Inhibitory effects of triptolide on cell proliferation and metastasis in Raji cells in vitro

AIM:To investigate the inhibitory effects of triptolide on cell proliferation and metastasis in Burkitts lymphoma cell line Raji cells.METHODS:The effects of triptolide on the growth of Raji cells were studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay. The effects of triptolide on the cell apoptosis of Raji cells were detected by using Annexin Ⅴ/PI double-labled cytometry. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α (rhSDF-1α）in vitro.RESULTS:Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24 h IC50 value of 43.06 nmol/L and a 36 h IC50 value of 25.08 nmol/L. Following the treatment of triptolide, the cell apoptosis rate was increased as the treatment concentration increased and the culture time extended. The effects were dose- and time- dependent. Triptolide could downregulate the expression of CXCR4 on Raji cells in a dose-dependent manner. Moreover, chemotaxis assay suggested that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent.CONCLUSION:Triptolide could inhibit the cell proliferation and induce the cell apoptosis of Raji cells. Furthermore， it could block the cell metastasis of Raji cells in vitro and the underlying mechanism might be related to the inhibition of the SDF-1/CXCR4 axis.     

Tetrandrine enhances radiosensitivity of human nasophayrngeal carcinoma cell lines

AIM: To study whether tetrandrine (Tet) enhances the radiosensitivity of human nasopharyngeal carcinoma cell lines in vitro and its mechanism.METHODS: The inhibitory effect on proliferation was evaluated by MTT assay. The radiosensitivity of the cells was compared by colony formation assay. The cell cycle was analyzed by flow cytometry. RESULTS: The maximum non-cytotoxic doses of Tet for CNE1 and CNE2 cells were 1.5 and 1.8 μmol/L, respectively. Compared with radiation group, the cell proliferation in Tet plus radiation group was significantly inhibited on the 4th to 6th days (P<0.01). The mean lethal doses for CNE1 and CNE2 cells in radiation group were (1.26±0.02) Gy and (2.27±0.04) Gy, respectively,and the values changed to (0.73±0.05) Gy and (1.61±0.08) Gy in Tet plus radiation group, respectively, resulting in the sensitivity enhancement ratio of 1.73 and 1.40, respectively (P<0.05). The CNE1 and CNE2 cells in G₂ phase of the cell cycle in radiation group were (42.62±2.07)% and (34.82±2.74)%, respectively, while those in Tet plus radiation group were (17.02±1.87)% and (19.64±4.82)%, respectively. CONCLUSION: Tetrandrine enhances the radiosensitivity of human nasopharyngeal carcinoma cell lines and the mechanism may be related to the abrogation of radiation-induced G₂ phase arrest.     

and laminin on adhesion and proliferation of human hepatocellular carcinoma cell

AIM:To explore the effects of PMA(phorbol-12-myristate-13-acetate, a tumor promoter, mimicking the action of diacylglycerol on PKC)and laminin on the adhesion and the proliferation of human hepatocellular carcinoma cells, and provide a new clue to liver cancer treatment.METHODS:Human hepatocellular carcinoma cell line(BEL-7402)was used to identify the endogenous laminin and protein kinase C-α(PKC-α) expression, and the effects of laminin and PMA on the adhesion and the proliferation were also investigatedin vitro.RESULTS:By the effect of exogenous laminin, human hepatocellular carcinoma cell (BEL-7402) possessed endogenous laminin expression and increased the adhesion and the proliferation, which was showed the synergistic action by the effect of PMA in combination. By the action of PMA alone, the proliferation and the PKC-α expression increased by exogenous laminin were decreased, and the adhesion and the endogenous laminin expression were increased.CONCLUSIONS:The finding suggested that the adhesion and the proliferation of human hepatocellular carcinoma cell were closely related to the effects of endogenous or exogenous laminin, which were associated with cPKC-α activity. Therefore, the application of anti-laminin antibody in combination with PKC antagonist might be a new clue to find out the therapy for liver cancer.     

Expression of human leukocyte antigen E in hepatocellular carcinoma cell lines

AIM: To investigate the human leukcyte antigen E (HLA-E) expression in hepatocellular carcinoma cell lines. METHODS: The techniques of real-time PCR and Western blotting were used to study the HLA-E expression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real-time PCR showed that no statistical difference of HLA-E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA-E mRNA expression in Hep3B2.1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA-E protein levels between L02 cells and the 5 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2.1-7), and no HLA-E protein in Hep3B2.1-7 cells was detectable. CONCLUSION: Asynchronization of HLA-E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.     

Inhibitory effects of nm23-H1 gene on proliferation and invasion of A549 cell line

AIM:To study the inhibitory effects of nm23-H1 gene on proliferation and invasion of human lung adenocarcinoma A549 cell line. METHODS:Recombinant eukaryotic expression vector pcDNA3.1-nm23-H1 containing full length of human nm23-H1 cDNA was constructed and transfected into a human lung adenocarcinoma A549 cell line by lipofectamine. Cell strain that expressed nm23-H1 stably was screened out by G418 and named pcDNA-nm23-A549. Expression of nm23-H1 was identified by RT-PCR and immunohistochemistry. Growth curves were drawn to detect the inhibitory effects on cell proliferation. Cell cycle of pcDNA-nm23-A549 was examined by flow cytometry. Atomic force microscopy was used to observe the filopodia on the surface of the cells. RESULTS:Introduction of nm23-H1 obviously inhibited the proliferation of A549. Expression of nm23-H1 did not induce apotosis in A549 cells but increased the percentage of phase G₁ cells and decreased phase S cells. Meanwhile, phase G₁ to phase S transition was restrained. Filopodia in the cell surface was much fewer and its structure changed in cells transformed. CONCLUSION:nm23-H1 is capable of inhibiting A549 proliferation and decreasing its metastatic ability, probably by interfering with cell cycle and cell surface structure.     

Association of miR-497 and prognosis in patients with renal cell carcinoma and its effects on proliferation,apoptosis and invasion of human renal cell carcinoma cell line 786-0

ATM: To investigate the association of microRNA-497 (miR-497) and prognosis in the patients with renal cell carcinoma (RCC) and its effects on the proliferation, apoptosis and invasion of human RCC cell line 786-0. METHODS: Paired RCC and adjacent non-tumor tissue specimens were surgically collected from 80 patients who were diagnosed with primary RCC between 2011 and 2015. The expression of miR-497 in the paired RCC and adjacent non-tumor tissue specimens was detected by real-time PCR. Recurrence-free survival was estimated using the Kaplan-Meier method and compared using the log-rank test. The 786-0 cells were transfected with miR-497 mimics or scramble control miRNA. The proliferation, apoptosis and invasion abilities of the transfected cells were assessed by MTT assay, Trypan blue exclusion, flow cytometry and Transwell chamber experiment. The protein expression of miR-497-targeted gene cyclin D1 in the transfected cells was quantified by Western blotting. RESULTS: miR-497 was down-regulated in the RCC specimens compared with the adjacent tissues. miR-497 was down-regulated in the RCC 786-0 cells compared with the HK-2 cells. By the end of the study, 74 cases were followed up. The follow-up rate was 92.5%. Median follow-up was 29 months (ranging from 2 months to 48 months). The 3-year recurrence-free survival rates of the patients with high and low miR-497 expression were 71.2% and 40.1%, respectively. Over-expression of miR-497 resulted in significant suppression effect on RCC cell proliferation, invasion and the expression of cyclin D1. CONCLUSION: Low expression of miR-497 was correlated with poor prognosis in the RCC patients. miR-497 inhibits proliferation and invasion of RCC 786-0 cells and its mechanism is associated with the down-regulation of cyclin D1.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

Inhibitory effect of ampelopsin combined with mitomycin on proliferation of gastric carcinoma cells

AIM: To investigate the synergistic effects of ampelopsin (AMP) and a chemotherapeutic drug mitomycin (MMC) on the proliferation of gastric cancer cell line SGC-7901.METHODS: SGC-7901 cells were cultured in vitro and divided into 4 groups: control group, AMP group, MMC group and AMP+MMC group. Cell proliferation was measured by MTT method. The apoptotic index was examined by flow cytometry. The expression of apoptotic proteins, Bcl-2 and survivin, was detected by Western blotting.RESULTS: AMP at the concentrations ranging from 2.2 mg/L to 14.84 mg/L exerted inhibitory effect on the growth of SGC-7901 cells. Cell proliferation in AMP (14.84 mg/L) group was inhibitory by (60.85±1.13) %, significantly higher than that in control group (P<0.05). The inhibitory rates of cell proliferation varied from (17.40±0.30) % to (72.23±1.36) % when the concentrations of MMC increased from 1×10^-3 g/L to 1×10^-2 g/L. AMP combined with MMC showed a synergistic effect on the growth of SGC-7901 cells. The inhibitory rates of cell proliferation varied from (21.83±2.50) % to (46.70±1.45) % when the concentrations of MMC increased from 1×10^-3g/L to 5×10^-3g/L. The inhibitory effect of AMP plus MMC was higher than that of MMC or AMP alone. The protein levels of Bcl-2 and survivin were inhibited in AMP group, MMC group and AMP+MMC group, and were significantly lower in AMP+MMC group than those in AMP group or MMC group.CONCLUSION: AMP enhances the inhibitory effect of MMC on the growth of SGC-7901 cells. The mechanism is related to the inhibition of apoptotic protein expression.     

Activated NK cells against human hepatocellular carcinoma cell lines in vitro and in vivo

AIM: To evaluate the role of natural killer(NK) cells against a variety of human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo, and to investigate the expression of MHC class I chain-related protein (MIC protein) in these HCC cell lines. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 50 mL peripheral blood donored by a healthy volunteer and cultured in the NK cell kit followed by amplification and cytokine activation. The release of lactate dehydrogenase (LDH) was detected by lysis experiment. Animal experiments were performed following vaccination with HCC cell lines (BEL7402, HepG2 and SMMC7721) in BALB/c-nu/nu mice. The diameters of the tumors were measured and the growth curves of difference cell lines were delineated. Three weeks later, these mice were sacrificed and the weight of the transplanted tumors was also detected. Furthermore, the expression of MIC protein was determined. RESULTS: The obvious differences of lysis effects of NK cells on different cell lines were observed, in which the lysis effect of NK cells on K562 cells was the strongest, the effect on BEL7402 cells was in the middle and the effect on SMMC7721 cells was the weakest. In animal experiments, NK cells also showed obvious and different restraining effects on a variety of HCC cell line-transplanted tumors in nude mice. The tumor inhibitory rates of NK cells were 43.5% in BEL-7402 cell-transplanted tumor, 40.7% in HepG2 cell-transplanted tumor and 36.0% in SMMC-7721 cell-transplanted tumor. The protein expression of MIC was 48.7%, 32.8% and 0.9% in the 3 cell lines,respectively. CONCLUSION: The lysis effects of NK cells on a variety of human HCC cell lines are different. The expression of MIC in the tumor cells may play distinct role in evaluating these differences.     

Effects of miRNA-25 on proliferation of human esophageal squamous-cell carcinoma cell line TE1

AIM: To investigate the effects and mechanisms of microRNA-25(miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1. METHODS: The abundance of miRNA-25 in different tissues was measured by RT-PCR. After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2(CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respectively. RESULTS: miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05). Over-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the entrance of the cells into S phase (P<0.05). Inverse results were obtained after down-regulation of miRNA-25(P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-expression of miRNA-25, but decreased after down-regulation of miRNA-25(P<0.05). CONCLUSION: miRNA-25 enhances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for treating esophageal squamous-cell carcinoma.     

Inhibitory effect of extract of Oratosquilla on migration and vasculogenic mimicry in human nasopharyngeal carcinoma cell line in vitro

AIM: To study the inhibitory effect of the extract of Oratosquilla (EOS) on the migration and vasculogenic mimicry in human poorly differentiated nasopharyngeal carcinoma cell line CNE-2.METHODS: CNE-2 cells were cultured in the medium with different concentrations of EOS (0 mg/L, 125 mg/L, 250 mg/L and 500 mg/L). The migration of CNE-2 cells and the formation of tube-like structures (TLSs) by CNE-2 cells were determined with wound healing assay and in vitro anti-angiogenesis test, respectively. The formation of TLSs by CNE-2 cells and their structural characteristics were observed by anti-angiogenesis test on the Matrigel. The protein expression of fascin 1 and vascular endothelial growth factor (VEGF) was detected by Western blotting.RESULTS: Compared with control group, EOS significantly decreased the migration velocity of CNE-2 cells in a dose-dependent manner. CNE-2 cells formed TLSs on the Matrigel, and the formation of TLSs by CNE-2 cells was inhibited by EOS in a dose-dependent manner. The expression of fascin 1 and VEGF in CNE-2 cells was also decreased after treatment with EOS. A positive correlation between the expression of fascin 1/VEGF and the formation of TLSs by CNE-2 cells was observed.CONCLUSION: CNE-2 cells form TLSs on the Matrigel, and EOS inhibits the migration and vasculogenic mimicry of CNE-2 cells, which are related with down-regulating the expression of fascin 1 and VEGF in CNE-2 cells.     

Effect of PTEN gene overexpression on cell cycle and tumor invasion in a human ovarian carcinoma SKOV3 cell line

AIM:To investigate the role of phosphatase and tensin homology deleted on chromosome(PTEN) gene in the cell cycle and invasion ability of human ovarian carcinoma SKOV3 cell line in vitro. METHODS:Human ovarian carcinoma SKOV3 cells were transfected with a eukaryotic expression plasmid vector containing PTEN gene in vitro,and then the positive cell clones were selected and amplified. MTT method was used to observe the inhibitory rate,flow cytometry was used to detect the cycle of transfected PTEN cells and apoptosis level. Western blotting analysis was used to determine PTEN gene expression. The invasiveness of transfected cells were measured quantitatively by Matrigel invasion assays (Transwell chamber). RESULTS:The expression of PTEN mRNA in SKOV3 cells increased after transfection with PTEN gene. Flow cytometry showed that the percentage of cells in S phase increased,but that in G₂/M phase decreased. Invasiveness of SKOV3 was significantly decreased. CONCLUSION:The transfection of PTEN gene into SKOV3 cells can inhibit human ovarian carcinoma SKOV3 cell proliferation,invasion and induce SKOV3 cell apoptosis.     

Effects of human recombinant acidic fibroblast growth factor on cell proliferation and apoptosis in NIH3T3 cells

AIM:Through studying the differences betwe en wild-type acidic fibroblast growth factor (waFGF) with recombinant aFGF (raFG F ),the biological effect of raFGF concerned with mitogenic activity was evaluate d.METHODS:NIH3T3 cell line was used.Cell proliferation with MTT method was used to study the mitogenic activity.Flow cytometry was also used fo r detection of apoptosis,cell membrane permeability and mitochondria potential.The role of heparin sulfate (HS) on aFGF biological effect was studied at the s ame time in this research.RESULTS:The enhancement of raFGF on cell proliferation was sign ificantly lower than that of waFGF.The restriction of raFGF on apoptosis and th e enhancement of it on cell membrane permeability were all lower than those of w aFGF significantly.The enhancement of raFGF on mitochondria potential was lower than that of waFGF significantly.The HS improved the biological effect of aFGF .CONCLUSION:The mitogenic activity of raFGF is lower than that of waFGF and raFGF has little effect on apoptosis.     

Overexpression of CLN3 gene in human ovarian carcinoma

AIM: To determine whether overexpression of CLN3 is involved in the tumorigenesis of ovarian cacinoma. METHODS: 10 specimens of ovarian carcinoma were screened for CLN3 gene expression level by semiquantitative RT-PCR and Western blotting. RESULTS: Overexpression of CLN3 mRNA and protein was found in all ovarian tumor tissues as compared with normal tissues. CONCLUSION: The overexpression of CLN3 may contribute to tumor development of ovarian cancer.     

Effect of quercetin on proliferation of human ovarian cancer SKOV-3 cells

AIM：To study the inhibitory effect of quercetin on the proliferation and apoptosis of human ovarian cancer SKOV-3 cells. METHODS：SKOV-3 cells were treated with different doses of quercetin. The inhibitory effect of quercetin on the proliferation was detected by MTT assay. The cell apoptosis was determined by immunocytochemistry. The cell cycle and apoptosis were assessed by flow cytometry. RESULTS：Quercetin inhibited the proliferation of ovarian cancer SKOV-3 cells in a time- and dose-dependent manner. Quercetin induced apoptosis of SKOV-3 cells. The proportion of S phase and the apoptotic rate were significantly increased, and the proportion of G2/M phase was reduced after treatment with quercetin. CONCLUSION：Quercetin inhibits the growth of ovarian cancer SKOV-3 cells in vitro, and promotes apoptosis through S phase arrest.     

Inhibition of human hepatocellular carcinoma cell proliferation by galactose receptor mediated c-myc antisense oligonucleotide

AIM: To evaluate the inhibitory effect of galactose (Gal)-polyethyleneimine (PEI)-c-myc antisense oligodeoxynucleotide (ASODN) complex on proliferation of human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cell line Bel-7402 was treated with Gal-PEI-ASODN complex. Cell proliferation was tested by trypan blue dye at different time points and with various concentrations of ASODN treatment. Cell morphology was observed under inverted microscope, cell hypodiploid percentage was analyzed by flow cytometry and cell ultrastructure was observed through electron microscopy. RESULTS: Compared with ASODN group (20 μmol/L) from 0 h to 96 h, Gal-PEI-ASODN complex (with ASODN 0.75 μmol/L) significantly suppressed Bel-7402 cells proliferation, the ASODN concentration within Gal-PEI-ASODN complex and time course acquired were significantly lower and shorter, respectively. Incubated with pure ASODN at different concentrations for 72 hours, cell proliferation was inhibited and IC₅₀ was 20.9 μmol/L; while mediated with galactose receptor for 48 hours, ASODN significantly inhibited cell proliferation and IC₅₀ was only 0.294 μmol/L, the inhibitory efficacy of ASODN enhanced 70.9 folds. While Bel-7402 cells were incubated with Gal-PEI-ASODN complex for 48 hours, cell hypodiploid percentage was much higher than ASODN groups and cell apoptosis was seen under electron microscopy. CONCLUSIONS: Galactose receptor mediated ASODN delivery may significantly increase proliferation inhibition efficacy on Bel-7402 cells.     

Effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45

AIM: To investigate the effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45. METHODS: The protein levels of CADM1 in 3 human gastric carcinoma cell lines were detected by Western blotting. Eukaryotic expression vector pcDNA-CADM1 was constructed and transfected into MKN-45 cells. The MKN-45 cells stably expressing CADM1 were selected by G418 and identified by Western blotting. Furthermore, CCK-8 assay and Boyden chamber were used to analyze the effects of CADM1 overexpression on the prolife ration and invasion of gastric carcinoma cells. Western blotting was also utilized to detect the levels of cell proliferation- and invasion-related proteins. RESULTS: Relative level of CADM1 protein in MKN-45 cells was significantly lower than that in MKN-28 cells and SGC-7901 cells. Additionally, eukaryotic expression vector pcDNA-CADM1 was successfully constructed and MKN-45 cells stably expressing CADM1 were obtained. Compared with non-treatment and pcDNA3.1 groups, the proliferation of MKN-45 cells was obviously inhibited in pcDNA-CADM1 group. The result of Boyden chamber showed that the migrated cell numbers in pcDNA-CADM1 group (52.35±3.89) were significantly lower than that in untreated group (101.53±6.89) and pcDNA3.1 group (98.77±7.03). Compared with non-treatment and pcDNA3.1 groups, the protein level of p21 was significantly up-regulated and protein expression of MMP-2 and MMP-9 was obviously down-regulated. CONCLUSION: Overexpression of CADM1 may markedly inhibit cell proliferation and reduce invasion ability, and thus may be a novel target for treating gastric carcinoma.     

Inhibitory role of epigallocatechin-3-gallate in proliferation of human nasopharyngeal carcinoma cells by targeting P53/miR-34a

AIM: To study the effect of epigallocatechin-3-gallate(EGCG) on the proliferation of human nasopharyngeal carcinoma(NPC) cells, and to explore its mechanism by targeting miR-34a.METHODS: Nasopharyngeal carcinoma CNE-2Z cells were treated with various concentrations of EGCG. The ability of cell proliferation was detected by CCK-8 assay, 5-ethynyl-2-deoxyuridine(EdU) incorporation assay and colony-forming assay. The cell cycle distributions were analyzed by flow cytometry. The protein levels of P53 and Notch1 were detected by Western blot. The expression of miR-34a and Notch1 mRNA was measured by real-time PCR.RESULTS: EGCG effectively inhibited the proliferation and colony formation of CNE-2Z cells in a dose-dependent manner, which was related to its induction of cell cycle arrest at G₀/G₁ phase. The expression of P53 and miR-34a in CNE-2Z cells was significantly increased after treated with EGCG, while the expression of Notch1 at mRNA and protein levels was markedly suppressed.CONCLUSION: EGCG induces cell cycle arrest and suppresses cell proliferation by regulating the P53/miR-34a/Notch1 pathway in NPC cells.