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.

The effect of celastrol in combination with 5-fluorouracil on proliferation and apoptosis of gastric cancer cell lines

Background:Despite the availability of chemotherapy drugs such as 5-fluorouracil(5-FU),the treatment of some cancers such as gastric cancer remains challenging due to drug resistance and side effects.This study aimed to investigate the effect of celastrol in combination with the chemotherapy drug 5-FU on proliferation and induction of apoptosis in human gastric cancer cell lines(AGS and EPG85-257).Materials and Methods:In this in vitro study,AGS and EPG85-257 cells were treated with different concentrations of celastrol,5-FU,and their combination.Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The synergistic effect of 5-FU and celastrol was studied using Compusyn software.The DNA content at different phases of the cell cycle and apoptosis rate was measured usingflow cytometry.Results:Co-treatment with low concentrations(10%inhibitory concentration(IC10))of celastrol and 5-FU significantly reduced IC50(p<0.05)so that 48 h after treatment,IC50 was calculated at 3.77 and 6.9μM for celastrol,20.7 and 11.6μM for 5-FU,and 5.03 and 4.57μM for their combination for AGS and EPG85-257 cells,respectively.The mean percentage of apoptosis for AGS cells treated with celastrol,5-FU,and their combination was obtained 23.9,41.2,and 61.9,and for EPG85-257 cells 5.65,46.9,and 55.7,respectively.In addition,the 5-FU and celastrol-5-FU combination induced cell cycle arrest in the synthesis phase.Conclusions:Although celastrol could decrease the concentration of 5-fluorouracil that sufficed to suppress gastric cancer cells,additional studies are required to arrive at conclusive evidence on the anticancer effects of celastrol.

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.

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.

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.

Effects of simulated carbon dioxide and helium peumoperitoneum on proliferation and apoptosis of gastric cancer cells

AIM:To investigate the effects of carbon dioxide (CO2) and helium insufflation administered at different pressures on the growth and apoptosis of cultured human gastric cancer cells. METHODS:The gastric cancer cells MKN-45 were exposed to a CO2 and helium environment maintained at different pressures (0, 5, 10 and 15 mmHg). The cells were exposed to simulated pneumoperitoneum environment for 4 h, and pH of the culture media was measured after it was moved to normal conditions for 0, 2, 4, 6 and 8 h. Proliferation viability of MKN-45 was examined by 3-4,5Dimethylthiazol-2-yl,5-diphenyltetrazolium bromide or triazolyl blue (MTT) assay after it was moved to normal conditions. Apoptotic ratio was measured by Annexin V-FITC/PI double labelled staining. RESULTS:The pH of media was acid and recovered to normal after 4 h in the CO2 group while it was basic in the helium group. There was no difference between CO2 groups (under 10 mmHg ) and control group (P > 0.05) in the proliferative viability of the cells. The cultured cells exposed to 15 mmHg CO2 environment grew more slowly than control group from 4 to 7 d (P < 0.01 ) while there was no difference from 1 to 3 d (P > 0.05). The proliferative viability in helium group was not obviously different from the control group (P > 0.05). The apoptotic ratio of the cultured cells was markedly higher than that of the control group (P < 0.01) at 10 and 15 mmHg CO2 insufflation pressure. In helium group, the apoptotic ratio was not obviously different from the control group (P > 0.05). CONCLUSION:There is no obvious effect in the proliferation and apoptosis of MKN-45 cells under 10 mmHg CO2 insufflation pressure and helium in any pressure. Fifteen mmHg CO2 insufflation pressure can inhibit the proliferation of the cells and improve apoptosis.

Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

AIM： Polo-like kinase 1 （PLK1） serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells. METHODS： PLK1 expression was blocked by small RNA interference（siRNA）. The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity, cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1 knockdown cells were observed. RESULTS： PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity （but not with the expression levels）, accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation, delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation, attenuated pro-caspase 3 levels and increased apoptosis. CONCLUSION： Blockage of to decreased mitosis or even PLK1 expression may lead apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

Intracellular chloride regulates the G_1/S cell cycle progression in gastric cancer cells

Recent studies show that ion channels/transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels/transporters on cell proliferation suggest that the intracellular chloride concentration ([Cl-]i) regulated by them would be one of critical messengers. We investigated whether the [Cl-]i controls cell proliferation and cell cycle progression in human gastric cancer cells. Our studies indicated that furosemide, a blocker of Na+ /K+ /2Cl- cotransporter (NKCC), diminished cell growth by delaying the G1-S phase progression in gastric cancer cells with high expression and activity of NKCC. Furthermore, we found that the culture in the low Cl- medium (replacement of Cl- by NO3-) decreased the [Cl-]i and inhibited cell growth of gastric cancer cells and that this inhibition of cell growth was due to cell cycle arrest at the G0/G1 phase caused by diminutionof CDK2 and phosphorylated Rb. The culture of cells in the low Cl- medium significantly increased expressions of p21 mRNA and protein. In addition, the low Cl- medium induced phosphorylation of mitogen activated protein kinases (MAPKs). Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium and rescued gastric cancer cells from the low Cl- -induced G1 cell cycle arrest. These findings revealed that the [Cl-]i affects the cell proliferation via activation of MAPKs through upregulation of p21 in gastric cancer cells. Our results suggest that the [Cl-]i regulates important cellular functions in gastric cancer cells, leading to the development of novel therapeutic strategies.

A study on arsenic trioxide inducing in vitro apoptosis of gastric cancer cell lines

INTRODUCTION Cell apoptosis,which involves the biologic regulation of the numbers and vital activity of cells,is an important metaboloc process in both normal cells and tumor cells.

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.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 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.

Acetyl-11-keto-β-boswellic acid inhibits proliferation and induces apoptosis of gastric cancer cells through the phosphatase and tensin homolog/Akt/cyclooxygenase-2 signaling pathway

BACKGROUND Gastric cancer is one of the most common malignant tumors of the digestive system worldwide,posing a serious danger to human health.Cyclooxygenase(COX)-2 plays an important role in the carcinogenesis and progression of gastric cancer.Acetyl-11-keto-β-boswellic acid(AKBA)is a promising drug for cancer therapy,but its effects and mechanism of action on human gastric cancer remain unclear.AIM To evaluate whether the phosphatase and tensin homolog(PTEN)/Akt/COX-2 signaling pathway is involved in the anti-tumor effect of AKBA in gastric cancer.METHODS Human poorly differentiated BGC823 and moderately differentiated SGC7901 gastric cancer cells were routinely cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin.Gastric cancer cell proliferation was determined by methyl thiazolyl tetrazolium colorimetric assay.Apoptosis was measured by flow cytometry.Cell migration was assessed using the wound-healing assay.Expression of Bcl-2,Bax,proliferating cell nuclear antigen,PTEN,p-Akt,and COX-2 were detected by Western blot analysis.A xenograft nude mouse model of human gastric cancer was established to evaluate the anti-cancer effect of AKBA RESULTS AKBA significantly inhibited the proliferation of gastric cancer cells in a dose-and time-dependent manner,inhibited migration in a time-dependent manner,and induced apoptosis in a dose-dependent manner in vitro;it also inhibited tumor growth in vivo.AKBA up-regulated the expression of PTEN and Bax,and downregulated the expression of proliferating cell nuclear antigen,Bcl-2,p-Akt,and COX-2 in a dose-dependent manner.The PTEN inhibitor bpv(Hopic)reversed the high expression of PTEN and low expression of p-Akt and COX-2 that were induced by AKBA.The Akt inhibitor MK2206 combined with AKBA downregulated the expression of p-Akt and COX-2,and the combined effect was better than that of AKBA alone.CONCLUSION AKBA inhibits the proliferation and migration and promotes the apoptosis of gastric cancer cells through the PTEN/Akt/COX-2 signaling pathway.

Carbon ion irradiation-induced DNA damage evokes cell cycle arrest and apoptosis via the pRb/E2F1/c-Myc signaling pathway in p53-deficient prostate cancer PC-3 cells

Carbon ion radiotherapy has the advantages of better therapeutic effect and fewer side effects compared with those of X-rays in many kinds of tumors,including prostate cancer,and thus is an attractive treatment approach for prostate cancer.However,the biological effects and underlying mechanisms of carbon ion irradiation in prostate cancer are not yet fully understood.Therefore,this study systematically compared the effects of carbon ion irradiation with those of X-ray irradiation on DNA damage response and found that carbon ion irradiation was more effective than X-ray irradiation.Carbon ion irradiation can induce a high level of DNA double-strand break damage,reflected by the number of y-H2 A histone family member X foci,as well as by the foci lasting time and size.Moreover,carbon ion irradiation exhibited strong and long-lasting inhibitory effect on cell survival capability,induced prolonged cell cycle arrest,and increased apoptosis in PC-3 cells.As an underlying mechanism,we speculated that carbon ion irradiation-induced DNA damage evokes cell cycle arrest and apoptosis via the pRb/E2 F1/c-Myc signaling pathway to enhance the radiosensitivity of p53-deficient prostate cancer PC-3 cells.Collectively,the present study suggests that carbon ion irradiation is more efficient than X-ray irradiation and may help to understand the effects of different radiation qualities on the survival potential of p53-deficient prostate cancer cells.

Wild pink bayberry free phenolic extract induces mitochondria-dependent apoptosis and G0/G1 cell cycle arrest through p38/MAPK and PI3K/Akt pathway in MDA-MB-231 cancer cells

Polyphenol-rich foods have been shown to be good for cancer prevention as powerful antioxidants. In this study, the mechanisms of wild pink bayberry free phenolic extract(WPBFE)inhibiting the proliferation and inducing apoptotic of MDA-MB-231 breast cancer cells was examined. The main phenolic acids and flavonols in WPBFE were gallic acid((18.83 ± 0.44)μg/g FW)and myricetin((1.52 ± 0.05)μg/g FW), respectively. The maximum inhibition rate of WPBFE at non-cytotoxicity dose(below 80 mg/mL)was 81%. Western blotting analysis showed that WPBFE could cause the arrest of cell cycle in G0/G1 phase by down-regulating expression levels of PCNA, CDK4, cyclin D1 and up-regulating the expression level of p21. Meanwhile, WPBFE induced apoptosis through initiating the mitochondrial death pathway by up-regulating cleaved caspase-3 and enhancing the ratio of Bax/Bcl-2, with the maximum expression levels of 1.29 and 2.03 folds that of control group, respectively. Further study of the upstream protein, we found that WPBFE down-regulated TRAF2, while upregulated p-ASK1, p-p38 and p-p53. Furthermore, WPBFE could down-regulate the expression of p-PI3K and p-Akt. These observations indicated that WPBFE might play an anticancer role through regulating the p38 MAPK together with PI3K/Akt pathway.

Effects of Ligustrazine Combined with Cisdichlorodiamine Platinum on Anti-proliferation,Cell Cycle and Apoptosis of Human Gastric Carcinoma SGC-7901 Cell Lines

[Objective] This study aimed to investigate the combination effects of ligustrazine and cis-dichlorodiamine platinum （DDP） on anti-proliferation, cell cycle and apoptosis of SGC-7901 cell lines in vitro. [Methed] SGC-7901 cells were treat- ed with ligustrazine and DDP alone or combined for 48 h for morphology assay. Anti-proliferative effects with the same treatment for 24, 48 and 72 h were assayed by MTT method, respectively. Cell cycle distribution and apoptosis assay of treated cells were performed in flow cytometry. Zhengjun Jin＇s protocol was used to assay the effect of drug combination. [Result] Ligustrazine significantly increased the prolif- eration inhibition rate of DDP on SGC-7901 cells in combination with DDP, com- paring with the effects of ligustrazine or DDP alone, and exhibited synergistic antitu- mor effect. The combination drug treatment induced cell cycle arrest occurred in S and G2 phase of the cell cycle and increased the apoptosis rate significantly. [Conclusion] Our results indicate that ligustrazine, as a low-toxic and natural herbal component, can significantly increase the anti-proliferative effect and apoptosis rate of antitumor drug DDP on human gastric carcinoma SGC：.-7901 cells.

Primula vulgaris extract induces cell cycle arrest and apoptosis in human cervix cancer cells

Primula vulgaris belongs to the genus Primula, members of which are frequently used in folk medicine. Various studies have investigated the cytotoxic effect of different Primula species, but there have been limited studies on the cytotoxic effect of P vulgaris. The aim of this study was to investigate the cytotoxic effects, and possible mechanisms involved, of P vulgaris flower extract on human cervical cancer （HeLa） cells. The cytotoxic effect of the extract on HeLa cells was revealed using the MTT assay. Mechanisms involved in the extract＇s cytotoxic effect were then investigated in terms of apoptosis, mitochondrial membrane potential, and the cell cycle, using fluorometric methods. P vulgaris flower extract exhibited selective cytotoxic effects against HeLa cells by arresting their cell cycle at the S phase, and inducing the number of apoptotic cells compared to normal fibroblast cells by reducing mitochondrial membrane potential in a concentration-dependent manner. This is the first study to reveal the antiproliferative effect ofP. vulgaris flower extract. Further studies are now needed to identify the cytotoxic molecules in the extract and their mechanisms.

Effects of allitridi on cell cycle arrest of human gastric cancer cells

AIM： To determine the effect of allitridi on cell cycle of human gastric cancer （HGC） cell lines MGC803 and SGC7901 and its possible mechanism. METHODS： Trypan blue dye exclusion was used to evaluate the proliferation, inhibition of cells and damages of these ceils were detected with electron microscope, Flow cytometry and cell mitotic index were used to analyze the change of cell cycle, immunohistochemistry, and RT- PCR was used to examine expression of the p21^WAF1 gene, RESULTS： MGC803 cell growth was inhibited by allitridi with 24 h ICso being 6.4μg/mL, SGC7901 cell growth was also inhibited by allitridi with 24 h IC50 being 7.3μg/mL, After being treated with allitridi at the concentration of 12μg/mL for 24 h, cells were found to have direct cytotoxic effects, including broken cellular membrane, swollen and vesiculated mitochondria and rough endoplasmic reticula, and mass lipid droplet, When cells were treated with allitridi at the concentration of 3, 6, and 9μg/mL for 24 h, the percentage of Go/G1 phase cells was decreased and that of G2/M phase cells was significantly increased （P = 0.002） compared with those in the group, When cells were treated with allitridi at the concentration of 6μg/mL, cell mitotic index was much higher （P = 0.003） than that of control group, indicating that allitridi could cause gastric cancer cell arrest in M phase, Besides, the expression levels of p21^WAF1 gene of MGC803 cells and p21^WAF1 gene of SGC7901 cells were remarkably upregulated after treatment, CONCLUSION： Allitridi can cause gastric cancer cell arrest in M phase, and this may be one of the mechanisms for inhibiting cell proliferation, Effect of allitridi on cells in M phase may be associated with the upregulation of p21^WAF1 genes. This study provides experimental data for clinical use of allitridi in the treatment of gastric carcinoma.

Anoctamin 5 regulates the cell cycle and affects prognosis in gastric cancer

BACKGROUND Anoctamin 5(ANO5)/transmembrane protein 16E belongs to the ANO/transmembrane protein 16 anion channel family.ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid scrambling and regulate other membrane proteins in numerous cell types.Previous studies have elucidated the roles and mechanisms of ANO5 activation in various cancer types.However,it remains unclear whether ANO5 acts as a plasma membrane chloride channel,and its expression and functions in gastric cancer(GC)have not been investigated.AIM To examine the role of ANO5 in the regulation of tumor progression and clinicopathological significance of its expression in GC.METHODS Knockdown experiments using ANO5 small interfering RNA were conducted in human GC cell lines,and changes in cell proliferation,cell cycle progression,apoptosis,and cellular movement were assessed.The gene expression profiles of GC cells were investigated following ANO5 silencing by microarray analysis.Immunohistochemical staining of ANO5 was performed on 195 primary tumor samples obtained from patients with GC who underwent curative gastrectomy between 2011 and 2013 at our department.RESULTS Reverse transcription-quantitative polymerase chain reaction(PCR)and western blotting demonstrated high ANO5 mRNA and protein expression,respectively,in NUGC4 and MKN45 cells.In these cells,ANO5 silencing inhibited cell proliferation and induced apoptosis.In addition,the knockdown of ANO5 inhibited G1-S phase progression,invasion,and migration.The results of the microarray analysis revealed changes in the expression levels of several cyclin-associated genes,such as CDKN1A,CDK2/4/6,CCNE2,and E2F1,in ANO5-depleted NUGC4 cells.The expression of these genes was verified using reverse transcription-quantitative PCR.Immunohistochemical staining revealed that high ANO5 expression levels were associated with a poor prognosis.Multivariate analysis identified high ANO5 expression as an independent prognostic factor for 5-year survival in patients with GC(P=0.0457).CONCLUSION ANO5 regulates the cell cycle progression by regulating the expression of cyclin-associated genes and affects the prognosis of patients with GC.These results may provide insights into the role of ANO5 as a key mediator in tumor progression and/or promising prognostic biomarker for GC.

Knockdown of GRHL3 Inhibits Activities and Induces Cell Cycle Arrest and Apoptosis of Human Colorectal Cancer Cells

The Grainyhead-like 3（GRHL3） is involved in epidermal barrier formation, neural tube closure and wound repair. Previous studies have suggested that GRHL3 has been linked to many different types of cancers. However, to date, its effects on human colorectal cancer（CRC） has not been clarified yet. Our microarray analysis has indicated predominant GRHL3 expression in CRC. The purpose of this study was to investigate the expression and significance of GRHL3 in CRC tumorigenesis using CRC tissues and paired paracancerous tissues, as well as using distinct CRC cell lines（HT29 and DLD1）. We observed increased GRHL3 expression at both m RNA and protein levels in CRC tissues and CRC cell lines using quantitative real-time polymerase chain reaction（q RT-PCR） and Western blotting. Moreover, silencing GRHL3 with si RNA could suppress CRC cell proliferation, viability and migration in vitro. We also found that knockdown of GRHL3 could promote cell cycle arrest at G0/G1 phase in HT29 cells and DLD1 cells, and induce cell apoptosis in HT29 cells. Together, our study revealed the down-regulation of GRHL3 in vitro could inhibit CRC cell activity and trigger cell cycle arrest at G0/G1 phase and apoptosis.