Zunzhen Zhang

Sodium arsenite and arsenic trioxide differently affect the oxidative stress, genotoxicity and apoptosis in A549 cells: An implication for the paradoxical mechanism

Although arsenic toxicity greatly depends on its chemical forms, few studies have taken into account the paradoxical phenomenon which is manifested by that sodium arsenite (NaAsO₂) acts as a potent carcinogen but arsenic trioxide (As₂O₃) serves as an effective therapeutic agent. In this study, we compared the in vitro effects of NaAsO₂ and As₂O₃ on cell viability, colony formation, cell cycle progression, apoptosis, genotoxicity and oxidative stress in human lung adenocarcinoma A549 cells. Our results demonstrated that both NaAsO₂ and As₂O₃ caused oxidative stress, genotoxicity, cytotoxicity, cell cycle arrest as well as apoptosis, while As₂O₃ induced higher production of reactive oxygen species (ROS) with a more remarkable decrease in superoxide dismutase (SOD) activities and intracellular levels of glutathione (GSH) than NaAsO₂. Moreover, the degree of DNA damage, chromosomal breakage, cell cycle arrest and apoptosis in As₂O₃-treated cells were more severe than those in NaAsO₂-treated cells. These findings suggest that differential effects and mechanisms of NaAsO₂ and As₂O₃ may responsible for the paradoxical effects of arsenic on the carcinogenesis and anticancer function.

Dual role of resveratrol in modulation of genotoxicity induced by sodium arsenite via oxidative stress and apoptosis.

The potential benefits of resveratrol as an anticancer (proapoptosis) and antioxidant (pro-survival) compound have been studied extensively. However, the role of resveratrol in modulation of the toxicity induced by sodium arsenite (NaAsO₂) is still unclear. In the present study, we examined the effects of resveratrol on NaAsO₂-induced cytotoxicity, DNA and chromosomal damage, cell cycle progression, apoptosis and oxidative stress in human lung adenocarcinoma epithelial (A549) cell line at concentrations from 1 to 20 μM after 24h exposure. Our results revealed that at 1 and 5 μM, resveratrol was found to exert benefit effects, promoting cell viability and proliferation over 24h NaAsO₂ exposure, whereas, resveratrol was showed to inhibit cell survival under the same condition at 20 μM. Corresponding to the opposing effect of resveratrol at low vs. high concentrations, DNA and chromosomal damage, cell apoptotic rate and level of oxidative stress were also alleviated by lower concentrations (1, 5 μM) of resveratrol, but exacerbated by higher concentration (20 μM) resveratrol. Our study implicates that resveratrol is the most beneficial to cells at 1 and 5 μM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its concentration dependent effect.

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

Antioxidant activities of Ganoderma lucidum polysaccharides and their role on DNA damage in mice induced by cobalt-60 gamma-irradiation

In this study, the radio-protective effects of Ganoderma lucidum polysaccharides (GLP) were investigated in a mouse animal model exposed to (60)Co gamma-irradiation. Each of three batches of mice were divided into five groups (negative control, positive gamma irradiated control, and low, middle and high dosage GLP groups). Different batches of animals were used to evaluate the impact of GLP on peripheral white blood cell count, immune organ index; DNA damage, lipid peroxidation; micronuclei formation, and nucleated cell count in bone marrow induced by (60)Co gamma-irradiation. DNA strand-break and micronuclei frequency were significantly reduced and glutathione peroxidase activity and nucleated cell count in bone marrow were significantly increased by GLP treatment in a dose-dependent manner. GLP intervention also increased the activity of superoxide dismutase and decreased the level of malondialdehyde in middle and high GLP treatment groups. No adverse effects were observed on peripheral white blood cells and immune organ or body weight in either the control groups or GLP treated gamma exposed mice. These findings suggest that GLP possesses marked antioxidant capacity which plays an important role in the prevention of radiation damage in mice induced by (60)Co gamma-irradiation.

Role of DNA polymerase beta in the genotoxicity of arsenic

Arsenic, an important hazard in the environment, is associated with human cancer and other degenerative diseases. However, the mechanisms underlying arsenic hazardous effects remain unclear. It has been reported arsenic exposure can result in increased cellular reactive oxygen species and oxidative DNA damage. This suggests DNA base excision repair (BER), the major pathway for repairing oxidative DNA damage, may be involved in combating arsenic hazardous effects. As a critical repair enzyme in BER, DNA polymerase beta (Pol β) might play an essential role in reducing arsenic toxicity. To test this hypothesis, we evaluated arsenic‐induced cytotoxic and genotoxic effects under Pol β deficiency. Our results demonstrated that the viability of Pol β‐deficient mouse embryonic fibroblasts was much lower than that of Pol β wild‐type cells after treatment with arsenite (As3+). An increased level of DNA damage and significantly delayed arsenite‐induced DNA damage repair in Pol β‐deficient cells indicated reduced repair of DNA lesions under Pol β deficiency. This was consistent with the increase in the frequency of micronuclei (MN), an indicator of chromosomal breakage, which was also observed in Pol β‐deficient cells treated with arsenite. In contrast, cells harboring overexpressed Pol β resulted in a lower level of DNA damage and MN than Pol β wild‐type cells, indicating overexpression of the enzyme can combat arsenic‐induced genotoxic effects. In conclusion, our results indicate an important role for Pol β in repairing arsenite‐induced DNA damage and maintaining chromosomal integrity and further suggest deficiency of BER may be involved in arsenic genotoxicity and carcinogenicity. Environ. Mol. Mutagen., 2011.

Resveratrol Synergistically Triggers Apoptotic Cell Death with Arsenic Trioxide via Oxidative Stress in Human Lung Adenocarcinoma A549 Cells

Arsenic trioxide (As2O3) is a potent anticancer drug for the treatment of acute promyelocytic leukemia. However, the clinical applications of the agent to treat solid tumors are largely compromised by the drug resistance. Our previous study demonstrated that resveratrol, a plant-derived natural product, could potentiate the toxicity of arsenite in lung adenocarcinoma A549 cells at relatively high concentration, indicating that combination of resveratrol and As2O3 may be a helpful strategy to solve the drug resistance of As2O3 in tumor cells. To test this possibility, in the present study, we determined the combined effects of resveratrol and As2O3 in cultured A549 cells. Our results showed that co-treatment of resveratrol with As2O3 resulted in a synergistic augmentation of cytotoxicity and apoptosis in cells at the tested concentration. To further reveal the detailed mechanism of this synergistic effect on cytotoxicity and apoptosis, apoptosis-related proteins, DNA and chromosomal damage, and the level of oxidative stress were also evaluated. Our data revealed that co-treatment with resveratrol and As2O3 caused more genotoxicity and serious oxidative stress in A549 cells than that of single agent treatment. Moreover, resveratrol and As2O3 could also corporately enhance the release of cytochrome c and the expressions of death receptor Fas and FasL. Together, our results suggest that resveratrol and As2O3 synergistically increase the apoptotic cell death in A549 cells through induction of oxidative stress, indicating that the combination of resveratrol with As2O3 may be a promising strategy to increase the clinical efficacy of As2O3 in the treatment of lung tumor.

Instability of CTG repeats is governed by the position of a DNA base lesion through base excision repair.

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 5′-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 3′-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase β and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.

Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells.

Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs‐induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs‐induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs‐induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549‐R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549‐R and A549 cell lines treated with COF condensate (COFC). RT‐PCR and Western blot results showed that the expression of hOGG1 in A549‐R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549‐R cell line was much lower than that in the A549 cell line, and more COFC‐induced DNA damage was detected in the A549‐R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549‐R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF‐induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1‐mediated repair pathways, and oxidative DNA damage may be involved in COF‐induced carcinogenesis. Environ. Mol. Mutagen., 2008.

Resveratrol protects against arsenic trioxide-induced oxidative damage through maintenance of glutathione homeostasis and inhibition of apoptotic progression

Arsenic trioxide (As2O3) is commonly used to treat acute promyelocytic leukemia and solid tumors. However, the clinical application of the agent is limited by its cyto‐ and genotoxic effects on normal cells. Thus, relief of As2O3 toxicity in normal cells is essentially necessary for improvement of As2O3‐mediated chemotherapy. In this study, we have identified a series of protective effects of resveratrol against As2O3‐induced oxidative damage in normal human bronchial epithelial (HBE) cells. We showed that treatment of HBE cells with resveratrol significantly reduced cellular levels of DNA damage, chromosomal breakage, and apoptosis induced by As2O3. The effect of resveratrol against DNA damage was associated with a decreased level of reactive oxygen species and lipid peroxidation in cells treated by As2O3, suggesting that resveratrol protects against As2O3 toxicity via a cellular anti‐oxidative stress pathway. Further analysis of the roles of resveratrol demonstrated that it modulated biosynthesis, recycling, and consumption of glutathione (GSH), thereby promoting GSH homeostasis in HBE cells treated by As2O3. This was further supported by results showing that resveratrol prevented an increase in the activities and levels of caspases, Fas, Fas‐L, and cytochrome c proteins induced by As2O3. Our study indicates that resveratrol relieves As2O3‐induced oxidative damage in normal human lung cells via maintenance of GSH homeostasis and suppression of apoptosis. Environ. Mol. Mutagen. 56:333–346, 2015.

Crosstalk between MSH2-MSH3 and polβ promotes trinucleotide repeat expansion during base excision repair.

Studies in knockout mice provide evidence that MSH2–MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2–MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol β fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2–MSH3 not only stimulates pol β to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.