Drug Resistance In Malignancies Research Articles

Insulin-resistant conditions: A favorable milieu for aggressive drug-resistant malignancies.

Insulin-resistant conditions: A favorable milieu for aggressive drug-resistant malignancies.

Induction of Apoptosis in Human Renal Cell Carcinoma Cells by Vitamin E Succinate in Caspase-independent Manner

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies, and an effective therapy is lacking for metastatic RCC. Vitamin E (VE) has been intensively studied as a chemopreventive agent for various cancer types. Preclinical investigations have suggested that VE succinate (VES) is the most effective analog of VE in cancer cells; however, no study of VES in RCC has been done. We investigated the anticancer activity of VES against RCC. Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell morphologic changes and cell viability were evaluated using phase-contrast microscopy and the trypan blue dye-exclusion test, respectively. Caspase activity was measured with a quantitative colorimetric assay. VES exerted dose- and time-dependent cytotoxicities against ACHN, a human RCC cell line, but VE and VE acetate did not. The cytotoxic effect was also observed in 2 other RCC cell lines, Caki-1 and Caki-2, and in primary RCC cells derived from 8 patients. Hoechst 33258 staining and DNA ladder analysis demonstrated that VES induced apoptosis in RCC cells. However, VES did not affect activation of caspase-3, -6, -8, or -9. Furthermore, inhibitors specific to caspase-8, -9, -6, and -3 did not block VES cytotoxicity and neither did the general caspase inhibitor VAD. VES might induce apoptosis and cytotoxicity against RCC cells in a caspase-independent manner and has potential in vivo applications in the treatment of drug-and/or immunotherapy-resistant RCC.

Involvement of caspase-8 in chemotherapy-induced apoptosis of patient derived leukemia cell lines independent of the death receptor pathway and downstream from mitochondria

Resistance of leukemic cells to chemotherapy frequently occurs in patients with acute leukemia, which may be caused by alterations in common apoptotic pathways. Controversy exists whether cytostatic agents induce the mitochondrial or death receptor pathway of apoptosis. In the mitochondrial pathway cytochrome C release and caspase-9 activation play a central role in the induction of apoptosis, while formation of a Death Inducing Signaling Complex (DISC) and caspase-8 activation have been reported to be essential in death receptor-induced apoptosis. Here, we show in human derived myeloid and lymphoblastic leukemia cell lines that caspase-8 plays a more important role than previously expected in apoptosis mediated via the mitochondrial pathway. We demonstrated in these malignant cells chemotherapy-induced apoptosis independent of the death receptor pathway, since blocking this pathway using a retroviral construct encoding Flice inhibitory protein (FLIP) did not inhibit drug-induced apoptosis or caspase-8 activation, while overexpression of Bcl-2 completely inhibited both events. Furthermore, we showed that activation of caspase-8 by cytostatic agents occurred downstream from mitochondria. Since caspase-8 plays a central role in both death receptor- and chemotherapy-induced apoptosis of malignant cells from patients with acute leukemia, therapeutic strategies focusing at modulation and activation of caspase-8 may be successful in the treatment of drug-resistant malignancies.

Screening of an annotated compound library for drug activity in a resistant myeloma cell line

Resistance to anticancer drugs is a major problem in chemotherapy. In order to identify drugs with selective cytotoxic activity in drug-resistant cancer cells, the annotated compound library LOPAC1280, containing compounds from 56 pharmacological classes, was screened in the myeloma cell line RPMI 8226 and its doxorubicin-resistant subline 8226/Dox40. Cell survival was measured by the Fluorometric Microculture Cytotoxicity Assay. Selective cytotoxic activity in 8226/Dox40 was obtained for 33 compounds, with the most pronounced difference observed for the glucocorticoids. A microarray analysis of the cells showed a difference in mRNA-expression for the glucocorticoid receptor suggesting potential mechanisms for the difference in glucocorticoid sensitivity. In the presence of the glucocorticoid-receptor antagonist RU486, the sensitivity to the glucocorticoids was reduced and a similar effect level in RPMI 8226 and 8226/Dox40 was achieved. In conclusion, screening of mechanistically annotated compounds on drug-resistant cancer cells can identify compounds with selective activity and provide a basis for the development of novel treatments of drug-resistant malignancies.

Hydrolytically activated etoposide prodrugs inhibit MDR-1 function and eradicate established MDR-1 multidrug-resistant T-cell leukemia

AbstractEffective therapy of high-risk leukemia with established cytotoxic drugs may be limited by poor antitumor efficacy, systemic toxicity, and the induction of drug resistance. Here, we provide the first evidence that hydrolytically activated prodrugs may overcome these problems. For this purpose, VP16 was functionally blocked by hydrolytically cleavable carbonate linkers with unique characteristics to generate 2 novel prodrugs of VP16. First, we established a more than 3-log higher efficacy of the 2 prodrugs compared with VP16 on a panel of naturally drug-resistant tumor cell lines. Second, the prodrugs did overcome VP16-induced multidrug resistance-1 gene (MDR-1)—mediated multidrug resistance in vitro in a newly established VP16-resistant T-cell leukemia cell line MOVP-3 by functionally blocking MDR-1—mediated efflux. Third, in vivo studies showed a maximum tolerated dose of ProVP16-II (> 45mg/kg), which was at least 3-fold higher than that of VP16 (15 mg/kg). Finally, tests of ProVP16-II in a multidrug-resistant xenograft model of T-cell leukemia expressing MDR-1 indicated that only the mice treated with this prodrug revealed a complete and long-lasting regression of established, drug-resistant leukemia. In summary, the hydrolytically activated etoposide prodrugs proved effective against multidrug-resistant T-cell leukemia in vitro and in vivo and provide proof of concept for a highly promising new strategy for the treatment of MDR-1 drug-resistant malignancies. (Blood. 2003;102:246-253)

Enhancement of TRAIL/Apo2L-mediated apoptosis by adriamycin through inducing DR4 and DR5 in renal cell carcinoma cells.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies in humans. We show that adriamycin (ADR) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L have a synergistic cytotoxic effect against RCC cells. This synergistic cytotoxicity was obtained in ACHN, A704, Caki-1 and Caki-2 human RCC cell lines and freshly derived RCC cells from 6 patients. This synergistic effect, however, was not achieved in 5 samples of freshly isolated normal kidney cells. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of TRAIL/Apo2L and ADR was realized by inducing apoptosis. Sequential treatment with ADR followed by TRAIL/Apo2L induced significantly more cytotoxicity than the reverse treatment. ADR increased the expression of DR4 and DR5 in RCC cells, but not in the normal kidney cells. Furthermore, the synergistic cytotoxicity was significantly inhibited by DR4:Fc and DR5:Fc fusion proteins, which inhibit TRAIL/Apo2L-mediated apoptosis. In addition, caspase activity assays and treatment of caspase inhibitors demonstrated that the combination treatment with ADR and TRAIL/Apo2L activated caspase cascade, including caspase-9, -8, -6 and -3, which were the downstream molecules of death receptors. These findings indicate that ADR sensitizes RCC cells to TRAIL/Apo2L-mediated apoptosis through induction of DR4 and DR5, suggesting that the combination therapy of TRAIL/Apo2L and ADR might be effective for RCC therapy.

Drug resistance and apoptosis in cancer treatment: development of new apoptosis-inducing agents active in drug resistant malignancies.

Modulation of multidrug resistance (MDR) has been extensively studied in vitro and in vivo. However, several clinical trials have failed to show any important benefits in terms of response to chemotherapy or the length of survival using MDR reversing agents. This may be due to the expression or co-expression of other drug resistance mechanisms in malignant cells. Several studies have shown that most, if not all, chemotherapeutic agents exert their anticancer activity by inducing apoptosis; therefore, resistance to apoptosis may be a major factor limiting the effectiveness of anticancer therapy. In the last few years, effort has been made to understand the biochemical alterations of apoptotic pathways in cancer. Many of these alterations confer a multidrug resistant phenotype to malignant cells. In this context, the new recently developed anticancer therapies based on drugs that modulate apoptosis may have importance for the treatment of tumors that are scarcely responsive to the conventional anticancer chemotherapy. In this review, we discuss the current knowledge about drug resistance, apoptosis and cancer and report the recently developed apoptosis modulating strategies that have potential therapeutic implications for the drug resistant tumors.

The role of caspase-8 in resistance to cancer chemotherapy.

Toxicity of chemotherapeutic agents against cancer cells is mediated through the initiation of programmed cell death. Apoptosis is an evolutionarily conserved cascade of intracellular proteolytic events propagated by a family of cysteine proteases called caspases. Many receptor- and non-receptor-mediated death signals induce apoptosis via activation of caspase-8 (FLICE/MACH). Mechanisms of tumor resistance to cytotoxic drugs through decreased apoptosis may occur by altered expression of caspase-8, upregulation of caspase-8 inhibitors like FLIP (FLICE-like Inhibitory Protein), or sequestration of caspase-8 by Bcl-2. Modulation of caspase-8 and apoptosis may be a therapeutic strategy for sensitization of drug-resistant malignancies to radiation or combination chemotherapy.

Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.

Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Drug-resistance enables selective killing of resistant leukemia cells: exploiting of drug resistance instead of reversal.

Drug resistance is a well recognized problem in cancer therapy. Despite the current dogma that drug resistance is always an obstacle for treatment, here I show that it provides opportunities for selective protection of non-resistant cells with killing of drug-resistant cancer cells. According to the proposed 'two-drug' strategy, the first drug should be ineffective against a target drug-resistant cell (ie the drug is a substrate of MRP or Pgp pumps). In addition, it must be cytostatic but not cytotoxic. The second drug, which is applied in sequence, must be a cycle-dependent apoptotic drug to which the target cell is not cross-resistant. Thus, low doses of adriamycin, etoposide and actinomycin D, used as the first drugs, were cytostatic to parental HL60 cells. Therefore, these drugs precluded Bcl-2/Raf-1 phosphorylation, PARP cleavage and cell death which are otherwise induced by paclitaxel, a mitosis-selective apoptotic drug for HL60 cells. In contrast, HL60/ADR cells which express MRP, a transporter which pumps out the first drugs from a cell, were insensitive to the first drugs and therefore readily underwent apoptosis following the second drug. This strategy also allowed a selective killing of HL60/TX cells which express MDR-1, with the only difference being that the second drug, paclitaxel, was substituted for epothilones, non-Pgp substrates. Lack of protection by the first drug, a Pgp substrate, resulted in HL60/TX killing by the second drug, whereas parental HL-60 cells were fully protected. Therefore, drug resistant cells can be selectively killed by a combination of drugs not killing sensitive cells. Lack of toxicity against normal cells will be clinically translated in reduction of adverse side-effects of chemotherapy against drug-resistant malignancies.

Modulation of multidrug resistance gene (mdr-1) with antisense oligodeoxynucleotides.

1. Multidrug resistance is the major obstacle to successful cancer chemotherapy. Circumventing multidrug resistance therefore represents a high priority for clinical anti-cancer treatment. Among many reversal strategies, antisense oligodeoxynucleotides may offer a molecular targeting tool for overcoming cellular multidrug resistance. 2. Two 17-mer phosphorothioate antisense oligomers, complementary to the 5' end of the ATG initiator codon-containing region and loop-forming site (located at nucleotides 991-1007 from the first ATG codon) in mdr-1 cDNA sequence, were synthesized. The purpose was to study their effects on the function and expression of P-glycoprotein and mdr-1 gene. 3. The results showed that 10 mumol/l antisense oligomers could significantly inhibit the growth of multidrug resistant K562/Adm cells cultured in adriamycin-containing medium. No such effect was observed for parental (sensitive) K562/S cells. Intracellular daunorubicin accumulation increased greatly in the K562/Adm cells after they were treated with oligomers for 48 h and P-glycoprotein synthesis was strikingly reduced. 4. Further investigation with [alpha-32P]dCTP incorporation by the reverse transcriptase-polymerase chain reaction method revealed that antisense oligomers could result in a reduction in the level of mdr-1 mRNA, probably through hindering mdr-1 gene transcription. 5. The high reversal efficiency and specificity of antisense oligomers in regulating mdr-1 gene expression suggest a potential clinical application in gene therapy for drug resistant malignancies.

ANALYSIS OF INTRACELLULAR RETENTION OF MORPHOLINYL ANTHRACYCLINES IN MULTIDRUG-RESISTANT CANCER-CELLS BY INTERACTIVE LASER CYTOMETRY

Interactive laser cytometry was applied to measure intracellular fluorescence of doxorubicin (DOX) accumulation in a uterine sarcoma cell line, MES-SA and a series of multidrug resistant sublines, Dx0.3, Dx1 and Dx5. Exposure of each cell line to 10 mu M DOX for 2 h resulted in an intracellular fluorescent level directly correlated to its sensitivity to the drug but inversely related to its cellular P-glycoprotein (P-gp) level. The morpholinyl anthracyclines, methoxymorpholinyl DOX (MMDX) and morpholinyl DOX (MRA), were equally highly cytotoxic against the multidrug sensitive and resistant cancer cells. After exposure to 10 mu M of MMDX or MRA for 2 h, the multidrug resistant cells, Dx5, retained as much intracellular fluorescence as the multidrug sensitive cells, MES-SA. In the resistant cells, the intracellular fluorescence of MMDX or MRA was 8 fold higher than that of DOX. Interactive laser. cytometer is a useful fool for screening cancer cells with the MDR phenotype and for identifying new anthracyclines effective against drug resistant malignancies.

Vitamin A derivatives in the prevention and treatment of human cancer.

Vitamin A is essential for normal cellular growth and differentiation. A vast amount of laboratory data have clearly demonstrated the potent antiproliferative and differentiation-inducing effects of vitamin A and the synthetic analogues (retinoids). Recent in-vitro work has led to the exciting proposal that protein kinase-C may be centrally involved in many of retinoids' anticancer actions including the effects on ornithine decarboxylase induction, intracellular polyamine levels, and epidermal growth factor receptor number. Several intervention trials have clearly indicated that natural vitamin A at clinically tolerable doses has only limited activity against human neoplastic processes. Therefore, clinical work has focused on the synthetic derivatives with higher therapeutic indexes. In human cancer prevention, retinoids have been most effective for skin diseases, including actinic keratosis, keratoacanthoma, epidermodysplasia verruciformis, dysplastic nevus syndrome, and basal cell carcinoma. Several noncutaneous premaligancies, however, are currently receiving more attention in retinoid trials. Definite retinoid activity has been documented in oral leukoplakia, laryngeal papillomatosis, superficial bladder carcinoma, cervical dysplasia, bronchial metaplasia, and preleukemia. Significant therapeutic advances are also occurring with this class of drugs in some drug-resistant malignancies and several others that have become refractory, including advanced basal cell cancer, mycosis fungoides, melanoma, acute promyelocytic leukemia, and squamous cell carcinoma of the skin and of the head and neck. This report comprehensively presents the clinical data using retinoids as anticancer agents in human premalignant disorders and outlines the ongoing and planned studies with retinoids in combination and adjuvant therapy.

A phase I-II study of continuous 5-day infusion mitomycin-C

A phase I-II study was undertaken to establish the maximum-tolerated dose of a continuous 5-day infusion of mitomycin-C and its efficacy in patients with advanced metastatic drug-resistant breast and gastrointestinal malignancies. The dose-limiting toxicity was myelosuppression, predominantly thrombocytopenia, and was severe and cumulative. Nonhematologic toxicity was infrequent, and no renal or cardiac toxicity was seen. For patients with breast cancer who had received extensive prior therapy, 3 mg/m2/day for 5 days repeated every 6-8 weeks were well-tolerated doses; and for patients with gastrointestinal cancer, the maximum-tolerated dose was 4 mg/m2/day. One patient with breast cancer had a partial response lasting 4 months, and no responses were observed in patients with gastrointestinal cancer. The administration of mitomycin-C by continuous infusion did not appear to have improved its therapeutic index.