Ovarian cancer is the second most frequently diagnosed gynecological cancer, and has the highest mortality among gynecological cancers worldwide (1). The standard therapy for patients with advanced ovarian cancer is complete cytoreductive surgery, followed by combination chemotherapy, consisting of taxane- and platinum-based regimens. Although patients with advanced ovarian cancer typically respond well to initial treatment with cisplatin-based chemotherapy, the majority of them (over 80%) eventually experience resistance to currently available treatment options, ultimately leading to therapeutic failure (2). Platinum-based anticancer drugs, including cisplatin, are DNA cross-linking agents. Their antitumor activities are mediated by binding with nuclear DNA, resulting in the formation of intra- and inter-strand adducts, which induce DNA damage response pathways that eventually block cell cycle progression and cause apoptotic cell death (3). The mechanisms related to induced resistance to platinum compounds are complex and numerous, and still not fully understood. Several underlying mechanisms of resistance at the cellular and molecular levels have been described, including 1) reduced intercellular drug accumulation or increased efflux of platinum agents mediated by copper-transporting P-type ATPases (ATP7A/7B) and multidrug-associated protein 2, 2) inactivation of intracellular platinum agents by forming adducts with detoxification components and antioxidants, such as glutathione and metallothionein, 3) increased DNA repair processing through five major pathways of nucleotide excision repair (NER), mismatch repair, homologous recombination repair, base excision repair (BER), and translesion synthesis (4). Recently, aberrant DNA methylation changes at promoter cytosine-phosphate-guanine (CpG) sites have been reported in cisplatin-resistant cells. These DNA methylation changes regulate the expression of genes critical for response to chemotherapeutic agents, thereby leading to anticancer drug resistance (5).
Poly (ADP-ribose) polymerases (PARPs) comprise a large protein family of 17 members that possess poly-ADP ribosyltransferase activity (6), and are involved in diverse cellular processes, including DNA repair (7). Some isoforms, including PARP1 and PARP2, have been well characterized during their involvement in DNA repair processes, such as BER in response to single-strand DNA breaks (8) and NER (9). Because of their roles in DNA repair, PARP inhibitors are attractive chemotherapy candidates for many cancer types, including ovarian cancer (10). PARP4 is one of the largest members of the PARP protein family, with a molecular mass of 193 kDa, and possesses poly (ADP-ribosyl) transferase activity (11). PARP4 is part of the cytoplasmic ribonucleoprotein complex, also known as the vault, which is known for its involvement in multidrug resistance (12, 13). Although PARP4 is known to be catalytically active, the biological functions of PARP4 remain largely undefined.
Herein, we investigated alterations of
Previously, we evaluated cisplatin-induced cytotoxicity in 11 human ovarian cancer cell lines, and classified the results into three groups: sensitive, intermediate, and resistant (14). In the present study, we conducted reverse-transcription quantitative polymerase chain reaction (RT-qPCR) on three cisplatin-sensitive cell lines (PA-1, TOV-112D, and A2780) and three cisplatin-resistant cell lines (OVCAR-3, OV-90, and SK-OV-3). The results showed that
It has been widely observed in various cancer cell lines that aberrant alteration of DNA methylation plays a critical role in the development of chemoresistance (15). We therefore performed epigenome-wide methylation profiling using the IlluminaⓇ HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA), to examine differences between cisplatin-sensitive and cisplatin-resistant cell lines in DNA methylation within the promoter region of the
To determine whether
To determine whether
Cisplatin sensitivity was evaluated in siNC or siPARP4 transfected cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Silencing of
Additionally, DNA damage (DNA fragmentation) was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by flow cytometry in siRNA-treated SK-OV-3 cells (Fig. 2D). Flow cytometry results revealed knockdown with siPARP4 led to significantly increased (∼21%) TUNEL-positive cells, compared to siNC-transfected control cells (Fig. 2E). Collectively, these results showed that
Patients with recurrence within 12 months from the last dose of platinum-based chemotherapy were defined as being cisplatin-resistant, whereas those without recurrence > 12 months from the last dose of platinum-based chemotherapy were defined as cisplatin-sensitive (Table 1).
We also investigated DNA methylation status at specific promoter CpG sites of the
PARP4 is a part of the cytoplasmic ribonucleoprotein complex, also known as the vault, composed of major vault protein (MVP), telomerase-associated protein (TEP1), PARP4, and vault-associated RNA (16). Although upregulation of the vault complex has been implicated in multidrug resistance against a broad spectrum of anticancer drugs, such as doxorubicin and cisplatin, the precise molecular functions of the vault complex remain largely unknown (12, 13). PARP4 loss in mice leads to an increased carcinogen-induced colon tumor incidence, multiplicity, and reduced tumor latency, indicating PARP4 potentially functions as a tumor suppressor. However, PARP4-deficient mice in a lung cancer model did not demonstrate any significantly increased carcinogen-induced tumor multiplicity, implying the tumor suppressive role of PARP4 could be cancer-type-dependent (16). PARP4 is thought to be involved in the BER pathway, due to its possession of the BRCA1 carboxy-terminal domain, which is common in other DNA repair pathway proteins, such as PARP1 and X-ray repair cross-complementing 1. Nevertheless, to date, there is no evidence demonstrating that PARP4 is involved in DNA repair processes (17).
In the present study, we investigated the alteration of
A previous study examining the expressions of vault proteins, including PARP4, in post-surgery ovarian cancer samples showed that mRNA levels of
Although the precise mechanisms underlying the protective process against cisplatin-induced DNA damage need to be further investigated, our results suggest aberrant
The human ovarian cancer cell lines studied were PA-1, TOV-112D, A2780, OVCAR-3, OV-90, and SK-OV-3. All cell lines were cultured using the complete growth media recommended by the suppliers (Supplementary Table 1) in a 37°C incubator with a humidified atmosphere of 95% air and 5% CO2.
Fifteen cisplatin-sensitive patients and nine cisplatin-resistant patients with ovarian cancer were included in this study. Tumor tissue samples were obtained from the Korea Gynecologic Cancer Bank at the time of staging the surgery. The clinicopathological characteristics are shown in Table 1. This study was approved by the Institutional Review Board (Permit Number: EUMC 2014-05-004-001), and written informed consent was obtained from all patients.
To inhibit DNA methylation, three cisplatin-sensitive ovarian cell lines (PA-1, A2780, and TOV-112D) were treated with 10 μM 5-aza-dc (Sigma-Aldrich, St. Louis, MO, USA) for 3 days at 37°C. The medium was freshly exchanged every day and supplemented with 10 μM 5-aza-dc.
Bisulfite-treated DNA was used as a template for qMSP. The primers of qMSP were designed for detecting the methylated or unmethylated forms of the specific CpG site (cg18582260). The sequences of the methylated/unmethylated-specific primers were as follows: 5’-TGGGAGGTATGGAAAGGC-3’ (methylated forward), 5’-TGGGAGGTATGGAAAGGT-3’ (unmethylated forward), 5’-AAAACATAAACTAACTCTATATTTAA-3’ (reverse). The qMSP was performed using a 7500 Fast Real-time PCR system (Thermo Fisher Scientific) as previously described (21). The percentages of methylation at the specific CpG site were calculated as follows (Ct represents the threshold-cycle): percent of methylation = 100 / [1 + 2(ΔCtmethylation − ΔCtunmethylation)]% (20).
The siNC or siPARP4-transfected SK-OV-3 cells were treated with 120 μM of cisplatin after 24 h transfection. The siNC-transfected SK-OV-3 cells without cisplatin treatment served as controls. After 48 h cisplatin treatment, the TUNEL assay (Abcam, Cambridge, MA, USA) was conducted following the manufacturer’s protocol. The fluorescent intensity of cells was analyzed using flow cytometry (NovoCyte 3000; Agilent Technologies, Santa Clara, CA, USA): Ex/Em = 488/576 nm for BrdU-Red and Ex/Em = 488/655 nm for 7-AAD.
The bisulfite pyrosequencing analysis was performed on two CpG sites within the promoter region of the
Results are expressed as the mean ± standard deviation of at least three independent experiments. All statistical analyses were performed using Prism 5 software (GraphPad Software, San Diego, CA, USA). A value of P < 0.05 was considered statistically significant.
The research resources (tumor tissues from patients with ovarian cancer) were provided by Korea Gynecologic Cancer Bank (KGCB) of the Infrastructure Project for Basic Science of the Ministry of Education, Science, and Technology (MEST), Korea.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B070 43451, NRF-2022R1I1A1A01068767, NRF-2020R1I1A3073845), and the Korean government (MSIT) (NRF-2020R1A5A2019210). The present study was also supported by RP-Grant 2021 of Ewha Womans University.
The authors have no conflicting interests.
Clinical characteristics of patients with ovarian cancer who were sensitive or resistant to primary chemotherapy
|Patient No.||Chemo-response||Histology||Stagea||Gradeb||PFI (month)|
aInternational Federation of Gynecology and Obstetrics (FIGO) stage.
R, ovarian tumor tissues from cisplatin-resistant patients; S, ovarian tumor tissues from cisplatin-sensitive patients; PFI, Platinum free interval; the time between the last dose of platinum-based therapy and documented relapse (month).