2.1. Materials
Human ovarian cell lines SK-OV3 and A2780 were purchased from Fuheng Biological Co.(Shanghai, China), and OV-CAR5 was stored in lab. The BALB/C nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) [License SCXK (Jing) 2021–0006]. NIR (toluenesulfonate nilaparil) capsules were provided by Zaiding Pharmaceutical Co., Ltd. (China). ANL (anlotinib hydrochloride) capsules were provided by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Jiangsu, China). Roswell Park Memorial Institute (RPMI) 1640, high-glucose DMEM, and 0.25% trypsin solution and penicillin-streptomycin solution were purchased from Grand Island Biological Co., Ltd. (NY, USA). Dulbecco's modified eagle medium nutrient mixture F-12 (DMEM/F-12, 1:1) was purchased from Thermfisher Biochemical Products Co., Ltd. (Beijing, China). Foetal bovine serum (FBS) was purchased from Lanzhou Minhai Biotechnology Co., Ltd. (Gansu, China). Dimethyl sulfoxide (DMSO), thiazolyl blue (MTT), crystal violet solution, phospholipid-binding protein (ANNEXIN V)-FITC/PI Apoptosis Detection Kit, and BCA Protein Concentration Measurement Kit were purchased from Solarbio Life Sciences (Beijing, China). FGF basic/bFGF and EGF were purchased from MedChemexpress Biotechnology Co., Ltd. (New Jersey, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The following antibodies, include phosphatidylinositol kinase (PI3K), p-PI3K, serine/threonine kinase (Akt), p-Akt, neural calcium mucin (N-cadherin), epithelial calcium adhesion protein (E-cadherin), B-lymphoblastoma-2 gene (Bcl-2), BCL2-associated X (Bax), SRY box-2 (SOX2), aldehyde dehydrogenase 1A1 (ALDH1A1), hypoxia-inducible factor 1 subunit alpha (HIF1-α), and microtubule protein (Tubulin) were purchased from Chengdu Zhengneng Biological Co., Ltd. (Sichuan, China).
2.2. Cell Culture
RPMI1640 medium was used for SK-OV3 cells, and OV-CAR5 and A2780 cells were cultured in high-glucose DMEM. The complete medium configuration was medium + 1% penicillin-streptomycin solution + 10% FBS. Cells were cultured at 37°C in a 5% CO2 incubator.
2.3. Determination of Drug Interaction
Drug interaction assays were performed to study the synergistic effects of the co-administration of NIR + ANL, as described by Chou and Talalay. Briefly, ovarian cancer cells were treated with different concentrations of NIR (1 µM) and ANL (8, 4, 2, 1, 0.5, and 0.25 µM), and their optimal dosages were selected using their respective semi-inhibitory concentration values. Cell growth viability after 24 h of treatment was determined using the Methyl thiazolyl tetrazolium (MTT) assay, and the effect categories were determined by calculating the combination index (CI) using the calculation software version 1.0 (CompuSyn, Paramus, NJ, USA). Antagonistic, additive, and synergistic effects are expressed as CI > 1, CI = 1, and CI < 1, respectively [11].
2.4. MTT Assay
Ovarian cancer cells were seeded at a density of 5 × 104 cells per well for 24 h. The cells were treated with different concentrations of the drug or DMSO for 24 h and incubated with 20 µL of MTT solution for 4 h at 37°C. After aspirating the medium, each well was filled with 100 µL of DMSO and incubated for 10 min. The absorbance was measured at 492 and 630 nm using an microplate reader (ThermoFisher Scientific, Shanghai, China) [12].
2.5. Colony Formation Assay
Cells were inoculated in 6-well plates at a density of 1,000 cells per well for 24 h and treated with 1 µM NIR, 1 µM ANL, or 1 µM NIR + 1 µM ANL. The cells in the control group were treated with an equal amount of DMSO. The medium was aspirated every 3 d, replaced with fresh DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. After 12 d of incubation, the cells were fixed with 4% paraformaldehyde, washed twice with 1 × phosphate buffer saline (PBS), dried at 37°C, and stained with 0.1% crystal violet at 25°C room temperature. Finally, the optimal number of clones was determined [13].
2.6. Wound Healing Assay
First, a horizontal line was drawn evenly at the bottom of a 6-well plate using a marker pen. Subsequently, ovarian cancer cells were uniformly inoculated in the marker plate at a density of 3 × 105 cells per well to allow adhesion and incubated with DMSO (control), 1 µM NIR, 1 µM ANL, or 1 µM NIR + 1 µM ANL at 37°C and 5% CO2. When the cells were close to fusion, the monolayers were scratched, and cell migration was recorded under an inverted microscope (Olympus, Tokyo, Japan) at 0, 24, and 48 h. Changes in wound width were analysed using the Sige Image Acquisition System software (version 4. 0. 3. 27) [14].
2.7. Transwell Assay
Ovarian cancer cells (1 × 105) were suspended in 200 µL of serum-free medium containing a mixture of 1 µM NIR, 1 µM ANL, 1 µM NIR + 1 µM ANL, and DMSO (control) and were added to the upper chamber of Transwell inserts. The lower chamber was filled with 600 µL of medium, and 20% FBS was added. After incubation at 37°C for 24 h, cells crossing the membrane were fixed in 4% formaldehyde at room temperature for 20 min, washed twice with PBS, soaked in 0.1% crystal violet for 30 min at room temperature, and counted under an inverted microscope [15].
2.8. In vitro Determination of Apoptosis
Ovarian cancer cells were homogeneously inoculated into 6-well plates at a density of 5 × 105 cells per well, allowed to adhere, and incubated with DMSO (control), NIR (1 µM), ANL (1 µM), or a combination of NIR and ANL for 48 h. The medium was aspirated, and the cells were washed thrice with pre-cooled PBS, followed by digestion with trypsin without EthyleneDiamine Tetraacetic Acid (EDTA). Centrifugation was performed after digestion. The cells were first incubated with membrane-linked protein V-FITC dye for 5 min at 4°C in the dark and finally with PI for 5 min at 4°C (also in the dark). Detection of apoptotic cells was performed using flow cytometry [16].
2.9. Tumorsphere Assay
Cells in the logarithmic growth phase were inoculated in well plates at 1 × 105 cells/well and incubated for 24 h. Different concentrations of compounds were added to the corresponding wells, and the cells were treated for 48 h. Cells in the corresponding wells were digested with trypsin, and centrifuged using normal medium, followed by secondary centrifugation with PBS. The supernatant was discarded, and each cell group was resuspended in a serum-free tumour ball-forming medium (20 ng/mL EGF, 20 ng/mL bFGF, and 2% B27 in DMEM:F12). Cells were counted for each group, inoculated at a density of 1,000 cells/well in a low-adherence 24-well plate and blown carefully for the cells in each well to show a single distribution without clustering. Three replicate wells were established for each concentration gradient. The plates were incubated in a cell culture incubator, photographed, and counted under a microscope after 7 d [17].
2.10. Stem Cell Spheroidal Limiting Dilution Assay
Cells in the logarithmic growth phase were inoculated at 1 × 105 cells/well and cultured in an incubator for 24 h. Different concentrations of compounds were added to the corresponding wells, and the cells were treated for 48 h. Cells in the corresponding wells were digested with trypsin, centrifuged using the normal medium, and centrifuged twice with PBS. The supernatant was discarded, and each group of cells was resuspended in a serum-free tumour ball formation medium. The number of cells was counted, and the cells were inoculated into 96-well plates. Each sample was divided into five groups according to the number of cells: 200, 100, 50, and 25, in a decreasing gradient, and each group was set up with 8–10 replicate wells. Counting was done after 7–10 d of incubation. Data were processed using the Limit Analysis online analysis program. [18]
2.11. Quantitative Real-time PCR (qRT-PCR)
Logarithmically grown ovarian cancer cells were taken and inoculated with 3 × 105 cells/well in 6-well plates. After 24 h, different drug concentrations were added and acted for 48 h. RNA was extracted, and reverse transcription was performed. CSC primers (ALDH1A1 and SOX2) and GAPDH (control) were synthesised by Beijing Prime Biotech. The primer sequences are as follows:
ALDH1A1
5'-ACAGGATCAACAGAGGTTGGC-3' (Forward)
5'-GGCTGTTGTCATACTTCTCATGG-3' (Reverse)
These values were used as threshold cycles for each gene and normalised to the internal reference gene (GAPDH). The 2−ΔΔCT method was used to calculate the relative changes of each gene in different cell lines [19].
2.12. Determination of Anti-Tumour Activity In Vivo
Four-week-old female BALB/c mice (15–20 g) were fed a standard diet and housed at a constant temperature (22 ± 2°C) and humidity (55 ± 15%) with a standard light/dark cycle (12:12 h light/dark). Animal procedures were approved by the Tianjin University of Science and Technology Institutional Animal Care Committee and performed in strict compliance with local and national ethical guidelines. After 7 d of normal feeding, mice were subcutaneously injected with a 5 × 107 A2780 cell suspension near the right axilla. The tumour size was measured every other day. The maximum longitudinal and transverse diameters of each tumour were measured using calipers, and the total tumour volume was calculated as (length × width2)/2. When the tumour volume approached 100–150 mm3, the mice were randomly divided into the following groups and administered the indicated treatments daily: 5% sodium carboxymethyl cellulose (CMC-Na) aqueous solution (control group), NIR (15 mg/kg), ANL (1.5 mg/kg), or NIR (10 mg/kg) + ANL (1 mg/kg). NIR and ANL were dissolved in 5% CMC-Na aqueous solution and intragastric administration for 16 d. The mice were killed painlessly, and blood, tumours, and different tissues were collected from all groups. The tumour was quickly taken out, weighed, and cleaned using a physiological saline solution and stored at -80°C.
2.13. Western Blotting
Cells or tumour tissue homogenates were incubated in RIPA (Radio Immunoprecipitation Assay) buffer containing phenylmethanesulfonyl fluoride (PMSF, 1 mM) for 0.5 h and centrifuged at 13,000 rpm for 10 min at 4°C. The protein concentration was determined by BCA Protein Assay Kit. After separating proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, samples were transferred onto polyvinylidene fluoride (PVDF) membranes, blocked using 5% skim milk diluted in PBST, and shaken for 1 h at 25°C. The PVDF membranes were washed thrice with PBST. The membranes were incubated with primary antibodies (1:1000) at 4°C overnight. The PVDF membranes were washed thrice with PBST and incubated with the secondary antibodies (1:5000) for 1 h at 25°C with shaking. Lastly, the PVDF membranes were visualized using an Odyssey western blotting system (Amersham Pharmacia Biotech). Image J software was used for grey analysis of protein bands[20].
2.14. Immunofluorescence
Tumour tissues were embedded in paraffin, dehydrated in a graded ethanol. Sections were treated with 3.0% hydrogen peroxide for 25 min to eliminate the effect of endogenous peroxidase. Sections were incubated overnight at room temperature with the primary antibodies diluted in PBS (pH 7.4): ALDH1A1 Rabbit mAb (ABclonal Technology Co., Ltd. Wuhan, China) at 4°C. Sections were washed in PBS and incubated with HRP-labelled goat anti-rabbit IgG secondary antibody (1:200; Servicebio, Wuhan, China) for 50 min and counterstained with DAPI. The sections were finally imaged under an upright fluorescence microscope (Nikon, Japan) [21].
2.15. Statistical Analysis
GraphPad Prism 8.0 was used for data analysis. Results are presented as means ± standard deviation (SD) or means ± standard error of the mean (SEM). Statistically significant differences between two or more groups were analysed by one-way analysis of variance (ANOVA), the least significant difference (LSD) test and two-tailed independent samples t-tests using IBM SPSS Statistics 25 [22].