Antitumor effect of imperatorin enhances cytotoxicity of doxorubicin to HeLa cells

AIM: To determine whether imperatorin would enhance the effect of doxorubicin therapy on cervical cancer in vitro.METHODS: The viability of HeLa cells treated with imperatorin and doxorubicin was determined by MTT assay in vitro. The expression of Bcl-2 protein family(Mcl-1, Bcl-2, Bcl-xL, Bad and Bax) in HeLa cells treated with imperatorin and doxorubicin was evaluated by Western blot analysis. The apoptosis and mitochondrial membrane potential(ΔΨ_m) in the HeLa cells treated with imperatorin and doxorubicin were analyzed by flow cytometry. A Mcl-1 expression vector was constructed, and its role in the cytotoxicity of imperatorin plus doxorubicin to HeLa cells was detected by MTT assay.RESULTS: Addition of imperatorin significantly enhanced the cytotoxicity of doxorubicin to HeLa cells in vitro. Mcl-1 expression was down-regulated by imperatorin but was not influenced by doxorubicin in the HeLa cells. A combination of imperatorin and doxorubicin induced apoptosis and ΔΨ_m collapse more significantly compared with the treatment with imperatorin or doxorubicin alone. Furthermore, the imperatorin-induced sensitization for doxorubicin cytotoxicity to HeLa cells was abolished by the transfection with Mcl-1 expression plasmid.CONCLUSION: The combination of doxorubicin with imperatorin enhances the antitumor effect of doxorubicin on cervical cancer cells via targeting Mcl-1.     

miR-125a-5p suppresses epithelial-mesenchymal transition via GSK-3β/Snail signaling pathway in breast cancer cells

AIM: To investigate the function of microRNA-125a-5p (miR-125a-5p) on epithelial-mesenchymal transition (EMT) of breast cancer cells via GSK-3β/Snail signaling pathway.METHODS: The expression of miR-125a-5p in normal breast epithelial cells and breast cancer cells, as well as the transfection efficiency of miR-125a-5p plasmid in MDA-MB-231 cells was detected by RT-qPCR. The chemotaxis ability and invasion ability were detected by chemotaxis assay and Transwell invasion assay. The changes of EMT-related markers, the protein level of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and the nuclear translocation of Snail were determined by Western blot. RESULTS: The expression of miR-125a-5p in the breast cancer cells was significantly lower than that in the normal breast epithelial cells. The expression of miR-125a-5p was significantly higher in MDA-MB-231/miR-125a-5p cells than that in MDA-MB-231/NC cells. The ability of epithelial growth factor (EGF) at 10 μg/L to induce chemotaxis of MDA-MB-231 cells was the strongest. Compared with MDA-MB-231/NC group, stimulation of EGF decreased the invasion ability of MDA-MB-231/miR-125a-5p cells, and resulted in the increase in E-cadherin expression, while significantly decreased the protein levels of vimentin and p-GSK-3β. Meanwhile, the nuclear localization of Snail was significantly inhibited. The invasion capacity of MDA-MB-231/miR-125a-5p+GAB2 cells was significantly enhanced compared with MDA-MB-231/miR-125a-5p+Con cells, the expression of E-cadherin was decreased, and the protein levels of vimentin and p-GSK-3β were significantly increased, while the nuclear localization of Snail was promoted. CONCLUSION: miR-125a-5p suppresses EMT via GSK-3β/Snail signaling pathway, thus inhibiting the invasion ability of breast cancer cells.     

MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

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.     

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.     

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.     

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.     

Expression and significance of miR-193b in cervical cancer

AIM: To investigate the expression of microRNA-193b(miR-193b) in the cervical tissues, and further to explore the effect of silencing miR-193b on diamminedichloroplatinum(DDP)-treated HeLa cell viability. METHODS: The expression levels of miR-193b in different cervical tissues were examined by qPCR. After transfection of miR-193b-inhibitor, the cell migration was determined by Transwell assay, the sensitivity of HeLa cells to DDP was measured by MTT assay, the protein levels of phosphate and tension homology deleted on chromsome ten(PTEN), protein kinase B(Akt), p-Akt and p-glycoprotein(P-gp) were determined by Western blot. RESULTS: The mRNA level of miR-193b was significantly increased in the cervical cancer tissues compared with normal cervical tissues(P<0.05). Knockdown of miR-193b obviously inhibited migration and enhanced sensitivity to DDP of HeLa cells(P<0.05). Additionally, after transfection of miR-193b-inhibitor, the expression of PTEN was increased, whereas the protein levels of p-Akt and P-gp were decreased(P<0.05). CONCLUSION: miR-193b is highly expressed in the cervical cancer tissues. Inhibition of miR-193b augments the sensitivity to DDP of HeLa cells, at least in part, through PTEN-PI3K/Akt signaling pathway.     

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.     

3-MA combined with TSA inhibits growth of triple-negative breast cancer cells

AIM: To investigate the effect of 3-methyladenine (3-MA) combined with trichostatin A (TSA) on triple-negative breast cancer cells and the mechanism involved. METHODS: The viability of MDA-MB-231 cells was detected by MTT assay, the migration ability was determined by scratch assay, and the expression of autophagy-related proteins was detected by Western blot. RESULTS: TSA significantly inhibited the viability of MDA-MB-231 cells in a time-and dose-dependent manner. The results of scratch assay showed that TSA inhibited cell migration ability. Western blot data indicated that TSA resulted in a moderate increase in LC3-Ⅱ expression. Moreover, 3-MA inhibited cell autophagy induced by TSA, and combination of 3-MA and TSA further inhibited the viability of MDA-MB-231 cells. CONCLUSION: Combination of 3-MA and TSA may effectively inhibit the growth of triple-negative breast cancer cells.     

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.     

Effect of dominant negative IκBα transfection on NF-κB pathway and cell motility in breast cancer

AIM: To observe the effect on NF-κB pathway and cell motility in breast cancer cell lines after transfection of dominant negative IκBα plasmid.METHODS: After stable transfection of mutant IκBα plasmid into highly metastatic breast cancer lines MDA-MB-231 and MDA-MB-435, we detected NF-κB binding activity by EMSA, cell growth ability by cell growth curve, colony forming test, and cell motility by millicell-PCF chamber.RESULTS: Constitutive activities in MDA-MB-231 and MDA-MB-435 were observed. Stable transfection of a dominant negative ⅠκBα resulted in downregulation of NF-κB binding activity, thus inhibited cell mobility without significant effect on cell growth.CONCLUSION: Cell migration ability is inhibited in highly invasive breast cancer cells by inhibition of NF-κB pathway in vitro.     

Construction of recombination vector of pcDNA3.1(+)-MER-Syk（L）and the effect of Syk(L) on the proliferation of breast cancer cells

AIM: To explore the effect of 4-hydroxytomacifen (4-OHT) on MER-Syk(L) cellular localization and the function of Syk(L) on cell proliferation in breast cancer cells.METHODS: pcDNA3.1(+)-MER-Syk(L) vector was constructed and the cell line MDA-MB-231, which expressed MER-Syk(L) stably, was established. Western blotting and immunofluorescence techniques were used to detect localization of MER-Syk(L) fusion protein in MDA-MB-231 cells cultured with or without 4-OHT. MTT assay was used to explore the proliferation ability of MDA-MB-231 stable cells.RESULTS: (1) MER-Syk(L) fusion protein, not MER-Syk(S) and MER protein, translocated from cytoplasm to nucleus in the presence of 4-OHT. (2) Nuclear not cytoplasmic MER-Syk(L) fusion protein inhibited MDA-MB-231 stable cell growth. (3) With or without the treatment of 4-OHT, MER-Syk（S）and MER protein always located in cytoplasm and did not suppress cell growth.CONCLUSION: With 4-OHT, MER-Syk(L) fusion protein translocates to nucleus and inhibits cell growth.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

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.     

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.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.