST-pi, without attributing a causal relationship to either event, and extends the range of anti-tumour drugs known to elicit modifications in glutathione metabolism.