斑蝥素應用在膀胱癌和口腔鱗狀細胞癌治療效用和分子機制之研究

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题名:	斑蝥素應用在膀胱癌和口腔鱗狀細胞癌治療效用和分子機制之研究 The Study of Therapeutic Effects and Molecular Mechanisms of Cantharidin in Bladder Cancer and Oral Squamous Cells Carcinoma
作者:	蘇金泉;Chin-Chuan Su
关键词:	斑蝥素;膀胱癌;口腔鱗狀細胞癌;內質網壓力;細胞凋亡;Cantharidin;Bladder carcinoma cells;OSCC;PKC;ER stress;apoptosis
日期:	2018-12-14
上传时间:	2019-11-11
出版者:	中國醫藥大學
摘要:	膀胱癌是一種全球性常見的惡性腫瘤。然而，至今仍然沒有有效治療膀胱癌的藥物。在本研究中 ,我們將探討的斑蟊素在人類膀胱癌細胞株(包含T24及RT4細胞)的細胞毒性。斑蟊素是一種純化合物，它是由中國斑蟊(Mylabris phalerata or Mylabris cichorii)或稱為西班牙蒼蠅(Cantharis vesicatoria)所產生的天然毒素。人類膀胱癌細胞株T24細胞經由斑蟊素處理後，可以顯著地降低其細胞活性並誘導其caspase-3活性表現增強，此現象可以藉由Z-DEVD-FMK (一種特異性的caspase-3抑制劑)所回復。在經由斑蟊素處理後的T24細胞，其裂解型態的caspase9/7/3表現也增加。並且，斑蟊素可以增加eIF2?悛瑣F酸化表現和Grp78蛋白的表現，並且可以降低 procaspase-12的蛋白質表現；這些現象並會伴隨著在T24細胞calpain酵素的活性增加。除此之外，斑蟊素可以增加細胞內鈣離子濃度和protein kinase C（PKC）的鄰酸化。添加BAPTA/AM (一種細胞內鈣離子螯合劑)和 RO320432 (一種選擇性細胞穿透性PKC抑制劑) 可以有效逆轉回復經由斑蟊素誘導的caspase-3和calpain的酵素活性表現增加，PKC和eIF2的鄰酸化程度與Grp78蛋白質表現增加，和 procaspase-12的蛋白質表現減少的等現象。重要的是，斑蟊素可以顯著地減少在裸鼠異體移植的T24細胞的腫瘤體積大小(在經過21天的治療後，斑蟊素可以顯著的減少百分之七十一腫瘤的體積大小)。總體而言，這些實驗結果顯示斑蟊素可以經由鈣離子和PKC調節內質網壓力訊息路徑促使人類膀胱癌細胞凋亡。這些發現顯現出斑蟊素可能是一種針對人類膀胱癌細胞全新並具潛力的抗癌藥劑。口腔癌是屬於頭頸部癌症的一種，也是全世界常見的惡性腫瘤。大部分口腔癌的病理診斷是口腔鱗狀細胞癌。口腔鱗狀細胞癌的特徵是具有高度的局部侵犯性與高比例的區域頸部淋巴結轉移。如何預防與治療口腔鱗狀細胞癌是重要並且迫切需要的。斑蟊素是由斑蟊分離出來的活性成分，在此研究中，我們探討斑蟊素在體外對口腔鱗狀細胞癌治療效果與其相關的分子機轉。研究結果顯示斑蟊素可以顯著地降低人類舌部鱗狀細胞癌細胞SAS, CAL-27,和SCC-4等細胞株的細胞活性。接續的機轉實驗主要以SAS細胞株來進行。研究結果發現斑蟊素可以顯著地增加細胞凋亡相關的訊息諸如caspase-9, caspase-7 and caspase-3蛋白質的表現。此外，斑蟊素可以降低粒線體膜電位並且誘導細胞色素c (cytochrome c)與凋亡誘導因子(AIF)的釋放。斑蟊素也可以增加Bax, Bid,和Bak蛋白質的表現並降低Bcl-2蛋白質的表現。斑蟊素亦增加包含eIF2?悛瑣F酸化表現和CHOP蛋白等內質網壓力訊息的表現但其並不包含Grp78 和Grp94等蛋白質的表現。並且斑蟊素可以降低pro-caspase-12蛋白質的表現。並且，在絲裂原活化蛋白激?(mitogen-activated protein kinases)訊息表現方面，斑蟊素可以增加JNK蛋白的鄰酸化但不包含ERK and p38蛋白。轉染JNK的短髮夾RNA（shRNA）至口腔鱗狀細胞癌細胞中，可以有效反轉斑蟊素所引起的粒線體與內質網壓力相關細胞凋亡訊息分子表現。總體而言，這些發現顯示斑蟊素可以經由JNK調節粒線體與內質網壓力的訊息路徑促使人類口腔鱗狀細胞癌細胞凋亡。 Bladder cancer is a common malignancy worldwide. However, there is still no effective therapy for bladder cancer. In this study, we investigated the cancer cell cytotoxicity of cantharidin, a natural toxin (pure compound) produced from Chinese blister beetles (Mylabrisphalerata or Mylabriscichorii) and Spanish flies (Cantharis vesicatoria) in human bladder cancer cell lines (including: T24 and RT4 cells). Treatment of T24 cells with cantharidin significantly decreased cell viability and induced an increase in caspase-3 activity, which could be reversed by Z-DEVD-FMK (a specific caspase-3 inhibitor). The expression of cleaved form of caspase-9/-7/-3 was also increased in cantharidin-treated T24 cells. Furthermore, cantharidin increased the levels of phospho-eIF2?? and Grp78 and decreased the protein expression of pro-caspase-12, which was accompanied by the increase in calpain activity in T24 cells. Cantharidin was capable of increasing the intracellular Ca2+ and the phosphorylation of protein kinase C (PKC) in T24 cells. Addition of BAPTA/AM (a Ca2+ chelator) and RO320432 (a selective cell-permeable PKC inhibitor) could effectively reversed the increases in caspase-3 and calpain activity, the phosphorylation levels of PKC and eIF2?? and Grp78 protein expression, and the decrease in pro-caspase-12 expression induced by cantharidin. Importantly, cantharidin significantly decreased the tumor volume (a dramatic 71% reduction after 21 days of treatment) in nude mice xenografted with T24 cells. Taken together, these results indicate that cantharidin induced human bladder carcinoma cell apoptosis through a calcium/PKC-regulated ER stress pathway. These findings suggest that that cantharidin may be a novel and potential anti-cancer agent targeting on bladder carcinoma cells. Oral cancer is a subtype of head and neck cancer and a common malignant cancer all around the world. Most of oral cancer is histopathologically diagnosed as oral squamous cell carcinoma (OSCC). OSCC is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. How to prevention and treatment of OSCC is important and imperative. Here, we investigated the therapeutic effect and molecular mechanism of cantharidin, an active compound isolated from blister beetles, on OSCC in vitro. Results showed that cantharidin significantly decreased cell viability in human tongue squamous carcinoma-derived SAS, CAL-27, and SCC-4 cell lines. The further mechanistic studies were carried out in SAS cells. Cantharidin also significantly increased apoptosis-related signals, including caspase-9, caspase-7 and caspase-3 proteins. Besides, cantharidin decreased mitochondrial transmembrane potential (MMP) and induced cytochrome c and apoptosis inducing factor (AIF) release. Cantharidin also increased Bax, Bid, and Bak protein expressions and decreased Bcl-2 protein expression. Cantharidin could also increase the endoplasmic reticulum (ER) stress signals, including the expressions of phosphorylated eIF-2?? and CHOP, but not Grp78 and Grp94. Furthermore, cantharidin reduced pro-caspase-12 protein expression. In signals of mitogen-activated protein kinases, cantharidin increased the phosphorylation of JNK, but not ERK and p38. Transfection of shRNA-JNK to OSCC cells effectively reversed the cantharidin-induced cell apoptotic signals, including the mitochondrial and ER stress-related signaling molecules. Taken together, these findings suggest that cantharidin induces apoptosis in OSCC cells via the JNK-regulated mitochondria and ER stress-related signaling pathways.
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