Therapeutic benefits of niraparib tosylate as radio sensitizer in esophageal squamous cell carcinoma: an in vivo and in vitro preclinical study

The proliferation of five human esophageal squamous cell carcinoma cells KYSE-30, KYSE-150, KYSE-450, KYSE-510, and TE-1 were detected by CCK8 assay. The cell line with the lowest and highest IC₅₀ value for niraparib and radiation sensitivity were selected for subsequent experiment. The human esophageal squamous cell carcinoma cell (ESCC) line KYSE-30 and KYSE-150 were purchased from Procell Life Technology Co., Ltd. (Wuhan, China), and the cells were cultured in DMEM culture medium (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin/streptomycin (Thermo Fisher, USA) at 37 °C in an atmosphere of 95% air and 5% CO₂.

To confirm the radiation sensitization effect of niraparib tosylate, CCK8 was used to detect the effect of different treatments at different time points [11]. Niraparib tosylate was obtained from the Zai Lab (Shanghai) Co., Ltd, China. The purity of compounds was more than 99%. KYSE-30 and KYSE-150 cells were cultured in 96-well plates at 3000 cells/well for 24 h. These were divided into four groups; blank control, radiation (4 Gy) treatment, niraparib tosylate (5 µmol/l) treatment and niraparib tosylate and radiation combined treatment groups (combination treatment). Following the treatment, 10 µl enhanced cell counting Kit-8 solution (CCK8, BIOSS Technologies, Inc., Beijing, China) was added to each well at different time points. Then, the absorbance at 450 nm for each well was measured and the cell survival rate was calculated (cell survival rate (%) = (value of experimental group—blank group value)/ (value of control group—blank group value). Each experiment was repeated thrice independently.

Cells were seeded in 60 × 60 mm cell culture plates at 1000 cells/plate, and were cultured for 24 h to allow the cells adhere to the wall. KYSE-30 and KYSE-150 cells were seeded and divided into four groups, as mentioned above. First, the cells were incubated for 8–10 days. Later, the colonies were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet solution for 15 min at room temperature. Subsequently, the colonies consisting ≥ 50 cells were counted under a microscope [12]. The colony formation rate (%) was calculated as follows: number of colonies counted/number of cells seeded × 100%. The surviving fraction (SF) was determined as follows: SF = number of colonies/(number of cells seeded under the same condition × colony formation rate) [13].

KYSE-30 and KYSE-150 cells were plated in 6-well plates and treated with or without 5 µmol/l niraparib tosylate for 6 h, and then exposed to X-rays (4 Gy). After 24 h, cells were trypsinized and washed with phosphate-buffered saline (PBS). The cell suspensions were stained using the Annexin V-FITC Apoptosis detection kit (BD Biosciences, Beijing, China), as per the manufacturer's protocol, followed by flow cytometric analysis. The apoptosis rate was analysed using FlowJo 7.6 software. All experiments were repeated thrice independently [14].

The cells were incubated on coverslips in 35 mm dishes and treated with 5 μmol/l niraparib tosylate and radiation at a dose of 4 Gy. Immunofluorescence staining of γ-H2AX (#7631), Bcl-2 (#3498), and Bax (#5023) was performed. The cells were then fixed (4% paraformaldehyde), permeabilized (0.1% Triton X-100), blocked (5% BSA), and then incubated with primary antibodies overnight at 4℃. Subsequently, the cells were incubated with Alexa Fluor pre-adsorbed secondary antibody for 1 h at room temperature (Abcam, ab150117). Nuclei were stained with DAPI (Solarbio, Beijing, China). Thereafter, the single-stained and merged images were acquired using laser confocal microscopy and Zen 3.0 imaging software [15].

Total RNA was extracted using Trizol (Invitrogen). RNA concentration was measured using the Qubit^® RNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA) and the purity was determined using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA). RNA degradation and contamination were assessed on 1% agarose gels. cDNA was synthesized with the Takara-RR047A, and the PCR amplification mixture was 10 μl in total, containing 1 μl cDNA [16]. Primer pairs used were as follows: β-actin: forward 5′-GGCACCACACCTTCTACAATG-3′, reverse 5′-GTGGTGGTGAAGCTGTAG-3′; Bcl-2: forward 5′-GGGAGGATTGTGGCCTTCTT-3′, reverse 5′-GGGCCAAACTGAGCAGAGTC-3′; Bax: forward 5′-CGGGGAGCAGCCCAGAG-3′, reverse 5′-TGAGACACTCGCTCAGCTTC-3′; Caspase-3: forward 5′-ATTTGGAACCAAAGATCATACATGG-3′, reverse 5′-CTGAGGTTTGCTGCATCGAC-3′. Every assay was performed in triplicate and the mRNA levels of β-actin were used as internal standards.

Western blotting was conducted to determine the effects of niraparib tosylate treatment on the expression of apoptosis-related genes and DNA double-strand breaks. Western blot was used to detect the expression of Bcl-2, Bax, and γ-H2AX in each group [11]. Experimental groupings and cell inoculation were as described above. Cells were collected 24 h after irradiation. The protein was extracted from cells and quantified using a BCA kit (catalog no. P0010s; Beyotime Institute of Biotechnology). A total of 30 µg protein was loaded per lane for western blot analysis. 10% SDS–PAGE and 5% stacking gels were prepared, and the protein samples were electrophoresed. The proteins were then transferred to a polyvinyl difluoride membrane and blocked with 5% BSA or 5% skim milk for 2 h at room temperature. The membranes were then incubated with the following primary antibodies: rH2AX (#7631), Anti-BAX (#5023), Anti-BCL-2 (#3498), Caspase-3 (#9664) (1:1000; Cell Signaling Technology, Inc.), FANCG (10215-1-AP) (1:500; Protein tech Group, Inc.) at 4 °C overnight.β-ACTIN (#4970) (1:1000; Cell Signaling Technology, Inc.) was used as an internal control. Subsequently, the membranes were incubated with anti-rabbit IgG secondary antibody (1:1000; Cell Signaling Technology, Inc) for 1 h at room temperature. Visualization was performed with enhanced chemiluminescence reagent. Image J 1.8.0 processing software was used for gray-scale analysis. Each experiment was repeated thrice independently.

Radiosensitization of niraparib tosylate in vivo was evaluated [11]. For this a tumor-bearing nude mouse model of esophageal squamous cell carcinoma KYSE-30 cells was established. Right subcutaneous flank injections were performed on twenty 4–5-week-old BALB/c-SCID female mice (obtained from Beijing Charles River Laboratory Animal Technology Co., Ltd.) with 2 × 10⁶ KYSE-30 cells resuspended in 100 μl PBS. When the tumors reached approximately 70 mm³, mice were randomly divided into four groups as follows: Control group (oral PBS), niraparib tosylate group, Radiation group and combined group (oral niraparib tosylate + irradiation). Mice in the treatment groups were anesthetized and subjected to local irradiation (12 Gy) of the tumors. Niraparib tosylate (50 mg/kg) was administered orally once daily for 10 days. Tumor volumes and body weights were measured every 3 days. Tumor volume was calculated as: 1/2 × length × width². Tumor inhibition rate = (average volume of control group—average volume of experimental group)/average volume of control group × 100%. All procedures were performed in a certified biosafety cabinet and carried out in accordance with protocols approved by the Committee for the Care and Use of Laboratory Animals of our institute.

To further observe the morphological structure and growth of tumor in each group, HE staining, Caspase-3 and FANCG immunohistochemical staining were used to detect tumor in each group [17, 18]. The mice were sacrificed at the end of the experiment and the tumor tissues were fixed in 10% formalin, paraffinized, and cut into 5 μm thick sections.

The slides were dewaxed and stained with hematoxylin–eosin for histological assessments.

After microwave pre-treatment in a citrate buffer (pH 6.0; for antigen retrieval), the slides were immersed in 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. After intensive washing with PBS, the slides were incubated with Caspase-3, FANCG antibodies and then incubated with HRP-conjugated antibodies. The slides were visualized with a DAB Horseradish Peroxidase Color Development Kit (Beyotime, China), and counterstained with hematoxylin. The images were analyzed by Image-Pro Plus 6.0 software.

Four groups of tumor-bearing nude mouse transplantation models were subjected to transcriptome high-throughput sequencing, 20 genes with significant expression differences were selected, relevant articles were queried through PubMed and related pathways were queried through KEGG database for analysis, so as to determine the modification and research of FANCG gene. Analyzed lentiviruses with overexpression of FANCG and negative control lentiviruses (Shanghai GeneChem Co., LTD.) were manufactured according to the instructions. In brief, cell suspensions (50,000 cells/ml, 2 ml/well) of KYSE-30 cells were inoculated into six-well plates for 24 h in DMEM with 10% FBS. Then, DMEM without FBS was replaced, lentivirus solution (20 μl 1 × 108 TU/ml) was added for another 16-h incubation and DMEM with 10% FBS was added back for another 72 h. The screening and purification of infected cells was performed by culture in a medium containing puromycin.

All data were presented as the mean ± standard deviation (SD) and were analysed with statistical software SPSS 21.0 (SPSS Inc). Comparisons of continuous variables between the groups were performed using the student’s t test. The criterion for statistical significance was taken at P < 0.05.

The Cancer Genome Atlas (TCGA) data were queried for esophageal squamous cell carcinoma. The Kaplan–Meier survival plot was generated to assess the association between FANCG expression on esophageal squamous cell carcinoma and overall survival [19].

The median inhibitory concentrations (IC50) of niraparib tosylate at 24 h for KYSE-30, KYSE-150, KYSE-450, KYSE-510, and TE-1, respectively, were 39.44 μmol/l, 36.87 μmol, 38.98 μmol/l, 36.69 μmol/l, 19.05 μmol/l (supplementary Fig. 1). TE-1 was highly sensitive to drugs, and the difference was statistically significant compared with the other four (P < 0.01). KYSE-30 had the highest radiation sensitivity, while KYSE-150 had the lowest sensitivity, and the difference was not statistically significant (P > 0.05). Therefore, two cell lines KYSE-30 and KYSE-150 were selected for the study (supplementary Fig. 2). The radiotherapy dose was 4 Gy, and niraparib tosylate was 5 μmol/l. The colony formation efficiency of combined group was much lower than that of single radiation group (P < 0.01) (Fig. 1). CCK8 assay confirmed that the addition of niraparib tosylate significantly reduced the proliferation of irradiated cells (Fig. 2). Therefore, both colony formation assay and CCK8 assay confirmed that niraparib tosylate could improve radiotherapy sensitivity by inhibiting cell proliferation.

Compared with the control group, although the late cell apoptosis was steadily increased in the niraparib tosylate and radiotherapy groups (P < 0.05), the increase in cell apoptosis was more significant in the combination group (P < 0.01) (Fig. 3).

It was observed that the combination of niraparib tosylate and radiation inhibited the expression of Bcl-2 (P < 0.01), up-regulated the expression of Bax and Caspase-3 (P < 0.01), and significantly increased the expression of γ-H2AX (P < 0.01). Compared with the radiation group, the addition of niraparib tosylate significantly increased the DNA DSBs of apoptotic cells. These results suggest that the role of niraparib tosylate in radiation-induced apoptosis is to promote apoptosis by regulating apoptosis-related genes (Fig. 4-1, -2, -3).

The results showed that in the xenograft tumor model before FANCG gene modification, niraparib tosylate alone had a weak inhibition effect on tumor growth (P > 0.05), while both radiotherapy and combination therapy could effectively inhibit tumor growth in the xenograft model (P < 0.05), and the combination group had a stronger inhibitory effect (P < 0.01). There was no significant difference in body weight among the four groups (P > 0.05). These results suggest that niraparib tosylate and radiation have a significant synergistic effect on KYSE-30 xenotransplantation and have little effect on quality of life in nude mice (Fig. 5).

HE staining showed increased tumor necrosis in all treatment groups, especially in the combination group, compared with the control group. Caspase-3 staining showed that the expression of Caspase-3 was significantly increased in the combined treatment group, indicating that tumor cell apoptosis was more significant after combined treatment. This reconfirmed the tumor suppressive effect of niraparib tosylate combined with radiotherapy at cellular and animal levels (Supplementary Fig. 3).

After extracting cell protein and tumor tissue protein, Western blotting results showed that FANCG expression decreased in the niraparib tosylate group and the radiotherapy group, and the FANCG expression was the lowest in the combined treatment group, with statistically significant differences (P < 0.01) (Fig. 4-1 and Supplementary Fig. 5). A total of 28,567 genes were detected by transcriptome sequencing of transplanted tumor, and 349 genes were significantly different, including 316 up-regulated genes and 33 down-regulated genes. The difference in FANCG expression was statistically significant (P < 0.01). (Supplementary Fig. 4). Western blotting results showed that FANCG expression was decreased in the niraparib tosylate group and the radiotherapy group, and the FANCG expression was the lowest in the combined treatment group, with statistically significant differences (P < 0.01) (Fig. 4-1). After overexpression of FANCG, the inhibition of xenograft tumor was significantly reduced in both the radiotherapy group and the combined group, and there was no statistical difference in tumor weight between the four groups (P > 0.05). There was statistically significant difference in tumor inhibition rate between the combined group and the control group (P < 0.05), but there was no statistically significant difference in other groups (Fig. 6). As revealed from Kaplan–Meier plot, the differential expression of FANCG in esophageal squamous cell carcinoma cells showed no difference in survival (Fig. 7). In vitro and in vivo experiments at the molecular level demonstrated that niraparib tosylate can target FA-BRCA signalling and reduce FANCG expression, thereby enhancing radiosensitivity. A graphical description of the proposed pharmacological mechanism of niraparib tosylate drug is provided in Fig. 8 and findings for the current proposal are illustrated in Fig. 9.