臺灣博碩士論文加值系統

在台灣，大腸癌的發生率已經連續數年位居首位，致死率則排名第三，爰亟需有效之大腸癌治療藥物。先前研究已指出STAT3主導之促存活路徑在大腸癌細胞中普遍呈現持續性活化，並且STAT3路徑之持續性活化與大腸癌之不良預後呈正相關性，因此STAT3訊息路徑為開發大腸直腸癌治療藥物的關鍵標的。Hispolon為藥用真菌桑黃(Phellinus linteus)之藥用成份之一且具抗癌活性，但近年研究發現某些Hispolon類似物較Hispolon之抗癌效應更強，而Dehydroxyhispolon methylether為Hispolon類似物之一，惟目前缺乏Dehydroxyhispolon methylether之生物活性(包括抗癌活性)之研究。
在我們研究中發現Dehydroxyhispolon methylether確實具有明顯之抗大腸癌效應，並且可有效抑制STAT3之持續性活化。具體而言，我們發現：Dehydroxyhispolon methylether可有效抑制大腸癌細胞株之存活率與細胞群落生成能力，但對正常人類大腸上皮細胞則較無毒性；Dehydroxyhispolon methylether的抗大腸癌效應顯著地高於Hispolon；Dehydroxyhispolon methylether可以誘發大腸癌細胞株之細胞凋亡以毒殺細胞。信號轉導和轉錄激活因子3（STAT3）的組成型激活與結直腸癌腫瘤發生和預後不良高度相關，暗示STAT3是結直腸癌治療的有希望的藥物靶標。 我們發現Dehydroxyhispolon methylether顯著抑制大腸癌細胞株之STAT3持續性活化，當我們在HCT116、HCT-15及LoVo細胞株大量表達持續性活化STAT3能夠顯著拮抗Dehydroxyhispolon methylether抑制之細胞凋亡，證實抑制STAT3活化是Dehydroxyhispolon methylether誘導大腸癌細胞株細胞凋亡之必要機制。研究中發現Dehydroxyhispolon methylether對JAK2活性沒有影響；但我們證實Dehydroxyhispolon methylether能抑制大腸癌細胞株JAK1、SRC的活性，並在大量表現SRC活性之穩定細胞株中，Dehydroxyhispolon methylether抑制STAT3活性能力受到拮抗，證實Dehydroxyhispolon methylether乃透過抑制SRC活性以壓抑STAT3活性進而促進細胞凋亡。西方墨點法結果顯示Dehydroxyhispolon methylether能抑制STAT3下游之抗凋亡蛋白BCL-2蛋白表現量；此外，在大量表達持續性活化STAT3之HCT116、HCT-15及LoVo穩定細胞株中， BCL-2 無法被Dehydroxyhispolon methylether所抑制，顯示Dehydroxyhispolon methylether乃透過抑制STAT3活性以抑制BCL-2之轉錄。我們將BCL-2在HCT116、HCT-15及LoVo中大量表現，西方墨點法及Annexin V assay結果顯示大量表達BCL-2能拮抗Dehydroxyhispolon methylether誘導之大腸癌細胞之凋亡，證明Dehydroxyhispolon methylether抑制STAT3以調降BCL-2，進而誘發細胞凋亡。
總之，我們在此提供了支持抑制JAK1 / SRC-STAT3-BCL-2介導的存活途徑的第一證據，其解釋了DHME的抗結腸癌作用。

Colorectal cancer is the most commonly diagnosed cancer and the third cause of cancer mortality in Taiwan. It is known that the signal transducer and activator of transcription 3 (STAT3) is a key signaling molecule implicated in the regulation of growth and malignant transformation, is aberrantly activated in colon cancer,implicating STAT3 as a promising drug target for colorectal cancer therapy.
Hispolon, a polyphenolic compound, is one of the medicinal components of the medicinal fungus Phellinus linteus .Several studies have demonstrated the antioxidant,anti-inflammatory, anti-estrogenic activity and anti-cancer properties of hispolon. However, in recent years, some Hispolon analogues have been found to have stronger anticancer effects than Hispolon, and Dehydroxyhispolon methylether is one of Hispolon analogues.However, the underlying mechanisms of anti-colorectal cancer effect of Dehydroxyhispolon methylether are still poorly understood .
Here, we reported that Dehydroxyhispolon methylether induce apoptosis and , less toxic to normal human large intestinal epithelial cells; the anti-colorectal cancer effect of Dehydroxyhispolon methylether is significantly higher than that of Hispolon ;markedly suppresse colony formation of colon cancer cell lines HCT116, HCT-15, and LoVo.
Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is highly correlated with colorectal cancer tumorigenesis and poor prognosis, implicating STAT3 as a promising drug target for colorectal cancer therapy. We found that Dehydroxyhispolon methylether suppresse constitutive activation of STAT3, as evidenced by reduced levels of phosphorylated tyrosine 705 of STAT3 (activated STAT3). Notably, overexpression of constitutively active STAT3 mutant (STAT3-CA) reversed apoptosis of Dehydroxyhispolon methylether -treated cells. It was found that Dehydroxyhispolon methylether had no effect on JAK2 activity; however, we confirmed that Dehydroxyhispolon methylether inhibited the activity of colorectal cancer cell lines JAK1 and SRC, and that the ability of Dehydroxyhispolon methylether to inhibit STAT3 activity was antagonized in a large number of stable cell lines expressing SRC activity. Dehydroxyhispolon methylether promotes apoptosis by inhibiting SRC activity by suppressing STAT3 activity. Western blotting results showed that Dehydroxyhispolon methylether inhibited the expression of anti-apoptotic protein BCL-2 protein downstream of STAT3; in addition, BCL-2 could not be expressed in HCT116, HCT-15 and LoVo stable cell lines expressing a large number of persistently activated STAT3 Inhibition by Dehydroxyhispolon methylether showed that Dehydroxyhispolon methylether inhibited the transcription of BCL-2 by inhibiting STAT3 activity. We showed a large number of BCL-2 in HCT116, HCT-15 and LoVo. Western blotting and Annexin V assay showed that BCL-2 could antagonize the apoptosis of colorectal cancer cells induced by Dehydroxyhispolon methylether, demonstrating that Dehydroxyhispolon methylether inhibits STAT3. To reduce BCL-2, and then induce apoptosis.
In conclusion, we herein provide the first evidence supporting that suppression of JAK1/SRC- STAT3-BCL-2-mediated survival pathway accounts for the anti-colon cancer effect of Dehydroxyhispolon methylether.

目次
中文摘要………………………………………………………………………………i
Abstract………………………………………………………………………………iii
前言（Introduction）………………………………………………………………..1
一、大腸癌…………………………………………………………………………1
二、桑黃衍生物 ( Dehydroxyhispolon methylether )………………………… ..10
三、STAT3 (Signaling transducer and activator of transcription 3)……………11
四、STAT3與大腸癌……………………………………………………………...13
五、JAK(Janus kinase)……………………………………………………………14
六、Src（Proto-oncogene tyrosine-protein kinase Src）………………………….16
七、細胞凋亡（Apoptosis）……………………………………………………….17
八、Bcl-2家族……………………………………………………………………19
研究目的（Aim）…………………………………………………………………..21
實驗材料及試劑配方 (Materials)………………………………………………….22
一、藥物……………………………………………………………………………22
二、試劑 (Buffer) …………………………………………………………22
三、質體建構（Plasmid construction）…………………………………………..24
四、抗體配製………………………………………………………………………25
實驗方法（Methods）……………………………………………….……………..27
一、細胞培養 （Cell lines and cell culture）……………………..…………….27
二、細胞冷凍保存與解凍……………………………………..…………………28
三、細胞存活率測試（Cell viability assay）………...…………………………28
四、細胞群落形成能力檢測（Colony formation assay）…………….…..…….29
五、細胞凋亡試驗 （Apoptosis assay）………………………………..………29
六、細胞總量蛋白萃取（Whole protein extraction）……………….….………29
七、蛋白濃度定量分析（Protein quantification）………………...……………30
八、西方墨點法（Western blotting）……………………..……………….……30
九、大腸桿菌質體轉型作用（Transformation）………..……………………...32
十、病毒製備與病毒感染（Retrovirus and Lentivirus production and infection） ………………………………………..…………………………………….…..32
結果 (Results)…………………………………………………………………….. 34
一、DHME有效抑制大腸癌細胞株之存活率………………............34
二、DHME能有效抑制人類大腸癌細胞株群落生成能力………………………34
三、DHME誘發大腸癌細胞株細胞凋亡以毒殺細胞……………….34
四、DHME抑制大腸癌細胞株內持續性之STAT3活化…………………35
五、DHME可抑制IL-6誘導之STAT3活化……………………………………..35
六、抑制STAT3活化為DHME誘發人類大腸癌細胞凋亡之必要機制……………………………………………………………………….…………36
七、DHME透過抑制JAK1、SRC以抑制STAT3活化……………………36
八、DHME透過SRC-STAT3路徑促使大腸癌細胞之細胞凋亡………………37
九、DHME抑制STAT3轉錄其下游基因BCL-2…………………37
十、DHME抑制STAT3以調降BCL-2以誘發大腸癌細胞之凋亡……………………………………………..……………………………….38
討論（Discussion）…………………………………………………………….…..39
一、本論文首度發現之結果………………………………………………………..39
二、探討Dehydroxyhispolon methylether (DHME)之抗大腸癌潛力…………39
三、探討DHME與 Hispolon對大腸癌細胞存活率之影響………………………40
四、抑制轉錄因子STAT3活性在DHME誘導之人類大腸癌細胞凋亡之意義…………………………………………………………………………………..40
五、DHME透過抑制SRC、JAK1以壓抑STAT3活化促使細胞凋亡…………………………………………………………………………………41
六、DHME抑制STAT3轉錄下游標的基因BCL-2，誘發大腸癌細胞凋亡………………………………………………………………………………..42
七、其餘可能機制之探討……………………………………………………………43
結論 (Conclusion) ………………………………………………………………44
實驗結果圖表 (Results and Figures) ……………………………………………..45
圖一(A)、Dehydroxyhispolon methylether ( DHME )有效抑制大腸癌細胞株
之存活率…………………………………………………………………45
圖一(B)、Hispolon 與Dehydroxyhispolon methylether ( DHME )毒殺大腸癌
細胞株的毒性相比有較差…………………………………………….46
圖二、Dehydroxyhispolon methylether( DHME )有效抑制人類大腸癌細胞群落
生成能力……………………………………………………………………47
圖三、Dehydroxyhispolon methylether促使PARP截切活化影響細胞生存….48
圖四、Dehydroxyhispolon methylether處理促進人類大腸癌細胞株細胞凋亡….49
圖五、Dehydroxyhispolon methylether抑制多株人類大腸癌細胞STAT3持續性
活化………………………………………………………………………..50
圖六、Dehydroxyhispolon methylether抑制人類大腸癌細胞株持續性STAT3活
化…………………………………………………………………………….51
圖七、Dehydroxyhispolon methylether有效抑制誘導性STAT3活化…….52
圖八、抑制STAT3之持續性活化為Dehydroxyhispolon methylether誘導大腸癌
細胞細胞凋亡之必要機制…………………………………………………53
圖九、抑制STAT3之持續性活化為Dehydroxyhispolon methylether抑制大腸癌
細胞存活率之必要機制……………………………………………………54
圖十、抑制STAT3之持續性活化為Dehydroxyhispolon methylether抑制大腸癌
細胞存活率之必要機制…………………………………………………..55
圖十一、抑制STAT3之持續性活化為Dehydroxyhispolon methylether誘導大腸
癌細胞細胞凋亡之必要機制………………………………………….56
圖十二、Dehydroxyhispolon methylether並非透過JAK2路徑影響人類大腸癌細
胞株中STAT3活性………………………………………………………57
圖十三 、Dehydroxyhispolon methylether抑制STAT3上游蛋白JAK1磷酸
化使人類大腸癌細胞株STAT3活性下降………………………….58
圖十四、Dehydroxyhispolon methylether抑制STAT3上游蛋白SRC磷酸化
使人類大腸癌細胞株STAT3活性下降…………………………………59
圖十五、Dehydroxyhispolon methylether透過SRC-STAT3路徑促使大腸癌細胞
細胞凋亡…………………………………………………………………60
圖十六、抑制SRC之持續性活化為Dehydroxyhispolon methylether抑制大腸
癌細胞存活率之必要機制………………………………………………61
圖十七、Dehydroxyhispolon methylether抑制STAT3下游人類大腸癌細胞株之
抗凋亡蛋白 BCL-2蛋白表現量…………………………………………62
圖十八、在STAT3持續性活化之人類大腸癌細胞株HCT-15、HCT 116及LoVo中Dehydroxyhispolon methylether抗凋亡蛋白BCL-2蛋白表現量……..63
圖十九、在大量表現constitutively active BCL-2之人類大腸癌細胞中，
Dehydroxyhispolon methylether所誘導之細胞凋亡受到抑制……..64
圖二十、在大量表現constitutively active BCL-2之人類大腸癌細胞中，BCL-2
之持續性活化為Dehydroxyhispolon methylether抑制大腸癌細胞存活
率之必要機制…………………………………………………………….65
圖二十一、在大量表現constitutively active BCL-2之人類大腸癌細胞中，
Dehydroxyhispolon methylether所誘導之細胞凋亡受到抑制…………66
參考文獻（References）…………………………………………………………….67

參考文獻(References)
1.行政院衛生福利部國民健康署. 104年癌症登記年報. （2018); Available from: https://www.hpa.gov.tw/Pages/Detail.aspx?nodeid=269&pid=8084.
2.Siegel RL, et al. Colorectal cancer statistics. CA Cancer J.Clin. 2017:67:177-93.
3.Arnold M, et al. Global patterns and trends in colorectal cancer incidence and mortality. Gut, 2017;66:683–91.
4.Van Cutsem E, et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016;27:1386–422.
5. Sanford D, et al. Molecular Basis of Colorectal Cancer. N Engl J Med, 2009; 361:2449-60.2.
6.M Ryan-Harshman, Diet and colorectal cancer. Clinical Review, 2007 Nov; 53(11): 1913–1920.
7.LM Hannan, et al. The association between cigarette smoking and risk of colorectal cancer in a large prospective cohort from the United States. Cancer Epidemiol Biomarkers Pre, 2009 Dec;18(12):3362-7.
8.Kruk, J., Physical activity in the prevention of the most frequent chronic diseases: an analysis of the recent evidence. Asian Pac J Cancer Prev, 2007. 8(3): p. 325-38.
9.Okamoto M, et al. Relationship between age and site of colorectal cancer based on colonoscopy findings. Gastrointest Endosc, 2002 Apr;55(4):548-51.
10.Wark, P.A., et al. Family history of colorectal cancer: a determinant of advanced adenoma stage or adenoma multiplicity? Int J Cancer, 2009. 125(2): p. 413-20.
11.Triantafillidis, J.K., G. Nasioulas, and P.A. Kosmidis, Colorectal cancer and inflammatory bowel disease: epidemiology, risk factors, mechanisms of carcinogenesis and prevention strategies. Anticancer Res, 2009. 29(7): p. 2727-37.
12.Church J1, et al. Risk of rectal cancer in patients after colectomy and ileorectal anastomosis for familial adenomatous polyposis: a function of available surgical options. Dis Colon Rectum. 2003 Sep;46(9):1175-81.
13.Jin Y, et al. Risk analysis of the canceration of colorectal large polyps. Zhonghua Wei Chang Wai Ke Za Zhi. 2018 Oct 25;21(10):1161-1166.
14.Saeed RS, et al. Knowledge and Awareness of Colorectal Cancer among General Public of Kuwait. Asian Pac J Cancer Prev. 2018 Sep 26;19(9):2455-2460.
15.American Cancer Society.Colorectal Cancer Stages; Available from: https://www.hpa.gov.tw/Pages/Detail.aspx?nodeid=269&pid=8084.
16.癌症希望基金會大癌照護網2017; Available from: http://www.crctw.org/crctw_web/QA_&xargs=0&pstart=1&b=11
17.Chiu , H. M. Screening, Diagnosis and Treatment of Early Colorectal Cancer. 中華民國癌症醫學會雜誌.2008 Jun 10:P148 – 156.
18.Romano, G., et al., The TGF-beta pathway is activated by 5-fluorouracil treatment in drug resistant colorectal carcinoma cells. Oncotarget, 2016. 7(16): p. 22077-91.
19.Wong, T.W. and A. Bose, Glyoxalated chitosan-5-fluorouracil/chitosan-folate as colon-specific and colon cancer cell-targeted device. J Control Release, 2015. 213: p. e105.
20.Gao, X.Y. and X.L. Wang, An adoptive T cell immunotherapy targeting cancer stem cells in a colon cancer model. J BUON, 2015. 20(6): p. 1456-63.
21.Taal BG, et al. Adjuvant 5FU plus levamisole in colonic or rectal cancer: improved survival in stage II and III. Br J Cancer. 2001 Nov 16;85(10):1437-43.
22.Messersmith, W.A. and D.J. Ahnen, Targeting EGFR in colorectal cancer. N Engl J Med, 2008. 359(17): p. 1834-6.
23.Zaniboni, A. and V. Formica, The Best. First. Anti-EGFR before anti-VEGF, in the first-line treatment of RAS wild-type metastatic colorectal cancer: from bench to bedside. Cancer Chemother Pharmacol, 2016. 78(2): p. 233-44.
24.Temraz, S, et al. Methods of overcoming treatment resistance in colorectal cancer. Crit Rev Oncol Hematol, 2014. 89(2): p. 217-30.
25.Chen W, et al. Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway. Free Radic Biol Med. 2008;45:60–72.
26.Mo S, et al. Phelligridins C-F: cytotoxic pyrano[4,3-c][2]benzopyran-1,6-dione and furo[3,2-c]pyran-4-one derivatives from the fungus Phellinus igniarius. Journal of natural products. 2004;67:823–828.
27.Ali NA, et al. Inhibition of chemiluminescence response of human mononuclear cells and suppression of mitogen-induced proliferation of spleen lymphocytes of mice by hispolon and hispidin. Die Pharmazie. 1996;51:667–670.
28.Yang LY, et al. Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages. Journal of ethnopharmacology. 2014;156:61–72.
29.Huang GJ, et al. Hispolon Protects against Acute Liver Damage in the Rat by Inhibiting Lipid Peroxidation, Proinflammatory Cytokine, and Oxidative Stress and Downregulating the Expressions of iNOS, COX-2, and MMP-9. Evidence-based complementary and alternative medicine : eCAM. 2012;2012:480714.
30.Wang J, et al. Estrogenic and anti-estrogenic activities of hispolon from Phellinus lonicerinus (Bond.) Bond. et sing. Fitoterapia. 2014;95:93–101.
31.Chen T, et al. Selenocystine induces S-phase arrest and apoptosis in human breast adenocarcinoma MCF-7 cells by modulating ERK and Akt phosphorylation. Journal of agricultural and food chemistry. 2008;56:10574–10581.
32.Lu TL, et al. Hispolon from Phellinus linteus has antiproliferative effects via MDM2-recruited ERK1/2 activity in breast and bladder cancer cells. Food Chem Toxicol. 2009;47:2013–2021.
33.Hsieh MJ, et al. Hispolon from Phellinus linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway. Phytomedicine : international journal of phytotherapy and phytopharmacology. 2014;21:1746–1752.
34.Wu Q, et al. The anticancer effects of hispolon on lung cancer cells. Biochemical and biophysical research communications. 2014;453:385–391.
35.Ravindran J, et al. Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro. Biochem Pharmacol. 2010;79:1658–1666.
36.Neduri V. Balaji, et al. Design, Synthesis and In Vitro Cell-based Evaluation of the Anti-cancer Activities of Hispolon Analogs. Bioorg Med Chem. Author manuscript; available in PMC 2016 May 1.Published in final edited form as:Bioorg Med Chem. 2015 May 1; 23(9): 2148–2158.Published online 2015 Mar 21.
37.Darnell JE, et al. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular proteins. Science. 1994;264:1415–1421.

38.Levy DE, et al. Interferon-dependent transcriptional activation: signal transduction without second messenger involvement? New Biol. 1990;2:923–928.
39.Rane, S.G. and E.P. Reddy, Janus kinases: components of multiple signaling pathways. Oncogene, 2000. 19(49): p. 5662-79.
40.David E. Levy and Chien-kuo Lee. What does Stat3 do? J Clin Invest. 2002 May 1; 109(9): 1143–1148.
41.Guschin D, et al. A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. EMBO J. 1995;14:1421–1429.
42.Yu, H., et al., Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat Rev Cancer, 2014. 14(11): p. 736-46.
43.Levy DE, Gilliland DG. Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene disruptions in mice. Oncogene. 2000;19:2505–2510.
44.Bromberg J. Stat proteins and oncogenesis. J Clin Invest. 2002;109:1139–1142.
45.Corvinus, F.M., et al., Persistent STAT3 Activation in Colon Cancer Is Associated with Enhanced Cell Proliferation and Tumor Growth. Neoplasia, 2005. 7(6): p. 545-555.
46.Corvinus, F.M., et al. Persistent STAT3 Activation in Colon Cancer Is Associated with Enhanced Cell Proliferation and Tumor Growth. Neoplasia, 2005. 7(6): p. 545-555.
47.Fan, L.C., et al., Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer. Neoplasia, 2015. 17(9): p. 687-96.
48.Kusaba, T., et al., Activation of STAT3 is a marker of poor prognosis in human colorectal cancer. Oncol Rep, 2006. 15(6): p. 1445-51.
49.Lin, L., et al., STAT3 is necessary for proliferation and survival in colon cancer-initiating cells. Cancer Res, 2011. 71(23): p. 7226-37.
50.Lin, Q., et al., Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells. Am J Pathol, 2005. 167(4): p. 969-80.
51.Tsareva, S.A., et al., Signal transducer and activator of transcription 3 activation promotes invasive growth of colon carcinomas through matrix metalloproteinase induction. Neoplasia, 2007. 9(4): p. 279-91.
52.Aghazadeh,S.and R. Yazdanparast, Activation of STAT3/HIF-1alpha/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN. Biochim Biophys Acta, 2017.
53.Fernandes, A., A.W. Hamburger, and B.I. Gerwin, ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells. Int J Cancer, 1999. 83(4): p. 564-70.
54.Kim, D.Y., et al., STAT3 expression in gastric cancer indicates a poor prognosis. J Gastroenterol Hepatol, 2009. 24(4): p. 646-51.
55.Mace, T.A., et al., Pancreatic cancer-associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. Cancer Res, 2013. 73(10): p. 3007-18.
56.Li, F., et al., CUEDC2 suppresses glioma tumorigenicity by inhibiting the activation of STAT3 and NF-kappaB signaling pathway. Int J Oncol, 2017.
57.Pencik, J, et al. IL-6/STAT3/ARF: the guardians of senescence, cancer progression and metastasis in prostate cancer. Swiss Med Wkly, 2015. 145: p. w14215.
58.Song, J.I. and J.R. Grandis, STAT signaling in head and neck cancer. Oncogene, 2000. 19(21): p. 2489-95.
59.Siveen, K.S., et al. Targeting the STAT3 signaling pathway in cancer: role of synthetic and natural inhibitors. Biochim Biophys Acta, 2014. 1845(2): p. 136-54.
60.Corvinus F M, Orth C, Moriggl R. et al Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth. Neoplasia 20057545–555.]
61.Lin Q, Lai R, Chirieac L R. et al.Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells.Am J Pathol 2005167969–980.
62.Ma X T, Wang S, Ye Y J, et al .Constitutive activation of Stat3 signaling pathway in human colorectal carcinoma. World J Gastroenterol 2004101569–1573.
63.Kusaba T, Nakayama T, Yamazumi K. et al Expression of p‐STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors. J Clin Pathol 200558833–838.
64.Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–57.
65.Kisseleva ,et al. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002-02-20, 285 (1–2): 1–24.
66.Yeatman, T.J., A renaissance for SRC. Nat Rev Cancer, 2004. 4(6): p. 470-80.
67.Chong, Y.P., T.D. Mulhern, and H.C. Cheng, C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)--endogenous negative regulators of Src-family protein kinases. Growth Factors, 2005. 23(3): p. 233-44.
68.Hunter, T., A tail of two src's: mutatis mutandis. Cell, 1987. 49(1): p. 1-4.
69.Dehm, S.M. and K. Bonham, SRC gene expression in human cancer: the role of transcriptional activation. Biochem Cell Biol, 2004. 82(2): p. 263-74.
70.Cartwright, C.A., et al., pp60c-src activation in human colon carcinoma. J Clin Invest, 1989. 83(6): p. 2025-33.
71.Yu, C.L., et al., Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science, 1995. 269(5220): p. 81-3.
72.Chen, J., et al., The role of Src in colon cancer and its therapeutic implications. Clin Colorectal Cancer, 2014. 13(1): p. 5-13.
73.Formigli L, et al. Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis .Cell Physiol. 2000 Jan; 182(1):41-9.
74.Sperandio S, et al. An alternative, nonapoptotic form of programmed cell death.Proc Natl Acad Sci U S A. 2000 Dec 19; 97(26):14376-81
75.Debnath J, et al. Does autophagy contribute to cell death?Autophagy. 2005 Jul; 1(2):66-74.
76.Norbury CJ, Hickson ID.Cellular responses to DNA damage.Annu Rev Pharmacol Toxicol. 2001; 41():367-401.
77.Hirsch T, et al.The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene. 1997 Sep 25; 15(13):1573-81.
78.Zeiss CJ, The apoptosis-necrosis continuum: insights from genetically altered mice. Vet Pathol. 2003 Sep; 40(5):481-95.
79.Susan Elmore. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. Author manuscript; available in PMC 2007 Dec 6.Published in final edited form as:Toxicol Pathol. 2007; 35(4): 495–516.
80.Desagher, S. and J.C. Martinou, Mitochondria as the central control point of apoptosis. Trends Cell Biol, 2000. 10(9): p. 369-77.
81.Elmore, S., Apoptosis: a review of programmed cell death. Toxicol Pathol, 2007. 35(4): p. 495-516.
82.Thomas, et al. Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3' Uridylated Intermediates Degraded by DIS3L2. Cell Reports. 11: 1079–89.
83.Böhm I (2003). "Disruption of the cytoskeleton after apoptosis induction by autoantibodies". Autoimmunity. 36: 183–189. doi:10.1080/0891693031000105617.
84. Susin, S, et al. Two Distinct Pathways Leading to Nuclear Apoptosis. Journal of Experimental Medicine. 192 (4): 571–80.
85.Madeleine Kihlmark et al. Sequential degradation of proteins from the nuclear envelope during apoptosis. Journal of Cell Science. 114 (20): 3643–53.
86.Nagata S. Apoptotic DNA fragmentation. Exp. Cell Res. 256 (1): 12–8.
87.Gong JP, et al. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem. 218: 314–319.
88.Muchmore SW, et al. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 381 (6580): 335–41.
89.Youle, Richard J. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology. 9 (1): 47–59.
90.Coultas, L. and A. Strasser, The role of the Bcl-2 protein family in cancer. Semin Cancer Biol, 2003. 13(2): p. 115-23.
91.Reed JC, et al.Structure-function analysis of Bcl-2 family proteins. Regulators of programmed cell death. Adv. Exp. Med. Biol. 406: 99–112.
92.Chen, J., et al., The role of Src in colon cancer and its therapeutic implications. Clin Colorectal Cancer, 2014. 13(1): p. 5-13.
93.Silva, C.M., Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene, 2004. 23(48): p. 8017-23.
94.Hague, A., et al., BCL-2 expression in human colorectal adenomas and carcinomas. Oncogene, 1994. 9(11): p. 3367-70.