珊瑚树Vibsane型二萜类化合物对人体HepG2细胞增殖的影响及其机制

目的：探讨珊瑚树vibsane型二萜类化合物对肝癌HepG2细胞增殖的影响及其机制,为研发新型天然植物类抗肿瘤药物提供实验依据。方法：采用噻唑蓝比色法及苔盼蓝染色计数法观察珊瑚树vibsane类二萜类化合物对不同肿瘤细胞增殖的影响;应用流式细胞仪检测细胞周期及细胞凋亡,利用Apo-ONE Homogeneous Caspase-3/7试剂盒检测vibsane二萜类化合物1#对HepG2细胞内Caspase-3酶活性的影响。结果：活性筛选发现vibsane型二萜类化合物1#显著抑制人肝癌HepG2细胞增殖,构效分析表明化合物C11位连接侧链的基团修饰影响其细胞增殖抑制活性。此外,HepG2细胞对1#化合物最敏感,1#化合物抑制其增殖具有剂量和时间依赖性。机制研究显示1#化合物诱导HepG2细胞发生明显的细胞周期G0/G1期阻滞,具有时间和剂量效应;同时,较高浓度1#化合物（5-10μmol/L）引起HepG2细胞凋亡明显增加,并剂量依赖性诱导细胞内Caspase3/7激活。结论：珊瑚树vibsane型二萜类化合物能够明显抑制人肝癌HepG2细胞增殖,其可能通过诱导细胞周期阻滞和细胞凋亡发挥抗肿瘤作用。     

Promising anti-growth effects of palladium(II) saccharinate complex of terpyridine by inducing apoptosis on transformed fibroblasts in vitro

Fibrosarcoma is one of the fatal cancer types and there is still not satisfactory success in its treatment despite new drugs. Therefore, the search for a new compound has been going on. It is currently known that some palladium-based anti-cancer compounds seem to have powerful apoptosis-inducing effects in cancer cells. For this purpose, a palladium(II)-saccharinate complex containing terpyridine which was synthesized by our research group was investigated in terms of its anti-tumor effects against mouse embryonic fibroblast NIH/3T3 (normal cell line) and rat embryonic fibroblast 5RP7 (H-ras transformed cell line) in vitro. The MTT and ATP viability assays were used to determine anti-growth/cytotoxic effects. Cytotoxic activity was confirmed by real time cytotoxicity analysis system. Flow cytometry analysis was further used to determine the mode of cell death (apoptosis/necrosis). Apoptosis was confirmed by triple-staining the cells with Hoechst 33342/PI/Calcein-AM triple and evaluated with fluorescence microscopy. It was found that the compound showed significant anti-growth activity by inducing apoptosis in a dose dependent manner. In conclusion, taking into account the cytotoxic activity of the compound at even relatively lower doses, in vivo experiments to elucidate its potential use for the treatment of fibrosarcoma are warranted.     

Rapid and highly sensitive detection of cadmium chloride induced cytotoxicity using the HSP70B' promoter in live cells

One unique to detect cytotoxicity is to utilize reporter gene assays for promoters that respond to stress-induced effects. In the present study, we discovered that the DNA sequence from nt -287 to +110 of the heat shock protein 70B' (HSP70B') gene could be used as a functional promoter to detect cytotoxicity of cadmium chloride. We thus detected cytotoxicity induced by cadmium chloride with the luciferase assay using this functional HSP70B' promoter, as well as the cell viability test based on the quantification of intracellular ATP. The luciferase assay using the functional HSP70B' promoter resulted in nearly maximal luciferase activity after only 12 h of exposure to cadmium chloride, however, with intracellular ATP quantification, the decrease in cell viability only reached a plateau after 24 h of exposure. Cytotoxicity detection limits for cadmium chloride with the functional HSP70B' promoter assay or cell viability based on ATP quantification were 130 ng/mL and 530 ng/mL, respectively. Our results therefore suggest that the novel reporter gene assay using a functional region of the HSP70B' promoter has significant advantages for the detection of cytotoxicity in terms of both speed and sensitivity, when compared to the cell viability test based on ATP quantification.     

A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death

The efficacy of distinct anti-cancer drugs used in the chemotherapy of human malignancies varies between tumor tissues and depends largely on the ability of the therapeutic agents to simultaneously inhibit cell proliferation and to eliminate malignant cells by apoptosis. Especially, detection of early apoptotic changes seems to be important because early stages of apoptosis differ from those of necrosis. Therefore, the development of a novel test allowing fast and concomitant screening of the anti-proliferative and pro-apoptotic action of a number of anti-cancer drugs is of great interest. For this purpose, we choose as an experimental model a well characterized anti-proliferative and pro-apoptotic effect of cisplatin (CP) on human cervical carcinoma HeLaS3 cells. As previously reported, exposure of HeLaS3 to CP resulted in a concomitant inhibition of cell proliferation and induction of apoptosis in a dose- and time-dependent manner. In the present study we performed two independent approaches. In the first approach, we examined the cell proliferation and activity of caspases-3/7 in two separate microtiter plates using the CellTiter-Glo Luminescent Cell Viability Assay and the Caspase-Glo 3/7 Assay, respectively. In the second approach, we determined the same parameters sequentially in one microtiter plate by a mutiplexing assay using CellTiter-Blue Cell Viability Assay and Caspase-Glo 3/7 Assay. The both approaches gave very similar results indicating that this new multiplexing assay offers an important advantage for simultaneous detection of cell number and activation of caspases-3/7. The new multiplexing assay offers a range of benefits over standard assays.     

香蕉皮抗氧化和抑制人肝癌HepG2细胞活性研究

对香蕉皮提取物（banana peel extraction,BPE）的体外抗氧化作用和抗肿瘤活性进行了研究。使用福林酚和硝酸铝法测定BPE中总多酚和总黄酮含量;通过检测DPPH和O₂^-自由基清除能力测定BPE的体外抗氧化活性;采用Alamar Blue法检测BPE对HepG2细胞活性的影响;荧光染色及流式技术检测HepG2细胞凋亡;免疫印迹技术检测凋亡蛋白的变化;Caspase活性检测试剂盒测定Caspase-3和Caspase-9的活性;Caspase-9和Caspase-3抑制剂验证BPE抗肿瘤相关的信号转导通路。结果表明,BPE中总多酚和总黄酮含量分别为39.23 ± 2.35 mg·L^-1和26.53 ± 1.97 mg·L^-1;BPE显示出一定的DPPH和O₂^-自由基清除能力（65.31%±3.82%和51.29%±4.23%）,可明显抑制HepG2细胞的增殖（IC₅₀=54.32 mL·L^-1）;BPE通过上调Caspase-3、Bax、Bid、Apaf-1、Bak、p53、Caspase-9的表达,下调Bcl-2、Bcl-xl的表达诱导HepG2细胞凋亡;Caspase-9和Caspase-3抑制剂（Z-LEHD-FMK、Ac-DEVD-CHO）在一定程度上逆转了BPE的增殖抑制活性。BPE具有一定的自由基清除能力,对HepG2细胞具有明显增殖抑制作用,这些结果为香蕉皮的进一步开发利用提供了数据支撑。     

Evaluation of viability assays for anthocyanins in cultured cells

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC(50) of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended.     

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.     

Synthesis and Antitumor Evaluation in Vitro of NO‐Donating Ursolic Acid‐Benzylidene Derivatives

Antitumor activity of triterpenoid and its derivatives has attracted great attention recently. Our previous efforts led to the discovery of a series of NO‐donor betulin derivatives with potent antitumor activity. Herein, we prepared eight compounds derived from ursolic acid (UA). All the compounds were evaluated for their in vitro cytotoxicity against four human cancer cell lines (HepG‐2, MCF‐7, HT‐29 and A549). Among the compounds tested, compound 4a was found to be most active against HT‐29 (IC₅₀=4.28 μm ). Further biological assays demonstrated that compound 4a could induce cell cycle arrest at G1 phase and apoptosis in a dose‐dependent manner. In addition, compound 4a was found to upregulate pro‐apoptotic Bax, p53 and downregulate anti‐apoptotic Bcl‐2. All these results suggested that compound 4a is a potential candidate drug for the therapy of colon cancer.     

荧光内参在校正Caspase-3／7活性检测偏差中的应用

目的改进Caspase-3／7活性检测方法。方法在顺铂诱导的HeLa细胞凋亡模型中,分别利用传统的和改良的实验方法检测Caspase-3／7的活性变化,并与Western印迹实验结果进行方法学比较。结果改良的实验方法显示不同剂量顺铂诱导下,HeLa细胞Caspase-3／7活性有剂量依赖性增高,与Western印迹实验结果相一致,但传统实验方法显示HeLa细胞Caspase-3／7活性呈现先增高后降低的趋势。结论由于参测细胞数不同,导致这种Caspase-3／7活性检测方法不能真实反映细胞凋亡程度。本研究成功建立了一种新的改良型Caspase-3／7活性检测方式,这种检测方法可以排除不同凋亡诱导方式诱导细胞凋亡时所产生的因参测细胞数不同所造成的Caspase-3／7活性检测的误差。     

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

Protocatechuic acid (PCA), chlorogenic acid (CA) and luteolin (LT) are plant phenols found in Chinese medicinal herbs such as Lonicera japonica. Cytotoxicity assays showed that PCA, CA and LT (at 100 μmol/L) effectively killed the HepG2 hepatocellular carcinoma cells. Among these three naturally occurring compounds, only PCA was capable of stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. Coincidently, PCA-induced cell death was rescued by specific inhibitors for JNK and p38, while the cytotoxicities of CA and LT were partially eliminated by the antioxidant effect of N-acetyl-L-cysteine (NAC). Further investigation demonstrated that the aqueous extract of Lonicera japonica also triggered HepG2 cell death in a JNK-dependent manner, but the amount of PCA alone in this herbal extract was insufficient to contribute the subsequent cytotoxic effect. Collectively, our results suggest that PCA is a naturally occurring compound capable of inducing JNK-dependent hepatocellular carcinoma cell death. An erratum to this article is available at.     

Chemotypes sensitivity and predictivity of in vivo outcomes for cytotoxic assays in THLE and HepG2 cell lines

In the present study, we demonstrate the utility of in vitro ATP depletion assays in both THLE and HepG2 cells for predicting the toxicological outcome in Exploratory Toxicology Studies across 446 Pfizer proprietary compounds. Our results suggest a higher likelihood of selecting suitable compounds for in vivo safety studies by using cytotoxicity assays in multiple cell-lines over a single cell line. In addition, we demonstrate that different cell-lines have different sensitivities to compounds depending on their ionization state, that is, acid, base or neutral. HepG2 cells are more sensitive for basic compounds, whereas THLE cells have a relatively higher sensitivity for the acidic and neutral compounds. These in vitro cytotoxicity assays when combined with physicochemical properties (c Log P >3 and topological polar surface area (TPSA) <75 Å²), are the most effective means to prioritize compounds having a lower probability of causing adverse events in vivo.     

Role of ATP5G3 in sodium nitroprusside-induced cell death in cervical carcinoma cells

We identified a gene, subunit C3 (ATP5G3) of mitochondrial ATP synthase, that displayed changes in gene expression under oxidative stress. We examined the role of ATP5G3 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using ATP5G3 small interfering RNA (siATP5G3)-transfected HeLa cells. A significant increase in cytotoxicity was observed in the transfected cells treated with SNP, which suggests a protective role of ATP5G3 in SNP-induced cytotoxicity in the cells. The transfected cells treated with photodegraded SNP showed equal cytotoxicity to SNP, and pretreatment with deferoxamine (DFO) completely inhibited this cytotoxicity. Further, cytotoxicity was significantly inhibited by pretreatment with a p38 inhibitor and was accentuated by the p38 activator in cells. Pretreatment with the Bcl-xL inhibitor also significantly accentuated cytotoxicity. The increase in p38 phosphorylation was significantly higher in siATP5G3-transfected cells treated with SNP in immunoblotting, which was inhibited by pretreatment with DFO. The increase in cytotoxicity with siATP5G3 transfection was completely blocked by cotransfection with sip38, and the blocking effect disappeared by cotransfection with additional siBcl-xL, which suggests that the protective role of ATP5G3 is mediated by Bcl-xL via the inhibition of p38 activity. Cytotoxicity was completely blocked by the cotransfection of siATP5G3 with siBax. No change in apoptotic parameters was observed during cytotoxicity. However, pretreatment with lysosomal inhibitors significantly inhibited cytotoxicity and increased p62 protein levels. These findings suggest that ATP5G3 plays a protective role in autophagic cell death/lysosome-associated cell death induced by SNP via the sequential signaling of ROS/p38/Bcl-xL/Bax in HeLa cells.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

Synthesis and structure-activity relationships of taxuyunnanine C derivatives as multidrug resistance modulator in MDR cancer cells

A series of new generation taxoids bearing a bulky group on different positions such as C-2, C-5, C-7, C-9, C-10 or C-14 were obtained by chemical modifications and biotransformation of taxuyunnanine C (1) and its analogs, 4, 5, and 10. Compounds 3, 5, 6, 8, and 9a showed significant activity toward calcein accumulation in MDR 2780AD cells. The most effective compound 9a with a cinnamoyloxy group at C-14 and a hydroxyl group at C-10 was actually efficient for the cellular accumulation of the anticancer agent, vincristine, in MDR 2780AD cells. The enhancing effects of 6 and 9a for taxol, adriamycin, and vincristine were at the same levels as those of verapamil toward MDR 2780AD cells. Thus, compounds 6 and 9a can modulate the multidrug resistance of cancer cells. The cytotoxicity (IC(50)) of the compounds was examined against human normal cell line, WI-38, and cancer model cell lines, VA-13 and HepG2. Since compounds 6 and 8 had no cytotoxicity, they were expected to be lead compounds of MDR cancer reversal agents. On the contrary, compounds 3, 5, and 9a showed cell growth inhibitory activity toward VA-13 and/or HepG2 as well as accumulation activity of calcein and/or vincristine in MDR 2780AD and they were expected to be lead compounds of new-type anticancer agents.     

Overexpressed ATP7B protects mesenchymal stem cells from toxic copper

Wilson’s disease (WD) is characterized by accumulation of high levels of copper in liver due to malfunction of copper transporter ATP7B which is central for copper homeostasis. Here we report for the first time that mesenchymal stem cells (MSC) derived from bone marrow express detectable levels of ATP7B. The role of ATP7B overexpression for MSC survival and selection in high copper was investigated. Hepatoma cell line HepG2 that has a high intrinsic expression of ATP7B served as a control. Using retroviral vector a significant higher expression level of ATP7B could be achieved in MSCs. Whereas copper treatment resulted in cell death in untransduced MSCs, viability assays demonstrated a unique copper resistance of ATP7B overexpressing MSCs that outcompeted HepG2. In long-term cell culture stable transgene expression for up to 9 weeks was shown for ATP7B overexpressing MSCs which rapidly overgrew untransduced cells. Our findings suggest that ATP7B overexpression provides an important selection advantage to MSCs in high copper microenvironments, and may represent novel cell transplants for therapy of WD.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

In vivo and in vitro activity and mechanism of action of the multidrug cytarabine-L-glycerylyl-fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 microM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 microM), but no activity in FM3A/TK(-) (IC50: 18.3 microM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK(-) (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7-70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

In Vivo and in Vitro Activity And Mechanism Of Action of the Multidrug Cytarabine-L-Glycerylyl-Fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 μM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 μ M), but no activity in FM3A/TK^? (IC50: 18.3 μM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK^? (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7–70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.     

A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT‐116 colon cancer cells

Colchicine is a tubulin‐binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C‐ring modified semi‐synthetic derivative of colchicine; N‐[(7S)‐1,2,3‐trimethoxy‐9‐oxo‐10‐(4‐phenyl‐piperidin‐1‐yl)‐5,6,7,9 tetrahydrobenzo[a]heptalen‐7‐yl]acetamide ( 4h ) on colon cancer HCT‐116 cell line. The compound 4h was screened for anti‐proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT‐116 and Colo‐205 with IC₅₀ of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time‐dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT‐116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3‐MA blocked the production of LC3‐II and reduced the cytotoxicity in response to 4h , but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell‐death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy. Copyright © 2016 John Wiley & Sons, Ltd.