The online version of this article (doi:10.1186/1477-7819-10-228) contains supplementary material, which is available to authorized users.
No potential conflicts of interest are disclosed.
ZZ and GM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. HS, YG, and XL carried out the immunoassays. YY and YL participated in the sequence alignment. QW, ZW, and EL participated in the design of the study and performed the statistical analysis. XQ and YL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
The purpose of the research is to investigate the roles of Jak-STAT3 signaling pathway in bufalin-induced apoptosis in colon cancer SW620 cells.
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.
(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.
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.
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. 10.1200/JCO.2009.27.6055. CrossRefPubMed
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. PubMed
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. PubMed
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. PubMed
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. PubMed
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. 10.1038/sj.onc.1207606. CrossRefPubMed
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.
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. PubMed
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. PubMed
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. PubMed
- Inhibition of Jak-STAT3 pathway enhances bufalin-induced apoptosis in colon cancer SW620 cells
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