Preparation of Two Organoselenium Compounds and Their Induction of Apoptosis to SMMC-7221 Cells

Two organoselenium compounds: xylitol selenious ester (xylitol-Se) and sucrose selenious ester (sucrose-Se) were synthesized, and their molecular structures were characterized in this study. In MTT assay, xylitol-Se and sucrose-Se showed cytostatic effects on human hepatocellular carcinoma cells SMMC-7221 in a dose-dependent manner, whereas they had no negative influences on the proliferation of human normal hepatic cells HL-7702 in the concentration range from 0.15 to 1.2 ppm Se. Morphological observation, agarose gel electrophoresis, and caspase-3 assay indicated that xylitol-Se and sucrose-Se induced mitochondrial apoptosis to SMMC-7221 cells, which is supported by the depletion of mitochondrial membrane potential and suppression of caspase-3 activity, indicating their ability of inducing apoptosis to cancer cells and great potentials as anticancer drugs.     

Daidzin inhibits growth and induces apoptosis through the JAK2/STAT3 in human cervical cancer HeLa cells

Daidzin, 4′, 7-dihydroxyisoflavone is an isoflavonic phytoestrogen present in leguminous plants. Traditional Chinese medicine utilizes daidzin to treat various diseases such diarrhea, fever, hepatitis, cardiac problems etc. In current study we examined the anticancer activity of daidzin against human cervical cancer in vitro. HeLa, human cervical cancer cell line was purchased from ATCC and the cells were cultured with DMEM medium. The cytotoxic effect of daidzin against HeLa cell line was analyzed with MTT assay. The IC-50 value was obtained at 20 µM hence the cells were treated with 20 µM of daidzin for further analysis. ROS generation was assessed with DCFH-DA staining and the induction of apoptosis was examined with Rhoadmine-123 staining. Acridine orange and ethidium bromide staining was done to examine the apoptotic and viable cells. Further the matrigel cell adhesion assay was done to analyze the inhibitory property of daidzin against cancer cell adhesion. Apoptotic induction of daidzin was examined by estimating the levels of Caspase 8 & 9 using ELISA technique. Inflammatory and cell proliferation signaling proteins were analyzed with qPCR analysis to confirm the anticancer activity of daidzin against human cervical cancer HeLa cell line. Daidzin significantly generated ROS and altered the mitochondrial membrane permeability in HeLa cell line. The results of AO/EtBr staining prove daidzin induced apoptosis in HeLa cell line and it also inhibited the cell adhesion property of HeLa which is reported in our matrigel cell adhesion assay. It also increased the caspases 8 & 9 which are key regulators of apoptosis. Daidzin significantly decreased the expression of inflammatory gene and cell proliferating signaling molecule. To, conclude our results confirm daidzin effectively decreased inflammation and induced apoptosis in human cervical cancer HeLa cell line.     

Antiproliferation and apoptosis of human tumor cell lines by a lectin (AMML) of Astragalus mongholicus

A lectin (AMML) from the roots of Astragalus mongholicus was extracted and purified by affinity chromatographic technique. Human cervical carcinoma cell line (HeLa), human osteoblast-like cell line (MG63) and human leukemia cell line (K562) were used to check the effects of AMML on cell proliferation, apoptosis and cell cycle. Maximum growth inhibition (92%) was observed with HeLa cells, followed by K562 cells (84%) and MG63 (48%) cells. Morphological observation showed that AMML-treated HeLa cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation and appearance of membrane-enclosed apoptotic bodies. The apoptosis of HeLa cells was confirmed by flow cytometry using Annexin V/FITC and propidium iodide (PI) staining technique. For the first time we also report a significant cell cycle arrest at S phase of HeLa cells by AMML. Therefore, the present investigation may lead to the possible therapeutic use of Astragalus mongholicus lectin.     

CEA启动子控制HSV—TK基因表达对人结直肠癌细胞的杀伤作用

An expression plasmid pCEA-TK, in which HSV-TK gene was under the control of CEA promoter, was constructed. The human colorectal carcinoma cell line LoVo or the human uterine cervical cancer cell line HeLa was co-transfected with pSV2-neo and pCEATK, respectively. After G418 selection, both transgenic cell clones (LoVo/CEATK and HeLa/CEATK) were obtained. LoVo/CEATK cells were 1300 times more sensitive to the cytotoxicity of ganciclovir than LoVo cells. However, the elevation of GCV sensitivity induced by pCEATK gene in HeLa line was only 8 times. Injection of GCV resulted in significant regression of HSV-TK transfected LoVo tumor in nude mice. These data suggested that the expression of TK gene driven by CEA promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electromicroscopy and DNA fragmentation assay demonstrated that GCV could induce apoptosis in LoVo/CEATK cells. The possibility of the CEATK/GCV system in the treatment of human colorectal carcinoma was discussed.     

CEA启动子控制HSV-TK基因表达对人结直肠癌细胞的杀伤作用

构建了以CEA启动子控制的HSV-TK基因表达质粒pCEA-TK。转染pCEA-TK的人结肠癌细胞LoVo对GCV的敏感性提高了1300倍。同样条件下,人宫颈癌细胞HeLa对GCV的敏感性仅提高8倍,且对低于血药浓度(20μmol/L)的GCV不敏感。以上结果显示在GCV存在时,CEA启动子控制下HSV-TK基因的表达使CEA阳性的人结直肠癌细胞获得专一性杀伤。此外,DNA片段分析和电镜观察表明GCV诱导转染pCEA-TK的LoVo细胞发生凋亡可能是这个系统杀死肿瘤细胞的机制之一。本工作还讨论了癌胚抗原(CEA)基因启动子用于人结直肠癌专一性自杀基因治疗的可能性。     

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.     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.     

Study of the effects of a new pyrazolecarboxamide: Changes in mitochondria and induction of apoptosis

Drug resistance of cancer cells is often correlated with the evasion of apoptosis, thus a major goal in cancer research is to search for compounds able to counteract cancer by promoting apoptosis. A variety of compounds with anticancer activity are characterised by the presence of the pyrazole as core nucleus. We synthesised a panel of pyrrolyl-pyrazole-carboxamides and we focused on the new compound RS 2780 (N-2-phenylethyl 1-(4-chlorophenyl)-3-methyl-5-pyrrolylpyrazole-4-carboxamide). The biological effects of RS 2780 on cell proliferation and viability were first evaluated on human HeLa cancer cells. As revealed by cell growth and viability experiments, a 24-h treatment of HeLa cells with increasing concentrations of RS 2780 (ranging from 0.1 to 100 μM) proved to inhibit cell proliferation and to affect cell viability. Notably, the new compound was effective also on colon carcinoma SW613-B3 cells, which are extremely resistant to most drugs, while it does not alter the proliferation of normal fibroblasts. We observed that RS 2780 interferes with the structural and functional properties of mitochondria, leading to the activation of the mitochondria-dependent apoptotic pathway. Apoptosis occurrence was supported by a number of morphological and biochemical hallmarks, including chromatin condensation, internucleosomal DNA fragmentation, PARP-1 cleavage and caspase activation. In conclusion, our results demonstrate for the first time the antiproliferative properties of the new compound RS 2780 on HeLa and SW613-B3 cancer cells and show that its effects on mitochondria lead to apoptosis.     

LIV-1 suppression inhibits HeLa cell invasion by targeting ERK1/2-Snail/Slug pathway

It was reported that expression of the estrogen-regulated zinc transporter LIV-1 was particularly high in human cervical cancer cell line HeLa. This result prompted us to study the role that LIV-1 played in human cervical cancer. The results of real-time PCR showed that LIV-1 mRNA was significantly higher in cervical cancer in situ than in normal tissues. RNAi mediated suppression of LIV-1 in HeLa cells significantly inhibited cell proliferation, colony formation, migration, and invasive ability, but had no effect on cell apoptosis. Furthermore, LIV-1 suppression is accompanied by down-regulation of p44/42 MAPK, phospho-p44/42 MAPK, Snail and Slug expression levels. Hence, our data provide the first evidence that LIV-1 mRNA is overexpressed in cervical cancer in situ and is involved in invasion of cervical cancer cells through targeting MAPK-mediated Snail and Slug expression.     

虎杖提取物对人胰腺癌细胞系Panc-1增殖与凋亡的影响

目的:通过虎杖提取物干预人胰腺癌细胞系Panc-1,探讨虎杖提取物对人胰腺癌细胞增殖凋亡表型的影响。方法:制备不同浓度(0、10、50、100、150、200μg/m L)的虎杖提取物,将各个浓度的虎杖提取物分别加入待处理的人胰腺癌Panc-1细胞系中持续培养24 h后,利用CCK-8(cell counting kit-8)法检测细胞株Panc-1的增殖活性;将100μg/m L虎杖提取物处理人胰腺癌细胞系Panc-124 h后,利用流式细胞术(FCM)检测其细胞周期及凋亡分布;100μg/m L虎杖提取物处理人胰腺癌细胞株Panc-124 h后,提取细胞总RNA及总蛋白,后续利用实时荧光定量PCR及Western blot分别检测人胰腺癌细胞株Panc-1增殖标志基因PCNA、CDK2及凋亡标志基因BAD、BAX的转录和翻译水平。结果:CCK-8结果表明虎杖提取物对人胰腺癌细胞系Panc-1细胞增殖的抑制率随浓度增加;流式细胞术结果显示虎杖提取物抑制人胰腺癌细胞增殖促进其凋亡;荧光定量PCR和Western blot结果显示虎杖提取物能使人胰腺癌细胞增殖标志基因PCNA,CDK2表达量下降,凋亡标志基因BAD,BAX表达量上升。结论:虎杖提取物能够抑制人胰腺癌细胞系Panc-1细胞增殖并促进其凋亡。     

Cellular and molecular mechanisms of silibinin induces cell-cycle arrest and apoptosis on HeLa cells

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models.     

Pardaxin-induced apoptosis enhances antitumor activity in HeLa cells

Pardaxin, a pore-forming antimicrobial peptide that encodes 33 amino acids was isolated from the Red Sea Moses sole, Pardachirus mamoratus. In this study, we investigated its antitumor activity in human fibrosarcoma (HT-1080) cells and epithelial carcinoma (HeLa) cells. In vitro results showed that the synthetic pardaxin peptide had antitumor activity in these two types of cancer cells and that 15 μg/ml pardaxin did not lyse human red blood cells. Moreover, this synthetic pardaxin inhibited the proliferation of HT1080 cells in a dose-dependent manner and induced programmed cell death in HeLa cells. DNA fragmentation and increases in the subG1 phase and caspase 8 activities suggest that pardaxin caused HeLa cell death by inducing apoptosis, but had a different mechanism in HT1080 cells.     

Benzothiazole thiourea derivatives as anticancer agents: Design,synthesis, and biological screening

In a systematic effort to identify a potent anticancer agent, we synthesized benzothiazole thiourea derivatives and examined their cytotoxic activity against five different human and animal cancer cell lines. Benzothiazolylthiocarbamides have been prepared in excellent yields by reaction of substituted 2-amino benzothiazoles with carbon disulfide and dimethyl sulfate followed by their ammonolysis. Cytotoxicity of the four compounds were screened for antitumor activity against human breast cancer cells (MCF-7), human cervix epithelial carcinoma (HeLa), human colon cancer cell line (HT-29), human leukemia cell line (K-562), and mouse neuroblastoma cell line (Neuro-2a) using cisplatin as a reference by MTT assay. Our results presented herein provide experimental evidence that benzothiazolylthiocarbamides induce apoptosis in cancer cell lines. According to flow cytometry results, treatment of HT-29 cells with 1-(6-ethoxy-1,3-benzothiazol- 2-yl)thiourea produced a large population of apoptotic cell (79.45%), which was 1.2-fold higher than that produced by cisplatin (65.28%) at the same concentration.     

Loss of macrophage migration inhibitory factor impairs the growth properties of human HeLa cervical cancer cells

Objectives: This study aims to determine the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with cell proliferation and tumour growth in vivo. Materials and methods: Our team used RNA interference technology to knock down MIF expression in human HeLa cervical cancer cells and to establish a stable cell line lacking MIF function. Results: Our results showed that long‐term loss of MIF had little effect on cell morphology, but significantly inhibited their population growth and proliferation. The HeLa MIF‐knockdown cells retained normal apoptotic signalling pathways in response to TNF‐alpha treatment; however, they exhibited unique DNA profiles following doxorubicin treatment, suggesting that MIF may regulate a cell cycle checkpoint upon DNA damage. Our data also showed that knockdown of MIF expression in HeLa cells led to increased cell adhesion and therefore impaired their migratory capacity. More importantly, cells lacking MIF failed to either proliferate in soft agar or form tumours in vivo, when administered to nude mice. Conclusion: MIF plays a pivotal role in proliferation and tumourigenesis of human HeLa cervical carcinoma cells, and may represent a promising therapeutic target for cancer intervention.     

Isolation of ergosterol peroxide from Nomuraea rileyi infected larvae of tobacco cutworm

In the search for potential cytotoxic substances produced by Nomuraea rileyi, an active compound was isolated from mycosed insects through an activity guided fractionation process. The compound, cytotoxic against the Sf9 insect cell line, was identified to be ergosterol peroxide (5α, 8α-epidioxy-24(R)-methylcholesta-6, 22-dien-3β-ol) using nuclear magnetic resonance techniques, infrared spectrometry, and mass spectroscopy. Anticancer screens demonstrated that ergosterol peroxide at micromolar concentrations inhibited the growth of hormone-dependent breast cancer cell line (T47D), hormone-independent breast cancer cell line (MDA-MB-231), human epidermoid carcinoma in mouth cell line (KB), human cervical carcinoma cell line (HeLa), lung cancer cell line (H69AR) and human cholangiocarcinoma cell line (HuCCA-1). Ergosterol peroxide showed moderate effects against Spodoptera litura larvae; 46.7% mortality via topical application after 7 day post-treatment whereas the insect’s death was not found in per os application. The amounts of ergosterol peroxide produced by N. rileyi cultures under in vitro and in vivo were determined. The physiological levels of ergosterol peroxide detected in mycosed and mummified cadavers were very low (0.011 and 0.386 μg/larva) less then levels that either inhibited insect cell proliferation or caused insecticidal activity.     

PLK1基因沉默抑制HeLa细胞凋亡

以高表达Polo样激酶1（Polo-like kinase 1,PLK1）的宫颈癌细胞系HeLa细胞为模型,观察针对PLK1基因的短发夹状RNA（short hairpin RNA,shRNA）对其凋亡和增殖的影响.设计并合成了针对PLK1的shRNA,将其导入构建的携带增强型绿色荧光蛋白（EGFP）的RNAi（RNA interference）表达载体中.通过 RT-PCR和Western 印迹分别检测HeLa细胞PLK1基因和蛋白水平的表达,以流式细胞仪和PI-Hochest双染法测细胞凋亡,MTT法检测细胞的增殖水平.成功构建了携带EGFP的RNAi表达载体pEGFP-H1.转染shRNA后,HeLa细胞PLK1的表达降低至30%.与对照组和空载体转染组相比,shRNA转染组的HeLa细胞凋亡率明显增加,其增殖活性则明显降低.本课题构建的RNAi表达载体便于观察靶基因的转染情况,且不影响H1启动子的体内转录.PLK1的基因沉默能明显增加HeLa细胞的凋亡,抑制该细胞的增殖,有可能为未来肿瘤的治疗找到新的靶点和有效途径.     

Bone marrow stromal cell-derived growth inhibitor inhibits growth and migration of breast cancer cells via induction of cell cycle arrest and apoptosis

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.