探討低氧及低氧模擬物對前列腺癌細胞之影響

UNDERSTANDING THE EFFECTS OF HYPOXIA AND HYPOXIA-MIMETIC AGENTS IN THE HUMAN PROSTATE CARCINOMA CELLS

鍾麗娟(Li-chuan Chung)

指導教授 ： 李綉鈴

大同大學/工程學院/生物工程學系所/博士(2011年)

摘要

L-mimosine及Dimethyloxalylglycine (DMOG)皆是酺胺酸羥基酶抑制劑 (prolyl hydroxylase inhibitors)，可抑制癌細胞生長。L-mimosine 及DMOG亦能穩定低氧誘導因子（HIF-1α）蛋白質，具有類似低氧的效果，常被用於體外及體內的低氧模擬實驗。B-cell translocation gene 2（BTG2）主要調控細胞週期中G1/S期。N-myc downstream-regulated gene 1 (NDRG1) 為一抑癌的基因，會促進細胞分化而抑制癌細胞生長、腫瘤形成及轉移。前列腺特異抗原（Prostate-specific antigen, PSA）是由前列腺上皮管腔細胞所分泌，而血清PSA 濃度是評估及診斷前列腺癌症的重要標誌。此博士論文主要探討低氧及低氧模擬物質對前列腺癌細胞之影響。以3H-thymidine incorporation及流式細胞儀分析發現L-mimosine 可促使PC-3 及 LNCaP 細胞週期分別停留於 G1 或 S 期，進而抑制細胞生長。從西方點墨法及短暫性基因報導分析法發現L-mimosine及低氧可穩定 PC-3 及 LNCaP 細胞的HIF-1α，進而誘導BTG2及NDRG1的蛋白質表現，但是降低週期素A (cyclin A) 的蛋白質表現。在LNCaP 細胞剔減 (knockdown) HIF-1α基因後，低氧或L-mimosine所誘導BTG2 及 NDRG1蛋白質表現增加的情形便減緩。該結果顯示L-mimosine 抑制前列腺癌細胞 PC-3 及 LNCaP 的生長是經由調控 BTG2 及 NDRG1 基因的轉錄，且此調控機轉需依賴細胞內HIF-1α的存在。西方點墨法及酵素連結免疫分析法（ELISA）結果亦發現，L-mimosine 及 DMOG穩定 HIF-1α蛋白質並誘導 PSA 的基因表達於LNCaP細胞。經由短暫性基因報導分析法也顯示低氧促使PSA基因表達需依賴細胞內 HIF-1α及雄性素受體(AR)的表現。薑黃素(curcumin) 可以降低因低氧所促進的AR蛋白質表現，但卻不影響HIF-1α 及血管內皮生長因子(VEGF) 蛋白質表現。ELISA及短暫性基因報導分析法也顯示薑黃素可阻斷L-mimosine 或 DMOG 對PSA基因的活化。綜合上述，此博士論文的研究結果顯示低氧及低氧模擬物質(L-mimosine及Dimethyloxalylglycine)可經由穩定HIF-1α蛋白質而促進 BTG2及NDRG1基因的表現，並抑制前列腺癌細胞 PC-3 及 LNCaP 的增生。同時，L-mimosine 及 DMOG與低氧處理亦可誘導PSA基因的表達。而藉由curcumin處理可經由抑制AR蛋白質的表現進而阻斷低氧及低氧模擬物質對PSA基因的活化。

關鍵字

細胞週期 ； 低氧模擬物 ； 低氧 ； 薑黃素 ； 前列腺癌 ； 前列腺特異性抗原

並列摘要

L-mimosine and dimethyloxalylglycine (DMOG) are prolyl 4-hydroxylase inhibitors stabilizing the hypoxia-inducible factor-1α (HIF-1α) and are used to mimic the effects of hypoxia in vivo and in vitro. The B-cell translocation gene 2 (BTG2) regulates the G1/S transition phases of the cell cycle. N-myc downstream-regulated gene 1 (NDRG1) is a differentiation-inducing gene upregulated by hypoxia. Prostate-specific antigen (PSA) is a well-known biomarker for diagnosing and evaluating the status of prostate cancer. Studies of this dissertation are to evaluate the effects of hypoxia and hypoxia mimetics on prostate carcinoma cells. The 3H-thymidine incorporation and flow cytometry assays revealed that the L-mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and L-mimosine stabilized HIF-1α and induced BTG2 and NDRG1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. The transient gene expression assay revealed that L-mimosine treatment or co-transfection with HIF-1α expression vector enhanced the promoter activities of BTG2 and NDRG1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both BTG2 and NDRG1 by hypoxia or L-mimosine in LNCaP cells. Immunoblot and enzyme-linked immunosorbent (ELISA) assays indicated that L-mimosine and DMOG stabilized HIF-1α and induced PSA gene expression in LNCaP cells. Further studies indicated that induction of the PSA expression by hypoxia is both HIF-1α- and AR-dependent. Immunoblot assays revealed that a curcumin treatment decreased the protein level of AR but did not significantly affect the protein levels of HIF-1α and vascular endothelial growth factor (VEGF) which were induced by hypoxia. ELISA and transient gene expression assays indicated that curcumin blocked the activation of L-mimosine or DMOG treatment on PSA expression. In conclusion, results of this dissertation indicated that hypoxia and L-mimosine modulated BTG2 and NDRG1 at the transcriptional level, which is dependent on HIF-1α. L-mimosine enhanced expression of BTG2 and NDRG1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells. L-mimosine and DMOG stabilized HIF-1α and induced PSA gene expression. The curcumin blocked the AR protein expression, which attenuated the enhanced effect of PSA expression by L-mimosine and DMOG that induce hypoxia condition.

並列關鍵字

hypoxia ； HIF-1a ； mimosine ； PSA ； dimethyloxalylglycine ； NDRG1 ； BTG2 ； androgen receptor ； curcumin

參考文獻

1. Aggarwal BB and Sung B (2009) Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci 30(2):85-94.

2. Bae MK, Kim SH, Jeong JW, Lee YM, Kim HS, Kim SR, Yun I, Bae SK and Kim KW (2006) Curcumin inhibits hypoxia-induced angiogenesis via down-regulation of HIF-1. Oncol Rep 15(6):1557-1562.

4. Bandyopadhyay S, Pai SK, Hirota S, Hosobe S, Takano Y, Saito K, Piquemal D, Commes T, Watabe M, Gross SC, Wang Y, Ran S and Watabe K (2004) Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression. Oncogene 23(33):5675-5681.

5. Chang HC, Lee TH, Chuang LY, Yen MH and Hung WC (1999) Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms. Cancer Lett 145(1-2):1-8.

6. Chen SS, Chen KK, Lin AT, Chang YH, Wu HH and Chang LS (2009) Correlation between pretreatment serum biochemical markers and treatment outcome for prostatic cancer with bony metastasis. J Chin Med Assoc 72(6):301-306.