Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells

Journal of Experimental & Clinical Cancer Research, Sep 2015

Background Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown. Methods Cell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays. Results We showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth. Conclusion Our results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.

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Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells

Wu et al. Journal of Experimental & Clinical Cancer Research Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells Jingjing Wu 1 2 4 Shunyu Zhao 1 2 4 Qing Tang 2 4 Fang Zheng 2 4 YuQin Chen 2 4 LiJun Yang 2 4 Xiaobing Yang 3 4 Liuning Li 3 4 WanYin Wu 3 4 Swei Sunny Hann 0 2 4 0 No. 55, Neihuan West Road , Higher Education Mega Center , Panyu District, Guangzhou, Guangdong Province 510006 , P. R. China 1 Equal contributors 2 Laboratory of Tumor Molecular Biology and Targeted Therapies, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine , Guangzhou 510120Guangdong Province , P. R. China 3 Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical 4 Medical Collage, University of Guangzhou Traditional Chinese Medicine , Guangzhou, Guangdong Province 510120 , P. R. China Background: Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown. Methods: Cell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays. Results: We showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and timedependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth. Conclusion: Our results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells. Human lung cancer cells; SAPK/JNK; DNMT1; EZH2; Ursolic acid; SP1 - Introduction Lung cancer is the most common malignancy worldwide and a leading cause of cancer-related deaths. Despite recent progress in understanding the tumorgenesis signaling network and in producing new therapeutic strategies, this malignancy especially non-small cell lung cancer (NSCLC) still shows poor prognosis and high incidence of recurrence [1]. Majority of lung cancer presents at an advanced stage and treatment results in poor outcome [1, 2]. As a result, there is a significant interest in developing adjunctive therapeutics to augment available treatment regiments without compromising therapeutic efficacy. Ursolic acid (UA) may be one such potential candidate. UA, a triterpene compound came from certain traditional medicinal plants [3], has been widely used for its anticancer properties via variety of biological functions involved in inducing cell apoptosis, anti-proliferation, chemo- and radiotherapy sensitization, anti-invasion and metastases [4–6]. In such, multiple signaling pathways and potential gene targets involved in UA-inhibited cancer cell growth including lung cancer have been reported in the past [5–8]. However, the molecular mechanisms underlying the beneficial effects of UA in the treatment of lung cancer remain largely unknown. DNA methylation is controlled by DNA methyltransferase (DNMT), an enzyme that catalyzes the transfer of a methyl group to DNA. DNMT1 [DNA (cytosine-5-)-methyltransferase 1] is primarily a DNA methyltransferase that maintains DNA methylation, which is one of the regulatory mechanisms of gene expression [9]. DNMT1 also participates in several cellular functions other than through DNA methylation [10, 11]. Overexpression of DNMT1 has been found in various cancer types including lung cancer and inhibition of DNMT1 suppressed growth and induced apoptosis of cancer cells through multiple signaling pathways and distinct mechanisms [12–14]. Thus, re-expression (...truncated)


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Jingjing Wu, Shunyu Zhao, Qing Tang, Fang Zheng, YuQin Chen, LiJun Yang, Xiaobing Yang, Liuning Li, WanYin Wu, Swei Hann. Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells, Journal of Experimental & Clinical Cancer Research, 2015, pp. 99, 34, DOI: 10.1186/s13046-015-0215-9