ABSTRACT
In recent years, the clinical treatment of colorectal cancer(CRC) has made great progress, but chemoresistance is still one of the main reasons for reducing the survival rate of patients with colorectal cancer. Therefore, ameliorating chemotherapy resis-tance is an urgent problem to be solved. The purpose of this study was to investigate the regulatory role and related molecular mechanisms of hydroxysafflor yellow A(HSYA) in colorectal cancer cell proliferation, migration, and 5-fluorouracil(5-FU) chemoresistance. In this study, HCT116 and HT-29 cells were used as research subjects. Firstly, methyl thiazolyl tetrazolium(MTT) assay and colony formation assay were used to detect and analyze the effect of HSYA on the proliferation of CRC cells. Secondly, the effect of HSYA on the cell cycle in CRC cells was analyzed by cell cycle assay. Furthermore, the effect of HSYA on the migration of CRC cells was analyzed by wound-healing assay and Transwell assay. Based on the above, the influences of HSYA on 5-FU chemoresistance of CRC cells and related molecular mechanisms were explored and analyzed. The results showed that HSYA significantly inhibited the proliferation and migration of CRC cells, and arrested the cell cycle in G_0/G_1 phase. In addition, HSYA significantly ameliorated the chemoresistance of CRC cells to 5-FU. The results of acridine orange staining and Western blot showed that the autophagy activity of CRC cells in the HSYA and 5-FU combined treatment group was significantly higher than that in the 5-FU single drug treatment group. As compared with the 5-FU single drug treatment group, the phosphorylation levels of protein kinase B(Akt) and mammalian target of rapamycin(mTOR) in the HSYA and 5-FU combined treatment group were significantly reduced, indicating that the Akt/mTOR signaling pathway in the combined treatment group was down-regulated in CRC cells. In conclusion, HSYA may upregulate autophagy activity through the Akt/mTOR signaling pathway, thereby inhibiting the proliferation and migration of CRC cells and ameliorating the chemoresistance to 5-FU.
Subject(s)
Humans , Proto-Oncogene Proteins c-akt/metabolism , Drug Resistance, Neoplasm , Cell Line, Tumor , TOR Serine-Threonine Kinases/metabolism , Fluorouracil/pharmacology , Cell Proliferation , Autophagy , Colorectal Neoplasms/drug therapyABSTRACT
Objective@#To evaluate the effect of X-ray radiation on cell proliferation, migration, survival ability and cell cycle of human esophageal squamous cell carcinoma after RNA interference-mediated down-regulation of HMGB1 gene expression.@*Methods@#The expression of HMGB1 at mRNA and protein levels in the human esophageal squamous cell carcinoma cell lines ECA109 and KYSE30 was determined using RT-PCR and Western blot assays. MTS and Transwell assays were employed to examine the proliferation and migration of ECA109 and KYSE30 cell lines. The cellular survival ability in vitro was assessed by clone formation assay. The cell cycle after X-ray radiation in different groups was detected by flow cytometry.@*Results@#The expression of HMGB1 at mRNA and protein levels in ECA109 and KYSE30 cells were markedly higher in a dose-dependent and time-dependent manner in the radiation group than that in the control group (all P<0.05). MTS results demonstrated that the proliferation of ECA109 and KYSE30 cells was obviously lower at each time point after radiation than that in the group without radiation (all P<0.01). The expression of HMGB1 at mRNA and protein levels was significantly inhibited in the HMGB1 siRNA group than those in the control and NC groups (both P<0.01). The data from the clone formation assay revealed that the radiosensitivity was significantly increased after down-regulation of HMGB1 expression (P<0.01). Transwell migration assay revealed that the number of migrating cells at the fourth hour after X-ray irradiation in the HMGB1 siRNA group was significantly lower than those in the control and negative groups (both P<0.01). In the HMGB1 siRNA group, the percentage of cells at G0/G1 phase was obviously higher, whereas the percentage of S phase was significantly lower than those in the control and NC groups, and the trend was even more significant after X-ray radiation (all P<0.01).@*Conclusion@#Inhibition of HMGB1 expression by siRNA can suppress the proliferation and migration of ECA109 and KYSE30 cells and enhance the radiosensitivity by increasing the cell cycle arrest at G0/G1 stage after X-ray irradiation in vitro.
ABSTRACT
@# Objective:To explore the prognostic significance of thrombospondin 2 (THBS2) expression and its effects on the proliferation and migration of pancreatic cancerASPC-1 cells for patients with pancreatic cancer, and to investigate its possible molecular mechanism. Methods: The expression of THBS2 in pancreatic cancer tissues and its effects on overall survival rate in patients were analyzed by online database. THBS2 expression in pancreatic cancerASPC-1 cells was detected by Western Blotting; RNAinterference was used to knockdown the expression of THBS2 inASPC-1 cells, and then the effects of THBS2 knockdown on cell proliferation and migration were detected by MTT and Transwell assays, while its effects on protein expression levels (MMP, E-cadherin,AKT and PI3K) were detected by Wb. Results: Expression of THBS2 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues (P<0.01), and the high expression of THBS2 could lead to the decrease of overall survival rate in pancreatic cancer patients. The expression of THBS2 in pancreatic cancer cell lines was significantly up-regulated; however, after interference on the expression of THBS2, the proliferation (P<0.01) and migration ability (P<0.01) of ASPC-1 cells were significantly decreased, and the expression of AKT and PI3K in cells was significantly down-regulated (P<0.01). Conclusion: THBS2 is highly expressed in pancreatic cancer tissues and cells, and is negatively correlated with the prognosis of patients. The mechanism is possibly related with the proliferation and migration of ASPC-1 cells that regulated byAKT/PI3K signaling pathway.
ABSTRACT
Objective To investigate the effects of semaphorin 4D(Sema4D) on the proliferation,migration and angiogenic of human pancreatic carcinoma cells.Methods Sema4D-siRNA was designed and synthesized and transfected into human pancreatic carcinoma cells.After 48 hours of transient infection,the changes of expression of Sema4D mRNA before and after transfection were detected by reverse transcription-polymeruse chain reaction method.And after 72 hours of transient infection,the changes of expression of Sema4D protein before and after transfection were detected by Western blot method.The changes of growth of the transfected cells were observed by methyl thiazolyl terazolium assay.Using transwell migration test and scratch repair test to detect the changes of migration ability of human pancreatic carcinoma cells after transfection.Using tubule formation assay to observe the effect of supernatant of pancreatic carcinoma cell cultures on angiogenesis after transfection.Results Compared with the negative control group and blank control group,the expression of Sema4D mRNA and Sema4D protein and the growth rate of pancreatic carcinoma cells decreased significantly (P ＜ 0.05).In transwell migration test and scratch repair test,it was observed that Pancreatic cancer cells penetrating cell number and scratch repair rate were significantly lower than that in negative control group and blank control group (P ＜ 0.05).Tubule formation assay showed that there were significant differences in angiogenesis numbers among siRNA transfection group(0.5 ± 0.02),negative control group(1.45 ± 0.60) and blank control group (1.37 ± 0.52) (P ＜ 0.05).Conclusion Sema4D-siRNA can induce RNA interference in pancreatic carcinoma cells and down-regulate the expression of Sema4D gene,which can inhibit the proliferation of pancreatic carcinoma cells,significantly reduce the migration ability of pancreatic carcinoma ceils and inhibit angiogenesis.
ABSTRACT
Objective To preliminarily explore the effects of human microRNA-181a on migration of gastric cancer cells and its mechanism.Methods The expression of miRNA-181a-5p in gastric cancer cell line GC9811 and peritoneal high metastasis gastric cancer cell line GC9811-P were tested by quantitative real-time polymerase chain reaction (qRT-PCR ). GC9811 cell line was transfected byendogenous synthetic analog mimic and its homologous negative control of miRNA-181a-5p,which were considered as up-regulated group and its control group respectively;GC9811-P were transfected by miRNA inhibitor and its homologous negative control of miRNA-181a-5p,which were considered as down-regulated group and its control group respectively.After miRNA-181a-5p was up or down-regulated,cell proliferation,migration and apoptosis capabilities of gastric cells were detected by matrix thiazolyl tetrazollium (MTT)assay,cloning-forming assay,Transwell migration test,wound healing assays and apoptosis test.After miRNA-181a-5p was up or down regulated,the changes of matrix metalloproteinase (MMP)14 expression were determined by Western blot.Independent sample t test was performed for mean comparison between samples and chi square test was used for rate comparison.Results The results of qRT-PCR showed the relative expression quantity of miRNA-181a-5p in GC9811 was 1 .000 00 ± 0.021 26 and in GC9811-P was 3.175 61 ±0.106 76,and the difference was statistically significant (t =34.620,P <0.01 ).The results of MTT assay indicated that the cell proliferation rate of up-regulated group was higher than that of up-regulated control group,and that of down-regulated group was lower than down-regulated control group.The cloning-forming assay demonstrated that the number of clone forming and clone forming rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 234.00±10.12 and 46.8%,93.00±9.61 and 18.6%,51 .00 ±7.96 and 10.2%,99.00±8.05 and 19.8%,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t = 17.500,7.344,χ2 = 12.27, 9.51 ,all P <0.01).The results of Transwell migration experiment showed the number of cells migrated through membrane hole of up-regulated group was 164.00±19.31 ,and which was higher than that of up-regulated control group (87.00±23.04,t=4.436,P <0.05);that of down-regulated group was 157.00± 11 .50,and which was lower than that of down-regulated control group (234.00 ±12.12,t =7.982,P <0.05).The result of wound healing assays indicated that the rate of migration distance of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 2.09 ± 0.18,1 .27 ±0.23,1 .15 ±0.15 and 1 .67 ±0.12,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t =4.863 and 4.689, both P <0.05).The results of apoptosis experiments demonstrated that the apoptosis rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were (6.10± 1 .02 )%,(9.10 ± 2.13 )%,(12.70 ± 1 .23 )%,(8.70 ± 2.54 )%,respectively,and there was no statistically significant difference between up-regulated group,down-regulated group and their control group (both P *0.05).The results of Western blot showed that the grey value of up-regulated group was 561 .881 ±35 .740,which was higher than that of up-regulated control group (275 .784±23.520);that of down-regulated group was 579.565 ±37.950,which was lower than that of down-regulated control group (1 312.760±51 .270),and the differnces were statistically significant (t =11 .580 and 19.910,both P <0.01).Conclusion miRNA-181a-5p highly expresses in peritoneal high metastasis gastric cancer cell line GC9811-P and promoted the proliferation and migration of gastric cancer cell line GC9811 and GC9811-P with a tendency to suppress apoptosis.
ABSTRACT
BACKGROUND: We investigate the influence of cell surface adhesion receptor integrin alphavbeta3, alpha5beta1 contributes to proliferation and migration of tumor cell in osteosarcoma for carves out a new treatment model by regulation of integrin roles in human osteosarcoma. MATERIALS AND METHODS: We performed proliferation assay, total 11 cell lines including 7 osteosarcoma cell lines established from patients and 4 osteosarcoma standard cell lines. Murine monoclonal anti-alpha5beta1 and anti-alphavbeta3 (Chemicon International Inc. Temecula, CA) were used for growth inhibition assays. We also performed cell motility assay by using the Boyden chamber to evaluate the effect of integrin mediated cell migration. We used the HOS standard osteosarcoma cell lines and each separates contained serum free media with mouse IgG1 negative control antibody, anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. RESULTS: Proliferation of cells decreased significantly in 10 out of 11 cell lines when blocking with alphavbeta3 or alpha5beta1 respectively. Blocking with anti-alphavbeta3 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 7 cell lines showed significantly more decrease of proliferation with anti-alphavbeta3 antibody than with anti-alpha5beta1antibody. Blocking with anti-alpha5beta1 antibody decreased significantly tumor cell proliferation in 10 cell lines. Among the 10 cell lines, 3 cell lines showed significantly more decrease of proliferation with anti-alpha5beta1 antibody than with anti-alphavbeta3 antibody. Including statistically not significant 2 cell lines the growth inhibition of osteosarcoma cell lines was more obvious (10 out of 11) in blocking with anti-alphavbeta3 antibody. The migration of cells was significantly decreased when blocked with anti-alpha5beta1 antibody and anti-alphavbeta3 antibody. CONCLUSION: Under the based on the integrin alphavbeta3, alpha5 beta1 are central role on proliferation and migration of osteosarcoma cells, we could be more approach to new therapeutic endeavors with antibody to integrin alphavbeta3, alpha5beta1 molecular target of osteosarcoma.