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
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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)