Inhibition of Jak-STAT3 pathway enhances bufalin-induced apoptosis in colon cancer SW620 cells

World Journal of Surgical Oncology

Yunlong Yu¹  Yunpeng Liu³  Xiujuan Qu³  Qingjun Wang⁴  Xiaomei Liu⁴  Zhenghua Wang⁴  Guangyou Ma⁴  Hongzhi Sun⁴  Yu Gao⁴  Yangyang Liu⁴  Enze Li⁴  Zhitu Zhu² 

[1] Department of Oncology, China National Offshore Oil Corporation Hospital, No 10, Shiyou Rd, Tanggu District, Tianjing, 300452, China;Department of Oncology, The First Affiliated Hospital of Liaoning Medical University, No 2, Wuduan Rengmin Street, Jinzhou, 121001, China;Department of Oncology, The First Affiliated Hospital of Chinese Medical University, No 155, Nanjingbei Street, Heping District, Shenyang, Liaoning, 11001, China;Department of Oncology, The First Affiliated Hospital of Liaoning Medical University, Jinzhou, Liaoning, 121001, China

关键词: Apoptosis;    M-phase arrest;    Caspase;    Livin;    Bufalin;    Jak-stat3;    Colon cancer;   

Others  :  827026
DOI  :  10.1186/1477-7819-10-228

 received in 2012-03-26, accepted in 2012-09-27,  发布年份 2012

【 摘 要 】

Background

The purpose of the research is to investigate the roles of Jak-STAT3 signaling pathway in bufalin-induced apoptosis in colon cancer SW620 cells.

Methods

The inhibitory effects of bufalin on cell proliferation were determined by MTT (Methyl thiazolyltetrazolium) assay. The morphological changes of cells were measured by Wright-Giemsa staining. The cell cycle arrest and apoptosis were tested by flow cytometry analysis. Western Blot was used to determine the protein expression of the apoptosis inhibitors livin and caspase-3, the apoptosis-related proteins Bax and Bcl-2, as well as the key protein kinases in the Jak-stat3 signaling pathway, stat3 and p-stat3.

Results

(1) Bufalin inhibited the proliferation of SW620 cells. IC50 at 24 h, 48 h and 72 h were 76.72 ± 6.21 nmol/L, 34.05 ± 4.21 nmol/L and 16.7 ± 6.37 nmol/L. (2) Bufalin induced SW620 cell cycle arrest and apoptosis, indicated by the appearance of apoptotic bodies; (3) The results from flow cytometry demonstrated that there was cell cycle G2/M phase arrest in 20 nmol/L bufalin treatment group (36.29 ± 2.11% vs 18.39 ± 1.74%, P<0.01); there was a sub-diploid apoptosis peak in 80 nmol/L bufalin treatment group (19.69 ± 1.63% vs 0.99 ± 0.23%, P <0.01). The apoptosis rate was 34.63 ± 2.57% (vs 19.69 ± 1.63%, P = 0.002) in JAK kinase inhibitor AG490 plus bufalin treatment group. (4) During the process of bufalin-induced apoptosis in SW620 cells, transient activation of p-stat3 inhibited the activation of stat3, up-regulated Bax expression, down-regulated livin and Bcl-2 expression (P<0.01), and activated caspase-3. Inhibition of Jak-stat3 signaling pathway by pre-treatment with AG490 significantly enhanced the bufalin-induced apoptosis (P<0.01), further up-regulated Bax protein expression, down-regulated livin and Bcl-2 protein expression and enhanced caspase-3 activation.

Conclusions

Bufalin not only inhibited the growth of colon cancer SW620 cells, but also induced apoptosis of SW620 cells. Activation of caspase-3, up-regulation of Bax, down-regulation of livin and Bcl-2, as well as inhibition of Jak-stat3 signaling pathway might be the important mechanisms for the bufalin-induced apoptosis.

【 授权许可】

   
2012 Zhu et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer statistics, 2009. CA Cancer J Clin 2009, 59:225-249.
• [2]Peeters M, Price TJ, Cervantes A, Sobrero AF, Ducreux M, Hotko Y, André T, Chan E, Lordick F, Punt CJ, Strickland AH, Wilson G, Ciuleanu TE, Roman L, Van Cutsem E, Tzekova V, Collins S, Oliner KS, Rong A, Gansert J: Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as second-line treatment in patients with metastatic colorectal cancer. J Clin Oncol 2010, 28:4706-4713.
• [3]Krenn L, Kopp B: Bufadienolides from animal and plant sources. Phytochemistry 1998, 48:1-29.
• [4]Cao-Hong , Shibayama-Imazu T, Masuda Y, Shinki T, Nakajo S, Nakaya K: Involvement of Tiam1 in apoptosis induced by bufalin in HeLa cells. Anticancer Res 2007, 27:245-249.
• [5]Tian X, Wang PP, Liu YP, Hou KZ, Jin B, Luo Y, Qu XJ: Effect of bufalin-inducing apoptosis on Bcl-2 and PKC in HL-60 cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2007, 15:67-71.
• [6]Liu Y, Qu X, Wang P, Tian X, Luo Y, Liu S, Lu X: WT1 downregulation during K562 cell differentiation and apoptosis induced by bufalin. Zhonghua Xue Ye Xue Za Zhi 2002, 23:356-359.
• [7]Tian X, Luo Y, Liu YP, Hou KZ, Jin B, Zhang JD, Wang S: Downregulation of Bcl-2 and survivin expression and release of Smac/DIABLO involved in bufalin-induced HL-60 cell apoptosis. Zhonghua Xue Ye Xue Za Zhi 2006, 27:21-24.
• [8]Watabe M, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y, Nakaya K: The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells. J Biol Chem 1996, 271:14067-14072.
• [9]Aggarwal BB, Kunnumakkara AB, Harikumar KB, Gupta SR, Tharakan ST, Koca C, Dey S, Sung B: Signal transducer and activator of transcription-3, inflammation, and cancer: how intimate is the relationship. Ann NY Acad Sci 2009, 1171:59-76.
• [10]Huang S: Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications. Clin Cancer Res 2007, 13:1362-1366.
• [11]Scheper MA, Nikitakis NG, Sauk JJ: Survivin is a downstream target and effector of sulindac-sensitive oncogenic Stat3 signalling in head and neck cancer. Int J Oral Maxillofac Surg 2007, 36:632-639.
• [12]Kanda N, Seno H, Konda Y, Marusawa H, Kanai M, Nakajima T, Kawashima T, Nanakin A, Sawabu T, Uenoyama Y, Sekikawa A, Kawada M, Suzuki K, Kayahara T, Fukui H, Sawada M, Chiba T: STAT3 is constitutively activated and supports cell survival in association with survivin expression in gastric cancer cells. Oncogene 2004, 23:4921-4929.
• [13]Haura EB, Turkson J, Jove R: Mechanisms of disease: Insights into the emerging role of signal transducers and activators of transcription in cancer. Nat Clin Pract Oncol 2005, 2:315-324.
• [14]Kawazoe N, Watabe M, Masuda Y, Nakajo S, Nakaya K: Tiam1 is involved in the regulation of bufalin-induced apoptosis in human leukemia cells. Oncogene 1999, 18:2413-2421.
• [15]Chen A, Yu J, Zhang L, Sun Y, Zhang Y, Guo H, Zhou Y, Mitchelson K, Cheng J: Microarray and biochemical analysis of bufalin-induced apoptosis of HL-60 Cells. BiotechnolLett 2009, 31:487-494.
• [16]Takai N, Ueda T, Nishida M, Nasu K, Narahara H: Bufalin induces growth inhibition, cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells. Int J Mol Med 2008, 21:637-643.
• [17]Li D, Qu X, Hou K, Zhang Y, Dong Q, Teng Y, Zhang J, Liu Y: PI3K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells. Anticancer Drugs 2009, 20:59-64.
• [18]Xie CM, Chan WY, Yu S, Zhao J, Cheng CH: Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation. Free Radic Biol Med 2011, 51:1365-1375.
• [19]Jing Y, Watabe M, Hashimoto S, Nakajo S, Nakaya K: Cell cycle arrest and protein kinase modulating effect of bufalin on human leukemia ML1 cells. Anticancer Res 1994, 14:1193-2008.
• [20]Watabe M, Ito K, Masuda Y, Nakajo S, Nakaya K: Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells. Oncogene 1998, 16:779-787.
• [21]Sun Y, Lin Y, Li H, Liu J, Sheng X, Zhang W: 2,5-Hexanedione induces human ovarian granulosa cell apoptosis through BCL-2, BAX, and CASPASE-3 signaling pathways. Arch Toxicol 2012, 86:205-215.
• [22]Yan B: Research progress on Livin protein: an inhibitor of apoptosis. Mol Cell Biochem 2011, 357:39-45.
• [23]Kitamura H, Honma I, Torigoe T, Hariu H, Asanuma H, Hirohashi Y, Sato E, Sato N, Tsukamoto T: Expression of livin in renal cell carcinoma and detection of anti-livin autoantibody in patients. Urology 2007, 70:38-42.
• [24]Chang H, Schimmer AD: Livin/melanoma inhibitor of apoptosis protein as a potential therapeutic target for the treatment of malignancy. Mol Cancer Ther 2007, 6:24-30.
• [25]Wang Q, Huang Y, Ni Y, Wang H, Hou Y: siRNA targeting midkine inhibits gastric cancer cells growth and induces apoptosis involved caspase-3,8,9 activation and mitochondrial depolarization. J Biomed Sci 2007, 14:783-795.
• [26]Pedranzini L, Dechow T, Berishaj M, Comenzo R, Zhou P, Azare J, Bornmann W, Bromberg J: Pyridone 6, a pan-Janus-activated kinase inhibitor, induces growth inhibition of multiple myeloma cells. Cancer Res 2006, 66:9714-9721.
• [27]Yao X, Zhu F, Zhao Z, Liu C, Luo L, Yin Z: Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway. J Cell Biochem 2011, 112:2837-2849.
• [28]Wang Z, Jin H, Xu R, Mei Q, Fan D: Triptolidedownregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression. Exp Mol Med 2009, 41:717-727.
• [29]Kusaba T, Nakayama T, Yamazumi K, Yakata Y, Yoshizaki A, Inoue K, Nagayasu T, Sekine I: Activation of STAT3 is a marker of poor prognosis in human colorectal cancer. Oncol Rep 2006, 15:1445-1451.
• [30]Lin L, Liu A, Peng Z, Lin HJ, Li PK, Li C, Lin J: STAT3 Is Necessary for Proliferation and Survival in Colon Cancer-Initiating Cells. Cancer Res 2011, 71:7226-7237.
• [31]Xi RC, Biao WS, Gang ZZ: Significant elevation of survivin and livin expression in human colorectal cancer: inverse correlation between expression and overall survival. Onkologie 2011, 34:428-432.