探討細胞壓力反應造成大腸腺癌細胞凋亡的分子機轉

The Molecular Mechanism of Cellular Stress Response-Mediated Apoptosis of Colonic Carcinoma Cells

吳明順(Ming-Shun Wu)

指導教授 ： 連吉時

共同指導教授 ： 陳彥州(Yen-Chou Chen)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2014年)

https://doi.org/10.6831/TMU.2014.00032

摘要

由於飲食和生活習慣的改變，大腸直腸癌的發生率在亞洲地區迅速竄升，也是國人十大癌症死因的第三名。其主要的治療為手術切除，以及術後的化學治療，甚至要併用標靶治療，然而高劑量的化學治療仍有很多全身性的副作用，因此食物中所含的自然而無毒的天然物就常被用來做為癌症治療的研究，以其合併飲食添加物的抗氧化劑或標靶治療或許可以做為輔助療法，藉以增強療效並降低藥物副作用。
細胞壓力反應是當細胞接觸到環境壓力時所產生一連串的分子變化。而這些變化是為了讓細胞更能夠適應環境。細胞壓力反應主要可以透過一些壓力蛋白質的作用，進而保護細胞免於死亡。例如熱壓力誘導熱休克蛋白，氧化壓力誘導產生第一型血紅素氧化脢，而內質網壓力可以減少未摺疊蛋白的聚集。如果可以合併多種方法阻斷這些機制，就可能促成癌細胞的死亡。
氧化壓力(oxidative stress)或過多的抗氧化物引發的氧化還原失衡都能造成細胞凋亡，NAC能夠抑制過氧化物 (reactive oxygen species, ROS)造成的細胞死亡，但卻無法抑制滅必治 (etoposide)這類抗癌藥物造成的細胞凋亡。此外，由於標靶治療目前已經廣泛地運用於癌症的治療，以漆黃素合併熱休克蛋白90抑制劑來探討熱休克蛋白對大腸癌細胞凋亡的機轉。因此，本研究主要的目的是希望透過併用藥物造成細胞的壓力反應來觀察大腸癌細胞凋亡的機轉。
以天然物漆黃素(fisetin)與抗氧化劑N-乙醯- L -半胱氨酸 (N-acetyl-L-cysteine, NAC)探討氧化壓力對大腸癌細胞凋亡的機轉，使用無毒的10 mM 及20 mM的NAC可以強化漆黃素所造成的大腸癌細胞凋亡。合併療法也會增加caspase-3的裂解與活性，產生更多的DNA ladder，同時也降低粒線體的膜電位(mitochondrial membrane potential, MMP)及Caspase-9的裂解與活性。合併療法更能夠有效地抑制ERK蛋白的磷酸化及形成DNA ladder，並增加裂解caspase-3與活性。此外，NAC也能增強一種類似漆黃素的化學物質Chrysin(白楊素)誘導COLO205細胞的凋亡，而且在不同的大腸癌細胞株實驗，包括HCT-116，HT-29 及HCT-15也同樣觀察得到這樣的現象。由此實驗得知NAC強化漆黃素(或白楊素)造成大腸癌細胞凋亡的原因，並不是透過ROS的系統而來，而是透過降低mitochondrial membrane potential (粒線體膜電位)及Bcl-2蛋白形成後續的凋亡途徑。
此外，我們發現漆黃素若併用熱休克蛋白90抑制劑，geldanamycin (GA)與Radicicol (RAD)，能夠造成大腸癌細胞凋亡，原因在於增加p53蛋白上的泛?旬擘訄O蛋白(ubiquitin-tagged protein)，所以加速裂解，降低p53蛋白細胞及其穩定度。
由細胞實驗的結果看來，併用天然物漆黃素(或白楊素)與NAC或休克蛋白90抑制劑具有抗大腸癌的潛力，值得進行後續的動物研究。

關鍵字

大腸癌 ； 漆黃素 ； 熱休克蛋白90抑制劑

並列摘要

The incidence of colorectal cancer (CRC) is dramatic increasing in Asia due to significant changes in diet and lifestyle over the past few decades. It’s also emerging as the top third leading mortality of cancer in Taiwan. Surgical resection and postoperative adjuvant chemotherapy or even target therapy is a mainstream of anti-cancer therapy. However, there are systematically side effects in such kind of chemical compounds. Novel dietary compounds are also being aggressively researched as cancer therapeutic agents. Combination of dietary compounds, antioxidant of supplement and targeted therapy may be an alternative anti-cancer therapeutic agents.
Cellular stress response is a general term covering a wide range of molecular change that cells undergo in response to environmental stress. These various processes serve the adaptive purpose to accommodate the environment. It is primarily mediated through stress proteins which can exhibit widely varied functions within a cell to protect cell death. Such as thermal stress induced heat shock protien (HSP) production, oxidative stress triggered heme oxygenase-1 (HO-1) production and endoplasmic reticulum (ER) stress attenuated the accumulation of unfolded protein aggregates. By using the combinatorial strategy to inhibit these processes may induce cancer cell death.
Oxidative stress or excessive antioxidant levels-caused redox imbalance can alter apoptotic responses, and N-acetyl-L-cysteine (NAC) was able to inhibit H2O2-mediated cell death, but unable to prevent apoptosis induced by other chemicals such as etoposide. Heat shock protein 90 (HSP90) inhibitors, a group of promising compounds for targeted therapy, have been vigorously investigated in clinical trials. However, their potential for anticancer effects in combination with dietary flavonoids is still unclear. The specific aim addressed in this study is to observe apoptosis of colon carcinoma cells under combination of chemicals. Combined fisetin and antioxidant, such as N-acetyl-L-cysteine (NAC) to investigate the mechanism of oxidative stress in apoptosis. Combined fisetin and heat shock protien 90 inhibitors (HSP inhibitors), geldanamycin (GA) and Radicicol (RAD) to study the mechanism of HSP on cancer treatment。
We demonstrate that 10 and 20 mM NAC, non-toxic concentrations, can enhance fisetin (FIS)-mediated apoptosis in colon cancer cells COLO205. Compared to treatment with FIS alone, combination treatment with NAC increased the expression of cleaved caspase-3 and PAPR protein, and produced greater density of DNA ladders. NAC reduced the mitochondrial membrane potential of FIS-treated COLO205 cells with induction of caspase 9 protein cleavage. DNA ladders induced by FIS+NAC were diminished by adding the caspase 3 inhibitor, DEVD-FMK, and the caspase 9 inhibitor, YVAD-FMK.
Combinatorial treatment COLO205 cells with NAC and FIS showed potent inhibition on ERK protein phosphorylation, compared with those from FIS or NAC-treated groups by Western blotting using specific antibodies. Addition of the chemical ERK inhibitors, PD98059 and U0126, significantly inhibited ERK protein phosphorylation, accompanied by induced DNA ladder formation, cleavage of caspase 3 and PARP protein in COLO205 cells. Furthermore, NAC showed an enhancement on a FIS-related chemical chrysin (CHR)-induced apoptosis of COLO205 cells, and NAC sensitization of colon cancer cells to FIS-induced apoptosis was also identify in colonic cancer cells HCT-116, HT-29, and HCT-15 cells. The evidence supports NAC sensitizing human colon cancer cells to FIS (or CHR)-induced apoptosis through decreased mitochondrial membrane potential (MMP) and Bcl-2 protein.
To investigate the cytotoxic effect of FIS on human COLO205 colonic cancer cells in the presence and absence of the HSP90 inhibitors, geldanamycin (GA) and radicicol (RAD) were studied. Compared to FIS treatment alone of COLO205 cells, GA and RAD significantly enhanced FIS-induced cytotoxicity through increasing ubiquitin-tagged proteins that reduced the stability of the p53 protein. The evidence supports HSP90 inhibitors possibly sensitizing human colon cancer cells to FIS-induced apoptosis.
According to the finding of our result, application of combination therapy with FIS (or CHR) and NAC or FIS and NAC as a strategy to treat colonic cancer deserved for further in vivo study.

並列關鍵字

Colon cancer ； Fisetin ； Heat shock protein inhibitors

參考文獻

2. Birt, D. F., Hendrich, S., & Wang, W. (2001). Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol Ther, 90(2-3), 157-177.

3. Arai, Y., Watanabe, S., Kimira, M., Shimoi, K., Mochizuki, R., & Kinae, N. (2000). Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J Nutr, 130(9), 2243-2250.

4. Higa, S., Hirano, T., Kotani, M., Matsumoto, M., Fujita, A., Suemura, M., . . . Tanaka, T. (2003). Fisetin, a flavonol, inhibits TH2-type cytokine production by activated human basophils. J Allergy Clin Immunol, 111(6), 1299-1306.

5. Sung, B., Pandey, M. K., & Aggarwal, B. B. (2007). Fisetin, an inhibitor of cyclin-dependent kinase 6, down-regulates nuclear factor-kappaB-regulated cell proliferation, antiapoptotic and metastatic gene products through the suppression of TAK-1 and receptor-interacting protein-regulated IkappaBalpha kinase activation. Mol Pharmacol, 71(6), 1703-1714. doi: 10.1124/mol.107.034512

6. Hanneken, A., Lin, F. F., Johnson, J., & Maher, P. (2006). Flavonoids protect human retinal pigment epithelial cells from oxidative-stress-induced death. Invest Ophthalmol Vis Sci, 47(7), 3164-3177. doi: 10.1167/iovs.04-1369