Drug-resistant Human Cancer Research Articles

Proteomic characterization of cytoskeletal and mitochondrial class III β-tubulin

Class III beta-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function.

Antiproliferative activity of ent-abietane lactones against resistant human cancer cell lines

Multidrug resistance (MDR) is considered to be one of the main causes of chemotherapy failure. The mechanisms of MDR could be related with the intracellular drug distribution through overexpression of certain transport proteins (e.g. Pgp/MDR1). This type of MDR is often known as classical or typical MDR. However, it does not completely explain the phenomenon. Alternative forms of MDR have been described, known as atypical MDR that included, among others, the change in drug targets (e.g. decreased activity of Topoisomerase II) [1]. Topoisomerases are nuclear enzymes that are essential for DNA replication, transcription, chromosomal aggregation and DNA recombination. For these reasons, Topo II is one of the possible targets for the commonly used anticancer drugs [2]. The aim of this work was to study the antiproliferative effect of several ent-abietane diterpenic lactones, isolated from Euphorbia tuckeyana, in human cancer cell lines, which were derived from three different tumour entities: gastric (EPG85–257), pancreatic (EPP85–181) and colon (HT-29) carcinomas. The classical and atypical MDR sublines of these cell models were also investigated. IC50 values were determined by proliferation assays, after continuous exposure for 5 days. Relative resistance (RR) was also determined as the relation between the IC50 of the resistance cell line and the IC50 of the parental drug-sensitive cell line. In parental drug sensitive cell lines, all the tested compounds showed a weak antiproliferative effect. However, most of the drug resistant cancer sublines showed increased sensitivities to the studied compounds when compared to the parental sublines. Some of the tested compounds were found to be highly effective against the classical MDR subline of gastric carcinoma, showing RR of 0.08 and 0.09. The results obtained lead to conclude that, ent-abietane lactones may be considered as potential new drugs for the treatment of drug-resistant human cancer cells.

Calebin-A induces apoptosis and modulates MAPK family activity in drug resistant human gastric cancer cells

This study is the first to investigate Calebin-A, a natural compound present in Curcuma longa, which inhibits cell growth and induce apoptosis in SGC7901/VINCRISTINE cells, a multidrug resistant (MDR) human gastric adenocarcinoma cell line. Our data suggest the drug efflux function of P-glycoprotein was inhibited by Calebin-A treatment, while the expression level of P-glycoprotein was not affected. Additionally, co-treatment of Calebin-A and vincristine resulted in a remarkable reduction in S phase and G2/M phase arrest in SGC7901/VINCRISTINE cells. Calebin-A was also found to modulate the activities of mitogen-activated protein kinase (MAPK) family members, which includes decreased c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and increased protein kinase of 38 kDa (p38) activity. These results suggest that Calebin-A might be an effective compound for the treatment of human gastric and other MDR cancers.

Multiple Drug Resistance Associated with Function of ABC-Transporters in Diabetes Mellitus: Molecular Mechanism and Clinical Relevance

ATP-binding cassette (ABC) transporters are involved in a variety of physiological processes such as lipid metabolism, ion homeostasis and immune functions. A large number of these proteins have been causatively linked to rare and common human genetic diseases including familial high-density lipoprotein deficiency, retinopathies, cystic fibrosis, diabetes and cardiomyopathies. Furthermore, genetic variations in ABC transporter genes and deregulated expression patterns significantly contribute to drug resistance in human cancer and pancreatic beta cells and alter the pharmacokinetic properties of a variety of drugs. Up-to-date 15 ABC transporters have been identified in human pancreatic beta cells, however only a few of them are identified to date as proteins/genes associated with multidrug resistance (MDR) in diabetes mellitus. Prominent members include the multidrug resistance protein 1 (MRP1/ABCC1), sulfonylurea receptor 1 (SUR1/ABCC8), the multi drug transporter TAP2 and member of the ATP-binding cassette transporter subfamily A (ABCA1). ABCC8 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion. Although the physiological role of these transporters to MDR is not yet fully understood, they play an important role in the blood-membrane barrier in pancreatic beta cells. The aim of this article is to provide an overview and to present few examples of drug treatment in MDR in diabetes mellitus associated with function of ABC-transporters.

Induction of drug resistance and transformation in human cancer cells by the noncoding RNA CUDR

Refractory to apoptosis induced by anticancer drugs is one of the major causes of drug resistance in human cancers. The involvement of noncoding RNA (ncRNA) in cancer cell drug resistance has not yet been reported. By using the technique of RT-PCR-based differential display, a novel gene, cancer up-regulated drug resistant (CUDR) gene, was found to be overexpressed in a doxorubicin-resistant subline of human squamous carcinoma A431 and A10A cells, which were also more resistant to drug-induced apoptosis. The full-length CUDR mRNA transcript is approximately 2.2 kb as detected by Northern blot analysis and has no sequence homology with other genes identified so far. Interestingly, no distinct open reading frame was found throughout the CUDR cDNA sequence, and no recombinant protein was detected from in vitro translation or from a protein lysate of human cancer cells after CUDR transfection. Therefore, CUDR is likely to exert its function as a noncoding RNA. Stable transfection with the CUDR gene was found to induce resistance to doxorubicin and etoposide as well as drug-induced apoptosis in A431 cells. By Western blot analysis, down-regulations of caspase 3 were observed in CUDR transfectants. On the other hand, overexpression of CUDR promoted anchorage-independent growth in A431 cells. Results from the present study suggest that CUDR may likely regulate the drug sensitivity and promote cellular transformation at least through caspase 3-dependent apoptosis.

Expression of major vault protein gene in osteosarcoma patients

Osteosarcoma (OS) is a primary malignant tumor of bone. Despite the successful use of multiple chemotherapeutic agents in the treatment of OS, more than 30% of OS tumors remain resistant to treatment. Elucidation of cellular resistance mechanisms may lead to better treatments for cancer patients. In this study, we used the low-density expression cDNA array, GEArray Q Series Human Cancer Drug Resistance and Metabolism Gene Array to screen genes related to drug resistance in 15 OS tumors. Expression patterns of the MPV gene were validated by real time PCR on 45 OS patient tumor samples and correlated with clinical and pathological data. Major vault protein (MVP) expression was present in 24 (53%) tumor samples and absent in 21 (47%). Samples from surgery showed correlation between the expression of MVP, metastatic disease at diagnosis and event free survival (EFS). The MVP gene expression correlates with metastatic disease at diagnosis after neoadjuvant chemotherapy (p=0.048), and is also associated with worse EFS (p=0.036). These findings suggest that MVP expression is involved in one of the mechanisms of drug resistance in OS and is induced by chemotherapy.

391 POSTER The significance of Pyk2 in hepatocellular carcinoma invasiveness

We previously demonstrated that the PPARγ agonist Troglitazone (TRG), a potent antiproliferative agent, in combination with the anthracycline antibiotic Doxorubicin (DOX), is an effective killer of multiple drug resistant (MDR) human cancer cells. Cell killing was accompanied by increased global histone H3 acetylation. Presently, we investigated the epigenetic and cell killing effects of TRG in estrogen receptor (ER) positive MCF7 breast cancer cells. MCF7 cells were treated with the Thiazolidinediones (TZDs) TRG and Ciglitazone (CIG), the non-TZD PPARγ agonist 15PGJ2, and the histone deacetylase inhibitors (HDACi’s) Trichostatin A (TSA), sodium butyrate and PXD101. Using MTT cell viability assays, Western analyzes and mass spectrometry, we showed a dose-dependent increase in cell killing in TRG and HDACi treated cells, that was associated with increased H3 lysine 9 (H3K9) and H3K23 acetylation, H2AX and H3S10 phosphorylation, and H3K79 mono- and di-methylation. These effects were mediated through an ER independent pathway. Using HDAC activity assays, TRG inhibited HDAC activity in cells and in cell lysates, similar to that observed with TSA. Furthermore, TRG and TSA induced a slower migrating HDAC1 species that was refractory to HDAC2 associations. Lastly, TRG and the HDACi’s decreased total and phosphorylated AKT levels. These findings suggest that TRG’s mode of killing may involve downregulation of PI3K signaling through HDAC inhibition, leading to increased global histone post-translational modifications.

Overcoming Drug-Resistant Cancer by a Newly Developed Copper Chelate through Host-Protective Cytokine-Mediated Apoptosis

Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines. Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)-bearing mice and doxorubicin-resistant sarcoma 180-bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay ex vivo. IFN-gamma and tumor necrosis factor-alpha were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-gamma and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-gamma and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells. CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-gamma and/or tumor necrosis factor-alpha, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-gamma-producing T cells in the ascetic tumor site. Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.

P73-dependent induction of 14-3-3 σ increases the chemo-sensitivity of drug-resistant human breast cancers

It has been well documented that tumor suppressor p53 is mutated in about 50% of all human tumors. p53 status might be one of the critical determinants for the chemo-sensitivity of human tumors. In the present study, we have found that p53 family member p73 as well as 14-3-3σ is down-regulated in response to adriamycin (ADR) in ADR-resistant human breast cancer-derived MBA-MD-436 cells which carry p53 mutation. Like p53, 14-3-3σ was transactivated by p73 and, in turn, stabilized p73. Luciferase reporter analysis and colony formation assays demonstrated that 14-3-3σ has an ability to enhance the p73-mediated transcriptional activity as well as its pro-apoptotic function. Furthermore, enforced expression of 14-3-3σ increased the ADR sensitivity of MBA-MD-436 cells. Taken together, our present results strongly suggest that p73-dependent induction of 14-3-3σ plays an important role in the regulation of chemo-sensitivity of breast cancers bearing p53 mutation.

Transport Mechanism-Based Drug Molecular Design: Novel Camptothecin Analogues to Circumvent ABCG2-associated Drug Resistance of Human Tumor Cells

Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

Recent Advances in the New Generation Taxane Anticancer Agents

AbstractFor Abstract see ChemInform Abstract in Full Text.

Recent Advances in the New Generation Taxane Anticancer Agents

Recent advances in the design and preclinical evaluations of promising new generation taxane anticancer agents are reviewed in this article. Paclitaxel and docetaxel are two of the most important anticancer drugs today. However, recent reports have shown that treatment with these drugs often encounters undesirable side effects as well as drug resistance. Therefore, it is important to develop new taxane anticancer agents with fewer side effects, superior pharmacological properties, and improved activity against drug-resistant human cancers. Structure-activity relationship (SAR) studies led to the discovery of a series of highly active second-generation taxanes. One of them, "Ortataxel" (SB-T-101131, IDN5109, BAY59-8862), exhibits excellent activity against a variety of drug-sensitive and drug-resistant cancer cell lines, as well as human tumor xenografts in mice. It is orally active and is currently in phase II clinical trials. Photoaffinity labeling of microtubules and P-glycoprotein using photoreactive radiolabeled taxoids has disclosed the drug-binding domain of tubulin as well as Pgp. Together with information on microtubule-bound fluorine-labeled taxoids obtained by solid-state NMR studies, the bioactive conformation of paclitaxel and taxoids appears to emerge. Novel taxane-monoclonal antibody (mAb) immunoconjugates, have shown highly promising results for the tumor-specific delivery and release of an extremely cytotoxic, second-generation taxane. Also, another novel series of second generation taxanes conjugated with n-3 polyunsaturated fatty acids, e.g. decosahexaenoic acid (DHA), has exhibited impressive antitumor activity with minimum general toxicity against the highly drug-resistant DLD-1 human colon cancer xenografts in SCID mice.

Novel camptothecin analogues that circumvent ABCG2-associated drug resistance in human tumor cells.

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN-38, the active form of CPT-11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2-overexpressing PC-6/SN2-5H cells transported SN-38 and its glucuronide conjugate in an ATP-dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827-32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell-free system, as did SN-38. Their antitumor activities in the SN-38-resistant PC-6/SN2-5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC-6/SN2-5H2 cells and ABCG2-transfected HEK-293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2-associated drug resistance, low-polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

Reversal of multidrug resistance in drug-resistant human gastric cancer cell line SGC7901/VCR by antiprogestin drug mifepristone.

To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcription-polymerase chain reaction(RT-PCR). Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2, Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells. Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis. The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular [(3) H]VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively. Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively. After treatment with 1, 5, 10, and 20 micromol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively. MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells. The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose. After 96 h of incubation with mifepristone 10 micromol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+/-0.65)% from (25.32+/-1.44)%, whereas the expression of Bax protein was increased to (19.69+/-1.13)% from (1.24+/-0.78)% (P<0.01). Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis. Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR.

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2).

Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance. In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found. The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells. BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2. Like all members of the ABC G (white) subfamily, BCRP is a half transporter. Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer. BCRP maps to chromosome 4q22, downstream from a TATA-less promoter. The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors. Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482. Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition. Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics. BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype. Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers. More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.

Medicinal chemistry and chemical biology of new generation taxane antitumor agents.

P-glycoprotein (P-GP)-based multidrug resistance (MDR) and undesirable side effects are significant drawbacks to the clinical use of paclitaxel and docetaxel. Extensive SAR studies of taxanes in these laboratories led to the discovery of new generation taxanes that are highly active against not only drug-sensitive but also drug-resistant human cancer cell lines as well as tumor xenografts in mice. One of these second generation taxanes, SB-T-110131 (IDN5109), exhibited excellent pharmacological profile in the preclinical studies and has been selected for clinical development (recoded as Bay 59-8862), which is currently in the phase II clinical trials. Bay 59-8862 is orally active with high bioavailability, showing excellent activity against a variety of drug-resistant tumors. "Advanced second generation taxanes" show essentially no difference in cytotoxicity against drug-resistant and drug-sensitive cell lines, virtually overcoming MDR. Photoaffinity labeling of P-GP using photoreactive radiolabeled paclitaxel analogs has disclosed the paclitaxel-binding domain of P-GP. Highly efficient taxane-based MDR reversal agents (TRAs) have also been developed, which can recover the cytotoxicity of paclitaxel to practically the original level against paclitaxel-resistant MDR expressing cancer cells. Highly promising results have emerged from the study of taxane-monoclonal antibody (MAb) immunoconjugates, which have been proved to specifically deliver extremely cytotoxic agents to tumor in an animal model.

LIPOSOMAL DAUNORUBICIN OVERCOMES DRUG RESISTANCE IN HUMAN BREAST, OVARIAN AND LUNG CARCINOMA CELLS

ABSTRACTMulti-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.

Reversal of Cisplatin and Multidrug Resistance by Ribozyme-Mediated Glutathione Suppression

γ-Glutamylcysteine synthetase (γ-GCS) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in intracellular detoxification, especially of anticancer drugs. Increased levels of GSH are commonly found in the drug-resistant human cancer cells. We designed a hammerhead ribozyme against γ-GCS mRNA (anti-γ-GCS Rz), which specifically down-regulated γ-GCS gene expression in the HCT-8 human colon cancer cell line. The aim of this study was to reverse the cisplatin and multidrug resistance for anticancer drugs. The cisplatin-resistant HCT-8 cells (HCT-8DDP cells) overexpressed MRP and MDR1 genes, and showed resistance to not only cisplatin (CDDP), but also doxorubicin (DOX) and etoposide (VP-16). We transfected a vector expressing anti-γ-GCS Rz into the HCT-8DDP cells (HCT-8DDP/Rz). The anti-γ-GCS Rz significantly suppressed MRP and MDR, and altered anticancer drug resistance. The HCT-8DDP/Rz cells were more sensitive to CDDP, DOX and VP-16 by 1.8-, 4.9-, and 1.5-fold, respectively, compared to HCT-8DDP cells. The anti-γ-GCS Rz significantly down-regulated γ-GCS gene expression as well as MRP/MDR1 expression, and reversed resistance to CDDP, DOX and VP-16. These results suggested that γ-GCS plays an important role in both cisplatin and multidrug resistance in human cancer cells.

Differential effects of mitomycin C and doxorubicin on P-glycoprotein expression

Previous studies have demonstrated that mitomycin C (MMC) and other DNA cross-linking agents can suppress MDR1 (multidrug resistance 1) gene expression and subsequent functional P-glycoprotein (Pgp) expression, whereas doxorubicin and other anthracyclines increase MDR1 gene expression. In the present study, with stably transfected Madin-Darby canine kidney C7 epithelial cells expressing a human Pgp tagged with green fluorescent protein under the proximal human MDR1 gene promoter, we demonstrated that MMC and doxorubicin have differential effects on Pgp expression and function. Doxorubicin caused a progressive increase in the cell-surface expression of Pgp and function. In contrast, MMC initially increased plasma membrane expression and function at a time when total cellular Pgp was constant and Pgp mRNA expression had been shown to be suppressed. This was followed by a rapid and sustained decrease in cell-surface expression at later times, presumably as a consequence of the initial decrease in mRNA expression. These studies imply that there are at least two independent chemosensitive steps that can alter Pgp biogenesis: one at the level of mRNA transcription and the other at the level of Pgp trafficking. Understanding the combined consequences of these two mechanisms might lead to novel chemotherapeutic approaches to overcoming drug resistance in human cancers by altering either Pgp mRNA expression or trafficking to the membrane.

Comparison of the accumulation and efflux kinetics of technetium-99m sestamibi and technetium-99m tetrofosmin in an MRP-expressing tumour cell line

The potential clinical use of technetium-99m labeled sestamibi (Tc-MIBI) and tetrofosmin (Tc-Tfos) to image tumours is currently being evaluated. In this study. the accumulation and efflux of Tc-MIBI and Tc-Tfos in the nasopharyngeal carcinoma cell line CNE-1 were examined in the presence or absence of various inhibitors of P-glycoprotein (PGP) and/or multidrug resistance associated protein (MRP) activity [GG918, PSC833, verapamil (Vrp), cyclosporin A (CsA) and buthionine sulfoximine (BSO)]. Reverse-transcriptase polymerase chain reaction analysis and immunodetection of the CNE-1 cells detected expression of MRP, MRPI and MRP2 but not PGP. Tc-MIBI and Tc-Tfos accumulation was increased (P < 0.0001) and efflux decreased (P < 0.05) in the presence of BSO, CsA, Vrp and PSC833 but not GG918, which is a specific inhibitor of PGP. The absolute accumulation of Tc-MIBI was approximately twofold higher than that seen with Tc-Tfos, whereas the addition of inhibitors caused a much greater suppression of Tc-Tfos transport (>2 times greater than for Tc-MIBI). However, no qualitative differences in inhibitors were seen between Tc-MIBI and Tc-Tfos. These results suggest that both Tc-MIBI and Tc-Tfos are substrates for the MRP transporter and that PSC833, Vrp, CsA and BSO but not GG918 can inhibit MRP activity. These results indicate that Tc-MIBI and Tc-Tfos may be suitable imaging agents for detecting MRP-mediated drug resistance in human cancers.