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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.[1] 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.[2] The hypoxic microenvironment is associated with a more aggressive malignant progression in gene mutations and genetic instability,[3, 4] local invasion and metastasis,[5] 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.[6]

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.[7] It involves deacetylation of various transcription factors and co-factors that trigger cell defenses and survival in response to stress and DNA damage.[8] 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.[10] It has been hypothesized that SirT1 can affect DNA damage repair by modifying the chromatin conformation at the site of damage.[11] Improper regulation of SirT1 proteins has been reported in a number of diseases including Alzheimer's disease,[12] Bowen's disease,[13] non-alchoholic fatty liver disease,[14] and type I diabetic nephropathy.[15]

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,[16] 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell culture and hypoxic incubation

HepG2 and SK-Hep-1 hepatoma cells were obtained from the Shanghai Cell Bank of China and maintained in DMEM (HyClone, Beijing, China) containing glucose (4.5 g/L) and supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), glutamate (4 mM) and 10% FBS (Gibco Invitrogen, Grand Island, NY, USA) in a fully humidified incubator with 5% CO2 at 37°C. Cultures at hypoxic conditions were maintained in an airtight humidified incubator (Huaxi Electronic Science and Technology, Hunan, China) containing 1% O2, 5% CO2, and 94% N2 at 37°C.

Transfection

Overexpression of SirT1 was carried out by transfection with a pRK7-SirT1 plasmid, which expressed wild-type SirT1 (human Sir2). The pRK7-control plasmid was used as negative control. The SirT1 RNAi plasmid (pSuperiorRetroPuro-SirT1) and its negative control plasmid (pSuperiorRetroPuro-control) were generously provided by Dr. Chuangui Wang (East China Normal University, Shanghai, China).[17] Stable inhibition of SirT1 was carried out by transfection with SirT1 siRNA plasmid and selected with 1 μg/mL puromycin for 14 days. Drug-resistant colonies were pooled for analysis. Cells infected with control plasmids were pooled and used as negative control. Transient inhibition of SirT1 and c-Myc was carried out by transfection with 50 nM SirT1 siRNA (siRNA-A, UGA AGU GCC UCA GAU AUU A; siRNA-B, GAU GAA GUU GAC CUC CUC A) and c-Myc siRNA (siRNA-A, CAT CAT CAT CCA GGA CTG TAT; siRNA-B, CGA GCT AAA ACG GAG CTT T), respectively, or a scrambled siRNA as control (Genepharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Cell irradiation

Cells were seeded on a 35-mm Petri dish (1 × 105 cells/dish) 1 day before irradiation. For the hypoxic experiment, the cells were incubated for 12 h in a humidified incubator containing 1% O2, 5% CO2, and 94% N2 at 37°C, then the cell dishes sealed in a box filled with N2 gas were irradiated with γ-rays at a dose rate of 0.79 Gy/min using a 137Cs irradiator (Gammacell-40; MDS Nordion, Toronto, Canada). Immediately after irradiation, the cells were washed three times with hypoxic PBS in an airtight hypoxic bench and maintained with fresh hypoxic medium, then transferred to the hypoxia incubator until further analysis. For the normoxic experiment, the cells were treated in the same way but under normoxic conditions. Non-irradiated control samples were treated in the same way except for irradiation.

Clonogenic survival assay

Cells were irradiated with 0, 2, 4, 6, or 8 Gy γ-rays, trypsinized and re-plated on 60-mm dishes at appropriate dilutions, and incubated for 14 days at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The colonies were stained with crystal violet, and the colonies containing at least 50 cells were counted for cell survival assay. Survival fraction (SF) was calculated as the ratio of the plating efficiency of irradiated cells to the plating efficiency of control cells without irradiation. Data from three separate experiments are presented as the mean ± SE, and the error bars for all survival data represent the 95% confidence intervals for normalized data points as calculated by Fieller's theorem.[18] The cell survival curves were fitted with the linear-quadratic equation of SF = exp[−(αD + βD2)] by optimizing variable parameters α and β.

Cell growth assay

The cell proliferation rate was measured using the SunBio Am-Blue Kit (Shanghai Sunbio Medical Biotechnology, Shanghai, China) according to the manufacturer's protocol. After irradiation, 100 μL each well of cells at a density of 5000 cells/mL was reseeded and cultured on a 96-well plate. At 5 days post irradiation, cell viability was assayed. Each experiment containing six replicates was repeated at least three times. The fluorescence intensity data were analyzed using GraphPad Prism 4 software (GraphPad, San Diego, CA, USA).

Micronuclei (MN) scoring

Micronuclei were measured using the cytokinesis block technique described by Xie et al.[19] Micronuclei were scored in at least 500 binucleated cells and the MN yield, YMN, was calculated as the ratio of the number of MN to the scored number of binucleated cells.

Western blotting and immunoprecipitation assays

SirT1, c-Myc, acetyl-Lys, p53, and phospho-p53 (Ser15) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Tubulin antibody was purchased from Beyotime Biotechnology (Jiangsu, China). The HRP-conjugated secondary antibodies were purchased from the Proteintech Group (Chicago, IL, USA). At the indicated time intervals after each treatment, cells (1.5 × 106) were harvested and the cell lysate was prepared as described by He et al.[20] An equal amount of total protein was subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Membranes were probed with primary antibodies as indicated. Tubulin was used for the loading control.

For the immunoprecipitate assay, the cells under the indicated treatments were extracted with lysis buffer, centrifuged for 10 min at 14 000g and the insoluble debris was discarded. Cell lysate was incubated with primary antibodies at a dilution recommended by manufacturer overnight at 4°C then further incubated with protein A agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 h. The beads were extensively washed with lysis buffer five times, boiled in SDS sampling buffer, then removed by centrifugation. Immunoprecipitated proteins were fractionated by SDS-PAGE and analyzed by Western blotting as described above.

Statistical analysis

Statistical analysis was compiled on the means of the data obtained from three independent experiments with three replicates in each case. All data are expressed as the means ± SE. The significance of differences in data of different groups were appropriately determined by the unpaired Student's t-test at < 0.05 using spss 13.0 software (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Genetic manipulation of SirT1 levels modulates radiation susceptibility

Many studies have indicated that SirT1 can promote cell survival after chemotherapy or irradiation. To concretely validate the role of SirT1 in the radiosensitivity of hepatoma cells, we transfected pRK7-SirT1 and pSuper-SirT1-RNAi plasmid into HepG2 cells in order to overexpress or knockdown the SirT1 gene, respectively. The transfection efficiency was identified by Western blotting assays. Figure 1(A) clearly shows that the expression of SirT1 was significantly enhanced in pRK7-SirT1-transfected cells (referred to as pRK7-SirT1 cells hereafter) but SirT1 expression was extensively reduced in the SirT1 RNAi-transfected cells (referred to as shSirT1 cells hereafter). When these derivative cell lines were exposed to γ-rays, a dose-dependent reduction in the clonogenic survival was induced. It was found that the pRK7-SirT1 cells with overexpressed SirT1 had a higher radioresistance than the pRK7-control cells (Fig. 1B). Interestingly, many more cells survived in the pRK7-SirT1 cells under hypoxia than under normoxia compared with the pRK7-control cells. Under normoxia and hypoxia conditions, the survival fractions at 4 Gy (SF4) of pRK7-SirT1 sublines were 0.18 ± 0.01 and 0.52 ± 0.06 and the SF4 of pRK7-control cells were 0.15 ± 0.02 and 0.29 ± 0.05, respectively. Hence, due to the transfection of SirT1, the SF4 were enhanced 1.2-fold and 1.8-fold for normoxic and hypoxic cells, respectively.

image

Figure 1. Effect of SirT1 on radiosensitivity in HepG2 and SK-Hep-1 hepatoma cells under different oxygen conditions. (A) Expression of SirT1 in pRK7-control, pRK7-SirT1, shControl, and shSirT1 of HepG2 cells. (B,C) Clonogenic survival of pRK7-control, pRK7-SirT1, shControl, and shSirT1 cells. (D) Expression of SirT1 in SK-Hep-1 cells infected with control siRNA, SirT1 siRNA-A, and SirT1 siRNA-B. (E) Clonogenic survival of SK-Hep-1 cells infected with control and SirT1 siRNA.

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The shSirT1 cells with a low SirT1 level had a higher susceptibility to irradiation compared with the shControl cells (Fig. 1C). Moreover, the shSirT1 cells under hypoxic conditions became more radiosensitive than those under normoxic conditions compared with the shControl cells. Under normoxia and hypoxia, the SF4 values of shSirT1 cells were 0.11 ± 0.01 and 0.18 ± 0.01, and the SF4 of shControl cells were 0.16 ± 0.001 and 0.51 ± 0.02, respectively. Hence, due to the silence of SirT1, the SF4 were reduced 1.45-fold and 2.83-fold for normoxic and hypoxic cells, respectively. These results indicate that the radiosensitivity of hypoxic cells could be regulated by SirT1 much more effectively than that of normoxic cells.

To ensure the generality of these findings, we also silenced SirT1 expression in SK-Hep-1 cells with two independent siRNAs, and efficient SirT1 knockdown was shown (Fig. 1D). In response to irradiation, knockdown of SirT1 resulted in a marked decrease of clonogenic survival (Fig. 1E). Furthermore, compared to the control siRNA transduced cells, SirT1 depletion cells under hypoxia showed much more radiosensitivity than those under normoxia. Collectively, these data supported the notion that SirT1 confers a higher radioresistance in hypoxic hepatoma cells than in normoxic cells.

We also measured the dose–response of cell proliferation in HepG2 cells. Figure 2 clearly shows that pRK7-SirT1 cells had a higher proliferation rate than pRK7-control cells, even with a high dose of irradiation, especially under hypoxic conditions (Fig. 2A,B). In contrast, the shSirT1 cells showed a lower proliferation rate than the shControl cells (Fig. 2C,D). In addition, radiation-induced cell growth suppression in shSirT1 cells under hypoxia was much more evident than under normoxia compared with shControl cells. Concordantly, downregulation of SirT1 led to a significant reduction of proliferation in SK-Hep-1 cells, and this decline effect under hypoxia manifested more severely than under normoxia (Fig. 2E,F). These results strongly indicated that reduced SirT1 expression aggravates radiation-induced cell growth suppression in hepatoma cells, especially under hypoxia.

image

Figure 2. Dose–response of the viability of HepG2 hepatoma cells transfected with pRK7-control and pRK7-SirT1 plasmid (A,B), shControl and shSirT1 plasmid (C,D), and SK-Hep-1 cells infected with control siRNA and SirT1 siRNA (E,F) under normoxic (left panels) and hypoxic (right panels) conditions.

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SirT1 regulates radiosensitivity through a c-Myc pathway

As a result of radiation damage, MN were induced in HepG2 cells and the effects of SirT1 on the damage in hypoxic cells were clearly more serious than in normoxic cells (Fig. 3). It was found that radiation-induced MN could be partly suppressed when the SirT1 gene was overexpressed, as shown in Figure 3(A), where the yield of MN in the pRK7-SirT1 transfected cells was lower than that in the pRK7-control cells. Importantly, this reduction of MN formation due to SirT1 overexpression was much more distinct in hypoxic cells than in normoxic cells. Compared with irradiated pRK7-control cells, the yield of MN in the irradiated pRK7-SirT1 cells was reduced by 34% under hypoxia but it was only reduced by 6% under normoxia. In contrast, when the SirT1 gene was knocked down, the yield of radiation-induced MN was obviously enhanced under both hypoxia and normoxia (Fig. 3B). Compared with irradiated shControl cells, the MN yield of irradiated shSirT1 cells increased by 141% and 35% under hypoxia and normoxia, respectively. These results further indicated that the SirT1 gene confers cell susceptibility to radiation, which is even more effective in hypoxic cells.

image

Figure 3. Influence of SirT1 and c-Myc on radiation-induced micronuclei (MN) formation in HepG2 hepatoma cells under different oxygen conditions. (A,B) Micronuclei formation in pRK7-control, pRK7-SirT1, shControl, and shSirT1 cells. (C) Expression of SirT1 and c-Myc in shControl and shSirT1 cells infected with c-Myc siRNA-A (lane A), c-Myc siRNA-B (lane B), c-Myc siRNA-A plus siRNA-B (lane A+B), and control siRNA. –, without siRNA treatment. (D) Micronuclei induction in shSirT1 cells infected with control and c-Myc siRNA. ***< 0.001 compared to non-irradiated control; ###< 0.001 between the indicated groups. YMN, micronuclei yield.

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To understand how SirT1 affects radiosensitivity, we further investigated the possible role of c-Myc in the above radiation effect by interfering with the function of c-Myc. It has been known that c-Myc plays an essential role in controlling cellular DNA damage and genetic stability,[16] and the activity and transformational capability of c-Myc can be compromised in the presence of SirT1.[21] Figure 3(C) shows that, when c-Myc siRNA-A and/or c-Myc siRNA-B was transfected into shSirT1 cells, the c-Myc protein expression was almost totally suppressed. Both c-Myc siRNA-A alone and siRNA-A plus siRNA-B reduced c-Myc to ~10% compared with the control siRNA, but c-Myc siRNA-B alone reduced the steady-state level of c-Myc by 98%. These data indicated a high efficiency of c-Myc knockdown, given the high level of c-Myc present in shSirT1 cells. Thus, we infected shSirT1 cells with siRNA-B against c-Myc in the following experiments. The results shown in Figure 3(D) show that the treatment of cells with c-Myc siRNA extensively decreased the MN yield of irradiated shSirT1 cells. With the c-Myc siRNA interference, the MN yield of 3 Gy-irradiated shSirT1 cells was diminished from 0.447 to 0.21 and from 0.526 to 0.15 under normoxic and hypoxic conditions, respectively. These data indicate that a SirT1–c-Myc regulatory axis may exist in radiosensitivity, especially under hypoxia.

The efficiency of SirT1 and c-Myc knockdown was shown in SK-Hep-1 cells (Fig. 4A). In line with previous observations, SirT1 silencing dramatically increased radiation-induced MN formation in SK-Hep-1 cells (Fig. 4B). However, this enhanced induction of MN could be strongly suppressed in the presence of c-Myc siRNA, which was much more prominent under hypoxia than normoxia. Taken together, the radiosensitivity of hepatoma cells was enhanced by SirT1 knockdown but it could be extensively eliminated by concomitant silencing of c-Myc. Moreover, the c-Myc interference strongly contributed to decreasing the radiosensitivity in hypoxic cells compared to normoxic cells.

image

Figure 4. Influence of SirT1 and c-Myc on radiation-induced micronuclei (MN) formation in SK-Hep-1 hepatoma cells under different oxygen conditions. (A) Expression of SirT1 and c-Myc in SK-Hep-1 cells infected with control siRNA, SirT1 siRNA, and SirT1 siRNA plus c-Myc siRNA. (B) Micronuclei formation of SK-Hep-1 cells infected with control siRNA, SirT1 siRNA, and SirT1 siRNA plus c-Myc siRNA. ***< 0.001 compared to non-irradiated control; ###< 0.001 between the indicated groups.

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SirT1 knockdown contributes to an increase in c-Myc and its acetylation

The observations in Figures 3 and 4 prompted us to detect the effect of SirT1 on the expression of c-Myc under different oxygen conditions. As shown in Figure 5, the expression of c-Myc protein in HepG2 cells was significantly increased when the SirT1 gene was knocked down by shSirT1 transfection, which was not altered after γ-ray irradiation under normoxic or hypoxic conditions. Although the expression of c-Myc in hypoxic cells was lower than in normoxic cells, the increment of c-Myc protein in shSirT1 cells was more striking under hypoxia than under normoxia (2.26-fold vs 1.47-fold), which was consistent with the effect of hypoxia on SirT1-regulated radiosensitivity.

image

Figure 5. Regulation of SirT1 in the expression of c-Myc. C-Myc was assayed in shControl and shSirT1 cells 2 h after 3 Gy irradiation under different oxygen conditions. (A) Expression of c-Myc and SirT1 protein. (B) Relative level of c-Myc protein calculated by normalizing its expression to tubulin then comparing to each control. ***< 0.001 compared to normoxic non-irradiated shControl; ###< 0.001 between the indicated groups.

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Acetylation is important for a protein's function. We then investigated whether SirT1 could influence the acetylation of c-Myc. Figure 6 illustrates that downregulation of SirT1 resulted in c-Myc hyperacetylation under both normoxia and hypoxia and the increase in acetylated c-Myc was concomitant with the increase in total c-Myc protein. Moreover, even though the acetylation of c-Myc in hypoxic cells was lower than in normoxic cells, the increased acetylation of c-Myc protein was more prominent in hypoxic shSirT1 cells than in normoxic cells (2.57-fold vs 1.40-fold).

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Figure 6. Immunoprecipitation analysis of the expression of acetylated c-Myc in shControl and shSirT1 cells under different oxygen conditions. (A) Acetylated proteins were immunoprecipitated by acetyl-lys (Ac-Lys) antibody and then acetylated c-Myc proteins were detected using c-Myc antibody (upper panel). Conversely, endogenous c-Myc was immunoprecipitated and acetylated c-Myc levels were detected (lower panel). (B) Relative expression level of acetylated c-Myc calculated by normalizing its expression to tubulin then comparing to each control. IP, immunoprecipitant. ***< 0.001 compared to normoxic shControl; ###< 0.001 between the indicated groups.

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Decreased radiosensitivity of hypoxic shSirT1 by c-Myc silencing correlates with depressed phospho-p53

Numerous studies have shown that SirT1 interacts with p53 protein in vitro and in vivo, thus negatively modulating p53 function. As show in Figure 7(A), the expression of p53 was barely influenced by irradiation in shControl and shSirT1 cells under both normoxia and hypoxia. However, as a potential biomarker of γ-radiation exposure, phosphorylated p53 on serine 15 (p-p53) was obviously upregulated in both normoxic shControl and shSirT1 cells, and the increased p-p53 in shSirT1 was slightly higher than in shControl cells. Nevertheless, compared to shControl cells, hypoxia treatment slightly promoted the expression of p-p53 in shSirT1 cells. Importantly, this increment of p-p53 became statistically significant in shSirT1 compared to shControl cells after irradiation, suggesting that the increase of p-p53 expression is related with the downregulation of SirT1 protein. Further study found that the upregulation of p-p53 in shSirT1 cells after irradiation was significantly decreased when the cells were infected with c-Myc siRNA under hypoxic conditions (Fig. 7B), indicating that c-Myc interference disrupts the enhancement of radiosensitivity in SirT1 knockdown cells due to the downregulation of p-p53 after irradiation. These results are quite consistent with our previous findings that radiation-induced DNA damage is dependent on the functionality of p53.[20]

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Figure 7. Influence of SirT1 and c-Myc on radiation-induced phospho-p53 (p-p53) expression. (A) Expression of total p53 and p-p53 in shControl and shSirT1 cells 2 h after 3 Gy irradiation under different oxygen conditions. (B) Expression of p-p53 in shControl and shSirT1 cells with or without c-Myc siRNA infected 2 h after 3 Gy irradiation under hypoxia. –, without siRNA treatment.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.[13] 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.[23] In colorectal cancer tissue, increase of SirT1 is frequently observed in cancer cells compared with adjacent normal epithelial cells.[24] It has been reported that SirT1-expressed tumor cells are resistant to ionizing radiation and chemotherapy.[25] 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.[16] 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.[19] 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.[21] 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.[30] 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.[31] 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.[20] 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.[42] 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.[43] 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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This study was supported by grants from the National Nature Science Foundation of China (Grant Nos. 31070758 and 11179002), the New Teacher Foundation of the Ministry of Education of China (Grant No. 20090071120096), and the Shanghai Nature Science Foundation (Grant No. 09ZR1403600).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References