Propolis from the Stingless Bee Trigona incisa from East Kalimantan,Indonesia, Induces In Vitro Cytotoxicity and Apoptosis in Cancer Cell lines

Background: Previously, stingless bee (Trigona spp.) products from East Kalimantan, Indonesia, weresuccessfully screened for in vitro antiproliferative activity against human cancer derived cell lines. It wasestablished that propolis from T. incisa presented the highest in vitro cytotoxicity against the SW620 coloncancer cell line (6% cell survival in 20 μg/mL). Materials and Methods: Propolis from T. incisa was extractedwith methanol and further partitioned with n-hexane, ethyl acetate and methanol. The in vitro cytotoxicity ofthe extracts was assessed by the MTT assay against human colon (SW620), liver (Hep-G2), gastric (KATO-III),lung (Chago) and breast (BT474) cancer derived cell lines. The active fractions were further enriched by silicagel quick column, absorption and size exclusion chromatography. The purity of each fraction was checked bythin layer chromatography. Cytotoxicity in BT-474 cells induced by cardanol compared to doxorubicin wereevaluated by MTT assay, induction of cell cycle arrest and cell death by flow cytometric analysis of propidiumiodide and annexin-V stained cells. Results: A cardol isomer was found to be the major compound in one activefraction (F45) of T. incisa propolis, with a cytotoxicity against the SW620 (IC50 of 4.51 ± 0.76 μg/mL), KATO-III(IC50 of 6.06 ± 0.39 μg/mL), Hep-G2 (IC50 of 0.71 ± 0.22 μg/mL), Chago I (IC50 of 0.81 ± 0.18 μg/mL) and BT474(IC50 of 4.28 ± 0.14 μg/mL) cell lines. Early apoptosis (programmed cell death) of SW620 cells was induced bythe cardol containing F45 fraction at the IC50 and IC80 concentrations, respectively, within 2-6 h of incubation.In addition, the F45 fraction induced cell cycle arrest at the G1 subphase. Conclusions: Indonesian stingless bee(T. incisa) propolis had moderately potent in vitro anticancer activity on human cancer derived cell lines. Cardolor 5-pentadecyl resorcinol was identified as a major active compound and induced apoptosis in SW620 cells inan early period (≤ 6 h) and cell cycle arrest at the G1 subphase. Thus, cardol is a potential candidate for cancerchemotherapy.     

Curcumin-Galactomannoside Complex inhibits the Proliferation of Human Cervical Cancer Cells: Possible Role in Cell Cycle Arrest and Apoptosis

Background: Cervical cancer is the most common cancer and has the highest morbidity rate of gynaecological malignancies in women worldwide. So, the development of effective anti-cancer agents to treat this condition is vital. Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade, free-curcuminoids (curcumin-galactomannoside complex; CGM) on cervical cancer cells (HeLa) of human origin. In this study, we examined the anticancer potential of CGM as well as its effects on the cell cycle and the apoptosis of HeLa cancer cell. Methods: Determination of anti-proliferative and apoptosis validation of CGM on HeLa cells was performed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT), acridine orange/propidium iodide and annexin-V-fluorescein isothiocyanate assays. Measurement of Reactive Oxygen Species (ROS) production, Caspase activities and protein expression experiments were performed to investigate the potential mechanisms of action in the apoptotic process. Results: The cytotoxic assays revealed that the CGM showed inhibition of cell survival and exhibited high cytotoxic activity against HeLa cells at 25 μg/mL. Further studies on morphological changes were done in CGM-treated cervical cancer cells contributing to apoptosis. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that CGM induced apoptosis in HeLa cell lines at 25 μg/mL. By the supplementation of CGM showed an increase in Bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. Conclusion: The evidence obtained from this study suggests that CGM is a potent and promising natural formulation against cervical cancer cells via induction of apoptosis through ROS mediated mitochondrial damage in HeLa cells. Hence, CGM could be further explored as a potential lead in treating cancer.     

The Cytotoxic,Apoptotic Induction,and Cell Cycle Arrest Activities of Solanum nigrum L. Ethanolic Extract on MCF-7 Human Breast Cancer Cell

Objective: The purpose of this research was to evaluate the cytotoxic, cell cycle arrest, and apoptotic induction activities of the fruit of S. nigrum L. ethanolic-70% extract against MCF-7 human breast cancer cell. Methods: S. nigrum L. ripe fruit was blended and macerated with ethanol 70% and the filtrate was evaporated. The semisolid extract was then analyzed phytochemically. Cytotoxic analysis was performed using MCF-7 cancer and Vero normal cell by MTT method and followed by apoptotic and cell cycle arrest analysis using flow cytometry. Results: The phytochemical analysis resulted that extract contained total phenolic and flavonoid compounds with the level of 1.545±0.080% and 0.212±0.002%, respectively. Glycitin was the highest level of isoflavone compound, namely, 375.0844 mg/100 g extract. The cytotoxic evaluation revealed that the extract exhibited a selectively toxic effect between cancer and normal cell. The extract inhibited MCF-7 proliferation with IC50 value about 40.77±4.86 μg/mL and conversely toward Vero cell at lower cytotoxic activity with an IC50 value of 298.96±27.28 μg/mL. Evaluation of MCF-7 cell cycles demonstrated that the extract arrested the cell cycle in the S phase and continued to the G2/M phase at the half of the IC50 value. The extract induced apoptotic of MCF-7 cell about 43.31% in which this activity was nearly the same with doxorubicin as a positive control (59.14%). However, solamargine was predicted as the most active anticancer compounds by a molecular docking study so that it was suggested to measure the level of this compound. Conclusion: It can be concluded that the fruit of S. nigrum L. ethanolic-70% extract demonstrated cytotoxic activity toward MCF-7 breast cancer cell and nontoxic on Vero normal cell. Solamargine was predicted as the most active anticancer compound. This extract had an opportunity to be developed as a potential anticancer agent to overcome breast cancer diseases.     

Dual antitumor effects of 5-fluorouracil on the cell cycle in colorectal carcinoma cells: a novel target mechanism concept for pharmacokinetic modulating chemotherapy

5-Fluorouracil (5-FU) is one of the most widely used anticancer agents for advanced colorectal carcinoma, but its response rate is only 15%. The "pharmacokinetic modulating chemotherapy" (PMC) regimen that we have advocated has proved to be highly effective in treating colorectal carcinoma. PMC consists of a continuous i.v. infusion of 5-FU over 24 h for 1day a week at 600 mg/m2/day, and an oral dose of uracil-tegafur (UFT), a 5-FU derivative, at 400 mg/day for 5-7 days per week, repeated every week for more than 6 months. Assays of 5-FU in 23 patients receiving this treatment showed serum concentrations ranging from 88 to 1,323 ng/ml. We then analyzed the effects of clinically relevant concentrations of 5-FU found in colorectal cancer patients treated with the PMC regimen on the growth of three human colorectal adenocarcinoma cell lines, SW480 and COLO320DM (mutant p53) and HCT116 (wild-type p53). Exposure of these three cell lines to 5-FU resulted in growth inhibition in a dose-dependent manner. Exposure to 100 ng/ml of 5-FU in SW480 and COLO320DM caused G1 arrest after 24 h and G2 arrest after 72-144 h, and only a minority of the cell population showed apoptotic features, which indicated that most of the cells were killed through mitotic catastrophe, nonapoptotic cell death. On the contrary, exposure to 1000 ng/ml of 5-FU in SW480 and COLO320DM resulted in G1-S-phase arrest and the induction of apoptosis throughout the experimental period. Nuclear cyclin B1 expression was markedly induced with exposure to 100 ng/ml of 5-FU in SW480 and COLO320DM; and expression of 14-3-3sigma protein, a cell cycle inhibitor in the GG phase, was induced in SW480. ICT116 responded to lower concentrations of 5-FU more rapidly: G2 arrest was seen after 24-72 h of exposure to 10 ng/ml of 5-FU, and G,1rrest was seen after 12-24 h of exposure to 100 ng/ml. These results show that 5-FU acts via two different pathways, depending on dose: (a) G,1S-phase cell cycle arrest and apoptosis at 1,000 ng/ml in SW480 and COLO320DM, and 100 ng/ml in HCT116; and (b) G2-M-phase cell cycle arrest and mitotic catastrophe at 100 ng/ll in SW480 and COLO320DM, and 10 ng/ml in HCT116. These results suggest that the efficacy of our PMC regimen is based on targeting at least two different phases of the cell cycle. In our clinical trial, we showed efficacy independent of p53 status, ascertained by cell kinetic analysis in vitro, which may lead to a novel concept of schedule-oriented biochemical modulation of this drug.     

褪黑素通过促凋亡协同增强顺铂对人喉癌细胞增殖的抑制作用

目的:研究褪黑素(melatonin,MT)对人喉癌细胞增殖与凋亡的影响及MT增强人喉癌细胞对顺铂(cisplatin,DDP)治疗的敏感性.方法:采用不同质量浓度MT和DDP单独或联合处理Hep-2细胞;通过CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡和细胞周期,采用两药相互作用指数(co-efficient of drug interaction,CDI)评估MT是否影响Hep-2细胞对DDP的敏感性.结果:CCK-8检测结果显示,单用MT或DDP可浓度依赖性抑制Hep-2细胞的增殖,MT可协同增强DDP对Hep-2细胞的增殖抑制作用(CDI＜1).流式细胞术检测细胞凋亡和细胞周期结果显示,MT可促进Hep-2细胞凋亡以及增加亚G1期细胞比例(P＜0.01),MT可协同DDP促进Hep-2细胞凋亡[0.5 mmol/L MT联合20μg/ml DDP组的细胞凋亡率显著高于20 μg/ml DDP组,(40.9±3.0)％vs(11.0±0.9)％,P＜0.01]以及亚G1期细胞比例[0.5 mmol/L MT联合20 μg/mlDDP组的亚G1期细胞比例显著高于20 μg/ml DDP组,(73.0±2.4)％vs(40.4±3.0)％,P＜0.01].加入Caspase抑制剂Z-VAD-fmk可逆转MT和/或DDP对Hep-2细胞的增殖抑制作用和凋亡诱导作用(均P＜0.01).结论:MT能以Caspase依赖的方式诱导人喉癌细胞Hep-2的凋亡,从而协同增强DDP对细胞的增殖抑制作用.     

Diallyl sulfide induces cell cycle arrest and apoptosis in HeLa human cervical cancer cells through the p53, caspase- and mitochondria-dependent pathways

Diallyl sulfide (DAS), one of the main active constituents of garlic, causes growth inhibition of cancer cells in vitro and promotes immune responses in vivo in experimental settings. However, its effects on the induction of cell cycle and apoptosis in human cervical cancer cells are still unclear. The aims of this study were to explore the anti-cancer effects of DAS in HeLa human cervical cancer cells and to investigate the underlying mechanisms in vitro. Cytotoxicity and apoptosis in HeLa human cervical cancer cells were examined by the morphological changes, viability assay, 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining, comet assay, Western blotting and confocal microscopy examination. The results showed that DAS treatment for 24-72 h resulted in a marked decrease in cell viability time- and dose-dependently. Flow cytometric analysis showed that a 48-h treatment of 75 μM DAS induced G0/G1 cell cycle arrest and sub-G1 phase (apoptosis) in HeLa cells. Typical apoptotic nucleus alterations were observed by fluorescence microscopy in HeLa cells after exposure to DAS using DAPI staining. Cells treated with different concentrations of DAS also showed changes typical of apoptosis such as morphological changes, DNA damage and fragmentation, dysfunction of mitochondria, cytochrome c release and increased expression of pro-caspase-3 and -9. DAS also promoted the release of AIF and Endo G from mitochondria in HeLa cells. In conclusion, DAS induced G0/G1 cell cycle arrest and apoptosis in HeLa cells through caspase- and mitochondria and p53 pathways providing further understanding of the molecular mechanisms of DAS action in cervical cancer. This study, therefore, revealed that DAS significantly inhibits the growth and induces apoptosis of human cervical cancer HeLa cells in vitro.     

Taxol Produced from Endophytic Fungi Induces Apoptosis in Human Breast,Cervical and Ovarian Cancer Cells

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasingdemand on the market because yews grow very slowly and are a rare and endangered species belonging to firstlevelconservation plants. Recently, increasing efforts have been made to develop alternative means of taxolproduction; microbe fermentation would be a very promising method to increase the production scale of taxol.To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibitingthe growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark ofyew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNAagarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breastcancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that thefungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner.IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were 0.1-1.0 μg/ml, 0.001-0.01 μg/ml and 0.01-0.1 μg/ml, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptoticcharacteristics, including morphological changes for chromatin condensation, chromatin crescent formation,nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the 0.01-1.0 μg/ml had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extractedfrom yews. This study offers important information and a new resource for the production of an importantanticancer drug by endofungus fermentation.     

青藤碱对肠癌细胞的抑制作用

目的:观察青藤碱对肠癌细胞的抑制作用。方法:选取HCT116细胞分3组，分别用DMSO（对照组）、20 μmol/L和40 μmol/L的青藤碱以及6 μmol/L的5氟尿嘧啶（5-FU）处理细胞。MTT检测不同浓度药物对HCT116细胞增殖的影响。PI染色技术和JC-1染色分别检测药物对细胞周期和凋亡的作用。通过Western blot检测青藤碱对HCT116细胞中Cyclin D1和bcl-2的影响。Transwell实验检测青藤碱对HCT116细胞迁移的影响。通过Western blot检测青藤碱对HCT116细胞中MMP2的影响。结果:与DMSO相比，20 μmol/L和40 μmol/L的青藤碱以及6 μmol/L的5-FU作用于HCT116细胞24 h后对细胞的抑制率分别为:(33.16±6.01)%、(47.48±2.32)%和(62.31±3.26)%。青藤碱抑制G1-S期转化，促进细胞凋亡。Western blot结果显示，青藤碱抑制Cyclin D1、bcl-2和MMP2在HCT116细胞中的表达。结论:青藤碱抑制HCT116细胞的生长和迁移。     

Anticancer Activity of Solvent Extracts of Hexogonia glabra against Cervical Cancer Cell Lines

Objective: In this study, we aimed to harness some solvent extracts of one wild mushroom Hexagonia glabra and test their anti-cancer activity against cervical human cell lines, namelyHeLa, SiHa, and CaSki. Methods: It includes cell morphological study by microscope, nuclear morphology by DAPI staining under fluorescence microscopy, apoptosis assay by fluorescence technique, anti-proliferation by MTT assay and expression of apoptotic and anti-apoptotic genes by Western blotting and cell cycle analysis was done. Results: The selected cervical cancer cells were treated separately with 150 µg/mL of three extracts, namely of ethanolic (EE), ethyl acetate (EAE), and water extract (WE) and exhibited features like round, shrink and dead. All extracts caused apoptosis in cell lines and EE had the highest effect in this regard. The percentages of apoptotic cells in HeLa, SiHa and CaSki, at the same concentration of EE were 79.23, 75.42, and 76.36% respectively. Cytotoxicity assay showed that all three extracts (50 – 250 μg/mL) were potent for inhibition of cell growth of three cell lines and again EE had the highest effect. The percentages of cell growth inhibition in HeLa, SiHa, and CaSki cells treated with EE at 24 h at 50 µg/mL were 45.79±4.11, 41.66±4.03, and 36.72±2.67, while they were 74.23±7.45, 62.31±5.97, and 54.23±5.04 at 150 µg/mL concentration. At 250 µg/mL concentration, the percentages of cell growth inhibition were 94.25 ±8.11, 90.02 ±8.67, and 85.43±6.21, respectively. The expression of apoptotic gene (Caspase 3, 9) and tumor guard gene (p53), as their proteins in Western blotting increased . However, anti-apoptotic BcL2 gene of all cell lines was decreased following treatment with extracts. In addition, the cell cycle analysis (CaSki cell) showed that treatment (EE) arrested at G2/M check point cell cycle. Conclusion: All extracts of this mushroom were active in arresting growth of three cell lines and EE had the highest effect, indicating that this mushroom can be a valuable source of anticancer agents.     

斑蝥提取物通过细胞周期G2/M期阻滞诱导人胆管细胞癌QBC939细胞凋亡

目的:研究斑蝥提取物(cantharis extract,CTE)体外诱导人胆管癌QBC939细胞凋亡作用及其对细胞周期分布的影响。方法:体外培养人胆管细胞癌QBC939细胞,CTE作用于QBC939细胞48小时,利用CCK-8法检测QBC939细胞增殖率并计算半数抑制浓度(IC50);光学显微镜下观察细胞形态的改变;AnnexinⅤ/PI双染法检测细胞凋亡情况;用PI染色检测对QBC939细胞周期分布的影响;Western blot法检测细胞凋亡相关蛋白及细胞周期相关蛋白表达变化。结果:CTE显著抑制QBC939细胞增殖,呈浓度及时间依赖性,处理48小时的IC50为4.16μg/ml。经光学显微镜下观察可见随着药物浓度增加,细胞密度减少,游离变圆的细胞明显增多。AnnexinⅤ/PI双染流式凋亡检测示,随着药物浓度的增加,凋亡细胞数量逐渐增加,药物处理组(1.25μg/ml、2.5μg/ml、5.0μg/ml)的细胞凋亡率分别为(9.52±1.01)%、(15.62±1.88)%、(46.03±4.88)%,与对照组(4.59±0.43)%比较,差异有统计学意义(P<0.05)。PI检测细胞周期示,CTE能将QBC939细胞周期阻滞在G2/M期,随药物浓度增加而增加,G2/M期细胞比例逐渐增加,与对照组比较差异有统计学意义,而G1期细胞比例逐渐减少。Western blot检测示,CTE处理组细胞增殖相关蛋白增殖细胞核抗原(PCNA)表达降低,相对蛋白表达量与对照组比较,差异有统计学意义(P<0.05);Bcl-2家族蛋白Bax和Bid表达增加,而Bcl-2、Mcl-1表达降低,抑制凋亡蛋白家族蛋白Survivin表达显著下降,凋亡相关蛋白半胱氨酸蛋白水解酶3(Caspase-3)表达下降,与对照组比较CTE处理组上述相对蛋白表达量差异有统计学意义(P<0.05);CTE处理组细胞周期相关蛋白Chk1表达无显著改变,相对蛋白表达量与对照组比较差异无统计学意义(P<0.05),磷酸化的Chk1蛋白及p53蛋白表达逐渐增加,相对蛋白表达量与对照组比较差异有统计学意义(P<0.05)。结论:CTE显著抑制QBC939细胞增殖,诱导QBC939细胞凋亡,其可能机制与CTE调节凋亡相关蛋白表达,同时调节肿瘤细胞周期相关。     

PXD101对人乳腺癌细胞MCF-7增殖及凋亡影响的机制探讨

  目的  探讨组蛋白去乙酰化酶抑制剂PXD101对人乳腺癌细胞MCF-7增殖、细胞周期及凋亡的影响及分子机制研究。  方法  应用不同浓度PXD101处理培养的乳腺癌细胞株MCF-7, 通过赛唑蓝比色(MTT)法和平板克隆形成实验检测药物对细胞增殖的影响; HoechSt33342荧光染色法观察细胞形态变化; 流式细胞仪PI染色法检测细胞周期变化以及AnnexinV-FITC/PI双染法检测细胞凋亡情况; Westen blot检测p21、CyclinB1、PARP、Bcl-2以及Bax的蛋白表达。  结果  PXD101以剂量时间依赖性抑制MCF-7细胞的增殖; 荧光显微镜观察发现细胞核碎裂, 出现凋亡小体; 0、0.1、1、10μmol/L PXD101作用24 h后, G₂/M期细胞比例增加, 分别为(12.66±1.55)%、(20.63±1.32)%、(23.20±1.82)%、(32.19±2.37)%(P < 0.05), 凋亡细胞也增加(P < 0.05);p21表达增多, CyclinBl表达减少, PARP剪切明显增加, Bcl-2表达减少, Bax表达增加。  结论  PXD101在体外条件下能够明显抑制乳腺癌MCF-7细胞的增殖, 诱导细胞周期阻滞及凋亡, 并呈剂量依赖性。      

单抗C225致人肺鳞癌H-520细胞生长抑制和凋亡增加的研究

目的：观察表皮生长因子受体单克隆抗体C225对人肺鳞癌细胞系（H-520）生长、凋亡和周期分布的影响。方法：流式细胞检测H-520细胞EGFR表达比例,将鼠抗人EGFR一抗和FITC标记的羊抗鼠二抗加入细胞悬液中,孵育、漂洗重悬细胞后流式检测。MTT法测定C225抑制H-520细胞生长最佳浓度和最佳时间,将不同浓度C225加入指数生长的细胞培养液中,继续培养一定时间（48h）,检测细胞生长抑制率。流式细胞仪检测细胞凋亡以及细胞周期。结果：H-520细胞中EGFR表达比例高达82.25%。C225抑制H-520细胞生长的最佳浓度是40nmol/L,最佳作用时间是72h。细胞凋亡实验结果显示,H-520细胞自然凋亡率为5.56%±0.62%,C225可使其凋亡百分率上升到13.75%±0.83%（P〈0.05）。细胞周期分布显示40nmol/L C225可使细胞阻滞于G0＋G1期,S期细胞比例下降。结论：C225对H-520细胞生长抑制作用,可能与细胞G0＋G1期阻滞后凋亡有关。     

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.     

In Vitro Cytotoxic Activity of Clinacanthus nutans Leaf Extracts Against HeLa Cells

Objective: This study was conducted to investigate the antiproliferative activity of extracts of Clinacanthus nutansleaves against human cervical cancer (HeLa) cells. Methods: C. nutans leaves were subjected to extraction using 80%methanol or water. The methanol extract was further extracted to obtain hexane, dichloromethane (DCM), and aqueousfractions. The antiproliferative activity of the extracts against HeLa cells was determined. The most cytotoxic extractwas furthered analyzed by apoptosis and cell cycle assays, and the phytochemical constituents were screened by gaschromatography-mass spectrometry (GC-MS). Results: All of the extracts were antiproliferative against HeLa cells, andthe DCM fraction had the lowest IC50 value of 70 μg/mL at 48 h. Microscopic studies showed that HeLa cells exposedto the DCM fraction exhibited marked morphological features of apoptosis. The flow cytometry study also confirmedthat the DCM fraction induced apoptosis in HeLa cells, with cell cycle arrest at the S phase. GC-MS analysis revealedthe presence of at least 28 compounds in the DCM fraction, most of which were fatty acids. Conclusion: The DCMfraction obtained using the extraction method described herein had a lower IC50 value than those reported in previousstudies that characterized the anticancer activity of C. nutans against HeLa cells.     

香加皮提取物抗肿瘤活性的研究

背景与目的： 研究中药香加皮提取物的抗肿瘤活性,探讨其抗肿瘤机制。 材料与方法： 采用噻唑蓝(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, MTT)法观察香加皮醇提物和水提物对MCF-7、TE-13、QG56、SMMC7721、T24、Hela、K562等肿瘤细胞增殖的抑制作用,应用流式细胞术检测肿瘤细胞的周期变化和对凋亡的影响。 结果： 香加皮醇提物和水提物均能明显抑制多种肿瘤细胞的增殖,呈浓度依赖性;醇提物的抑瘤作用(IC50<10 μg/ml)强于水提物(IC50<40 μg/ml),并且差异具有统计学意义(P<0.05)。香加皮醇提物可将MCF-7细胞阻滞于G0/G1期,并呈时间依赖性地诱导MCF-7细胞发生凋亡。10 μg/ml香加皮醇提物作用MCF-7细胞24 h,与对照组相比,G0/G1期细胞显著增多,作用48 h,凋亡率从对照组的0.70％±0.13％升高到13.54％±2.12％,两者之间的差异有统计学意义(P<0.05)。 结论： 香加皮提取物对体外肿瘤细胞的增殖具有显著的抑制作用,醇提物强于水提物,其抗瘤机制可能与阻滞肿瘤细胞周期和诱导凋亡有关。     

Toll样受体8在人宫颈癌细胞株HeLa中的表达及其激动剂对HeLa细胞增殖和凋亡的影响

目的 观察Toll样受体8(TLR8)在人官颈癌细胞株HeLa中的表达,探讨TLR8激动剂CL075对HeLa细胞增殖和凋亡的影响。方法 采用实时荧光定量聚合酶链反应(PCR)法,检测13种肿瘤细胞系中TLR8 mRNA和经CL075作用后HeLa细胞中环氧化酶2(COX-2)、Bcl-2和血管内皮生长因子(VEGF) mRNA的表达水平;采用免疫荧光技术对HeLa细胞中TLR8蛋白的表达进行定位观察;采用流式细胞术检测不同浓度CL075作用下HeLa细胞的细胞周期和凋亡的变化;采用四甲基偶氮唑蓝(MTT)法检测HeLa细胞的增殖状态。结果 与其他肿瘤细胞株相比,HeLa细胞中TLR8mRNA的表达水平最高,达703.7±20.6。TLR8蛋白主要定位于HeLa细胞的胞浆中。0.1、0.5和1.0 μg/ml的CL075分别作用于HeLa细胞48 h后,G2/M+S期细胞所占的比例逐渐增高,其中1.0μg/ml CL075作用组G2/M+S期细胞所占的比例最高,达(57.67±1.73)％,明显高于空白对照组[(39.02±2.33)％,P＜0.01]。经不同浓度的CL075处理后,HeLa细胞的凋亡水平与空白对照组相比,无明显改变(P＞0.05);但经顺铂处理后,凋亡细胞明显增多(P＜0.01)。MTT法检测结果显示,与空白对照组比较,CL075作用于HeLa细胞48和72 h后,HeLa细胞的增殖能力明显增强(P＜0.01)。CI075作用于官颈癌HeLa细胞24和48 h后,COX-2、Bcl-2和VEGF mRNA的表达水平明显升高(P＜0.05)。结论宫颈癌HeLa细胞中TLR8的表达水平及其与配体相互作用的信号,可能是肿瘤发生和发展的重要因素之一。TLR8可能是宫颈癌治疗的一个潜在靶点。     

Growth Inhibitory Activities of Crude Extracts Obtained from Herbal Plants in the Ryukyu Islands on Several Human Colon Carcinoma Cell Lines

There is increasing interest in the use of herbs for the treatment of human diseases including cancer. Therefore, ‍the purpose of this study was to determine whether crude extracts obtained from 44 herbal plants in the Ryukyu ‍Islands might contain components capable of inhibiting the growth of a variety of human colon carcinoma cell lines. ‍Leaves, roots and other parts of the plants were extracted with chloroform, and the crude extracts were dissolved in ‍dimethylsulfoxide and used for the experiments. Extracts of Hemerocallis fulva, Ipomoea batatas, Curcuma longa, ‍and Nasturium officinale caused marked dose-dependent growth inhibition, with IC50 values in the range of 10-80 ì žg/ml. With the HCT116 cell line, the extracts of Hemerocallis fulva and Ipomoea batatas induced G1 cell cycle arrest ‍after 48 h of treatment. In addition, we found that extracts of Curcuma longa, and Nasturium officinale induced ‍apoptosis in these cells after 48 h of treatment. The present studies are the first systematic examination of the growth ‍inhibitory effects of crude extracts obtained from herbal plants in the Ryukyu Islands. The findings provide evidence ‍that several plants in the Ryukyu Islands contain components that may have anticancer activity. ‍     

Bio-Effects of TiO2 Nanoparticles on Human Colorectal Cancer and Umbilical Vein Endothelial Cell Lines

Background: Due to the possible biomedical potential of nanoparticles, titanium dioxide nanoparticles (TiO2 NPs)have received great attention in cancer research. Although selectivity of cytotoxicity with TiO2 NPs in various cells isclinically significant comparisons of cancer and non-cancer cells have been limited. Therefore, we here studied exposureto TiO2 NPs in colorectal cancer cells (CRCs) and human umbilical vein endothelial cells (HUVECs). Methods: Aftercharacterization of TiO2 NPs, culture and treatment of cells (HCT116, HT29 and HUVEC), viability was assessed byMTT assay and in terms of morphological features. Acridine orange (AO) and propidium iodide (PI) assays were carriedout to estimate the incidence of apoptosis. The RT-PCR method was also employed to evaluate the expression of P53,Bax, Bcl-2 and Caspase 3. Results: Exposure to increasing concentrations of TiO2 NPs enhanced overall cell survivalof HCT116 cells and reduced the Bcl-2 and Caspase 3 expression while the ratio of Bax/Bcl-2 was down-regulated.TiO2 NPs at 400 and 50 μg/ml concentrations suppressed cell proliferation and induced apoptosis of HT29 cells andalso up-regulated P53 and Bax at the mRNA level, enhanced the Bax/Bcl-2 ratio and eventually up-regulated Caspase3 mRNA. Although, inhibition of cell proliferation in HUVECs was seen at 200 and 400 μg/ml TiO2 NPs, it was notmarked. Conclusion: TiO2 NPs have selective bio-effects on exposed cells with dose- and cell-dependent influence onviability. Cell proliferation in HCT116 as a metastatic colorectal cancer cell line appeared to be stimulated via multiplesignaling pathways, with promotion of apoptosis in less metastatic cells at 50 and 400 μg/ml concentrations. This wasassociated with elevated P53, Bax and Caspase 3 mRNA and reduced Bcl-2 expression. However, TiO2 NPs did notexert any apparent significant effects on HUVECs as hyperproliferative angiogenic cells.     

Diallyl trisulfide‐induced apoptosis in human cancer cells is linked to checkpoint kinase 1‐mediated mitotic arrest

Growth suppressive effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, against cultured human cancer cells correlates with checkpoint kinase 1 (Chk1)‐mediated mitotic arrest, but the fate of the cells arrested in mitosis remains elusive. Using LNCaP and HCT‐116 human cancer cells as a model, we now demonstrate that the Chk1‐mediated mitotic arrest resulting from DATS exposure leads to apoptosis. The DATS exposure resulted in G₂ phase and mitotic arrest in both LNCaP and HCT‐116 cell lines. The G₂ arrest was accompanied by downregulation of cyclin‐dependent kinase 1 (Cdk1), cell division cycle (Cdc) 25B, and Cdc25C leading to Tyr15 phosphorylation of Cdk1 (inactivation). The DATS‐mediated mitotic arrest correlated with inactivation of anaphase‐promoting complex/cyclosome as evidenced by accumulation of its substrates cyclinB1 and securin. The DATS treatment increased activating phosphorylation of Chk1 (Ser317) and transient transfection with Chk1‐targeted siRNA conferred significant protection against DATS‐induced mitotic arrest in both cell lines. The Chk1 protein knockdown also afforded partial yet statistically significant protection against apoptotic DNA fragmentation and caspase‐3 activation resulting from DATS exposure in both LNCaP and HCT‐116 cells. Even though DATS treatment resulted in stabilization and Ser15 phosphorylation of p53, the knockdown of p53 protein failed to rescue DATS‐induced mitotic arrest. In conclusion, the results of the present study indicate that Chk1 dependence of DATS‐induced mitotic arrest in human cancer cells is not influenced by the p53 status and cells arrested in mitosis upon DATS exposure are driven to apoptotic DNA fragmentation. © 2009 Wiley‐Liss, Inc.     

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells

Purpose  Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Methods  Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Results  Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20–100 μM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 μM) for 24 and 48 h. Panaxadiol (25 μM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 μM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Conclusions  Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.