Abstract
BackgroundUrsolic 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.MethodsCell 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.ResultsWe 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.ConclusionOur 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.
Highlights
Lung cancer is the most common malignancy worldwide and a leading cause of cancer-related deaths
We showed that Ursolic acid (UA) suppressed growth and induced apoptosis of non small cell lung cancer (NSCLC) cells through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)-mediated reduction of SP1 transcription factor; this in turn results in inhibition of [DNA-methyltransferase 1] (DNMT1) and EZH2 expression
Exogenous expression of DNMT1 overcame the effect of UA on EZH2 protein expression and cell growth we showed that exogenous expression of DNMT1 transfected into the cells reversed the effect of UA on EZH2 protein expression (Fig. 5a) and cell growth in H1299 and A549 cells (Fig. 5b), while overexpression of EZH2 had little effect on DNMT1 expression in H1299 and A549 cells (Fig. 5c)
Summary
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]. Multiple signaling pathways and potential gene targets involved in UA-inhibited cancer cell growth including lung cancer have been reported in the past [5,6,7,8]. The molecular mechanisms underlying the beneficial effects of UA in the treatment of lung cancer remain largely unknown. Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). The molecular mechanisms underlying the action of UA remain largely unknown
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