Autophagy suppresses radiation damage by activating PARP-1 and attenuating reactive oxygen species in hepatoma cells

Purpose: The radiation-induced bystander effect (RIBE) has important implications for the efficiency of radiotherapy but the underlying role of cellular metabolism is widely unknown. The roles of synthesis of cytochrome c oxidase 2 (SCO2), a key effector for respiratory chain, and related signaling factors in α-particle-induced bystander damage were currently investigated in a liver cell co-culture system.Materials and methods: Human hepatoma cells of HepG2 with wild-type p53 (wtp53) and Hep3B (p53 null) were irradiated with 0.4 Gy of α-particles and co-cultured with non-irradiated normal liver cells HL-7702 for 6 h, then the incidence of micronucleus (MN) in the bystander HL-7702 cells was analyzed. The expressions of total P53, phospho-P53 (p-P53), SCO2, and reactive oxygen species (ROS) in the irradiated hepatoma cells were detected. In some experiments, the hepatoma cells were respectively treated with p53 siRNA, SCO2 siRNA, or dimethyl sulfoxide (DMSO) before irradiation.Results: Bystander damage in HL-7702 cells was induced by α-irradiated HepG2 cells but not by α-irradiated Hep3B cells, and this bystander effect was diminished when the irradiated HepG2 cells were pretreated with p53 siRNA, SCO2 siRNA, or DMSO. Meanwhile, the expressions of p-P53 protein and SCO2 mRNA, the activity of SCO2 protein, and intracellular ROS were all increased in the irradiated HepG2 cells but not Hep3B cells and these expressions were eliminated by p53 siRNA treatment. Moreover, the radiation-enhanced expressions of SCO2 and ROS were inhibited by SCO2 siRNA.Conclusion: α-particle-induced bystander effect was regulated by p53 and its downstream SCO2 in the irradiated hepatoma cells, and ROS generation could be an early event for triggering this bystander response.

Purpose: Autophagy plays a crucial role in cellular response to ionizing radiation, but it is unclear whether autophagy can modulate radiation-induced bystander effect (RIBE). Here, we investigated the relationship between bystander damage and autophagy in human hepatoma cells of HepG2.Materials and methods: HepG2 cells were treated with conditioned medium (CM) collected from 3 Gy γ-rays irradiated hepatoma HepG2 cells for 4, 12, or 24 h, followed by the measurement of micronuclei (MN), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 in the bystander HepG2 cells. In some experiments, the bystander HepG2 cells were respectively transfected with LC3 small interfering RNA (siRNA), Beclin-1 siRNA or treated with 1% dimethyl sulfoxide (DMSO).Results: Additional MN and mitochondrial dysfunction coupled with ROS were induced in the bystander cells. The expressions of protein markers of autophagy, LC3-II/LC3-I and Beclin-1, increased in the bystander cells. The inductions of bystander MN and overexpressions of LC3 and Beclin-1 were significantly diminished by DMSO. However, when the bystander cells were transfected with LC3 siRNA or Beclin-1 siRNA, the yield of bystander MN was significantly enhanced.Conclusion: The elevated ROS have bi-functions in balancing the bystander effects. One is to cause MN and the other is to induce protective autophagy.

本研究旨在探讨羧甲基-β-1，3葡聚糖（CMG）对人肝癌HepG2细胞X射线或12C6+离子束辐射敏感性的影响。首先用CCK-8法检测CMG对HepG2细胞的生长抑制情况，得到半数抑制浓度（IC50）为120.6μg/mL。用浓度为0.1×IC50的CMG预处理HepG2细胞24 h，再给予2 Gy X射线或12C6+离子束辐照（CMG+辐照组）；CMG未处理组直接接受2 Gy X射线或12C6+离子束辐照（辐照组）。对比分析辐照组和CMG+辐照组细胞的克隆存活、DNA损伤、凋亡与周期分布、细胞内活性氧（ROS）水平。发现：与X射线辐照组相比，相同剂量的12C6+离子辐照组克隆存活率更小，DNA损伤和周期阻滞更加严重，细胞凋亡率和细胞内ROS水平也更高。与单独X射线或12C6+离子束辐照组相比，CMG+辐照组克隆存活率明显降低，细胞凋亡率随辐照后CMG作用时间的延长而明显增加，CMG使辐照后细胞内ROS维持在一个较高的水平，同时CMG明显加重了单独辐照诱导的DNA损伤和周期阻滞。结果表明，与X射线相比，HepG2细胞对相同剂量的12C6+离子辐射更敏感；CMG可增加HepG2细胞对X射线或12C6+离子辐射的敏感性；CMG可能通过增加受照HepG2细胞内的ROS水平，加剧辐照诱导的DNA损伤，促进辐射诱导细胞凋亡而起到辐射增敏作用。 This study aims to investigate the effect of carboxymethy-β-1, 3-glucan (CMG) on the sensitivity of human hepatoma HepG2 cells to X-rays or 12C6+ ions irradiation. First, the inhibitory effect of CMG on the growth of HepG2 cells was detected by CCK-8 assay, and the half maximal inhibitory concentration (IC50) was 120.6 μg/mL. HepG2 cells were pretreated with CMG at a concentration of 0.1×IC50 for 24 h and then irradiated with 2 Gy X-ray or 12C6+ ion beams (CMG + irradiation group). CMG untreated group was directly irradiated by 2 Gy X-rays or 12C6+ ions beam (irradiation group). The clone survival, DNA damage, cell apoptosis, cell cycle distribution, and intracellular reactive oxygen species (ROS) levels in irradiation group and CMG + irradiation group were comparatively analyzed. The results showed that the clone survival rate was lower, DNA damage and cycle arrest were more serious, and the rate of apoptosis and intracellular ROS levels were higher in 12C6+ ions irradiation group than those in the same dose of X-rays irradiation group. Compared with X-rays or 12C6+ ions irradiation group, the clone survival rate of CMG + irradiation group was significantly decreased, and the apoptosis rate significantly increased with the prolongation of CMG treatment post-irradiation; CMG maintained intracellular ROS at a higher level after irradiation, CMG also significantly aggravated radiation-induced DNA damage and cycle arrest. These results indicated that HepG2 cells were more sensitive to 12C6+ ions radiation than those at the same dose of X-rays. CMG increased the sensitivity of HepG2 cells to X-rays or 12C6+ ions irradiation by increasing intracellular ROS level, exacerbating radiation-induced DNA damage and promoting radiation-induced apoptosis in irradiated HepG2 cells.

The transcription factor Sp1 is implicated in the activation of G0/G1 phase genes. Modulation of Sp1 transcription activities may affect G1-S checkpoint, resulting in changes in cell proliferation. In this study, our results demonstrated that activated poly(ADP-ribose) polymerase 1 (PARP-1) promoted cell proliferation by inhibiting Sp1 signaling pathway. Cell proliferation and cell cycle assays demonstrated that PARP inhibitors or PARP-1 siRNA treatment significantly inhibited proliferation of hepatoma cells and induced G0/G1 cell cycle arrest in hepatoma cells, while overexpression of PARP-1 or PARP-1 activator treatment promoted cell cycle progression. Simultaneously, inhibition of PARP-1 enhanced the expression of Sp1-mediated checkpoint proteins, such as p21 and p27. In this study, we also showed that Sp1 was poly(ADP-ribosyl)ated by PARP-1 in hepatoma cells. Poly(ADP-ribosyl)ation suppressed Sp1 mediated transcription through preventing Sp1 binding to the Sp1 response element present in the promoters of target genes. Taken together, these data indicated that PARP-1 inhibition attenuated the poly(ADP-ribosyl)ation of Sp1 and significantly increased the expression of Sp1 target genes, resulting in G0/G1 cell cycle arrest and the decreased proliferative ability of the hepatoma cells.

Objective To investigate radiation induced bystander effect and its mechanism on hepatoma HepG2 cells under hypoxia condition. Methods Non-irradiated bystander hepatoma cells were co-cultured with irradiated cells or treated with the conditioned medium (CM) from irradiated cells, then micronuclei (MN) were measured for both irradiated cells and bystander cells. Results The MN yield of irradiated HepG2 cells under hypoxic condition was significantly lower than that under normoxia, the oxygen enhancement ratio of HepG2 cells of MN was 1.6. For both hypoxic and normoxic condition, the MN yield of bystander cells were obviously enhanced to a similar high level after co-culturing with irradiated cells or with CM treatment, and it also correlated with the irradiation dose. When the hypoxic HepG2 cells were treated with either DMSO, a scavenger of reactive oxygen species (ROS), or aminognanidine, an iNOS inhibitor, the yield of bystander MN was partly diminished, and the reducing rate of DMSO was 42.2%-46.7 %, the reducing rate of aminognanidine was 42 %. Conclusion ROS, NO and their downstream signal facets are involved in the radiation induced bystander effect of hypoxic HepG2 cells. Key words: Hypoxia; Hepatoma cell; Radiation-induced bystander effect; Micreuncleus; Signaling factor

Role of Phagocyte Oxidase in UVA-Induced Oxidative Stress and Apoptosis in Keratinocytes

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

PurposeFunctional analysis was performed to elucidate the mechanism by which hepatocellular carcinoma (HCC) outcome-associated mutation in the hepatitis B virus X (HBx) gene modifies the HCC process.MethodsProliferation, invasion, migration, and apoptosis assays were performed, and changes in fibrosis, intracellular reactive oxygen species (ROS), and cytokine levels were measured. The differences between variables were evaluated by Student’s t-test.ResultsThe influence of two previously identified nonsynonymous mutation, C1653T and T1753C, on HCC cells was assessed. With regard to HBX-induced promotion of proliferation (p < 0.01), invasion (p < 0.01) and migration (p < 0.01), the C1653T mutation displayed a significant additive effect in these assays (P < 0.05). The subsequent apoptosis assay indicated that HBX could inhibit apoptosis (P < 0.01), whereas the C1653T mutation markedly amplified this effect in HCC cells (P < 0.01). Furthermore, the tumor growth-promoting effect of HBX was confirmed in a mouse xenograft model of HCC (P < 0.05), and the C1653T mutation was observed to amplify this effect (P < 0.05). To further investigate the mechanism by which the C1653T mutation enhances malignancy in HCC cells, fibrosis, intracellular ROS, and cytokine levels were measured. The C1653T mutant increased fibrosis and intracellular ROS level, and altered monocyte chemotactic protein-1 and interleukin-18 expression in HepG2 cells. Drug sensitivity test revealed that the C1653T mutation is sensitive to apatinib treatment and that overexpression of vascular endothelial growth factor might be involved in this process.ConclusionOur data indicate that the C1653T mutation of HBx promotes HCC malignancy by altering the levels of fibrosis, ROS, and some cytokines. This mutation could serve as a potential biomarker for screening HCC patients to determine apatinib treatment efficacy.

Hepatocellular carcinoma (HCC) is the most common primary malignancy found in the liver. Autophagy is the intracellular bulk degradation process for long-lived proteins and dysfunctional organelles. In this study, we report that autophagy plays a role in HCC cell proliferation in response to ischemia-hypoxia (I/H) and reperfusion and discuss its potential therapeutic implications. By establishing a simulated model in cultured HepG2 (p53 wild-type) and Hep3B (p53 null) hepatoma cells in vitro, we found that exposure to I/H induced a significant increase in microtubule-associated protein 1 light chain 3 (LC3) lipidation and subsequent LC3 puncta formation. While the proliferation of HCC cells was stimulated upon acute I/H exposure compared to that of control, inhibition of autophagy by autophagy-related protein 7 interference abolished it. In addition, the steady-state levels of sequestosome 1 (p62) in both HepG2 and Hep3B cells were reduced following I/H exposure, supporting the notion that acute I/H induces autophagy. Intriguingly, the p62 level further decreased during reperfusion following I/H, accompanied by increased LC3 lipidation. The intracellular reactive oxygen species (ROS) accumulated during acute I/H exposure and persisted through reperfusion in both HepG2 and Hep3B cells and the ROS levels increased at a much faster rate during reperfusion than during I/H periods in both cells. Autophagy functions as a promoter for HCC cell survival during acute I/H and reperfusion and this also points to potential therapy for hepatoma by perturbing the acute I/H-reperfusion-autophagy axis.

Introduction: It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. We evaluated the combination of 1’-acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate (NaB), a major short chain fatty acid, on the growth of HepG2 cells. ACA exhibits chemopreventive effects on chemically induced tumors in mouse skin as well as on rat oral, colonic, esophangeal, and pancreatic tumors. ACA also exerts antitumor activity by inducing apoptosis in various tumor cells. On the other hand, sodium butyrate has multiple effects on tumor cells cultured in vitro, including, most notably, inhibition of cell proliferation and induction of apoptosis, as well as initiating the differentiation of various carcinoma cells. The aim of our study was to elucidate the synergistic interaction of ACA and NaB on cell viability in the human hepatocellular carcinoma cell line HepG2 and to examine the mechanism of the anti-cancer effect of combined ACA and NaB treatment. Methods: HepG2 cells or HT-29 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Results: The number of HepG2 cells was synergistically decreased via apoptosis induction with the combined treatment of ACA and NaB. In ACA- and NaB-treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and NaB was improved with the treatment of catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP-activated protein kinase (AMPK) plays an essential role in controlling processes related to tumor development. In ACA- and NaB-treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation. Conclusion: These findings are consistent with the hypothesis that combination treatments can sensitize cancer cells more effectively than individual treatments. We reported that combination treatment of ACA and NaB synergistically induced apoptotic cell death via an increase in intracellular ROS and an increase in pAMPK levels. Our findings may provide new insight into the development of novel combination therapies against hepatocellular carcinoma. Citation Format: Isao Matsui-Yuasa, Akiko Kojima-Yuasa. The synergistic anticancer activity of 1’-acetoxychavicol acetate and sodium butyrate in human hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2813.

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

ABSTRACTMacranthoside B (MB), a saponin compound in Lonicera macranthoides, can block cell proliferation and induce cell death in several types of cancer cells; however, the precise mechanisms by which MB exerts its anticancer effects remain poorly understood. MB blocked A2780 human ovarian carcinoma cell proliferation both dose- and time-dependently. MB induced apoptosis, with increased poly (ADP-ribose) polymerase (PARP) and caspase-3/9 cleavage. MB also caused autophagy in A2780 cells, with light chain 3 (LC3)-II elevation. Inhibiting MB-induced autophagy with the autophagy inhibitor 3-methyladenine (3-MA) significantly decreased apoptosis, with a reduction of growth inhibition; inhibiting MB-induced apoptosis with the pan-caspase inhibitor Z-VAD-FMK did not decrease autophagy but elevated LC3-II levels, indicating that MB-induced autophagy is cytotoxic and may be upstream of apoptosis. Furthermore, MB increased intracellular reactive oxygen species (ROS) levels, with activated 5′ adenosine monophosphate-activated protein kinase (AMPK), decreased mammalian target of rapamycin (mTOR) and P70S6 kinase phosphorylation, and increased PARP and caspase-3/9 cleavage, and LC3-II elevation; treatment with the ROS scavenger N-acetyl cysteine and the AMPK inhibitor Compound C diminished this effect. Therefore, the ROS/AMPK/mTOR pathway mediates the effect of MB on induction of apoptosis via autophagy in human ovarian carcinoma cells.

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulatingPI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1β (IL-1β) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis.