Astragalus polysaccharides sensitize ovarian cancer stem cells to PARPi by inhibiting mitophagy via PINK1/Parkin signaling

Summary

Ovarian cancer (OC) remains the most lethal gynecological malignant tumor. PARP inhibitors (PARPi) have significantly improved survival, particularly in patients with OC with BRCA1/2 mutations. However, the majority of patients eventually develop resistance to PARPi. Cancer stem cells (CSCs) are considered the source of drug resistance in cancer. Our study found that the synergistic effect of astragalus polysaccharides (APSs) and PARPi was observed in ovarian cancer stem cells (OCSCs) by decreasing cell viability and self-renewal potential while inducing apoptosis. The present study also demonstrated that OCSCs had increased mitophagy. Furthermore, it was observed that APS in combination with PARPi inhibits mitophagy and downregulates the PINK1 protein level in OCSCs. The overexpression of PINK1 via the pEGFP(+)-PINK1 plasmid resulted in a partial reversal of the increased susceptibility of OCSCs when PARPi were administrated concurrently with APS. In conclusion, APS increases OCSC sensitivity to PARPi by inhibiting mitophagy via the PINK1/Parkin pathway regulation.

Graphical abstract

Highlights

• •Ovarian cancer stem cells (OCSCs) were resistant to PARPi
• •The combination of APS and PARPi has synergistic cytotoxic effects on OCSCs
• •APS increases OCSC sensitivity to PARPi by inhibiting mitophagy via the PINK1/Parkin pathway

Biological sciences; Molecular biology; Cancer

Introduction

Ovarian cancer (OC), a highly lethal gynecological malignancy, usually manifests at an advanced stage in approximately 75% of patients. Moreover, the 5-year survival rate for advance-stage patients is less than 25%.¹ Recurrence and drug resistance are the primary causes of increased mortality rates in patients with OC.² However, there is some hope because PARP inhibitors (PARPi) have revolutionized the treatment landscape for epithelial OCs. Notably, the US Food and Drug Administration has approved using PARPi as maintenance therapy for patients with OC following chemotherapy.³ However, as the use of PARPi continues to rise, more patients are developing PARPi resistance, limiting the potential clinical efficacy.⁴ Therefore, it is imperative to develop novel strategies to address the issue of PARPi resistance in OC. Previous research has implicated ovarian cancer stem cells (OCSCs) as the underlying cause of chemotherapy resistance,⁵ but their significance in PARPi resistance is unknown.

Cancer stem cells (CSCs) are widely acknowledged as the driving force underlying cancer metastasis, recurrence, and treatment resistance.⁶ Numerous studies have unequivocally confirmed the presence of cancer stem-like cells, including OCSCs, within solid tumors, which are highly associated with therapeutic resistance.⁷ However, the precise mechanism remains unknown. Our previously published research revealed that OCSCs exhibit increased autophagy, a critical step for conferring resistance to chemotherapy and preserving stem cell properties.⁸ Earlier studies have demonstrated that autophagy inhibition can reverse innate resistance in breast cancers proficient in homologous recombination (HR) repair when treated with PARPi.⁹ Furthermore, PARPi-induced autophagy serves as an adaptive resistance mechanism in cancer cells with wild-type Breast cancer susceptibility gene (BRCA), and combining PARPi with autophagy inhibitors may improve outcomes for patients with OC.¹⁰ Nevertheless, the precise characterization of the role and pathogenesis of PARPi in OCSCs remains unclear.

Mitophagy, a type of selective autophagy that facilitates mitochondrial turnover and inhibits the accumulation of impaired mitochondria to maintain cellular homeostasis, has been implicated in drug resistance in cancer.¹¹ Recent research indicates that mitophagy contributes to chemoresistance in human OC cells¹² and sensitizes breast cancer cells to chemotherapy by inhibiting mitophagy via Dynamin 1-like (DNM1L)-mediated mitochondrial fission.¹³ A decrease in mitochondrial density induces mitophagy, resulting in decreased levels of reactive oxygen species (ROS) and cellular energy. Consequently, cells develop stem cell-like quiescence features, which enable their survival in hypoxic conditions and ultimately result in drug resistance and cancer relapses.¹⁴ Doxorubicin-induced mitophagy, rather than non-selective autophagy, has contributed to drug resistance in HCT8 human colorectal CSCs.¹⁵ Therefore, targeting mitophagy with anti-mitophagy therapy could be a promising strategy to overcome therapy resistance in OCSCs.

Chinese herbal medicine has received significant attention in oncology due to its safety, cost-effectiveness, and multi-target therapeutic efficacy. Our previous research has demonstrated that arsenic compounds increase the sensitivity of platinum-resistant and HR-proficient OC to PARPi.^16,17 However, the severe toxicity of these chemicals hindered their clinical applications. Therefore, there is a continual need to explore and identify Chinese herbal medicines with improved safety profiles and exceptional therapeutic potential. Astragalus membranaceus, commonly known as Huangqi, is a popular Chinese herbal medicine renowned for its various therapeutic effects, such as anti-fatigue, anti-sepsis, anti-inflammatory, and anti-hypertensive.¹⁸ It is currently considered a promising anti-tumor agent; extensive research has demonstrated the significant anti-tumor effects of Astragalus membranaceus in many tumors including ovarian, cervical, colon, liver, and stomach cancers.¹⁹ The proposed mechanisms underlying its anti-tumor effects include restoring depressed immune cell function, modulating tumor microenvironment, and enhancing of the efficacy of chemotherapeutic drugs.²⁰ Astragalus polysaccharides (APSs) is the most important natural active component in Astragalus membranaceus. It is a type of water-soluble heteropolysaccharide with bioactive effects that is extracted from the stems or dried roots of Astragalus membranaceus; the components are complex and diverse, and polymeric carbohydrates are mainly linked by a-type glycosidic bonds between the monosaccharides. APSs exerts multiple pharmacological effects including the regulation of immune function, anti-aging, anti-tumor, reducing blood sugar, lowering blood lipid, anti-fibrosis, anti-bacterial, radiation protection, and anti-viral effects.^21,22 Recent studies have revealed that Astragalus membranaceus efficiently inhibits the growth of A549 lung cancer cells by regulating autophagy.²³ However, the effect of Astragalus membranaceus on mitophagy is still unknown.

The present study aims to comprehensively understand the mechanism underlying combination therapy in addressing PARPi resistance in OC using Western and traditional Chinese medicine (TCM) approaches. Therefore, it was observed that OCSCs are resistant to PARPi therapy. Subsequently, the synergistic effects of combining PARPi with APSs (principal bioactive components of Astragalus membranaceus) in OCSCs were assessed, and the underlying mechanism was studied further. In light of these findings, a novel strategy for overcoming PARPi resistance in OC has been proposed.

Results

OCSCs exhibited greater resistance to PARPi

CSCs have been associated with tumor recurrence, metastasis, and resistance to chemotherapy. Previously, we successfully isolated OC (3AO and SKOV3 cells) stem-like cells using a serum-free suspension culture system called CSC spheres. These spheres have stem cell characteristics, such as self-renewal capacity, tumor-initiating ability, and chemotherapy resistance.^8,24 Therefore, we analyzed PARPi resistance in two paired cell lines: 3AO cells and 3AO spheres, and SKOV3 cells and SKOV3 spheres. IC₅₀ of olaparib was 4.56-fold higher in 3AO spheres than in 3AO cells (349.7 vs. 76.64 μM). Similarly, the IC₅₀ of olaparib in SKOV3 spheres was 3.90-fold higher than in SKOV3 cells (371.7 vs. 95.29 μM). Furthermore, the IC₅₀ of niraparib in 3AO spheres was 4.18-fold higher compared to 3AO cells (137.7 vs. 32.97 μM) and 6.36-fold higher in SKOV3 spheres than in SKOV3 cells (67.6 vs. 10.62 μM; Figure 1A).

Figure 1.

The sensitivity of OCSCs to PARPi

(A) The drug-response curves of survival and IC₅₀ values for cell viability were determined after treating 3AO cells, SKOV3 cells, and their sphere cells to various concentrations (0, 1, 10, 100, and 1,000 μM) of olaparib or niraparib or APS (Figure S1), as measured by the CCK-8 assay after 72 h.

(B) 3AO spheres and 3AO cells, and SKOV3 spheres and SKOV3 cells were treated for 48 h with DMSO, olaparib (200 μM, approximately 50% IC₅₀), and niraparib (30 μM, approximately 50% IC₅₀). The findings, represented by a histogram and representative data, depict the apoptotic rates after Annexin V-FITC/PI staining. Error bars indicate the mean ± SD of three independent repetitions, with statistical significance denoted by ∗p < 0.05; ∗∗p < 0.01.

Annexin V-FITC/PI dual staining assays were performed to measure the effects of olaparib or niraparib on cellular apoptosis to analyze the influence of PARPi on apoptosis in 3AO and SKOV3 spheres. The number of apoptotic 3AO and SKOV3 spheres was significantly lower than in 3AO and SKOV3 cells when treated with olaparib or niraparib (Figure 1B), indicating that OCSCs were resistant to PARPi.

APS significantly enhances the sensitivity of OCSCs to PARPi

Our findings in Figures 1 and S2 demonstrate that PARPi or APS alone has negligible effects on OCSCs. Therefore, we aimed to investigate whether the combination therapy of APS and PARPi might synergistically affect OCSCs. In Figure 2A, we compared the cell viability of 3AO and SKOV3 spheres treated with various drug combinations at different IC₅₀ doses. The synergistic effects of the drug combination were assessed using combination indices. The combination of olaparib and APS at various concentrations (75%, 100%, and 125% of IC_50s) significantly affected cell viability in 3AO spheres. Similarly, the combination of niraparib and APS at different concentrations (50%, 75%, 100%, and 125% of IC_50s) also revealed a significant impact on cell viability in 3AO spheres. These results were comparable with those observed in SKOV3 spheres, where treatment with olaparib or niraparib and APS at various concentrations (25%, 50%, 75%, 100%, and 125% of IC_50s) had a significant influence on cell viabilities. Based on these findings, it may be concluded that adding APS sensitizes OCSCs to PARPi. As shown in Figure S2, after treated with different concentrations of APS alone, the self-renewal capacity of OCSC can be suppressed when the concentration reaches a higher concentration (300 mg/L), but with no significant effect in lower concentration. So we use APS with a lower concentration (200 mg/L) to further evaluate the synergistic effects of combining PARPi on cell viability, apoptosis, and self-renewal.

Figure 2.

APS significantly enhances the sensitivity of OCSCs to PARPi

(A) Synergy analysis was performed to evaluate the effects of APS, olaparib, or niraparib on 3AO and SKOV3 spheres. The cells were treated with the indicated concentrations (25%, 50%, 75%, 100%, and 125% IC₅₀s) of APS and olaparib or niraparib for 72 h. Cell viability was measured using the CCK-8 assay. The combination index (CI) value was determined by CompuSyn software. CI value indicates the following: >1, antagonism; = 1, additive effect; and <1, synergism. The bars represent the mean ± standard deviation (SD) of three independent biological experiments.

(B) 3AO and SKOV3 spheres were treated with APS (200 mg/L), olaparib (200 μM), niraparib (30 μM), APS-olaparib, or APS-niraparib. The cell viability was measured using the CCK-8 assay after being monitored for 120 h using time-lapse imaging in the presence of the drug. The values represented are the mean ± SD of three technical replicates.

(C) 3AO and SKOV3 spheres were treated for 48 h with DMSO, APS (200 mg/L), olaparib (200 μM), niraparib (30 μM), or their respective combinations. Following Annexin V-FITC/PI staining, the obtained results are presented on the left, along with the histogram illustrating apoptotic rates.

(D) The sphere formation assay revealed a synergistic effect in 3AO and SKOV3 spheres when APS was combined with either olaparib or niraparib for 7 days. Scale bars, 100 μm. The drug concentrations used in this experiment were 10% of the IC₅₀. Representative images from three independent experiments are displayed. The bars represent the mean ± SD of three biological experiments. ∗p < 0.05; ∗∗p < 0.01.

We further evaluated the inhibitory effects of APS, PARPi, and their combination on cell viability using CCK-8 assay (Figure 2B). Cells were exposed to drug conditions at the indicated concentrations for different durations. Notably, 3AO and SKOV3 spheres demonstrated increased sensitivity to the combination of APS with either olaparib or niraparib compared to individual treatments. Annexin V-FITC/PI dual staining was performed to measure the effect of each drug on the apoptosis of OCSCs. Figure 2C illustrates that administering APS alone had no discernible influence on apoptosis in 3AO or SKOV3 spheres. Similarly, the individual treatment with olaparib or niraparib demonstrated minimal effect on apoptosis in 3AO or SKOV3 spheres. However, combination therapy of APS with olaparib or niraparib resulted in a significant increase in the number of apoptotic 3AO spheres and SKOV3 spheres. Furthermore, a sphere formation assay was performed to assess the self-renewal capacity of OCSCs. The initial generation of spheres was dissociated enzymatically into single cells and then reseeded. The size of the second generation of spheres was determined after treatment with APS, PARPi alone, or their combination. Figure 2D depicts that the self-renewal capacity of OCSCs was significantly reduced in combination groups compared to the groups treated with either drug individually. In conclusion, the synergistic effect of APS and PARPi was observed in OCSCs by decreasing cell viability and self-renewal potential while inducing apoptosis.

Mitophagy was enhanced in OCSCs

The protein expression levels of TOMM20, an outer mitochondrial membrane complex, and cytochrome c oxidase IV (COX IV), a widely used surrogate marker of mitochondrial content, were evaluated. TOMM20 and COX IV were both degraded in 3AO spheres and SKOV3 spheres compared to their respective cell counterparts (Figure 3A). To confirm the presence of mitophagy, cells were transfected with the autophagosome marker GFP-LC3 and co-stained with MitoTracker Red to label mitochondria in all cells. The mitophagy levels were quantified by measuring the co-localization of GFP-LC3 and MitoTracker Red using confocal imaging. Our findings indicate a significant increase in the co-localization (yellow) of mitochondria (red) with LC3 puncta (green) in 3AO and SKOV3 spheres (Figure 3B). Furthermore, transmission electron microscopy revealed a greater number of bilayer membrane-bound autophagosomes enclosing condensed mitochondria in 3AO and SKOV3 spheres than in 3AO and SKOV3 cells (Figure 3C).

Figure 3.

Mitophagy was enhanced in OCSCs

(A) Western blotting was used to measure the levels of mitochondrial proteins TOMM20 and COX IV in 3AO cells, 3AO spheres, SKOV3 cells, and SKOV3 spheres. GAPDH was used as a loading control. The presented data indicate one of three experiments that all produced comparable outcomes.

(B) 3AO and SKOV3 cells were transfected with GFP-LC3, and the co-localization of mitochondria (red) and GFP-LC3 (green) was assessed by Mitotracker Red staining. Representative images were displayed on the left side, and the number of co-localization puncta (merge, yellow) in 50 cells for each group was quantified using the Metamorph offline 7.7.8.0 software package. The co-localization puncta per cell were then calculated and presented on the right. Scale bars, 5 μm.

(C) TEM was used to visualize bilayer membrane-bound autophagosomes containing condensed mitochondria (indicated by red arrows) in 3AO cells, 3AO spheres, SKOV3 cells, and SKOV3 spheres. Scale bars, 0.5 μm. The results of three independent experiments were expressed as the means ± SD. ∗p < 0.05 and ∗∗p < 0.01.

APS inhibited mitophagy in OCSCs

The 3AO and SKOV3 spheres were treated with DMSO, APS, olaparib, niraparib, APS + olaparib, and APS + niraparib. The expression of mitochondrial proteins TOMM20 and COX IV was then assessed using western blotting. Western blotting results indicate that treatment with APS + olaparib or APS + niraparib results in increased levels of TOMM20 and COX IV proteins as compared to treatment alone (Figure 4A). This indicates that mitophagy is inhibited when APS is administered. To further validate the inhibition of mitophagy, the cells were stably transfected with the autophagosome marker GFP-LC3 and co-stained with MitoTracker Red to label mitochondria in all cells. At 72 h after transfection, the aforementioned therapy was administered to the cells. Mitophagy levels were measured by assessing the co-localization of GFP-LC3 and MitoTracker Red using confocal imaging. Our findings indicate that treatment with APS + olaparib or APS + niraparib significantly reduces the co-localization (yellow) of mitochondria (red) with LC3 puncta (green) in both 3AO and SKOV3 spheres (Figure 4B).

Figure 4.

APS inhibited mitophagy in OCSCs

(A) 3AO and SKOV3 sphere cells were treated with treatments including DMSO, APS (200 mg/L), olaparib (200 μM), niraparib (30 μM), APS + olaparib, and APS + niraparib for 72 h. After treatment, TOMM20 and COX IV expression levels were determined using western blotting, with GAPDH serving as a loading control. The data represented reflect a representative experiment out of three with comparable results.

(B) The 3AO and SKOV3 cells were transfected with GFP-LC3 and stained with Mitotracker Red. After 72 h of transfection, the cells were treated as previously described, and confocal microscopy was used to examine the co-localization of mitochondria (red) and GFP-LC3 (green). The figure above displays representative images, while the figure below quantifies the co-localization puncta per cell (merge, yellow). Scale bars, 5 μm. This analysis was performed in triplicate, and the results were reported as means ± SD. ∗p < 0.05; ∗∗p < 0.01.

Inhibition of mitophagy by APS is mediated through the PINK1/Parkin pathway

Three distinct pathways can promote mitophagy: PINK1/Parkin, BNIP3L/Nix (including BNIP3), and FUNDC1. Western blot analysis demonstrated a significant upregulation of PINK1 protein expression in 3AO and SKOV3 spheres compared to 3AO and SKOV3 cells. However, no differences in the protein expression of BNIP3 and FUNDC1 were identified (Figure 5A left). PINK1 and Parkin play crucial roles in regulating mitophagy, with Parkin being translocated to the mitochondrial surface by PINK1 upregulation, thereby initiating mitophagy. Furthermore, it was revealed that the expression of PINK1 significantly decreased when the concentration of APS reached high concentration (300 mg/L), but with no significant effect at low concentration (Figure 5A right). However, as shown in Figure S3, the combination of low-dose APS (200 mg/L) and PARPi synergistically decreased the PINK1 expression, indicated that the concentration of APS inversely correlated with the protein expression level of PINK1, and shown a link between APS-mediated mitophagy and the PINK1/Parkin pathways. To validate the participation of the PINK1/Parkin pathways in APS-mediated mitophagy, 3AO and SKOV3 cells were transfected with pEGFP(+)-PINK1(+) or pEGFP(+) plasmids. Western blot and quantitative reverse-transcription PCR were used to confirm the findings (Figure 5B). The cell viability inhibitory effects of cells transfected with pEGFP(+)-PINK1(+) or pEGFP(+) plasmids and treated with a combination of APS and PARPi were assessed using the CCK-8 assay. At various time intervals, the cells were exposed to APS-olaparib or APS-niraparib using the indicated concentrations of each drug. Notably, both 3AO and SKOV3 spheres were less sensitive to the combination of APS and olaparib or niraparib after PINK1 upregulation (Figure 5C). This suggests that APSs inhibits mitophagy through the PINK1/Parkin pathway.

Figure 5.

APS-inhibited mitophagy is mediated by PINK1/Parkin

(A) Protein levels of PINK1, BNIP3, and FUNDC1 were evaluated by western blotting in 3AO spheres and SKOV3 spheres compared to 3AO cells and SKOV3 cells, respectively (left); protein levels of PINK1 were determined by western blotting in 3AO spheres and SKOV3 spheres after treating with different concentrations of APS (0, 25, 50, 100, 200, and 300 mg/L) (right) for 72 h. GAPDH was analyzed as the loading control.

(B) 3AO and SKOV3 cells were transfected with pEGFP(+)-PINK1(+) or pEGFP(+) (NC) plasmid and the PINK1 expression levels were measured using western blot and qRT-PCR.

(C) The cells transfected with pEGFP(+)-PINK1(+) or pEGFP(+) (NC) plasmid were treated with APS (200 mg/L)-olaparib (200 μM) or APS (200 mg/L)-niraparib (30 μM). The cell viability was analyzed by time-lapse imaging for 120 h in the continued presence of the drug by CCK-8 assay. The values are the mean of three technical replicates.

Discussion

Our research provides initial evidence that the combination of PARPi and Chinese herbal medicine APSs has a strong synergistic effect on OCSCs despite their modest individual therapeutic efficacy.

The extensive use of PARPi has resulted in the development of acquired resistance in patients with cancer, particularly patients with OC.²⁵ Therefore, there is a growing need for therapeutic interventions that can overcome PARPi resistance in cancer therapy. CSCs play a crucial role in the cancer progression and development of drug resistance.²⁶ Our team has successfully identified and isolated OCSCs from the parental 3AO and SKOV3 cells in the previous studies.⁸ PARP-1 is implicated in the maintenance of induced pluripotent stem cells and CSCs,^27,28 but the sensitivity of OCSCs to PARPi is unknown. SKOV3 and 3AO cancer stem-like cells are resistant to PARPi, including olaparib and niraparib. Consequently, targeting OCSCs is a promising therapeutic strategy to overcome PARPi resistance in OC.

Previous research has demonstrated that OCSCs have higher levels of autophagy, a critical process for maintaining stemness and chemotherapy resistance.⁸ Autophagy is a cellular response mechanism to various stress-inducing conditions, including nutrient deprivation, hypoxia, starvation, chemotherapy, and radiation.²⁹ Recent studies revealed that autophagy can protect cancer cells from chemotherapy or enhance the efficacy of specific drugs, thereby regulating the efficacy of anti-cancer drugs and the development of drug resistance.³⁰ Previous research revealed that olaparib induces autophagy/mitophagy in breast cancer cells with BRCA mutations, which is implicated in the cell death mechanism.³¹ PARPi-induced autophagy has been identified as an adaptive resistance mechanism in OC cells. Combining PARPi with autophagy inhibitors can potentially improve therapeutic outcomes in patients with OC.¹⁰ In addition, it has been demonstrated that autophagy confers intrinsic resistance to PARPi and that suppressing autophagy improves the therapeutic efficacy of PARPi treatment in preclinical mice models with HR-proficient breast tumors. These findings support the consideration of autophagy inhibition in clinics.⁹ These findings indicate that the advent of PARPi resistance poses a significant concern in clinical practice. Consequently, we have investigated the impact of autophagy on the susceptibility of OCSCs to PARPi. Our findings reveal increased mitophagy in 3AO and SKOV3 OCSCs, with a significant association between mitophagy and PARPi resistance. Therefore, scientists are now compelled to explore potential combinations of PARPi with other pharmaceutical agents as novel therapeutic strategies to combat PARPi resistance in OC.

Our previous research described that arsenic trioxide (ATO) can sensitize HR-proficient and platinum-resistant OC to PARPi.^16,17 Acute promyelocytic leukemia is now treated with ATO, a TCM. However, the clinical use of high-dose ATO is limited due to the occurrence of severe adverse reactions.³² Therefore, it is imperative to investigate alternative TCMs with improved safety profiles, fewer adverse reactions, and superior therapeutic outcomes in cancer therapy. TCMs have received significant attention due to their potential anti-tumor properties; however, the underlying mechanisms remain elusive. TCM-combination therapy has developed as a novel treatment approach and an important therapeutic strategy for cancer, owing to its promising anti-tumor potential. Consequently, a growing number of researchers are optimistic that TCMs may offer a breakthrough solution to the persistent challenges of drug resistance and cancer relapse, thereby resolving the “bottleneck problem” in cancer treatment.

Curcumin increases the anti-tumor effect of cisplatin in A549 cells in vitro. It increases the sensitivity of prostate cancer cells to the oncolytic effects of vesicular stomatitis virus by modulating anti-viral responses and components of the intrinsic apoptotic pathway.³³ Furthermore, it has been established that the combination of resveratrol and doxorubicin reduces tumor volume and extends the lifespan of mice with Ehrlich ascitic carcinoma cells.³⁴ Ginsenoside Rg3 has improved the cytotoxic and apoptotic effects of paclitaxel on triple-negative breast cancer by suppressing nuclear factor κB activity and regulating Bax/Bcl2 expression.³⁵ Berberine can increase the sensitivity of drug-resistant breast cancer to doxorubicin and induce apoptosis in vitro and in vivo through dose-orchestrated AMPK signaling.³⁶ Combining low-dose cisplatin or doxorubicin with ATO can improve the anti-hepatocarcinoma effect.³⁷ Previous studies conducted by our group revealed that ATO sensitizes homologous recombination-proficient and platinum-resistant OC to PARPi.^16,17 The present study indicates that the combined administration of PARPi and low-dose APSs has superior efficacy in suppressing the proliferation of 3AO and SKOV3 OCSCs while significantly increasing the rate of cellular apoptosis.

Astragalus membranaceus is frequently used in TCM to treat various cancers. APS is the primary bioactive constituent of Astragalus membranaceus that inhibits OC cell proliferation through the microRNA-27a/FBXW7 signaling pathway.³⁸ Furthermore, novel APS-based nano-pomegranates have been developed for targeting estrogen receptor-positive breast cancer and multidrug resistance.³⁹ APS also enhances the susceptibility of cervical cancer HeLa cells to cisplatin by regulating cellular autophagy.⁴⁰ However, the role of APSs in autophagy remains debatable. APSs demonstrated cytotoxic action in MCF-7 cells by blocking autophagy and facilitating mitochondrial pathway-mediated apoptosis.⁴¹ Furthermore, a recent study revealed that selenizing APSs can prevent autophagy activated by H₂O₂ through PI3K/Akt activation, thereby lowering the oxidative stress-induced promotion of porcine circovirus type 2 replication.⁴² However, other studies indicated that APSs suppresses tumor cell proliferation by activating autophagy. For instance, APSs may improve the sensitivity of cervical cancer HeLa cells to cisplatin by promoting autophagic activity by upregulating Beclin1.⁴⁰ It has been documented to exhibit anti-oxidative activity in various cellular contexts, partially attributed to its protective effects against mitochondrial dysfunction.^43,44 Mitochondria, the principal cellular source and target of ROS, can be removed by mitophagy during mitochondrial impairment. Our research revealed that APSs and PARPi-combined treatment hindered mitophagy in 3AO and SKOV3 OCSCs, although the precise mechanisms behind this phenomenon remain unclear.

Mitophagy occurs predominately through three receptor-mediated pathways: PINK1/Parkin-mediated, FUNDC1-mediated, and BNIP3-mediated.⁴⁵ To further investigate the mechanism by which APSs and PARPi-combined treatment inhibits mitophagy in OCSCs, the protein levels of three mitophagy regulatory proteins, PINK1, BNIP3, and FUNDC1, were measured using western blotting in 3AO cells, SKOV3 cells, 3AO OCSCs, and SKOV3 OCSCs. Our findings indicate that PINK1 was significantly upregulated in 3AO and SKOV3 OCSCs. However, no significant differences were observed between BNIP3 and FUNDC1 protein expression. Furthermore, the protein expression level of PINK1 and the self-renewal capacity of OCSC was significantly suppressed when the APSs treatment alone reaches a higher concentration, but with no significant effect in lower concentration. Interestingly, the combination of PARPi and low-concentration APSs synergistically decreased the PINK1 expression and self-renewal of OCSC. Moreover, it is noteworthy that the upregulation of PINK1 partially restores the sensitivity of 3AO and SKOV3 CSCs to the combination of APSs and olaparib or niraparib. This suggests that the PINK1/Parkin pathway inhibits mitophagy to contribute to the APSs-induced sensitization of OCSCs to PARPi. This finding is consistent with a previous study that inhibiting PINK1/Parkin-dependent mitophagy increases the sensitivity of multidrug-resistant cancer cells to B5G1, a novel betulinic acid analog.¹¹ Another study demonstrated a correlation between elevated levels of the mitophagy-associated protein PINK1 and adverse outcomes in patients with esophageal squamous cell carcinoma who underwent neoadjuvant chemotherapy, indicating a poor response to treatment and a bleak prognosis.⁴⁶ These findings suggest that APSs may overcome PARPi resistance in OCSCs by inhibiting mitophagy via the PINK1/Parkin signaling pathway.

In summary, this study demonstrates that the combination of APSs and PARPi has synergistic cytotoxic effects on OCSCs in vitro. The underlying mechanism involves the suppression of mitophagy, potentially mediated by the PINK1/Parkin signaling pathway, resulting in increased sensitivity. Although the clinical implications of this finding are currently unknown, our findings support the potential clinical application of this combination therapy in patients with PARPi-resistant OC. The combination of TCM and Western medicine has the potential to increase the efficacy of anti-tumor therapy while mitigating toxic side effects. This collaborative approach has great promise for improving the quality of life and prognosis of patients with OC.

Limitations of the study

A limitation is that our study was performed using only two kinds of OC cell lines (3AO cell and SKOV3 cell); additional research in vivo and clinical investigations are required to validate these findings, and further exploration is required to elucidate the underlying mechanisms.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-TOMM20 antibody	Abcam	Cat#ab-186735; RRID: AB_2889972
Anti-COX IV antibody	Abcam	Cat#ab-14744; RRID: AB_301443
Anti-GAPDH antibody	Santa Cruz	Cat#sc-47724; RRID: AB_627678
Anti-PINK1 antibody	Santa Cruz	Cat#sc-518052; RRID: AB_2861352
HRP Goat Anti-Mouse IgG(H+L)	ABclonal	Cat#AS003; RRID: AB_2769851
HRP Goat Anti-Rabbit IgG(H+L)	ABclonal	Cat#AS014; RRID: AB_2769854
Critical commercial assays
basic fibroblast growth factor (bFGF)	Peprotech	Cat#100-18B
Epidermal growth factor (EGF )	Peprotech	Cat#100-15
Insulin	Sigma–Aldrich	Cat#I9278
B27	Life Technologies	Cat#17504044
Ultralow attach plates	Corning	Cat#3471
CCK8	Dojindo	Cat#CK04
The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit	Multi Sciences	Cat#AP101
GFP-LC3 plasmid	Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University	N/A
X-tremeGENE HP DNA Transfection Reagent	Roche	Cat#Rh-06366236001
MitoTracker™ Red FM	Invitrogen	Cat#M22425
Chemicals, peptides, and recombinant proteins
Niraparib	Cayman	Cat#CAY-20842-5
Olaparib	Cayman	Cat#CAY-10621-10
Dimethyl sulfoxide (DMSO)	Sigma–Aldrich	Cat#D2650
Astragalus polysaccharides(APS)	Yuanye Biological, Shanghai, China	Cat#B20562
Glutaraldehyde solution	Sigma-Aldrich	Cat#G5882
Experimental models: Cell lines
SKOV3 human ovarian cancer cell lines	American Type Cell Culture	N/A
3AO human ovarian cancer cell lines	Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China.	N/A
Software and algorithms
Microsoft PowerPoint	Microsoft	https://www.microsoft.com/en-us/microsoft-365/microsoft-office
Microsoft Word	Microsoft	https://www.microsoft.com/en-us/microsoft-365/microsoft-office
Microsoft Excel	Microsoft	https://www.microsoft.com/en-us/microsoft-365/microsoft-office
Adobe Illustrator	Adobe	https://www.adobe.com/uk/
GraphPad Prism 9	GraphPad	https://www.graphpad.com/
SPSS Statistics 26	SPSS	https://www.ibm.com/spss
CompuSyn software	CompuSyn	http://www.combosyn.com/index.html

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Weiguo Lu (lbwg@zju.edu.cn).

Materials availability

This study did not generate any new unique reagents and all materials in this study are commercially available.

Data and code availability

• •Data: All data generated or analyzed during this study are included in this published article. All relevant data are available from the lead contact upon request.
• •Code: This paper does not report original code.
• •Any additional information required to re-analyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Cell lines

Ovarian cancer cell lines

The SKOV3 human OC cell lines were obtained from the American Type Cell Culture, while the 3AO OC cell lines were obtained from the Women's Hospital, School of Medicine, Zhejiang University. 3AO and SKOV3 cell lines were cultured in RPMI 1640 media (Corning, Steuben County, NY, USA), supplemented with 10% fetal bovine serum (FBS).

Ovarian cancer stem-like cells culture

3AO and SKOV3 cells were cultured at 50,000 cells/mL density in serum-free DMEM/F12 medium (Cellgro, Virginia, USA) to form spheres. This medium included 10 ng/mL basic fibroblast growth factor (bFGF, Peprotech, Rocky Hill, NJ, USA), 20 ng/mL epidermal growth factor (EGF, Peprotech), 1 mg/mL insulin (Sigma–Aldrich, St Louis, MO, USA), and 1×B27 supplement (Life Technologies, Carlsbad, CA, USA) on ultralow attach plates (Corning, Steuben County, NY, USA). The cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Method details

Drug sensitivity assay and cell viability assay

Stock solutions of niraparib (Cayman, Cat No.CAY-20842-5) and olaparib (Cayman, Cat No.CAY-10621-10) were prepared using dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Cat No. D2650) and stored in aliquots at –80°C. The solution of APS (Yuanye Biological, Shanghai, China, Cat No.B20562) was stored at –4°C. Appropriate dilutions were prepared in the culture medium. In viability assays, 2000 cells were seeded in each well of 96-well plates for subsequent treatment with either DMSO or different concentrations(25% IC50, 50% IC50, 75% IC50, 100% IC50, 125% IC50) of niraparib, olaparib, or a combination of PARPi-APS drugs for 72 h; 2000 cells were seeded in each well of 96-well plates for subsequent treatment with APS(200 mg/L), olaparib(200 μM), niraparib(30 μM), APS-olaparib, or APS-niraparib,and incubated at 37°C for various lengths of time (0, 24, 48, 72, 96 and 120 hours). By adding 10 μl of CCK-8 solution (DOJINDO, Japan) to each well of the plate, cell viability was determined. A microplate reader with a 450 nm setting was used to calculate the absorbance of each well. Three duplicates of each experiment were carried out. Dose-response curves or the half maximal inhibitory concentration (IC₅₀) plots were constructed using GraphPad Prism 9.0 to measure cell viability. CompuSyn software assessed the potential synergy between the PARPi and APS.

Cell apoptosis measurement

The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (Multi Sciences, Cat No. AP101) was used to quantify cellular apoptosis. Cells were cultured in 6-well plates at an appropriate density per well for 24 h before being treated with DMSO, olaparib, niraparib, APS, or a combination of PARPi-APS drugs for 48 h. All cells were then suspended in pre-chilled 1 × binding buffer, stained with FITC and PI for 5 min at room temperature (RT) in the dark, and subsequently analyzed using a flow cytometer (BD Biosciences, FACS Verse).

Western blot analysis

Using Cell Lysis buffer (Beyotime, China), the protein was extracted from the cells. Each group received the proper amount of cell lysate, which was added, thoroughly mixed with a vortex, and then put on ice for 30 minutes before being centrifuged for 10 minutes at 4C at 12,000 rpm. Using a BCA protein quantification kit, the protein concentration was quantified. The proteins were loaded and separated on a 10% SDS-PAGE (GenScript Biotech, Piscataway, NJ, USA) gel before being transferred to 0.2-μm polyvinylidene fluoride(PVDF) membranes (1620177, Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% nonfat dry milk in tris buffered saline tween(TBST), membranes were incubated with the primary antibodies at 4°C overnight, followed by secondary antibody incubation. The membranes were then washed with TBST and treated with the EZ-ECL kit (BI Biological Industries, Cromwell, CT, USA) to visualize the bands with the ImageQuant LAS 4000 mini (Cytiva, Marlborough, MA, USA). All experiments were performed in a triplicate manner.

The primary antibodies used were as follows: Anti-TOMM20 (Cat No. ab-186735, 1:1000) and anti-COX IV (Cat No. ab-14744, 1:1000), were purchased from Abcam; anti-GAPDH (Cat No. sc-47724, 1:1000) and anti-PINK1 (Cat No. sc-518052, 1:500) were obtained from Santa Cruz Biotechnology. The secondary antibodies used were: HRP-linked anti-rabbit IgG(Cat No.AS014, 1:5,000) and HRP-linked anti-mouse IgG (Cat No.AS003, 1:5,000), were purchased from ABclonal (Wuhan, China).

Plasmids transfection

The construction of the pEGFP(+)-PINK1 plasmid involved the synthesis of the PINK1 ORF by Sangon Biotech (Shanghai, China), which was then inserted into the EcoRI/BamHI sites of a pEGFP-N1 vector. The mock control vector was an empty pEGFP-N1. The GFP-LC3 plasmid was generously provided by Dr. Hong-He Zhang from the Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University (Hangzhou, China). SKOV3 and 3AO cells were grown to 80% confluency before plasmid transfection. Using the DNA: X-tremeGENE HP DNA Transfection Reagent (Roche, Cat No. Rh-06366236001), 2 mg of DNA was added to each well of a 6-well plate at a ratio of 1:2. The transfection protocol was followed in accordance with the manufacturer's instructions. Following plasmid transfection for 24 h, SKOV3, and 3AO cells were diluted to 10% to 15% confluency and then treated for ten days with 400 mg/mL G418 (Sigma-Aldrich).

Fluorescence microscopy

3AO spheres, SKOV3 spheres, 3AO cells, and SKOV3 cells were transfected with GFP-LC3 overnight. The cells were then transferred to coverslips and treated for 30 min at 37°C with MitoTracker Red dye (Invitrogen, Cat No. M22425) to facilitate mitophagy detection. Cells were washed in PBS, fixed in 4% ice-cold paraformaldehyde for 15 minutes, and then washed again in PBS for immunofluorescence, and permeabilized with 0.1% Triton X-100/PBS for 10 minutes. The nuclear counter stain was performed using 4’6-Diamidino-2-phenylindole (DAPI). The presence of GFP-LC3 dots indicated the presence of autophagosomes. Additionally, MitoTracker Red dye was used to visualize the mitochondria. The degree of co-localization between GFP-LC3 and MitoTracker Red was measured as a mitophagy indicator. The images were acquired with a spinning disk confocal fluorescence microscope, namely a CSU-X1 spinning disk from Yokogama, an IX81 microscope from Olympus (Tokyo, Japan), and an IXON3 CCD from Andor at a magnification of 600×. The Metamorph offline 7.7.8.0 software package was used to quantify the number of yellow puncta in 50 cells per group.

Transmission electron microscopy (TEM)

The 3AO spheres, SKOV3 spheres, 3AO cells, and SKOV3 cells were immobilized overnight in a 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH7.0) for more than 4 h, washed thrice in PBS for 15 min at each step, post-fixed with 1% OsO4 in phosphate buffer for 1 h and washed thrice in PBS for 15 min at each step; and the cells were first dehydrated via an ethanol gradient (30%, 50%, 70%, 80%; approximately 15 min at each step), and then dehydrated using an acetone gradient (90%, 95%; approximately 15 min at each step). Finally, the cells were dehydrated twice with absolute acetone for 20 min each time; and placed in a 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h at room temperature and then transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3 h and the final Spurr resin mixture overnight. After using a series of ethanol fractions and being embedded in 812 resin (Ted Pella, Redding, CA, USA), then staining them with 2% uranyl acetate for 5 to 10 min, they were examined with a Tecnai 10 transmission electron microscope (Philips, Amsterdam, NED).

Quantification and statistical analysis

Experiments were performed according to standard protocols, with a minimum of three biological replicates, and independently performed in triplicate. Compusyn software (Compusyn, Paramus, NJ, USA) calculated the drug combination index (CI) between the PARPi and APS. The CI value indicates the nature of the interaction, with values more than one indicating antagonism, values equal to one indicating an additive effect, and values less than one indicating synergism. The statistical analyses of these were performed using the paired Student’s t-test. The obtained data were presented as the mean ± standard deviation (SD) and analyzed using GraphPad Prism 9.0 and SPSS 26.0 Software. It was considered statistically significant when the P-value was less than 0.05. Statistical significance was determined at the ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 levels.

Published: June 27, 2024