Jianghong Zhang

Extracellular miR-1246 promotes lung cancer cell proliferation and enhances radioresistance by directly targeting DR5

MiRNAs in the circulation have been demonstrated to be a type of signaling molecule involved in intercellular communication but little is known about their role in regulating radiosensitivity. This study aims to investigate the effects of extracellular miRNAs induced by ionizing radiation (IR) on cell proliferation and radiosensitivity. The miRNAs in the conditioned medium (CM) from irradiated and non-irradiated A549 lung cancer cells were compared using a microarray assay and the profiles of 21 miRNAs up and down-regulated by radiation were confirmed by qRT-PCR. One of these miRNAs, miR-1246, was especially abundant outside the cells and had a much higher level compared with that inside of cells. The expressions of miR-1246 in both A549 and H446 cells increased along with irradiation dose and the time post-irradiation. By labeling exosomes and miR-1246 with different fluorescence dyes, it was found that the extracellular miR-1246 could shuttle from its donor cells to other recipient cells by a non-exosome associated pathway. Moreover, the treatments of cells with miR-1246 mimic or its antisense inhibitor showed that the extracellular miR-1246 could enhance the proliferation and radioresistance of lung cancer cells. A luciferase reporter-gene transfer experiment demonstrated that the death receptor 5 (DR5) was the direct target of miR-1246, and the kinetics of DR5 expression was opposite to that of miR-1246 in the irradiated cells. Our results show that the oncogene-like extracellular miR-1246 could act as a signaling messenger between irradiated and non-irradiated cells, more importantly, it contributes to cell radioresistance by directly suppressing the DR5 gene.

SirT1 regulates radiosensitivity of hepatoma cells differently under normoxic and hypoxic conditions.

Intratumoral hypoxic cells are more resistant to radiotherapy due to a reduction in lifespan of DNA‐damaging free radicals and augmentation of post‐irradiation molecular restoration. SirT1, a member of the mammalian sirtuin family, deacetylates various transcription factors to trigger cell defense and survival in response to stresses and DNA damage. In this study, we provide new evidence indicating that overexpression of SirT1 in hepatoma HepG2 cells allowed the cells to become much more resistant to irradiation under hypoxia than under normoxia. When SirT1 was knocked down in both HepG2 and SK‐Hep‐1 cells, the radiosensitivity was increased, especially under hypoxia. But this enhanced radiosensitivity in SirT1‐deficient cells was extensively decreased by infecting cells with c‐Myc siRNA. Furthermore, the expression of c‐Myc protein and its acetylation were increased in the SirT1 knockdown cells and these increments under hypoxic conditions were much more notable than under normoxia. In addition, c‐Myc interference significantly suppressed phosphorylated p53 protein expression after irradiation, especially under hypoxic conditions. The current findings indicate that SirT1 confers a higher radioresistance in hypoxic cells than in normoxic cells due to the decreased levels of c‐Myc protein and its acetylation, and that a c‐Myc‐dependent radiation‐induced phosphorylated p53 may be involved. SirT1 could serve as a novel target of radiation damage and thus as a potential strategy to advance the efficiency of radiotherapy in hepatoma entities. (Cancer Sci 2012; 103: 1238–1244)

SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation.

Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved.

Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells.

Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H2S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H2S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H2S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

Radiation Exposure Promotes Hepatocarcinoma Cell Invasion through Epithelial Mesenchymal Transition Mediated by H2S/CSE Pathway.

There is growing evidence to suggest that radiotherapy can paradoxically promote tumor invasion and metastatic processes, however, the underlying molecular mechanisms remain obscure. In this study, we found that exposure to X rays promoted cell invasion by triggering the epithelial mesenchymal transition (EMT) in two hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. This was made evident by a reduced expression of E-cadherin and enhanced expressions of N-cadherin, Vimentin and Snail. Moreover, exposure to radiation stimulated the signaling of hydrogen sulfide (H2S), a newly found gas transmitter, by upregulating the expressions of H2S-producing proteins of cysthionine-γ-lyase (CSE), cystathionine-β-synthase (CBS). Inhibition of CSE by siRNA or inhibitor not only increased the radiosensitivity but also strongly suppressed radiation-enhanced invasive properties of HCC cells. Interestingly, we found that H2S/CSE inhibition attenuated radiation-enhanced EMT, and the above effect was an end result of blockage of the radiation-activated pathway of p38 mitogen-activated protein kinase (p38MAPK). Collectively, our findings indicate that radiation could promote HCC cell invasion through EMT mediated by endogenous H2S/CSE signaling via the p38MAPK pathway.

Role of ROS-mediated autophagy in radiation-induced bystander effect of hepatoma cells

Abstract 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.

Role of nuclear factor-κB and P53 in radioadaptive response in Chang live cells

Understanding the mechanism governing radioadaptive response (RAR) has important implication for cancer risk assessment of a low-dose radiation (LDR). However the related knowledge especially the key gene of RAR is still limited. In this study, Chang liver cells were irradiated with a priming dose of 0.016 Gy, 0.08 Gy, or 0.16 Gy of gamma-rays, and with 4 h interval, they were irradiated again with a challenging dose of 2 Gy or 3 Gy. It was found that only 0.08 Gy, but not 0.016 Gy or 0.16 Gy, induced RAR of micronuclei induction to the challenging irradiation. This RAR could be slightly reduced by pifithrin-alpha, an inhibitor of P53, however it was completely suppressed by BAY11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB). Further assays using western blotting and luciferase reporter gene found that nuclear NF-kappaB and its activity could be triggered by the priming irradiation of 0.08 Gy so that the expressions of them in the primed cells were higher than those in the cells exposed to the challenging dose alone. In contrast, LDR neither influenced the expressions of both P53 and phospho-P53 (ser15) nor enhanced P53 activity; the expression of phospho-P53 and the activity of P53 in the primed cells were lower than that in the non-primly challenged cells. Our results demonstrate that the induction of RAR relays on an optimum priming irradiation dose and it is NF-kappaB rather than P53 that contributes to RAR.

Cadmium induced radioadaptive response via an ATM-independent H2S/cystathionine γ-lyase modulation

The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. Here we explored cadmium induced radioadaptive response (RAR) and investigated the role of hydrogen sulfide (H(2)S) and ATM kinase in this response. Our data showed that the cadmium ions with a sub-lethal concentration could induce RAR in Chang liver cells towards subsequent γ-irradiation and this response could be abrogated by DL-propargylglycine (PPG), the endogenous H(2)S synthetase inhibitor of cystathionine γ-lyase (CSE), but not by aminooxyacetic acid (AOAA), the inhibitor of cystathionine β-synthase (CBS). Moreover, the pretreatment of cells with NaHS also stimulated cellular adaptive response to radiation. Both cadmium treatment and irradiation up-regulated the expression of CSE protein in a time-dependent manner but had no influence on the expression of CBS protein. In the primed cells, the time course of CBS expression showed no significant difference with the cells treated with 2Gy irradiation alone, however, the CSE expression was easier to reach the maximum level, indicating a more efficient H(2)S production by CSE. Moreover, the cadmium-induced RAR was totally suppressed by KU-55933, a specific ATM inhibitor that did not change the CSE expression after radiation. However, exogenous H(2)S decreased the phosphorylation level of radiation-induced ATM. In conclusion, the present results demonstrate firstly that H(2)S is involved in the cadmium induced cross-adaptive response to challenging radiation. CSE, rather than CBS, may mainly responsible for the H(2)S production during this RAR which may also be mediated by ATM pathway. However, the activation of CSE is independent of ATM but could negatively regulate the phosphorylation of ATM.

Suppression of Endogenous Hydrogen Sulfide Contributes to the Radiation-Induced Bystander Effects on Hypoxic HepG2 Cells

Radiation-induced bystander effects may have important implications in radiotherapy, but it is still not well known if radiation-induced bystander effects can be triggered in hypoxic tumor cells and what are the related bystander signals. Using human hepatoma cells of HepG2, the present study found that micronuclei (MN) could be induced in the nonirradiated cells after treatment with conditioned medium (CM) harvested from irradiated cells under hypoxic conditions. Bystander effects were diminished when the irradiated cells were pretreated with sodium hydrosulfide (NaHS, an exogenous H2S donor) (≤100 μM). However, the bystander effects were increased when the irradiated cells were pretreated with an inhibitor of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), the synthases of endogenous hydrogen sulfide (H2S). Western blotting showed that the expressions of CSE and CBS were increased in the irradiated hypoxic cells, but were reduced in the CM treated bystander cells. The ratio of Bcl-2/Bax, a molecular marker of apoptosis, decreased with CM treatment time. However, the activity of caspase-3 increased in the hypoxic bystander cells, and this could be regulated by both NaHS and the inhibitor of endogenous H2S. These results demonstrate that under hypoxic conditions irradiated hepatoma cells induce bystander responses by depressing the generation of endogenous H2S and altering Bcl-2/Bax ratios as well as caspase-3 dependent damage in the bystander cells.

Radioprotective role of H2S/CSE pathway in Chang liver cells

Radiation-induced liver cell damage may be life-threatening. Here, we investigated whether hydrogen sulfide (H(2)S)/cystathionine γ-lyase (CSE) pathway could serve the protective role toward radiation in normal human liver cells. Our data showed that pretreatment of cells with H(2)S donor, sodium hydrosulfide (NaHS) significantly attenuated radiation induced micronuclei formation and improved cell viability. However, the use of dl-propargylglycine (PPG), a potent inhibitor of CSE, markedly enhanced the cell-killing effect induced by radiation. Exposure of cells to 2Gy γ-radiation led to significant increases of the endogenous H(2)S content. The mRNA and protein expressions of CSE also increased after radiation in a time-dependent manner, while the expression of cystathionine β-synthase (CBS), another endogenous H(2)S synthetase, did not change significantly. Notably, radiation induced production of reactive oxygen species (ROS) was significantly reversed by the pretreatment of NaHS, while blockage of CSE activity resulted in an enhanced ROS production in irradiated cells. Moreover, NaHS markedly suppressed radiation-induced phosphorylation of P53, decrease of Bcl-2/Bax, and activity of nuclear factor kappaB (NF-κB). In conclusion, our finding demonstrates that H(2)S/CSE pathway plays a radioprotection role by inhibiting radiation-induced ROS production, P53 phosphorylation, NF-κB activation and decrease of Bcl-2/Bax, indicating that modulation of H(2)S may be a novel protection strategy for liver radiation injury in radiotherapy.