Cell lines and cell culture

American Typical Cell Culture (ATCC) was the source of ovarian cancer cells SKOV3, PEO-1, and CAOV3. As a culture medium, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin were added to RPMI-1640 medium for SKOV3 cells. In addition to 10% fetal bovine serum, 10% glucose, and 1% penicillin/streptomycin, cell culture medium was used for PEO-1 and CAOV3 cells. 37 °C, 5%CO2, and 95% humidity were the culture environments for these cell lines. The source of ATRA (all-trans retinoic acid) and TAM (tamoxifen) was MedChemExpress.

Cell proliferation assay

Cell proliferation assays were performed at 37˚C. SKOV3, PEO-1 and CAOV3 cells were inoculated into 96‑well plates with a density of 5 × 103 cells per well. The cells were incubated overnight in a 37˚C, 5%CO2 incubator, observed to assess adhesion and respectively treated with ATRA and TAM after cell adhesion, for 72 h. A total of 20 µl MTS was added to each well away from light and incubated in a 37˚C incubator for 20‑50 min. The optical density (OD) of each well was measured at 490 nm using an Spectra Max 190 enzyme spectrometer (Molecular Devices LLC), and the OD of all samples were recorded when the OD of the control reached 0.8‑1.2. MTS viability assays measures cell proliferation by measuring cell metabolism.

Colony formation rates

Five hundred and three cells per well were seeded in 6-well cell culture plates for SKOV3 and PEO-1 cells, adding 10% FBS to medium and incubating overnight in triplicate. In the experimental group, fresh medium containing drug was added to maintain a certain drug concentration, and the control group was also treated with DMSO at the same volume. A visible colony was observed by naked eye after one week at 37 °C. After cloning had occurred, cells were fixed with paraformaldehyde for about 25 min, in the subsequent step, the cells were washed with PBS and stained for 15 to 20 min with 2% crystal violet solution to color them. Finally, an excess of staining solution was washed off the surface of the cells and dried by air. A count of cell colonies was performed in the Wells, rate of colony formation (%)/rate of colony formation (control) (%) was used to calculate colony formation rates.

Flow cytometry

SKOV3 and PHO-1 cells in the experimental group were seeded in medium containing a certain concentration of ATRA and TAM, and In the control group, DMSO was added at the same volume. A 48-h treatment period was followed by the collection of supernatants and digested cell suspensions. At room temperature in the dark, whole cells in the binding buffer suspension were stained with 1 µL RNA enzyme (Sigma, USA), 2 μL annexin V–FITC (BD, USA), and 2 μL propidium iodide (PI) (Sigma, USA) for 15 min. Cells unstained and those stained once served as controls. Flow cytometry cups were used to examine these samples, and FACS Calibur (BD) was used to analyze stained cell. FlowJo software (v10) was used to analyze the data.

Western blot analysis

10-cm dishes were used to seed and culture SKOV3 and PEO-1 cells. Cells were collected after 48 h of drug treatment. RIPA lysis buffer (Sigma) was used to extract proteins, and BCA assay was used to determine protein concentrations. SDS-PAGE (Sanguang Bioengineering Technology Services, Shanghai, China) was performed by means of the operating instructions on the Cell Signaling Technologies website. Western blotting was performed using antibodies against BCL2 (T40056F, ABWAYS), BCL-XL (2764S, Cell Signaling Technology), CyclinB1 (12231S, Cell Signaling Technology), CyclinE1 (4129 T, Cell Signaling Technology), c-MYC (CY5150, ABWAYS), γ-H2AX (9718S, Cell Signaling Technology), GAPDH (AB0036, ABWAYS). Anti-rabbit 800 and mouse 800 (LI-COR Biosciences). Imaging of the membrane was carried out using an Odyssey infrared imaging system (LI-COR Biosciences), and measurements were made using Image Studio Lite software.

Immunofluorescence

Eighty-three thousand cells were seeded into each well of a 24-well plate, and a cover slip was placed on top. Different concentrations of ATRA and Tamoxifen were used to treat the cells. After treatment, incubation was started at 37 °C, 5%CO2 and 95% humidity for 48 h. 0.2%Triton (Sangon, China) in 1 × PBS was used to fix the cells and permeabilized for 30 min. 1%BSA (Sangon) in 0.2%Triton/PBS was used to incubate the cells for 30 min. Primary rabbit anti-γH2AX antibody (1:400) was then used to incubate the cells overnight at 4° C. 0.2%triton/PBS was then used to wash the cells three times for 3 min each, and a second anti-rabbit 800 antibody was used to incubate with the cells for 1 h in the dark. DAPI (D9542, Sigma) was used to counterstain the nuclei for 5 min, and for three 5 min-washings, 0.2% triton/PBS was used. An Olympus inverted fluorescence microscope was used to capture images.

Quantitative real-time PCR (RT-qPCR)

ATRA or TAM was used to treat SKOV3, PEO-1, and CAOV3 cells for 24 h, and isolation of total RNA was performed using TRIzol reagent. RNA was extracted, and a reverse transcription reaction using Prime Script RT kit was used to produce cDNA from the extracted RNA. Reverse transcription of cDNA served as the template for the RT-qPCR reaction, and this reaction was detected using SYBR Green on the QuantStudio® 3 real-time PCR system. The internal control was GAPDH. The conditions for the reaction were as follows: In the first step, 25 °C is kept for 5 min, in the second step, 42 °C is kept for 30 min, in the third step, 85 °C is kept for 5 min, and in the final step, 16 °C is kept. In the PCR profile, the first reaction took place for 2 min at 95 °C, followed by 40 cycles at 95 °C for 10 SEC and 60 °C for 30 SEC. GraphPad Prism was used to analyze the data, and the 2-ΔΔCT method was used to calculate relative gene expression.

Xenograft tumor growth

1 × 107 SKOV3 cells were injected subcutaneously with Matrigel at a ratio of 1:1 in 6 ~ 8 week old female nude mice. When the average tumor volume reached 100 mm3, the mice were randomly grouped and then administered by intraperitoneal injection for 36 d. The body weights and tumor sizes of the rats were measured. Tumour volume was calculated by the formula: tumor volume = length × width2 × 0.52. Mice were executed after drug administration, and the tumors and major organs were dissected and collected for subsequent experiments.

Statistical analysis

The mean ± SD is presented as the result. All experiments presented in this article were performed at least three times to make the results more reliable, except for the animal experiments. In order to determine whether the differences between the two groups were statistically significant, we used the Student t test. Statisticians used GraphPad Prism 8.0 to analyze the data. In *P < 0.05, P < 0.01, P < 0.001 and ******P < 0.0001 level to determine the mean significant difference.