Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

Cheng Hsiao-Ling, Su Shu-Jem, Huang Li-Wen, Hsieh Bau-Shan, Hu Yu-Chen, Hung Thu-Ching, Chang, Chang Kee-Lung

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Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. Methods HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface β1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. Results A low concentration of arecoline (≤ 100 μg/ml) caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased β1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-X L and Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline. Conclusions This study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased RhoA/Rock activation and that the STAT3 and RhoA/Rock signaling pathways are connected.

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• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 2 Mo

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Chenget al.Molecular Cancer2010,9:126 http://www.molecular-cancer.com/content/9/1/126

R E S E A R C H Open Access Research Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

1 2 3 1 1 1 Hsiao-Ling Cheng , Shu-Jem Su , Li-Wen Huang , Bau-Shan Hsieh , Yu-Chen Hu , Thu-Ching Hung and Kee- 4 Lung Chang*

Abstract Background:Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. Methods:HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface β1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. Results:A low concentration of arecoline (≤ 100 μg/ml) caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased β1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-X and L Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline. Conclusions:This study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased RhoA/Rock activation and that the STAT3 and RhoA/Rock signaling pathways are connected.

Background Arecoline has been suggested as a possible cognition enhancer in Alzheimer's type dementia [1,2]. Recent studies have shown that it decreases interleukin-6 (IL-6) production in keratinocytes and KB cancer cells [3,4]. In addition, Changet al. [3] reported that arecoline elicits cell cycle deregulation in KB cancer cells. Moreover, our previous study [Changet al.: Arecoline decreases inter-

* Correspondence: [e-mail] 4 Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Full list of author information is available at the end of the article

leukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells (BCC/KMC), submitted] showed that, in basal cell carcinoma cells, are- coline reduces levels of the tumor cell survival factor IL-6, increases levels of the tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis, showing that arecoline interferes with cancer cell cycle progres- sion. Our preliminary data showed that arecoline induces detachment of the hepatoma cell line HA22T/VGH from the extracellular matrix (ECM). Adherence of epithelial cells to the ECM is important for cell growth and survival and detachment from the

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