探討去甲斑蝥素誘導人類腎臟癌細胞凋亡分子機制

Study on molecular mechanism of Norcantharidin induces apoptosis in human renal cancer cells

吳敏華(Min-Hua Wu)

指導教授 ： 楊順發 ； 謝逸憲

中山醫學大學/醫學院/醫學研究所/博士(2018年)

摘要

先前研究報告證實去甲斑蝥素(Norcantharidin , NCTD)具有抗腫瘤的能力。本研究探討NCTD在人類腎臟癌的抗腫瘤功能和分子機制。利用MTT的結果證實NCTD明顯減少人類腎臟癌細胞生長能力和Annexin V/PI染色以流式細胞儀分析的結果證實NCTD明顯誘導細胞凋亡。NCTD活化caspase和PARP蛋白表現。此外，NCTD誘導粒線體去極化(depolarization)，伴隨著Bax蛋白量上升，Bcl-2和Mcl-1 蛋白量表現下降。NCTD也會誘導內質網壓力增加是經由活化Grp78、p-elF2α、ATF4和CHOP 蛋白表現增加。接下來，用內質網壓力抑制劑（Salubrinal）明顯減弱NCTD抑制786-0細胞生長、細胞凋亡和內質網壓力相關蛋白的表現。NCTD 也會抑制AKT 途徑活化。當AKT過度表現會減緩NCTD誘導的細胞凋亡能力，同時也會減少NCTD誘導的Bcl-2和Mcl-1蛋白表現量下降及Bax、cleaved-caspase-9、cleaved-PARP和p-elF2α蛋白量表現上升。最後動物實驗研究證明NCTD抑制腎臟癌腫瘤生長。綜合以上結果證實NCTD在人類腎臟癌治療上是一個可能的抗腫瘤藥劑。

關鍵字

人類腎臟癌 ； 去甲斑蝥素 ； 細胞凋亡

並列摘要

Previous studies reported that norcantharidin (NCTD) has anti-tumor effects. We investigated the antitumor effects and underlying mechanism of NCTD on human renal cancer in vitro and in vivo. NCTD significantly decreased renal cancer cell viability by induction of apoptosis, as determined by the MTT assay and annexin V/PI staining. NCTD treatment of 786-O and A-498 cells altered the expression of caspase family proteins and PARP. Moreover, NCTD induced mitochondrial depolarization, which was accompanied by an increased level of Bax and decreased levels of Bcl-2 and Mcl-1. NCTD induced endoplasmic reticulum (ER) stress by increasing the expression of Grp78, p-elF2α, ATF4, and CHOP. Pretreatment with an ER stress inhibitor (salubrinal) significantly attenuated the effect of NCTD. NCTD also induced inactivation of the AKT pathway in 786-O and A-498 cells. Overexpression of AKT partly reversed the effect of NCTD on apoptosis. NCTD treatment led to decreased expression of Bcl-2 and Mcl- 1, and increased expression of Bax, cleaved-caspase-9, cleaved-PARP, and p-elF2α. Our in vivo studies demonstrated that NCTD significantly inhibited tumor growth in a nude mouse xenograft model. Taken together, our results suggest that NCTD is a potential anti-tumor agent for treatment of renal carcinoma.

並列關鍵字

Human renal cell carcinoma ； Norcantharidin ； Apoptosis

參考文獻

1. Kraut JA, Madias NE (2017) Adverse Effects of the Metabolic Acidosis of Chronic Kidney Disease. Adv Chronic Kidney Dis 24: 289-297.

2. Sprangers B, Nair V, Launay-Vacher V, Riella LV, Jhaveri KD (2018) Risk factors associated with post-kidney transplant malignancies: an article from the Cancer-Kidney International Network. Clin Kidney J 11: 315-329.

3. Sokoloff MH, Daneshmand S, Ryan CW (2005) Current clinical trials in renal cell carcinoma. Urol Oncol 23: 289-292.

4. Choudhury D, Luna-Salazar C (2008) Preventive health care in chronic kidney disease and end-stage renal disease. Nat Clin Pract Nephrol 4: 194-206.

5. Lipworth L, Tarone RE, McLaughlin JK (2006) The epidemiology of renal cell carcinoma. J Urol 176: 2353-2358.