Hydrangea serrata extract exerts tumor inhibitory activity against hepatocellular carcinoma HepG2 cells via inducing p27/CDK2-mediated cell cycle arrest and apoptosis

Objective:To examine the inhibitory effect of Hydrangea serrata extract against hepatocellular carcinoma HepG2 cells and its underlying mechanisms.Methods:The effects of Hydrangea serrata extract on growth inhibition of tumor cells and spheroids were assessed using MTT and 3D culture assays.Quantitative real-time PCR and Western blot analyses were employed to investigate the changes in mRNA and protein expression levels of molecules related to cell cycle and apoptosis.Results:Hydrangea serrata extract effectively inhibited the growth of both tumor cells and spheroids.The extract also significantly upregulated p27 mRNA expression and downregulated CDK2 mRNA expression,leading to cell cycle arrest.Moreover,increased BAX/Bcl-2 ratio as well as caspase-9 and-3 were observed after treatment with Hydrangea serrata extract,indicating the induction of tumor cell apoptosis.Conclusions:Hydrangea serrata extract has the potential to alleviate tumors by effectively modulating cell-cycle-related gene expressions and inducing apoptosis,thereby inhibiting tumor growth.

Induction of apoptosis and G2/M cell cycle arrest by oridonin in human gastric cancer BGC-823 cells

Aim To investigate in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca^2＋ to shed light on the mode of its anticancer action. Methods The MTT method was used to investigate the inhibitory effect of oridonin on BGC-823 cells. The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry. The change of mitochondrial membrane potential and the increase of intracellular Ca^2＋ were assessed by fluorescence probe rhodamine123 and Fluo 3-AM, respectively, with flow cytometry. The expression of apoptosis and cell cycle related proteins was studied using western blotting. Results Oridonin inhibited BGC-823 cells growth with IC50 of 22.21 p, mol.L^-1. It induced apoptosis in a dose-dependent manner. In addition, it decreased mitochondria membrane potential, increased intracellular Ca^2＋, and activated pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. Conclusion Oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca^2＋, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A.

Cell cycle arrest and apoptotic cell death in cultured human gastric carcinoma cells mediated by arsenic trioxide

AIM: To investigate the effect of arsenic trioxide on human gastric cancer cell line MKN45 with respect to both cytotoxicity and induction of apoptosis in vitro. METHODS: MKN45 cells were treated with arsenic trioxide (As2O3) at the concentration of 1, 5, and 10 μmol/L, respectively, for three successive days. Cell growth and proliferation were observed by cell counting and trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Morphologic changes were studied with light microscopy. Flow cytometry was used to assay cell DNA distribution and apoptotic cells were confirmed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and DNA electrophoresis. RESULTS: The growth of MKN45 cells was significantly inhibited by As2O3 which was confirmed by colony-forming assay. After 7 d of culture with various concentrations of As2O3, colony-forming capacity of MKN45 cells decreased with As2O3 increment in comparison with that of control group. The inhibitory rate of colony-formation was 38.5%, 99.1%, and 99.5% when the concentration of As2O3 was 1, 5, and 10 μmol/L in culture medium, respectively. The cell number of a single colony in drug treatment groups was less than that of control group. The cell-killing rate of As2O3 to MKN45 cells was both dose- and time-dependent with an IC50 of (11.05±0.25) μmol/L After incubation in 10 μmol/L As2O3 for 24 h, the cell-killing rate was 27.1%, and it was close to 50% after 48 h. The results showed that As2O3 induced time- and dose-dependent apoptosis in MKN45 cells, blocked at G2/M phase. The apoptotic peak (sub-G1 phase) appeared and cell apoptotic rate in MKN45 cells was 18.3-32.5% after treatment by 10 umol/L As2O3 for 48 h. The percentage of G2/M cell of the experimental groups was 2.0-5.0 times than that of the control group. Gel electrophoresis of DNA from cells treated with each concentration of As2O3 for 48 h revealed a 'ladder' pattern, indicating preferential DNA degradation at the internucleosomal, linker DNA sections. TUNEL also demonstrated strand breaks in DNA of MKN45 cells treated with As2O3, while control cells showed negative labeling. CONCLUSION: As2O3 can induce apoptosis of human gastric carcinoma cells MKN45, which is the basis of its effectiveness. It shows great potential in the treatment of gastric carcinoma.

Down-regulation of Hsp90 could change cell cycle distribution and increase drug sensitivity of tumor cells

AIM:To construct Hsp90 antisense RNA eukaryotic expression vector, transfect it into SGC7901 and SGC7901/VCR of MDR-type human gastric cancer cell lines, HCC7402 of human hepatic cancer and Ec109 of human esophageal cancer cell lines, and to study the cell cycle distribution of the gene transected cells and their response to chemotherapeutic drugs.METHODS:A 1.03kb cDNA sequence of Hsp90beta was obtained from the primary plasmid phHSP90 by EcoR I and BamH I nuclease digestion and was cloned to the EcoR I and BamH I site of the pcDNA by T4DNA ligase and an antisense orientation of Hsp90beta expression vector was constructed. The constructs were transfected with lipofectamine and positive clones were selected with G418. The expression of RNA was determined with dot blotting and RNase protection assay, and the expression of Hsp90 protein determined with western blot. Cell cycle distribution of the transfectants was analyzed with flow cytometry, and the drug sensitivity of the transfectants to Adriamycin (ADR), vincrinstine (VCR), mitomycin (MMC) and cyclophosphamide (CTX) with MTT and intracellular drug concentration of the transfectants was determined with flow cytometry.RESULTS:In EcoR I and BamH I restriction analysis, the size and the direction of the cloned sequence of Hsp90beta remained what had been designed and the gene constructs were named pcDNA-Hsp90.AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones all expressed Hsp90 anti-sense RNA. The expression of Hsp90 was down-regulated in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 cell clones. Cell cycle distribution was changed differently. In AH-SGC7901/VCR and AH-Ec109 cells, G(1) phase cells were increased; S phase and G(2) phase cells were decreased as compared with their parental cell lines. In AH-SGC7901 cell, G(1)phase cells were decreased, G(2) phase cells increased and S phase cells were not changed, and in AH-HCC7402 cells G(1), S and G(2) phase cells remained unchanged as compared with their parental cell lines. The sensitivity of AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 to chemotherapeutic drugs, the sensitivity of AH-SGC7901/VCR to ADR, VCR, MMC and CTX the sensitivity of AH-HCC7402 to ADR and VCR, and the sensitivity of Ec109 to ADR, VCR and CTX all increased as compared with their parental cell lines. The mean fluorescence intensity of ADR in AH-SGC7901, AH-SGC7901/VCR, AH-HCC7402 and AH-Ec109 was also significantly elevated (P 【 0.05).CONCLUSION: Down-regulation of Hsp90 could change cell cycle distribution and increase the drug sensitivity of tumor cells.

Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells

AIM： The genes were divided into seven categories according to biological function; apoptosis-related, immune response-related, signal transduction-related, cell cyclerelated, cell growth-related, stress response-related and transcription-related genes. METHODS： We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated （5 mg/mL, 24 h） and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT- PC R. RESULTS： Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category： exonuclease 1 （EXO1）, ATP-binding cassette, sub-family F, member 2 （ABCF2）, MRE11 meiotic recombination 11 homolog A （MRE11A）, topoisomerase （DNA） Ⅰ （TOP1）, and FK506 binding protein 12-rapamycin-associated protein 1 （FRAP1）. RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells. CONCLUSION： These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells, and might be used for therapeutic anticancer drug.

DNA damage,apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes

AIM： To study the effect of fluoride on oxidative stress, DNA damage and apoptosis as well as cell cycle of rat oral mucosal cells and hepatocytes.METHODS： Ten male SD rats weighing 80N120 g were randomly divided into control group and fluoride group, 5 animals each group. The animals in fluoride group had free access to deionized water containing 150 mg/L sodium fluoride （NaF）. The animals in control group were given distilled water. Four weeks later, the animals were killed. Reactive oxygen species （ROS） in oral mucosa and liver were measured by Fenton reaction, lipid peroxidation product, malondialdehyde （MDA）, was detected by thiobarbituric acid （TBA） reaction, reduced glutathione （GSH） was assayed by dithionitrobenzoic acid （DTNB） reaction. DNA damage in oral mucosal cells and hepatocytes was determined by single cell gel （SCG） electrophoresis or comet assay. Apoptosis and cell cycle in oral mucosal cells and hepatocytes were detected by flow cytometry.RESULTS： The contents of ROS and MDA in oral mucosa and liver tissue of fluoride group were significantly higher than those of control group （P〈 0.01）, but the level of GSH was markedly decreased （P〈 0.01）. The contents of ROS, MDA and GSH were （134.73 ＋ 12.63） U/mg protein, （1.48 ＋ 0.13） mmol/mg protein and （76.38 ~ 6.71） mmol/ mg protein in oral mucosa respectively, and （143.45＋ 11.76） U/mg protein, （1.44：1：0.12） mmol/mg protein and （78.83±7.72） mmol/mg protein in liver tissue respectively. The DNA damage rate in fluoride group was 50.20% in oral mucosal cells and 44.80% in hepatocytes, higher than those in the control group （P 〈0.01）. The apop- tosis rate in oral mucosal cells was （13.63 ＋ 1.81） % in fluoride group, and （t2.76＋ 1.67） % in hepatocytes, higher than those in control group. Excess fluoride could differently lower the number of oral mucosal cells and hepatocytes at G0/G1 and S G2/M phases （P〈 0.05）.CONCLUSION： Excess fluoride can induce oxidative stress and DNA damage and lead to apoptosis and cell cycle change in rat oral mucosal cells and hepatocytes.

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri（L.） Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism

Aim： To explore the effect of androgen receptor （AR） on the expression of the cell cycle-related genes, such as CDKNIA and BTG1, in prostate cancer cell line LNCaP. Methods： After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction （RT- PCR） tests were carried out to confirm the results of the chips. Results：After AR antagonist flutamide treatment, three hundred and twenty-six genes （3.93 %） expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDCIO, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKNIA and BTG2. The CDKNIA and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. Conclusion： Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

Effect of p27^(KIP1) on cell cycle and apoptosis in gastric cancer cells

AIM： To elucidate the effect of p27^KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells. METHODS： The whole length of p27^KIP1 cDNA was transfected into human gastric cancer cell line SCG7901 by lipofectamine. Expression of p27^KIP1 protein or mRNA was analyzed by Western blot and RNA dot blotting, respectively. Effect of p27^KIP1 on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27^KIP1. Flow cytometry, TUNEL, and electron microscopy were used to assess the effect of p27^KIP1 on cell cycle and apoptosis. RESULTS： Expression of p27^KIP1 protein or mRNA increased evidently in SCG7901 cells transfected with p27^KIP1. The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently （0.55±0.14 cm vs 1.36±0.13cm, P〈0.01）. p27^KIP1 overexpression caused cell arrest with 36% increase （from 33.7% to 69.3%, P〈0.01） in G1 population. Prolonged p27^KIP1 expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy. CONCLUSION： p27^KIP1 can prolong cell cycle in G1 phase and lead to apoptosis, p27^KIP1 may be a good candidate for cancer gene therapy.

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

AIM： To investigate the effects of Chrysanthemum indicum extract （CIE） on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma （HCC） MHCC97H cell line. METHODS： Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide （PI）, mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS： CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION： CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

Cell cycle and radiosensitivity of progeny of irradiated primary cultured human hepatocarcinoma cells

AIM： To evaluate the change of growth characteristics and radiosensitivity of irradiated primary cultured human hepatocarcinoma cells. METHODS： All tumor tissue samples were obtained from 39 hepatocarcinoma patients with a mean age of 49.6 years （range 22-76 years）. We divided the samples into irradiated group and non-irradiated group and measured their plating efficiency （PE）, population doubling time （PDT）, radiosensitivity index SF2 and cell RESULTS： The PDT of primary culture of hepatocardnoma cells was 91.0±6.6 h, PE was 12.0±1.4%, SF2 was 0.41±0.05%. The PDT of their inadiated progeny was 124.8±5.8 h, PE was 5.0±0.7%, SF2 was 0.65±0.09%. The pdmary cultured human hepatocarcinoma cells showed significant S reduction and G^2 arrest in a dose-dependent manner. The progeny of irradiated primary cultured hepatocarcinoma cells grew more slowly and its radiosensitivity increased. CONCLUSION： The progeny of irradiated primary cultured human hepatocarcinoma cells grows more slowly and its radiosensitivity increases.

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.

Effects of Rapamycin on Expression of Bcl-2 and Bax in Human Lens Epithelial Cells and Cell Cycle in Rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells（LECs） and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations（20,40,60 and 80 ng/mL） of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance（A） values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction（RFQ-PCR） and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.

Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells

AIM： To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells. Possible molecular mechanisms were explored. METHODS： [3- （4, 5）-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide （MTT） assay, plasmid transfection, reporter assay, FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells. RESULTS： C2-ceramide was found to inhibit the growth of Bel7402 cells by indudng cell cycle arrest. During the process, the expression of p21 protein increased, while that of cyclinD1, phospho-ERKl/2 and c-myc decreased. Furthermore, the level of CDK7 was downregulated, while the transcriptional activity of PPARy was upregulated. Addition of GW9662, which is a PPARy specific antagonist, could reserve the modulation action on CDK7. CONCLUSION： Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7, at least partly, through PPARy activation. The ERK signaling pathway was involved in this process.

Pterocarine, a New Diarylheptanoid from Pterocarya tonkinesis, its Cell Cycle Inhibition at G_0/G_1 Phase and Induction of Apoptosis in HCT-15 and K562 Cells

Pterocarine (1), a new diarylheptanoidal compound, was isolated from Pterocaryatonkinesis (Franch.) Dode. together with a known diarylheptanoid, myricatomentogenin (2), througha bioassay-guided fractionation procedure. The structure of 1 was elucidated as (+)-3', 4''-epoxy-1-(4'-hydroxyphenyl)-7-(3''-hydroxyphenyl)-heptane-3-one by the spectroscopic methods.Pterocarine (1) inhibited the proliferation of tsFT210, HCT-15 and K562 cells with the inhibitionrates of 20.2±2.4, 23.8±2.4 and 50.5±1.2% at 100 μg/mL, respectively. Flow cytometric analysisindicated that 1 could inhibit the cell cycle of tsFT210, HCT-15 and K562 cells at the G0/G1 phaseand could also induce apoptosis in HCT-15 (19%) and K562 (11%) cells.

Paris Saponin Ⅰ Induces G_2/M Cell Cycle Arrest and Apoptosis in Human Gastric Carcinoma SGC7901 Cells

The aim of this study was to investigate the effect of Paris saponin I （PSI ） on human gastric carcinoma cell growth and apoptosis and to explore the potential mechanisms. The proliferation of SGC7901 cells was monitored by the MTT cell viability assay, while the nuclear morphology of apoptotic cells was assessed by Hoechst 33258 staining. Flow cytometry was performed to analyze the cell cycle progression of propidium iodide （PI）-stained SGC7901 cells and the apoptotic rate of annexin V/PI-stained ceils. Western blotting was used to examine the expression of several cell cycle proteins, including cyclin 131 and Cdkl, and the apoptosis-regulated proteins Bcl-2, Bax, cytochrome c, procaspase-9, and procaspase-3. The MTT assay demonstrated that PSI could induce significant dose- and time-dependent inhibition of SGC7901 cell proliferation. Marked morphological changes, including condensation of chromatin, nuclear fragmentation and apoptotic bodies were clearly shown on Hoechst 33258 staining. PS I treatment also resulted in the disruption of the cell cycle at Gz/M and the induction of apoptosis. Following PSI treatment, the cell cycle-related proteins cyclin B 1 and Cdkl were down- regulated. Expression of the pro-apoptotic protein Bax was increased, while anti-apoptotic protein Bcl-2 decreased. PSI treatment resulted in elevated cytoplasmic cytochrome c and activation of the apoptotic proteases caspase-9 and caspase-3. These data indicate that PS I acts as an inhibitor of proliferation in SGC7901 cells by inducing cell cycle arrest and mitochondria-dependent apoptosis. PSI is a potential therapeutic agent against human gastric carcinoma.

Downregulation of SNRPG induces cell cycle arrest and sensitizes human glioblastoma cells to temozolomide by targeting Myc through a p53-dependent signaling pathway

Objective:Temozolomide(TMZ)is commonly used for glioblastoma multiforme(GBM)chemotherapy.However,drug resistance limits its therapeutic effect in GBM treatment.RNA-binding proteins(RBPs)have vital roles in posttranscriptional events.While disturbance of RBP-RNA network activity is potentially associated with cancer development,the precise mechanisms are not fully known.The SNRPG gene,encoding small nuclear ribonucleoprotein polypeptide G,was recently found to be related to cancer incidence,but its exact function has yet to be elucidated.Methods:SNRPG knockdown was achieved via short hairpin RNAs.Gene expression profiling and Western blot analyses were used to identify potential glioma cell growth signaling pathways affected by SNRPG.Xenograft tumors were examined to determine the carcinogenic effects of SNRPG on glioma tissues.Results:The SNRPG-mediated inhibitory effect on glioma cells might be due to the targeted prevention of Myc and p53.In addition,the effects of SNRPG loss on p53 levels and cell cycle progression were found to be Myc-dependent.Furthermore,SNRPG was increased in TMZ-resistant GBM cells,and downregulation of SNRPG potentially sensitized resistant cells to TMZ,suggesting that SNRPG deficiency decreases the chemoresistance of GBM cells to TMZ via the p53 signaling pathway.Our data confirmed that SNRPG suppression sensitizes GBM cells to TMZ by targeting Myc via the p53 signaling cascade.Conclusions:These results indicated that SNRPG is a probable molecular target of GBM and suggested that suppressing SNRPG in resistant GBM cells might be a substantially beneficial method for overcoming essential drug resistance.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.