Inactivation of PI3-K/Akt and reduction of SP1 and p65 expression increase the effect of solamargine on suppressing EP4 expression in human lung cancer cells

Journal of Experimental & Clinical Cancer Research, Dec 2015

Background Lung cancer is the most common cause of cancer-related deaths worldwide. Natural phytochemicals from traditional medicinal plants such as solamargine have been shown to have anticancer properties. The prostaglandin E2 receptor EP4 is highly expressed in human cancer, however, the functional role of EP4 in the occurrence and progression of non small cell lung cancer (NSCLC) remained to be elucidated. Methods Cell viability was measured by MTT assays. Western blot was performed to measure the phosphorylation and protein expression of PI3-K downstream effector Akt, transcription factors SP1, p65, and EP4. Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of EP4 gene. Exogenous expression of SP1, p65, and EP4 genes was carried out by transient transfection assays. EP4 promoter activity was measured by Dual Luciferase Reporter Kit. Results We showed that solamargine inhibited the growth of lung cancer cells. Mechanistically, we found that solamargine decreased the phosphorylation of Akt, the protein, mRNA expression, and promoter activity of EP4. Moreover, solamargine inhibited protein expression of SP1 and NF-κB subunit p65, all of which were abrogated in cells transfected with exogenous expressed Akt. Intriguingly, exogenous expressed SP1 overcame the effect of solamargine on inhibition of p65 protein expression, and EP4 protein expression and promoter activity. Finally, exogenous expressed EP4 feedback reversed the effect of solamargine on phosphorylation of Akt and cell growth inhibition. Conclusion Our results show that solamargine inhibits the growth of human lung cancer cells through inactivation of Akt signaling, followed by reduction of SP1 and p65 protein expression. This results in the inhibition of EP4 gene expression. The cross-talk between SP1 and p65, and the positive feedback regulatory loop of PI3-K/Akt signaling by EP4 contribute to the overall responses of solamargine in this process. This study unveils a novel mechanism by which solamargine inhibits growth of human lung cancer cells.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://jeccr.biomedcentral.com/track/pdf/10.1186/s13046-015-0272-0?site=jeccr.biomedcentral.com

Inactivation of PI3-K/Akt and reduction of SP1 and p65 expression increase the effect of solamargine on suppressing EP4 expression in human lung cancer cells

Chen et al. Journal of Experimental & Clinical Cancer Research Inactivation of PI3-K/Akt and reduction of SP1 and p65 expression increase the effect of solamargine on suppressing EP4 expression in human lung cancer cells YuQing Chen 1 Qing Tang 1 JingJing Wu 1 Fang Zheng 1 LiJun Yang 1 Swei Sunny Hann 0 1 0 Higher Education Mega Center , No. 55, Neihuan West Road, Panyu District, Guangzhou, Guangdong Province 510006 , PR China 1 Laboratory of Tumor Biology, Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine , Guangzhou, Guangdong Province 510120 , China Background: Lung cancer is the most common cause of cancer-related deaths worldwide. Natural phytochemicals from traditional medicinal plants such as solamargine have been shown to have anticancer properties. The prostaglandin E2 receptor EP4 is highly expressed in human cancer, however, the functional role of EP4 in the occurrence and progression of non small cell lung cancer (NSCLC) remained to be elucidated. Methods: Cell viability was measured by MTT assays. Western blot was performed to measure the phosphorylation and protein expression of PI3-K downstream effector Akt, transcription factors SP1, p65, and EP4. Quantitative realtime PCR (qRT-PCR) was used to examine the mRNA levels of EP4 gene. Exogenous expression of SP1, p65, and EP4 genes was carried out by transient transfection assays. EP4 promoter activity was measured by Dual Luciferase Reporter Kit. Results: We showed that solamargine inhibited the growth of lung cancer cells. Mechanistically, we found that solamargine decreased the phosphorylation of Akt, the protein, mRNA expression, and promoter activity of EP4. Moreover, solamargine inhibited protein expression of SP1 and NF-κB subunit p65, all of which were abrogated in cells transfected with exogenous expressed Akt. Intriguingly, exogenous expressed SP1 overcame the effect of solamargine on inhibition of p65 protein expression, and EP4 protein expression and promoter activity. Finally, exogenous expressed EP4 feedback reversed the effect of solamargine on phosphorylation of Akt and cell growth inhibition. Conclusion: Our results show that solamargine inhibits the growth of human lung cancer cells through inactivation of Akt signaling, followed by reduction of SP1 and p65 protein expression. This results in the inhibition of EP4 gene expression. The cross-talk between SP1 and p65, and the positive feedback regulatory loop of PI3-K/Akt signaling by EP4 contribute to the overall responses of solamargine in this process. This study unveils a novel mechanism by which solamargine inhibits growth of human lung cancer cells. Human lung cancer cells; Solamargine; PI3-K/Akt; SP1; EP4; p65 Background Lung cancer is the most common cancer and the leading cause of cancer mortality worldwide for both men and women [ 1 ]. Most patients present with incurable advanced or metastatic disease with poor 5-year survival rate. Among them, more than 80 % of lung cancers are non-small cell lung carcinoma (NSCLC) with adenocarcinoma as the most prevalent subtype [ 1 ]. The choice of treatment for patients with advanced disease remains dilemma and challenge, which dependent on the histological types, tumor characteristics, stages, co-morbidities and prior therapies history. Inspire of the advance in understanding the molecular mechanism and treatment options, the poor patient survival still remain no changes and the caused debilitating symptoms seriously affect the quality of life of patients. Therefore, searching for more effective alternative treatment strategies in order to strengthen the therapeutic efficacy with negligible side effects is urgently needed. Natural compounds, known as phytochemicals, obtained from traditional medicinal plants have gained more attention in the prevention and intervention of human illness including cancers [ 2 ]. solamargine (SM), the typical metabolites of solanum lycocarpum fruit glycoalkaloid extract from traditional herbal medicine, demonstrated not only anti-viral, anti-inflammatory but also antiproliferative activity against the several types of human cancers including lung [ 3–7 ]. Report showed that SM induced apoptosis and inhibited growth of hepatoma SMMC-7721 cells through activation and induction expression of caspase-3 [ 8 ]. Early study found that combination of low concentrations of SM with low-toxic topoisomerase II inhibitor epirubicin synergistically accelerated apoptotic cell death through up-regulation of Fas expression and down-regulated the expression of human epidermalgrowth factor receptor-2 (HER2) and topoisomerase II alpha (TOP2A) in NSCLC A549 and H441 cells [ 9 ] However, the detailed mechanisms and potential therapeutic benefices by which this agent in controlling the growth of human lung cancer cells have not been well determined. As a central to multiple signalin (...truncated)


This is a preview of a remote PDF: https://jeccr.biomedcentral.com/track/pdf/10.1186/s13046-015-0272-0?site=jeccr.biomedcentral.com

Fang Zheng, JingJing Wu, LiJun Yang, Qing Tang, Swei Sunny Hann, YuQing Chen. Inactivation of PI3-K/Akt and reduction of SP1 and p65 expression increase the effect of solamargine on suppressing EP4 expression in human lung cancer cells, Journal of Experimental & Clinical Cancer Research, 2015,