Bcl‐2 protects TK6 cells against hydroquinone‐induced apoptosis through PARP‐1 cytoplasm translocation and stabilizing mitochondrial membrane potential

B cell leukemia/lymphoma‐2 (Bcl‐2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl‐2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)‐induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration‐dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ‐H2AX, and production of the DNA damage‐responsive enzyme poly(ADP‐ribose)polymerase‐1 (PARP‐1). Exposure of TK6 cells to HQ leads to an increase in Bcl‐2 and co‐localization with PARP‐1 in the cytoplasm. Inhibition of Bcl‐2 using the BH3 mimetic, ABT‐737, suppressed the PARP‐1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ‐induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT‐737 combined with HQ increased the levels of cleaved PARP and γ‐H2AX, but significantly decreased the level of P53. Thus, ABT‐737 can influence PARP‐1 translocation and induce apoptosis via mitochondria‐mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP‐1 attenuated the HQ‐induced production of cleaved PARP and P53. These results identify Bcl‐2 as a protective mediator of HQ‐induced apoptosis and show that upregulation of Bcl‐2 helps to localize PARP‐1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49–59, 2018. © 2017 Wiley Periodicals, Inc.     

Induction of macrophage cell‐cycle arrest and apoptosis by humic acid

Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25–200 µg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25–200 µg/mL) induces cell‐cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G₂/M phase of the cell cycle by reducing cyclin A/B₁, Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub‐G₁ accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase‐3 or caspase‐9 activation, and Bcl‐2/Bax dysregulation. In addition to the mitochondrial pathway, HA‐induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase‐8, caspase‐4, and caspase‐12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage‐mediated immune system. Environ. Mol. Mutagen. 55:741–750, 2014. © 2014 Wiley Periodicals, Inc.     

2‐(Pro‐1‐ynyl)‐5‐(5,6‐dihydroxypenta‐1,3‐diynyl) Thiophene Induces Apoptosis Through Reactive Oxygen Species‐Mediated JNK Activation in Human Colon Cancer SW620 Cells

2‐(Pro‐1‐ynyl)?5‐(5,6‐dihydroxypenta‐1,3‐diynyl) thiophene (PYDDT) is a naturally occurring thiophene isolated from the roots of Echinops grijsii, a Chinese herbal medicine used to treat colon cancer, breast cancer, and lung cancer. There are many reports on the clinical use of Echinops grijsii alone or in combination with other herbs to treat malignant tumors. We previously reported that the expression and activity of phase II enzymes including GSTs and NQO1 could be induced through the activation of Keap1‐Nrf2 pathway by the treatment of PYDDT. In this study, we reported the anticancer effect and mechanism of PYDDT against human colon cancer SW620 cells. Our results demonstrate that treatment of SW620 cells with PYDDT leads to induction of mitochondrial‐mediated apoptosis, which is characterized by the cleavage of PARP, activation of caspase 9 and caspase 3, release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, down‐regulation of Bcl‐2, and mitochondrial translocation of Bax. The PYDDT treatment caused the production of reactive oxygen species (ROS), and the activation of JNK but not p38 mitogen‐activated protein kinases and ERK1/2. Specific JNK inhibitor SP600125 prevented the PYDDT‐induced down‐regulation of Bcl‐2, mitochondrial translocation of Bax, activation of caspase 3, and apoptosis of SW620 cells. Moreover, PYDDT‐induced apoptosis as well as activation of JNK was abrogated by the pretreatment with antioxidant N‐acetylcysteine. Taken together, these findings suggest that PYDDT induces apoptosis in SW620 cells through a ROS/JNK‐mediated mitochondrial pathway. Anat Rec, 298:376–385, 2015. © 2014 Wiley Periodicals, Inc.     

5‐Nitro‐2‐(3‐Phenylpropylamino) Benzoic Acid Induced Drug Resistance to Cisplatin in Human Erythroleukemia Cell Lines

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel‐3 (ClC‐3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin‐induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl‐2, decreased the protein expressions of cytochrome C and caspase‐3, and increased the mRNA expressions of cyclin D1 and ClC‐3 in cells treated with cisplatin. The caspase‐3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC‐3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC‐3. NPPB‐induced CIC‐3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin‐induced apoptosis in human erythroleukemia K562 and RK562 cells. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Supplementation With Quercetin Attenuates 4‐Nitrophenol‐Induced Testicular Toxicity in Adult Male Mice

The beneficial effects of quercetin on reproductive damage elicited by 4‐nitrophenol (PNP) were studied in adult male mice. A six‐week treatment of weekly intraperitoneal injections of PNP (50 mg/kg) resulted in severe damage to the seminiferous tubules, a remarkable increase in both hydroxyl radical and malondiadehyde production, and notably decreased glutathione peroxidase and superoxide dismutase activities. Moreover, PNP treatment induced germ cell apoptosis, inhibited Bcl‐xl expression, and then activated Bax expression and the caspase‐3 enzyme. Exposure to PNP also increased XBP‐1 and HO‐1 mRNAs levels. However, simultaneous supplementation with quercetin (75 mg/kg) attenuated the toxicity induced by PNP through renewal of the antioxidant enzyme's status, alleviating apoptosis by regulating the expressions of Bax and Bcl‐xl, XBP‐1 and HO‐1mRNAs, and the regulation of caspase‐3 activity. Taken together, these findings indicated that the antioxidant quercetin displays a potential preventive effect on PNP‐induced oxidative damage in mouse testes and may represent an efficient supplement to attenuate reproductive toxicity from environmental toxicants in order to ensure reproductive health and sperm production. Anat Rec, 296:1650–1657, 2013. © 2013 Wiley Periodicals, Inc.     

米托蒽醌对HL-60细胞的促凋亡效应

目的 :探索米托蒽醌 (MTN)促HL 6 0细胞凋亡效应及其机理。方法 :取对数生长期HL 6 0细胞 ,不同浓度MTN作用不同时间后 ,利用MTT法、透射电镜、DNA电泳、流式细胞术观察MTN对HL 6 0细胞的抑制效应、促凋亡效应及凋亡相关基因Bcl 2、Bax蛋白表达情况。结果 :(1)毫微克级的MTN对HL 6 0细胞有明显的抑制效应 ,且效应具有时间、剂量依赖关系。(2 )MTN作用后的HL 6 0细胞透射电镜观察胞膜完整、出现核碎裂、凋亡小体等形态学特征。DNA电泳出现 2 0 0bp左右的梯形条带。 (3)Bcl 2、Bax蛋白表达 :MTN作用于HL 6 0细胞相同时间 ,Bcl 2蛋白及Bcl 2蛋白与Bax蛋白比值 (Bcl 2 Bax)随MTN作用时间延长而下降。结论 :MTN具有诱导HL 6 0细胞凋亡作用 ,此过程中伴Bcl 2下调和Bcl 2 Bax的降低。提示MTN可能通过Bcl 2途径诱导HL 6 0细胞凋亡     

Protective Effect of Quercetin on Cadmium‐Induced Oxidative Toxicity on Germ Cells in Male Mice

Cadmium is a toxic heavy metal that is widely distributed in the environment. As a critical process, oxidative toxicity mediates the morphological and functional damages in germ cells after cadmium exposure. In this study, the protective effect of quercetin on cadmium‐induced oxidative toxicity was investigated in mouse testicular germ cells. After oral administration of cadmium chloride at 4 mg/kg body weight for 2 weeks, damages in spermatozoa occurred in the early stage of spermatogenesis. Cadmium treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione (GSH) level, superoxide dismutase (SOD), and GSH peroxidase (GSH‐Px) activities. Moreover, exposure to cadmium resulted in an increase of hydrogen peroxide production and lipid peroxidation in testes. In addition, cadmium provoked germ cell apoptosis by upregulating expression of the proapoptotic proteins Bax and caspase‐3 and downregulating expression of the antiapoptotic protein Bcl‐XL. However, combined administration of a common flavonoid quercetin at 75 mg/kg body weight significantly attenuated cadmium‐induced germ cell apoptosis by suppressing the hydrogen peroxide production and lipid peroxidation in testicular tissue. Simultaneous supplementation of quercetin markedly restored the decrease in GSH level and SOD and GSH‐Px activities elicited by cadmium treatment. Additionally, quercetin protected germ cells from cadmium‐induced apoptosis by downregulating the expression of Bax and caspase‐3 and upregulating Bcl‐XL expression. These results indicate that quercetin, due to its antioxidative and antiapoptotic characters, may manifest effective protective action against cadmium‐induced oxidative toxicity in mouse testicular germ cells. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

MLH1‐mediated recruitment of FAN1 to chromatin for the induction of apoptosis triggered by O6‐methylguanine

O⁶‐Methylguanines (O⁶‐meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein‐dependent manner. To understand the molecular mechanism of O⁶‐meG‐induced apoptosis, we performed functional analyses of FANCD2 and FANCI‐associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N‐methyl‐N‐nitrosourea (MNU), indicating the formation of a FAN1‐MMR complex. In comparison with control cells, FAN1‐knockdown cells were more resistant to MNU, and the appearances of a sub‐G₁ population and caspase‐9 activation were suppressed. FAN1 formed nuclear foci in an MLH1‐dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single‐stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU‐induced formation of ssDNA was dramatically suppressed in FAN1‐knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O⁶‐meG.     

Exposure of HepG2 cells to low levels of PAH‐containing extracts from contaminated soils results in unpredictable genotoxic stress responses

Contaminated soil is a serious environmental problem, constituting a risk to humans and the environment. Polycyclic aromatic hydrocarbons (PAHs) are often present at contaminated sites. However, risk levels are difficult to estimate because of the complexity of contaminants present. Here, we compare cellular effects of extracts from contaminated soils collected at six industrial settings in Sweden. Chemical analysis showed that all soils contained complex mixtures of PAHs and oxy‐PAHs. Western blotting and immunocytochemistry were used to investigate DNA damage signaling in HepG2 cells exposed to extracts from these soils. The effects on phosphorylated Mdm2, p53, Erk, H2AX, 53BP1, and Chk2, cell cycle regulating proteins (cyclin D1 and p21), and cell proliferation were compared. We found that most soil extracts induced phosphorylation of Mdm2 at the 2A10 epitope at low concentrations. This is in line with previous studies suggesting that this endpoint reflects readily repaired DNA‐damage. However, we found concentration‐ and time‐dependent γH2AX and 53BP1 responses that were sustained for 48 hr. These endpoints may reflect the presence of different types of persistent DNA‐damage. High concentrations of soil extracts decreased cyclin D1 and increased p21 response, indicating cell cycle arrest. Phosphorylation of Mdm2 at Ser166, which attenuates the p53 response and is induced by many tumor promoters, was induced in a time‐dependent manner and was associated with Erk phosphorylation. Taken together, the PAH extracts elicited unpredictable signaling responses that differed between samples. More polar compounds, i.e., oxy‐PAHs, also contributed to the complexity. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Berbamine Enhances the Antineoplastic Activity of Gemcitabine in Pancreatic Cancer Cells by Activating Transforming Growth Factor‐β/Smad Signaling

Drug‐resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine‐based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc‐3 and Panc‐1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time‐ and dose‐dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine‐induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down‐regulation of anti‐apoptotic proteins (Bcl‐2, Bcl‐xL) and up‐regulation of pro‐apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor‐β/Smad (TGF‐β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor‐β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up‐regulation of p21 and down‐regulation of c‐Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis‐related proteins and the activation of TGF‐β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment. Anat Rec, 297:802–809, 2014. © 2014 Wiley Periodicals, Inc.     

Proteinase activated receptor‐2‐mediated dual oxidase‐2 up‐regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase‐activated receptor 2 (PAR‐2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX‐2, have been shown to be involved in airway inflammation in response to Toll‐like receptor activation. However, the association between PAR‐2 and DUOX‐2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX‐2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR‐2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX‐2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl‐2, caspase‐3). Challenge with CE led to up‐regulation of DUOX‐2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD‐1068, a small molecule antagonist of PAR‐2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX‐2/ROS pathway; however, these effects were less pronounced than PAR‐2 antagonism. The current study suggests that PAR‐2 activation leads to up‐regulation of the DUOX‐2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.     

IFN‐γ regulates survival and function of tumor‐induced CD11b+Gr‐1high myeloid derived suppressor cells by modulating the anti‐apoptotic molecule Bcl2a1

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b⁺Gr‐1^high granulocytic MDSCs. Coculture of CD11b⁺Gr‐1^high granulocytic MDSCs with antigen‐stimulated T cells and simultaneous blockade of IFN‐γ by the use of anti‐IFN‐γ blocking antibody, IFN‐γ^?/? effector T cells, IFN‐γR^?/? MDSCs or STAT1^?/? MDSCs led to upregulation of Bcl2a1 in CD11b⁺Gr‐1^high cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM‐CSF released by antigen‐stimulated CD8⁺ T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN‐γ by a direct interaction of phosphorylated STAT‐1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN‐γ/ STAT1‐dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.     

Expression of Bcl‐2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma

Coutinho‐Camillo C M, Lourenço S V, Nishimoto I N, Kowalski L P & Soares F A
(2010) Histopathology 57 , 304–316 Expression of Bcl‐2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma Aims: To characterize the expression of proteins that inhibit (Bcl‐2, Bcl‐x, Bcl‐xL, Bcl‐2‐related protein A1, BAG‐1) or promote (Bak, Bax, Bim/Bod, Bim‐Long, Bad, Bid, PUMA) apoptosis and determine possible correlations between the expression of these proteins and clinicopathological features of oral squamous cell carcinoma (OSCC). Methods and results: Two‐hundred and twenty‐nine cases of OSCC, arranged in a tissue microarray, were immunohistochemically analysed. The results demonstrated that the absence of vascular invasion was associated with increased expression of Bak, Bax, Bcl‐xL, Bcl‐2‐related protein and PUMA. Increased expression of Bim/Bod and BAG‐1 was associated with the presence of perineural infiltration. An increase in Bid and Bim‐Long expression was associated with moderately to well‐differentiated tumours. Increased expression of the Bcl‐2‐related protein and PUMA was associated with tumours occurring in the floor of mouth and increased expression of PUMA was also associated with recurrence of the tumour. Multivariate Cox analysis demonstrated that PUMA and Bim‐Long were independent factors in prognosis of OSCC. Conclusions: Our results showed the involvement of the Bcl‐2 family of proteins in OSCC tumorigenesis and suggest that the expression of apoptotic molecules might be used as a prognostic indicator for OSCC.     

Reducing‐Autophagy Derived Mitochondrial Dysfunction during Resveratrol Promotes Fibroblast‐Like Synovial Cell Apoptosis

In rheumatoid arthritis patients, the fibroblast‐like synovial cells (FLS) growth is not controlled normally, but is similar to the tumor cells proliferation in histology. Our previous studies have shown that resveratrol inhibits the proliferation of FLS and promotes FLS apoptosis. However, the molecular mechanisms involved in resveratrol‐induced FLS apoptosis have not been determined yet. Here, we showed that the FLS cell viability (following pretreatment with 5 µM H₂O₂ for 24 hr) exhibited better proliferation performance than at other concentrations via the CCK‐8 assay. The cell apoptotic rate increased with the increasing concentration of resveratrol (0, 40, 80, 160, 320 μM), as detected by TdT‐mediated dUTP nick‐end labeling (TUNEL) staining and western blotting. Furthermore, the expression level of autophagy‐related proteins (LC3A/B, ATG‐5) decreased with the increased concentration of resveratrol, as determined by immunofluorescence and western blot analysis. We also showed that resveratrol induced FLS mitochondrial morphology change. Moreover, mitochondrial function detection showed that the mitochondrial membrane potential was lost with the increased concentration of resveratrol as examined by the JC‐1 assay. The production of ATP in cells was positively and negatively correlated with the resveratrol concentration. Simultaneously, the intracellular calcium release and calcium influx decreased gradually with the increase in resveratrol concentration. Therefore, we proposed that resveratrol can reduce the level of autophagy in FLS. The decrease in the autophagy level can lead to the accumulation of reactive oxygen species, which may result in mitochondrial dysfunction and promotion of FLS apoptosis. Anat Rec, 301:1179–1188, 2018. © 2018 Wiley Periodicals, Inc.     

Three structurally homologous isothiocyanates exert “Janus” characteristics in human HepG2 cells

In this study, we used the single cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test to investigate the DNA damaging effects and the antigenotoxic potencies of three structurally related ITCs in human HepG2 cells. The results show that all three ITCs possess the characteristic of a “Janus” compound, i.e., they exert both significant genotoxicity and antigenotoxicity, depending on the concentrations used in the test systems applied. Regression line analysis of the results derived by SCGE analysis showed genotoxic potency of the ITCs in the following order: 3‐methylthiopropyl ITC (MTPITC) > 4‐methylthiobutyl ITC (MTBITC) > 5‐methylthiopentyl ITC (MTPeITC); however, this order in genotoxic potency was not confirmed by MN analysis. Additionally, the MN test showed significant mutagenicity of the test substances at higher concentrations when compared with the SCGE assay. Twenty‐four hour‐treatment of the cells with the ITCs, followed by a 1‐hr recovery period, showed significant DNA repair in the SCGE assay at a concentration ≥10 μM MTPITC, ≥3 μM MTBITC, and ≥0.1 μM MTPeITC, respectively. In antigenotoxicity studies, the most effective concentration of MTPITC and MTPeITC toward B(a)P‐induced DNA damage was 0.1 μM in both test systems. MTBITC suppressed MN formation in B(a)P‐treated cells to the background level at a concentration of 1 μM. The ambivalent character of the ITCs under studymust be further clarified, especially in the possiblecontext of high dose therapeutic applications. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Modification of radiation‐induced DNA double strand break repair pathways by chemicals extracted from Podophyllum hexandrum: An in vitro study in human blood leukocytes

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre‐treatment with G‐002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G‐002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ‐H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose‐dependent kinetics. Semiquantitative RT‐PCR was performed at various time points to measure gene expression of DNA‐PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre‐treatment of blood with G‐002M resulted in reduction of γ‐H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non‐G‐002M‐treated irradiated cells. These results confirm suppression in radiation‐induced DNA DSBs. Samples pre‐treated with G‐002M and then irradiated also showed significant up‐regulation of DNA‐PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G‐002M plays a protective role against DNA damage. The protective effect of G‐002M may be due to its ability to scavange radiation‐induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G‐002M on signaling molecules involved in radiation‐induced DNA damage repair pathways. Environ. Mol. Mutagen. 55:436–448, 2014. © 2014 Wiley Periodicals, Inc.     

蝌蚪提取液诱导HL60细胞分化和凋亡的实验研究

目的 为了寻找新的肿瘤细胞分化诱导剂,探讨蝌蚪醚提取液（Ｔ８７１２）的抗肿瘤作用。方法 将ＨＬ６０细胞培养于Ｔ８７１２和ＲＰＭＩ１６４０按１：２００（ｖ／ｖ）混合的培养基中。结果 Ｔ８７１２能诱导ＨＬ６０细胞向单核／巨噬细胞方向分化,表现为生长抑制,ＮＢＴ还原能力提高,酸性非特异性酯酶（ＡＮＡＥ）活性增加,形态学观察类似单核／巨噬细胞。当Ｔ８７１２与ＲＰＭＩ１６４０培养基按１：１００（ｖ／ｖ）混合     

MTBITC mediates cell cycle arrest and apoptosis induction in human HepG2 cells despite its rapid degradation kinetics in the in vitro model

Despite the great variety of structure homologous, experimental research on the cancer preventive properties of isothiocyanates (ITCs) is limited to only a fractional amount thereof so far. Especially the degradation of these compounds in the experimental system has not been investigated so far. In this study, we investigated the effect of 4‐methylthiobutyl isothiocyante (MTBITC) on the proliferation of human hepatoma (HepG2) cells and underlying mechanisms. A concentration and time‐dependent reduction in proliferation activity could be observed in cells treated with MTBITC exceeding 10 μM. At these concentrations MTBITC‐induced apoptosis in HepG2 cells could be observed by internucleosomal DNA fragmentation, flow cytometry analysis, and the detection of single‐stranded apoptotic DNA. In all the three assays, clear apoptotic events were present after 6‐hr exposure to MTBITC. Apoptosis induction was accompanied by a time‐dependent arrest of HepG2 cells at the G2/M phase of the cell cycle. This study shows for the first time the inhibitory potency of MTBITC on metabolically competent hepatoma cells, whereas the loss of reduced glutathione and its impact on mitochondria seem to be the major processes involved in the initiation and execution of the apoptotic cell death. The results of this study also showed that irrespective of the intense degradation kinetics of MTBITC, the strong cytostatic effect of the ITC was not markedly affected by it and suggests that although ITCs are only present at maximum concentrations in a living system for a rather short time, this might be sufficient to exert their therapeutic effects. Environ. Mal. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

The protective properties of melatonin against aluminium‐induced neuronal injury

Aluminium (Al) toxicity is closely linked to the pathogenesis of Alzheimer's disease (AD). This experimental study investigated the neuroprotective effect of melatonin (Mel; 10 mg/kg bwt) on aluminium chloride (AlCl₃; 34 mg/kg bwt) induced neurotoxicity and oxidative stress in rats. Adult male albino Wistar rats were injected with AlCl₃ for 7 days. The effect on brain structure, lipid peroxidation (LPO), nitric oxide (NO) levels, glutathione (GSH) content, antioxidant enzymes (SOD, CAT, GPx and GR), apoptotic proteins (Bax and Bcl‐2) and an apoptotic enzyme (caspase‐3) was investigated. No apparent changes occurred following the injection of melatonin. Melatonin pretreatment of the AlCl₃‐administered rats reduced brain damage, and the tissues appeared like those of the control rats. Compared to treatment with AlCl₃, pretreatment with melatonin decreased LPO and NO levels and increased the GSH content and antioxidant enzyme activity. Moreover, melatonin increased the levels of the anti‐apoptotic protein, Bcl‐2, decreased the levels of the pro‐apoptotic protein, Bax, and inhibited caspase‐3 activity. Therefore, our results indicate that melatonin may provide therapeutic value against aluminium‐induced oxidative stress and histopathological alternations in the rat brain and that these effects may be related to anti‐apoptotic and antioxidant activities.