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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)
Hepatocellular carcinoma (HCC) has become the leading cause of cancer-related deaths, especially in the Asia–Pacific region. Hepatocellular carcinoma develops from a setting of chronic hepatitis or cirrhosis induced by impairment of the liver blood system, which constrains oxygen supplies and leads to hypoxia. Excessive proliferation of cancerous cells also results in local hypoxia inside HCC. The hypoxic microenvironment is associated with a more aggressive malignant progression in gene mutations and genetic instability,[3, 4] local invasion and metastasis, and increased angiogenesis. The cells in hypoxic regions are more resistant to radiotherapy due to a reduction in lifespan of DNA-damaging free radicals and augmentation of post-irradiation molecular restoration.
Effective gene therapy using a hypoxia-susceptible suicide gene should specifically kill the tumor cells that are resistant to conventional treatment, and SirT1 develops the basis for a compelling gene therapy. SirT1 is a member of the mammalian sirtuin family. It involves deacetylation of various transcription factors and co-factors that trigger cell defenses and survival in response to stress and DNA damage. The mechanism of SirT1 in regulating these processes is due to its ability to deacetylate histones and non-histone proteins, such as p53, FOXO3, Ku70, NF-κB,[7, 9] and eNOS. It has been hypothesized that SirT1 can affect DNA damage repair by modifying the chromatin conformation at the site of damage. Improper regulation of SirT1 proteins has been reported in a number of diseases including Alzheimer's disease, Bowen's disease, non-alchoholic fatty liver disease, and type I diabetic nephropathy.
To better understand the function of SirT1 in hypoxic cells, we analyzed the radiosensitivity of HepG2 and SK-Hep-1 cell lines following SirT1 overexpression or SirT1 deficiency under both normoxic and hypoxic conditions. Given that c-Myc plays an essential role in controlling numerous cellular effects such as proliferation, angiogenesis, senescence, oncogenesis, metabolism, DNA damage response, and genetic stability, the present study shows that SirT1 regulates the radiosensitivity of HepG2 cells by modulating the accumulation of c-Myc and its acetylation. This reveals a negative regulatory pathway of c-Myc-mediated DNA damage and provides a mechanistic insight into how SirT1 regulates c-Myc in modulating cell radiosensitivity under hypoxia.
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Also known as a potential tumor suppressor gene, SirT1 plays an important role in balancing intracellular proliferation, defenses and survival, senescence, and apoptosis.[8, 22] Overexpression of SirT1 has been reported in various malignant cells and tissues from patients with leukemia and glioblastoma, suggesting that SirT1 may be involved in tumorigenesis and thus is a useful marker for tumor diagnosis. SirT1 is frequently upregulated in all kinds of non-melanoma skin cancers including squamous cell carcinoma, basal cell carcinoma, Bowen's disease, and actinic keratosis. Similarly, SirT1 could be significantly elevated in transgenic mouse model of prostate cancer with poorly differentiated adenocarcinomas compared with the normal counterpart, and it also had a high level in human prostate cancer cells. In colorectal cancer tissue, increase of SirT1 is frequently observed in cancer cells compared with adjacent normal epithelial cells. It has been reported that SirT1-expressed tumor cells are resistant to ionizing radiation and chemotherapy. Nevertheless, the present study showed that overexpression of SirT1 in hepatoma HepG2 and SK-Hep-1 cells contributed to a higher radioresistance under hypoxic conditions than under normoxic conditions, and depletion of SirT1 was more effective in reducing the radiosensitivity of hypoxic cells than normoxic cells.
The c-Myc oncoprotein is a crucial regulator of cell cycle progression, proliferation, apoptosis, tumorigenesis, DNA damage response, and genetic stability in numerous human cancers. In response to cellular stress such as ionizing radiation, hypoxia, or growth factor deprivation, deregulation of c-Myc often induces cell apoptosis.[26, 27] Our previous data documented that pretreatment of cells with 10058-F4, a specific c-Myc inhibitor, decreased the radiosensitivity of HepG2 cells. In this study, we found that, when c-Myc was downregulated, the radiosensitivity was extensively decreased in the SirT1 knockdown of HepG2 and SK-Hep-1 cells. These observations were consistent with the phenomenon that cells expressing a higher c-Myc level were more susceptible to irradiation, and that the loss of c-Myc expression might confer cell resistance to radiotherapy.[28, 29] However, when the c-Myc gene was knocked down, the yield of micronuclei in the irradiated SirT1 depletion cells was still not completely eliminated to the background level, suggesting that more radiation-responsible factors other than c-Myc might be involved in the radiation-induced damage.
Yuan and colleagues have documented that SirT1 could deacetylate c-Myc, resulting in a decrease of c-Myc stability, thus the activity and transformational capability of c-Myc are compromised in the presence of SirT1. Consistent with this observation, our results showed that knockdown of the SirT1 gene significantly enhanced the expression of c-Myc protein, thus augmenting radiation-induced DNA damage. In particular, increases in c-Myc protein in the SirT1-silenced cells were more striking under hypoxia than under normoxia compared with their control cells, which indicates further that SirT1 exerts radioresistance through the modulation of c-Myc expression. In addition, radiation itself did not alter the expression of c-Myc protein in HepG2 cells under either normoxia or hypoxia. In fact, there are inconsistent conclusions from several studies comparing cellular sensitivity with c-Myc depletion or overexpression. We speculate that this outcome could be cancer-specific and cell type-dependent. However, the nature of the relationship between c-Myc status and irradiation effect remains to be established.
We than investigated whether the expression of c-Myc protein was associated with its acetylation degree and SirT1 status. As shown in Figure 6, downregulation of SirT1 resulted in c-Myc hyperacetylation for both normoxic and hypoxic HepG2 cells. The increased acetylation of c-Myc protein was more prominent in hypoxic shSirT1 cells than normoxic ones compared with shControl cells. Importantly, the increase in c-Myc acetylation was accompanied by an increase in total c-Myc protein. Indeed, a higher c-Myc acetylation was concomitant with higher irradiation cytotoxicity, suggesting that a higher c-Myc acetylation level may lead to a higher radiosensitivity. Based on the evidence of the inducibility of acetylated c-Myc protein following the downregulation of SirT1, it can be proposed that SirT1 may promote hypoxic tumor cells to be resistant to radiation by inhibiting c-Myc acetylation and subsequently decreasing its accumulation.
Our previous studies, as well as other reports, have shown that p53 is a key molecule involved in the cellular response to irradiation and regulates the radiosensitivity of mammalian cells. Early studies reported that p53 was a direct target gene of c-Myc, which drives p53-mediated apoptosis and acts as a safeguard mechanism against aberrant oncogenic activation.[32-34] The present study showed that c-Myc silencing disrupts the enhancement of radiosensitivity on SirT1 knockdown hypoxic cells due to down regulation of p-p53 after irradiation. These results are in keeping with a previous study showing that hypoxia pretreatment inhibited the activation of p53 by radiation and that radiation-induced DNA damage depends on the functionality of p53.[20, 35, 36] On the basis of the present and other findings, we propose that SirT1 confers a higher radioresistance in hypoxic HepG2 cells than in normoxic cells due to decreased levels of c-Myc protein and its acetylation, and that a c-Myc-dependent pathway of radiation-induced p-p53 may be involved.
Given the regulation of the cellular response to DNA damage, we could conclude that SirT1 is a sensor of radioresistance in hypoxic cells, but limited published reports are available regarding the role of SirT1 in human cancers. SirT1 inhibition in combination with radiation is being increasingly appreciated as a promising option for anticancer strategies.[22, 37, 38] Considering the antitumor effect of inhibiting SirT1 function, the inclusion of adjuvant chemotherapy and radiotherapy may be a better treatment approach for patients with SirT1-overexpressed hepatoma.[37, 39-41] In addition, recent studies have revealed that a decrease in hypoxia-inducible factor-1 (HIF-1) results in the appearance of acetylated HIF-2α through the SirT1 pathway, which conversely augments HIF-2 mediated transcriptional activation of the isolated SirT1 promoter. However, as cell growth can be suppressed by HIF-1α but promoted by HIF-2α, SirT1 might activate HIF-2α to conduct cellular responses in the early phase of hypoxia treatment, then facilitate HIF-1α to take over hypoxic signaling. Although the current experiments indicate an important correlation between hypoxic stress, SirT1 status, and radiosensitivity, it should be emphasized that we cannot exclude that other hypoxic gene changes caused by knockdown of SirT1, such as many hypoxia-depressed genes, might also contribute to the observed radiation-induced damage. Nonetheless, our results along with other published data suggest that SirT1 can function as an oncogene in hepatoma by inhibiting c-Myc acetylation and c-Myc accumulation-mediated p53 activation. SirT1 could be the focus for a potential strategy in blocking hypoxia-induced progression of tumor development for advancing the efficiency of radiotherapy.