Synergistic antitumor efficacy of rMV-Hu191 and Olaparib in pancreatic cancer by generating oxidative DNA damage and ROS-dependent apoptosis

Highlights

• •The oncolytic virus rMV-Hu191 synergistically enhanced the sensitivity of HR-proficient PDAC cells to PARP inhibitor olaparib through synthetic lethal.
• •rMV-Hu191 and olaparib synergistically enhanced oxidative stress in human PDAC cells.
• •Combination therapy with rMV-Hu191 and olaparib produced oxidative DNA damage in human PDAC cells.
• •rMV-Hu191 collaborated with olaparib to induce ROS-dependent apoptosis in human PDAC cells.
• •Combination therapy with rMV-Hu191 and olaparib generated a profound antitumor effect in vivo with minimal toxicity.

Abstract

Background

Malignancies with BRCA1/2 deficiencies are particularly sensitive to PARP inhibitors. Thus, combining PARP inhibitors with agents that impair DNA damage repair to treat BRCA1/2 wild-type PDAC could broaden the clinical use of these promising PARP inhibitors. Here we examined the synergism and mechanism of oncolytic measles virus (rMV-Hu191) with a PARP inhibitor (Olaparib) in vitro and in vivo.

Methods

The cell viability assay, cell cycle analysis, colony formation assay, TCID 50 method, western blotting, flow cytometry, DNA comet assay, Mice bearing PDAC xenografts, IF, IHC and TUNEL assay were performed to explore the antitumor efficacy and underlying mechanisms.

Results

In this study, we explored the antitumor activities of rMV-Hu191 and Olaparib in two PDAC cell lines harboring wild-type BRCA1/2 genes. Compared to monotherapy, the combination of rMV-Hu191 and Olaparib was able to synergistically cause growth arrest, apoptotic cell death and DNA damage, accompanying with excessive oxidative stress. Mechanistically, the data indicated that the observed synergy depended on the oxidative DNA damage and ROS-dependent apoptosis generating by rMV-Hu191 combined with Olaparib in human PDAC cells. Tumor inhibition and prolonged survival of PDAC mice xenografts in vivo confirmed the synergism of combinational treatment with trivial side-effects.

Conclusions

Our findings firstly suggested that combination treatment with rMV-Hu191 and Olaparib had a profound and synergistic therapeutic effect against human PDAC through synthetic lethality. In conclusion, we recommend combining oncolytic rMV-Hu191 with a PARP inhibitor (Olaparib) as a novel therapeutic strategy and provided a potential mechanism for advanced PDAC regardless of BRCA mutation status.

Abbreviations

PDAC

pancreatic ductal adenocarcinoma

DDR

DNA damage repair

PARP

poly (ADP-ribose) polymerase

SSBs

DNA single-strand break

DSBs

DNA double-strand break

HR

homologous recombination

MV

measles virus

rMV-Hu191

recombinant Chinese Hu191 measles virus

FDA

the U.S. Food and Drug Administration

ROS

reactive oxidative species

ATCC

American Type Culture Collection

TCID 50

the median tissue culture infective dose

MOI

multiplicity of infection

PFU

plaque forming unit

CCK-8

cell counting kit-8

DCF

dichlorofluorescein

IF

immunofluorescence

IHC

immunohistochemistry

H&E

hematoxylin and eosin

ALB

albumin

GLB

globulin

AST

aspartate aminotransferase

ALT

alanine aminotransferase

BUN

blood urea nitrogen

CREA

creatine

RCD

regulated cell death

Introduction

Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority (90 %) of pancreatic neoplasms, which is considered as one of the most aggressive cancers with a poor prognosis and high mortality rate [1]. Due to lack of symptoms at early stages and proper screening methods for precancerous lesions, most patients present with locally advanced (30 %–35 %) or metastatic (50 %–55 %) disease at diagnosis [2]. The 5-year survival rate approaches 10 %, which is the lowest among all types of cancer [3]. Surgical resection is the only strategy that can potentially cure PDAC at early stage, and adjuvant chemotherapy has increased survival rate for pancreatic cancer patients [4]. Unfortunately, chemotherapy such as gemcitabine (a first-line chemotherapeutic agent of PDAC) alone or in combination for treating advanced and metastatic pancreatic cancer frequently limits its clinical success due to unexpected gastrointestinal toxicity and drug resistance [5]. Therefore, new multiagent cytotoxic therapies with minimum side effects for this extremely aggressive disease are urgently needed.

One particularly aggressive form of PDAC, known as the unstable isoform, is frequently mutated in DNA damage repair (DDR) genes such as BRCA1/2 and ATM (ATM Serine/threonine kinase) [6,7]. Therefore, dozens of ongoing DDR inhibitors trials demonstrated that targeting DDR genes and eventually making tumors DDR defective is an objective in molecular oncology. The poly (ADP-ribose) polymerase (PARP), a DNA-binding protein involved in apoptosis as well as DNA single-strand break (SSBs) and DNA double-strand break (DSBs) repair, has become a popular therapeutic target for many different malignancies, such as ovarian cancer [8], breast cancer [9], pancreatic cancer [10,11] and prostate cancer [12]. By blocking PARP activity, PARP inhibitors convert unrepaired SSBs into DSBs requiring DNA homologous recombination (HR) repair pathway, which ultimately lead to synthetic lethality effects in BRCA1/2 mutant cells [13]. Of the PARP inhibitors, Olaparib has been approved by the U.S. Food and Drug Administration (FDA) for BRCA-mutated metastatic PDAC treatment [10,14]. However, patients with non-BRCA mutations may not benefit from it. Thus, combining PARP inhibitors with agents that impair DNA damage repair to treat BRCA1/2 wild-type pancreatic cancer could broaden the clinical use of these promising PARP inhibitors.

Measles virus (MV), one of the oncolytic viruses that has been identified as selectively replicating in and killing tumor cells while retaining normal cells, represents a promising alternative in the light of the limited efficacy and severe side effects in conventional cancer therapeutics [15,16]. And it is usually used in combination with chemotherapy or radiotherapy to achieve greater efficacy [17], [18], [19], [20]. Notably, previous studies have shown that virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic viruses activities, and promoting DNA synthesis and/or impeding HR repair could potentiate the effects of oncolytic viruses in the treatment of cancers [21], [22], [23], [24]. Due to the stability of MV genome and the long history of vaccination application in China [25,26], the recombinant Chinese Hu191 measles virus (rMV-Hu191) with enhanced safety and immunogenicity modified by the virus reverse genetic system in our laboratory [27] has been demonstrated to have synergistic effect in gastric cancer combined with the chemotherapy drug cisplatin [28]. However, whether rMV-Hu191 could synergize with Olaparib in PDAC cells has not been reported to the date.

Furthermore, the live-attenuated MV vaccine has been elaborated to generate oncolytic effects on ovarian cancer via ROS-induced apoptosis [29]. Reactive oxidative species (ROS), which are produced ubiquitously in mammalian cells, are important for normal biological processes in physiological concentrations, whereas excessive ROS can damage cellular components such as lipids, proteins, DNA, carbohydrates, and finally lead to cell death [30,31]. Recent evidences suggest that PDAC commonly has intratumoral hypoxia and high ROS production which could activate the oncogenic signaling, drive DNA damage and genetic instability [32,33]. PARP inhibitors have also been determined as targeted therapeutic agents that can generate antitumor effect by stimulating ROS accumulation and ROS-induced DNA damage [34,35]. Thus, we proposed a hypothesis that oncolytic rMV-Hu191 could synergistically aggrandize the antitumor effect of a PARP inhibitor (Olaparib) by generating excessive ROS and oxidative DNA damage in PDAC cells.

In the present study, we investigated the combination of rMV-Hu191 and Olaparib in two PDAC cell lines (PANC-1 and MIA-PaCa2) harboring wild-type BRCA1 and BRCA2 genes [36,37]. When combined simultaneously, the two agents caused additive to synergistic growth arrest both in vitro and in vivo, cooperatively resulted increased DSBs of DNA and apoptotic cell death induced by oxidative stress enhancement in PDAC cells. As described herein, for the first time, we recommend combining oncolytic rMV-Hu191 with a PARP inhibitor (Olaparib) as a novel therapeutic strategy and provided a potential mechanism for advanced PDAC regardless of BRCA mutation status.

Materials and methods

Cell culture and reagents

African green monkey kidney Vero cells (RRID: CVCL_0059) were purchased from the American Type Culture Collection (ATCC, USA), and cultured in DMEM (Life Technologies, USA) supplemented with 10 % FBS. The human pancreatic cancer cells PANC-1 (RRID: CVCL_0480) and MIA-PaCa2 (RRID: CVCL_0428) were purchased from Cell Bank, Chinese Academy of Sciences, and were cultured in RPMI 1640 medium (Life Technologies, USA) supplemented with 10 % FBS. All cells were cultured in an incubator, at 37 °C with 5 % CO2 and saturated moisture. Reagents: Olaparib (Selleck, S1060), Z-VAD (ApexBio, A1902), NAC (Beyotime, S0077).

Virus construction, titration and infection assays

rMV-Hu191 was constructed by our laboratory as previously described [26,38]. Virus titer was measured on Vero cells using the TCID 50 method [39]. For virus infection assay, cells were seed at 3 × 10⁵ per cell in six-well plates. After 24 h, cells were washed once in PBS and incubated with rMV-Hu191 at multiple MOIs for 1.5 h at 37 °C. Then the unabsorbed virus supernatant was replaced with fresh medium.

Cell viability assays

Cells were seeded in 96-well plates at 2000 cells per well in 100 μl medium. After treatment with rMV-Hu191 at different multiplicity of infection (MOI) and Olaparib at different concentrations, cell viability was calculated by Cell Counting Kit-8 assay (CCK-8, Dojindo, CK04). At indicated time, cells were incubated with 90 μL RPMI 1640 medium and 10 μL reagent for 30 min at 37 °C, and then determined the optical absorbance at 450 nm using a microplate reader (PowerPacTM Basic, BIO-RAD).

Bliss independence analysis

The complete combination matrix for antagonism or synergy were determined by using the Bliss independence method (www.synergyfinder.fimm.fi) [40] (Synergy Finder, RRID: SCR_019318). This model is an effect-based strategy that compares the effect of combination to the effect of its individual components, and quantifies the degree of synergy that multiplicative effect of single drugs as if they acted independently. Positive synergy score indicated that the two drugs had synergistic effect, while negative synergy score indicated antagonistic effect. A synergy score of zero indicated no drug interactions.

Colony formation assay

PANC-1 and MIA-PaCa2 cells were plated at 3 × 10⁵/well in six-well plates and exposed to rMV-Hu191 (MOI=1), Olaparib (5 μM, 10 μM), or the combination. After 36 h, 1000 cells per well were planted in a new six-well plate. On day 7, cells were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. Cell groupings composed of at least 50 cells were regarded as colonies. Colonies were imaged under light microscope (Zeiss, Germany) with companion software and counted using ImageJ 1.45S software (National Institutes of Health). Three independent experiments were performed.

Flow cytometry

For cell cycle analysis, cells were trypsinized, washed once in ice-cold PBS, and fixed in 70 % ethanol overnight at 4 °C. Then, cells were centrifuged, washed once in ice-cold PBS, and resuspended in 500 μl buffer solution per sample consisted of 25 μl PI and 10 μl RNase A for 30 min at 37 °C in the dark (Beyotime, C1052). Samples were detected by flow cytometry within 1 h (Navios, Beckman Coulter, USA) and analyzed using FlowJo 10.0 software (Tree Star Inc, USA).

For apoptosis analysis, 1 × 10⁶ cells at indicated treatments were collected, washed twice in ice-cold PBS, double-stained using an Annexin V FITC Apoptosis Detection Kit (BD Biosciences, 556,547) following the vendor's instructions, and then detected by a flow cytometer (Navios, Beckman Coulter, USA) and analyzed using Flowjo 10.0 software (Tree Star Inc, USA).

For intracellular ROS measurement, cells were incubated with DCFH-DA (10 μM), which could be oxidized to fluorescent dichlorofluorescein (DCF) by intracellular ROS, for 40 min at 37 °C in the dark by using a Reactive Oxygen Species Assay Kit (Beyotime, S0033M). And then the fluorescent emission was measured at 525 nm with the excitation wavelength of 488 nm by a flow cytometer (Navios, Beckman Coulter, USA) and analyzed using Flowjo 10.0 software (Tree Star Inc, USA).

Immunofluorescence (IF)

Cells after indicated treatment were fixed with 4 % paraformaldehyde for 30 min at 4 °C, permeabilized with 0.1 % Triton X-100 for 15 min, washed twice with ice-cold PBS, and then blocked with 2 % BSA for 1 h at 37 °C. Cells were then incubated with primary antibody against γ-H2AX (CST, 9718, RRID: AB_2,118,009) diluted with 2 % BSA at 4 °C overnight, washed three times with ice-cold PBS, followed with Alexa Fluor 594 goat anti-rabbit IgG (H + L) (Invitrogen, A-11,012) diluted with PBS for 30 min at 37 °C in the dark. DAPI reagent (Beyotime, C1002) was employed to stain cell nuclei. A cell was identified as γ-H2AX positive only if >10 foci per nucleus. Images were visualized using Lecia TCS SP8 laser scanning microscope and analyzed using Flowjo 10.0 software (Tree Star Inc, USA).

DNA comet assay

After desired treatment, cells were resuspended in PBS, and combined with 0.5 % low melting point agarose, then immediately pipetted onto slides pre-coated with 1 % normal melting point agarose. Slides with coverslip were solidified at 4 °C for 5 min, lysed in cell lysis solution at 4 °C for 1 h, unwound in prechilled alkaline electrophoretic buffer at 4 °C for 20 min, and then electrophoresed for 25 min in the dark (25 V/350 mA/7 W). The slides were stained with Gel Red (Invitrogen, GR502) after neutralization. Fifty cells per slide were counted. The length of DNA tail was considered as a parameter to evaluate DNA strand breaks. Images were captured using fluorescent microscopy and analyzed by CASP software (v1.1.2).

Western blotting

Human pancreatic cancer cells after designed treatment were harvested at indicated times, washed once with ice-cold PBS, and then lysed in RIPA buffer supplemented with phosphatase inhibitor (cocktail, Thermo, 87,785) and protease inhibitor (PMSF, Beyotime, ST506–2). 30 μg of lysates per sample were separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, 1,620,177). The PVDF membranes were blocked with 5 % non-fat milk at room temperature for 1 h, incubated with specific primary antibodies diluted in TBST at 4 °C overnight, and then followed by secondary antibodies diluted in TBST at room temperature for 1 h. Membranes were visualized by using an enhanced chemoluminescence kit (Biological Industries, 20–500–120). ImageJ 1.45S software was employed for quantitative density analysis of blots. For Western blot analysis: anti-β-actin (CST, 4970, RRID: AB_2,223,172), anti-caspase-3 (CST, 9665, RRID: AB_2,069,872), anti-PARP (CST, 9542, RRID: AB_2,160,739), anti-measles nucleoprotein (MV-N, abcam, ab106292, RRID: AB_10,863,975), anti-γ-H2AX (CST, 9718, RRID: AB_2,118,009), secondary HRP-linked anti-rabbit IgG (CST, 7074, RRID: AB_2,099,233), secondary HRP-linked anti-mouse IgG (CST, 7076, RRID: AB_330,924).

In vivo PDAC xenograft studies

Female 3–4 weeks old athymic nude mice were purchased and housed in the animal center of Zhejiang Chinese Medical University, and all the following animal experiments were approved by its animal ethics committee (IACUC-20,200,330–01). 7 × 10⁶ MIA-PaCa2 cells suspended in 100 μl PBS were injected subcutaneously into the right flank of athymic nude mice to construct xenograft tumor models. When tumor reached an average volume of 30–50 mm³ (V = length × width²/2), mice were randomized into four groups (mock group, rMV-Hu191 group, rMV-Hu191+Olaparib group, Olaparib group, n = 10), injected intratumorally with 100 μl Opti-MEM within rMV-Hu191 (1 × 10⁷ PFU) and injected intraperitoneally with 200 μl PBS within Olaparib (50 mg/kg) six times on day 5, 6, 7, 9, 11 and 13 post implantation. Tumor volume and weight were measured every 3–4 days. Mice were euthanized when tumor diameter exceeded 1.5 cm in any direction, body weight loss over 20 %, natural death or tumor burst occurred.

Immunohistochemistry (IHC)

The mice were randomly sacrificed (n = 3) after the last administration, and tumor tissues were excised, fixed in 4 % formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E) or probed with the anti-γ-H2AX (CST, 9718, RRID: AB_2,118,009) and primary anti-cleaved-caspase3 (CST, 9579, RRID: AB_10,897,512) antibodies. In situ apoptotic cells in tumor slides were measured by an Apoptosis Detection Kit (TUNEL, Takara, MK500). Color changes of the stained slides were photographed and analyzed with a microscope (Leica, Germany).

In vivo assessment of side-effects

The day after last injection, mice (n = 3) were sacrificed to harvest angular vein blood for liver (ALB, GLB, ALT, AST) and kidney (BUN, CREA) functions analysis on an automatic blood chemical pipeline analyzer (Beckman Coulter, USA).

Statistical analysis

All data exhibited representatively were repeated at least three times and presented as mean ± SD. Statistical significance was analyzed by one-way ANOVA or two-way ANOVA using GraphPad Prism 9.0. Survival analysis was calculated using Kaplan–Meier method and log-rank test. P value 〈 0.05 was considered statistically significant. ^ns P〉 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P<0.001.

Results

rMV-Hu191 synergized with olaparib to enhance antitumor effects without virus progeny alteration in human PDAC cells

The oncolytic effects of rMV-Hu191 in human PDAC cells were quantified with a dose- and time-dependent manner by CCK8 assay (Fig. S1). To determine if rMV-Hu191 and Olaparib synergize to induce PDAC cytotoxicity, cells were treated simultaneously with rMV-Hu191 at multiple MOIs and Olaparib at various concentrations. As shown in Fig. 1A, compared to treatment with rMV-Hu191 or Olaparib alone, their combination significantly augmented the cytotoxicity in PDAC cells. Drug synergism was detected by calculating the combination index values for the combination of rMV-Hu191 with Olaparib in PANC-1 (Bliss score, 14.11) and MIA-PaCa2 (Bliss score, 9.90) (Fig. 1B).

Fig. 1.

Cytotoxic effects and cell cycle arrest of rMV-Hu191 and Olaparib in human PDAC cells. (A) Relative cell viability of PANC-1 and MIA-PaCa2 cells after combinational treatment with rMV-Hu191 at indicated MOIs and Olaparib at different doses for 72 h. * indicated comparison between the combinational and rMV-Hu191 treated groups, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001; ^§ indicated comparison between the combinational and Olaparib treated groups, ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001. (B) Inhibition and Bliss synergy plots for serial dilutions of rMV-Hu191 in combination with Olaparib in PDAC cells. The circle region represented the strongest synergy. Bliss scores >0 indicated drug synergism, while negative values indicated drug antagonism. (C) Cell cycle profiles of PANC-1 (MOI=0.1, Olaparib=5 μM) and MIA-PaCa2 (MOI=1, Olaparib=2.5 μM) cells for 36 h were assessed by flow cytometry. (D) Representative pictures of colony formation assay and statistical analysis diagrams of PDAC cells with rMV-Hu191, Olaparib, or combinational treatments. (E) Virus titer was quantified by TCID 50 method in PDAC cells treated with rMV-Hu191 alone or combination of rMV-Hu191 (MOI=0.1) and Olaparib (5 μM) for 24 h, 48 h, 72 h, 96 h. Experiments were repeated independently for three times. Data presented as mean ± SD. ^nsP > 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 by two-way ANOVA for (A), (C), (E) or one-way ANOVA for (D).

Our data also showed that compared to monotherapy, rMV-Hu191 synergized with Olaparib to significantly arrest the cell cycle in the S phase, producing proliferation inhibition by inhibiting DNA division in PDAC cells (Fig. 1C). Consistently, the colony forming ability was suppressed remarkably by the combination therapy compared with monotherapy (Fig. 1D). And the virus titer results showed that there was no alternation in the virus growth dynamics of rMV-Hu191 and combined with Olaparib (Fig. 1E). In conclusion, combined treatment of rMV-Hu191 and Olaparib could excite synergistical antitumor effects and proliferation inhibition in PDAC cells.

rMV-Hu191 and olaparib cooperated to trigger caspase‐dependent apoptosis in human PDAC cells

In order to explore the mechanism of cell death responsible for this enhanced sensitivity, we examined the levels of apoptosis induction. The expression levels of cleaved-caspase-3 and cleaved-PARP proteins were determined with a concentration-dependent increase in combination group compared to rMV-Hu191 or Olaparib group (Fig. 2A). Moreover, we investigated the altered expressions of apoptosis-associated proteins after incubated with Z-VAD, a pan-caspase inhibitor. As shown in Fig. 2B, Z-VAD prominently decreased the protein expressions of cleaved-caspase-3 and cleaved-PARP in PDAC cells received rMV-Hu191 or combination treatment.

Fig. 2.

Combination of rMV-Hu191 and Olaparib increased caspase‐dependent apoptosis in human PDAC cells. (A) Expression of apoptosis marker proteins in PDAC cells with rMV-Hu191 (MOI=1), Olaparib (5 or 10 µM), or combinational treatments for 48 h. Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. (B) Expression of apoptosis marker proteins in PDAC cells with indicated treatment (MOI=1, Olaparib=10 µM), in the presence or absence of Z-VAD (50 µM) for 48 h. Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. (C) Ratio of apoptotic cells by flow cytometry analysis in PDAC cells with indicated treatment (MOI=1, Olaparib=10 µM, Z-VAD=50 µM) for 48 h. (D) Relative cell viability of PDAC cells with indicated treatment (MOI=1, Olaparib=10 µM, Z-VAD=50 µM) for 72 h. Experiments were repeated independently for three times. Data presented as mean ± SD. ^nsP > 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P<0.001 by one-way ANOVA.

Consistent with the results of apoptosis-associated proteins, the proportion of apoptotic cells detected by flow cytometry was apparently increased in combination therapy compared with monotherapy, and also remarkably decreased with Z-VAD incubation (Fig. 2C). And the representative microscopic images showed that Z-VAD restrained rMV-Hu191 virus-specific syncytium formation in PDAC cells with the above treatments, which implying inhibition of rMV-Hu191 virus amplification (Fig. S2). Similarly, the cell viability (Fig. 2D) quantified by CCK-8 assay and the cell density (Fig. S3) observed under microscope of PDAC cells received rMV-Hu191 or combination treatment were both reversed in the presence of Z-VAD. These data suggested that rMV-Hu191 combined with Olaparib strengthened caspase‐dependently pro-apoptotic capacity than their monotherapy in PDAC cells.

rMV-Hu191 facilitated olaparib-induced DNA damage in human PDAC cells

Of note, Olaparib as a PARP inhibitor had been reported to impair DNA damage repair, which providing a theoretical basis for the synergistic effect of PARP blocking with drugs that cause DNA damage [41]. Phosphorylation of histone H2AX (γ-H2AX) typically occurred in response to early DNA damage and activation of DNA double-strand breaks (DSBs) repair pathways. As shown in Fig. 3A, the γ-H2AX foci formation and the percentage of γ-H2AX positive cells presented a dose-dependent increase in combination treatment compared to rMV-Hu191 or Olaparib treatment alone, respectively. As anticipated, the protein levels of γ-H2AX were consistent with previous observations of immunofluorescence staining (Fig. 3B). To further confirm amplification effect of rMV-Hu191 upregulation on Olaparib-induced DNA damage, comet assay was applied to determine DSBs in PANC-1 cells with different treatments. Either rMV-Hu191 or Olaparib treatment alone had an obviously induction of DSBs, while the combination of both increased tail length more significantly (Fig. 3C). Above all, our data demonstrated that rMV-Hu191 synergized Olaparib to induce DNA damage in PDAC cells.

Fig. 3.

rMV-Hu191 and Olaparib cooperated to induce DNA damage in human PDAC cells. (A) Representative images showing DAPI-stained cell nuclei and the corresponding immune-labeling of γ-H2AX in PDAC cells with rMV-Hu191 (MOI=1), Olaparib (5 or 10 µM), or combinational treatments for 24 h. A cell was identified as γ-H2AX positive only if >10 foci per nucleus. The percentage of γ-H2AX-positive cells were estimated from 10 different fields of view in each cell line. Scale bar, 20 µm. (B) γ-H2AX protein expression in PDAC cells after indicated treatments for 48 h. Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. (C) Representative images of comet assay in PANC-1 cells treated with rMV-Hu191 (MOI=1), Olaparib (10 µM), or combination. Tail length of comet assay were scored in each group with 50 cells. Scale bar, 50 µm. Experiments were repeated independently for three times. Data presented as mean ± SD. *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P<0.001 by one-way ANOVA.

rMV-Hu191 and olaparib synergistically produced excessive ROS and oxidative DNA damage in human PDAC cells

Increased ROS production had been detected in a variety of cancers and approved to play an important role in driving DNA damage and genetic instability. Hence, we reasoned whether the rMV-Hu191 and Olaparib treatment could cause aberrant ROS signaling in PDAC cells. We used DCFH-DA staining to measure intracellular ROS production, with H2O2, a universal cellular ROS inducer, as positive control. As expected, massive ROS generation in PDAC cells was noted in combination treatment with a dose-dependent manner compared to rMV-Hu191 or Olaparib treatment alone (Fig. 4A), which could be reversed by the use of ROS scavenger NAC (Fig. 4B). In addition, γ-H2AX protein expression decreased significantly after scavenging ROS with NAC which mean most of the forementioned DNA damage was rescued (Fig. 4C). These results implied that rMV-Hu191 and Olaparib synergistically induced oxidative DNA damage in human PDAC cells by producing excessive ROS.

Fig. 4.

rMV-Hu191 and Olaparib synergistically produced excessive ROS and oxidative DNA damage in human PDAC cells (A) PDAC cells with indicated treatments (MOI=1, Olaparib=5 or 10 µM, 36 h) were incubated with DCFH-DA (10 µM) for 40 min in dark. Cellular ROS levels were determined using flow cytometry. All cell lines were treated with H2O2 (0.1 mg/ml) for 30 min as positive control. (B) Cellular ROS levels were determined of PDAC cells after indicated treatments (MOI=1, Olaparib=10 µM, 36 h), with or without NAC (15 mM, pre-treated 2 h and then incubated for 36 h). (C) Expression of γ-H2AX protein after the same treatment as above. Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. Experiments were repeated independently for three times. Data presented as mean ± SD. ^nsP > 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 by one-way ANOVA.

ROS was required for viral replication and rMV-Hu191- and combination- induced apoptosis

Because ROS inhibition decreased rMV-Hu191- and combination- induced oxidative DNA damage in human PDAC cells, we further determined whether ROS was involved in the generation of rMV-Hu191 replication and membrane fusion. As shown in Fig. 5A and B, NAC (a ROS scavenger) observably suppressed virus titer of rMV-Hu191 quantified using TCID50 method and MV-N protein detected by Western blotting. Consistent with the above findings, NAC also significantly blocked rMV-Hu191 virus-specific syncytium formation in PDAC cells with the above treatments (Fig. S4). To test the association of the excessive ROS and oxidative DNA damage in the rMV-Hu191- and combination- induced apoptosis, we investigated the altered apoptosis production after incubated with NAC. As we suspected, NAC significantly inhibited the expressions of cleaved-caspase-3 and cleaved-PARP proteins (Fig. 5C) and the proportion of apoptotic cells (Fig. 5D) in PDAC cells received rMV-Hu191 or combination treatment. We subsequently examined the impacts of ROS inhibition on rMV-Hu191- and combination- generated antitumor effects. Compared with the mock control, NAC significantly restrained rMV-Hu191- and combination- generated cytotoxicity and reversed the cell density, which remained resistant (Fig. S5). Taken together, these findings indicated that ROS played a vital role in viral replication and rMV-Hu191- and combination- induced apoptosis.

Fig. 5.

Ros was required for viral replication and rMV-Hu191- and combination- induced apoptosis. (A) Virus titer was quantified by TCID 50 method in human PDAC cells after rMV-Hu191 (MOI=1) treatment for indicated times, with or without NAC (15 mM). Expression of MV-N protein (B) and apoptosis marker proteins (C) in PDAC cells with indicated treatment (MOI=1, Olaparib=10 µM) for 36 h, in the presence or absence of NAC (15 mM). Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. (D) Ratio of apoptotic cells by flow cytometry analysis in PDAC cells with indicated treatment (MOI=1, Olaparib=10 µM, NAC=15 mM) for 36 h. (E) Proposed mechanism for the role of ROS, DNA damage and apoptosis of rMV-Hu191- and combination-induced antitumor effects in human PDAC cells. Experiments were repeated independently for three times. Data presented as mean ± SD. ^nsP > 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 by two-way ANOVA for (A) or one-way ANOVA for (B), (C), (D).

rMV-Hu191 synergistically inhibited growth of pancreatic cancer xenografts in combination with olaparib without toxicity

To evaluate the anticancer activity of rMV-Hu191 and Olaparib on pancreatic tumor growth in vivo, mice with subcutaneous MIA-PaCa2 tumors were intratumorally injected with rMV-Hu191, intraperitoneally injected with Olaparib or a combination of the two. Compared with monotherapy, rMV-Hu191 plus Olaparib treatment significantly restricted tumor growth (Fig. 6A and B). Meanwhile, there were no significant difference in body weight among these groups (Fig. 6C and D). Administration of combination treatment resulted significantly prolonged survival compared with rMV-Hu191 (median survival = 46 days) and Olaparib (median survival = 32 days) treatment alone (Fig. 6E).

Fig. 6.

Combination therapy of rMV-Hu191 and Olaparib inhibited growth of pancreatic cancer xenografts without toxicity. (A) Tumor volumes and (C) body weights of human PDAC xenografts were measured every 3–4 days (n = 7). Distributions of tumor volumes (B) and body weights (D) on day 21 post implantation. (E) The survival rate of tumor-bearing mice was supervised by Kaplan-Meier analysis and statistical difference was administrated with log-rank test (P < 0.001). (F) Protein expressions of γ-H2AX, pro-caspase-3 and cleaved-caspase-3 extracted from tumor tissues. Band intensity quantification was relative to the expression of β-actin and then relative to mock group by ImageJ. (G) Representative images of HE staining, IHC of γ-H2AX and cleaved-caspase-3, TUNEL assays in tumor tissues. The quantifications of positive area (%) of γ-H2AX, cleaved-caspase-3 and TUNEL were estimated from 3 different fields of view. Scale bar, 50 µm. (H) Evaluation of liver and kidney functions (ALT, AST, ALB, GLB, BUN, CREA) in mice. Experiments were repeated independently for three times. Data presented as mean ± SD. ^nsP > 0.05, *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 by one-way ANOVA for (B), (D), (F), (G), (H), two-way ANOVA for (A), (C) or log-rank test for (E).

At the endpoint, the levels of DNA damage and apoptosis were further examined in the subcutaneous xenograft tumors by western blotting, H&E staining, IHC staining and TUNEL staining. As shown in Fig. 6F and G, we observed the larger variation of center necrosis and apoptosis areas, and the higher levels of γ-H2AX and cleaved-caspase-3 proteins in the combined treatment group compared with the single treatment groups. And the original figures of Western Blot design of this study were shown in Fig. S6. We further evaluated the liver and kidney functions of mice angular vein blood in each group by measuring the levels of ALT, AST, ALB, GLB, BUN, and CREA, and demonstrated that there was no statistical difference between treatment and control groups (Fig. 6H). Collectively, these data indicated that the combinatorial therapeutic strategy of pancreatic cancer was effective and tolerable with no side-effects in vivo, and was in accordance with the mechanism in vitro.

Discussion

Data in this manuscript first documented the novel observations that the intrinsic resistance of HR-proficient PDAC cells to PARP inhibitor monotherapy could be improved by combination with oncolytic virus rMV-Hu191, manifested as inhibiting growth and inducing death in human PDAC cells. Meanwhile, the results in human PDAC xenografts showed that combined treatment with rMV-Hu191, Olaparib has generated a profound antitumor effect in vivo with minimal toxicity. The central findings in the present study were that rMV-Hu191 collaborated with Olaparib to induce ROS formation, leading to ROS-mediated DNA damage accompanied with γ-H2AX (Ser139) phosphorylation and mediating the increased activation of ROS-induced caspase-dependently apoptosis accompanied with PARP and caspase-3 cleavage in human PDAC cells (Fig. 5E). The in vivo data also supported this conclusion.

The sensitivity of PARP inhibitors in tumor cells is usually dependent on genetic alterations in DDR genes. Many literatures have demonstrated that PARP inhibitors could significantly prolong the survival of patients with multiple malignancies, including ovarian cancer, breast cancer and pancreatic cancer, especially with HR deficiency, such as BRCA1/2 mutation [42,43]. And deficiency in additional genes implicated in HR deficiency (such as ATM, CHEK2, BARD1, BRIP1, RAD51C, and DNA-PK) are considered as BRCAness, which also confers sensitivity to PARP inhibitors. At that point, the inhibition of PARP could even represent the therapeutic strategy of sporadic cancers with BRCA-like properties, known as BRCAness [44]. At the end of 2019, the Pancreas Cancer Olaparib Ongoing (POLO) trial was approved by FDA to use Olaparib as a maintenance therapy to improve progression-free survival in metastatic PDAC patients with germline BRCA1/2 mutations [10]. However, less than 10 % of PDAC patients carry BRCA mutations [45]. Despite complex rearrangement patterns and mitotic errors are universal characteristics of PDAC, most pancreatic cancer patients have intact DDR pathway that do not benefit from PARP inhibitors.

Recent literatures also document encouraging results for the potential utility of PARP inhibitors in combination with chemotherapy having complementary mechanisms of cytotoxicity [46]. As some preclinical studies have confirmed that tumor cell growth inhibition can be achieved using high-dose PARP inhibitors combined with relatively low doses of chemotherapy [47]. Therefore, in cancer cells without identified DDR deficiencies, the synthetic lethality of PARP inhibitors can be generated by combined with pharmacological inhibition of DDR [48]. Multiagent cytotoxic therapies that cooperate PARP inhibitors with treatment modalities such as drugs (ATR inhibitors [49], SIK2 inhibitors [50], CHK1 inhibitors [51], etc.) and genomic alterations [52] that cause DDR deficiency are being extensively investigated for a variety of malignant tumors. It has been demonstrated that oncolytic viruses, as novel targeted therapeutic agents (NDV [22], BoHV-1 [53], Zika virus [54], etc.), developed effective malignancies therapeutic effects by inducing DNA damage and activating DDR pathways. Carmela Passaro et al. have previously shown that the oncolytic adenovirus dl922–947 induced extensive PARP activation and DNA damage, and also confirmed that PARP inhibition exerted a synergistic antitumoral effect of the PARP inhibitor Olaparib in association with dl922–947 [55]. From our data, we explored for the first time that rMV-Hu191 in combination with PARP inhibitor Olaparib showed a synergistic time- and dose-dependent growth inhibition compared to monotherapy, together with arresting the cell cycle of PDAC cells in the S phase and inhibiting the cell's monoclonal ability. However, whether combinational drugs would hinder viral replication of oncolytic measles virus lacked consistency [28,56]. In this study, we found that there was almost no alternation in viral growth dynamics of rMV-Hu191 when combined with Olaparib.

Previous studies have demonstrated that PARP primarily governed the detection of DNA damage and the initiation of DDR [57], and the cleavage of PARP has been considered as the evidence of caspase activation and apoptosis [58]. γ-H2AX, a sensitive marker of DSBs, is the first step in recruiting and locating DDR proteins. DNA damage caused by HSV and KSHV has been clarified to be associated with phosphorylation of H2AX (Ser139) [59,60]. Consistent with these reports, rMV-Hu191 cooperated Olaparib to generate more significant apoptosis, as shown by increased cleavage of PARP and caspase-3 and augmented apoptotic cells detected by flow cytometry. At the same time, rMV-Hu191 and Olaparib synergistically aggravated DNA damage in PDAC cells, which was manifested by accumulated γ-H2AX foci formation, increased γ-H2AX phosphorylation, and lengthened DNA tails measured by comet assay.

Excessive oxidative stress derived from ROS accumulation, a group of short-lived, highly reactive, oxygen-containing molecules, deregulates the antioxidative defense system, which is closely associated with various diseases [61,62], especially cancers [63]. Increased ROS production has been detected in various cancers and has been shown to have several roles. For example, they can continuously attack the structure and function of DNA and affect the DDR to drive DNA damage and genetic instability [64]. As we know, oncolytic viruses as environmental factors can enhance oxidative stress in a variety of cancer cells by stimulating ROS production to impeding tumor cell growth and promoting oxidative DNA damage [65]. Consistent with these reports, we found that rMV-Hu191 combined with Olaparib produced more significant oxidative stress than monotherapy, accompanied by a concentration-dependent increase of ROS production in PDAC cells as measured by DCFH-DA assay, which was reversed by NAC treatment, a ROS scavenger. Our results demonstrated, for the first time, that rMV-Hu191 cooperated Olaparib to produce the phosphorylation of γ-H2AX (Ser139) which was inhibited with NAC treatment, suggesting that rMV-Hu191 combined with Olaparib synergistically induced oxidative DNA damage in PDAC cells through ROS generation.

When ROS accumulation exceeds the tipping point, the carcinogenic effects of ROS are shifted to antitumor effects via the generation of regulated cell death (RCD) programs (mainly including apoptosis, necroptosis and ferroptosis), which providing ROS manipulation as a potential target for cancer therapies [66]. The role of ROS in modulating apoptosis is well recognized. ROS stimulate the extrinsic [67] and intrinsic [68] apoptotic pathway to form apoptosome, subsequently activate the caspase signaling cascade and induce apoptosis. A noteworthy study have demonstrated that oncolytic NDV-induced apoptosis of cervical cancer cell is mediated by ROS production [69]. We found that NAC suppressed rMV-Hu191 replication in a time-dependent manner. This finding was consistent with the replication characteristic of BoHV-1 [65], suggesting that ROS was required for rMV-Hu191 replication. Corresponding apoptosis-related proteins and apoptotic cells were also remarkably reversed in the presence of NAC. Similarly, the rMV-Hu191- and combined with Olaparib- induced cytotoxicity in PDAC cells was also diminished with NAC treatment. These results further indicated that rMV-Hu191 potentiated the antitumoral effects of Olaparib through ROS-dependent apoptosis mechanism.

Surprisingly, in our in vivo studies, externally measuring tumor volume of pancreatic cancer xenografts in nude mice indicated that the combined drug treatment of rMV-Hu191 and Olaparib caused a significant decrease of tumor volume, compared to the other treatment groups, indicating treatment response. Upon further investigation, TUNEL assay and H&E staining revealed that rMV-Hu191 cooperate Olaparib to induce larger center apoptosis and necrosis areas, and had a cavity in the center of the tumors compared to the individual drug treatments. And IHC and Western blotting examination showed that the combined treatment group had higher γ-H2AX and cleaved-caspase-3 expression than the other treatment groups, indicating that the two drugs synergized in generating DNA damage and apoptosis in vivo, which was consistent with in vitro results. An important criterion to evaluate the clinical potential of a multiagent cytotoxic therapy is the toxicity and side effects of the combination therapy. Thus, we further validated that there were no alternations in body weights and liver and kidney functions of pancreatic cancer xenografts in nude mice between treatment and control groups.

As outlined above, our findings firstly suggested that combination treatment with rMV-Hu191 and Olaparib had a profound and synergistic therapeutic effect against human PDAC both in vitro and in vivo with minimal toxicity. Mechanistically, the data indicated that the observed synergy and efficacy depended on the oxidative DNA damage and ROS-dependent apoptosis generating by rMV-Hu191 combined with Olaparib in human pancreatic cancer. In conclusion, our goal is to extend PARP inhibitors-based therapy to PDAC patients, regardless of BRCA mutation status, by combining PARP inhibitors with other novel targeted therapeutic agents.

CRediT authorship contribution statement

Chu-di Zhang: Conceptualization, Methodology, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Li-hong Jiang: Methodology. Xue Zhou: Data curation. Yong-ping He: Formal analysis. Ye Liu: Data curation. Dong-ming Zhou: Methodology. Yao Lv: Formal analysis. Ben-qing Wu: Writing – review & editing. Zheng-yan Zhao: Conceptualization, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.