探討 DHTS 誘導結腸直腸癌細胞凋亡之分子機轉

Study of the molecular mechanisms of DHTS-induced apoptosis in colorectal cancer cells

曾方瑜(Fang-Yu Tzeng)

指導教授 ： 梁有志

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00015

摘要

由中草藥萃取出來之天然物，可做為發展新抗癌藥物的來源。本篇論文所使用的藥物15,16-Dihydrotanshinone I (DHTS) 為傳統中草藥丹参之萃取物，是具有細胞毒殺活性的成分之一。根據本實驗室先前的研究發現，DHTS 透過降低 cyclin D 和 cyclin E 的表現，讓乳癌細胞停滯於 G1 期，接著再透過粒線體途徑導致細胞凋亡。另外，實驗室也證實 DHTS 可誘導細胞內質網壓力與未折疊蛋白反應造成前列腺癌細胞凋亡。在本篇論文中探討 DHTS 作用在人類結腸直腸癌細胞 SW480 和 SW620 是否具有抗癌活性，並且進一步分析其分子機制。實驗結果首先發現，SW480 及 SW620 細胞在 1 ?慊/ml DHTS 的處理之下，細胞存活率明顯的下降。利用流式細胞儀偵測 Annexin V/propidium iodide (PI)雙染的螢光強度，得知隨著 DHTS 濃度的增加而造成細胞凋亡。接著我們研究 DHTS 使結腸直腸癌細胞凋亡的分子機制，從西方墨點法的結果發現，DHTS 誘導結腸直腸癌細胞 activating transcription factor 3 (ATF3) 的表現，其為 ATF/CREB 蛋白家族成員之一。ATF3 可藉由不同刺激使其表現，並由多種訊息途徑調控之。為了闡明 DHTS 調控的訊息傳遞路徑，我們分析 mitogen-activated protein kinase (MAPK) 的活化情形，並以 MAPK 抑制劑觀察其對 ATF3 之影響。結果顯示，DHTS 活化 MAPK 路徑早於 ATF3，並且呈現持續活化的情形，而前處理 JNK 或 p38 抑制劑，可略為阻斷 DHTS 誘導 ATF3 之表現。ATF3 過量表現可使結腸直腸癌細胞 SW480 細胞凋亡顯著增加。相較之下，結腸直腸癌細胞 SW620 過度表現 ATF3，則保護細胞因 DHTS 誘導之細胞凋亡作用。直到目前為止，實驗證明 DHTS 刺激 ATF3 表現，接著引發細胞凋亡。而 ATF3 是透過 p38-, JNK- 途徑調控之。我們証實 ATF3 在癌症發展過程中具有雙重角色，由結果顯示細胞惡性度能影響 ATF3 之功能。

關鍵字

DHTS ； 結腸直腸癌 ； 細胞凋亡 ； ATF3

並列摘要

Natural products derived from Chinese medicinal plant provide a source for development of new anticancer drugs. 15,16-dihydrotanshinone I (DHTS) was found to be an active cytotoxic component from the well-known traditional Chinese medicinal plant Salvia miltiorrhiza Bunge (Tanshen). According to our previous studies have shown that DHTS downregulated cyclin D and cyclin E, resulted in G1 phase arrest in breast cancer cell lines, and then induced apoptosis through mitochondrial apoptosis pathway. Besides, another study demonstrated that DHTS could induce apoptosis of prostate cancer cells via induction of ER stress and unfold protein response. In this study, we have investigated the molecular mechanism of DHTS anti-tumor activity in human colorectal cell lines SW480 and SW620. Preliminary data exhibited that DHTS treatment 1 ?慊/ml of SW480 and SW620 cells significantly decreased cell viability. Using Annexin V/propidium iodide (PI) staining by flow cytometry, showed DHTS resulted in a concentration-dependent apoptotic cell death. We further examined the molecular mechanism of DHTS on apoptosis in colorectal adenocarcinoma, DHTS alone induced activating transcription factor 3 (ATF3) expression, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, in colorectal adenocarcinoma as determined by Western blot analysis. Expression of ATF3 is stimulated by diverse stimuli and is likely to be regulated by many signaling pathway. To elucidate the signal transduction by DHTS, the activation of mitogen-activated protein kinase (MAPK) and the effects of ATF3 expression by MAPK inhibitors were examined. We showed that DHTS-induced ATF3 was preceded by a rapid and sustained activation of MAPK. The ATF3 expression by DHTS was blocked by JNK or p38 inhibitor pretreatment. Overexpression of ATF3 resulted in significant augment of
DHTS-activated apoptosis in SW480 cells. In contrast, ectopic expression of ATF3 in SW620 cells protected the cells from DHTS-induced reduction in cell numbers. Thus far, the current study shows that DHTS stimulates ATF3 expression and subsequently induces apoptosis. These pathways are mediated through p38-, JNK-dependent pathways. We present evidence that ATF3 has a dichotomous role in cancer development, our result suggests that the degree of malignancy of the cells affects ATF3 function.

並列關鍵字

DHTS ； colorectal cancer cells ； apoptosis ； ATF3

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