Introduction
Epithelial ovarian cancer (EOC), as the most lethal gynaecological malignancy, shows a higher mortality rate than other common gynaecological malignancies, such as endometrial cancer [
1]. Extensive cytoreductive surgery and platinum-based chemotherapy are routine upfront treatments [
2]. Early-stage ovarian cancer is highly curable, but 70% of women present with stage III/IV disease, resulting in a low overall survival rate of ~ 40% [
3]. Extending the survival period and improving the quality of life of patients are the main goals of current EOC treatment.
Poly (ADP-ribose) polymerase inhibitors (PARPi) are expected to be an effective drug for the treatment of ovarian cancer by targeting DNA repair. PARP-1 is involved in DNA repair through different pathways, such as homologous recombination (HR), nucleotide excision repair (NER), alternative nonhomologous end-joining (alt-NHEJ) and single-strand DNA break (SSB) repair [
4]. PARPi induces PARP DNA entrapment and destabilizes replication forks, exacerbating replication deficiencies and motor catastrophe [
5]. Niraparib is an approved oral PARPi for the maintenance treatment of ovarian cancer. PARPi was initially used to treat ovarian cancer patients with BRCA1 mutations. Regardless of the occurrence of BRCA mutations, the effectiveness of PARPi for the maintenance treatment of ovarian cancer has been verified in many clinical studies [
6,
7]. Nevertheless, a large number of patients still fail to benefit from PARPi, and drug resistance is one of the main reasons [
8‐
10]. Overcoming drug resistance is one of the keys to maintenance treatment of ovarian cancer, and combination strategies are a scientifically rational way to target alternative DNA repair pathways to improve PARPi sensitivity. Some research has already explored strategies of PARPi combined with VEGFR inhibitors [
11], PI3K inhibitors [
12], CDK inhibitors [
13], PD-L1 blockade [
14] and so on, and some of them have entered clinical trials.
The effectiveness of short-term starvation (STS) or calorie restriction combined with chemotherapy in the treatment of malignant tumours has been widely demonstrated. Studies have shown that STS selectively protects normal cells while making malignant cells more sensitive to chemotherapy, which is called differential stress resistance [
15,
16]. In addition, STS can synergistically reduce the side effects of chemotherapy, making STS more attractive [
17]. However, the effect of STS on the maintenance treatment of ovarian cancer and the efficacy of PARPis have not yet been elucidated. This study evaluated the preclinical efficacy of STS in combination with niraparib in the chemotherapy of BRCA1 wild-type ovarian cancer cells. We performed in vivo and in vitro experiments and showed that STS synergistically enhanced the cytotoxicity of niraparib, including intensive double-strand DNA damage repair, inhibition of cell proliferation, apoptosis induction, cell cycle arrest and enhanced autophagic flux, and Akt/mTOR signalling was involved in this process [
18]. Our research indicates that STS combined with the PARPi niraparib is a promising treatment of EOC.
Material and methods
Cell culture and reagents
SKOV3 and A2780 cell lines were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in RPMI-1640 (GIBCO-BRL, Gaithersburg, MD, USA) with 10% fetal bovine serum (FBS, Yeasen, Shanghai, China) and 1% antibiotics (penicillin and streptomycin, Yeasen, Shanghai, China) in an incubator with 5% CO2 at 37 °C. Niraparib was obtained from Zai Lab Co.,Ltd (Suzhou, China), and dissolved in dimethyl sulfoxide (DMSO, Yeasen, 60313ES60). The glucose content in the cell culture medium of different groups is dissimilar. Compared with the control culture medium with 2.0 g/L glucose and 10% FBS, STS group was performed in RPMI-1640 medium with 0.5 g/L glucose and 1% FBS for 24 h and then incubated back to control medium for niraparib or other follow-up experiments. Other reagents sources were listed below: trypsin/EDTA solution (HyClone, Utah, USA), Cell Counting Kit (CCK)-8 (Multiscience Biotech, China), MK-2206 (MCE, HY10358) and AZD8055 (MCE, HY-10422).
Clonogenic assay and cell viability analysis
SKOV3 and A2780 cells were seeded to 60–70% confluency and treated for indicated time. After treatment, cells were digested and plated evenly on 6-well plates at density of 500 cells/well. After growing 8–14 days for colony formation, cells were washed with PBS, fixed with methylalcohol for 20 min and stained with 0.5% crystal violet for 20 min. The number of colonies was quantified by Image J software in three independent experiments.
PerkinElmer Victor3 1420 Multilabel Counter (Waltham, MA) was used to performed the cell viability assay. Cell Counting Kit (CCK)-8 was obtained from Multiscience Biotech, China. Cells were plated with a density of 5000 cells/well into 96-well plates and then incubated with different concentration of niraparib (0, 1.25, 2.5, 5, 10, 20, 40 μM) for 24 or 48 h. 10μL CCK-8 was added into each well and cells were incubated in incubator with 5% CO2 at 37 °C for 2 h. All assays were conducted in triplicate. The absorbance of each group of cells was detected at 450 nm wavelength.
Western blot
Cells were treated with RIPA Lysis Buffer, and protein concentrations were quantitated by BCA assay kit (P0013B, Beyotime, Shanghai, China). Primary antibodies used were: anti-PARP-1 (13371-1-AP, Proteintech, 1:1000), anti-cleaved caspase3(#9664, CST, 1:1000), anti-GAPDH (10494-1-AP, Proteintech, 1:5000), anti-Bcl2 (12789-1-AP, Proteintech,1:1000), anti-Bax (50599-2-Ig), anti-γH2AX (ab229914, abcam, 1:2000), anti-RAD51 (ab133534, abcam,1:2000), anti-phospho-Akt (phospho S473) (ab18622, abcam, 1:500),anti-Akt (10716-2-AP, Proteintech,1:1000), anti-phospho-mTOR (phospho S2448) (abcam,ab109268, 1:3000), anti-mTOR (abcam,ab134903, 1:10,000), anti-p62 (MBL, M162-3, 1:5000), anti-LC3I/II (14600-1-AP, Proteintech, 1:1000). Secondary antibodies used were: HRP goat anti-rabbit IgG (BL003A, Biosharp, 1:5000), HRP goat anti-mouse IgG (BL001A, Biosharp, 1:5000).
Samples of whole-cell lysate containing equal amounts of protein was loaded on 10% SDS-PAGE and then transfer to PVDF membranes (0.45 μm, EMD Millipore, Billerica, MA, USA). After being blocked in TBST with 5% non-fat milk, the PVDF membranes were incubated with primary antibody overnight at 4 °C and then with horseradish peroxidase (HRP)-conjugated secondary for 1 h. PVDF membranes were visualized using a chemiluminescence substrate kit (Pierce™ ECL Western Blotting Substrate; Thermo Scientific Fisher, Inc.). Immunoblots were quantitated using Image J (v1.8.0).
Autophagy flux analysis
mRFP-GFP-LC3 adenoviral (HanBio Technology, Shanghai, China) was used to transfect SKOV3 and A2780 cells. Then, the cells were incubated in control medium containing 5 μM niraparib or STS medium for indicated time. Autophagy flux assay were performed with a laser scanning confocal microscopy (FV1200, Olympus Corp, Tokyo, Japan).
Flow cytometry for apoptosis and cell cycle analysis
For apoptosis analysis, cells were suspended with 100 μl of 1 × binding buffer and stained with Annexin V-PE and 7-AAD (Annexin V-PE Apoptosis Detection kit; BD Pharmingen; SanDiego, CA, USA) for 15 min at 37 °C and 400 μl binding buffer was added. For cell cycle analysis, cells were harvested, centrifuged and added into 70% ice-cold ethanol and incubated at 4 °C overnight. In the second day, cells were incubated in cell cycle staining solution (Biosharp, BL105A) with 20 μg/ml propidium iodide, 200 μg/ml RNAase A and 0.1% Triton X-100 for 1 h. Apoptosis and cell cycle analysis were carried by BD FACSAria (BD Biosciences, Franklin Lakes, NJ, USA). Data were visualized using Flowjo Software (Flowjo 10.6.2, LLC, Ashland, OR, USA). All assays were conducted in triplicate.
Immunofluorescence staining
SKOV3 and A2780 cells grown on glass coverslips and were fixed in 4% formaldehyde for 10 min and then permeabilized for 15 min in 1% Triton X-100. After blocked with 5% bovine serum albumin for 1 h at room temperature, cells were incubated in primary antibodies including γH2AX (1:100) and RAD51 (1:500) overnight at 4 °C and then in Alexa 488-conjuated secondary antibody (1:500, Thermo Fisher Scientific, A-11070) for 1 h at room temperature. Glass coverslips were mounted in anti-fade mounting medium containing DAPI (Biosharp, BL739A) and analyzed under laser scanning confocal microscopy (FV1200, Olympus Corp, Tokyo, Japan).
Comet assay
Comet assay were performed under alkaline conditions using the Comet Assay Kit (Abcam, ab238544) according to the manufacturer’s instructions. Briefly, cells were treated with STS for 24 h or 5 μM niraparib or their combination, harvested and blended with 0.5% low melting point agarose at a ratio of 1:10 (volume/volume). Spread the mixture evenly on a slide and immersed in lysis solution for 20 min. After that, slides were electrophoresis in a horizontal electrophoresis apparatus under conditions of 25 V, 300 mA and then stained with Acridine orange to visualize cellular DNA. The image acquisition was performed with orthographic microscope (BX53F2, Olympus, Tokyo, Japan). All assays were conducted in triplicate. Comet assay software project (CASP) was used to analysis the Tail Moment of each comet, which represents the level of DNA damage.
ATP assay
According to the manufacturer’s instructions, ATP assay Kit (S20026, Beyotime, Shanghai, China) was used to measure the intracellular ATP level. Cells were treatment as mentioned above in this study. Lysis cell homogenate were centrifuged at 12,000 rpm at 4 °C for 10 min and 100 μl ATP detection reagent was mixed with 20 μl supernatant in a 96-well plate and the luminescence (RLU) of each group was detected by PerkinElmer Victor3 1420 Multilabel Counter (Waltham, MA).
Tumor xenograft study
All animal experiments were conducted in compliance with the National Institute of Health guidelines for animal research and approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University. 6 weeks old female BALB/c nude mice (nu/nu) were purchased from the Beijing Vital River Laboratory Animal Technology Cooperation (Beijing, China). Logarithmic growth phase A2780 tumor cells (1 × 107) were subcutaneous injections into female mice aged 7–8 weeks under the skin of neck and back. When the volume of tumor were approximately 50mm3 (day 17), the mice were randomly grouped and used in subsequent experiments (n = 7). niraparib group was given niraparib intragastric injections (50 mg/kg) once a day and 5 days a week. STS group fasted for 48 h per week. The STS + Niraparib group was given a short-term starvation for 2 days before the intragastric injection of niraparib for 5 days per week. After three weeks treatment, nude mice were anaesthetized with isoflurane and sacrificed by the cervical dislocation method, important organ and tumor tissues were collected for further analysis.
Histopathology and immunohistochemistry
Briefly, tissues were fixed with 10% formalin and embedded in paraffin, 4 μm paraffin embedded were performed and stained with H&E or IHC. The primary antibodies used were Ki67 (27309–1-AP, Proteintech, 1:200), γH2AX (1:100) and RAD51 (1:500). Major organ sections and primary tumors were performed with H&E staining. Images were collected using Microscope (BX53F2, Olympus, Tokyo, Japan) and IHC stained images were analyzed by Image J 1.8.0.
Statistical analysis
Data were means ± standard deviation of three independent experiments or 7 independent samples. GraphPad Prism software version 7.0 (San Diego CA, USA) were used to perform statistical analysis. One-way analysis of variance (ANOVA) was used for multiple comparisons in three or more groups and unpaired t-tests were applied to determine significance for comparisons in two groups. P values < 0.05 were considered significant statistically.
Discussion
The value of fasting during cancer therapy has gradually attracted attention. Fasting can prolong the lifespan of organisms ranging from yeast to mice [
23,
24]. Compared with long-term calorie restriction, STS is easier to implement and complete because of less weight loss and better enforceability. The efficacy of STS combined with chemotherapy or radiotherapy in the treatment of malignant tumours has been confirmed, such as neurocytoma [
16], malignant multiforme [
24,
25], breast cancer [
26] and lung carcinoma [
27]. A study has shown that in prostate cancer and pancreatic cancer, STS increases the radiosensitivity of metastatic tumour cells but not normal fibroblasts [
28]. In several clinical studies of gynaecological malignancies, STS in combination with chemotherapy is feasible and well tolerated without significant side effects [
29,
30]. STS for 48 or 60 h did not result in a significant decrease in body mass, and QOL scores during chemotherapy were improved. In this study, we confirmed that STS enhances the effectiveness of niraparib in ovarian cancer chemotherapy.
Our study showed that STS increased the sensitivity of SKOV3 and A2780 cells to PARPi niraparib treatment. In our experiments, niraparib induced cytotoxicity in SKOV3 and A2780 cells in a time- and dose-dependent manner. Although STS had no effect on the malignant behaviour of ovarian cancer cells, it enhanced the inhibition of cell proliferation and apoptosis induced by niraparib chemotherapy. In addition, consistent with previous research, niraparib induced G2/M phase arrest [
31]. A comet assay was used to detect the degree of DNA lesions, including single- and double-strand breaks [
32]. Compared with the niraparib group, higher alkali-labile sites and higher tail moments illustrated the efficacy of the combination of STS and niraparib in the treatment of ovarian cancer. In addition, western blot and immunofluorescence analyses demonstrated that compared with the niraparib group, STS pretreatment resulted in an increase in γH2AX and a decrease in RAD51, which meant that high levels of DNA damage and inhibition of HR repair and overloading stress make tumour cells more sensitive to chemotherapy by exhausting the stress response pathway [
33].
Mechanistically, overactive PARP induces a derangement of ATP production, accounting for the overconsumption of β-nicotinamide adenine dinucleotide (NAD
+), resulting in necrosis and autophagy [
34,
35]. In our study, autophagic vacuoles were observed by confocal microscopy, and the expression of LC3II and p62 was quantified by western blot analysis. Consistent with other research, niraparib induced enhanced autophagy and high expression of LC3II [
36], and the mechanisms of autophagy may be triggered by cellular stress, such as ROS, or genomic instability rather than protective effects. The Akt/mTOR signalling pathway regulates cell growth, proliferation, motility, metabolism and cell size, and inhibition of this pathway promotes tumour regression [
37,
38]. Previous studies have shown the main mechanism by which STS enhances the effect of chemotherapy is by inhibiting IGF1/Akt/mTOR signalling [
21,
39,
40], and the coordinated repression of p-Akt and m-TOR may be the main reason for the enhanced effect of niraparib. Enhanced autophagy flux and inhibition of the AKT/mTOR signalling pathway were observed after STS and niraparib were used in combination, indicating the important role of AKT/mTOR in this process. Moreover, MK2206 and AZD8055 were applied on the basis of STS and niraparib combination treatment. Our results revealed that inhibition of AKT/mTOR induced stronger cell proliferation inhibition and higher expression of the DNA damage-related protein γH2AX, while the reduction in RAD51 was consistent with impairment of the HR repair mechanism.
Furthermore, we performed in vivo experiments to detect the effect of STS and niraparib in combination in a xenograft nude mouse model. No mice died during the experiment, and STS alone showed no significant effect on tumour growth compared to the control. We found that STS significantly enhanced the antitumour effect of niraparib, and the combination therapy did not show significant major organ-related toxicity (lung, liver, spleen, heart, and kidney). In addition, although STS alone or in combination with niraparib for 48 h caused a sharp drop in the weight of mice, when their normal diet was restored, the weight of the mice returned to normal, which indicated that STS had no effect on weight in the long term. Gastrointestinal toxicity is one of the side effects of most chemotherapeutics, including niraparib. In this study, we observed the protective effect of STS on the small intestine after niraparib application.
In conclusion, our research showed that STS synergistically enhanced the toxicity of niraparib on ovarian cancer cells through the Akt/mTOR signalling pathway. These findings reveal that the combined application of STS and niraparib is a potential maintenance treatment strategy for ovarian cancer. However, our experimental study on the synergistic effect of STS and niraparib was carried out in vitro and at the animal level. In the future, it is necessary to further explore the effect of this combination therapy in clinical research.
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