| Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and has become the leading cause for cancer-related deaths, especially in the Asia-Pacific region. HCC develops from cirrhosis induced by a setting of chronic injury, such as chronic hepatitis. This chronic injury causes fibrogenesis to demolish the normal liver blood system, which constraints oxygen supplies and leads to hypoxia. Excessive proliferation of cancerous cells also results in local hypoxia inside HCC. These microenvironments provide an opportunity for tumor progression, and contribute to the development of aggressive malignant phenotype in gene mutations and genetic instability, local invasion and metastasis, local relapse, and increased angiogenesis. Meanwhile, the cells in the hypoxic regions are more resistant to radiotherapy due to a reduction in life span of DNA-damaging free radicals and augmentation post-irradiation molecular restoration. Hypoxia has been identified as a potent stimulus for several transcription factors. Therefore, new strategies for hypoxia-specific gene therapy and targeting these transcription factors are certainly promising in anti-cancer efficacy. However, during cancer radiotherapy, the irradiated-cancer cells can transmit responses to surrounding non-irradiated normal cells through cell medium or gap junctional intercellular communication and then cause biological damage effects to the normal cells, which was termed as radiation-induced bystander effect (RIBE). The fraction of DNA damage induced by RIBE in hypoxic cells is greater than in normoxic cells. Therefore, deeper understanding the mechanisms of RIBE in hypoxic hepatoma cells as well as the self-protective mechanisms in normal cells, will help us to enhance the efficiency of cancer radiotherapy and provide new ideas for the protection of normal tissues.In this study, hepatoma HepG2 cells pre-treated with SirTl activator (Resveratrol) or inhibitor (Nicotinamide), were irradiated with γ-rays under hypoxic condition. Using cell survival fraction and micronuclei (MN) formation as the biological endpoints, the influence of SirT1 in radiation response was observed. We also explored the role of SirTl in regulating the expression of c-Myc protein to elucidate the function of deacetylation in hypoxia-induced radioresistance. Furthermore, using genetic manipulation, we investigated the differences and underlying mechanisms of SirTl in regulating radiosensitivity, radiation-induced proliferation inhibition and apoptosis in hepatoma HepG2 and SK-Hep-1 cells under normoxic and hypoxic conditions. Meanwhile, the expression of c-Myc and its acetylation, apoptosis-related factors Bax and Bcl-2, and P53 and its phosphorylation were measured under different treatments. Moreover, using medium transfer strategy, we investigated the bystander effect from irradiated hepatoma cells to normal liver cells, as well as the role of SirT1 in this RIBE. The transcriptional activity of c-Myc protein and intracellular ROS level in irradiated cells, as well as the MN formation in bystander cells were detected, to illuminate the function of SirT1 in regulating RIBE and the pathways of transmitting DNA damage activity factors to normal liver cells. Finally, the role of endoplasmic reticulum stress in RIBE on normal liver cells was investigated. Micronuclei formation, induction of apoptosis, and the expression of endoplasmic reticulum stress response-related signaling factors were determined to clarify the mechanism of endoplasmic reticulum stress mediated cell protection in RIBE.The results showed that the administration with Resveratrol, a putative SirTl activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxic condition; while Nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated Nicotinamide-induced radiosensitive effect. Further studies revealed that Resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, Nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity.Further study using gene manipulation indicating that overexpression of SirT1 in hepatoma HepG2 cells allowed the cells to become much more resistant to radiation under hypoxia than under normoxia. When SirTl 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. Meanwhile. SirT1 interference also enhanced the phosphorylation level of P53 protein, while c-Myc interference significantly suppressed phosphorylated P53 protein expression after irradiation, especially under hypoxic condition. In addition, post-irradiation, SirT1 interference significantly promoted the expression of pro-apoptotic factor Bax protein and inhibited the expression of anti-apoptotic factor Bcl-2, and the ability of radiation-induced apoptosis due to SirTl depletion was much more prominent in hypoxic hepatoma cells that of normoxic cells.RIBE has important implications for secondary cancer risk assessment during cancer radiotherapy. Our results showed that MN formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being cultured with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was diminished when SirTl gene was overexpressed, but it was enhanced when SirTl gene was knocked down. Importantly, the protective function of SirTl on RIBE was much more prominent in hypoxia than normoxia. But this increased bystander responses on HL-7702 cells induced by irradiated SirT1-knockdown HepG2 and SK-Hep-1 cells were almost completely abolished by concomitant blockade of endogenous c-Myc. Moreover, SirTl knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation especially in hypoxic HepG2 cells, and this accumulation of ROS could be interrupted by c-Myc interference. ROS scavenger treatment effectively abolished the RIBE triggered by irradiated HepG2 cells even with SirTl depletion.Previous study found that RIBE could be induced among tumor cells, the present results illustrates that RIBE also existed between tumor cells and normal cells, suggesting that RIBE not only enhanced the effect of radiotherapy, but might also increase the degree of DNA damage to the surrounding normal cells. Our results showed that initiation the endoplasmic reticulum stress response in normal liver cells significantly suppressed the DNA damage of MN formation and cells apoptosis induced by irradiated hepatoma cells under either normoxia or hypoxia, while the BiP interference significantly enhanced MN formation and the induction of apoptosis. Moreover, depletion of BiP was more effective in promoting chromosomal damage and apoptosis induced by irradiated-hypoxic hepatoma cells than that of normoxic cells. In addition, BiP interference also enhanced the bystander apoptosis even the target cells were not irradiated, while initiation the endoplasmic reticulum stress response could relieve the spontaneous apoptosis. Further studies revealed that initiation the endoplasmic reticulum stress response significantly up-regulated the expression of BiP protein induced by irradiated hepatoma cells, but BiP interference obviously depressed BiP protein expression. In the process of the endoplasmic reticulum stress response, BiP protein augmented PERK protein level then promoted the phosphorylation level of eIF2a; simultaneously, enhanced the expression of XBP-1 mRNA, and up-regulated the expression of IRE la protein for splicing of XBP-1 mRNA. However, activation of the endoplasmic reticulum stress response after BiP interference significantly enhanced the expression of CHOP protein and bystander apoptosis induced by irradiated hepatoma cells.In summary, the present results indicated that applying its own deacetylase activity, SirTl negatively regulated the levels of c-Myc protein expression and acetylation to affect the transcriptional activity of c-Myc protein, thereby inhibiting the phosphorylation levels of P53 protein post-irradiation, promoting the expression of apoptotic factor Bax, and suppressing the accumulation of anti-apoptotic factors Bcl-2, thus achieving its specific regulation of radiosensitivity in hypoxic hepatoma cells. Meanwhile, by affecting the activity of c-Myc protein, SirTl suppressed the accumulation of intracellular ROS for inhibition the bystander effect induced by irradiated hypoxic hepatoma cells, and reduce the DNA damage of normal liver cells. Moreover, normal liver cells could initiate the endoplasmic reticulum stress response and activate the three important signaling pathways of PERK-p-eIF2α, ATF6-XBP-1 mRNA, and IREla-of XBP-1 mRNA splicing to protect themselves from chromosome damage and apoptosis against RIBE. Therefore, intervention the SirTl gene expression in hepatocellular carcinoma during radiotherapy to enhance the radiosensitivity of tumor cells, as well as moderate activation the endoplasmic reticulum stress response mediated self-protective mechanisms in normal cells,will help us to enhance the efficiency of cancer radiotherapy and minimize the hazard to normal tissues. |
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