| Prostate cancer (prostate cancer, PCa) is the most common malignant cancer in Europe and America countries. Although its incidence in China is lower than Europe countries, PCa has become more important cause of death in the old men as the average lifespan increasing in our country.PCa is an androgen-dependent tumor, and androgen ablation serves as a standard treatment for patients who present advanced, androgen-dependent metastatic PCa for many years. However, the high failure rate for castration causes progresses to hormone refractory prostate cancer (HRPC), that is resistant to radiation, surgery, and chemotherapy, and leading to high mortality. Despite numerous advances including aberrant regulation of androgen/androgen receptor leading to constitutive active androgen receptor, and androgen-independent signaling pathways which contribute cell proliferation in addition to androgen stimuli in PCa, few therapeutic options are available. to treate this disease. Docetaxel is the standard of care in men with HRPC. However,～50%of patients do not respond to Docetaxel, and are exposed to significant toxicity without direct benefit or devolpment of multidrug resistance (MDR). Although evidence has been demonstrated that aberrant activation of some genes appears to be essential for development of docetaxel or paclitaxel resistance, it still requires a further step towards understanding of how resistance is activated and how to reverse the resistance in PCa. In the present study, we devolped paclitaxel-resistant cells PC3/Tax from androgen-independent PC-3cells by st epwise increased concen tration s of paclitaxel. In addition, reversal effect of Riccardin D (RD) on PC3/Tax were preliminarily investigated.Objective:To understand mechanisms of taxol resistance in hormone-refractory prostate cancer (HRPC), a taxol-resistant cell line PC3/Tax was developed over one year induction.Methods:The human androgen-independent prostate cancer cell line PC3was used to establish taxol resistance subline via stepwise elevation of the taxol concentrations in a culture medium. The cells were considered resistant when they were freely divided in medium containing of75μM taxol. The morphology changes and cell cycle analysis of PC3/Tax were performed by morphometry and flow cytometry. Cell growth rate and drug resistance index were evaluated using MTT assay. Changes in mRNA and protein levels of P-glycoprotein were analyzed by RT-PCR and flow cytometry.Results:Section I Characteristics of paclitaxel-resistant cells1. The taxol resistant subline PC3/Tax could be stably cultured in the presence of taxol. Cells became longer compared to the parental taxol-sensitive PC3cells. The IC50for taxol in the PC3/Tax cells was52.86-fold higher than that in PC3cells, for vinblastine and doxorubicin were60.42-fold and40.24-fold higher, respectively. But cisplatin resistance in the PC3/Tax cells remained unchanged when compared to the parental cells.2. Morphologic changes were further observed in PC3/Tax cells. Paclitaxel-resistant cells became longer and thinner than that of PC-3cells by phase contrast microscopy. Changes in nuclear and cytoplasmic morphology in PC3/Tax cells were evaluated by TEM. PC3/Tax cells showed slight luminal swelling in endoplasmic reticulum (ER) and Golgi. Additionally, high electron-dense was observed in mitochondrial matrix in PC3/Tax cells.3. Analysis of cell cycle in both cell lines demonstrated that paclitaxel slightly delayed PC3/Tax cells in G1phase and a corresponding decrease in S-phase fraction. Molecular regulators of cyclin Dland A, associated with G1/S phase, remained unchanged in PC3/Tax cells by Western blotting, whereas the expression level of Cdc25A was marginal increased in PC3/Tax cells compared to parent PC3cells. The doubling time as a parameter of growth capacity was2.46days in PC3/Tax cell line, slightly increased in comparison with1.93days in the parental cell line. Since compelling evidence has demonstrated that PI3K/Akt and NF-κB signaling is constitutively activated and plays a critical role in proliferation of PC3cell line, we observed that phosphorylation of Akt and p-65was decreased in paclitaxel-resistant cells, while the total proteins of Akt and p65maintained at same levels in both cell lines.4. Flow cytometry assays demonstrated that no detectable P-glycoprotein expressions were observed both in the PC3and PC3/Tax cells, while expressions of mRNAs of mdrl were presented in both cell lines, and more pronounced in PC3/Tax cell line, consistent with reports that the resistance of PC3/Tax cells is not MDR-1mediated. Additionally, ABCC1/MRP1, another important transporter which confers resistance, remained unchanged, whereas ABCC2/MRP2, ABCC3/MRP3and ABCC10/MRP7were upregulated significantly in the paclitaxel-resistant cell line, log ratio is5.6,3.9and2.1for ABCC2/MRP2, ABCC3/MRP3and ABCC10/MRP7, respectively. It has been reported that ABCC10/MRP7has a structural configuration similar to MRP1and induces resistance to paclitaxel and docetaxel.5. Gene expression profiles obtained by microarray revealed that a total of3814genes were upregulated, and4370genes were doworegualted in resistant cell line. Of those genes, a-tubulin8(2.8-fold) andβ-tubulin6(8.0-fold) isotypes were upregulated significantly, whereasa-tubulinla, α-tubulinlb, β-tubulin, β-tubulin2a remained unchanged in resistant cells compared to the parent cells. In addition, other MDR related genes including glutathione S-transferase pi (GST-pi), Topoisomerase Ⅱ alpha (TOPO-2α), thioredoxin reductase3(TXNRD3), antiapoptotic gene Bcl2were observed to be upregulated in paclitaxel resistant cells, the fold changes were6.9,7.4,13.9,14.9, respectively.Conclusion:Taxol resistant PC3/Tax cells were developed from androgen-independent, taxol-sensitive PC3cells by taxol dose escalation. Multiple MDR genes are altered in resistant cells, and combination of mechanisms underlying the resistance to chemotherapy may exist in PCa. P-glycoprotein may not be involved in the development of taxol resistance. Further investigation is aimed to understand of proteins and signaling pathways associated with taxol resistance by PC3/Tax.Section II Effects of Riccardin D (RD) on PC3/Tax cells1. Treatment of RD caused a significant decrease in cell viability in parent PC3and drug-resistant PC3/Tax cell lines, the values of IC50were14.0±1.2,12.1±0.89, respectively.2. Riccardin D does not increase the sensitivity of resistant cells to paclitaxel: Riccardin D can not reverse paclitaxel-mediated resistance of multiple drugs; there is no significant change in IC50of the paclitaxel when riccardin D was used together with paclitaxel.3. The effects of riccardin D on drug accumulation:with experiments of Rh123accumulation, the results showed that riccardin D can increase the accumulation of Rh123in the drug resistant PC3/Tax cells, but the generic drug pump Inhibitors verapamil treated PC3/Tax cells showed significant increase of Rh123accumulation, much higher than the riccardin D treated group, indicating inhibition of riccardin D on drug efflux pump in resistant cell may be weak. This result suggests that riccardin D may not influence the function of the drug pump P-gp, but may, to a certain extent, inhibit other transmembrane transporter protein; in the mean while, riccardin D significantly inhibited proliferation of the drug resistant cells.4. Administration of RD to xenograft mice was associated with loss of tumorigenicity, and leading to a prolonged suppression of tumor growth. Analysis of tumor tissues revealed that RD coordinately caused an increase in the expression of BAX gene, and a decreased Bcl2involved in apoptosis.Conclusion:These observations suggested that Riccardin D was able to inhibit drug-resistant cell proliferation, and exerted antitumor activity via induction of cellular apoptosis. The reversal of resistance upon exposure to RD might not be transporter-dependent. |
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