Apoptosis assays for quantifying the bioactivity of anticancer drug products

The goal of cancer therapy is to kill cancer cells. Many anticancer drugs are designed to kill cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. There are a number of commercial assays available to measure apoptosis; however, many of these assays are not appropriate for use in high-throughput screening formats preferred by industry to measure drug activity, also known as potency, due to their inherent low robustness and/or high variability. This review outlines the strengths and weaknesses of current apoptosis assays and highlights new promising assay developments for evaluation of anticancer therapeutics, such as the design of fluorescent and luminescent constructs to be applied as caspase substrates.     

Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays

Here we show the results of comparing cell viability, cytotoxicity, and apoptosis assays for measuring the time- and dose-dependent toxic effects of tamoxifen on HepG2 cells. The quantitation of adenosine 5'-triphosphate (ATP), 5-(3-carboxymethoxyphenyl)-2-(4,5- dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium, inner salt (MTS) tetrazolium reduction, and resazurin reduction methods used to estimate the number of viable cells all showed a similar trend of decreased cell viability after longer periods of tamoxifen exposure to HepG2 cells. The release of lactate dehydrogenase (LDH) as a marker for cells with a compromised membrane and the increase in caspase-3/7 activity as a marker for apoptosis were both shown to increase using the same tamoxifen exposure conditions that caused a decrease in HepG2 cell viability. The longer the duration of exposure of tamoxifen, the lower the concentration required to kill or induce apoptosis in HepG2 cells. In contrast, there was no change in LDH release from HL-60 cells using conditions of vinblastine treatment that caused an increase in caspase activity and a decrease in ATP content, suggesting a difference in the mechanism of cell death between the two model systems. Both the density of parent stock cultures used as a source of cells to prepare assay plates and the density of cells per well in the assay plates were demonstrated to be factors than can influence the apparent potency of a toxin in viability, toxicity, and apoptosis assays. These results illustrate the importance of understanding the kinetics and mechanism of cell death of each in vitro model system as prerequisites for choosing the most appropriate assay method.     

Different viabilities and toxicity types after 6-OHDA and Ara-C exposure evaluated by four assays in five cell lines.

Cell viability studies are useful when screening novel drugs for the diseases that are related to either increased cell death or enhanced cell survival. There are numerous assays but the results that they produce are rarely unanimous. Here we compared the performance of (1) morphological microscopic assay with double DNA staining, (2) propidium iodide-digitonin assay, (3) MTT-assay, and (4) ATP-assay in human neuroblastoma (SH-SY5Y), rat glioma (C6), rabbit smooth muscle (SMC), Chinese hamster ovary (CHO) and monkey fibroblast cells (CV1-P) exposed to cytosine arabinoside (Ara-C) and 6-hydroxydopamine (6-OHDA). We found that neuronal SH-SY5Y cells were most sensitive to both toxins and the results in all viability tests correlated well. All the other cell lines were much more resistant, particularly to Ara-C but also to 6-OHDA. Toxicity of the compounds was best revealed by MTT and ATP assays, measuring the metabolic activity of the cells, and only occasionally by morphological observations or with the propidium iodide-digitonin assay which is based on the cell membrane integrity. In this research, Ara-C induced pure apoptosis whereas the toxicity type of 6-OHDA was dose-dependent. The use of several viability tests and cell lines is recommended when studying cell death, particularly apoptosis, and performance of antiapoptotic compounds.     

Anticancer effects of seaweed compounds fucoxanthin and phloroglucinol,alone and in combination with 5-fluorouracil in colon cells

ABSTRACT

Colorectal cancer therapy with 5-fluorouracil (5-Fu) frequently become ineffective due to resistance to this drug; and thus other effective compounds are essential for therapy. It is well-known marine brown seaweeds contain antioxidant compounds the carotenoid fucoxanthin (Fx) and polyphenolic compound phloroglucinol (Ph) which exerted diverse biological activities including antioxidant and anticancer. The aim of this study was to determine the anticancer activities of Fx or Ph alone as well as combination of each chemical with 5-Fu on two human colorectal cancer cell lines (HCT116 and HT29), with comparison to responses in a normal colon cell line (CCD-18Co). Effects of these compounds on cell viability, induction of DNA damage, and cell death were evaluated using MTT assay, comet assay, nuclear condensation assay, and Western blot. 5-Fu decreased cell viability in a concentration-dependent manner in HCT116 and HT29 cells but was not cytotoxic in CCD-18Co cells. 5-Fu induced DNA damage in HCT116 cells with induction of cell death, while no marked effects on DNA damage and cell death were observed in HT29 cells. Fx or Ph alone also reduced cell viability in both cancer cell lines but no apparent cytotoxic effect in CCD-18Co cells, except for Fx at 50 and 100 µM. Diminished cell viability was accompanied by induction of DNA damage (by Fx) and induction of cell death (by Ph). In combination with 5-Fu, Fx at 10 µM (in HCT116 and HT29 cells), and Ph at 300 µM (in HT29 cells) enhanced the cytotoxic effect of 5-Fu; however, no marked cytotoxicity was noted in CCD-18Co cells. Since Fx and Ph alone reduced cancer cell line viability without an effect on normal cells and when in combination enhanced the cytotoxic effect of 5-Fu only in colon cancer cells, these compounds seem promising as anticancer agents.     

MiR-19反义核酸与CD133+HT29细胞亚群对多柔比星敏感性关系的研究

目的 探讨miR-19反义核酸与CD133⁺HT29细胞亚群对多柔比星敏感性的关系并研究其机制。方法 用RT-qPCR方法检测miR-19在结直肠癌细胞中的表达水平。流式细胞术检测miR-19反义核酸和多柔比星对HT29细胞系的CD133⁺细胞亚群种群比例的影响。MTT法检测miR-19反义核酸对多柔比星杀伤CD133⁺HT29细胞亚群能力的影响。利用生物信息学及Western blot法验证miR-19是否调节CD133⁺HT29细胞亚群中PTEN的表达。运用Western blot、免疫共沉淀、流式细胞术研究miR-19反义核酸影响多柔比星疗效的信号通路。结果 结直肠癌细胞系的miR-19表达水平显著高于正常结直肠上皮细胞系,并且CD133⁺细胞中的miR-19表达水平显著高于常规结直肠癌细胞。多柔比星体外单独治疗能提高HT29细胞系中CD133⁺细胞亚群的比例,然而联用miR-19反义核酸后CD133⁺HT29细胞亚群的种群比例显著下降。MTT结果表明miR-19反义核酸可显著增强多柔比星对CD133⁺HT29细胞亚群的杀伤活性。Western blot实验表明miR-19的靶基因可能为PTEN。MiR-19反义核酸可显著抑制CD133⁺HT29细胞亚群PI3K、AKT和Bad的磷酸化,增强Bad与Bcl-2和Bcl-xl的结合,从而提高CD133⁺HT29细胞亚群对多柔比星依赖的凋亡信号的敏感性,促进caspase-9和caspase-3的活化。结论 MiR-19反义核酸通过PTEN/PI3K/AKT/Bad途径提高CD133⁺HT29细胞亚群对多柔比星的敏感性。     

Cytotoxic activity of the seaweed compound fucosterol,alone and in combination with 5-fluorouracil,in colon cells using 2D and 3D culturing

ABSTRACT

Colorectal cancer (CRC) is one of the most frequently occurring carcinomas which require effective therapies. Fucosterol is a sterol present in marine brown seaweeds with several biological activities. However, the influence of fucosterol in CRC remains to be determined. Thus, the aim of this study was to examine the anticancer activity of fucosterol alone and in combination with 5-fluorouracil (5-Fu) on two human CRC cell lines (HCT116 and HT29) and compared with cytotoxicity in one normal colon fibroblast cell line (CCD-18co) in monolayer (2D). The effect of fucosterol alone or in combination with 5-Fu was further assessed using HT29 multicellular spheroids (3D). Data demonstrated that fucosterol alone or combined with 5-Fu decreased cell viability in HT29 cells in 2D cultures without inducing cytotoxic in normal colon cells. The combination, fucosterol, and 5-Fu, also inhibited cell proliferation, clonogenic potential and cell migration without producing cell death in 2D. In multicellular spheroids, the combination fucosterol plus 5-Fu at the same concentrations used in 2D was not effective demonstrating that under the tested conditions the 3D model was more resistant than the 2D model. Taken together, these findings suggest that fucosterol might be a promising alternative to enhance the cytotoxic and anti-invasive actions of 5-Fu in colon cancer cells without consequent major adverse effects in normal cells. Our results also reinforce the need to include more complex 3D culture models in the initial stages of drug screening.     

Cell viability assessment: toward content-rich platforms

Importance of the field: Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete end point measurements such as membrane permeability.

Areas covered in this review: This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function.

What the reader will gain: We describe current approaches for assessing cell viability and, using case studies, emphasize their limitations. As an alternative, we propose the use of live, multiplexed readouts to accurately record cell death induction.

Take home message: Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately.     

Permeability Comparison between Hepatocyte and Low Efflux MDCKII Cell Monolayer

Determination of passive permeability is not only important for predicting oral absorption and brain penetration, but also for accurately predicting hepatic clearance. High throughput (HT) measurement of passive permeability across hepatocyte cell membrane is technically more challenging than using monolayer cell-based permeability assays. In this study, we evaluated if the HT Madin-Darby canine kidney II-low efflux (MDCKII-LE) cell monolayer permeability assay can be used as a surrogate to predict the passive permeability of hepatocytes. Apparent passive permeability of MDCKII-LE is well correlated to passive diffusion clearance of human and rat hepatocytes, suggesting that the HT MDCKII-LE assay can be used as a surrogate to estimate the passive permeability of hepatocytes. In addition, lipophilicity (Log D determined at pH 7.4) was also found to be well correlated with both MDCKII-LE and hepatocyte permeability for most compounds, hence it may serve as another permeability surrogate.     

Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells

To screen cost‐effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC₅₀ values by the alamar blue assay in the DEF cells had a high correlation (R² = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC₅₀ values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC₅₀ for aflatoxin B₁, fumonisins B₁ and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml^–1, respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC₅₀ values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species‐specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Functional drug screening assay reveals potential glioma therapeutics

Here we describe a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells. Using this assay, we identified small-molecule drugs that sensitized brain tumor cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. Human glioblastoma multiforme cells were engineered by gene transfer to express Gluc as a reporter for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay for screening and validated it using the National Institute of Neurological Disorders and Stroke (NINDS) custom collection II library consisting of 1,040 drugs and bioactive compounds, most of which are Food and Drug Administration-approved and are able to cross the blood-brain barrier. We found that the cardiac glycosides family sensitized glioblastoma multiforme cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. In conclusion, the Gluc secretion assay is a robust tool for functional drug screening and can be applied to many different fields including cancer.     

A screen for apoptotic synergism between clinical relevant nephrotoxicant and the cytokine TNF-α

Nephrotoxicity remains one of the main reasons for post-market drug withdrawal. Tumour necrosis factor α (TNF-α) secretion has been shown to underlie the nephrotoxicity induced by some of these drugs. Yet, there is currently no reliable and sensitive in vitro assay available to screen for nephrotoxicants of which toxicity largely depends on TNF-α secretion. Therefore, we developed and applied a sensitive fluorescence-based in vitro assay for TNF-α-mediated nephrotoxicity screening using mouse immortalized proximal tubular epithelial cells (IM-PTECs). Our assay allows rapid evaluation of TNF-α-mediated toxicant-induced apoptosis and necrosis using fixed endpoint and live cell measurements. To evaluate our assay, sixteen nephrotoxicants and two control non-nephrotoxicants were used. Out of the sixteen nephrotoxicants, eight induced cell death, of which five induced apoptosis as well as necrosis. Moreover, TNF-α significantly enhanced apoptotic cell death induced by cisplatin, cyclosporine A, tacrolimus and azidothymidine. These nephrotoxicants are known to induce inflammation in vivo which has been linked to an enhancement of nephrotoxicity for cisplatin, cyclosporine A and tacrolimus, confirming the functionality of our assay. Overall, our assay allows rapid and sensitive measurement of apoptosis and necrosis induced by a combination of nephrotoxicants and inflammatory components such as TNF-α and can be used as an alternative assay for nephrotoxicity prediction in vitro.     

Screening for apoptosis--classical and emerging techniques

There has been a rapid development of cell-based assays and screening methods to identify promising apoptosis-inducing drug candidates for the treatment of cancer. Distinguishing between the complex processes involved in apoptosis and other forms of cell death requires information on both biochemical and morphological processes in the cell. Traditionally, many assays have been limited to measuring, for example, caspase activity using fluorogenic substrates. However, these screening assays provide only limited information on the complex processes involved in apoptosis. In this review we describe some of the available apoptosis assays amenable to high-throughput screening. In particular, image-based high-content screening assays to evaluate multiple biochemical and morphological parameters in apoptotic cells are described. Through combining the imaging of cells in microtiter plates with powerful image analysis algorithms, one can acquire deeper knowledge on multiple biochemical or morphological pathways at the single-cell level at an early stage in the development of novel anti-cancer drugs.     

Investigation of the cytotoxicity and insulin transport of acrylic-based copolymer protein delivery systems in contact with Caco-2 cultures.

Microparticles or nanospheres of hydrogels of crosslinked poly(methacrylic acid) grafted with poly(ethylene glycol) as well as crosslinked poly(acrylic acid) grafted with poly(ethylene glycol) were prepared for use as oral insulin delivery carriers. The copolymer carriers were synthesized by precipitation/dispersion polymerization that led to gel nanospheres or by bulk polymerization and subsequent size reduction of thin films to obtain gel microparticles. The cytotoxicity of these copolymers was investigated in contact with Caco-2 cell cultures using a metabolic assay to measure the effect of the presence of copolymers on the cell viability. The copolymers were found to exhibit no cytotoxic effect on the cell cultures. Insulin-loaded formulations were also tested for cytotoxicity and insulin transport studies across cell monolayers. The copolymers were shown to open the tight junctions between cells, increasing the available area for diffusion across the cell monolayer, and thus increasing the permeability of insulin across the monolayer.     

High-throughput fluorescence imaging approaches for drug discovery using in vitro and in vivo three-dimensional models

Introduction: High-resolution microscopy using fluorescent probes is a powerful tool to investigate individual cell structure and function, cell subpopulations and mechanisms underlying cellular responses to drugs. Additionally, responses to drugs more closely resemble those seen in vivo when cells are physically connected in three-dimensional (3D) systems (either 3D cell cultures or whole organisms), as opposed to traditional monolayer cultures. Combined, the use of imaging-based 3D models in the early stages of drug development has the potential to generate biologically relevant data that will increase the likelihood of success for drug candidates in human studies.

Areas covered: The authors discuss current methods for the culturing of cells in 3D as well as approaches for the imaging of whole-animal models and 3D cultures that are amenable to high-throughput settings and could be implemented to support drug discovery campaigns. Furthermore, they provide critical considerations when discussing imaging these 3D systems for high-throughput chemical screenings.

Expert opinion: Despite widespread understanding of the limitations imposed by the two-dimensional versus the 3D cellular paradigm, imaging-based drug screening of 3D cellular models is still limited, with only a few screens found in the literature. Image acquisition in high throughput, accurate interpretation of fluorescent signal, and uptake of staining reagents can be challenging, as the samples are in essence large aggregates of cells. The authors recognize these shortcomings that need to be overcome before the field can accelerate the utilization of these technologies in large-scale chemical screens.     

Methylamine irisolidone,a novel compound,increases total ATPase activity and inhibits apoptosis in vivo and in vitro

We propose to further research the protective effect of MMI on myocardium ischemic rat model and H9c2 cells that underwent cell apoptosis induced by hypoxia. We established the myocardium ischemic rat model via the cardiac surgical procedures in vivo and treated the model rats with different concentration of MMI. In vitro, with the pretreatment of MMI for 12 h in the model of Na₂S₂O₄-induced hypoxia injury, the H9c2 cells viability was determined by MTT assay. We found that MMI had significantly improved cardiac function of the myocardial ischemia, and significantly decreased the reactive oxygen species level. The expression of P53, Bcl-2, Bax, and caspase-9 was also induced by MMI. In vitro study revealed a concentration-dependent increase in cell viability associated with MMI pretreatment. Annexin V-FITC and PI staining results showed that MMI had a preventive effect on hypoxia-induced apoptosis in H9c2 cells. MMI also inhibited the mitochondrial membrane potential decrease and increased total ATPase activity during hypoxia in H9c2 cells. In conclusion, MMI can enhance the cardiac function in myocardial ischemic rat and increase cell viability and attenuate the apoptosis in H9c2 cells induced by hypoxia, which was associated with inhibiting MMP decreasion and increasing total ATPase activity.     

Gallic Acid,an Active Constituent of Grape Seed Extract,Exhibits Anti-proliferative,Pro-apoptotic and Anti-tumorigenic Effects Against Prostate Carcinoma Xenograft Growth in Nude Mice

Purpose  Gallic acid, a natural agent present in a wide-range of fruits and vegetables, has been of potential interest as an anti-cancer agent; herein, we evaluated its efficacy in androgen-independent DU145 and androgen-dependent-22Rv1 human prostate cancer (PCa) cells. Materials and Methods  Cell viability was determined by MTT and apoptosis by Annexin V-PI assays. In vivo anti-cancer efficacy was assessed by DU145 and 22Rv1 xenograft growth in nude mice given normal drinking water or one supplemented with 0.3% or 1% (w/v) gallic acid. PCNA, TUNEL and CD31 immunostaining was performed in tumor tissues for in vivo anti-proliferative, apoptotic and anti-angiogenic effects of gallic acid. Results  Gallic acid decreased cell viability in a dose-dependent manner in both DU145 and 22Rv1 cells largely via apoptosis induction. In tumor studies, gallic acid feeding inhibited the growth of DU145 and 22Rv1 PCa xenografts in nude mice. Immunohistochemical analysis revealed significant inhibition of tumor cell proliferation, induction of apoptosis, and reduction of microvessel density in tumor xenografts from gallic acid-fed mice as compared to controls in both DU145 and 22Rv1 models. Conclusion  Taken together, our findings show the anti-PCa efficacy of gallic acid and provide a rationale for additional studies with this naturally-occurring agent for its efficacy against PCa.     

Three-dimensional hydrogel constructs for exposing cells to nanoparticles

In evaluating nanoparticle risks to human health, there is often a disconnect between results obtained from in vitro toxicology studies and those from in vivo activity, prompting the need for improved methods to rapidly assess the hazards of engineered nanomaterials. In vitro studies of nanoparticle toxicology often rely on high doses and short exposure periods due to the difficulty of maintaining monolayer cell cultures over extended time periods as well as the difficulty of maintaining nanoparticle dispersions within the culture environment. In this work, tissue-engineered constructs are investigated as a platform for providing doses of nanoparticles over different exposure periods to cells within a three-dimensional environment that can be tuned to mimic in vivo conditions. Uptake of quantum dots (QDs) by model neural cells was first investigated in a high-dose exposure scenario, resulting in a strong concentration-dependent uptake of carboxyl-functionalised QDs. Poly(ethylene glycol) hydrogel scaffolds with varying mesh sizes were then investigated for their ability to support cell survival and proliferation. Cells were co-encapsulated with carboxyl-functionalised poly(ethylene glycol)-coated QDs at a lower dose than is typical for monolayer cultures. Although the QDs leach from the hydrogel within 24 h, they are also incorporated by cells within the scaffold, enabling the use of these constructs in future studies of cell behaviour and function.     

Nonylphenol‐induced apoptotic cell death in mouse TM4 Sertoli cells via the generation of reactive oxygen species and activation of the ERK signaling pathway

Nonylphenol (NP), a representative endocrine disruptor, interferes with reproductive function in aquatic organisms and animals. Although many previous studies have focused on apoptotic cell death by NP, the fundamental mechanism of NP on apoptosis remains poorly understood. Here, we investigated the molecular mechanism on NP‐induced apoptotic cell death in mouse TM4 Sertoli cells. To evaluate NP treatment on cell viability, formazan and lactate dehydrogenase (LDH) assays were performed. Results indicate that NP reduced cell viability and increased the release of LDH in dose‐ and time‐dependent manners. The reduction of cell viability by NP treatment appeared to involve necrosis as well as apoptosis based on nuclear fragmentation, an increase in the sub G1 population, and the detection of poly(ADP ribose) polymerase and caspase‐3 cleavage. Additionally, the anti‐apoptotic protein Bcl‐2 diminished, whereas the pro‐apoptotic protein Bax increased in a time‐dependent manner. Note that NP‐induced apoptotic cell death was enhanced by the generation of reactive oxygen species (ROS) and activation of extracellular signal‐regulated kinase (ERK) signaling. Pretreatment with N‐acetylcysteine, an antioxidant, attenuated NP‐induced apoptotic cell death. Moreover, NP caused a transient activation of the MAPK pathway. In particular, NP‐induced cell death was significantly suppressed by U0126, a specific inhibitor of ERK. Taken together, our results suggest that NP induces apoptosis in mouse TM4 Sertoli cells via ROS generation and ERK activation. Copyright © 2013 John Wiley & Sons, Ltd.     

AKT/TSC2/p70S6K signaling pathway is involved in quinocetone-induced death-promoting autophagy in HepG2 cells

Quinocetone (QCT, 3-methyl-2-quinoxalin benzenevinylketo-1, 4-dioxide) is widely used as a veterinary drug and animal feed additive in China. Although it promotes growth and improves feed efficiency, QCT’s in vitro and in vivo toxicities remain uncertain. This study was conducted to explore the mechanism of QCT-induced autophagy in HepG2 cells. By the results obtained from monodansylcadaverine (MDC) staining, ultrastructural observation by transmission electron microscopy (TEM), as well as Western blotting analysis for LC3, p62, and Beclin-1, it was demonstrated that QCT induced autophagy in HepG2 cells. Furthermore, PI3K/AKT inhibitor significantly enhanced QCT-induced autophagy, while TSC2 knockdown attenuated this process. In addition, inhibition of autophagy by pharmacological approach remarkably increased the viability of QCT-treated cells detected by MTT assay, suggesting that QCT-triggered autophagy may play as a promotion mechanism for cell death. Meanwhile, apoptosis was markedly downregulated after autophagy blockage, and evaluated by flow cytometry and Western blotting analysis for caspase-3 cleavage. Consequently, these results suggested that QCT-induced autophagy was mediated by AKT/TSC2/p70S6K signaling pathway, and inhibition of autophagy promoted QCT-treated cell survival by attenuating apoptosis.     

可诱导型及Bak基因过度表达对多柔比星诱导HCC-9204细胞凋亡的增敏作用(英文)

目的:研究Bak基因过度表达在HCC-9204细胞凋亡途径中的作用以及对多柔比星和长春瑞滨的可能的增敏作用.方法:采用MT-Ⅱ可调控性表达载体系统,通过外加ZnSO_4(100 μmol/L)诱导Bak基因表达,并获得稳定转染子.以形态学标准并结合TUNEL或流式细胞仪检测细胞凋亡.克隆形成实验反映克隆细胞存活率,MTF法检测细胞活力.结果:Bak基因过度表达的细胞出现显著的细胞死亡,TUNEL证实为一种凋亡性细胞死亡.流式细胞仪的结果显示Bak能够显著地诱导细胞在G_1期聚积并在阿霉素诱导后24 h 19.26％的细胞发生凋亡.Bak基因过度表达只能显著降低阿霉素处理组的克隆存活率,而对长春花碱组没有显著效果.MTT实验的结果类似,提示Bak基因能够选择性对阿霉素诱导的细胞死亡具有增敏作用,而对长春花碱没有这种作用.结论:Bak基因的过度表达使HCC-9204细胞的细胞周期在G_1延长,导致细胞凋亡并选择性对化疗药物具有增敏作用.