Geranium thunbergii extract-induced G1 phase cell cycle arrest and apoptosis in gastric cancer cells

ABSTRACT

Geranium thunbergii is a traditional East Asian medicine for stomach diseases including dysentery and stomach ulcers in East Asia and has been reported to possess biological activity. The benefits of G. thunbergii in gastric cancer are unknown. In this study, we demonstrate that G. thunbergii extract suppresses proliferation and induces death and G1/S cell cycle arrest of gastric cancer cells. Proliferation was significantly inhibited in a time- and dose-dependent manner. Cell cycle arrest was associated with significant decreases in CDK4/cyclinD1 complex and CDK2/cyclinE complex genes expression. In addition, the protein expression of caspase-3 was decreased and that of activated poly (ADP-ribose) polymerase (PARP) was increased, which indicated apoptosis. The expressions of the Bax and Bcl-2, which are apoptosis related proteins, were upregulated and down-regulated, respectively. The results indicate that G. thunbergii extract can inhibit proliferation and induce both G/S cell cycle arrest and apoptosis of gastric cancer cells. Also, the induction of apoptosis involved the intrinsic pathways of the cells. Take the results, we suggest that G. thunbergii extract has anti-gastric cancer activity and may be a potential therapeutic candidate for gastric cancer.     

Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells

Background

Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.

Methods

OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H₂O₂) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.

Results

The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H₂O₂ or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H₂O₂ or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).

Conclusion

Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H₂O₂ or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.     

A Novel Compound,L34, Induced Apoptosis in Human Gastric Cancer Cells

Selectively apoptosis-targeting compounds in gastrointestinal cancers attract broad interest. Here, we investigated a synthetic sulfonamide, 4-bromo-N-(5-ethyl-5H-pyrido[4,3-b]indol-8-yl)benzenesulfonamide (L34). It showed high activity against gastric cancer cells SGC-7901, causing apoptosis, which was associated with downregulation of caspase-3 and XIAP, upegulation of cleaved caspase-3, and cleavage of PARP. Hence, L34 might be a potent chemotherapeutic agent against human gastric cancer.     

gamma-Tocotrienol induces mitochondria-mediated apoptosis in human gastric adenocarcinoma SGC-7901 cells

Tocotrienols are naturally occurring isoprenoid compounds highly enriched in palm oil, rice bran, oat, wheat germ, barley and rye. Tocotrienols have antioxidant properties as well as potent anticancer properties. In this study, the mechanisms underlying the apoptosis of gamma-tocotrienol on human gastric adenocarcinoma SGC-7901 cells were further studied, especially in correlation with the involvement of the apoptotic pathway. gamma-Tocotrienol inhibited SGC-7901 cell growth in a concentration- and time-dependent manner. The inhibitory effects of SGC-7901 cells were correlated with the DNA damage and arresting cell cycle at G(0)/G(1) phase in a time-dependent manner at 60 mumol/L concentration of gamma-tocotrienol. gamma-Tocotrienol induced activation of caspase-3 and increased the cleavage of the downstream substrate poly(ADP-ribose) polymerase. Furthermore, gamma-tocotrienol-induced apoptosis on SGC-7901 cells was mediated by activation of caspase-9. The data in this study suggested that gamma-tocotrienol could induce the apoptosis on human gastric cancer SGC-7901 cells via mitochondria-dependent apoptosis pathway. Thus, our findings revealed gamma-tocotrienol as a potential, new chemopreventive agent for human gastric cancer.     

3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1,2,4] triazolo [3,4-b][1,3,4] thiadiazine,a novel tubulin inhibitor,evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells

Gastric cancer and cervical cancer are two major malignant tumors that threaten human health. The novel chemotherapeutic drugs are needed urgently to treat gastric cancer and cervical cancer with high anticancer activity and metabolic stability. Previously we have reported the synthesis, characterization and identification of a novel combretastatin A-4 analog, 3-(3-methoxyphenyl)-6-(3-amino-4- methoxyphenyl) -7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine (XSD-7). In this study, we sought to investigate its anticancer mechanisms in a human gastric cancer cell line (SGC-7901 cells) and human cervical carcinoma cell line (HeLa cells). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that XSD-7 induced cytotoxicity in SGC-7901 and HeLa cells with inhibitory concentration 50 values of 0.11 ± 0.03 and 0.12 ± 0.05 µM, respectively. Immunofluorescence studies proved that XSD-7 inhibited microtubule polymerization during cell division in SGC-7901 and HeLa cells. Then, these cells were arrested at G2/M cell cycle and subsequently progressed into apoptosis. In further study, mitochondrial membrane potential analysis and Western blot analysis demonstrated that XSD-7 treatment-induced SGC-7901 cell apoptosis via both the mitochondria-mediated pathway and the death receptor-mediated pathway. In contrast, XSD-7 induced apoptosis in HeLa cells mainly via the mitochondria-mediated pathway. Hence, our data indicate that XSD-7 exerted antiproliferative activity by disrupting microtubule dynamics, leading to cell cycle arrest, and eventually inducing cell apoptosis. XSD-7 with novel structure has the potential to be developed for therapeutic treatment of gastric cancer and cervical cancer.     

A novel compound, L34, induced apoptosis in human gastric cancer cells

Selectively apoptosis-targeting compounds in gastrointestinal cancers attract broad interest. Here, we investigated a synthetic sulfonamide, 4-bromo-N-(5-ethyl-5H-pyrido[4,3-b]indol-8-yl)benzenesulfonamide (L34). It showed high activity against gastric cancer cells SGC-7901, causing apoptosis, which was associated with downregulation of caspase-3 and XIAP, upegulation of cleaved caspase-3, and cleavage of PARP. Hence, L34 might be a potent chemotherapeutic agent against human gastric cancer.     

白扁豆多糖对人胃癌细胞凋亡的作用及其机制

目的：探讨白扁豆多糖对人胃癌细胞凋亡的作用及其相关机制。方法：胃癌细胞HGC-27和SGC-7901经终浓度为16、8、4、2、1和0 μg/ml的白扁豆多糖作用24、48和72h,各设3个复孔。MTS法检测其增殖活性;分别取经4、0 μg/ml白扁豆多糖作用24h的HGC-27和SGC-7901细胞（各3个复孔）,JC-1染色观察线粒体膜电位,流式细胞仪分析细胞周期和凋亡率,QPCR法探讨Bcl-2、caspase-3和Bax基因的mRNA转录水平。结果：白扁豆多糖作用后,HGC-27和SGC-7901细胞线粒体膜电位降低;细胞增殖显著受抑制（P<0.01）,且效果与药物作用浓度和时间有关;细胞凋亡率分别为53.15%和38.77%,均较PBS处理组（8.07%和6.03%）明显增加（P<0.01）,而细胞周期无显著变化;同时,细胞内Bcl-2基因的转录水平明显受抑制,Bax和caspase-3基因的转录明显上调（P<0.01）。结论：白扁豆多糖可通过调节Bax-Bcl-2-caspase3通路,诱导胃癌细胞HGC-27和SGC-7901凋亡。     

Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Inhibition of two gastric cancer cell lines induced by fucoxanthin involves downregulation of Mcl-1 and STAT3

Fucoxanthin is a natural carotenoid that had never been previously demonstrated to have anti-tumor effect on human gastric adenocarcinoma SGC-7901 or BGC-823 cells. Here it was found to inhibit proliferation and induce apoptosis through JAK/STAT signal pathway in these cells; the mechanism by which this occurred was investigated. We find that fucoxanthin significantly increased the number of apoptotic cells by propidium iodide (PI) dye staining and flow cytometry. Fucoxanthin (50 or 75 μM) induced SGC-7901 cells cycle arrest at S phase, while BGC-823 cells arrest at G2/M phase. RT-PCR and western blot analysis revealed that the expressions of Mcl-1, STAT3 and p-STAT3 were obviously decreased by fucoxanthin in a dose-dependent manner. Synthetic siRNA targeting Mcl-1 was transfected into cells which had no effect on expressions of STAT3. After pretreatment with AG490 (50 μM) which led to blocking of the JAK/STAT signal pathway, the reductive expressions of Mcl-1, STAT3 and p-STAT3 caused by fucoxanthin were inhibited. This is the first analysis of effects on SGC-7901 and BGC-823 cells by fucoxanthin. Fucoxanthin can induce cell-cycle arrest and apoptosis in these cells. These effects involved downregulation of Mcl-1, STAT3 and p-STAT3. This work is significant for better understanding of mechanisms leading to human gastric adenocarcinoma formation and informing exploitation of anti-tumor marine drug, and for providing Mcl-1 and STAT3 as potential therapeutic targets for gastric adenocarcinoma.     

蓖麻根提取物对HepG2,NCI-H460和SGC-7901细胞增殖及凋亡作用的影响

探讨蓖麻根不同提取物对肝癌HepG2细胞株、肺癌NCI-H460细胞株和胃癌SGC-7901细胞株增殖及其凋亡的影响.采用MTT法检测蓖麻根不同提取物处理48h、72h对HepG2细胞、NCI-H460细胞和SGC-7901细胞增殖的抑制率;Hoechst 33258荧光染料染色法观察HepG2细胞凋亡,流式细胞术检测...     

Melatonin inhibits cell growth and migration,but promotes apoptosis in gastric cancer cell line,SGC7901

Abstract

The pineal hormone, melatonin (MLT), has been shown to have therapeutic effects in patients with gastric cancer; however, the mechanisms for the anti-cancer effects are unknown. We investigated the effects of melatonin on cell proliferation, apoptosis, colony formation and cell migration in the gastric adenocarcinoma cell line, SGC7901, using MTT assay, Hoechst 33258 staining, flow cytometry, western blot, caspase-3 activity assay, soft agar colony formation assay, and scratch-wound assay. Our results showed that melatonin could inhibit cell proliferation, colony formation and migration efficiency, and it promoted apoptosis of SGC7901 cells. Our findings suggest that the anti-cancer effects of melatonin may be due to both inhibition of tumor cell proliferation and reduction of the metastatic potential of tumor cells.     

Upregulation of periostin prevents P53-mediated apoptosis in SGC-7901 gastric cancer cells

Periostin is frequently upregulated in human cancers including gastric cancer and implicated in cancer cell proliferation, invasion, and epithelial–mesenchymal transition. This study was undertaken to investigate the effects of periostin overexpression on the chemosensitivity of gastric cancer cells. We constructed a stable cell line overexpressing periostin in SGC-7901 human gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that periostin had no influence on the proliferation of SGC-7901 cells. Compared to empty vector-transfected cells, overexpression of periostin rendered SGC-7901 cells more resistant to cisplatin or 5-fluorouracil (5-FU)-induced apoptosis, accompanying with less release of cytochrome c from mitochondria and diminished cleavage of caspase-3 and poly (ADP-ribose) polymerase. Periostin-overexpressing cells treated with cisplatin or 5-FU showed significantly (p < 0.05) decreased expression of Bax and p53 proteins and increased expression of Bcl-2 protein, when compared to drug-treated mock counterparts. Restoration of p53 expression by delivering wild-type p53 gene resulted in a marked increase in drug-induced apoptosis in periostin-overexpressing SGC-7901 cells. Periostin overexpression elevated the phosphorylation of Akt. Pretreatment of periostin-overexpressing cells with an Akt inhibitor, MK-2206, partially rescued periostin-mediated inhibition of p53 expression and drug resistance. Taken together, our data indicate that periostin confers protection against cisplatin or 5-FU-induced apoptosis in SGC-7901 cells, likely through modulating the Akt/p53 pathway, and thus represents a potential therapeutic target in gastric cancer.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

Casticin Potentiates TRAIL-Induced Apoptosis of Gastric Cancer Cells through Endoplasmic Reticulum Stress

Background

Casticin is one of the main active components obtained from Fructus Viticis and has been reported to exert anti-carcinogenic activity on a variety of cancer cells but the precise mechanism underlying this activity remains unclear.

Materials and Methods

Apoptotic activities of casticin (1.0 µmol/l) and TRAIL (25, 50 ng/ml) alone or in combination in the gastric cancer cell lines BGC-823, SGC-7901 and MGC-803 were detected by the use of a cell apoptosis ELISA detection kit, flow cytometry (FCM) with propidium iodide (PI) staining and activities of caspase-3, -8 and -9 by ELISA and cleavage of polyADP-ribose polymerase (PARP) protein using western blot analysis. Death receptors (DR) expression levels were evaluated using FCM analysis and western blotting. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to measure the increase in reactive oxygen species (ROS) levels in cells. Multiple interventions, such as siRNA transfection and pharmacological inhibitors were used to explore the mechanisms of these actions.

Results

Subtoxic concentrations of casticin significantly potentiated TRAIL-induced cytotoxicity and apoptosis in BGC-823, SGC-7901 and MGC-803 cells. Casticin dramatically upregulated DR5 receptor expression but had no effects on DR4 or decoy receptors. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by the co-application of TRAIL and casticin. Gene silencing of the CCAAT/enhancer binding protein homologous protein (CHOP) and pretreatment with salubrinal, an endoplasmic reticulum (ER) stress inhibitor, attenuated casticin-induced DR5 receptor expression, and apoptosis and ROS production. Casticin downregulated the expression levels of the cell survival proteins cFLIP, Bcl-2, XIAP, and survivin. In addition, casticin also induced the expressions of DR5 protein in other gastric cancer cells (SGC-7901 and MGC-803).

Conclusion/Significance

Casticin enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway.     

低氘环境对胃癌细胞生长影响的体外实验

目的：研究低氘环境对人胃癌细胞（SGC-7901）增殖的影响并初步探讨其相关机制。方法：用含不同氘浓度的蒸馏水（实验组：25 ppm;对照组：150 ppm）配制的RPMI-1640培养基培养胃癌细胞SGC-7901。分别在不同的时间点对两组细胞的增殖率、细胞周期及凋亡情况进行检测,用Western blot法对两组细胞的增殖细胞核抗原蛋白（PCNA）的表达进行检测。结果：低氘环境下SGC-7901细胞的增殖率比对照组低10%左右。低氘环境对细胞的划痕愈合能力及集落形成能力也有显著抑制作用（P<0.05）。流式细胞术检测结果显示,与对照组相比,低氘组的细胞G1期细胞的比例增加（P<0.01）,而其所处S期细胞的比例下降（P<0.05）,两组细胞间早凋及晚凋比率差异无统计学意义。Western blot的结果显示低氘环境下培养的胃癌细胞的PCNA的表达明显下降。结论：低氘环境能够抑制胃癌细胞的生长,这可能与低氘环境下胃癌细胞阻滞于G1期及下调其PCNA的表达有关。     

桦木酸对胃癌SGC-7901细胞凋亡的影响*

目的: 以人胃癌SGC-7901细胞为研究对象,探究桦木酸对其凋亡的影响。方法: 将人胃癌SGC-7901细胞分为4组,每组设置3个复孔,对照组未加入桦木酸,而三组实验组分别加入浓度为10 mg/L、20 mg/L及30 mg/L的桦木酸,将各组细胞放入5%的CO₂培养箱中培养48 h,激光共聚焦显微镜观察细胞形态变化;流式细胞术检测细胞凋亡率和线粒体膜电位变化;qRT-PCR和Western blot分别检测SGC-7901细胞凋亡相关基因Bcl-2、Bax和Caspase-3在mRNA和蛋白水平的表达。结果: 与对照组相比,终浓度为10 mg/L、20 mg/L、30 mg/L的桦木酸处理组,细胞发生皱缩、细胞核裂解并出现凋亡小体;细胞早期凋亡与晚期凋亡率显著增加(P＜0.05 or P＜0.01),线粒体膜电位明显降低(P＜0.05 or P＜0.01);细胞凋亡相关基因Bax和Caspase-3的mRNA与蛋白表达水平均显著上升(P＜0.01),而Bcl-2的mRNA与蛋白表达水平显著降低(P＜0.01)。结论: 在一定浓度范围内,桦木酸通过调节凋亡相关基因Bcl-2、Bax和Caspase-3的表达诱导人胃癌SGC-7901细胞凋亡。     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.