Rationale and clinical implication of combined chemotherapy with cisplatin and oestrogen in prostate cancer: primary evidence based on methylation analysis of oestrogen receptor-alpha.

OBJECTIVE: To determine whether oestrogen enhances platinum sensitivity, and if promoter CpG methylation of the oestrogen receptor-alpha (ER-alpha) gene determines the potential of cisplatin-induced apoptosis in prostate cancer, as the high-mobility group 1 (HMG1) preferentially binds to cisplatin-modified DNA and is up-regulated after oestrogen treatment in breast cancer cell line MCF-7. MATERIALS AND METHODS: The study comprised prostate cancer cell lines (LNCaP and PC-3), 156 pathologically confirmed 156 radical prostatectomy samples and eight hormone-refractory prostate cancer (HRPC) samples (from needle biopsy). Expression of HMG1 in cell lines was analysed by Western blotting or differential reverse-transcription-polymerase chain reaction (PCR). The methylation status of ER-alpha was analysed by methylation-specific PCR using bisulphite DNA as a template or bisulphite DNA sequencing. RESULTS: In LNCaP cells, treatment with oestrogen increased HMG1 expression and co-treatment with cisplatin and oestrogen reduced cell viability by accelerating apoptosis, compared with cisplatin alone. However, in PC-3, oestrogen did not up-regulate HMG1 or accelerate the cisplatin-induced apoptosis. Although ER-beta was expressed in both LNCaP and PC-3, ER-alpha was expressed only in LNCaP. Bisulphite DNA sequencing of the ER-alpha promoter showed partial methylation in LNCaP but complete methylation in PC-3. ER-alpha AS transfection diminished the cisplatin-induced apoptosis in oestrogen-treated LNCaP cells. In clinical samples there was ER-alpha hypermethylation in 40% of prostate cancers this correlated with Gleason score (GS, 31% for GS < 7, 50% for GS = 7 and 56% for GS > 7). In addition, five of eight HRPC samples showed ER-alpha hypermethylation. CONCLUSION: These findings suggest that HMG1 induction as an enhancer of platinum sensitivity is mediated through interaction between oestrogen and ER-alpha. As CpG hypermethylation of the ER-alpha promoter is a frequent event in aggressive prostate cancer, negative conversion of ER-alpha methylation is essential to achieve the most beneficial effect when combined chemotherapy of cisplatin with oestrogen is used in patients with prostate cancer.

Blockade of paclitaxel-induced thymidine phosphorylase expression can accelerate apoptosis in human prostate cancer cells.

Recently, survival benefit by chemotherapy using paclitaxel (PTX) and the induction of thymidine phosphorylase (TP) by PTX have been reported in several solid tumors. On the other hand, TP confers antiapoptotic effect on tumor cells through inhibition of caspase-8 activation in vitro. On the basis of these previous observations, we hypothesized that (a) TP can be induced after PTX treatment in human prostate cancer (PC) and (b) blockade of PTX-induced TP expression can enhance the apoptotic processes in human PC cells. PTX was used to find TP expression in all eight hormone-refractory PC cases after chemotherapy; however, cleaved caspase-8 was not expressed after chemotherapy in the six hormone-refractory PC cases with strong TP expression. In PC cell lines (PC-3, DU 145, and LNCaP), TP expression after PTX treatment was clearly up-regulated in a dose-dependent manner. Cell viability of PC cell lines treated with PTX and TP antisense was significantly reduced in a time-dependent and dose-dependent manner compared with the PTX treatment alone. Likewise, apoptotic index of PC cells treated with PTX and TP antisense was significantly increased in comparison with PTX alone. After complete blockade of PTX-induced TP translation by TP antisense transfection, cleaved form of caspase-3 and poly(ADP-ribose) polymerase was increased, and this exaggeration of apoptosis also ran parallel with caspase-8 activation in a PTX dose-dependent manner. However, in PC cell lines treated with TP antisense alone, neither caspase-3 nor poly(ADP-ribose) polymerase was cleaved despite caspase-8 activation. These results indicate that PTX-induced TP up-regulation is associated with decreased caspase-8 activation. This study is the first report showing that blockade of PTX-induced TP expression could exaggerate the processing of apoptosis in PC cells treated with PTX. Our results provide preclinical evidence that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells.

Decreased survival of prostate cancer cells in vitro by combined treatment of heat and an antioxidant inhibitor diethyldithiocarbamate (DDC).

The aim of this study was to examine a modulation of thermotolerance by treatment with combination of heat and the antioxidant inhibitor diethyldithiocarbamate (DDC) of the PC-3 prostate cancer cells. To determine thermotolerance, cells were heated once or twice. Two 1 h exposures at 43 degrees C, with a recovery period in between, revealed better survival/recovery of cells after the second exposure than after the first (fig. 1A + 1B). Additional experiments were performed, heating cells twice (fig. 1B + 1C). First, cells were heated at 43 degrees C for 1 h and, after various recovery times (intervals) at 37 degree C, subsequently reheated at 44 degrees C for 1 h. To ensure effective cell killing, efficiency of the combined treatments of 1 mM DDC and heating at 43 or 44 degrees C for 1 h was estimated by measuring cell survival, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and heat shock protein 70 (hsp 70) expression. To obtain a more effective method for subsequent heat exposure, cells were heated twice after a 24 h interval in the presence or absence of 1 mM DDC. ROS generation and SOD activity immediately increased correlating with duration of heating, but their levels gently decreased with time after discontinuation of heating. On the other hand, hsp 70 levels slowly increased, also correlating with duration of heating but continued to increase with time after discontinuation of heating for a certain period. DDC administration coupled with heating at 43 or 44 degrees C significantly decreased cell survival compared to heating alone (p < 0.05). Furthermore, significant decreases in numbers of viable cells were observed for cells after the first heat exposure when combined with DDC as compared to heat alone at 43 and 44 degrees C (p < 0.05). These findings suggest that heat combined with DDC could have potential benefits in the treatment of prostate cancer.