山苦瓜活性物質TCD抑制人類胃癌細胞增生之作用機轉

Abstract

動機: 先前文獻報導葫蘆烷三萜類化合物能抑制乳癌、子宮頸癌及前列腺癌細胞增生，具抗腫瘤的潛力。本研究室先前證實一種葫蘆烷三萜類化合物3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD)能延滯細胞週期、引起細胞凋亡而抑制人類胃癌細胞株AGS細胞增生。然而其作用機轉未明，因此本研究接續探討TCD對於人類胃癌細胞株(AGS、MKN28、MKN45)增生的抑制效應及其誘導細胞凋亡之分子機制。

方法: 使用MTT測定TCD處理對於AGS、MKN45、MKN28存活率的影響。以流式細胞儀和西方墨點分析TCD處理AGS細胞對於細胞週期與細胞凋亡之相關蛋白表現量的影響。另外以西方墨點分析內質網壓力相關蛋白(GRP78 、p-PERK、IRE-1、XBP-1、ATF4、CHOP、p-JNK)的變化。

結果: 本實驗結果發現TCD可抑制胃癌細胞株AGS、MKN28、MKN45的增生，其IC50值分別為41, 40 和 45 μM。以TCD (30 μM)處理AGS細胞24 小時後，發現TCD經由抑制Cyclin E、Cyclin D1、CDK2、CDK4和p-Rb蛋白表現，造成細胞週期停滯於G1期。經TCD(30μM，24小時) 處理後顯著提升GRP78蛋白表現，激活未折疊蛋白反應中p-PERK、ATF-4、CHOP、p-JNK，抑制抗凋亡蛋白Bcl-2及激活凋亡蛋白Bax，活化下游 cleaved caspase 9、cleaved caspase 3和 cleaved PARP，而引起細胞凋亡。另 TCD亦可促使MKN28和MKN45細胞的提升GRP78蛋白表現，推測亦可激活這兩株細胞的未折疊蛋白反應、引起內質網壓力。以外，從MTT細胞存活分析結果發現化療藥物SN38和TCD以等比例(AGS --1:200，MKN45--1:2000，MKN28--1:300) ，合併使用時，能有效抑制AGS、MKN28和MKN45細胞增生，分別具協同 (synergism)或加成(additive)作用。

結論: 由上述結果推論TCD可經由延滯細胞週期、引起內質網壓力導致細胞凋亡而抑制胃癌AGS細胞增生，具抗胃癌的潛力 。而TCD與SN38併用可協同或加成性的抑制胃癌細胞株增殖，推測TCD具有增強化療藥物作用的潛力，但仍待未來更多研究證實。Purpose: 3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD), a dietary triterpenoid, was isolated from wild bitter melon leaf (Momordica charantia Linn. var. abbreviata Ser.). Our previous studies showed that TCD inhibited cell cycle and induced DNA breaks and apoptosis in human AGS gastric adenocarcinoma cells. However, the inhibitory mechanism of TCD on AGS cell proliferation is unclear. Therefore, the aim of this study was to investigate the mechanism of anti-proliferative effect of TCD on human gastric cancer cell lines including AGS, MKN28, and MKN45 cells.

Methods: The MTT assay was used to determined cell viability. The effects of TCD on cell cycle regulation and apoptosis were analyzed using flow cytometric analysis and Western blot assay. Specific cellular protein expressions (GRP78, p-PERK, IRE-1, XBP-1, ATF4, CHOP, and p-JNK) were measured using Western blot analysis.

Results: TCD significantly decreased AGS, MKN28 and MKN45 cell viability, with IC50 values of 41, 40, and 45 μM, respectively. Flow cytometric analysis indicated that TCD (30 μM) induced G1 cell-cycle phase arrest and apoptosis in AGS cells. Western blot assay showed that the reduction of Cyclin E, Cyclin D1, CDK2, CDK4 and p-Rb levels after TCD treatment in AGS cells may paly critical roles in the G1 cell cycle arrest that blocked cell proliferation. TCD treatment resulted in an increase of Bax level and a decrease of Bcl-2. Similarly, caspase dependent poly (ADP) ribose polymerase (PARP) cleavage by TCD with which to induced apoptosis in AGS cells was observed. In addition, TCD activates the GRP78/ p-PERK/ATF-4/CHOP arm and p-JNK of endoplasmic reticulum (ER) stress. To evaluate the chemo-sensitizing potential of TCD, combination of TCD with SN-38 resulted in synergistic or additive cytotoxicity in all three gastric cancer cell lines type.

Conclusion: TCD treatment increased GRP78 to activate unfolding protein response and induction of apoptosis, and caused cell-cycle arrest to inhibit gastric cancer cell proliferation. Our results suggest that TCD may be a promising candidate agent used in the treatment of gastric cancer. However, further work is still needed to investigate its chemo-sensitizing effect on chemotherapy drug-resistant gastric cancer cells.