Investigation On Estrogen Receptor Alpha 36 In Signet Ring Gastric Cancer

Cancer is one of the leading cause of deaths worldwide.Gastric cancer is the third primary cause of cancer-related deaths globally.In China,gastric cancer is the second leading cause of death,and 1/3 of all the gastric cancer patients are from China.Estrogen is a class of hormone,which is essential in the physiological development,and differentiation of the body.Its functions are mediated by estrogen receptor(ER).Estrogen receptor has two subtypes ERa and ER3 and has various isoforms.Estrogen receptors have crucial roles in the events of various cancers and most importantly in breast cancer.Estrogen receptor alpha 36(ERa36)an isoform of ERa is extensively studied in breast cancer,but our understanding is limited regarding the role of ERa36 in gastric cancer.Signet ring gastric cancer(SRGC)is a poorly differentiated discrete histological sub-type of gastric cancer.It showed worse prognosis in the clinic.SRGC is more resistant to the first line chemotherapeutic drugs used in the treatment of gastric cancer.Therefore,discovering novel therapeutic marker and novel lead compounds that can then be used as a therapeutic agent in signet ring gastric cancer is the need of time.Molecular docking is used to screen the lead compounds from an extensive database based on the ligand binding interactions.Epigallocatechin Gallate(EGCG)belongs to the flavonoid group and is a constituent of green tea leaves.Traditionally it is used in Chinese medicines,and it is also tested against a variety of cancer cell lines.EGCG showed a significant effect on various tumors,yet their exact underlying mechanism is not understood yet.In this study,we determined that ERa36 is highly expressed in 9 gastric cancer cell lines at mRNA level and at the protein level.Immunohistochemistry analysis of gastric cancer tissues showed ERa36 is highly expressed in signet ring gastric cancer tissues.ERa36 is present in all the gastric cancer cell lines at mRNA level,expressed in all 3 signet ring gastric cancer cell lines at protein levels and in 80%of the signet ring gastric tissues.ERa36 localization is mainly to the membrane in tissues.Similarly,we determined the expression of ER? in gastric cancer.ER? is less expressed at mRNA and protein level analyzed by RT-PCR and western blot analysis respectively.Similarly,ER? is expressed in only 41%of the gastric cancer tissues analyzed via immunohistochemistry and is localization is to the nucleus.As ERa36 is highly expressed,we investigated the role of ERa36 in signet ring gastric cancer and as a potential therapeutic target.ERa36 was knockdown by using the multi-hairpin miR30 artificial miRNA incorporated into a recombinant adenovirus.The artificial miRNA sequence against ERa36 was sub-cloned into adenovirus expression vector and was used for the down-regulation of ERa36 in signet ring gastric cancer cell line GCSR-1-6.Knockdown of ERa3 6 showed that ERa3 6 suppression is associated with decreased proliferation,migration,and invasion.ERa36 knockdown also showed altered expression of the various protein responsible for the proliferation,differentiation,migration,invasion,and angiogenesis of gastric cancer.Which suggests that ERa36 is essential for the proliferation of signet ring gastric cancer cells.Furthermore,we investigated the downstream signaling pathway associated with ERa36;ERa36/EGFR positive feedback loop mechanism,ERa36/MAPK/ERK,Akt/ERa36 pathways modulate ERa3 6 downstream signaling pathway.ERa3 6 suppression is associated with the compromised ERa36 downstream signaling pathways.ERa36 depletion was also associated with autophagy and production of reactive oxygen species that can lead to apoptosis.Hereafter,we found that ERa36 suppression leads to the activation of caspase,which in turn can lead to the cell death.Cell death induced by ERa36 suppression was further confirmed by the protein expression of various important proteins and by flow cytometry.ERa3 6 knockdown showed a significant decrease in the tumor volume,weight and decreased proliferation in signet ring based tumor in vivo.Our results showed that ERa36 is highly expressed in signet ring gastric cancer and down-regulation of ERa36 can drastically inhibit cell growth,proliferation,migration,invasion,and survival in vitro and in vivo.Moreover,we tried to discover a therapeutic agent that can directly target ERa36.As there is no commercially available inhibitor against ER?36,we decided to find the direct inhibitor of ERa36 by using molecular docking simulations to predict the possible inhibitor/s of ERa36.We screened 2295 compounds via molecular docking,and 6 compounds were shortlisted as lead compounds by molecular docking scores.The resulted compounds were all flavonoids suggesting flavonoids can potentially bind to ERa36.The compounds were then screened for their drugability properties by Lipinski's rule of five,important for the calculation of drugability properties of the drug.Results showed EGCG is the most suitable compound with lesser deviations to the Lipinski's rule of five and more bioactive properties.Based on molecular docking,molinspiration and literature survey we used EGCG for further investigations.We tested EGCG on GCSR-1-6 and GCSR-1-11 two signet ring gastric cancer cell lines and MDA-MB231 and MCF-7 are breast cancer cell lines with varying expression of ERa36.Our results showed EGCG is more effective against the cell lines with higher expression of ERa36 and less effective against cell lines with lower ERa36 expression or presence of other ER isoforms.We further observed that EGCG treatments downregulate the expression of ERa36;suggesting EGCG can be used as an inhibitor of the ERa36 downstream cascade.Furthermore,GCSR-1-6 cells treated with EGCG showed decreased proliferation,migration,and invasion in a dose-dependent manner.Similarly,EGCC treatment compromised the ERa36 downstream signaling pathway associated with the growth and proliferation of cancer cells.EGCG also induced apoptosis in a dose-dependent manner in signet ring gastric cancer.Taken together our results showed that ERa36 is localized mainly to the plasma membrane in gastric cancer.It is highly expressed in signet ring gastric cancer,and it might be used as a therapeutic target for the treatment of signet ring gastric cancer.EGCG a novel inhibitor can down-regulate ERa3 6 expression and modulate underlying signaling pathways result in a suppressed proliferation of signet ring gastric cancer cells in vitro and in vivo and thus may be used as a therapeutic agent for therapeutic purposes.