sdz-psc-833 and Liver-Neoplasms

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclosporins; Diketopiperazines; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; Gene Silencing; Heterocyclic Compounds, 4 or More Rings; Humans; Liver Neoplasms; Neoplasm Proteins; Neoplastic Stem Cells; Up-Regulation

Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors.. Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity.. Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice.. The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells.

Topics: Adenocarcinoma; Aldehyde Dehydrogenase; Animals; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Blotting, Western; Camptothecin; Cell Differentiation; Colonic Neoplasms; Cyclosporins; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Flow Cytometry; Humans; Irinotecan; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Spheroids, Cellular; Time Factors; Topoisomerase I Inhibitors; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The present study explores the hypothesis that over-expression of P-glycoprotein (Pgp, product of mdr1) is intimately associated with liver cancer development and therefore inhibitors of Pgp should inhibit the development of liver cancer. Accordingly, we determined the effect of PSC833 (PSC), a potent inhibitor of Pgp, on experimental liver carcinogenesis in rats. To study the effects of PSC on liver cancer development, a daily dose of 30 mg PSC/kg body wt (PSC30) was chosen based on an initial dose-response experiment. Accordingly in experiment 1, PSC30 was fed to rats initiated by 1,2-dimethylhydrazine coupled with two-thirds partial hepatectomy and promoted for 22 weeks with 1% dietary orotic acid. Surprisingly, in contrast to our earlier observations in rats without hepatic nodules, in rats bearing hepatic nodules, PSC30 was found to be toxic. Because of this, PSC30 diet was discontinued after 5 weeks and the rats were transferred to basal diet (BD). The rats were killed 10 and 25 weeks thereafter. Cumulative results indicate that PSC30 exhibited a 40% decrease in the incidence of hepatocellular carcinoma (HCC; 15 of 18 in the BD group compared with eight of 17 in the PSC30 group; P = 0.08) coupled with significant reduction of tumor multiplicity (54%; P < 0.05) and tumor burden (61%; P < 0.005) compared with controls. In experiment 2, 15 mg PSC/kg body wt (PSC15) was fed for 20 weeks to rats similarly initiated and promoted for 35 weeks. PSC15 inhibited the incidence of HCC by 75% (four of four in the BD group compared to one of four in the PSC30 group; P = 0.15) and significantly reduced tumor burden by 55% (P < 0.05). The lack of statistical significance of inhibition on tumor incidence reflects the small sample size. Taken together the results indicate a possible intrinsic role for Pgp in liver cancer development and introduce another promising unexplored therapeutic approach in liver cancer treatment.

Topics: 1,2-Dimethylhydrazine; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Body Weight; Carcinogens; Cyclosporins; Dimethylhydrazines; Dose-Response Relationship, Drug; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Orotic Acid; Rats; Rats, Inbred F344; Time Factors

The P-glyprotein (P-gp), which is a membrane channel encoded by the MDR1 gene, represents a possible explanation for multidrug resistance in human hepatoblastoma (HB). P-gp shows up-regulation in tumor cells after chemotherapy; however, to date, its exact role in HB has not been described. The authors investigated the role of the MDR1 gene in the clinical course of patients with HB and in an in vivo model of HB. They also studied the effects of the MDR1 antagonizer PSC 833 on chemotherapy in mice xenotransplanted with HB.. Resected tumor specimens, including both primary tumors and recurrent tumors, from a child suffering from HB were investigated histologically. Cell suspensions from the originally removed tumor were incorporated subcutaneously into nude mice. Animals were treated with cisplatin (CDDP) plus PSC 833. MDR1 gene expression levels in the different resected tumors from the patient and in the xenotransplants after treatment were determined with polymerase chain reaction analysis.. MDR1 gene expression was increased in the patient's tumors after every course of chemotherapy from 30% to > 190%. In the xenotransplants, MDR1 gene expression was enhanced significantly after chemotherapy (P(CDDP) = 0.008; P(CDDP+PSC) = 0.002). Tumor volumes (P < 0.001) and serum alpha-fetoprotein levels (P = 0.0002) were significantly lower in the animals that were treated with CDDP + PSC compared with the animals that were treated with CDDP alone.. The current results suggest that MDR1 gene expression and P-gp are a potential mechanism of drug resistance in HB. The chemosensitizer PSC 833 significantly improved the effects of chemotherapy in animals xenotransplanted with HB. These data encourage further studies concerning the role of chemosensitizers in overcoming multidrug resistance in patients with HB.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; Cisplatin; Cyclosporins; Drug Resistance, Multiple; Gene Expression Regulation, Neoplastic; Genes, MDR; Hepatoblastoma; Humans; Infant; Liver Neoplasms; Male; Mice; Neoplasms, Experimental; Transplantation, Heterologous; Up-Regulation

P-glycoprotein, a membrane efflux pump encoded by the MDR1 gene, plays an important role in the development of multidrug resistance in human hepatoblastoma (HB). Chemosensitizers antagonize the efflux action of P-glycoprotein. This study investigates the effects of 3 chemosensitizers (the cyclosporin analogue SDZ PSC 833 (PSC 833), the acridone carboxamide derivative GG 918, and verapamil) on the chemotherapy of HB in vitro.. The doxorubicin (DOXO) concentration that produces 50% growth inhibition (IC50) in a HB cell line was determined and additional effects of PSC 833, GG 918, and verapamil were investigated in a cytotoxicity assay. The MDR1 gene expression after treatment was determined in a semiquantitative reverse transcription polymerase chain reaction approach.. The IC50 of DOXO is 2.5 microg/mL, 0.61 microg/mL for DOXO + PSC 833, 1.17 microg/mL for DOXO + verapamil, and 1.47 microg/mL for DOXO + GG 918. In combination with DOXO, cell growth was inhibited 4.1-fold by PSC 833, 2.1-fold by verapamil, and 1.9-fold by GG 918. The MDR1 gene expression was enhanced significantly in all treated cells, with and without modulator.. MDR1 modulators significantly improve the response of HB to DOXO in vitro. The combination of anticancer agents and MDR1 modulators might be a possible contribution to overcome multidrug resistance in HB.

Topics: Acridines; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclosporins; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression; Genes, MDR; Hepatoblastoma; Humans; Inhibitory Concentration 50; Isoquinolines; Liver Neoplasms; Tetrahydroisoquinolines; Tumor Cells, Cultured; Verapamil

Topics: Aclarubicin; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cyclosporins; Daunorubicin; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Epirubicin; Humans; Liver Neoplasms; Tumor Cells, Cultured

We previously showed that combined neoadjuvant doxorubicin (DOX) treatment and orthotopic liver transplantation produced a 3-year tumor-free survival rate of 54% in stage II-IVa nonresectable hepatocellular carcinomas (HCCs). These patients received posttransplant immunosuppressive doses of cyclosporin A (CsA). CsA has been shown to modify the function of a membrane P-glycoprotein (Pgp) whose overexpression is associated with a multidrug-resistant (MDR1) phenotype. This study utilized HCC cell lines to characterize the in vitro chemomodulatory properties of CsA as found in posttransplant patient plasma to consider the hypothesis that CsA may prolong posttransplant survival by enhancing the therapeutic efficacy of DOX against multidrug-resistant hepatoma cells. We characterized Pgp expression in the HCC lines Hep3B, Hep G2, and SK-HEP-1 by immunohistochemistry and the reverse transcription-polymerase chain reaction. The combined cytotoxicity of DOX + CsA was examined by [3H]thymidine uptake and flow cytometric drug-retention assays. Pgp expression was assessed further after prolonged (10-day) treatment with CsA. Hep3B and Hep G2 cells expressed low to moderate levels of Pgp. The effective DOX dose required for inhibiting MDR1(+) Hep3B and Hep G2 cell proliferation by 50% (DOX IC50) was 44.5 ng/ml and 43.5 microgram/ml, as compared with 10.7 ng/ml for Pgp-negative SK-HEP-1 cells. Optimal concentrations of CsA (0.8 micrometer) lowered DOX IC50 for Hep3B cells and Hep G2 cells by 6-fold and 4-fold, respectively. Similarly, plasma from patients containing immunosuppressive levels of CsA lowered DOX IC50 of the MDR1(+) Hep G2 cells by up to 4-fold. Prolonged exposure to CsA did not affect its chemosensitizing capacity or Pgp expression of HCC cells. PSC-833, a nonimmunosuppressive analogue of CsA, was equally effective in reducing the DOX IC50 of MDR1(+) HCC cells. CsA and PSC-833 increased drug retention by approximately 75%, but did not significantly affect hepatoma cell viability or Pgp expression. Pharmacological concentrations of cyclosporin analogues, including one nonimmunosuppressive form, enhance DOX cytotoxicity of MDR1(+) HCC cells by modulating drug retention. CsA as found in posttransplant patient plasma enhanced DOX cytotoxicity to human MDR1(+) hepatoma cells in vitro, albeit at less than optimal chemosensitizing concentrations. Prolonged exposure to CsA did not affect its chemosensitizing properties or block Pgp expression of HCC cells. These fi

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cyclosporine; Cyclosporins; Doxorubicin; Humans; Immunosuppressive Agents; Liver Neoplasms; Liver Transplantation