HDAC Inhibitor 4SC-202 Increases Sensitivity to Epirubicin in Liver Cancer Cells by Inhibition of ATM


 Background: Histone deacetylase (HDAC) inhibitors can enhance the anticancer effect of epirubicin in liver cancer. However, the molecular mechanism remains unknown. In our study, we aimed to examine the effect of the combination of HDAC inhibitor 4SC-202 and epirubicin and the underlying mechanism in liver cancer cells. Methods: Cell viability assay, flow cytometry, western blotting, colony formation assay, animal experiment, immunohistochemistry, PCR, and chromatin immunoprecipitation were conducted. Results: We found that 4SC-202 and epirubicin inhibited the growth and induced cell cycle arrest and apoptosis in of HepG2 and Huh7 cells. The combination treatment also reduced tumorigenicity of liver cancer cells in vitro and in vivo. Moreover, 4SC-202 promoted tumor protein D52 (TPD52) transcription and induced TPD52 expression by inhibiting HDAC1/2. TPD52 downregulated ataxia telangiectasia mutated (ATM) expression caused by epirubicin, and liver cancer cells were more sensitive to epirubicin after ATM inhibition. Conclusions: Our results show that 4SC-202 enhances chemosensitivity to epirubicin in liver cancer cells via inhibiting ATM and suggest that the combination may serve as a novel strategy for treatment of liver cancer.


Introduction
Liver cancer is the third leading cause of death and a major public health problem in China [1]. In 2007, sorafenib was approved by the FDA for the treatment of liver cancer and is currently the rst-line drug for liver cancer [2]. However, sorafenib is often associated with drug resistance and serious adverse side effects, limiting its clinical application [2]. Therefore, the development of new effective strategies for liver cancer treatment is required. Epirubicin (Fig. 1a) is a type of anthracycline and shows cytotoxic effects on a variety of solid tumors [3,4]. Epirubicin induces DNA double-strand breaks (DSBs), causing toxic DNA damage and cell death [3].
Previous studies reported that epirubicin shows less cardiotoxicity compared with doxorubicin, another anthracycline [3,5]. However, the clearance of DSBs and DNA damage repair leads to chemoresistance to epirubicin, which hampers the clinical use of this agent [3,5].
HDAC inhibitors are a series of chemicals targeting HDACs that show anticancer effects in many types of cancer [6][7][8]. Four HDAC inhibitors have been approved by the FDA for cancer treatment, and several clinical trials of HDAC inhibitors in liver cancer are ongoing [9,10]. 4SC-202 (domatinostat) (Fig. 1b) is a novel selective class I HDAC inhibitor (HDACi) that mainly targets HDAC1/2/3 [11]. This compound has been reported to exert antineoplastic effects on various types of cancer in vitro and in vivo [12][13][14][15][16][17]. A previous study showed that 4SC-202 exhibits antitumor activity in liver cancer by activating the ASK1dependent mitochondrial apoptosis pathway [18]. A phase I study demonstrated that patients with advanced hematological malignancies bene ted from 4SC-202 administration [19]. Some studies have shown bene ts of combination anticancer drug treatments including HDACi. We previously reported that another HDACi, vorinostat, enhanced chemosensitivity to anticancer agents such as 5-uorouracil and oxaliplatin in liver cancer [7,8]. The HDACi valproic acid also augmented the cytotoxic effect of epirubicin on liver cancer HepG2 cells [20].
In this study, we investigated the anticancer effect of the combination administration of 4SC-202 and epirubicin on liver cancer cells. The underlying mechanism of the combination treatment was also explored.

Materials And Methods
Cell lines, cell culture, and reagents Cytotoxicity assay, combination index (CI) analysis, and dose-reduction index (DRI) analysis Cytotoxicity assay was performed as described previously [7,8]. Brie y, HepG2 (1,000 cells/well) and Huh7 (1,500 cells/well) cells were seeded and treated with increasing concentrations of 4SC-202 and epirubicin. IC50 values of these two drugs were calculated using SPSS software. CI and DRI were analyzed as described previously to evaluate the synergistic effect of the two drugs [7,8].

Flow cytometry
Cell cycle and apoptosis analysis was carried out as described previously [7,8]. HepG2 and Huh7 cells were treated with 4SC-202 and epirubicin for 48 h and analyzed using a FACSAria Cell Cytometer (BD Biosciences, San Jose, CA, USA). Cell cycle distribution and apoptotic rate were analyzed using CellQuest software (BD Biosciences).

Colony formation assay
In vitro tumorigenicity assay was carried out as described previously [7,8]. HepG2 (500 cells/well) and Huh7 (500 cells/well) cells were seeded in 6-well plates and treated with 4SC-202 and epirubicin. The numbers of colonies were analyzed using an Alpha Innotech Imaging system (Alphatron Asia Pte Ltd, Singapore).

Animal studies
Animal studies were performed as previously described [8]. All procedures in animal studies were approved by the Committee on the Ethics of Animal Experiments of Zhongnan Hospital, Wuhan University. Animal Experiments were carried out in the Animal Research Center of Wuhan University.
HepG2 cells were subcutaneously injected into mice. At day 9 after injection, mice were treated with 4SC-202, epirubicin or control and divided into 4 groups (n = 6). 4SC-202 (100 mg/kg) was dissolved in DMSO and dosed per os (p.o.) daily. Epirubicin (3 mg/kg) was dissolved in DMSO and injected intraperitoneally (i.p.) every third day. On day 27, all the animals were sacri ced by cervical dislocation. Tumors were harvested for growth and apoptosis analysis. Biochemical functions of liver and kidney including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), and blood urea nitrogen (BUN) in serum were measured by the Re otron Plus system (Roche, IN, USA). Organs such as lung, spleen, liver, kidney, and heart were sectioned for analyses.
Quantitative PCR analysis PCR was performed as described previously [8]. The sequences of primers for ATM, TPD52, HDAC1, HDAC2, HDAC3, and ACTB genes are listed in Table 1. ACTB was used as an internal control for normalization. The relative change folds were analyzed using the 2 -ΔΔCq method [21].
Statistical analysis SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses. All experiments were repeated at least three times. The nal data were expressed as the mean ± SD.
Comparisons among the different groups were analyzed using one-way ANOVA, and P < 0.05 was considered as statistically signi cant.

4SC
-202 elevated the level of acetylated histone H3 in liver cancer cells We rst examined the impact of the HDACi 4SC-202 on AcH3 level in two liver cancer cell lines. Western blotting result showed that 4SC-202 administration for 48 h enhanced the levels of AcH3 in HepG2 and Huh7 cell lines in a dose-dependent manner (Fig. 1c).

4SC-202 and epirubicin inhibited the proliferation of liver cancer cells
We then explored the cytotoxic effect of 4SC-202 and epirubicin on the two liver cancer cell lines. HepG2 and Huh7 cells were treated with increasing concentrations of 4SC-202 or epirubicin. CCK-8 assays showed that 4SC-202 and epirubicin each exerted time-dependent and dose-dependent inhibitory effects on HepG2 and Huh7 cell proliferation ( Fig. 1d and e). We also examined 4SC-202 in combination with epirubicin and found that all CI values were lower than 1 (Fig. 1f) and all DRI values were higher than 1 ( Fig. 1g), indicating a synergistic effect of the combination of 4SC-202 and epirubicin.
4SC-202 and epirubicin led to cell cycle arrest and apoptosis in liver cancer cells Epirubicin treatment resulted in S phase arrest in both HepG2 and Huh7 cells (Fig. 2a). Co-treatment with 4SC-202 reduced the epirubicin-mediated S phase arrest, promoting S phase slippage of epirubicintreated cells and inducing transition into G2/M phase (Fig. 2a). Both 4SC-202 and epirubicin caused apoptosis in HepG2 and Huh7 cells (Fig. 2b). Co-treatment with the two drugs signi cantly increased the apoptotic level compared with either 4SC-202 or epirubicin alone. Western blotting demonstrated that the expression of the apoptotic factor cleaved-PARP was elevated after 4SC-202 and/or epirubicin (Fig. 2c).

4SC-202 and epirubicin attenuated the tumorigenicity of liver cancer cells in vitro
We next performed colony formation assay to evaluate the effect of 4SC-202 and epirubicin on the tumorigenicity of liver cancer cells. The results showed that 4SC-202 and epirubicin decreased both the size and number of colonies in HepG2 and Huh7 cells (Fig. 2d). Combined treatment of the two drugs signi cantly inhibited colony formation compared with treatment of 4SC-202 or epirubicin alone.
4SC-202 increased the antitumor effect of epirubicin in vivo We next evaluated the in uence of 4SC-202 and epirubicin on the growth of liver cancer cells in an animal model. On day 27, mice were sacri ced. Treatment with 4SC-202 or epirubicin caused a slight decrease in tumor growth, while the combination led to a signi cantly increased reduction in tumor size and weight compared with the single treatments alone (Fig. 3a-c). Immunohistochemistry of Ki-67 and cleaved caspase-3 was used to analyze proliferation and apoptosis in tumor sections. Treatment with either 4SC-202 or epirubicin caused a slight decrease in the percentage of Ki-67-positive cells and a slight increase in cleaved caspase-3-positive cells (Fig. 3d). Notably, the combination treatment led to a signi cant reduction of Ki-67-positive cells and a signi cant accumulation of cleaved caspase-3-positive cells compared with the single treatment groups. No differences in body weight of animals were observed among groups, indicating minimal drug toxicity or 4SC-202 and/or epirubicin treatment (Fig. 3e). Moreover, biochemical functions of liver and kidney were not affected by administration of the two drugs ( Fig. 3f). No obvious histological changes were observed in the organs such as lung, spleen, liver, kidney, and heart (Fig. 3g). Therefore, the administration doses of 4SC-202 and epirubicin used in this study were safe and effective.

4SC-202 downregulated ATM expression caused by epirubicin in liver cancer cells
Epirubicin treatment of in HepG2 induced elevation of both ATM mRNA and protein levels ( Fig. 4a and b).
In contrast, ATM mRNA and protein expressions were downregulated following 4SC-202 treatment ( Fig. 4c and d). Co-treatment of both drugs showed that 4SC-202 reversed the effect on ATM upregulation caused by epirubicin ( Fig. 4e and f).
To examine the role of ATM in epirubicin-mediated effects on liver cells, we inhibited ATM expression using siRNA and veri ed the knock-down by qPCR and western blotting ( Fig. 4g and h). We found that after ATM inhibition, HepG2 cells were much more sensitive to epirubicin, indicating the role of ATM in the chemosensitivity of liver cancer cells to epirubicin (Fig. 4i).

TPD52 was repressed by HDAC1/2 and downregulated ATM
We next evaluated TPD52 expression after 4SC-202 and epirubicin treatment. The mRNA and protein expressions of TPD52 were upregulated by 4SC-202 but not in uenced by epirubicin ( Fig. 5a and b). We con rmed effective don regulation of TPD52 expression by siRNA (Fig. 5c). While ATM mRNA levels were not affected upon TPD52 knockdown, ATM protein level was upregulated in cells transfected with TPD52 siRNA (Fig. 5d and e), which was consistent with previous research in breast cancer cells [23].
TPD52 mRNA was increased and ATM mRNA was decreased followed by HDAC1/2 inhibition (Fig. 5i and  j). Western blot results of TPD52 and ATM levels were consistent with these results (Fig. 5k).
AcH3 and HDAC1/2 competitively bound to TPD52 promoter We next performed ChIP assay to evaluate whether AcH3 or HDAC1/2 directly bound to the promoter of the TPD52 gene. First, we designed three pairs of primers for the TPD52 promoter region ( Fig. 6a and b). We observed strong binding of AcH3 to the TPD52 promoter region, and binding was signi cantly enriched with 4SC-202 treatment (Fig. 6c). We also detected binding of HDAC1/2 to the TPD52 promoter region, but binding of both HDACs was attenuated by 4SC-202 treatment (Fig. 6d and e). These results suggest that 4SC-202 upregulates TPD52 expression by inhibiting HDAC1/2 and increasing AcH3 at the TPD52 promoter region.

Discussion
In this study, we investigated the effects of the combination treatment of the HDACi 4SC-202 with epirubicin on liver cancer cells. We found that 4SC-202 augmented AcH3 levels in liver cancer cells. The combination treatment of 4SC-202 and epirubicin synergistically inhibited the proliferation of liver cancer cells. Epirubicin caused S phase arrest and 4SC-202 partially reversed this effect; 4SC-202 mediated S phase slippage of cells after epirubicin treatment and led to transition into G2/M phase. We also found that 4SC-202 and epirubicin synergistically triggered apoptosis and showed that 4SC-202 and epirubicin synergistically attenuated the tumorigenicity of liver cancer cells in vitro and in vivo. Together these results indicate 4SC-202 as an e cacious chemosensitizer of epirubicin in liver cancer cells.
As a DNA-damaging reagent, epirubicin causes DSBs, and the DNA damage then activates the ATM pathway. ATM plays a key role in regulation of the DNA repair. ATM phosphorylates and then activates BRCA1, BRCA2, FANCD2, BLM, Nbs1, Chk1, H2AX, WRN, p53, RPA, SMC1, and c-Abl to induce homologous recombination [24]. Consequently, ATM promotes chemoresistance to epirubicin, and our study showed that liver cancer cells were much more sensitive to epirubicin after ATM inhibition.
Previous studies demonstrated the synergistic effects of HDACi and epirubicin on various types of cancers such as breast cancer, gastric cancer, colorectal cancer, melanoma, lung cancer, and osteosarcoma in vitro and in vivo [25][26][27][28]. In a clinical trial, the HDACi valproic acid followed by epirubicin has shown promise as an effective treatment for advanced solid tumors [29]. Some studies showed that HDACi can function as a negative regulator of ATM pathway. TPD52 is an inhibitor of ATM pathway [23]. A direct interaction between ATM and TPD52 protein was con rmed by co-immunoprecipitation and GST pull-down assays, and overexpression of TPD52 led to reduced ATM protein levels [23]. Previous studies reported an involvement of TPD52 in the tumorigenesis of liver cancer and TPD52 has bee considered as a tumor suppressor in liver cancer [27]. In our study, we hypothesized that 4SC-202 may upregulate TPD52 expression to inhibit ATM expression. We found that 4SC-202 decreased ATM expression upregulated by epirubicin and increased TPD52 expression. Consistent with previous studies, we also found that TPD52 downregulated the protein level of ATM but not mRNA expression. Through knockdown experiments, we found that HDAC1/2 inhibited the mRNA and protein levels of TPD52 but induced those of ATM, suggesting that HDAC1/2 in uenced the mRNA level of ATM independent of TPD52. The in uence of HDAC1/2 on ATM transcription needs to be further explored. ChIP assay further demonstrated that AcH3 and HDAC1/2 competitively bound to the TPD52 promoter to in uence its transcription.
In summary, our study showed that 4SC-202 enhanced the chemosensitivity of liver cancer cells to epirubicin (Fig. 7). 4SC-202 promotes TPD52 transcription and expression via inhibition of HDAC1/2. TPD52 attenuates ATM expression caused by epirubicin. These ndings suggested that 4SC-202 may serve as a chemosensitizer to epirubicin for patients with liver cancer.   for 48 h. All the wells were stained and images were acquired on day 14. Colony formation rate was calculated. *P < 0.05, **P < 0.01, ***P < 0.001.    AcH3 and HDAC1/2 competitively bind to the TPD52 promoter. a TPD52 promoter. Sequences of primers are in blue letters. b Schematic diagram of the TPD52 promoter region. c ChIP assay was performed to evaluate the binding of AcH3 to the promoter of TPD52. Three different DNA regions (A, B, and C) from the TPD52 promoter were analyzed by qPCR. Cells were treated with 4SC-202 or vehicle. d, e ChIP assay was performed to evaluate the binding of HDAC1 and HDAC2 to the promoter of TPD52. *P < 0.05, **P < 0.01.

Figure 7
Overview of the molecular mechanism by which 4SC-202 increases sensitivity to epirubicin in liver cancer cells.