Preliminary study of apatinib combined with fluzoparib in the treatment of HRP ovarian cancer via the HR pathway

Introduction

Ovarian cancer is the most common malignant tumor among women, and a high number of new cases are reported every year (1). Approximately half of ovarian cancer cases have homologous recombination deficiency (HRD) while the other half have normal homologous recombination proficiency (HRP) (2,3).

Most patients with early-stage ovarian cancer achieve clinical remission after surgery and chemotherapy; however, the short-term recurrence rate remains very high, primarily due to the development of chemotherapy resistance. Moreover, conventional chemotherapy is not suitable for every patient. Patients who do not wish to undergo chemotherapy or who do not tolerate the toxicity of chemotherapy can opt for “chemotherapy-free”, and current “chemotherapy-free” therapies are mostly focused on PARP inhibitors (4).

PARP inhibitors have shown promising antitumor activity in patients with impaired HR repair. Single-agent PARP inhibitors are particularly effective in ovarian cancer patients with BRCA1 or BRCA2 mutations, or those positive for HRD. However, the benefit for HRP ovarian cancer patients is very limited (5). Thus, research needs to be conducted to determine how HRP ovarian cancer patients can benefit from PARP inhibitors.

A previous study has shown that the PARP inhibitor olaparib combined with the antiangiogenic agent cediranib has an antitumor activity similar to that of platinum-based chemotherapy agents (6). The National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology also note that PARP inhibitors can be used in combination with other non-chemotherapy drugs, such as antiangiogenic targeted therapy, to establish an effective individualized “chemotherapy-free” regimen (7,8).

Targeting the DNA damage response in tumors with defective DNA repair is a successful clinical strategy. The RAS/RAF/MEK/ERK signaling pathway is frequently dysregulated in human cancers. The MEK1/2 branch of the MAPK pathway primarily stimulates cell proliferation, migration, and differentiation, which is the target of MAPK signal transduction inhibition. MEK1/2 signaling hyperactivation occurs frequently in malignant tumors, and thus can be used as a target for anti-cancer therapy (9,10). Targeting MEK has been shown to impair DNA damage repair pathways, and sensitize tumor cells to radiotherapy and chemotherapy. It has been shown that in pancreatic cancer, MEK inhibitors have been shown to interfere with the HR and non-homologous end-joining (NHEJ) pathways, thereby making pancreatic cancer cells more sensitive to radiotherapy and chemotherapy (11).

Apatinib can highly selectively inhibit vascular endothelial growth factor receptor 2 (VEGFR-2), and the inhibition of VEGFR-2 leads to the inhibition of downstream phosphorylated extracellular signal-regulated kinases (12). Research has shown that the inhibition of VEGF-induced VEGFR2/RAF/MEK/ERK1/2 signaling affects tumor cell growth in cholangiocarcinoma cells and pancreatic cancer cells (13,14).

RAD51 is an important protein for HR repair. The increased number and size of foci are associated with genomic damage (15). Research has shown that MEK inhibition reduces the formation of the HR marker RAD51, thereby enhancing DNA damage after olaparib treatment (16).

Thus, we sought to combine the antiangiogenic drug apatinib and PARP inhibitor fluzoparib to preliminarily analyze the mechanism of their combined therapeutic effect. We present this article in accordance with the ARRIVE reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-666/rc).

Methods

Cell experiments

Cell culture and drugs

The human ovarian cancer cell line SKOV3 was purchased from Shanghai Mingjin Biological Co., LTD. (Shanghai, China). SKOV3 is an HRP-type ovarian cancer cell (17). The cells were maintained in McCoy’s 5A (Gibco, Massachusetts, USA) medium supplemented with 1% double antibody (Seven Biotech Co., Ltd., Beijing, China) and 10% fetal bovine serum (Gibco). The cells were placed and cultured in an incubator at a constant temperature of 37 ℃ and 5% CO₂.

Both apatinib and fluzoparib were donated by Jiangsu Hendray Pharmaceutical Co., LTD. (Jiangsu, China). Cisplatin for injection (the freeze-dried type) was purchased from Qilu Pharmaceutical Co., LTD. (Shandong, China).

Cell viability was measured by MTT assay

The SKOV3 cells were spread into 96-well plates at 7×10³ cells per well. According to the results of the preliminary experiment, the optimal concentration and time of administration were determined. The adherent cells were then treated with apatinib (8, 16, 32, and 64 µmol/L) and fluzoparib (148.15, 222.22, 333.33, and 500.00 µmol/L) for 48 hours. Cell viability was measured by MTT assay.

Drug grouping and cell specimen collection

The drug combination concentration was determined based on the cell viability assay results described above. The SKOV3 cells in the logarithmic growth phase were spread in 96- and 6-well plates for culturing. After adherence, the cells were divided into the control group, cisplatin group (10 µg/mL) (18), apatinib group (64 µmol/L), fluzoparib group (300 µmol/L), and apatinib (64 µmol/L) combined with fluzoparib (300 µmol/L) group. The drug acts for 48 hours. Apatinib is easy to precipitate and has a poor dose-response relationship. For the western blot experiments, apatinib was used at three different concentration gradients (16, 32, and 64 µmol/L) to enable a comprehensive molecular analysis to be conducted. Finally, the cell precipitates in the 6-well plates were collected and stored at −80 ℃ for the subsequent western blot experiments.

MTT and scratch assays were used to evaluate the effects of the drug combination

Experimental grouping was performed as described in “Drug grouping and cell specimen collection”. The effect of the drug combination on cell proliferation was evaluated by MTT assay. The effect of the drug combination on cell migration was evaluated by scratch assay. In the cell-scratch experiment, the cells were cultured in an incubator for 24 h and then placed under an inverted digital biomicroscope to observe the cell migration in the same field of view at 0 h after scratching, and take pictures. Software was used to calculate the cell-scratch area to compare cell mobility between drug combination group and the rest of the group.

Western blot was used to detect the expression of p-MEK, RAD51, and γH2AX in each group

The cell precipitates collected in “Drug grouping and cell specimen collection” were lysed on ice and centrifuged to collect the supernatant. The protein concentration was measured in accordance with the instructions of the bicinchoninic acid assay (BCA) protein quantification kit (Seven Biotech Co., Ltd, Beijing, China) for subsequent experiments. The proteins P-MEK, RAD51, and γH2AX were incubated to obtain corresponding band pictures (antibody from Abcam, Washington, USA).

Animal experiments

Establishment of subcutaneous tumor model and the in vivo study

In total, 30 female nude mice (Vital River Laboratory Animal Technology Co., Ltd, Beijing, China), aged 4 to 6 weeks, were used to establish cell-derived xenograft models. The SKOV3 cells in the logarithmic growth phase were measured. The cells were digested with trypsin and resuspended in phosphate buffered saline. A suspension of 2×10⁷ cells per mL was prepared. The nude mice were subcutaneously injected with 2×10⁶ cells (i.e., 100 uL of the cell suspension) in the mid-posterior axilla. Randomization was performed approximately 2 weeks after inoculation when the tumor diameter reached approximately 0.5 cm (n=6). The groups received normal saline, intraperitoneal cisplatin (3 mg/kg, Q3D), apatinib (100 mg/kg/bid) and fluzoparib (30 mg/kg/bid), or apatinib (100 mg/kg/bid) combined with fluzoparib (30 mg/kg/bid). The treatment period was 14 days.

Tumor growth status was observed daily during the drug intervention. Body weight, and long and short tumor diameters were measured regularly. Tumor volume (V) was calculated as follows: V = long diameter × short diameter 2 × 0.5 (unit: mm³). The volume was plotted as a function of days. At the end of the treatment period, the nude mice in each group were sacrificed after drug anesthesia, and the tumor tissues were isolated and weighed. The tumor growth inhibition (TGI) rate was calculated as follows: TGI = (1 – T/C) ×100%, where T represents the mean tumor weight of the treatment group, and C represents the mean tumor weight of the control group. The liver and kidney were dissected and weighed to calculate the organ index, which was calculated as follows: organ index (%) = [(organ weight)/(mouse weight)] × 100%. The organs were embedded in paraffin sections, and stained with hematoxylin and eosin (HE). After dissection, half of the subcutaneous tumor tissue was stored at –80 ℃ for the subsequent western blot experiments, and the other half was fixed for 24 h and used for immunohistochemical detection. Animal experiments were performed under a project license (No. 2023035) granted by ethics board of Shanxi Province Cancer Hospital Animal Experiment, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Western blot was used to detect the expression of p-MEK, RAD51, and γH2AX in the subcutaneous tumors of each group

The subcutaneous tumor tissue was ground and lysed on ice to collect the supernatant by centrifugation. The concentration was measured in accordance with the instructions of the BCA protein quantification kit for the subsequent experiments. The proteins p-MEK, RAD51, and γH2AX were incubated to obtain corresponding band pictures.

Immunohistochemistry (IHC) was used to detect the expression of γH2AX, p-ERK, and BRCA1 in the subcutaneous tumors

The subcutaneous tumor tissue fixed with fixative was made into paraffin sections. The expression of p-ERK, HR pathway-related protein BRCA1, and DNA damage marker γH2AX in the tumor tissues was detected by IHC. The immunostaining for BRCA1, p-ERK, and γH2AX was evaluated at ×400 magnification in at least three regions. Immunoreactivity was assessed semi-quantitatively based on staining intensity and proportion. There are divided into 4 grades according to the intensity of cell staining: high positive (sepia): 3 points; positive (brown): 2 points; low positive (faint yellow): 1 points; and negative (no positive coloration): 0 point. The different scores were then multiplied with the corresponding percentages of positive cells to determine the immunoreactivity score for each sample.

Statistical analysis

The western blot and IHC results were processed with Fiji-ImageJ software, version 1.53c (https://imagej.net/software/fiji/). GraphPadPrism (GraphPadPrism, version 9.5.0(525) for MacOSX, GraphPadSoftware, SanDiego, CA, USA) was used for the data analysis and mapping. The quantitative data are expressed as the mean ± standard deviation. The t-test was used for comparisons between two groups, and an analysis of variance was used for comparisons between multiple groups. A P value <0.05 was considered statistically significant.

Results

Effects of apatinib combined with fluzoparib on the ovarian cancer SKOV3 cells

Effects of different concentrations of apatinib and fluzoparib on the proliferation of the ovarian cancer SKOV3 cells

Both apatinib and fluzoparib inhibited cell proliferation in a dose-dependent manner (Figure 1). According to the MTT results, the half maximal inhibitory concentration (IC50) values of apatinib and fluzoparib at 48 h were 77.35 and 284.8 µmol/L, respectively. Therefore, 64 µmol/L of apatinib and 300 µmol/L of fluzoparib were selected for the subsequent experiments according to the IC50 values of cells.

Figure 1 Effects of apatinib and fluzoparib at different drug concentrations on the proliferation rate of SKOV3 cells for 48 h (N=5). A, apatinib; F, fluzoparib; IC50, half maximal inhibitory concentration.

Effects of apatinib combined with fluzoparib on the proliferation and migration ability of ovarian cancer SKOV3 cells

The effects of the different drugs on the proliferation of SKOV3 cells are shown in Figure 2. Compared with a single drug, apatinib combined with fluzoparib significantly inhibited cell proliferation (P<0.05). These results indicate that combination therapy is more effective than monotherapy.

Figure 2 MTT assay results of the effects of different drugs on the cell proliferation rate at 48 h (N=5). When compared with the fluzoparib plus apatinib group: ****, indicates a statistically significant difference of P<0.0001. MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.

The effect of apatinib combined with fluzoparib on the migration ability of SKOV3 cells was verified by cell-scratch assay. The results are shown in Figure 3. Cisplatin, apatinib, and fluzoparib significantly inhibited the migration ability of the SKOV3 cells. Among the treatments, the combined treatment had the most significant inhibitory effect on the migration ability of the tumor cells. Additionally, the effect of apatinib combined with fluzoparib was better than that of cisplatin, apatinib, and fluzoparib alone, and the difference was statistically significant (P<0.05).

Figure 3 Wound-healing results of the SKOV3 cells treated with different drugs (N=3). Observe the same position at ×100 magnification. Compared with the combination drug group (i.e., the apatinib plus fluzoparib group): **, indicates a statistically significant difference of P<0.01; ***, indicates a statistically significant difference of P<0.001; ****, indicates a statistically significant difference of P<0.0001.

Effects of apatinib combined with fluzoparib on p-MEK, RAD51, and γ-H2AX in the SKOV3 cells

The western blot results are shown in Figure 4. Compared with the apatinib combined with fluzoparib group, γH2AX protein expression was significantly decreased in the apatinib monotherapy group (P<0.05). Compared with the control group, RAD51 and p-MEK expression was significantly decreased in the apatinib combined with fluzoparib group (P<0.05). Thus, we hypothesized that the combination of apatinib and fluzoparib may down-regulate the expression of HR pathway-related protein RAD51 by reducing the expression of MEK phosphorylation signaling and up-regulate the expression of DNA damage-related protein γH2AX.

Figure 4 Effects of different drugs on the protein expression levels of γH2AX, p-MEK, and RAD51 in the SKOV3 cells (N=3). (A) Raw protein expression for each indicator. (B) Expression statistics of the protein γH2AX. (C) Expression statistics of the protein p-MEK. (D) Expression statistics of the protein RAD51. A1: 16 µmol/L; A2: 32 µmol/L; A3: 64 µmol/L of apatinib; F: 300 µmol/L of fluzoparib; L: 64 µmol/L of apatinib combined with 300 µmol/L of fluzoparib. Compared with the control group, ****, indicates a statistically significant difference of P<0.0001.

Effects of apatinib combined with fluzoparib in the SKOV3 cell-line xenograft tumor model

Apatinib combined with fluzoparib inhibits the growth of the SKOV3 cell-line transplanted tumor model

The results of the in vivo experiments are shown in Figure 5. At the end of the treatment, the nude mice were sacrificed. Figure 5A shows an anatomical diagram. Figure 5B shows the tumor weight after dissection. Compared with that of the control group, the tumor weights of the cisplatin group, apatinib group, and apatinib combined with fluzoparib group were significantly reduced (P<0.05). As Figure 5C shows, the tumor volume of the control and drug-treated mice increased progressively as the treatment days increased. As Figure 5D shows, in the final tumor volume determined after the end of the final experiment, the tumor volume of the mice receiving the combined treatment was significantly smaller than that of the other three treatment groups (P<0.05), and there was no significant difference between the combined treatment group and the cisplatin group. These results indicate that apatinib combined with fluzoparib effectively inhibited the growth of the SKOV3 cell-line xenograft tumor model.

Figure 5 Effects of the different drugs in the subcutaneous tumor models in nude mice (typical picture) (N=6). Data are presented as the mean ± standard deviation. (A) Photographs of exfoliated tumors obtained at the end of treatment. (B) Plot of tumor weight by drug group. Compared with the control group: ns indicates no statistically significant difference; *, indicates a statistically significant difference of P<0.05; **, indicates a statistically significant difference of P<0.01. (C) Plot of tumor volume versus days of treatment. (D) The last tumor volume map of different drug groups. Compared with the apatinib combined with fluzoparib group: ns indicates no statistically significant difference, * indicates a statistically significant difference of P<0.05; ***, indicates a statistically significant difference of P<0.001.

In vivo toxicity analysis of apatinib combined with fluzoparib

The results of the in vivo toxicity analysis are shown in Figure 6A,6B. Figure 6A shows the organ index of the liver and kidney, which is an indicator to evaluate the relative weight of organs, which can quickly assess the effect of drugs on specific organs. There was no significant difference in organ index between groups. HE staining in Figure 6B showed that there was no obvious organ toxicity in the drug treatment, and only partial liver damage was observed in the cisplatin group.

Figure 6 In vivo toxicity analysis of drugs (N=3). The kidneys of each group were observed at ×200 magnification; the livers were observed at ×100 magnification, and the liver damage in the cisplatin group was observed with ×200 magnification. (A) Kidney/liver organ index in the nude mice. The data are presented as the mean ± standard deviation. (B) HE staining of liver and kidney of nude mice. The HE staining of nude mice organs showed liver damage in the cisplatin group. HE, hematoxylin and eosin.

Western blot was used to detect the effects of apatinib combined with fluzoparib on p-MEK, RAD51, and γ-H2AX in the xenograft model of the SKOV3 cell line

Western blot was used to detect the changes of the corresponding indexes in the transplanted tumor model. The western blot results are shown in Figure 7. Compared with the single-drug groups, the expression of the DNA damage marker γH2AX protein in the apatinib combined with fluzoparib group was significantly increased (P<0.05), indicating that the combination of apatinib and fluzoparib had the greatest effect on DNA damage in the mice. Compared with the control group, the apatinib combined with fluzoparib group showed a significant reduction in the expression of p-MEK (P<0.05).

Figure 7 Effects of different drugs on γH2AX (A), p-MEK (B) and RAD51 (C) protein expression in vivo (N=3). (A) Raw protein expression for each indicator. (B) Expression statistics of the protein γH2AX. (C) Expression statistics of the protein p-MEK. (D) Expression statistics of the protein RAD51. Compared with the control group: *, indicates a statistically significant difference of P<0.05; **, indicates a statistically significant difference of P<0.01; ***, indicates a statistically significant difference of P<0.001; ****, indicates a statistically significant difference of P<0.0001.

IHC was used to detect the effects of apatinib combined with fluzoparib on γ-H2AX, p-ERK, and BRCA1 in the xenograft model of SKOV3 cell line

Changes in the corresponding indexes of the transplanted tumor model were detected by IHC. The IHC experiment results are shown in Figure 8. Compared with the control group, the apatinib combined with fluzoparib group showed a significant increase in the expression of the DNA damage marker γH2AX protein (P<0.05), and a significant reduction in the expression of p-ERK and BRCA1 (P<0.05).

Figure 8 Effects of different drugs on γH2AX, p-ERK, BRCA1, and protein expression in vivo. The overall figure was observed at ×400 magnification and the local at ×800 magnification (Nuclear staining) (N=3). (A) Raw protein expression for each indicator. (B) Expression statistics of the protein γH2AX. Compared with the control group, * indicates statistically significant difference, P<0.05, **** indicates statistically significant difference, P<0.0001. (C) Expression statistics of the protein p-ERK. (D) Expression statistics of the protein BRCA1. Compared with the control group: *, indicates statistically significant difference of P<0.05; ****, indicates statistically significant difference of P<0.0001.

Discussion

At present, “chemotherapy-free” therapies are mostly focused on PARP inhibitors. PARP inhibitors are not only used in the maintenance treatment of ovarian cancer, but are also expected to “replace chemotherapy” as a new “chemotherapy-free” treatment mode for patients. Moreover, compared with traditional chemotherapy, targeted therapy has the advantages of strong specificity and clear targets. The FZOCUS-3 trial, which involved patients with gBRCA-mutated platinum-sensitive recurrent ovarian cancer who had received 2–4 lines of platinum-based chemotherapy, reported a promising treatment effect with fluzoparib (19). Nearly 75% of the non-gBRCA patients have a urgent need for diagnosis and treatment. Therefore, the combination of fluzoparib and anti-vascular drugs will be an effective treatment.

Our cell experiments showed that apatinib combined with fluzoparib had a better inhibitory effect on cell growth than either drug alone. It also significantly inhibited SKOV3 cell migration. The western blot results showed that the combination of apatinib and fluzoparib significantly inhibited the phosphorylation of the MEK protein, and then down-regulated the expression of the HR pathway-related protein RAD51. Finally, the expression of DNA damage-related protein γH2AX in the ovarian cancer cells was aggravated.

Similarly, our experimental results showed that apatinib combined with fluzoparib significantly inhibited subcutaneous tumor growth in the SKOV3 mice. No significant in vivo toxicity was observed, and the corresponding liver injury was only observed in the cisplatin group. The western blot results showed that the combined treatment significantly increased the expression of DNA damage-related protein γH2AX, and had a weak inhibitory effect on the expression of p-MEK in the MAPK pathway.

The IHC results also confirmed that apatinib combined with fluzoparib significantly increased the expression of DNA damage-related protein γH2AX. The phosphorylation of ERK, a downstream protein of MEK, and BRCA1, a HR pathway-related protein, were down-regulated.

Taken together, we concluded that the effect of apatinib combined with fluzoparib on the HR pathway was mediated by the inhibition of MEK signaling in the MAPK pathway, which resulted in the down-regulation of the HR pathway-related proteins RAD51 and BRCA1. Finally, combinations medications enhanced the expression of the DNA damage-related protein γH2AX and exerted a killing effect on the tumor cells. However, our research results and research depth are preliminary, and the expression of downstream signals in the MAPK pathway and the deep mechanism need to be further verified by corresponding experiments.

Conclusions

In this study, we demonstrated by in vivo and in vitro experiments that apatinib combined with fluzoparib significantly inhibited the growth of SKOV3 lineage ovarian cancer, producing a better tumor suppression effect than apatinib and fluzoparib alone. The mechanism of action may be through the inhibition of MEK signaling in the MAPK pathway, which led to the down-regulation of the HR pathway-related proteins RAD51 and BRCA1, and significantly increased the expression of the DNA damage-related protein γH2AX. We provide a rationale for clinical drug combinations that expand the scope of PARP inhibitors and provide an emerging “chemotherapy-free” option for HRP ovarian cancer patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-666/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-666/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-666/prf

Funding: This work was supported by Shanxi Provincial Key Research and Development Program (No. 201903D321174 to H.Z.) and the Scientific and Educational Cultivation Fund of the National Oncology Regional Medical Center (SD2023029 to H.Z.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-666/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. 2023035) granted by ethics board of Shanxi Province Cancer Hospital Animal Experiment, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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