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.     

Notch-1 gene silencing promotes phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells

AIM: To investigate the effect of Notch1 gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells.METHODS: shRNA-Notch1 eukaryotic expression plasmid was constructed and transfected into MCF-7 cells. The expression of Notch1 and Hes-1 was observed by Western blotting after transfction. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blotting was also used to determine the protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 after Notch1 silencing was performed in MCF-7 cells.RESULTS: Silencing of Notch1 significantly reduced the expression of Notch1 and Hes-1 in MCF-7 cells (P<0.01). In shNotch1 group, the number of apoptotic cells was much higher (P<0.01) and mitochondrial membrane potential was much lower (P<0.05) than those in shControl group. The protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 increased obviously after silencing of Notch1 was performed in MCF-7 cells (P<0.05).CONCLUSION: Notch1 silencing induces apoptosis of human breast cancer MCF-7 cells through promoting phosphorylations of JNK1 and p53, and increasing the production of PUMA, NOXA and cleaved caspase-3.     

Effects of the grub extract on apoptosis of MCF-7 human breast cancer cell line

AIM: To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS: MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope, light microscope, AO/EB double fluorescent staining under fluorescent microscope. FCM was used to assay the change of apoptotic rate. The expression of Bcl-2, Fas, caspase-9, caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored.RESULTS: (1) The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners. The inhibitory rate in exposure group was significantly different from that in control group (P<0.01). (2) Morphological changes of apoptosis including nuclear condensation, fragment and apoptosis body formation were observed by invert microscope. (3) The MCF-7 human breast cancer cells in experimental group by HE staining showed nuclear condensation and blue-black, cytoplasm slight red, nuclear chromatin condensation and fragment shape, apoptosis body formations. (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope. (5) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. (6) The expression of Bcl-2 was down-regulated, while that of Fas, caspase-3, caspase-9 was up-regulated, compared with control group (P<0.01).CONCLUSION: (1) The proliferation of MCF-7 human breast cancer cell line can be inhibited significantly by the grub extract in vitro. (2)The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be mediated by down-regulation of Bcl-2 and up-regulation of Fas, caspase-3, caspase-9. This type of apoptosis starting and performing is through death receptor pathway and mitochondrial 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.     

Pulsatilla saponin A affects proliferation and radiosensitivity of breast cancer cells by regulating miR-24-3p/RNF2 expression

AIMTo investigate the effect of Pulsatilla saponin A on proliferation and radiosensitivity of breast cancer cells and its mechanism. METHODSHuman breast cancer MCF-7 cells were treated with Pulsatilla saponin A at concentrations of 0, 5, 10, 15 and 20 mg/L and transfected with microRNA-24-3p (miR-24-3p) over-expression vector or inhibitory expression vector. The proliferation and radiosensitivity of the MCF-7 cells were measured by MTT assay and colony formation assay. The miR-24-3p expression and ring finger protein 2 （RNF2） mRNA level were detected by RT-qPCR. The protein expression of RNF2 was determined by Western blot. The luciferase reporter assay was used to detect the targeting relationship between miR-24-3p and RNF2. RESULTSCompared with control group (0 mg/L), the proliferation inhibitory rate of the MCF-7 cells was significantly increased in 5, 10, 15 and 20 mg/L Pulsatilla saponin A groups (P<0.05). The survival score of the MCF-7 cells treated with Pulsatilla saponin A was significantly decreased after irradiation, and the expression of RNF2 was significantly decreased (P<0.05). miR-24-3p targeted RNF2 and negatively regulated its expression. When the MCF-7 cells were simultaneously treated with Pulsatilla saponin A and miR-24-3p, the cell survival curve significantly shifted down. Inhibition of miR-24-3p expression reversed the proliferation-inhibiting and radiation-sensitizing effects of Pulsatilla saponin A on the MCF-7 cells. CONCLUSION Pulsatilla saponin A may affect the proliferation and radiosensitivity of breast cancer cells through miR-24-3p/RNF2 signaling pathway.     

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.     

Effects of p21-activated protein kinase 2 down-regulation on proliferation and apoptosis of human breast cancer cells

AIM：To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interference on the proliferation and apoptosis of human breast cancer cells. METHODS：The short hairpin RNA (shRNA) targeting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection. The knockdown efficiency was assessed by Western blotting. The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays. The cell apoptosis induced by staurosporine was detected by flow cytometry. RESULTS：The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells (P<0.01). Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01). Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION：Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemotherapeutic drug-induced cell apoptosis, suggesting that PAK2 might be a new therapeutic target in breast cancer treatment.     

Effects of WNT5B over-expression on viability and apoptosis of human breast cancer cell line MCF-7

AIM: To detect the expression of WNT5B in normal breast epithelial cells and different breast cancer cell lines, and to investigate the effects of WNT5B over-expression on the viability and apoptosis of human breast cell line MCF-7.METHODS: The mRNA expression of WNT5B was detected by RT-PCR in different breast cancer cells. MCF-7 cells were transfected with plasmid pcDNA3.1/WNT5B or pcDNA3.1, and the expression of WNT5B at mRNA and protein levels was examined in the 2 groups by real-time PCR and Western blotting, respectively. Subsequently, the changes of cell viability and cell apoptosis were analyzed by CCK-8 assay and flow cytometry, respectively. RESULTS: The expression of WNT5B in the breast cancer cell lines was lower than that in MCF10A cells. The WNT5B expression in the MCF-7 cells in experimental group was significantly higher than that in vector group (P<0.05). However, the cell viability in experimental group decreased significantly as compared with vector group (P<0.05). The number of the cells in S-phase obviously increased, while the percentage of the cells in G₁-phase and G₂/M-phase decreased compared with vector group. The number of apoptotic cells in WNT5B group was significantly higher than that in vector group.CONCLUSION: The expression of WNT5B is decreased in breast cancer cells. WNT5B over-expression significantly inhibits the cell growth and promotes the cell apoptosis in breast cancer MCF7 cells.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

iASPP on apoptosis in breast cancer cells which expressed wild type p53

AIM: To investigate the RNAi effect of the inhibitory member of the ASPP family (iASPP) on the apoptosis of human breast cancer cell MCF-7 which expressed the wild type p53 gene. METHODS: The recombinant plasmid pAd-iASPP-RNAi was transfected into MCF-7 cells. The expression of iASPP mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell apoptosis was detected by FCM, and then the MCF-7 cells were transplanted into nude mice to set up transplantation model. The expression of iASPP RNA and protein in transplanted neoplasm were determined by RT-PCR and Western blotting, the apoptosis index was detected by FCM at the same time. RESULTS: The results showed that the expression of iASPP descended in MCF-7 cells (mRNA 95.4% and protein 96.8%, respectively, P<0.01) and the apoptosis rate and necrosis rate of MCF-7 cells increased (P<0.01) after transfection. As treated with pAd-iASPP-RNAi, the expression of iASPP in transplantation tumor cells descended 87.4% (mRNA) and 89.2% (protein), respectively (P<0.01), and the apoptosis rate and necrosis rate increased accordingly (P<0.01, P<0.05). CONCLUSION: The inhibition of iASPP may resume the ability of p53 to induce apoptosis in breast cancer cells which is able to express wild type p53.     

Expression of CUG-binding protein 1 in breast cancer and its effect on cell viability and invasion ability

AIM: To investigate the expression of CUG-binding protein 1 (CUGBP1) in breast cancer tissues, and to explore the effect of CUGBP1 gene silencing on the viability and invasion ability of human breast cancer MCF-7 cells. METHODS: A total of 96 cases of patients with breast cancer undergoing surgical treatment were selected in the Second Affiliated Hospital of Zhengzhou University from March 2015 to September 2017. Immunohistochemical staining was used to detect the protein expression of CUGBP1 in the breast cancer and adjacent tissues. MCF-7 cells were cultured and divided into CUGBP1 interference sequence group, control sequence group and blank group. Western blot was used to detect the protein expression of CUGBP1, Twist, E-cadherin and vimentin in the cells. The cell viability was measured by MTT assay. The cell invasion ability was detected by Transwell assay. RESULTS: The positive expression rate of CUGBP1 protein in the breast cancer tissues was higher than that in the adjacent tissues (χ²=28.900, P<0.001). The differences of CUGBP1 protein expression in the breast cancer tissues among TNM staging, histological grading and lymph node metastasis were statistically significant (P<0.05). The relative protein expression levels of CUGBP1, Twist and vimentin in CUGBP1 interference sequence group were lower than those in control sequence group and blank group, while the relative protein expression of E-cadherin was higher than that in control sequence group and blank group (P<0.05). The cell viability at 24 h, 48 h, 72 h and 96 h in CUGBP1 interference sequence group was lower than that in control sequence group and blank group (〖P<0.05). The invasive cells in CUGBP1 interference sequence group were less than those in control sequence group and blank group (P<0.05). CONCLUSION: CUGBP1 protein is highly expressed in the breast cancer tissues. Specific silencing of 〖STBX〗CUGBP1〖STBZ〗 gene expression in breast cancer MCF-7 cells effectively inhibits the cell viability and invasiveness, and its mechanism may be related to inhibiting the process of epithelial-mesenchymal transition.     

Construction of eukaryotic expression vector miRNA let-7a1 and its effect on the proliferation of lung cancer A549 cells

AIM: To construct a recombinant eukaryotic expression vector, pSilencer 4.1-let-7a1 and to express it in lung cancer A549 cells for detecting its effect on the proliferation of A549 cells. METHODS: The pre-let-7a1 sequence was amplified by RT-PCR using RNA from human lung cancer A549 cells, and then inserted into pSilencer 4.1-CMV neo vector to generate pSilencer 4.1-let-7a1 which was transfected into lung cancer A549 cells. The expression of miRNA let-7a1 was verified by RT-PCR. Its activity in A549 cells was determined by luciferase reporter assay after cotransfection of let-7a1 target sequence-reporter gene plasmid with pMIR-report let-7a1T, which was constructed by inserting let-7a1 target sequence into the luciferase reporter 3’UTR of pMIR-report luciferase vector. The effect of pSilencer 4.1-let-7a1 transfection on A549 cell proliferation was detected by MTT method. RESULTS: The sequences of cloned pre-let-7a1 were correct. RT-PCR results indicated that pSilencer 4.1-let-7a1 was effectively expressed in the transfected A549 cells. The relative luciferase activity was decreased significantly after A549 cells were co-transfected with pSilencer 4.1-let-7a1 and pMIR-report let-7a1T, indicating that let-7a1 was expressed effectively and had biologic activity in A549 cells that were transfected with pSilencer 4.1-let-7a1. MTT results showed that miRNA let-7a1 gene overexpression in A549 inhibited cell proliferation. CONCLUSION: The eukaryotic expression vector pSilencer 4.1-let-7a1 is successfully constructed and effectively expresses in A549 cell. The overexpression of miRNAlet-7a1 gene inhibits lung cancer A549 cell proliferation.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Effect of gonadotropin-releasing hormone analogue on human breast cancer cell line in vitro

AIM: To investigate the effects of gonadotropin-releasing hormone (GnRH) analogue on the growth of breast cancer cell lines MCF-7 and MDA-MB-231 in vitro and to explore the related mechanisms with PI3K/Akt or ERK/MAPK pathways. METHODS: The proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 treatment with triptorelin was detected by MTT assay and the distribution of the cell cycle was determined by flow cytometry. The phosphorylation of the ERK1/2 and Akt was evaluated by Western blotting. RESULTS: Triptorelin inhibited the proliferation of MCF-7 cells at concentration of 10^-5 mol/L after treated for 192 h or at concentration of 10^-4 mol/L after treated for 168 h and 192 h. Triptorelin inhibited the proliferation of MDA-MB-231 cells at concentration of 10^-4 mol/L after treated for 192 h (P<0.05).Treatment with triptorelin for 192 h at concentration of 10^-4 mol/L had no statistical significance effect on phosphorylation of ERK1/2 and Akt(P>0.05).CONCLUSION: Inhibitory effect of GnRH analogue triptorelin on human breast cancer cells is not just the connection with the down-regulation of pituitary hormone, but also a direct inhibitory effect. The role may not be involved in the activation of ERK/MAPK and PI3K/Akt signaling pathways.     

Combination therapy of FK228 with rapamycin synergistically promotes human breast cancer cell apoptosis by DNA damage and cell cycle arrest

AIM: To investigate the depressant effect of FK228 combined with rapamycin on the human breast cancer cell line MCF-7 and MDA-MB-435.METHODS: FK228, a new histone deacetylase inhibitor, and rapamycin, the specific inhibitor of the mammalian target of rapamycin (mTOR) protein, were used in the study. MCF-7 cells and MDA-MB-435 cells were exposed to different concentrations of FK228 and rapamycin. The inhibitory rate of cell growth was determined by SRB assay. Combination index (CI) was used to evaluate the interaction between FK228 and rapamycin. The expression of the apoptotic proteins, cycle proteins and nucleic acid proteins were detected by Western blotting. The cell cycle was analyzed by flow cytometry.RESULTS: Both FK228 and rapamycin showed growth inhibitory effects on the breast cancer cell lines in a time-and dose-dependent manner. CI of the 2 drugs was less than 1 when the inhibitory rate of the cell growth was 50% effective dose (ED50)~ED70, indicating a synergistic effect. The combination therapy of FK228 with rapamycin increased the apoptotic proteins, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 compared with a single use of the drugs. The combination therapy of FK228 with rapamycin reduced the cycle proteins, and the cell cycle was arrested in G₂/M. The levels of phosphorylated H2AX and acetylated H3 were ob-viously increased after combination therapy.CONCLUSION: The combination therapy of FK228 with rapamycin inhibits the cell proliferation and increases apoptosis with a synergistic effect, which may become a new trend for treating endometrial 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.