Clone Formation Test Research Articles

Effect of microRNA-451 on proliferation and migration of colorectal carcinoma via inhibition the expression of macrophage migration inhibitory factor

Objective To explore effect of microRNA (miRNA, miR)-451 on proliferation and migration of colorectal carcinoma via macrophage migration inhibitory factor (MIF). Methods miR-451 mimics and miR-451 NC were transfected into colorectal carcinoma cell by liposome Lipofectamine™3000. The expression of miR-451 was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell cycle was detected by flow cytometry. Cell migration ability was detected by Transwell method. The expression of MIF protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-451 in miR-451 mimics group (2.46±0.5) was higher than that in miR-451 NC group (1.00±0.11, t=52.374, P<0.05). Cell viability in miR-451 mimics group (0.37±0.03) was lower than that in miR-451 NC group (0.58±0.06, t=30.672, P<0.05). Cell clone number in miR-451 mimics group (41.56±4.17) was lower than that in miR-451 NC group (132.37±11.29, t=73.927, P<0.05). The G1 period in miR-451 mimics group was longer than that in miR-451 NC group (t=4.093, P<0.05), and the G2 period in miR-451 mimics group was shorter than that in miR-451 NC group (t=6.349, P<0.05). The number of cell invasion in miR-451 mimics group (56.59±5.66) was less than that in the miR-451NC group (184.73±18.47, t=64.992, P<0.05). The fluorescence intensity in miR-451 mimics+ pGL4 MIF 3’untranslated regions (3’UTR)-Wt group was lower than other transfection groups (F=2 875.12, P<0.05). The expression of MIF mRNA in miR-451 mimics (0.35±0.03) group was lower than that in miR-451 NC group (1.00±0.10, t=61.001, P<0.05). The western blot results showed that expression amount of MIF protein in mir-451 mimics group (0.23±0.02) was lower than that in miR-451 NC group (0.92±0.10, t=66.293, P<0.05). Conclusion Over-expression of miR-451 could inhibit colorectal carcinoma cell proliferation and migration by targeting down-regulation expression of MIF. Key words: Carcinoma carcinoma; MicroRNA-451; Macrophage migration inhibitory factor; Proliferation; Migration

Effect of microRNA-181 on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10

Objective To explore effect of microRNA (miRNA, miR)-181b on proliferation and apoptosis of hepatocellular carcinoma cells via regulation of phosphatase and tensin ho-molog deleted from chromosome 10 (PTEN). Methods miR-181b inhibitor and miR-181b NC were transfected into HepG2 cell by liposome Lipofectamine™3000. The expression of miR-181b was detected by real-time quantitative polymerase chain reaction (Real-time PCR). Cell viability was measured by methyl thiazol tetrazolium (MTT) assay. Cell clone ability was detected by cell clone formation test. Cell apoptosis and cell cycle was detected by flow cytometry. The expression of PTEN protein and mRNA was measured by Western blotting and Real-time PCR. Luciferase reporter analysis was performed. Results The expression of miR-181b in miR-181b inhibitor group (0.23±0.02) was lower than that in miR-181b NC group (1.00±0.01, P<0.05). Cell viability in miR-181b inhibitor group (0.37±0.03) was lower than that in miR-181b NC group (0.59±0.06, P<0.05). Clone formation test showed cell clone number in miR-181b inhibitor group (37.64±3.70) was lower than that in miR-181b NC group (132.56±12.37, P<0.05). Cell early apoptotic rate and late apoptotic rate in miR-181b inhibitor group was higher than that in miR-181b NC group (P<0.05). At the same time, compared with miR-181b NC group, cell cycle was made arrested at G1 phase (P<0.05), the expression of PTEN protein and mRNA was up-regulated in miR-181b inhibitor group (P<0.05), miR-181b inhibitor targeted regulation the expression of PTEN. Conclusion miR-181b inhibitor could inhibit HepG2 cell proliferation and induce cell apoptosis by targeting up-regulation expression of PTEN. Key words: Hepatocellular carcinoma; MicroRNA-181b; Phosphatase and tensin ho-molog deleted from chromosome 10; Proliferation; Apoptosis

The oxamate effect and mechanism in inhibiting the proliferation of anaplastic thyroid cancer

Objective To evaluate the antitumor effect of oxamate as a lactate dehydrogenase (LDH) inhibitor on Anaplastic human thyroid cancer, and to explore the therapeutic effect of inhibiting cancer metabolism on Anaplastic thyroid cancer (ATC). Methods Immunohistochemistry were used to detect the expression of LDHa in 12 cases of human ATC and adjacent tissues. Methyl thiazol tetrazolium (MTT) assay and plate cloning assay was used to detect the effect of Oxamate on the proliferation of ATC cell lines. The effects of Oxamate on the activity of LDH and the formation of lactate were detected by lactate dehydrogenase kit and lactate detection kit. Western blotting was used to detect changes in protein expression related to cell proliferation. Results Immunohistochemical staining showed that the positive rates of LDHa protein in cancer and adjacent tissues were 46.0% and 14.1% respectively, and the difference was statistically significant (P=0.004) LDH inhibitor oxamate was used to treat Hth83 and SW1736 cell lines in 0, 5, 10, 20, 40, 60, 80, 100 mmol/L, MTT assay showed that the 50% growth inhibitory concentration (IC50) of the Hth83 cell group was 64.3, 24.4, and 10.1 mmol/L, while in the SW1736 cell group, the IC50 values were 82.5 and 51.2, 25.3 mmol/L at 24, 48, and 72 h. The clone formation test indicated that two cell lines clone formation ability was also significantly declined, and the degree of decrease was positively correlated with the drug concentration (P=0.012). This inhibition was accompanied by a decrease in the activity of lactate dehydrogenase and the production of lactate. Western blotting analysis showed that the expression of phosphorylated protein kinase B (p-Akt), Cyclin D1, and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) was down-regulated. Conclusion LDHa is highly expressed in human ATC tissues. Oxamate, as an inhibitor of LDH enzyme, can decrease the proliferation potential of Hth83 and SW1736 cell lines. The possible molecular mechanism is that the Akt-Cyclin D1 protein pathway is inhibited. Key words: Oxamate; Lactate dehydrogenase; Thyroid carcinoma; Proliferation

Effect of microRNA-148b and microRNA-152 on proliferation and apoptosis of cholangiocarcinoma cells

Objective To investigate the microRNA (miRNA, miR)-148b and miR-152 expression and their biological functions in cholangiocarcinoma cells. Methods The expression levels of miR-148b and miR-152 in cholangiocarcinoma simples and QBC939 cells were detected using real-time quantitative polymerase chain reaction (Real-time PCR). After transfection with miR-148b/miR-152 mimics, cell proliferation, cell cycle and apoptosis were assessed using methyl thiazol tetrazolium (MTT) method, clone formation test and flow cytometry, respectively. Results The expression levels of miR-148b and miR-152 were significantly down-regulated [(2.75±0.16) folds and (3.05±0.18) folds] in cholangiocarcinoma samples as compared with normal samples (P=0.013, 0.019, respectively), and [(2.52±0.03) folds and (3.96±0.02) folds] in QBC939 cells as compared with H-69 cells (P=0.018, 0.012, respectively). Overexpression of miR-148b or miR-152 could significantly inhibit cell proliferation [(38.49±0.31)% and (52.69±1.01)%, respectively], and suppress cell clone formation (P=0.027, 0.017, respectively), block cell cycle in the S phase and induce cell apoptosis. Conclusion MiR-148b and miR-152 were down-regulated in cholangiocarcinoma cells, and overexpression of miR-148b and miR-152 could inhibit cell proliferation and induce cell apoptosis. Key words: MicroRNA-148b; MicroRNA-152; Cholangiocarcinoma; Proliferation; Apoptosis

Functional transformation of N-myc downstream-regulated gene 4 in gastric cancer cells under oxygen stress

Objective To investigate the influence of hypoxia on the function of N-myc downstream-regulated gene 4 (NDRG4) in gastric cancer cells. Methods The hypoxia model in gastric cancer cells was constructed to investigate the effect of hypoxia on metastasis of gastric cancer cells using scratch healing assay and Transwell assay. The mRNA and protein expression levels of NDRG4 in gastric cancer cells subjected to hypoxia were also detected using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. NDRG4 overexpression was conducted in gastric cancer cells and the effect of NDRG4 on malignant phenotype of gastric cancer cells was explored through scratch healing assay, Transwell assay, cell growth curve and clone formation test under normoxic conditions. Results Hypoxia could obviously enhance the metastasis capability of gastric cancer cells. As compared with the normoxic state, the number of invasive cells in both BGC-823 and MGC-803 subjected to hypoxia for 24 h was notably increased (174.20±9.04 vs. 48.20±11.76, and 229.00±34.22 vs. 28.75±2.50, respectively, both P=0.000). Besides, hypoxia could also significantly increase the scratch healing rate in both BGC-823 and MGC-803 cells [(25.00±0.02)% vs. (15.69±0.04)%, and (32.03±0.04)% vs. (11.90±0.04)% respectively, P=0.025, 0.006 respectively]. Accompanied with enhanced metastasis potential of gastric cancer cells induced by hypoxia for 24 h, the protein level of NDRG4 was dramatically increased. However, the ectopic overexpression of NDRG4 could dramatically inhibit the metastasis capacity, sustain cell proliferation and decrease the clone-forming ability under the normoxic condition. Conclusion Upregulation of NDRG4 induced by hypoxia might play an important role during the progression of gastric cancer trigged by hypoxia. We suspected that abnormal accumulation and functional protein modification of NDRG4 induced by hypoxia maybe contribute to the function changes of NDRG4 under different oxygen concentrations. Key words: Gastric cancer; N-myc downstream-regulated gene 4; Hypoxia; Metastasis

Far Upstream Element-Binding Protein 1 Facilitates Hepatocellular Carcinoma Invasion and Metastasis

Objective: To identify the role of FUBP1 in hepatocellular carcinoma (HCC) progression, invasion and metastasis. Methods: The expression and clinical significance of FUBP1 in HCC was analyzed by university of California santa cruz (UCSC) Xena online bioinformatics analysis tool and TMA from 451 HCC patients who underwent surgery that removed the primary tumor. CD45-EpCam circulating tumor cell was analyzed by CellSearchTM system. Gain-of-function and loss-of-function studies for FUBP1 were performed by implementing lentivirus infection system. HCC cell proliferation, migration and long term survival were measured by CCK-8, transwell assay and clone formation test respectively. The effect of tumor cell growth in vivo was measured by using tumor xenograft mice. Results: mRNA expression level of FUBP1 is higher in HCC tumors compared to adjacent normal liver tissue. Statistical analysis shows that higher expression of FUBP1 is related with hepatitis B virus (HBV) infection background and often with microvascular invasion. Kaplan-Meier analysis revealed significantly shorter median TTR (Time to Recurrence) and overall survival (OS) in patients with high FUBP1 compared with those patients with low FUBP1. The 2-year and 5-year recurrence rate is higher, corresponding with lower OS, in patients with high FUBP1 expression than low FUBP1. Univariate and multivariate analysis revealed that FUBP expression level was an independent indicator for both TTR and OS. In addition, FUBP1 expression in tumors positively correlated with the percentage of CD45-EpCam circulating tumor cell in blood before operation. Overexpression of FUBP1 enhances HCC proliferation, invasion and metastasis. By using shRNAs to deplete FUBP1 expression, HCC cells displayed decreased cell proliferation, soft-agar growth and invasion. In contrast, FUBP1 overexpression promotes primary tumor growth and lung metastasis. qPCR and Western Blot confirmed that the FUBP1 overexpression induces upregulation of N-cadherin and Vimentin whereas inhibiting E-cadherin, important epithelial mesenchymal transition (EMT) markers. By performing gene expression profiling, we identified that FUBP1 activates the TGF-β/smad pathway through promoting the expression of THBS1 (Thrombospondin-1, TSP-1). LSKL (Peptide antagonist of Thrombospondin-1) could inhibit HCC proliferation and invasion in vitro and vivo through blocking activation of TGF-β/smad pathway mediated by THBS1. Conclusions: High level of FUBP1 in HCC predicts poor prognosis after surgery. Overexpression of FUBP1 promotes HCC proliferation, invasion and metastasis, at least partially through enriching the generation of CTCs by activates TGF-I²/smad pathway and enhancing EMT in vitro and vivo. LSKL could inhibit HCC proliferation and invasion in vitro and vivo through blocking activation of TGF-β/smad pathway mediated by THBS1. Funding: National Science and Technology Major Project (2013ZX10002011-004, 2018ZX10203204-006-002). National Key Research and Development Program (2016YFC0902400), National Natural Science Foundation of China (81572823, 81572884). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: This study was approved by the Clinical Trail Ethics Committee of Zhongshan Hospital and all other four clinical centers. Approval for research protocol and use of human subjects was obtained from the research ethics committee of Zhongshan Hospital. Informed consent was obtained from each patient.

Screening glioma stem cells in U251 cells based on the P1 promoter of the CD133 gene.

Cluster of differentiation (CD)133 is an important cell surface marker of glioma stem cells (GSCs). The transcription of the CD133 gene is controlled by five alternative promoters (P1, P2, P3, P4 and P5), which are expressed in a tissue-specific manner. In the present study, gene recombination technology was used to construct two types of gene expression vectors that contained the P1 promoter of the CD133 gene, which regulated either the neomycin-resistance gene or the herpes simplex virus thymidine kinase (HSV-TK) gene. Following the stable transfection of U251 glioblastoma cells with these two gene vectors, the cells expressing the P1 promoter that regulated the neomycin-resistance gene were named CD133 (+) cells, while the cells expressing the P1 promoter regulating the HSV-TK gene were called CD133 (−) cells. The expression of CD133 was detected by flow cytometry and reverse transcription-quantitative polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess cell proliferation ability, while the cell cycle was analyzed by flow cytometry, and a clone formation test was performed to evaluate the invasive capability of the cells. The results demonstrated that, due to CD133 expression, the cell proliferation ability and the invasive capability of CD133 (+) cells were significantly higher than those of CD133 (−) cells. In conclusion, the present study successfully established a novel method of screening GSCs in U251 cells based on the P1 promoter of the CD133 gene.

Effect of NF-κB activation on the radiation response of esophageal cancer cells

To investigate the effect of NF-κB activation on radiation response of esophageal carcinoma. The expression of NF-κB was detected in pretreatment and posttreatment specimens of patients with ESCC by immunohistochemistry. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activation of NF-κB in esophageal cancer cell line KYSE150 cells. SN50, a specific NF-κB inhibitor, was applied to inhibit the activation of NF-κB. Clone formation test was used to detect the radiosensitivity of esophageal cancer cells. The median survival time of patients with activated and inactivated NF-κB in the pretreatment specimens were 16 and 19 months, respectively, with a non-significant difference between the two groups (P > 0.05). As to the patients with activated and inactivated NF-κB in posttreatment specimens, the median survival times were 13 and 35 months, respectively, with a significant difference (P < 0.01) between them. Western blot showed that the cytoplasmic expression of NF-κB was reduced with increasing radiation dose at 1.5 and 3 hours after radiation treatment. However, the expression of NF-κB in the cell nuclei was increased under the same condition, showing a trend of increased nucleus/cytoplasm ratio. The clone number in SN50 group was 96.66, 64.66, 76.66 and 10.00 under 0, 2, 4 and 12 Gy irradiation, which demonstrated a significant difference compared with the control groups (P < 0.001). Our results show that activation of NF-κB is induced by radiotherapy. Activation of NF-κB reduces the outcome of radiation treatment of esophageal cancer patients.

Harmine induces apoptosis in HepG2 cells via mitochondrial signaling pathway

Harmine has antitumor and antinociceptive effects, and inhibits human DNA topoisomerase. However, no detailed data are available on the mechanisms of action of harmine in hepatocellular carcinoma. This study aimed to investigate the effects of harmine on proliferation and apoptosis, and the underlying mechanisms in the human hepatocellular carcinoma cell line HepG2. The proliferation of HepG2 cells was determined by the cell counting kit-8 (CCK-8) assay and the clone formation test. The morphology of HepG2 cells was examined using fluorescence microscopy after Hoechst 33258 staining. Annexin V/propidium iodide (PI) was used to analyze apoptosis and PI to analyze the cell cycle. Western blotting was used to assess expression of the apoptosis-regulated genes Bcl-2, Bax, Bcl-xl, Mcl-1, caspase-3, and caspase-9. Mitochondrial transmembrane potential (ψm) was determined using JC-1. Harmine inhibited the proliferation of HepG2 cells in a dose-dependent manner. Hoechst 33258 staining revealed nuclear fragmentation and chromosomal condensation, cell shrinkage, and attachment loss in HepG2 cells treated with harmine. The percentage of the sub/G1 fraction was increased in a concentration-dependent manner, indicating apoptotic cell death. PI staining showed that harmine changed the cell cycle distribution, by decreasing the proportion of cells in G0/G1 and increasing the proportion in S and G2/M. Harmine induced apoptosis in a concentration-dependent manner, with rates of 20.0%, 32.7% and 64.9%, respectively. JC-1 revealed a decrease in ψm. Apoptosis of HepG2 cells was associated with caspase-3 and caspase-9 activation, down-regulation of Bcl-2, Mcl-1, and Bcl-xl, and no change in Bax. Harmine had an anti-proliferative effect in HepG2 cells by inducing apoptosis. Mitochondrial signal pathways were involved in the apoptosis. The cancer-specific selectivity shown in this study suggested that harmine is a promising novel drug for human hepatocellular carcinoma.

Effect of RhoC on hepatocellular carcinoma cell growth and related molecular mechanisms

To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth. siRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection. The inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05). RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.

Construction of Caveolin-1 RNA interfering eukaryotic expressing vector and its influence on biological behavior of human gastric cancer cells

Objective To design siRNA for Caveolin-1(CAV1) and construct eukaryotic expression vector Dsilence4.1-CMV-neo-CAV1, which is transfected into gastric cancer cell line SGC7901 to study the effects of inhibition of CAV1 expression with siRNA on biological behavior of gastric cell line SGC7901.Methods Design and chemosynthesize three pairs of double strands siRNA based on the neucleotide sequence of CAV1 gene provided by GeneBank.The siRNAs were transient transfected into SGC7901.The CAV1 expression of transfected cells were detected respectively by semi-quantity RT-PCR to sieve a valid siRNA sequence.Design and synthesized DNA sequence which can express small hairpin RNA(shRNA),construct RNA interfering eukaryotic expression vector of CAV1 gene.The SGC7901 Was stably transfected and was detected the expression of CAV1 gene by RT-PCR.The influence on the gastric cell growth and pmliferation after CAV1 was inhibited Was detected by cell growth curve, clone formation test and FCM.Results The CAV1 RNA interfering eukaryotic expression vector psilence4.1-CMV-neo-CAV1 was successfully constructed,which can significantly reduce the expression of CAV1 after stably transfected to target cells,and in contrast to Dsilence4.1-CMV-neo transfection group,PI and colony for mution rate were increased(t=7.98,P<0.05;t=13.19,P<0.01),the growth speed Was up.Conclusion The growth and proliferation ability of tumor is remarkablv accelerated by inhibiting the expression of CAV1 of gastric cell by RNAi. Key words: caveolin-1; RNA interference; Stomach neoplasms

The Lethal Effects of Nicotinamide Combined With Nonfocused Ultrasound on Human Pulmonary Carcinoma Cell Line A549 in Suspension

Objective: To explore the lethal effects of nicotinamide combined with nonfocused ultrasound (NFU) on human pulmonary carcinoma A549 cell live in suspension and provide new evidence for killing the cancer cells in hydrothorax and hydroperitonia or malignant cells infiltrating in tissues. Patients and Methods: Six groups were set in our experiment: blank control group, NA group, NFU group in 0 C, NFU group in 37 C, NA-combined NFU group in 0 C, and NA-combined NFU group in 37 C. Instant and 24-hour survival rates of A549 cells treated by different methods were determined based on the trypan blue exclusive assay, growth rates were detected by MTT after 24 hours, capability of cell colony formation was detected by clone formation test, changes in construction and form of Climbing film cells could be observed under the light microscope after 24 hours, and the percentage changes of apoptosis and the distributional percentage of the cell cycle were analyzed with flow cytometry. Results: Instant and 24-hour survival rates, growth rates, and capability of cell colony formation of A549 cells decreased more obviously in NA-combined NFU group than those in the other four groups. On the contribution of NA-combined NFU, the number of karyokinesis decreased significantly, while karyopyknosis and nuclear fragmentation increased greatly. Flow cytometry showed that the apoptotic index in NA-combined NFU group was higher than that in the NA or NFU group alone, and the cell cycle was held up in G1 stage. Conclusion: Certain concentration of NA has enhancement property on NFU and can increase the irradiated effectiveness of focused ultrasound markedly, which can provide reliable evidence for clinical application of NA as an ultrasound sensitizer.

The flavonoid component isorhamnetin in vitro inhibits proliferation and induces apoptosis in Eca-109 cells

Isorhamnetin is one member of flavonoid components which has been used in the treatment of heart disease. Recently the in vitro anti-cancer effect of isorhamnetin on human esophageal squamous carcinoma cell line Eca-109 was investigated in our lab. When Eca-109 cells were in vitro exposed to the graded doses of isorhamnetin (0–80 μg/ml) for 48 h, respectively, isorhamnetin exhibited cytostatic effect on the treated cells, with an IC 50 of 40 ± 0.08 μg/ml as estimated by MTT assay. Inhibition on proliferation by isorhamnetin was detected by trypan blue exclusion assay, clone formation test, immunocytochemical assay of PCNA and 3H-thymidin uptake analysis. Cell cycle distribution was measured by FCM. It was found that the viability of Eca-109 cells was significantly hampered by isorhamnetin. Compared with the negative control group, the treated group which was exposed to isorhamnetin had increased population in G 0/G 1 phase from 74.6 to 84 while had a significant reduction in G 2/M phase from 11.9 to 5.8. In addition to its cytostatic effect, isorhamnetin also showed stimulatory effect on apoptosis. Typical apoptotic morphology such as condensation and fragmentation of nuclei and blebbing membrane of the apoptotic cells could be observed through transmission electron microscope. Moreover, the sharp increase in apoptosis rate between the control and treated group were detected by FCM from 6.3 to 16.3. To explore the possible molecular mechanisms that underlie the growth inhibition and apoptosis stimulatory effects of isorhamnetin, the expressions of six proliferation- and death-related genes were detected by FCM. Expressions of bcl-2, c-myc and H-ras were downregulated whereas Bax, c-fos and p53 were upregulated. However, the in vivo experiments were required to further confirm the anti-cancer effects of isorhamnetin. In conclusion, isorhamnetin appears to be a potent drug against esophageal cancer due to its in vitro potential to not only inhibit proliferation but also induce apoptosis of Eca-109 cells.

Biological impact of the COOH-terminal 40 amino acid deletions of hepatitis B virus X protein in hepatocellular carcinoma cells

To investigate the biological impact of the wild type hepatitis B virus X protein (HBx) and HBx3'-40, an engineered deletion mutant of HBx lacking the last 40 C-terminal amino acids and a transcriptional transactivator on hepatoma cells. Human hepatocellular cells of the lines Huh7 and SMMC-7721 were transfected with HBx3'-40 or HBx constructs. The integration of the plasmid DNA and the expression of HBx3'-40 and HBx were confirmed by Neo gene PCR and Western blotting respectively. The growth curves of different cells were obtained by MTT method. Plate clone formation test to calculate the clone formation rate. Flow cytometry (FCM), and Chloramphenicol acetyl transferase (CAT)-ELISA. Male BALB/c mice were divided into 3 groups to be inoculated subcutaneously with plasmids pcDNA3HBx, pcDNA3HBx3'-40, and blank plasmas pcDNA3 as controls. The mice were killed 15 and 30 days after and the tumors were taken out to undergo measurement and immunohistochemistry. Growth curve showed that after transfection the HBx3'-40 group cells grew faster than HBx and pcDNA3 transfected group cells. FCM analysis showed that HBx3'-40 transfection enhanced the progression of G(1) --> S phase in Huh7 cell cycle and HBx did not show such impact. The clone formation rates of pcDNA3HBx3'-40 group was 12.2% +/- 1.4%, significantly higher than those of the pcDNA3HBx, blank vector, and control groups (6.3% +/- 0.7%, 4.1% +/- 0.9%, and 6.1% +/- 1.1% respectively, all P < 0.05) in the Huh7 cells. The situation was the similar in the SMMC-7721 cells: 83% +/- 6% vs.49% +/- 8%, 27% +/- 3%, and 51% +/- 5% respectively (all P < 0.05). The apoptotic rates of SMMC-7721 cells under no serum condition transfected with HBx and HBx3'-40, especially the former, were 12.57% and 9.15%, significantly higher than that of the pcDNA3HBx group. The CAT expression of the pcDNA3HBx-transfected SMMC-7721 cells was 2.913 and 3.652 times those of the pcDNA3HBx3'-40 group and pcDNA3 group. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected Huh7 cells were 1.2 g +/- 0.17 g, 0.55 g +/- 0.12 g, and 0.48 g +/- 0.15 g respectively. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected SMMC-7721 cells showed similar features. HBx3'-40 protein significantly promote the proliferation of hepatocellular carcinoma cells and is involved in cell apoptosis and invasion, thus supporting the hypothesis that HBx mutants play a key role in hepatocarcinogenesis by modifying the biological functions of HBx. Besides, C-terminal mutants of HBx makes p53 tumor suppressor gene lose its cancerostatic function.