Diverse resveratrol sensitization to apoptosis induced by anticancer drugs in sensitive and resistant leukemia cells

Naturally occurring dietary compound resveratrol (RES), possessing chemopreventive and cytostatic properties, has been shown as potent sensitizer for apoptosis induced by a variety of anticancer drugs. Cell cycle analysis in sensitive promyelocytic leukemia HL60 cell line and its multidrug-resistant variant HL60/VCR (P-gp positive) treated with RES resulted in cell cycle arrest in S-phase in both cell variants. Flow cytometry measurements showed diverse activities of RES in combination with anticancer drugs doxorubicin (DOX), cycloheximide (CHX), busulfan (BUS), gemcitabine (GEM) and paclitaxel (PTX), in some cases resulting in apoptosis induction, preferentially at the expense of S-phase. Thus, RES could become a candidate to enhance the efficacy of combination anticancer therapy in a variety of human cancer cells inclusive leukemias.     

Induction of apoptosis by norcantharidin in human colorectal carcinoma cell lines: involvement of the CD95 receptor/ligand

PURPOSE: Cantharidin, a natural toxin, is the active substance of mylabris and has antitumor effects in man. Norcantharidin, the demethylated analogue of cantharidin, has been used in the treatment of patients with primary hepatoma and those with leukopenia in China. The present study was designed to investigate whether norcantharidin exerts cytotoxic activity against colorectal cancer cells by inducing apoptosis and to examine the possible mechanism in the phenomenon. METHODS: Inhibition of proliferation of norcantharidin on Colo205, HT-29, and SW480 colorectal cancer cells was determined by the trypan blue dye exclusion test. Apoptosis of norcantharidin-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis, and quantitated by flow cytometry after staining with propidium iodide. Cell cycle and the cell surface expression of the CD95/CD95 ligand were evaluated by flow cytometry. Caspase 8-like protease and protein phosphatase 1 and 2A activities were also analyzed. RESULTS: Treatment with norcantharidin of colorectal cancer cells not only inhibited cell proliferation, but also induced apoptosis. Norcantharidin induced apoptosis mainly in two phases: rapid apoptosis in S-phase cells and delayed apoptosis in G2/M arrested cells. Treatment with norcantharidin resulted in an upregulation of the CD95 receptor and CD95 ligand on the cell surface. Furthermore, stimulation with anti-CD95 monoclonal antibody (mAb) resulted in further induction of apoptosis after treatment with norcantharidin. In addition, the apoptosis-inducing effect of norcantharidin was almost completely inhibited by anti-CD95 ligand mAb. Norcantharidin-treated cells showed the activation of caspase 8. Both zVAD-FMK (a broad range caspase inhibitor) and IETD-FMK (a caspase-8 inhibitor) showed apparent inhibition of the apoptosis-inducing effect. Norcantharidin did not show an inhibitory effect on protein phosphatase. CONCLUSIONS: These results suggest that norcantharidin triggers apoptosis in colorectal cancer cell lines via the activation of the CD95 receptor/ligand system, and that this agent may be useful for developing new therapeutic regimens for the treatment of colorectal carcinoma.     

Antiproliferation and apoptosis induction of paeonol in HepG2 cells

AIM To investigate the antiproliferative effect of paeonol (Pae) used alone or in combination with chemotherapeutic agents [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG2 and the possible mechanisms.METHODS The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay.Morphologic changes were observed by acridine orange (AO) fluorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). Drug-drug interactions were analyzed by the coefficient of drug interaction (CDI).RESULTS Pae (7.81-250 mg/L) had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of (104.77 7.28) mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae (15.63, 31.25, 62.5 mg/L) was combined with CDDP (0.31-2.5 mg/L), DOX (0.16-1.25 mg/L), or 5-FU (12.5-100 mg/L) at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae (P ＜0.01). Of the three different combinations, the sensitivity of cells to drugs was considerably different.CONCLUSION Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2,which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.     

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.     

Curcumin cytotoxicity is enhanced by PTEN disruption in colorectal cancer cells

AIM:To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10(PTEN) deficiency on the cytotoxicity of chemotherapeutic agents toward colorectal cancer cells.METHODS:PTEN-deficient colorectal cancer(CRC) cells were generated by human somatic cell gene targeting using the adeno-associated virus system. The cytotoxic effects of compounds including curcumin,5-fluorouracil(5-FU),dihydroartemisinin(DHA),irinotecan(CPT-11)and oxaliplatin(OXA) on cancer cells were determined using the MTT assay. Enhanced cytotoxicity of curcumin in PTEN-deficient CRC cells was observed,and this was confirmed using clonogenic assays. Apoptosis and cell cycle progression were analyzed by flow cytometry.Levels of apoptosis and cell cycle-related proteins were examined by Western blotting.RESULTS:We developed an isogenic set of CRC cell lines that differed only in their PTEN status. Using this set of cell lines,we found that disruption of the PTEN gene had no effect on the sensitivity of CRC cells to5-FU,CPT-11,DHA,or OXA,whereas PTEN disruption increased the sensitivity of CRC cells to curcumin. Loss of PTEN did not alter the curcumin-induced apoptosis in CRC cells. However,PTEN deficiency led to an altered pattern of curcumin-mediated cell cycle arrest.In HCT116 PTEN+/+cells,curcumin caused a G2/M phase arrest,whereas it caused a G0/G1 phase arrest in HCT116 PTEN-/-cells. Levels of cell cycle-related proteins were consistent with these respective patterns of cell cycle arrest.CONCLUSION:Curcumin shows enhanced cytotoxicity toward PTEN-deficient cancer cells,suggesting that it might be a potential chemotherapeutic agent for cancers harboring PTEN mutations.     

5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint

BACKGROUND & AIMS: Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. METHODS: CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. RESULTS: We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. CONCLUSIONS: Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.     

Tyrosine kinase of insulin-like growth factor receptor as target for novel treatment and prevention strategies of colorectal cancer

INTRODUCTION Colorectal cancer (CRC) is the third most common cancer and the fourth most frequent cause of cancer-related deaths worldwide. Long-term survival of colorectal cancer is related to the stage of disease. Once distal metastases develop the prog…     

舒林酸抑制结肠癌LOVO细胞增殖作用机制研究

目的 研究舒林酸对结肠癌细胞株LOVO生长的影响,并探讨其作用机制。方法 采用四甲基偶氮唑蓝（MTT）比色法检测舒林酸对结肠癌细胞增殖的抑制作用,流式细胞仪（FCM）分析其对细胞凋亡及细胞周期的影响,放免法分析其对前列腺素E2（PGE2）的影响。结果 MTT比色显示舒林酸能抑制LOVO细胞增殖,呈剂量和浓度依赖性;FCM显示舒林酸能促进细胞凋亡,使G0／G1期细胞比例升高,S期细胞比例降低;PGE2表达水平呈剂量依赖性下调。结论 舒林酸可抑制结肠癌细胞株LOVO生长,促进其凋亡;其作用机制可能与阻止细胞周期进展、抑制COX-2活性及PGE2合成有关。     

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 calci...     

大蒜素联合奥沙利铂对HCT-8细胞生长和增殖的影响及机制

目的探讨大蒜素与奥沙利铂联合应用治疗大肠癌的可行性及机制。方法将大蒜素和奥沙利铂单独及联合作用于人大肠癌细胞系HCT-8细胞,MTT法检测HCT-8细胞生长抑制率,流式细胞术检测细胞周期及细胞凋亡率,免疫细胞化学SP法检测Caspase-3蛋白表达,苏木素复染观察异常分裂细胞。结果联合大蒜素后,奥沙利铂对HCT-8的增殖抑制作用增强（P〈0．01）;细胞阻滞于G2／M期,且凋亡率升高（P〈0．01）;Caspase-3表达上凋（P〈0．01）;异常分裂细胞数增多（P〈0．01）。结论大蒜素能增强HCT-8细胞对奥沙利铂的化疗敏感性,此可能与大蒜素对HCT-8细胞的抑制增殖、细胞周期阻滞及促进凋亡作用有关。     

新疆蜂胶挥发油对结直肠癌HCT-116细胞增生、周期及凋亡的影响

目的:探讨新疆蜂胶挥发油对体外培养的大肠癌细胞HCT-116细胞增生、细胞周期和凋亡的影响.方法:采用四甲基偶氮唑盐(MTT)显色法,检测不同浓度蜂胶挥发油作用不同时间对HCT-116细胞生长所产生的不同影响;倒置显微镜观察凋亡细胞的形态特点:PI染色、流式细胞仪检测细胞周期分布;Annexin V-FITC/PI双染...     

ZNF278 siRNA抑制胃癌细胞株AGS增殖的实验研究

背景：ZNF278属C2H2型锌指蛋白,为参与生长发育的重要转录因子,其异常表达可能参与肿瘤发生。目的：研究ZNF278siRNA对胃癌细胞株AGS增殖和周期的影响。方法：以蛋白质印迹法检测正常胃上皮细胞株GES-1和胃癌细胞株AGS中ZNF278表达。构建ZNF278siRNA片段,并转染胃癌AGS细胞,转染阴性对照siRNA和RPMll640分别作为阴性对照组和空白对照组。以定量RT-PCR和蛋白质印迹法分别检测ZNF278mRNA和蛋白表达,利用MTT和细胞计数法检测细胞增殖情况,流式细胞术检测细胞周期变化。结果：胃癌AGS细胞中ZNF278表达明显高于正常胃上皮细胞GES-1。ZNF278siRNA转染胃癌ACTS细胞后,ZNF278mRNA和蛋白表达均明显下调,而阴性对照组与空白对照组无明显差异。MTT法示ZNF278siRNA组AGS细胞增殖率显著低于阴性对照组（0．64±0．03对0．81±0．03,P〈0．01）,细胞计数法示细胞数量亦显著降低[（4．11±0．35）×10^5对（5．78±0．50）×10^5,P〈0．01],流式细胞术显示ZNF278siRNA组G0／G1期细胞明显增加而s期细胞明显减少（P〈0．05）。结论：转染ZNF278siRNA可抑制胃癌AGS细胞增殖,细胞周期阻滞于G0／G1期。     

Effects of lysophosphatidic acid on human colon cancer cells and its mechanisms of action

AIM： To study the effects of lysophosphatidic acid （LPA） on proliferation, adhesion, migration, and apoptosis in the human colon cancer cell line, SW480, and its mechanisms of action. METHODS： Methyl tetrazolium assay was used to assess cell proliferation. Flow cytometry was employed to detect cell apoptosis. Cell migration was measured by using a Boyden transweU migration chamber. Cell adhesion assay was performed in 96-well plates according to protocol. RESULTS： LPA significantly stimulated SW480 cell proliferation in a dose-dependent and timeependent manner compared with the control group （P 〈 0.05） while the mitogen-activated protein kinase （MAPK） inhibitor, PD98059, significantly blocked the LPA stimulation effect on proliferation. LPA also significantly stimulated adhesion and migration of SW480 cells in a dosedependent manner （P 〈 0.05）. Rho kinase inhibitor, Y-27632, significantly inhibited the upegulatory effect of LPA on adhesion and migration （P 〈 0.05）. LPA significantly protected cells from apoptosis induced by the chemotherapeutic drugs, cisplatin and 5-FU （P 〈 0.05）, but the phosphoinositide 3-kinase （PI3K） inhibitor, LY294002, significantly blocked the protective effect of LPA on apoptosis. CONCLUSION： LPA stimulated proliferation, adhesion,migration of 5W480 cells, and protected from apoptosis. The Ras/Raf-MAPK, G12/13-Rho-RhoA and PI3K- AKT/PKB signal pathways may be involved.     

普伐他汀对人结肠癌细胞增殖及COX-2蛋白表达的影响

目的体外观察普伐他汀对人结肠癌细胞HT-29、Ls-174-T细胞增殖、细胞周期及COX-2蛋白表达的影响。方法采用噻唑蓝（MTT）法观察普伐他汀对HT-29和Ls-174-T细胞增殖的影响，流式细胞仪（FCM）研究普伐他汀对细胞周期的作用，免疫细胞化学观察COX-2蛋白的表达。结果体外普伐他汀可抑制HT-29和Ls-174-T细胞增殖，各处理组内G0／G1期细胞增多，但诱导凋亡不明显，体外普伐他汀可减少HT-29和Ls-174-T细胞株COX-2蛋白的表达。结论体外普伐他汀对HT-29和Ls-174-T细胞增殖有抑制作用，该作用可能与使细胞生长阻滞于G0／G1期及抑制COX-2蛋白表达有关。     

生存素反义寡核苷酸诱导肝癌细胞凋亡的实验研究

目的　生存素 (survivin)是近年来发现的凋亡抑制蛋白家族新成员 ,在多数恶性肿瘤组织中丰富表达。因此 ,观察生存素反义寡核苷酸转染对肝癌细胞凋亡、增殖、细胞对化疗药物敏感性的影响。方法　设计合成特异性靶向生存素的反义寡核苷酸 (ASODN)。肝癌细胞株hepG2 分为 6组 :空白对照组、脂质体转染对照组、正义链转染对照组、2 0 0、40 0和 6 0 0nmol/LASODN转染组。作用 2 0h后收获各组细胞。倒置显微镜观察细胞形态变化 ,Westernblot法检测各组细胞生存素表达情况 ,流式细胞术检测各组细胞增殖和凋亡指数 ,MTT法检测 5 氟尿嘧啶 (5 FU)和顺铂 (DDP)对各组细胞的生长抑制率。结果　各ASODN转染组细胞生存素表达有不同程度减弱 ,细胞变圆、折光增强、漂浮、细胞碎片形成等 ,而各对照组细胞生长良好 ;各ASODN转染组细胞凋亡指数明显高于各对照组 (P <0 .0 5 ) ,以 6 0 0nmol/L转染组最为明显 (P <0 .0 5 ) ,而各对照组间差异无显著性 (P >0 .0 5 ) ;各ASODN转染组细胞增殖指数明显低于各对照组 (P <0 .0 5 ) ,以 6 0 0nmol/L转染组最为明显 (P <0 .0 5 ) ,而各对照组间差异无显著性 (P >0 .0 5 ) ;等浓度化疗药物 5 FU和DDP对各ASODN转染组细胞的抑制率明显高于各对照组(P <0 .0 5 ) ,以 6 0 0nmol/L     

Selective killing of cancer cells by beta -lapachone: direct checkpoint activation as a strategy against cancer

Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that beta-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer.     

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of colorectal cancer cells in vitro

ABM: To study the effect of caffeic add phenethyl ester (CAPE) on proliferation, cell cycle, apoptosis and expression of β-catenin in cultured human colorectal cancer (CRC) cell line HCT116. METHODS: HCT116 cells were treated with CAPE at serial concentrations of 80,40,20,10,5,2.5 mg/L. The proliferative status of HCT116 cells was measured by using methaben-zthiazuron (MTT) assay. Cell cycle was analyzed by using flow cytometry (FCM) with propidium iodide (PI) labeling method. The rate of apoptosis was detected by using FCM with annexin V-FITC and PI double labeling method, β-catenin levels were determined by Western blotting, β-catenin localization in HCT116 was determined by indirect immunofluorescence. RESULTS: After HCT116 cells were exposed to CAPE (80, 40, 20, 10, 5, and 2.5 mg/L) for 24, 48, 72, 96 h, CAPE displayed a strong growth inhibitory effect in a dose- and time-dependent manner against HCT116 cells. FCM analysis showed that the ratio of G0/G1 phase cells increased, S phase ratio decreased and apoptosis rate increased after HCT116 cells were exposed to CAPE (10, 5, and 2.5 mg/L) for 24 h. CAPE treatment was associated with decreased cytoplasmic β-catenin, nuclear p-catenin and a concurrent increase in β-catenin protein expression at cell-cell junctions. CONCLUSION: CAPE could inhibit HCT116 cell proliferation and induce cell cycle arrest and apoptosis. Decreased β-catenin protein expression may mediate the anti-proliferative effects of CAPE.