Study On The Mechanism Of PHII - 7 Inhibiting Tumor Invasion And Migration In

Background:Invasion and migration are major characters of malignant cancers, and are the main problems which lead to the failure, recurrence and the most deaths in cancer patients. However there has been lack of effective clinical treatments for invasion and migration. Danggui Longhui Wan is a useful traditional Chinese herb to treat with chronic myelogenous leukemia and its active agent is called indirubin. We used the structure of indirubin to design, systhesize a range of derivatives and we screened a derivative called PHII-7which had a promising anti-tumor activity on a broad range of human cancers. Previously, we found that PHII-7inhibited cell growth in human cancers, including human immortalised myelogenous leukemia cell line k562and its multidrug resistant cell line k562/A02, promyelocytic leukemia cell line HL60and its multidrug resistant cell line HL60/ADR, breast cancer cell line MCF-7and its multidrug resistant cell line MCF-7/ADR. Our aim is to investigate the effects and mechanism of PHII-7on invasion and migration in vitro.Methods:Three human metastatic cancer cell lines were used to investigate the effects and mechanism of PHII-7:breast cancer cell line MDA-MB-231, breast multi-drug resistant cancer cell line MCF-7/ADR and non-small cell lung carcinoma cell line A549. Methods were used as following:MTT assay was used to test cell proliferation; Cell count assay was used to make cell growth curve; Cell colony formation was used to examine cell colony ability and cell proliferation; Transwell assay and wound healing assay were performed to test invasion and migration; Flow cytometry was used to test apoptosis and necrosis; Realtime PCR and western blot assay were used to test gene expression level on mRNA level and protein level, respectively. All results were repeated at least three times, and SPSS13.0software and Graphpad software were used to analyze data.Results:Firstly, MCF-7/ADR cells showed more invasive and migratory ability, and several EMT-related genes expression were significant different from MCF-7cells. Among these genes, the expression of E-cadherin decreased, however, expressions of Slug, β-catenin and vimentin increased. Secondly, PHII-7inhibited invasion and metastasis in a lower dose wich a dose-dependment manner. Higher dose of PHII-7suppressed cell growth, colony formation and induced apoptosis with a dose-and time-dependent manner. PHII-7induced apoptosis through activating caspase-related genes which were associated with apoptosis. Furthermore, we demonstrated that inhibitory effects of PHII-7on invasion and migration were related with EMT process. PHII-7could increase expression of genes which were epithelial markers and decrease expression of genes which were mesenchymal markers, including E-cadherin, Slug, β-catenin and vimentin. Taken together, PHII-7regulated EMT-related gene expression to inhibit invasion and migration in cancer cells.Conclusion:Clinical therapy with anti-cancer drugs is a usual strategy in cancer treatment, however it brings about severe side effects. Now, it is necessary to find a useful way to treat with cancer and to development clinical treatments. PHII-7is a promising anti-cancer derivative on research. PHII-7can not only increase sensitivity of multidrug resistant cells to anti-cancer drugs, inhibit cell proliferation, but also inhibit invasion and migration of human cancers, and showed potent anti-cancer effects in vitro and in vivo. The research of PHII-7will afford important data for developments of anti-cancer drugs, adjuvant therapy drugs and molecular targeted therapeutic drugs. Background:In recent years, anti-cancer drugs have developed rapidly, especially those anti-cancer drugs with effective targets. PHII-7is a promising anti-cancer derivative which was designed from active agent of traditional Chinese herb Danggui Longhui Wan. PHII-7shows anti-cancer activity on a broad range of human cancers and drug resistant cancers on cell growth, however, there was no data on the mechanism of PHII-7. Previously, using chemical modification and proteomics methods, we found that the mechanism of PHII-7was related to a-enolase (ENO1) which was from lysis of k562. In order to research the relationship between PHII-7and its special target protein ENO1, we established and screened ENO1stable silencing cell line k562-shENO1and its control cell line k562-shCON. In this section, stable silencing ENO1cell line was confirmed, and our aim is to investigate the mechanism of PHII-7and effects of PHII-7by silencing ENO1.Methods:Two stable silencing cell lines k562-shCON and k562-shENO1were used to investigate the mechanism of PHII-7in vitro. Methods were used as following:Cell morphology was observed under inverted microscope; MTT assay was used to test cell proliferation; Cell counting was used to make cell growth curve; Flow cytometry was used to test apoptosis and necrosis; Methyl cellulose semisolid colony forming experiment was used to examine cell colony ability and growth ability; Stem cell potential was assayed by flow cytometry; Realtime PCR and western blot assay were used to test gene expression level on mRNA level and protein level, respectively; Tumorigenesis was tested with nude mice in vivo. All results were repeated at least three times and SPSS13.0software and Graphpad software were used to analyze data.Results:Compared with k562cells, stable silencing k562-shCON cells and k562-shENO1cells showed no significant different on cell morphology. And stable silencing cells were suspended growth. Data demonstrated that k562-shENO1cells performed more sensitivity than k562-shCON cells on proliferation inhibition of PHII-7. Cell count assay showed that there was no significant difference on cell growth rate between k562-shCON cells and k562-shENO1cells, however, k562-shENO1cells performed more sensitivity to lower dose PHII-7. PHII-7induced apoptosis in k562-shCON cells and k562-shENO1cells and its mechanism was related to caspase family which was one of the major apoptosis-related pathways. The dissociations of cell nucleus was observed under confocal microscopy which were induced by PHII-7, and k562-shENO1cells showed more sensitivity than k562-shCON cells. Tumorigenesis assay demonstrated that all nude mice showed tumor formation in k562-shCON group with larger tumor volumn, however, nude mice showed little tumor formation activity with smaller tumor volumn.Conclusion:Using chemical modification and proteomics methods, PHII-7could interact with ENO1in vitro. Further research with cell biology demonstrated that stable silencing ENO1in k562cells would increase cytotoxicity of PHII-7. Data indicated that PHII-7might inhibit or affect ENO1to suppress cell growth, and the mechanism and target of PHII-7needed more data to confirm. Research on mechanism and target of PHII-7will afford important data for design and research of novel anti-cancer drugs, and will be beneficial to further research and clinical treatment of traditional Chinese herb Danggui Longhui Wan. Background:Drug treatment is one of the cancer clinical treatment strategies, however, some drugs can induce drug resistance, such as tamoxifen which is usually used to treat with breast cancer. Drug resistance is one of the failures for cancer treatments, and is one of hinders for clinical treatments. It has been reported that a-enolase(ENO1) is related to drug resistance, and its expression is usually normal in drug resistant cancer cells. Our aim is to research the mechanism of ENO1in human chronic leukemia multidrug resistant cancer cell line k562/A02.Methods:Plasmids pSilencer U6-shCON and pSilencer U6-shENO1were stably transfected into k562/A02cells, and we screened monocolony cell by hygromycin to establish stable silencing cell line k562/A02-shENO1and control cell line k562/A02-shCON. Cell morphology was observed under inverted microscope; MTT assay was used to test cell proliferation; Cell counting was used to make cell growth curve; Flow cytometry was used to test apoptosis and necrosis; Methyl cellulose semisolid colony forming experiment was used to examine cell colony ability and growth ability; Realtime PCR and western blot assay were used to test gene expression level on mRNA level and protein level, respectively; All results were repeated at least three times and SPSS13.0software and Graphpad software were used to analyze data.Results:ENO1was over-expressed in k562/A02cells and its expression was increased by (2.85±0.56) times and (1.43±0.05) times on mRNA level and protein level compared with k562cells. However, cell growth rate in k562/A02cells was no difference with k562cells. We screened three ENO1stable silencing cells k562/A02-shENO1and its control cells k562/A02-shCON. k562/A02-shENO1cells showed lower cell growth rate and higher drug sensitivity to anti-cancer drugs taxol and doxorubicin. Moreover the Rho123content was increased in k562/A02-shENO1cells while the expression of MDR1was decreased on both mRNA level and protein level in k562/A02-shENO1cells. On cell stem potential assay, k562-shENO1cells showed less stem potential compared with k562-shCON cells according to methyl cellulose semisolid colony forming experiment.Conclusion:ENO1silencing suppressed cell growth, decreased stem cell potential and increased sensitivity of k562/A02cells to anti-cancer drugs by decreasing MDR1expression. Data confirmed that there was a relationship between ENO1and drug resistant genes in k562/A02cells and ENO1could be regarded as a target for overcoming drug resistance.