Effect of EGCG on invasion of breast cancer cells and its possible mechanism

AIM: To study the effects of (-)-epigallocatechin-3-gallate (EGCG), a tea extract, on the invasion and metastasis of breast cancer cell line MDA-MB-231 and the possible mechanisms in vitro. METHODS: The expression of MUC1 in breast cancer cells treated with or without EGCG was detected by immunohistochemistry. The effect of EGCG on invasion of MDA-MB-231 cells was evaluated using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel). Gelatin zymography was performed to detect the secretion of collagenase-Ⅳ. RESULTS: EGCG reduced the expression of MUC1, significantly suppressed the invasion of tumor cells to basement membrane and reduced the secretion of collagenase-Ⅳ. CONCLUSION: In vitro, EGCG may suppress invasion, metastasis, and collagenase-Ⅳ secretion in MDA-MB-231 cells by inhibiting the production of MUC1.     

The inhibitory effects of endostatin gene transfer on the growth of breast cancer cells in vivo and in vitro

AIM: To investigate the therapeutic action of secreted endostatin (ES) on breast cancer cells. METHODS: Retroviral-mediated endostatin gene was transferred to breast cancer cell line MDA-MB-231. The ES biological properties and function were evaluated by polymerase chain reaction (PCR), MTT and a murine xenograft model. RESULTS: After retroviral transduction, endostatin genetically modified breast tumor cells were confirmed by PCR, and the integration and durative expression of endostatin gene was successfully committed. Compared with controls, endostatin secreted by genetically modified cells markedly inhibited endothelial cell proliferation (P<0.05) while the influences on the growth of MDA-MB-231 cell line in vitro were not found (P>0.05). The results of the transplanted subcutaneous tumor model in nude mice suggested that the subcutaneous growth of MDA-MB-231 was significantly inhibited by the expression of endostatin gene (P<0.05). In experimental groups, the tumor microvascular density (MVD) and VEGF expression were decreased. CONCLUSION: Retroviral- mediated overexpression of endostatin inhibits the proliferation of vascular endothelial cells and angiogenesis that associated with tumor growth in vivo via the paracrine pathway, which has a potential effect in the angiostatic gene therapy for breast cancer.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Cloning,transfer and anti-human breast cancer cell growth of the extracellular domain of cadherin 5

AIM: In order to study the effect of the extracellular domain of cadherin 5 on the growth of a human breast cancer cell line MDA-MB435.METHODS: Cadherin extracellular domain repeats 1 to 4(CED1-4) was cloned by using RT-PCR technique,and inserted into the plasmid vector pMSCV.pMSCV-CED1-4 was propagated in XL-blue strain of Escherichia coli,extracted and purified.CED1-4 was cut by restriction endonuclease,examined by using agar gel electrophoresis,and finally sequenced.CED1-4 gene was transferred into MDA-MB435 cell line.The expression of CED1-4 gene in MDA-MB435 cell was analyzed by methods of RT-PCR and Western blotting.The effect of CED1-4 on the growth of MDA-MB435 cell was observed by the methods of proliferation experiments in vitro and the experiments in nude mice in vivo.RESULTS: The recombinant vector pMSCV-CED1-4 was successfully constructed.CED1-4 band appeared between the 1 636 bp and 1 018 bp in agar gel electrophoresis.The sequence result showed that CED1-4 had 1 452 bp and codes 484 amino acids.PCR and Western blotting identified that CED1-4 mRNA and protein were expressed in the transfected MDA-MB435 cells.Cell proliferation experiments showed that the proliferation rate of MDA-MB435 cells was lower in the experimental group than that in the experimental control group and the blank control group.The mean volume and weight of tumors in nude mice were lower in the experimental group than those in the experimental control group and the blank control group.CONCLUSION: The growth of a human breast cancer cell line MDA-MB435 is inhibited in vitro and in vivo by cadherin 5 extracellular domain CED1-4.     

Effect of HC-1119 on biological behaviors of triple-negative breast cancer BT549 cells

AIM: To explore the effect of new artificially synthesized androgen receptor (AR) antagonist HC-1119 on the biological function of triple-negative breast cancer (TNBC) BT549 cells and the molecular mechanism. METHODS: The AR expression was assessed in different human breast cancer cell lines MDA-MB-231, T47D, MCF-7, SKBR3 and BT549 by Western blot. The TNBC BT549 cells with AR positive expression were treated with HC-1119. The cell viability was measured by CCK-8 assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The migration and invasion abilities were detected by Transwell assay in vitro. The protein expression of E-cadherin, vimentin and P21 was determined by Western blot. RESULTS: AR was positively expressed in BT549 cells. HC-1119 inhibited the cell viability in a time-and dose-dependent manner (P<0.05), increased the percentage of apoptotic cells and the percentage of S-phase cells significantly, repressed the migration and invasion abilities (P<0.05), and decreased P21 expression at protein level (P<0.01). No influence on the expression of E-cadherin and vimentin in the BT549 cells was observed. CONCLUSION: AR antagonist HC-1119 decreases the viability, migration ability and invasion ability, enhances the apoptosis, and arrests the cell cycle distribution of TNBC BT549 cells. HC-1119 represses the viability of BT549 cells by down-regulating P21 expression, while the process of epithelial-mesenchymal transition is not involved in the inhibition of cell migration.     

Metformin sensitizes human breast cancer MDA-MB-231 cells to tamo-xifen through down-regulation of c-Myc

AIM: To investigate whether metformin enhances the sensitivity of human breast cancer MDA-MB-231 cells to tamoxifen by down-regulating c-Myc. METHODS: The cell viability, colony formation, apoptosis, and migration and invasion abilities of MDA-MB-231 cells were detected by CCK-8 assay, colony formation experiment, flow cytometry and Transwell assay. The expression level of c-Myc was quantified by Western blot and immunohistochemical analysis. The antitumor effects of metformin and tamoxifen were investigated in vivo in a MDA-MB-231 triple-negative breast cancer xenograft model in the SCID mice. RESULTS: Metformin in combination with tamoxifen exerted synergistic effects on inhibition of the viability, colony formation, migration and invasion, and induced the apoptosis compared with the controls and either agent treatment alone in the MDA-MB-231 cells. The levels of c-Myc was down-regulated in vitro by treatment with metformin and/or tamoxifen (P<0.01). Moreover, metformin or in combination with tamoxifen also reduced the growth of MDA-MB-231 breast cancer tumors in the SCID mice by down-regulation of c-Myc in vivo. CONCLUSION: Metformin in combination with tamoxifen exerts synergistic effects on inhibition of the proliferation, migration, invasion and tumor growth of human triple-negative breast cancer MDA-MB-231 cells by down-regulating c-Myc expression, suggesting that metformin in combination with tamoxifen merits further evaluation as a target.     

Effect of neuregulins on mtp53 and HIF-1α in MDA-MB-231 cells

AIM: To explore the effect and significance of neuregulins /ErbB2 receptor signal transduction pathway on mtp53 and hypoxia-iducible factor-1α (HIF-1α) in none-overexpression ErbB2 breast cancer cell MDA-MB-231. METHODS: The expression of neuregulin was detected by immunocytochemistry and Western blotting. MDA-MB-231 cells were treated with ErbB2 kinase inhibitor AG825. Proliferation was measured by MTT assay. The cell cycle and apoptosis were determined by flow cytometry. The expressions of mtp53 and HIF-1α were detected by Western blotting. The mRNA expression of HIF-1α was detected by RT-PCR. RESULTS: MDA-MB-231 cells expressed a relative higher level of neuregulin. In the results of Western blotting, the positive reaction band was found in 44 kD which coincides with the molecular weight of neuregulin. When MDA-MB-231 cells were treated with AG825, the proliferation was inhibited in time and dose dependent manners (P<0.01). The cell cycle was arrested in G0/G1 phase (P<0.05). The apoptosis rate was increased (P<0.05). The protein expression levels of mtp53 and HIF-1α were decreased (P<0.05), and the mRNA level of HIF-1α was also decreased (P<0.05).CONCLUSION: Our study indicates that neuregulins are synthesized in MDA-MB-231 cells as transmembrane proteins. Neuregulins activate ErbB2 receptor signal transduction pathway by ligand autocrine or paracrine actions, and play an important role in proliferation of none-overexpression ErbB2 breast cancer cell MDA-MB-231. Proliferation and survivorship, and inhibition apoptosis can be induced with up-regulation of mtp53 and HIF-1α level.     

Role of VEGF-C in lymphangiogenesis and lymphatic metastasis of breast cancer

AIM: To study the inhibitory effect of VEGF-C/Flt-4 system on lymphangiogenesis and lymphatic metastasis of breast cancer. METHODS: Lymphatic endothelial cells (LEC) were cultured in vitro, the effects of VEGF-C and anti-Flt-4 antibody on the proliferation of treated cells were observed. The antisense oligodeoxynucleotides (ASODN) targeting VEGF-C was designed and its effect on VEGF-C gene expression in vitro experiments was observed. The nude mice transplantation tumor model was made and the effects of VEGF-C ASODN on lymphangiogenesis and metastasis in the model were determined. RESULTS: The supernatant of cultured PC3 cells promoted LEC proliferation obviously while the cells treated with anti-Flt-4 antibody were obviously decreased whenever cell counting. The mRNA and protein expression of VEGF-C in MCF-7 cells treated with ASODN were significantly lower than that in control groups in vitro. In vivo ASODN also significantly reduced the VEGF-C mRNA expression detected by RT-PCR. The result of 5-Nase-ALPase enzyme -histochemistry showed that ASODN had obvious inhibitory effect on tumor lymphangiogenesis. Tumor growth velocity in ASODN group was much slower than that in control group. ASODN also inhibited tumor volume and lymphatic metastasis. CONCLUSION: The strong relationships between VEGF-C/Flt-4 system and lymphangiogenesis and lymphatic metastasis of breast cancer have been observed. If the expression of Flt-4 is blocked, the proliferation of LEC induced by tumor cells can be blocked in some degree. ASODN inhibits tumor lymphangiogenesis and lymphatic metastasis by down-regulating VEGF-C expressions.     

Effects of c-Met on proliferation of triple negative breast cancer and sensitivity to doxorubicin

AIM: To investigate the effects of c-Met on the proliferation and the sensitivity to chemotherapeutic drugs of triple negative breast cancer cells. METHODS: Doxorubicin-resistant cells (MDA-MB-231/ADR) were established. The expression of c-Met at mRNA and protein levels in the MDA-MB-231/ADR cells and parental MDA-MB-231 cells was detected by real-time PCR and Western blotting. c-Met siRNA and plasmid or AKT siRNA were transfected into the cancer cells. The cell proliferation and the sensitivity to doxorubicin were determined by MTT assay. RESULTS: The expression of c-Met at mRNA and protein levels in MDA-MB-231/ADR cells was significantly higher than that in parental MDA-MB-231 cells. Transfection with pBABE-puro TPR-MET plasmid into the MDA-MB-231 cells induced cell proliferation and resistance to doxorubicin. Meanwhile, inhibition of c-Met in the MDA-MB-231/ADR cells by siRNA reversed the doxorubicin-resistance. In addition, over-expression of c-Met led to higher phosphorylation level of AKT, which was involved in the effects of c-Met on the MDA-MB-231 cell proliferation and doxorubicin-resistance. CONCLUSION: c-Met may have the potential as a therapeutic target in the treatment of triple negative breast cancer.     

Effect of norcantharidin on proliferation and invasion of human breast cancer cell line SKBR3 in vitro

AIM: To investigate the effect of norcantharidin（NCTD）on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test，Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC₅₀ value of 12.5 mg/L at 24 h．The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G₁ phase. The cell cycle was arrested at G₂/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G₂/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Effect of nerve growth factor on proliferation and survival of breast cancer cell line MDA-MB-231 and MCF-7

AIM: To study the effect of nerve growth factor (NGF) on the proliferation and survival of breast cancer cell lines MDA-MB-231 and MCF-7. METHODS: Two breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiment. The expression of NGF and its receptor tyrosine kinase A (TrkA) was determined by the method of immunofluorescence. The NGF autocrine was detected by the method of enzyme linked immunosorbent assay (ELISA). The expression of TrkA was measured by Western blotting. The effect of NGF blocking agent Ro 08-2750 on the proliferation of the cells was evaluated by MTT assay (mono-nuclear cell direct cytotoxicity assay). The cell apoptosis and the change of the cell cycle after exposed to Ro 08-2750 were observed by flow cytometry. RESULTS: Both cell lines expressed NGF and TrkA. Ro 08-2750 inhibited the proliferation of both cell lines in a dose-dependent manner. According to the results of flow cytometry, the S-phase cells in both cell lines increased but the G₂/M-phase cells decreased after treated with Ro 08-2750. An apoptotic peak occurred in MDA-MB-231 cells. CONCLUSION: The results suggest that proliferation of MDA-MB-231 and MCF-7 cell lines is dependent on NGF. NGF promotes the survival of MDA-MB-231 cells.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

miR-137 regulates invasion,migration and apoptosis of breast cancer MDA-MB-231 cells through TWIST1

AIM: To investigate the effect and its molecular mechanism of microRNA-137(miR-137) on the invasion, migration abilities and apoptosis of breast cancer cells. METHODS: miR-137 mimimics were transfected into the breast cancer MDA-MB-231 cells. The expression of miR-137 was detected by RT-qPCR. Apoptosis was analyzed by flow cytometry. The invasion and migration abilities were detected by Transwell assays. The protein levels of matrix metalloproteinase 9 (MMP-9), cleaved caspase-3 (C-caspase-3) and Bax were determined by Western blot. Bioinformatics software was used to predict that TWIST1 might be the target gene of miR-137 and then it was conformed by luciferase reporter gene identification. The effect of miR-137 mimics on TWIST1 protein expression was evaluated by Western blot. TWIST1 over-expression vector and miR-137 mimics were co-transfected into the MDA-MB-231 cells, and then the apoptosis, invasion, migration abilities and the protein levels of MMP-9, C-caspase-3 and Bax were determined. RESULTS: In the miR-137 mimics transfected MDA-MB-231 cells, the expression level of miR-137 and the apoptosis rate were increased, the cell invasion and migration abilities were decreased, the protein levels of C-caspase-3 and Bax were increased, the protein expression of MMP-9 was decreased (P<0.05). In addition, the target regulation of TWIST1 by miR-137 was identified by luciferase reporter assay. Moreover, the expression of TWIST1 in the MDA-MB-231 cells was inhibited by miR-137 mimics. Compared with the MDA-MB-231 cells co-transfected with negative control vector and miR-137 mimics, the protein expression levels of TWIST1 and MMP-9 in the MDA-MB-231 cells co-transfected with TWIST1 over-expression vector and miR-137 mimics were increased, the protein levels of C-caspase-3 and Bax and the apoptosis rate were decreased, the cell invasion and migration abilities were increased. CONCLUSION: miR-137 inhibits the invasion, migration abilities and induces apoptosis of breast cancer cells through targeting TWIST1.     

AMembrane-bound ligand-dependent bystander effect of the TNF-related apoptosis-inducing ligand (TRAIL)

AIM：To investigate the mechanism of bystander effect caused by TRAIL gene.METHODS：Bystander target cells were transduced with an adenovector expressing the lacZ gene (Ad/Lac-Z),while effector cells were transduced with an adenovector expressing the green fluorescent protein (GFP)/TRAIL fusion gene (Ad/g-TRAIL).Effector and target cells were cocultured in the same well with or without effector and target cell contact using a Transwell plate.RESULTS：A series of different cancer cell lines of different tissue origin were tested for the bystander effect,including human colon cancer cell lines DLD1,human lung cancer cell line A549,human hepatoma cell line Hep G2,human breast cancer cell line MDA-MB231 and human ovarian cancer cell line DOV13.No detectable soluble TRAIL (s-TRAIL) was found from the TRAIL-expressing cell cultures in all of the cell lines.Target cells were killed only when effector and target cells were mixed.The bystander effect and apoptosis induction of TRAIL was dramatically reduced if cells were seeded at a very low density.FACS analysis showed a high apoptosis percentage in high density group in DOV13,morphologically,the effector cells were surrounded by the apoptotic bystander cells.CONCLUSION：The bystander effect of the TRAIL gene is mainly mediated by membrane-bound TRAIL on the surface of transduced cells through cell-cell contacts.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.