Cell cultures and reagents
Human small cell lung cancer (SCLC) cell lines were kept in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO2. RPMI-1640 media, FBS, and penicillin/streptomycin were purchased from Gibco, Life Technologies (Carlsbad, CA, USA). CUDC-907 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Olaparib was obtained from Selleck Chemical (Shanghai, China). The compounds were first dissolved in DMSO and then diluted in medium (Sigma-Aldrich, Saint Louis, MO, USA).
Cell viability assay
The small cell lung cancer cells were seeded into a 96-well dish in triplicates for each condition at an initial density of 3 × 103 cells per well and cultured for 24 hours. The following day the cells were treated with increasing concentrations of CUDC-907 or olaparib alone or in combination for 72 hours. The cells were then harvested and analyzed using the CellTiter-Glo luminescent assay by following the manufacturer’s instructions. The IC50 values of CUDC-907 were calculated from the sigmoidal dose-response curve fits of data using the GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). Combination drug synergy was assessed using the CalcuSyn software (Biosoft) as previously described. In general, “CI < 1” denotes synergism, whereas “CI > 1” suggests antagonism [25].
Cell cycle and apoptosis
After indicated treatment, the cells were harvested and fixed by the dropwise addition of ice-cold EtOH. The fixed cells were stained with PI/RNase staining buffer (BD Biosciences, Franklin, NJ, USA), and then analyzed by a FACS Calibur (Sony Biotechnology, San Jose, CA, USA). The cell-cycle profiles were determined using ModFit software (Verity Software House, Topsham, ME, USA). To measure cell apoptosis, the cells were exposed to CUDC-907 or olaparib or in combination for 48 hours following which Annexin assays were conducted by incubating cells with Annexin V–fluorescein isothiocyanate and propidium iodide. Cell apoptosis was analyzed by a FACS Calibur (CytExpert, Beckman Coulter, Brea, CA, USA) and the data was analyzed using FlowJo V10 software (FlowJo LLC, Ashland, Oregon, USA)
Western blotting and antibodies
Western blot samples were prepared as described previously[17]. Antibodies against the following proteins were used in this study: Phospho-Akt (Ser473) (1:1000, CST, #9271), Phospho-4E-BP1 (Ser65) (1:1000,CST, #9456), Phospho-S6 Ribosomal Protein (Ser235/236) (1:1000, CST, #4858 ), acetyl-Histone H3(lys9)(1:1000, CST, #9649 ), PARP (1:1000, CST, #9542), Phospho-CHK1 (Ser317) (1:1000, CST, #12302), p-DNA-PK (S2056) (Abcam, ab124918), p-RPA2/32 (Ser4/Ser8) (1:5000, Novus, NB100-544), c-MYC (1:1000, CST, #5605), N-MYC (1:1000, CST, #9405), γ-H2AX(1:1000, CST, #9718), Rad51 (1:10000, Abcam, ab133534), BCL-2(1:1000, CST, #4223), BCL-XL(1:1000, CST, #2764 ), BRCA1(1:1000, CST, #14823),53BP1(1:1000, CST, #88436), FoxM1(1:1000, CST, #5436) and β-actin (1:10000, Transgen, HC201-02).
Immunofluorescence microscopy
Immunofluorescence staining was performed as described previously [23]. After blocking and permeabilizing, the cells were incubated with anti-γH2AX (1:500, CST, #2577) and anti-Rad51 (1:500, Abcam, ab133534), followed by a fluorophore-conjugated secondary antibody for 1 hour in a dark humidified chamber. After washing, the cells were counterstained using SlowFade Gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA) and imaged using a Zeiss fluorescence microscope.
Comet assay
Comet assay was performed as previously described[26]. Briefly, the cells were inoculated in a 6-well plate. After 48 hours of drug treatment, the cells were collected and subjected to neutral single-cell gel electrophoresis. Following electrophoresis, the cells were stained with SYBR gold and viewed using a Zeiss fluorescence microscope.
Quantitative real-time PCR
The total RNA from each sample was isolated with Trizol reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. cDNA synthesis was carried out with the cDNA Synthesis Kit (Roche, Mannheim, Germany). Q-PCR was performed using FastStart Essential DNA Green Master (vazyme, China) on a Roche LightCycler 96 Real-Time PCR System and following primers:
Rad51 | Forward primer | 5’-CAACCCATTTCACGGTTAGAGC-3’ |
Reverse primer | 5’-TTCTTTGGCGCATAGGCAACA-3’ |
MCM5 | Forward primer | 5’-ATGTCGGGATTCGACGATCCT-3’ |
Reverse primer | 5’-CCAGGTTGTAATGCCGCTTG-3’ |
CDC7 | Forward primer | 5’- GAGGCGTCTTTGGGGATTCAG-3’ |
Reverse primer | 5’- GGTCCTACTTGTAACTGTGCTG-3’ |
HK2 | Forward primer | 5’-GAGCCACCACTCACCCTACT-3’ |
| Reverse primer | 5’-CCAGGCATTCGGCAATGTG-3’ |
LDHA | Forward primer | 5’-ATGGCAACTCTAAAGGATCAGC-3’ |
Reverse primer | 5’-CCAACCCCAACAACTGTAATCT-3’ |
Patients-derived-xenograft (PDX) mouse model of small cell lung cancer
The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences. The tumors were subcutaneously transplanted into the dorsal flanks of mice. When the tumor volume reached about 100mm3 in about two to three weeks, the mice were randomized into four groups and treated with DMSO control, CUDC-907, olaparib, and CUDC-907/olaparib combination for 15 days. For drug treatment, CUDC-907 was administrated to mice by oral gavage at a dose of 75 mg/kg/day. Olaparib was administrated to mice by intraperitoneal administration at a dose of 55 mg/kg/day. The tumor volume and weight of mice were measured once a week. All mouse experiments were carried out according to a protocol approved by the Institutional Animal Care and Use Committee of Hefei Institutes of Physical Science. Female NOD/SCID (4–5-week old)) was purchased from Beijing Weitong Lihua Experimental Animal Limited Company.
Histological and immunohistochemical analyses
The tumor tissues were fixed overnight with 4% formalin, followed by dehydration and paraffin embedding. Histopathological analysis was carried out on 4-micrometer sections stained with hematoxylin and eosin. Immunohistochemical analyses were carried out according to the immunohistochemical (IHC) method described earlier [27]. Antibodies against the following proteins were used in this study: Ki67 (1:1000, CST #9449), Cleaved-Caspase 3 (1:300, CST, #9661), RAD51 (1:200, Abcam, ab133534) and γH2AX (Ser139) (1:500, CST, #2577), c-myc (1:500, abcam, ab32072). Four to five random 40X fields were scored for each tumor sample. The staining intensity was quantified as the percentage of nuclear positive cells.
Quantification and statistical analysis
The quantitative results were analyzed by double-tailed unpaired t-test using GraphPad Prism version 6.00 for Mac GraphPad Software. For in vitro analytical experiments, each test was repeated at least three times, with experiments being performed at least twice. Error bars for SD or SEM are shown. Where indicated in the figures, degrees of p-value significance are as follows: *p < 0.05, **p < 0.01, ***p < 0.001.