Cell lines, cell culture, and reagents
The HepG2 hepatoma cell line and Huh7 hepatocellular carcinoma cell line were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). Both cell lines were cultured in high-glucose DMEM (Hyclone, Logan, UT, USA) containing 10% FBS (Gibco, Grand Island, NY, USA) at 37°C and 5% CO2. Epirubicin and 4SC-202 were purchased from Selleck Chemicals (Houston, TX, USA). Crystal violet and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). The CCK-8 kit was purchased from Dojindo (Kumamoto, Japan).
Cytotoxicity assay, combination index (CI) analysis, and dose-reduction index (DRI) analysis
Cytotoxicity assay was performed as described previously [7, 8]. Briefly, 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).
Western blotting
Western blotting was performed as described previously [7, 8]. Primary antibodies against acetylated histone H3 (AcH3), β-actin, and TPD52 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against PARP, ATM, pATM, HDAC1, HDAC2, and HDAC3 were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). All secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).
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 sacrificed 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 Reflotron Plus system (Roche, IN, USA). Organs such as lung, spleen, liver, kidney, and heart were sectioned for analyses.
Immunohistochemistry analysis
Immunohistochemistry was performed as previously described [7, 8]. Ki-67 antibody was purchased from Dako (Golstrup, Denmark) and cleaved caspase-3 was from CST.
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].
Table 1
Primer sequences for qPCR
Gene
|
Primer sequences
|
ATM
|
F: 5’-GTGCCTAAACAAAGCTCTCAG-3’
|
|
R: 5’-CCTCAACACTTCTGACCATCT-3’
|
TPD52
|
F: 5'-GAGGAAGGAGAAGATGTTGC-3'
|
|
R: 5'-GCCGAATTCAAGACTTCTCC-3'
|
HDAC1
|
F: 5'-GCTCCACATCAGTCCTTCC-3'
|
|
R: 5'-GGTCGTCTTCGTCCTCATC-3'
|
HDAC2
|
F: 5’-AGGCAAATACTATGCTGTC-3’
|
|
R: 5'-TGAAACAACCCAGTCTATC-3’
|
HDAC3
|
F: 5’- CGGGATGGCATTGATGAC-3’
|
|
R: 5'- GGGCAACATTTCGGACAG-3’
|
ACTB
|
F: 5'-CAAGGCCAACCGCGAGAAGAT-3'
|
|
R: 5'-CCAGAGGCGTACAGGGATAGCAC-3'
|
RNA interference
Knockdown of ATM, TPD52, HDAC1, HDAC2, and HDAC3 was carried out using siRNA. HDAC1, HDAC2, and HDAC3 siRNAs were purchased from Santa Cruz Biotechnology. The sequence for siATM 1 was 5′-GCAAAGCCCUAGUAACAUA-3′ and the sequence for siATM 2 was 5′-GGGCAUUACGGGUGUUGAA-3′. The sequence for siTPD52 1 was 5′- GCGGAAACUUGGAAUCAAU-3′ and the sequence for siTPD52 2 was 5′- GGAGAAGUCUUGAAUUCGG-3′. The siRNAs were synthesized by Ribobio (Guangzhou, China). Transfection of siRNAs was performed using Lipofectamine™ 2000 Transfection Reagent according to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay
Quantitative ChIP assay was performed as described previously [22]. HepG2 cells were immunoprecipitated with anti-AcH3, anti-HDAC1, anti-HDAC2, or IgG. The primers for region A of the TPD52 promoter were 5′-CACCAGATTCAGGAGCCCAC-3′ (forward) and 5′- GTCCCATCGATCACCCAAGG-3′ (reverse). The primers for region B of the TPD52 promoter were 5′- AAGTGCTTGGATGACTGGCA-3′ (forward) and 5′-GCGCAACCATCACCGTAATC-3′ (reverse). The primers for region C of the TPD52 promoter were 5′-CCATGCTTAGTGGCAAGAGG-3′ (forward) and 5′-GTTAGTTGCATCGTCAGCCT-3′ (reverse).
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 final 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 significant.