| Background: Ovarian cancer is one of the most common gynecological malignancies,whose morbidity ranking third behind cervical cancer and corpus carcinoma.Due to the absence of obvious symptoms in the early stage and the lack of effective means of early detection and screening,about 70% of patients are diagnosed at an advanced stage.At present,the main therapy of ovarian malignancy is combination of surgery and chemotherapy;however,chemotherapy is toxic to the body of patients and is prone to relapse with drug resistance.Phosphorylation is one of the most extensively studied post-translational modifications(PTMs),which regulates a number of cellular functions like cell growth,differentiation and apoptosis,etc.Alterations in phosphorylation pathways are strongly associated with cancer,at the same time they serve as the potential targets for drug development against cancer.Proteomic investigations reveal that the signaling pathways,mediated by PTMs such as phosphorylation,plays a critical role in the development of ovarian cancer and its chemoresistance.Matrine is an alkaloid isolated from Sophora flavescens Aiton,possessing a wide spectrum of biological activities,such as anti-inflammatory,anti-viral,anti-cancer,anti-nociceptive,anti-bacterial,anti-oxidant,reducing blood lipid and anti-liver fibrosis,etc.A large number of studies have shown that matrine has a clear anti-cancer effect on lung cancer,breast cancer,gastric cancer,pancreatic cancer,prostatic cancer and many other types of cancers.However,the effect of matrine on ovarian cancer and its potential molecular mechanisms have not been reported yet.Objective: The purpose of this study was to explore the effect of matrine on ovarian cancer and its molecular mechanism,providing clues and basis for further work on matrine as a novel anticancer agent.Methods: 1.The effects of matrine on the proliferation of ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP were determined by MTT assay and colony formation assay.2.Flow cytometry was conducted to detect the influences of matrine on cell cycle and apoptosis of ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP.3.Transmission electron microscopy and fluorescence microscopy were used to observe the effect of matrine on autophagy of ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP.4.The impacts of matrine on the migration and invasion of ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP were evaluated by wound healing assay,transwell invasion assay and immunofluorescence assay.5.In vitro angiogenesis assay was performed to investigate the effect of matrine on tumor angiogenesis.6.Enzyme linked immunosorbent assay(ELISA)was used to examine the effect of matrine on VEGFA contents secreted by ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP.7.The effects of matrine on the m RNA levels of VEGFA and ANG-1 were detected by reverse transcription-polymerase chain reaction(RT-PCR).8.Western blot was used to determined the influences of matrine on the expression of proteins related to the proliferation,apoptosis,autophagy,migration and invasion,angiogenesis and cancer associated phosphorylation signaling pathways in ovarian cancer cells A2780 and SKOV3.9.Ovarian cancer cell A2780 and ovarian cancer cisplatin-resistant cell A2780/DDP subcutaneous xenograft model and tail veins mouse model were established,regular intraperitoneal injection of matrine was performed to observe the effect of matrine on the proliferation and metastasis of ovarian cancer cells in vivo.10.Hematoxylin-eosin(H&E)staining was used to observe the effect of matrine on tumor necrosis,pulmonary metastasis and hepatic injury in mice.The effect of matrine on tumor apoptosis was analyzed by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)assay.11.The effect of matrine on the total protein phosphorylation levels of ovarian cancer cell A2780 and tumor tissues,as well as the protein phosphorylation levels of signaling pathways related to proliferation,apoptosis,autophagy,migration and invasion,angiogenesis in tumor tissues were analyzed by Western blot,respectively.12.Immunohistochemistry(IHC)was used to further verify the impact of matrine on the protein phosphorylation levels of signaling pathways in tumor tissues.13.The contents of alanine aminotransferase(ALT)and aspartic aminotransferase(AST)in serum were measured by automatic biochemical analyzer to evaluate the effect of matrine on hepatic function in mice.Results: 1.Matrine significantly inhibited the proliferation of ovarian cancer cells A2780 and SKOV3 in a dose-and time-dependent manner.2.Matrine prominently induced cell cycle arrest in G0/G1 phase of ovarian cancer cells A2780 and SKOV3,accompanied by increased expression of p21 and decreased expression of cyclin D1 and CDK4.Matrine drastically reduced the phosphorylation levels of ERK1/2 and MEK1/2 in ovarian cancer cells A2780 and SKOV3.3.Matrine induced apoptosis of ovarian cancer cells A2780 and SKOV3,down-regulating the expression of Bcl-2 and up-regulating the expression of Bax.Matrine significantly reduced the phosphorylation levels of PI3 K and Akt without effect on their expression levels.4.Matrine induced autophagy in ovarian cancer cells A2780 and SKOV3,increased the expression of LC3-II and decreased the expression of SQSTM1.The phosphorylation levels of Akt and m TOR were significantly inhibited by matrine.5.Matrine apparently suppressed the migration and invasion of A2780 and SKOV3 cells in a dose-dependent manner.Matrine remarkably decreased the levels of active FAK(p-FAK)and Rho A(Rho A-GTP)in A2780 and SKOV3 cells.6.Matrine notably impaired the stimulation effect of angiogenesis induced by ovarian cancer cells.The levels of VEGFA secreted into the medium were significantly reduced after matrine treatment in a dose-dependent manner in A2780 and SKOV3 cells.Furthermore,the m RNA levels and protein expressions of VEGFA were significantly decreased in A2780 and SKOV3 cells with the addition of matrine.7.The m RNA levels and protein expressions of ANG-1 in HUVECs were significantly downregulated during the HUVEC/A2780 or HUVEC/SKOV3 co-culture relative to the HUVECs monoculture.The phosphorylation levels of VEGFR2 and Tie2 in HUVECs were found to dramatically decline after matrine treatment.8.Matrine inhibited the growth and metastasis of ovarian cancer cell A2780 in vivo,and the global phosphorylation levels of tyrosine and serine/threonine in matrine-treated tumor tissue lysates and in A2780 cell exposed to matrine were significantly reduced.9.Western blot and IHC analysis showed that matrine administration obviously decreased the phosphorylation levels of ERK1/2,MEK1/2,PI3 K,Akt,m TOR,FAK,Rho A,VEGFR2 and Tie2 in tumor tissues compared with the control group.10.Hepatic tissues of matrine-treated mice showed normal cellular morphology as the control group.Moreover,there was no obvious difference in serum activity of ALT and AST between marine treated and control mice.11.Matrine significantly inhibited the proliferation,apoptosis,autophagy,migration and angiogenesis of ovarian cancer cisplatin-resistant cell A2780/DDP.12.Matrine inhibited the proliferation and metastasis of ovarian cancer cisplatin-resistant cell A2780/DDP in vivo.Conclusion: Matrine inhibited the proliferation,induced the apoptosis and autophagy,inhibited the migration and invasion,impaired angiogenesis of ovarian cancer cells A2780,SKOV3 and ovarian cancer cisplatin-resistant cell A2780/DDP.Taken together,the present study demonstrated that matrine suppressed the development and progression of ovarian cancer by regulating proliferation,apoptosis,autophagy,migration,invasion and angiogenesis via inhibiting the cancer associated phosphorylation signaling pathways.Background: In recent decades,molecular targeted therapy has significantly improved therapeutic effects in patients with many types of malignant tumors,such as breast cancer,gastric carcinoma,colon cancer,lung cancer and ovarian cancer.Identification of ideal targets is essential for a successful development of molecular targeted therapies in cancer.According to the targets,they act on cell surface antigens,receptors and growth factors,and can regulate signal transduction pathways related to cell proliferation and metastasis,cell cycle progression and angiogenesis.Many of the currently available compounds target specific receptors,which regulate the signal transduction pathways,thus inhibiting the cell proliferation and eventually leading to cell death.The proto-oncogene c-Src(Src)is a non-receptor tyrosine kinase that is closely associated with the development,maintenance,progression and metastasis of a variety of human cancers.Src plays a key role in signal transduction pathways by interacting with and phosphorylating multiple proteins and protein complexes.Matrine is one of the alkaloids extracted from the traditional Chinese medicine Sophora flavescens,possessing a wide spectrum of pharmacological activities such as anti-inflammation,anti-tumor and anti-fibrosis.Studies have shown that matrine has antitumor activity against many types of cancers,including lung cancer,breast cancer,liver cancer,gastric carcinoma,pancreatic cancer,ovarian cancer and leukemia.Matrine exerts anti-tumor activity mainly through inhibiting tumor cell proliferation,inducing cell apoptosis and autophagy,inhibiting tumor cell migration,invasion and angiogenesis.At present,the antitumor effect of matrine has been widely studied,whereas the direct target of its anticancer activity has not been reported.Objective: The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action,so as to provide experimental basis for the development of targeted drugs for cancer.Methods: 1.Six types of cancer cells,including human colon cancer cell HT-29,human breast cancer cell MCF7,human non-small cell lung cancer A549,human pancreatic cancer cell Bx PC-3,human ovarian cancer cell SKOV3 and human cervical cancer cell He La were selected.The effects of matrine on the proliferation of cancer cells were determined by MTT assay.2.Human non-small cell lung cancer A549,human pancreatic cancer cell Bx PC-3 and human ovarian cancer cell SKOV3 subcutaneous xenograft models were established,regular intraperitoneal injection of matrine or normal saline were performed to observe the effect of matrine on tumor proliferation in vivo.3.Sophocarpine and ethanolamine were used as raw materials for chemical reaction to synthesize 13α-(2-amino)ethoxymatrine.The structure of13α-(2-amino)ethoxymatrine were characterized by infrared spectrum(IR)and mass spectrometry(MS).4.The matrine coupling resin was prepared by coupling reaction of 13α-(2-amino)ethoxymatrine with aminolink coupling resin.The concentration of13α-(2-amino)ethoxymatrine consumed in the coupling reaction was detected by ultraviolet light-visible light absorption spectrum,which indirectly reflected the efficiency of coupling reaction.5.The protein was extracted from ovarian cancer cell SKOV3,and the matrine coupling resin was used for pull down assay.Coomassie bright blue staining was used to find the specific binding protein of matrine,which was identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS).6.Cellular thermal shift assay(CETSA)was performed to verify the target of matrine.GST-Src recombinant protein was constructed,and the target of matrine was further verified by pull down experiment.7.Src truncated plasmids were constructed and transfected into HEK293 T cells,and the binding domain of matrine was verified by pull down experiment.8.GST-Kinase recombinant protein was constructed,and the binding domain of matrine was further verified by pull down assay.9.The molecular docking of matrine and Src kinase domain was conducted by Discovery Studio 4.5 software,exploring the binding amino acids of matrine in Src kinase domain.10.The optimal concentration of matrine in each cancer cell were selected and the effect of matrine on Src kinase activity in cancer cells were determined,respectively.11.The effect of matrine on the protein phosphorylation levels of MAPK/ERK,JAK2/STAT3 and PI3K/Akt signaling pathways were detected by Western blot.Results: 1.Matrine significantly inhibited the proliferation of cancer cells in a doseand time-dependent manner.2.Matrine inhibited the proliferation of cancer cells in vivo.3.13α-(2-amino)ethoxymatrine was synthesized,yield 65%.IR and MS results showed that the structure characterization of product was the same as 13α-(2-amino)ethoxymatrine.4.The matrine coupling resin was prepared,and the concentration of13α-(2-amino)ethoxymatrine consumed in the coupling reaction was detected by ultraviolet light-visible light absorption spectrum.The efficiency of coupling reaction was 54%.5.The results of Coomassie bright blue staining showed that there was an obvious protein band between 55-70 k Da.The band was significantly weakened after matrine added,suggesting that this protein might be the specific binding protein of matrine.Moreover,qualitative analysis by LC-MS/MS revealed that Src was the specific binding protein of matrine.6.CETSA results showed that the thermal stability of Src in matrine treatment group was significantly enhanced compared with the control group.Pull down assay was conducted by GST-Src recombinant protein and the results indicated that Src was detected in the matrine coupling resin group.With the addition of matrine,Src was significantly reduced,confirming that Src was the target of matrine.7.Src truncated plasmids USH3,SH4-UD,SH3,SH2 and Kinase were constructed and transfected into HEK293 T cells,then the total protein were extracted for pull down assay.Western blot analysis showed that Src kinase domain was the binding region of matrine.8.GST-Kinase recombinant protein was constructed for pull down experiment.Western blot analysis indicated that the target protein was detected in matrine coupling resin group and was significantly reduced after matrine was added,further confirming that Src kinase domain was the binding region of matrine.9.The molecular docking of matrine with Src kinase domain was predicted by Discovery Studio 4.5 software.The results revealed that matrine binded to Src kinase domain,mainly due to the strong hydrogen bond force formed between matrine and Ala392.van der Waals forces and other forces were also formed between matrine and surrounding amino acids.10.After treated with the optimal concentration of matrine,the Src kinase activity in cancer cells were significantly decreased.11.Western blot results showed that the protein phosphorylation levels of MAPK/ERK,JAK2/STAT3 and PI3K/Akt signaling pathways were down-regulated in cancer cells.Conclusion: Matrine inhibited the proliferation of cancer cells.The target of matrine was Src,and Src kinase domain was the binding region of matrine.Meanwhile,matrine significantly down-regulated the protein phosphorylation levels of MAPK/ERK,JAK2/STAT3 and PI3K/Akt signaling pathways in cancer cells.Therefore,matrine targeted the Src kinase domain by down-regulating the phosphorylation signaling pathways,thus inhibiting the proliferation of cancer cells. |
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