Proliferation Of HCCLM3 Cells Research Articles

Ribosome-inactivating Protein MAP30 Isolated from Momordica Charantia L. Induces Apoptosis in Hepatocellular Carcinoma Cells.

Ribosome-inactivating proteins (RIPs) have been reported to exert antitumor and anti-virus activities. A recent patent CN202011568116.7 has developed a new method to prepare Momordica anti-HIV protein of 30 kDa (MAP30). MAP30 is a type I RIP, which kills various tumor cells through the N-glycosidase activity and irreversibly inhibits protein synthesis. To assess the potential role of MAP30 in inducing apoptosis of human hepatocellular carcinoma HCC-LM3 cells and elucidate the molecular mechanism of MAP30. CCK-8 assay was used to assess the proliferation of HCC-LM3 cells. Flow cytometry was used to measure the cycle, the level of ROS and apoptosis in HCC-LM3 cells. Western blots was used to measure protein levels. Treatment with MAP30 reduced survival and proliferation of human liver cancer HCCLM3 cells in a dose-dependent manner. PI staining showed cell cycle arrest in G0/G1 phase. Furthermore, MAP30 increased the level of ROS in HCC-LM3 cells in 24 h treatment. To further confirm the role of MAP30 in inducing cell apoptosis, immunoblotting was carried out to detect the change of apoptosis-related proteins including PARP poly (ADP-ribose) polymerase (PARP- 1), Casepase3 and Cleaved-Caspase9. We found that PARP-1 and Caspase-3 were downregulated, whereas Cleaved-Caspase9 was up-regulated in HCC-LM3 cells treated with MAP30. This study indicated that MAP30 has the potential to be a novel therapeutic agent for human hepatocellular carcinoma.

UBE2O promotes hepatocellular carcinoma cell proliferation and invasion by regulating the AMPKα2/mTOR pathway.

The ubiquitin-conjugating enzyme (E2) is a critical component of the ubiquitin-proteasome system and regulates hepatocarcinogenesis by controlling protein degradation. Ubiquitin-conjugating enzyme E2 O (UBE2O), a member of the E2 family, functions as an oncogene in human cancers. Nevertheless, the role of UBE2O in hepatocellular carcinoma (HCC) remains unknown yet. Here, we demonstrated that the UBE2O level was markedly upregulated in HCC compared with adjacent noncancerous tissues. UBE2O overexpression was also confirmed in HCC cell lines. UBE2O overexpression was prominently associated with advanced tumor stage, high tumor grade, venous infiltration, and reduced HCC patients' survivals. UBE2O knockdown inhibited the migration, invasion, and proliferation of HCCLM3 cells. UBE2O overexpression enhanced the proliferation and mobility of Huh7 cells. Mechanistically, UBE2O mediated the ubiquitination and degradation of AMP-activated protein kinase α2 (AMPKα2) in HCC cells. UBE2O silencing prominently increased AMPKα2 level and reduced phosphorylated mechanistic target of rapamycin kinase (p-mTOR), MYC, Cyclin D1, HIF1α, and SREBP1 levels in HCCLM3 cells. UBE2O depletion markedly activated the AMPKα2/mTOR pathway in Huh7 cells. Moreover, AMPKα2 silencing reversed UBE2O downregulation-induced mTOR pathway inactivation. Rapamycin, an inhibitor of mTOR, remarkably abolished UBE2O-induced mTOR phosphorylation and HCC cell proliferation and mobility. To conclude, UBE2O was highly expressed in HCC and its overexpression conferred to the poor clinical outcomes of patients. UBE2O contributed to the malignant behaviors of HCC cells, including cell proliferation, migration, and invasion, by reducing AMPKα2 stability and activating the mTOR pathway.

Expression and function of transmembrane 4 superfamily proteins in digestive system cancers

BackgroundAlthough the medical level is constantly improving, cancer is still a major disease that threatens human health, and very effective treatments have not been found. In recent years, studies have found that four-transmembrane superfamily proteins are involved in multiple stages of tumorigenesis and development, but their expression and function in tumors have not been systematically studied.MethodsWe used the Oncomine database to analyze the mRNA expression levels of TSPAN family in various cancers. Then differentially expressed genes were screened out and verified by liver cancer, colorectal cancer, and gastric cancer cells by q-PCR and Western blot analysis. CCK8 and EDU analysis are used to detect cell proliferation, Cell wound scrape assay and Cell invasion assay are used to analyze cell invasion and metastasis. Nude tumor formation test used to verify the tumor suppressive effect of TSPAN7 in vivo.ResultsDifferential analysis of 33 TSPAN proteins revealed that a total of 11 proteins showed differential expression in 10% of independent analyses, namely TSPAN1, TSPAN3, TSPAN5, TSPAN6, TSPAN7, TSPAN8, TSPAN13, TSPAN25, TSPAN26, TSPAN29, TSPAN30. TSPAN7 is the only four-transmembrane protein with reduced expression in three types of digestive tract tumors, so we chose TSPAN7 to be selected for cellular and molecular level verification. We found that compared with normal cells, the expression of TSPAN7 in liver cancer cells was significantly reduced, while the expression of gastric and colon cancer was not significantly different from that of normal cells. In addition, we also found that the high expression of Tspan7 not only inhibited the proliferation of HCC-LM3 cells, but also inhibited its invasion and metastasis.ConclusionsOur study evaluated the expression and function of the TSPANs family in digestive cancers and explored TSPAN7 in hepatoma cells in detail. We found some members of the TSPAN family show significant expression differences between cancer and normal tissues, of which TSPAN7 may be a potential biomarker for liver cancer.

Effect of alanine-glyoxylate aminotransferase 2-like 1 on proliferation, apoptosis and cell-division cycle of human hepatoma cell lines 97H and HCCLM3

Objective To investigate the effect of alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1) on the proliferation, apoptosis and cell-division cycle of hepatoma cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of AGXT2L1 in 97H, HCCLM3 and LO2 cells. The siRNA targeting AGXT2L1 was designed, synthetized and transfected into 97H and HCCLM3 cells to construct AGXT2L1 knockdown group, and the efficiency of knockdown was validated by RT-qPCR. The cell proliferation of each group at 0, 12, 24, 48, 72 and 96 h was tested by cell counting kit-8 (CCK-8) assay. Cell cycle assay was applied to detect the distribution of cells in G1, S and G2 phases. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis rate of cells in each group. Results The expression of AGXT2L1 in 97H and HCCLM3 cells was significantly higher than that in normal liver cells (1 350.04±91.36, 1 710.64±92.34, t97H=25.574, P<0.01; tHCCLM3=32.068, P<0.01). The AGXT2L1 expression level in 97H and HCCLM3 cells was decreased significantly after transfection with AGXT2L1-siRNA (0.19±0.04 vs. 1.10±0.04, t97H =-28.722, P<0.01; 0.20±0.01 vs. 0.93±0.09, tHCCLM3=-13.887, P<0.01). The CCK-8 assay showed that proliferation rate of 97H and HCCLM3 cells decreased significantly after knocking down AGXT2L1 (0.71±0.01 vs. 0.95±0.12, t97H=-31.243, P<0.01; 0.67±0.13 vs. 0.87±0.17, tHCCLM3=-17.083, P<0.01). After transfection of AGXT2L1-siRAN into cells, the cell cycle assay showed that the ratio of S phase cells was lower than that in the control group [(13.45±1.20)% vs. (18.73±0.28)%, t97H=-7.451, P<0.01; (15.26±0.52)% vs. (20.45±1.08)%, tHCCLM3=-7.483, P<0.01], and the ratio of G2 phase cells was lower than that in the control group [(16.90±0.54)% vs. (19.99±0.54)%, t97H=-6.992, P<0.01; (16.23±0.28)% vs. (23.36±0.39)%, tHCCLM3=-25.720, P<0.01]. The knockdown of AGXT2L1 could inhibit the transformation of 97H and HCCLM3 cells from G1 to S phase and inhibit the proliferation of human hepatoma cells. Compared with the control group, after the knockdown of AGXT2L1, the apoptosis rate of 97H and HCCLM3 cells was increased [(13.06±1.86)% vs. (1.06±0.75)%, t97H=14.673, P<0.01; (14.41±2.21)% vs. (0.80±0.32)%, tHCCLM3=14.947, P<0.01]. Conclusion The knockdown of AGXT2L1 can inhibit the proliferation of 97H and HCCLM3 cells and promote apoptosis. Key words: Carcinoma, hepatocellular; Alanine-glyoxylate aminotransferase 2-like 1; Proliferation; Apoptosis

Effect and mechanism of curcumin on proliferation and apoptosis of human hepatoma HCC-LM3 cells

Objective To investigate the effects of curcumin on the growth and apoptosis of human hepatoma HCC-LM3 cells and explore the molecular mechanisms. Methods The human hepatoma HCC-LM3 cells were treated with different concentrations of curcumin and the cell growth inhibition rate was detected by CCK-8. The effect of curcumin on human hepatoma HCC-LM3 cells was observed. Refer to the relevant literature, the human hepatoma HCC-LM3 cells were treated with the concentration of 2.5, 5.0, 10.0, 15.0, 20.0, 40.0, 60.0 μmol/L of curcumin for 48 hours, taking the 0 μmol/L curcumin as control group, and the cell growth inhibition rate was detected by CCK-8. According to the results of CCK-8, selecting the concentration of 0 μmol/L as control group and the concentration of 10.0, 20.0, 40.0 μmol/L as experimental groups, which has significant difference on growth inhibition rates. Cell cloning assay was used to detect cell cloning ability, Flow cytometry was used to detect apoptosis, and Western blotting to detect the protein expression levels of Mcl-1, Bax, Bcl-2 and Bcl-xL. The measurement data were expressed in (±s), and the single factor analysis of variance was used for comparison between groups. Results CCK-8 assay showed that with treated by the concentration of 2.5, 5.0, 10.0, 15.0, 20.0, 40.0, 60.0 μmol/L, the growth inhibition rates were(6.71±3.45)%, (12.33±5.02)%, (20.07±5.60)%, (57.80±7.34)%, (78.37±6.53)%, (91.73±6.14)% and (96.18±3.45)%, suggesting that curcumin could inhibit the growth of human hepatoma HCC-LM3 cells in a dose-dependent manner. Cell clone formation experiment showed that curcumin could inhibit the clone of the human hepatoma HCC-LM3 cells, and the clone of the cells was inhibited significantly when the concentration of the curcumin was over 20.0μmol/L. The result of Annexin V-FITC/PI double staining analysis showed that the apoptotic rates of experimental groups and control groups were (5.20±1.44)%, (9.90±3.31)%, (55.67±5.29)%, (79.63±4.71)%, with all the apoptotic rates of experimental group over the control groups (P<0.05), suggesting curcumin could induce the apoptosis of human hepatoma HCC-LM3 cells.The Westen blotting showed that curcumin increased the expression of Bax protein while decreasing expression of Mcl-1 protein significantly in concentration-dependent manner (P<0.05), but have no effect on the expression of Bcl-2 and Bcl-xL proteins. Conclusion Curcumin could inhibit the proliferation and clone of human hepatoma HCC-LM3 cells, and induce apoptosis in a dose dependent manner. Key words: Curcumin; Carcinoma, hepatocellular; Cell proliferation; Apoptosis; HCC-LM3

Genistein Reinforces the Inhibitory Effect of Cisplatin on Liver Cancer Recurrence and Metastasis after Curative Hepatectomy

The high recurrence rate after hepatic resection in hepatocellular carcinoma (HCC) is a major obstacle to improving prognosis. The objective of the present study was to explore the function of genistein, a soy-derived isoflavone, in enhancing the inhibitory effect of cisplatin on HCC cell proliferation and on tumor recurrence and metastasis in nude mice after curative hepatectomy. Proliferation of human HCC cells (HCCLM3) was detected by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Synergistic effects of genistein and cisplatin were evaluated with the median-effect formula. Nude mice bearing human HCC xenografts underwent tumour resection (hepatectomy) 10 days post implantation, then received intraperitoneal administration of genistein or cisplatin alone or the combination of the two drugs. 33 days after surgery, recurrent tumours and pulmonary metastasis were evaluated individually. MMP-2 level in recurrent tumours was detected by immunohistochemistry and real-time PCR; MMP-2 expression in HCCLM3 was detected by immunocytochemistry. Genistein and cisplatin both suppressed the growth and proliferation of HCCLM3 cells. The two drugs exhibited synergistic effects even at relatively low concentrations. In vivo, mice in the combined genistein and cisplatin group had a smaller volume of liver recurrent tumors and fewer pulmonary metastatic foci compared with single drug treated groups. Cisplatin upregulated the expression of MMP-2 in both recurrent tumours and HCCLM3, while genistein abolished cisplatin-induced MMP-2 expression. Genistein reinforced the inhibitory effect of cisplatin on HCC cell proliferation and tumour recurrence and metastasis after curative hepatectomy in nude mice, possibly through mitigation of cisplatin-induced MMP-2 upregulation.

Effect of let-7c on the proliferation of human hepatocellular carcinoma cell HCCLM3

To investigate let-7c's effect on the proliferation of human hepatocellular carcinoma cell HCCLM3 by transient transfection and the mechanism inside. Lipofectamine 2000 was used to transfect miRNAs into HCCLM3 cells. The cells were divided into three groups, let-7c group: let-7c was transfected, negative control group: negative control miRNA was transfected, blank control group: nothing was transfected. The proliferation of HCCLM3 cells was evaluated using Cell Counting Kit-8 (CCK-8). The cell cycles of each group were assayed by flow cytometry. Western blot and Real time PCR were used to analyze the protein and mRNA expressions of cyclin D1. Statistical analysis was performed with SPSS 17.0. The absorbances of let-7c group were 0.70 ± 0.05, 0.77 ± 0.09 at 48 h and 72 h after transfection, lower than that of blank control group (0.97 ± 0.10, 1.21 ± 0.12) and negative control group (0.91 ± 0.07, 1.12 ± 0.09), 48 h: F = 14.431, P < 0.05, 72 h: F = 21.146, P < 0.05. The flow cytometry at 72 h after transfection revealed that let-7c increased the percentage of cells in G1 phase. The percentage of blank control group was 43.53% ± 0.86%, the negative control group was 44.82% ± 0.77%, and the let-7c group was 54.52% ± 0.13%, F = 240.739, P < 0.05. let-7c suppressed expressions of cyclin D1 at both protein and mRNA levels. The protein levels of cyclin D1 were 0.48 ± 0.09, 0.47 ± 0.06 and 0.23 ± 0.06 (F = 11.316, P < 0.05) in blank control group, negative control group and let-7c group, respectively. The mRNA levels were 1.03% ± 0.29%, 1.01% ± 0.11% and 0.63% ± 0.14% (F=6.315, P < 0.05) in the above three groups, respectively. Let-7c can inhibit proliferation of HCCLM3 cells and increase the proportion of cells in G1 phase. The mechanism may be that let-7c represses the expressions of cyclin D1 at both protein and mRNA levels.