Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

Fengjie Wang; Xianbing Chen; Depei Yuan; Yongfen Yi; Yi Luo

doi:10.1371/journal.pone.0225450

. 2019 Nov 26;14(11):e0225450. doi: 10.1371/journal.pone.0225450

Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

Fengjie Wang ^1,², Xianbing Chen ², Depei Yuan ², Yongfen Yi ¹, Yi Luo ^1,^3,^*

Editor: Yi-Hsien Hsieh⁴

PMCID: PMC6879129 PMID: 31770410

Abstract

Golgi reassembly and stacking protein 65 (GRASP65), which has been involved in cancer progression, is associated with tumor growth and cell apoptosis. Dihydromyricetin (DHM) has demonstrated antitumor activity in different types of human cancers. However, the pharmacological effects of DHM on ovarian cancer (OC) and the molecular mechanisms that underlie these effects are largely unknown. The present study showed that DHM reduced cell migration and invasion in a concentration- and time-dependent manner and induced cell apoptosis primarily through upregulation of Cleaved-caspase-3 and the Bax/Bcl-2 ratio in OCs. To further clarify the cancer therapeutic target, we assessed the effect of DHM on the expression of GRASP65, which is overexpressed in human ovarian cancer tissues. DHM activated caspase-3 and decreased GRASP65 expression to promote cell apoptosis, implying that downregulation of GRASP65 was related to DHM-induced cell apoptosis. Additionally, the knockdown of GRASP65 by siRNA resulted in increased apoptosis after DHM treatment, while western blot and flow cytometry analysis demonstrated that overexpression of GRASP65 attenuated DHM-mediated apoptosis. In addition, the JNK/ERK pathway may be involved in DHM-mediated caspase-3 activation and GRASP65 downregulation. Taken together, these findings provide novel evidence of the anti-cancer properties of DHM in OCs, indicating that DHM is a potential therapeutic agent for ovarian cancer through the inhibition of GRASP65 expression and the regulation of JNK/ERK pathway.

Introduction

Electron microscopy has been used to demonstrate Golgi fragmentation (GF) in tumor cells [1], and we have only just begun to understand the significance of GF in tumor biology. GF serves as a catalyst for the cell signaling pathways that drive cancer progression and metastasis. However, the causal relationship between GF and cancer pathogenesis remains largely unexplored. For example, swainsonine, an inhibitor of Golgi alpha-mannosidase II, has been shown to have antitumor activity in gastric carcinoma [2]. Another anti-Golgi agent, Brefeldin A, showed antiproliferative effects in vitro and inhibition of tumor growth in vivo [3]. Golgi reassembly and stacking proteins (GRASPs) are Golgi membrane proteins involved in cell migration, division, and apoptosis. Specifically, GRASP65, a target of polo-like kinases (PLK1) and Cdc2 during mitosis [4,5], mediates Golgi morphological changes to fulfill physiological functions [6–8]. In addition, the upregulation of Golgi proteins has been observed in many types of tumors, including ovarian cancer (OC). Golgi phosphoprotein3L (GOLPH3L) was overexpressed in epithelial ovarian cancer (EOC) tissues and cell lines [9] and associated with poor prognosis of patients with EOC [10]. GOLPH3 may promote EMT progression through the activation of Wnt/β-catenin pathway and act as a novel and independent prognostic factor of EOC [11]. Furthermore, silencing GM130 decreased angiogenesis and cell invasion in vitro and in a lung cancer mouse model, suggesting that it may be a potential therapeutic target for lung cancer [12]. Restoration of compact Golgi morphology in advanced prostate cancer may increase the susceptibility to Galectin-1-induced apoptosis [13], strengthening the notion of the “oncological Golgi” and its role in cancer progression and metastasis [1]. Therefore, targeting the Golgi proteins may be a potential therapeutic intervention for multiple cancers [14].

OC is one of the most common gynecological malignancies with high rates of metastasis and disease relapse worldwide. The invasion and progression of OC cells are presumed to be a multistep process involving multiple genetic changes. Consequently, numerous studies have focused on the identification of specific molecular markers that may serve as reliable prognostic biomarkers for ovarian cancer. Additionally, the current standard of care treatment for patients with ovarian cancer is surgery coupled with platinum and/or Taxane-based chemotherapy. While most patients are initially responsive to chemotherapy, the 5-year survival rate of OC patients is approximately 15–30% [15]. Therefore, there is an urgent need to improve the techniques employed for early disease detection, and to identify effective therapies to improve clinical outcomes for OC patients.

Recently, researchers have turned their attention to natural active compounds extracted from medicinal plants for the treatment of cancer patients [16]. Most natural compounds have shown cytotoxicity only in cancerous cells and are therefore potential therapeutic agents for future clinical development [17]. In addition, several studies have demonstrated that these components can substantially inhibit tumor formation and induce apoptosis [18,19]. Dihydromyricetin (DHM), a 2,3-dihydroflavonol compound, is the main bioactive component extracted from Ampelopsis grossedentata [20] and has attracted considerable attention in cancer research for its antitumor effects [21–23]. DHM has been shown to be an effective anticancer agent in various cancers and is also considered to have great antitumor potential for the treatment of OC [24]. However, the mechanism underlying the antitumor effect of DHM needs to be investigated.

In response to stress, the transcription of Golgi-associated genes can be upregulated to restore homeostasis or induce apoptosis, which gave rise to the term Golgi stress response (GSR) [25,26]. The role of GSR and cell apoptosis in chemotherapy can be quite complex [27] and their connection has made them an intriguing target that may improve anti-cancer treatment. Furthermore, morphological studies have shown that the Golgi complex is fragmented during apoptosis [28], and GF in apoptotic cells may be attributed to GRASP65 cleavage [29]. GRASP65 is phosphorylated by Cdc2 and PLK-1 during cell mitosis, which leads to GRASP65 deoligomerization and then Golgi unstacking [5,30]. Additionally, as a potential small molecular inhibitor of PLK-1, DHM may prevent cancer progression by inhibiting PLK-1 enzymes [31]. Therefore, we hypothesized that DHM possesses anti-tumor activity by regulating GRASP65 function. We also investigated the mechanisms and effects of DHM on OCs in order to provide preliminary evidence for future clinical applications.

Materials and methods

Reagents

Dihydromyricetin (CAS No. 27200-12-0, Bellancom) was ordered from Beijing Universal Materials Co., Ltd. (Beijing, China), with purity >98%, as detected by high performance liquid chromatography. DHM was dissolved in 100% dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution and was stored at −20°C. DHM solutions used in cell cultures were freshly prepared daily and the final concentration of DMSO did not exceed 0.1% throughout the study.

Apoptotic cells were quantified using an Annexin V-FITC/PI cell apoptosis detection kit from Becton Dickinson and Company (Franklin Lakes, NJ, USA) and monitored using flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA).

JNK inhibitor SP600125, ERK inhibitor U0126, and Caspase-3 inhibitor Ac-DEVD-CHO were purchased from Beyotime (Shanghai, China) and dissolved in DMSO at concentrations of 20, 10 and 30 μM, respectively.

Antibodies for p-JNK/JNK, p-ERK/ERK, and GRASP65 were bought from Abcam (Cambridge, MA, USA), antibody for Actin from Beyotime (Shanghai, China), and antibodies for p-p38/p38MAPK, cleaved-caspase-3, Bcl-2, and Bax from Cell Signaling Technology (Danvers, MA, USA).

Cell culture

The human ovarian cancer SKOV3 cell line was purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). The A2780 cell line was obtained from the Molecular Medicine and Cancer Research Center of Chongqing (Chongqing, China) and cultured in DMEM (Hyclone, Logan, Utah, USA), and supplemented with 10% FBS (Gibco, Invitrogen Life Technologies, Carlsbad, USA), 100 unit/mL penicillin, and 100 mg/mL streptomycin (Beyotime, Shanghai, China) in a humidified chamber containing 5% CO₂ at 37°C.

In vitro cell viability assay (CCK8)

SKOV3 and A2780 cells were seeded in 96-well microtiter plates (Corning, NY, USA) with 1×10⁴ cells per well and pretreated with various concentrations of DHM for 24 h and 48 h to select the most effective concentration and time point for the assessment of cell viability using the Cell Counting Kit-8 (Beyotime, Shanghai, China) following the manufacturer’s recommendations. Six reduplicate wells were used for each treatment and the experiments were performed three times. At each time point, the absorbance (A) was measured at 450 nm using a microplate reader (BioTek synergy HT, VT, USA). The concentration required to inhibit cell growth by 50% (IC₅₀, half maximal inhibitory concentration) was calculated using GraphPad Prism (San Diego, CA, USA).

The percentage of viable cells was calculated as follows:

Cell viability (%) = (A_{treated} / A_{control}) \times 100 % .

Wound healing assay

Wound healing assay was performed and the closure of the scratched area was calculated as previously described [32]. Cells were seeded at a high density (1 × 10⁶ cells/mL) in each well of a 6-well culture plate and allowed to adhere. Confluent cells were scratched with a 200 μL pipette tip and treated with the various concentrations of DHM diluted by serum-free DMEM medium and were cultured for the indicated time points. Cells were then photographed with a digital camera (IX70, Olympus) and the wound width was measured using an image analysis software (ImagePro Premier). Three fields were randomly selected from each wound.

Migration/Invasion assay

Cell migration assay was performed and determined using Corning Transwell insert chambers (Cat No. 3422, Corning, NY, USA). Thaw the Matrigel^® Matrix (Becton Dickinson, Oxford, UK; Cat No. 354234) overnight at 4°C and mix Matrigel with serum-free cold DMEM. 0.1ml of the diluted Matrigel was pre-coated directly onto each 24-well Transwell insert at 37°C for at least 1h for invasion assay [33].

Apoptosis assay

Apoptotic cells were assessed using an Annexin V-FITC/PI kit (BD Pharmingen, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Apoptotic cells were detected by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo cell analysis software (FlowJo, Ashland, OR, USA).

Caspase-3/9 activity assay

Caspase-3/9 activity was measured using a commercial kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. After treatment with different concentrations of DHM, cells were lysed and then incubated with the caspase reagent and its substrates, Ac-DEVD-pNA (caspase-3) and Ac-LEHD-pNA (caspase-9), for 1–2 h at 37°C. Absorbance (A) at 405 nm was measured using a microplate reader. The caspase activity (Unit) = A_treated / A_control × 100%.

Cellular immunofluorescence (IF)

Cells were seeded on slides in a 24-well plate and allowed to attach for 24 h. The cells were subsequently treated with different concentrations of DHM. For IF analysis, the samples were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 10 min. After being blocked with 10% goat serum albumin for 60 min at 37°C, the slides were incubated with primary antibodies against Caspase-3 (1:500, Rabbit mAb), Actin (1:400, mouse mAb) and GRASP65 (1:300, Rabbit mAb) overnight at 4 °C. The following day, the slides were washed three times with PBS and then incubated with FITC-labeled Goat anti-mouse or Cy3-labeled Goat anti-rabbit secondary antibodies for 1 h at 37°C before being labelled with DAPI for 1 min at room temperature. Finally, three fields per slide were randomly selected for observation under a fluorescence microscope (Olympus Inc., Tokyo, Japan). Staining intensities were measured by observers blinded to the experimental groups using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).

Gene knockdown using siRNA and transient transfection

Two GRASP65 siRNAs (siRNA1: GGUUGGUUCGGACCAGAUUTT; siRNA2: GGAACCAUCUUCACCUGCUTT) and one overexpression plasmid (NCBI Reference Sequences of GRASP65/GORASP1(human): NM_031899.3), as well as the corresponding negative control plasmids, were all designed and synthesized by Shanghai GenePharma Co., Ltd. Cells were transiently transfected with siRNAs or plasmid using the Lipofectamine^® 2000 Reagent kit (Invitrogen; Thermo Fisher Scientific, Inc., MA, USA) according to the manufacturer’s protocol.

Following a 6 h transfection, the cell culture solution was changed to a normal medium. After transfection for 24–48 h, cells were processed for further analysis and subsequent experiments, and non-transfected cells served as blank controls. Transfection efficiency was verified using western blot analysis.

Preparation of total cell extracts and western blot analysis

The cells were lysed with lysis buffer containing protease inhibitors. The soluble cell lysates were collected after centrifugation at 14000 g for 15 min. Equal amounts of protein (20–30μg) were subjected to 10 or 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Belford, MA). After blocking with 5% skim milk in PBS containing 0.1% Tween20 (TBST), membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBST three times for 10 min, the membranes were incubated at room temperature for 2 h with secondary antibodies. An enhanced chemiluminescent substrate (ECL, Thermo fisher, MA, USA) was added to the membranes, which were photographed using a protein analysis system (Tanon 5200, Shanghai, China).

Statistical analysis

All data are presented as the mean ± standard deviation (SD). The statistical significance of differences between groups was analyzed by one-way Analysis of Variance (ANOVA) followed by Dunnett’s or Tukey’s post hoc tests using SPSS 17.0 software (SPPS Software, Inc., Chicago, IL, USA). A value of P<0.05 was considered statistically significant.

Results

DHM reduced cell migration and invasion in SKOV3 and A2780 cells

The inhibitory effects of DHM on A2780 and SKOV3 ovarian cancer cells were assessed using CCK8, wound healing, and Transwell assays. A previous study showed that DHM had no significant cytotoxicity in human ovarian surface epithelial cells [24]. Firstly, the cell viability was detected by CCK-8 assay after treatment with DHM. DHM treatment significantly decreased the cell viability of SKOV3 and A2780 cells in a time- and dose-dependent manner. The IC₅₀ values of DHM for SKOV3 and A2780 cells were 213.4 and 157.2 μM, respectively, after DHM treatment for 24 h. The IC₅₀ values were 132.3 and 98.2 μM, respectively, after DHM treatment for 48 h. We selected 120 and 80 μM of DHM for 48 h in SKOV3 and A2780 cells based on the determined IC₅₀ values for subsequent studies, respectively.

Next, to examine whether DHM inhibited the migration of ovarian cancer cells, wound healing assays were performed using non-cytotoxic concentrations of DHM. The closure of the wound in the DHM-treated group was calculated and normalized to that of the control. As shown in Fig 1A and 1B, 40 μM DHM suppressed approximately 50% of wound closure in A2780 cells, and significantly reduced the closure of the wound after DHM treatment at both 80 μM and 120 μM in SKOV3 cells.

Fig 1 — **A/B**. Wound healing and Transwell assay were conducted in. SKOV3 cells (A) treated with DMSO (control), 80, and 120 μM of DHM, and A2780 cells (B) with DMSO (control), 40, and 80 μM for 48 h. Each experiment was repeated at least three times. * p < 0.05, **p < 0.01 vs the DMSO (control) group.

Additionally, Transwell assays were carried out to confirm the inhibitory effect of DHM on cell migration. The number of migratory DHM-treated cells was calculated and then normalized to that of the control cells. As shown in Fig 1A and 1B, DHM dose-dependently inhibited the migration of both ovarian cancer cell lines. Transwell assays were also performed to explore the effect of DHM on the invasion of cancer cells. Our results clearly showed that DHM significantly attenuated the invasion of SKOV3 and A2780 cells.

These results suggest that DHM could decrease cell viability and reduce the migration and invasion of different ovarian cancer cells at non-cytotoxic doses, implying that DHM may be a potent therapeutic agent for ovarian cancer.

DHM induced cell apoptosis in A2780 and SKOV3 cells

The apoptotic process is executed by a member of the highly conserved caspases, and modulation of the mechanisms of caspase activation and suppression is a critical molecular target in chemoprevention, since these processes lead to apoptosis [34]. To identify the mechanisms, cell nuclei were evaluated after DHM treatment by DAPI and Caspase-3 (Red) and Actin (Green) double staining was observed using fluorescence microscopy. Activation of Caspase-3/9 was also detected.

A2780 and SKOV3 cells were treated with 80 and 120 μM DHM, respectively, for 48 h to test the effects of DHM on cell apoptosis. DHM treatment resulted in an increase in nuclear disassembly, chromatin condensation, and the expression of Caspase-3 using IF double staining (Fig 2A), and the activation of caspases (Caspase-3 and -9) (Fig 2B), indicating apoptotic cell death in SKOV3 and A2780 cells.

Fig 2 — A. SKOV3 cells were treated with 120 μM DHM and A2780 with 80 μM for 48 h. Caspase-3 (Red) and Actin (Green) double staining were characterized by IF staining and observed under fluorescent microscopy. B. Caspase-3/9 activity was determined after exposure to DHM for 48 h. C. Expression of Bcl-2, cleaved-caspase-3, and Bax was determined by western blot analysis. Scale bar equals 50 μm. Each experiment was repeated at least three times. * p < 0.05, **p < 0.01 vs the control group.

Upon further investigation, we performed western blot analysis to evaluate the expression of apoptosis-related proteins following DHM treatment. As shown in Fig 2C, exposure to 80 μM DHM for 48 h resulted in an increase in Bax and cleaved-caspase-3 protein levels, and a decrease in the expression of Bcl-2 in A2780 cells. Similar DHM-induced apoptotic effects were also observed in SKOV3 cells (Fig 2C).

These results suggest that DHM can induce apoptosis in ovarian cancer cells.

DHM downregulated GRASP65 expression in SKOV3 and A2780 cells

We examined the expression of GRASP65 to determine its role in the inhibitory effect of DHM in ovarian cancer cells. SKOV3 and A2780 cells were treated with DHM for 48 h, and the expression of GRASP65 was detected by IF staining and western blot. In comparison to the control group, DHM induced cell apoptosis as shown in Fig 2, followed by the downregulation of GRASP65 expression in a concentration-dependent manner in Fig 3C and 3D. This finding was further supported by IF analysis (Fig 3A and 3B), indicating that DHM might downregulate the expression of GRASP65 during DHM-induced apoptosis.

Fig 3 — A. IF analysis of GRASP65 expression in DHM-treated SKOV3 cells. B. IF analysis of GRASP65 expression in DHM-treated A2780 cells. C. Western blot analysis of GRASP65 expression in DHM-treated SKOV3 cells. D. Western blot analysis of GRASP65 expression in DHM-treated A2780 cells. Scale bar equals 50 μm. Each experiment was repeated at least three times. * p < 0.05, **p < 0.01 vs the control group.

DHM-induced caspase-3 activation was crucial for suppression of GRASP65 expression in SKOV3 and A2780 cells

Morphological studies have shown that the Golgi complex is fragmented and GRASP65 is cleaved by caspase-3 during apoptosis [29]. Additionally, the expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited the breakdown of the Golgi ribbon in apoptotic cells [29]. To further explore the significance of caspase-3 activation and the relationship between caspase-3 activation and GRASP65 suppression in DHM-induced cell apoptosis, OCs were pre-treated with a specific caspase-3 inhibitor, Ac-DEVD-CHO, for 30 min to suppress the activity of caspase-3 to evaluate the contribution of caspase-3 in the effects of DHM. As shown in Fig 4A, Ac-DEVD-CHO dramatically attenuated DHM-induced increases in cleaved caspase-3, subsequently resulting in an increase in GRASP65 in SKOV3 and A2780 cells. This finding was further supported by flow cytometry analysis (Fig 4B).

Fig 4 — A. Western blot analysis showed that the caspase-3 inhibitor Ac-DEVD-CHO dramatically attenuated DHM-induced effects in SKOV3 and A2780 cells. B. SKOV3 and A2780 cells were treated with 120 and 80 μM DHM for 48 h with or without pretreatment with Ac-DEVD-CHO for 30 min and analyzed by flow cytometry after Annexin V-FITC/PI staining. Annexin-V-FITC−/PI− populations in Q4 were living cells, while Annexin-V-FITC+/PI−cells in Q3 were undergoing necrosis, and Annexin-V-FITC+/PI+ cells in Q2 were either in the end stage of apoptosis or were already dead. The total populations in Q2 and Q3 were considered as apoptotic cells. Quantitative analysis of total apoptotic cells is shown. Each experiment was repeated at least three times. * p < 0.05, **p < 0.01 vs the control group. ^#p < 0.05, ^##p < 0.01 vs the DHM group.

The present results revealed that activated caspase-3 was crucial for suppression of GRASP65 in DHM-induced cell apoptosis. Therefore, we speculated that suppression of GRASP65 might be related to caspase-3 cleavage during DHM-mediated cell apoptosis.

Effects of GRASP65 on DHM-induced cell apoptosis in A2780 cells

Previous studies have reported that GRASP65 is involved in cancer cell migration [35], polarity, and apoptosis [36]. To further determine whether DHM triggered cell apoptosis by decreasing GRASP65 expression, we silenced or overexpressed GRASP65 in A2780 cells to determine the role of GRASP65 in DHM-induced cell apoptosis. The efficacy of transfection was confirmed by western blot analysis as shown in Fig 5A. Therefore, we selected the most effective siRNA2 to silence and OE plasmid to overexpress GRASP65 in the following experiments.

As shown in Fig 5B, cells transfected with siGRASP65 showed a lower expression of GRASP65 and a higher level of cleaved caspase-3 than those in the control group, implying that GRASP65 depletion might lead to apoptosis in A2780 cells. Meanwhile, the expression of GRASP65 in cells transfected with siGRASP65 was lower than that in the control group when treated with 80 μM of DHM and the expression of cleaved caspase-3 was higher. The results of Annexin V-FITC/PI dual staining showed that there was more apoptosis in cells transfected with GRASP65 siRNA in comparison to controls when treated with 80 μM of DHM (Fig 5B). The finding suggests that combination of DHM and GRASP65 depletion could further promote apoptosis.

On the contrary, the level of cleaved caspase-3 decreased in the DHM-treated OE-GRASP65 group in comparison to the DHM-treated group (Fig 5C). Interestingly, the percentage of apoptotic cells was reduced to 8.59% in GRASP65-overexpressing cells after DHM treatment for 48 h, which was 5.94% lower than that observed in DHM group (Fig 5C). The above result revealed that overexpression of GRASP65 attenuated DHM-mediated apoptosis.

Effects of GRASP65 on DHM-mediated cell viability and migration in A2780 cells

Additionally, we also tested the effects of GRASP65 on cell viability and migration by CCK8 assay and wound healing assay, respectively, using GRASP65 siRNA and OE-GRASP65 transfection in A2780 cells. As shown in Fig 6A, the cell viability of A2780 cells transfected with GRASP65 siRNA was lower than that in the control group with or without treatment with 80 μM of DHM. In comparison to the DHM-treated group, DHM suppressed wound closure in A2780 cells after transfected with GRASP65 siRNA (Fig 6B).

On the contrary, overexpression of GRASP65 could improve DHM-induced inhibition of cell viability (Fig 6A) and induce cell migration when treated with 80 μM of DHM (Fig 6B).

These results suggested that GRASP65 depletion using siRNA combined with DHM treatment had an additive effect on DHM-induced inhibition of cell viability and cell migration.

JNK/ERK pathway participated in DHM-mediated apoptosis in A2780 cells

Previous studies have shown that the MAPK signaling pathway plays an important role in chemotherapy-induced apoptosis [37]. Three major MAPKs, namely ERK, JNK, and p38 MAPK, are activated by various stresses, including reactive oxygen species (ROS) [38] and can influence apoptosis either positively or negatively. Therefore, we first determined whether MAPKs were activated in DHM-treated A2780 cells. Western blot analysis showed that DHM induced activation of JNK and ERK in a dose-dependent manner, but no obvious changes in p38 level (Fig 7A).

We also found that there was no significant difference in p-JNK/ERK levels between the siGRASP65 group and the control group (Fig 7B), additionally, no difference between the DHM group and the DHM+siGRASP65 group (Fig 7B).

These results suggested that ERK/JNK signaling pathway involved in DHM-mediated cell apoptosis in A2780 cells, however, GRASP65 depletion had no effects on the p-JNK/ERK levels in A2780 cells.

Suppression of the ERK/JNK pathway attenuated DHM-induced apoptosis in A2780 cells

To make the mechanism involved in inhibiting GRASP65 further clear, protein expression of GRASP65 and apoptosis-related proteins in A2780 cells were examined with the use of JNK and ERK inhibitors, respectively.

A2780 cells were pre-treated with 20 μM SP600125 (a JNK inhibitor) or 10 μM U0126 (an ERK inhibitor) for 2 h, followed by 80 μM DHM treatment for another 48 h and then were analyzed by western blot. Compared with the Control group, DHM increased the p-JNK/ERK levels and the caspase-3 cleavage and downregulated the expression of GRASP65 simultaneously. Interestingly, in comparison to the DHM group, SP600125 and U0126 significantly inhibited DHM-induced caspase-3 activation and increased GRASP65 levels after treatment with 80 μM DHM, followed by the inhibition of p-JNK/ERK levels (Fig 8A and 8B).

Fig 8 — **A/B**. GRASP65, p-JNK/ERK and cleaved-caspase-3 levels were analyzed by western blot, following treatment with 80 μM of DHM in combination with SP600125 (8A) and U0126 (8B). Each experiment was repeated at least three times. * p < 0.05, **p < 0.01 vs the control group. ^#p < 0.05, ^##p < 0.01 vs the DHM group.

Taken together, these findings showed that the JNK/ERK pathway might be involved in DHM-mediated caspase-3 activation and GRASP65 inhibition in A2780 cells and suppression the ERK/JNK pathway could attenuate DHM-induced apoptosis, followed by an increase of GRASP65 expression.

Discussion

The Golgi complex has been demonstrated to undergo fragmentation during apoptosis in several cancers. Due to the role of ER and Golgi during induction/execution of apoptosis, Golgi proteins have garnered significant interest as novel targets for selective anti-cancer therapies [3]. Cleavage of GRASP65 by caspase-3 correlates with Golgi fragmentation [7], and the fragmentation partially prevented by the expression of a caspase-resistant form of GRASP65 during apoptosis [29, 39]. GRASP65 also seems to be the important target of signaling events leading to Golgi breakdown during apoptosis [35]. Furthermore, GRASP65 and GRASP55 have been recently used as tools to disrupt the Golgi structure and thereby determine the functional consequence of Golgi structural disruption [40], which prompt numerous researchers to explore the underlying mechanisms of Golgi structure formation and function. Additionally, the Golgi-localized caspase-2 and -3 are generally accepted as central players in the execution phase of apoptosis, as they mediate cleavage of several golgins and GRASPs, including GM130 [41] and GRASP65 [29]. Therefore, these studies indicated that Golgi proteins are potential therapeutic targets, as Golgi disruptive agents may facilitate Golgi fragmentation and induce apoptosis. In the present study, we mainly focused on the potential effects of GRASP65 in DHM-mediated ovarian cancer cell apoptosis.

DHM, a natural flavonoid derived from Ampelopsis grossedentata, has received considerable interest as a potential candidate for cancer therapy. Many studies have demonstrated that DHM has functions in cancer prevention and development [21–23]. Interestingly, an inverse relationship between the intake of dietary flavonoid and cancer risk has been observed in different studies [42]. Therefore, we sought to further investigate the antitumor activity of DHM. Cell apoptosis is an active process of endogenous programmed cell death, which is a standard benchmark for the selection of anticancer drugs [43], and its deregulation is a fundamental hallmark of cancer development and progression [44]. The results of our study showed that DHM treatment significantly promoted cell apoptosis by upregulating the proportion of Bax/Bcl-2 and activating caspase-3 in SKOV3 and A2780 cells. Similarly, many chemotherapies, such as cisplatin and paclitaxel, aim to cure or control cancer by inducing apoptosis of human carcinoma cells [45].

To further determine the effects of GRASP65 on DHM-induced cell apoptosis, we first examine GRASP65 expression using immunoblotting analysis. GRASP65 is a specific substrate of caspase-3[36], and cleavage of GRASP65 correlates with Golgi fragmentation, which can be inhibited by the expression of a caspase-resistant form of GRASP65[29]. And caspase cleavage of Golgi structural proteins may be the downstream result of effector caspase activation, allowing the packaging of Golgi remnants into apoptotic blebs for disposal [29]. Our findings showed that GRASP65 expression decreased during DHM-induced apoptosis, likely due to caspase-3 cleavage according to the reports that showed caspase-3 cleavage of GRASP65 is necessary for apoptotic Golgi fragmentation [29,36]. Furthermore, we found inhibition of caspase-3 activity by Ac-DEVD-CHO could mitigate DHM-induced cell apoptosis to delay or reduce cell death, followed by an increase of GRASP65 level. Therefore, we speculated that DHM might activate caspase-3, which is closely related to the suppression of GRASP65 expression during DHM-induced apoptosis. However, the significance of GRASP65 suppression and its relationship with DHM-mediated effects remain obscure.

Then, we performed GRASP65 siRNA and overexpression plasmid transfections to further detect the potential role of GRASP65 in DHM-induced cell apoptosis. Previous research reported that pharmacological intervention or overexpression of the C-terminal fragment of GRASP65 inhibits GF and decreases or delays neuronal cell death [46]. Depletion of GRASP65 by siRNA reduced the number of cisternae in the Golgi stacks, which can be rescued by expressing exogenous GRASP65 [47]. In addition, depletion of GRASP65 reduced cell attachment and migration [48], even resulted in cell death [36,49]. Numerous researches have indicated that the Golgi apparatus and GA fragmentation play important roles in apoptosis [28, 50] and GA is a sensor and common downstream effector of stress signals in cell death pathways [51]. Similarly, flow cytometry and western blot analysis in our study showed that overexpression of GRASP65 reduced cell apoptosis and inhibition of GRASP65 by siRNA induced apoptosis in A2780 cells. On the other hand, overexpression of GRASP65 mitigated DHM-mediated cell apoptosis. Conversely, GRASP65 depletion combined with DHM treatment could further promoted cell apoptosis, suggesting that DHM combined with GRASP65 intervention may elicit an antitumor response in ovarian cancer cells. Therefore, GRASP65 downregulation may have a critical role in DHM-induced apoptosis in OCs, while its role in DHM-induced Golgi morphological changes may be complex.

However, the molecular mechanism of DHM-mediated suppression of GRASP65 expression is unclear. Several studies have shown that the mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in chemotherapy-induced apoptosis [36,52–53]. JNK can function as pro-apoptotic and anti-apoptotic kinases in different cell types [54]. Accumulating evidence suggests that phosphorylation of GRASP65 by kinases, such as ERK [55, 56], JNK2[57], Cdk1 and PLK1, or cleavage by caspase, is required for mitotic or apoptotic Golgi fragmentation [8,58]. JNK2–GRASP65 signaling has a prominent role in the identification of novel anti-cancer agents that block cell cycle progression [57]. Once activated, JNKs in turn phosphorylate several transcription factors and cellular proteins that are associated with apoptosis, including Bcl2[53], capase-3[59], and others. In the present study, DHM could activate JNK/ERK signaling pathway in a concentration-dependent manner. On the other hand, inhibition of JNK and ERK signaling suppressed the cleavage of caspase-3 as well as increased the expression of GRASP65 in A2780 cells treated with DHM, indicating that DHM might activate JNK/ERK-caspase-3 pathway and the activation of caspase-3 was crucial for the downregulation of GRASP65. Moreover, we found DHM combined with GRASP65 siRNA intervention couldn’t further affect the p-JNK and p-ERK levels, only induce apoptosis. Increasing evidence suggests that the GA is a crucial downstream effector [51], even as a downstream target organelle of endoplasmic reticulum and mitochondria associated with GRASP65 phosphorylation when oxidative stress occurs [60,61]. Based on the findings, we speculated that GRASP65 suppression might be the downstream effector during DHM-mediated apoptosis in ovarian cancer cells, which was at least partially due to the activation of the JNK/ERK pathway.

Taken together, the present study demonstrated that DHM treatment promoted JNK/ERK activation and the cleavage of Caspase-3, which was crucial for the suppression of GRASP65 in ovarian cancer cells. Furthermore, DHM treatment combined with GRASP65 depletion had an additive role in DHM-mediated anticancer effects. However, the role of GRASP65 in DHM-induced Golgi distribution and morphology remains unclear. In the long term, future studies employing techniques, such as electron microscopy and confocal scanning microscopy, are needed to further reveal the structure-function relationships of the GA in DHM-induced cell apoptosis.

Supporting information

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Acknowledgments

We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the Health and Family Planning Commission of Hubei Province (No. WJ2019M104).

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PLoS One. doi: 10.1371/journal.pone.0225450.r001

Decision Letter 0

Yi-Hsien Hsieh

30 Aug 2019

PONE-D-19-21799

Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

PLOS ONE

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Reviewer #1: the present article entitled: Golgi reassembly and stacking protein 65 downregulation is required for the anticancer effect of dihydromyricetin on human ovarian cancer cells, the authors focused to investigate the anti-tumor effects of DHM in OC cells and also elucidated the associated underlying molecular mechanisms. Overall, the manuscript was clearly written and the data was obviously presented. However, several points should be seriously taken in consideration for the following raisons:

1- In the discussion section, the authors should cite more references.

2- The authors should add n=? in each legend to figure.

3- Why the authors did not investigated the expression of cell cycle regulatory proteins such as CDK?

4- In the flow cytometry analysis of apoptotic cells figures 4B and 5C, the Y-axis must be moved to be on 102 for control, DHM and DHM+Ac-DEVD-CHO-treated cells.

Reviewer #2: This manuscript presents the results of studies examining whether DHM-induced antitumor activity was through the downregulation of GRASP65 in SKOV3 and A2780 cells. They found that the suppressive effects of proliferation, migration, and the promotion of apoptosis induced by DHM was regulated by GRASP65. Since research on the involvement of Goldi proteins with DHM-induced antitumor effects has not been done so far, the topic is of interest. However, the studies are not sufficiently verified.

1. Although authors explained why they focused on the involvement of GRASP65 in DNM-induced anti-tumor activity, it was difficult to understand. Authors should explain how GRASP65 is regulated by ERK, CDK1, and PLK-1 specifically and whether the regulation is about the expression or morphological changes.

2. Fig. 4 showed that DHM-induced caspase-3 activation was crucial for suppression of GRASP65 expression and induction of apoptosis by DHM. This result did not imply that activated caspase-3 mediated cleavage and reduction of GRASP65 was crucial for DHM-induced cell apoptosis. The caption [GRASP65 was essential for the anti-cancer effects of DHM in A2780 cells] was not correct.

3. In Fig. 5, the results of western blotting and its quantitative results did not match. For example, in Fig. 5A, the inductive effect of OE2 was not obvious by western blotting, but the 1.5-fold inductive effect was observed by a densitometric analysis. In Fig. 5B, the combination effect of siGRASP65 and DHM on GRASP65 expression was not obvious by western blotting, but the additive effect was shown by a densitometric analysis.

4. In Fig. 5, both siGRASP65 and OE-GRASP65 increased the number of apoptotic cells. However, the combination of DHM and OE-GRASP65 attenuated the DHM-induced apoptotic effects. Is this correct?

5. In Fig. 6, authors should examine the effects of OE-GRASP65 on cell viability and migration. Furthermore, the result of invasion should be included.

6. In Fig. 7, the results of western blotting and its quantitative results did not match. Authors showed p38 level did not change, but it looks like that DHM suppressed the phosphorylation of p38 by western blotting. Furthermore, the addictive effects of siGRASP65 plus DHM on the expression of p-JNK and p-ERK were not observed.

7. As compared with the results of Fig. 7, the inductive effects on the expression of p-JNK and p-ERK by DHM were weak.

Reviewer #3: In this manuscript, Wang et al. aimed to identify a functional connection between the antitumor activity of Dihydromyricetin (DHM) and Golgi reassembly-stacking protein of 65 kDa (GRASP65) through a mechanism that involves activation of apoptosis in ovarian cancer cell lines. DHM is a flavonoid found in several species, and it has proapoptotic activity on several cancer cell lines, including of hepatoma, melanoma, osteosarcoma, gastric cancer, and ovarian cancer. GRASP65 is a Golgi apparatus protein implicated in several aspects of protein trafficking and in the structure of the Golgi apparatus including the stacking of Golgi cisternae and the linking of Golgi stacks to form a Golgi ribbon. In addition to confirming published data indicating that DHM has proapoptotic activity on ovarian cancer cells, this manuscript provides evidence that it also negatively affects cell migration and invasion of these cells. As for the proapoptotic activity, the data indicate that DHM-induced apoptosis proceeds with an increase in the levels of cleaved caspase-3, which correlates with a decrease in the levels of GRASP65. This is an expected correlation, because it is well known that GRASP65 is a target of activated caspase-3. The manuscript present data of GRASP65 RNAi and overexpression experiments designed to demonstrate the causality in the DHM-induced apoptosis that this Golgi protein might be involved in. Finally, the authors explore the signaling pathways that might be implicated in the apoptotic response to DHM. Overall, the manuscript shows data of good standard. However, the major conclusion that GRASP65 is required for the anti-cancer effect of DHM is not supported by the data provided. In addition, a number of major and minor issues do not warrant publication of the manuscript as it is.

Major issues:

1) As mentioned, the main conclusion of the manuscript that GRASP65 is required for the anti-cancer effect of DHM has no experimental support. Moreover, the results of the GRASP65 RNAi experiments show the opposite, which is that the treatment with DHM in cells knocked down of GRASP65 expression resulted in increased levels of cleaved caspase-3 and apoptotic rate, compared to control cells or to each of the individual treatments. If any, the conclusion here is that the reduction in the levels of GRASP65 cooperates in the proapoptotic effect of DHM, or vice versa. In fact, the data show that the knocking down of GRASP65 has also a proapototic effect, and that the treatment with DHM has an additive proapoptotic effect. Considering the results presented, the testing of the requirement of GRASP65 function for the proapoptotic activity of DHM needs a different experimental design.

2) An intriguing set of results is that the overexpression of GRASP65 also resulted in increased levels of cleaved caspase-3 and apoptotic rate, but somehow combined with DHM resulted in less proapoptotic effect compared to the treatment with DHM alone. The authors should comment on these results providing a possible explanation.

Minor issues:

3) The revised version should contain line numbering, as it is an editorial request, otherwise the revision process is time consuming.

4) Although overall the manuscript is read and understood, it is advised an additional professional scientific proofreading as some of the statements are incorrect and thus are potentially misleading.

5) In the "Abstract" the statement "...DHM inhibited cell migration..." is incorrect; DHM reduced cell migration.

6) In "Introduction" the statement "...leads to depolymerization and division of the Golgi..." is incorrect; the Golgi is not a polymer and it does not divide.

7) The sequence of each GRASP65 siRNA oligonucleotide should be provided, as well as a description of each of the plasmids used for the overexpression of GRASP65.

8) The results shown in Fig 1A should be removed, because similar results are already published.

9) In "Results" the statement "...and almost completely blocked the closure..." is incorrect; at the most, the treatments reduced SKOV3 migration or invasion to ∼25% the respective levels observed in control conditions.

10) The title of Fig. 1 legend is incorrect, because DHM did not inhibit cell viability, cell migration and cell invasion; it reduced the extent of these processes.

11) In "Results" the statement "...DHM downregulated the expression of GRASP65 in a concentration-dependent manner, followed by activation of Caspase-3"... is odd. The published data indicate that the process is the opposite, meaning that during apoptosis the activation of caspase-3 results in cleavage of GRASP65 and thus in GRASP65 downregulation.

12) In the legend of Fig. 3 " #p < 0.05, ##p < 0.01 vs the DHM group" should be removed.

13) In "Results" the title "GRASP65 was essential for the anti-cancer effects of DHM in A2780 cells" is incorrect, because the data does not show at all that GRASP65 is essential for the effects of DHM.

14) In the same section of "Results" the statement "... cells were pre-treated with a specific caspase-3 inhibitor, Ac-DEVD-CHO, for 30 min to suppress the effects of DHM..." is odd, because the experiment should not have been designed to suppress the effects of DHM, but instead to evaluate the contribution of caspase-3 in the effects of DHM.

15) The title of Fig. 4 legend is odd; please revise.

16) In "Results" the statement "These results suggested that downregulation of GRASP65 could promote DHM-induced inhibition of cell viability and cell migration" is at least speculative, and should be revised. The data indicate that the effects of GRASP65 RNAi and DHM treatment are additive and thus very unlikely to be mechanistically related.

17) Please revise the magnitude informed of the scale bar in the legend of Fig. 6 as it seems very similar to that of Figures 2 and 3.

18) Please, provide a rationale for not performing all the subsequent analyses in SKOV3 cells.

19) Please, explain why it was not performed the analysis of the levels of p-p38 in cells transfected with siGRASP65.

20) In "Discussion" the statement "The Golgi is essential for the endoplasmic reticulum and mitochondria..." is odd; please revise.

21) In "Discussion" the statement "GRASP65, a peripheral Golgi membrane protein, is required for mitotic or apoptotic Golgi fragmentation when specifically cleaved by caspases" is odd; please revise.

22) In "Discussion" the statement "...Golgi execution phase of apoptosis..." is odd; please revise.

23) In "Discussion" the statement "This implies that the Golgi is a potential therapeutic target, as Golgi disruptive agents may facilitate Golgi fragmentation and induce apoptosis" is a hypothesis already tested by several groups with several published examples in the literature. The authors should discuss their findings in the context of the published data.

24) In "Discussion" the statement "...Golgi formation may be carcinogenic, or a consequence of cancer progression" is immensely odd; please provide more explanations or revise.

25) In "Discussion" the statement "... inhibiting cleaved caspase-3 can block apoptotic cell death and increasing Caspase-3-like protease activity may be responsible for the delayed cell death" is odd; please revise.

26) In "Discussion" the statement "... DHM may activate caspase-3, which then cleaves and reduces GRASP65 expression to promote cell apoptosis" is an overstatement, because the data do not support the conclusion that the proapoptotic effects of DHM are mediated by the reduction in the levels of GRASP65.

27) In "Discussion" the statement "... activated caspase-3-mediated cleavage and the reduction of GRASP65 was crucial for DHM-induced cell apoptosis" is redundant, and, again, is not supported by the data.

28) The complete "Conclusion" section should be revised, because many statements are misleading (considering that some conclusions are incorrect).

29) In Fig. 3A and 3B the expected effect of DHM is the fragmentation of the Golgi apparatus. Because DHM resulted in a decrease in the levels of GRASP65, a different Golgi resident protein should be analyzed by immunofluorescence. Also, to diagnose Golgi apparatus fragmentation (instead of Golgi vesiculation) simultaneous immunofluorescence of at least cis and trans Golgi resident proteins should be provided. Higher magnification of the Golgi ribbon in control-treated cells and DHM-treated cells should be also included to properly assess Golgi fragmentation.

**********

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Reviewer #2: No

Reviewer #3: No

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PLoS One. 2019 Nov 26;14(11):e0225450. doi: 10.1371/journal.pone.0225450.r002

Author response to Decision Letter 0

8 Oct 2019

Dear Editor and Reviewers:

Thank the editor and the reviewers for your positive comments and constructive suggestions for our manuscript entitled “Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells” (Manuscript ID: PONE-D-19-21799). Your positive comments and useful suggestions that are beneficial for the further improvement of this manuscript are highly appreciated. Based on the reviewer’s suggestions, we have made double check and careful modifications for this manuscript. The modified or revised parts in the revised manuscript are shown in tracking changes.

We’re sure that our manuscript meets PLOS ONE's style requirements and have provided the original uncropped and unadjusted images underlying all blot or gel results reported in Supporting Information files.

Meanwhile, the point-to-point responses to address the concerns of the reviewers are listed as follows:

Review Comments to the Author

1- In the discussion section, the authors should cite more references.

Response: Thank the reviewer for giving this useful suggestion. We have added and cited more references and further discussed the issue in the discussion section as shown in the revised manuscript.

2- The authors should add n=? in each legend to figure.

Response: Thank the reviewer for pointing out the issue. We have added the sentence as “Each experiment was repeated at least three times” in each legend to figures according to the suggestion.

3- Why the authors did not investigated the expression of cell cycle regulatory proteins such as CDK?

Response: We would like to express our highest appreciation to the reviewer for this useful suggestion. The effects of DHM on the expression of cell cycle regulatory proteins, such as CDK, haven’t been detected, then we would like to further examine and provide the results in the future research.

4- In the flow cytometry analysis of apoptotic cells figures 4B and 5C, the Y-axis must be moved to be on 102 for control, DHM and DHM+Ac-DEVD-CHO-treated cells.

Response: Thank the reviewer for pointing out the issue. We have modified and re-analyzed the results of flow cytometry in the revised manuscript.

Response: We really appreciate the reviewer to point out the important issue. Most studies on GRASP65 were focused on the regulation of morphological changes by ERK, CDK1, and PLK-1. And we found DHM could affect the expression of GRASP65, we investigated the involvement and function of GRASP65 in DNM-induced anti-tumor activity firstly. Meanwhile, we want to further explore the morphological changes of GA and the relation between the morphology and function in DHM-induced effects in OCs in the future research.

Response: Thank the reviewer for pointing out the issue. We have modified the caption as “DHM-induced caspase-3 activation was crucial for suppression of GRASP65 expression” for Fig.4 in the revised manuscript and further discussed the results again.

Response: Thank the reviewer for pointing out the issue. We have exchanged the relative figure of western blot analysis and re-analyzed the results. Each experiment was repeated at least three times. Therefore, we’re sure that the effects of siRNAs to suppress GRASP65 and OE plasmid to overexpress GRASP65 were obvious by a densitometric analysis as shown in Fig.5 in the revised manuscript.

4. In Fig. 5, both siGRASP65 and OE-GRASP65 increased the number of apoptotic cells. However, the combination of DHM and OE-GRASP65 attenuated the DHM-induced apoptotic effects. Is this correct?

Response: We really appreciate the reviewer for pointing out this issue. We have performed the experiment again in this part, then we found that OE-GRASP65 plasmid transfection could reduce cell apoptosis. In addition, compared with DHM treatment group, combination of DHM treatment and OE-GRASP65 attenuated the DHM-induced apoptosis. Therefore, we re-analyzed the statistic results of flow cytometry as shown in Fig.5 in the revised manuscript.

5. In Fig. 6, authors should examine the effects of OE-GRASP65 on cell viability and migration. Furthermore, the result of invasion should be included.

Response: Thank the reviewer giving us this useful suggestion. We have added the detection of the effects of OE-GRASP65 on cell viability and migration according to the reviewer’s suggestion as shown in Fig.6 in the revised manuscript. However, we only performed the test of cell invasion using Transwell assay in the previous study to test DHM-mediated effects for some reasons. We would like to further detect the effects of siGRASP65 and OE-GRASP65 on cell invasion and provide better results in the following study.

Response: Thank the reviewer give us the suggestion for modifying this confusing result. Firstly, we’re sure that there was no obvious difference in the phosphorylation of p38 levels among the DHM treated groups based on the results, then we provided better results and figures of western blotting analysis as shown in Fig.7 in the revised manuscript.

In addition, according to the reviewer’s suggestion, we have re-performed the experiment to examine the effects of siGRASP65 plus DHM on the expression of p-JNK and p-ERK and found there was no obviously additive effects. Simultaneously, we speculated that GRASP65 downregulation might be the downstream effector in DHM-mediated activation of JNK/ERK-caspase-3 pathway according to the relative reference. But it is needed to confirm the relation in the following study.

7. As compared with the results of Fig. 7, the inductive effects on the expression of p-JNK and p-ERK by DHM were weak.

Response: Thank the reviewer give us the suggestion for modifying this confusing result. In fact, we have repeated the experiments and analyzed the results again according to your suggestion. Then we found that there was no obvious difference in p-JNK/ERK levels between the DHM group and DHM+siGRASP65 group, suggesting that GRASP65 depletion had no effects on the p-JNK/ERK levels.

Therefore, we have modified them as shown in Fig.7 in the revised manuscript.

Major issues:

Response: We would like to express our highest appreciation to the reviewer for pointing out the confusing conclusion. Frankly speaking, we might not provide a proper description to the present results in the manuscript. According to the reviewer’s suggestion, we couldn’t confirm the main conclusion of the manuscript based on the present results and we would like to further design the experiment comprehensively. However, considering the results presented, we could confirm that GRASP65 depletion has a proapoptotic effect and siGRASP65 combined with DHM treatment has an additive proapoptotic effect in ovarian cancer cells. We have modified the results in the revised manuscript.

We would like to receive your useful and constructive suggestion again.

Response: Thank the reviewer give us the suggestion for modifying this confusing result. In fact, the experiment data provided previously are misleading. We have repeated the experiment according to the suggestion and modified the results. The present results showed that overexpression of GRASP65 reduced cell apoptosis with the use of western blotting and FCM analysis and overexpression GRASP65 combined with DHM attenuated the proapoptotic effect compared to the DHM treatment alone. This is consistent with many reports about the effects of overexpression of GRASPs. We have modified the results and discussion sections as shown in the revised manuscript.

Minor issues:

3) The revised version should contain line numbering, as it is an editorial request, otherwise the revision process is time consuming.

Response: Thank the reviewer for the useful suggestion. We have added the line numbering in the revised manuscript.

Response: Thank the reviewer for pointing out the issue. We have made double check and careful modifications for this manuscript and got linguistic assistance from LetPub.

5) In the "Abstract" the statement "...DHM inhibited cell migration..." is incorrect; DHM reduced cell migration.

Response: Thank the reviewer for pointing out the issue. We have modified the statement as “The present study showed that DHM reduced cell migration and invasion….” in the “Abstract and Results” parts according to the reviewer’s suggestion.

6) In "Introduction" the statement "...leads to depolymerization and division of the Golgi..." is incorrect; the Golgi is not a polymer and it does not divide.

Response: Thank the reviewer for pointing out the issue. The statement provided is misleading. Therefore, we refer to the original literature to modify the description as “GRASP65 is regulated by Cdc2 and PLK-1 during cell mitosis, which leads to GRASP65 deoligomerization and then Golgi unstacking” in the revised manuscript.

7) The sequence of each GRASP65 siRNA oligonucleotide should be provided, as well as a description of each of the plasmids used for the overexpression of GRASP65.

Response: Thank the reviewer for the useful suggestion. We have provided the sequence of every GRASP65 siRNA oligonucleotide and the plasmid for overexpression of GRASP65 from NCBI (GRASP65/GORASP1(human): NM_031899.3), which were all designed and synthesized by Shanghai GenePharma Co., Ltd.

8) The results shown in Fig 1A should be removed, because similar results are already published.

Response: Thank the reviewer for the useful suggestion. We have deleted the results in Fig 1A according to the reviewer’s suggestion.

Response: Thank the reviewer for pointing out the issue. We have modified the results as “As shown in Fig. 1A and 1B, 40 μM DHM suppressed approximately 50% of wound closure in A2780 cells, and significantly reduced the closure of the wound after DHM treatment at both 80 μM and 120 μM in SKOV3 cells” in the revised manuscript.

10) The title of Fig. 1 legend is incorrect, because DHM did not inhibit cell viability, cell migration and cell invasion; it reduced the extent of these processes.

Response: Thank the reviewer for pointing out the issue. We have modified the title of Fig.1 as “DHM reduced cell migration and invasion in SKOV3 and A2780 cells” in the revised manuscript.

Response: Thank the reviewer for pointing out the incorrect description. According to the reviewer’s suggestion, we have modified the statement as “In comparison to the control group, DHM induced cell apoptosis as shown in Fig.2, followed by the downregulation of GRASP65 expression in a concentration-dependent manner in Fig. 3C/D.” in the manuscript.

12) In the legend of Fig. 3 " #p < 0.05, ##p < 0.01 vs the DHM group" should be removed.

Response: Thank the reviewer for pointing out the issue. We have deleted the unnecessary part in the legend of Fig.3.

Response: Thank the reviewer giving us the useful suggestion. Frankly speaking, it’s not so clear to clarify the relation between the GRASP65 suppression and DHM-induced apoptosis at first. According to the suggestion, we have modified the title as “DHM-induced caspase-3 activation was crucial for suppression of GRASP65 expression in SKOV3 and A2780 cells” in the revised manuscript. In addition, we have read and cited more reference to testify our findings.

Response: Thank the reviewer for pointing out the misleading issue. We have modified the statement as “OCs were pre-treated with a specific caspase-3 inhibitor, Ac-DEVD-CHO, for 30 min to suppress the activity of caspase-3 to evaluate the contribution of caspase-3 in the effects of DHM” in the revised manuscript.

15) The title of Fig. 4 legend is odd; please revise.

Response: Thank the reviewer giving us the useful suggestion. We have revised the title of Fig.4 as “Caspase-3 activation is crucial for suppression of GRASP65 expression in SKOV3 and A2780 cells” in the revised manuscript.

Response: We really appreciate the reviewer for providing these suggestions due to the inaccurate statement. According to the reviewer’s suggestion, we have modified the statement as “These results suggested that GRASP65 depletion using siRNA combined with DHM treatment had an additive effect during DHM-induced inhibition of cell viability and cell migration” in the “results” part and restated again in the “Discussion” part.

17) Please revise the magnitude informed of the scale bar in the legend of Fig. 6 as it seems very similar to that of Figures 2 and 3.

Response: Thank the reviewer for pointing out the issue. We have revised the results in Fig. 6 and provided the statistical analysis of cell viability and migration.

18) Please, provide a rationale for not performing all the subsequent analyses in SKOV3 cells.

Response: Thank the reviewer giving us the useful suggestion. In fact, we performed the experiment design including the analysis in two cell lines at first. Until now, we have achieved some results all provided in the manuscript and we thought the findings could indicate the present issue. Surely, we would like to support the subsequent results in the future.

19) Please, explain why it was not performed the analysis of the levels of p-p38 in cells transfected with siGRASP65.

Response: Thank the reviewer giving us the useful suggestion. Firstly, our results showed that there was no obvious difference in the phosphorylation of p38 levels among the DHM treated groups as shown in Fig.7A in the revised manuscript. However, we tested again the levels of p-38 in cells transfected with siGRASP65 according to the reviewer’s suggestion, and the results showed no difference among the four groups as shown in Fig.7B.

20) In "Discussion" the statement "The Golgi is essential for the endoplasmic reticulum and mitochondria..." is odd; please revise.

Response: Thank the reviewer giving us the useful suggestion. We have deleted the sentence due to the misunderstanding.

21) In "Discussion" the statement "GRASP65, a peripheral Golgi membrane protein, is required for mitotic or apoptotic Golgi fragmentation when specifically cleaved by caspases" is odd; please revise.

Response: Thank the reviewer for pointing out the issue. We have modified the statement as “Cleavage of GRASP65 by caspase-3 correlates with Golgi fragmentation [7], and the fragmentation partially prevented by the expression of a caspase-resistant form of GRASP65 during apoptosis [29, 39]” and added relative reference as shown in the “Discussion” section.

22) In "Discussion" the statement "...Golgi execution phase of apoptosis..." is odd; please revise.

Response: Thank the reviewer for pointing out the issue. We have modified the section as “ Additionally, the Golgi-localized caspase-2 and -3 are generally accepted as central players in the execution phase of apoptosis, as they mediate cleavage of several golgins and GRASPs, including GM130[41] and GRASP65[29]”in the revised manuscript and added the reference.

Response: Thank the reviewer giving us the useful suggestion. We have modified the statement and added more references to further discuss our findings as shown in the revised manuscript.

24) In "Discussion" the statement "...Golgi formation may be carcinogenic, or a consequence of cancer progression" is immensely odd; please provide more explanations or revise.

Response: Thank the reviewer for pointing out the issue. We have deleted the misleading statement.

Response: Thank the reviewer for pointing out the issue. We have modified as “Furthermore, we found inhibition of caspase-3 activity by Ac-DEVD-CHO could mitigate DHM-induced cell apoptosis to delay or reduce cell death, followed by an increase of GRASP65 level” in the revised manuscript.

Response: We really appreciate the reviewer for providing these suggestions due to the inaccurate statement. We have modified the statement as “Therefore, we speculated that DHM might activate caspase-3, which is closely related to the suppression of GRASP65 expression during DHM-induced apoptosis. However, the significance of GRASP65 suppression and its relationship with DHM-mediated effects remain obscure” in the revised manuscript.

Response: Thank the reviewer giving us the useful suggestion. We have deleted the redundant sentence.

28) The complete "Conclusion" section should be revised, because many statements are misleading (considering that some conclusions are incorrect).

Response: Thank the reviewer giving us the useful suggestion. We have removed the “Conclusion” part and further discuss our findings according to the published data.

Response: Thank the reviewer giving us the useful suggestion. In the present study, we want to indicate that DHM could induce Golgi apparatus fragmentation as well as a decrease in GRASP65 level, and then result in cell apoptosis in OCs. At present, we didn’t perform IF analysis of another Golgi resident protein for other reasons. We have designed experiment to confirm GF and the relationship between GF and Golgi function in DHM-mediated effects and we are doing experiment using confocal microscopy and TEM, no better results yet. However, we need some time to solve the issue. We would like to provide the subsequent results at any time.

Finally, we would like to appreciate the editor and the reviewers again for positive comments and constructive suggestions that are benefit for the improvement of our manuscript. We believe our revised manuscript is greatly improved and it will be satisfactory with you and reviewers. Meanwhile, we hope our revised manuscript can meet the requirements of your journal for the publication in Plos one.

Please do not hesitate to contact me if you still have any questions and I am looking forward to hearing from you.

Sincerely yours,

Fengjie Wang

Attachment

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PLoS One. doi: 10.1371/journal.pone.0225450.r003

Decision Letter 1

Yi-Hsien Hsieh

6 Nov 2019

Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

PONE-D-19-21799R1

Dear Dr. luo,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors properly answered all my raised questions and the revised version in now suitable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0225450.r004

Acceptance letter

Yi-Hsien Hsieh

13 Nov 2019

PONE-D-19-21799R1

Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

Dear Dr. Luo:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Supplementary Materials

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Golgi reassembly and stacking protein 65 downregulation is required for the anti-cancer effect of dihydromyricetin on human ovarian cancer cells

Fengjie Wang

Xianbing Chen

Depei Yuan

Yongfen Yi

Yi Luo

Roles

Abstract

Introduction

Materials and methods

Reagents

Cell culture

In vitro cell viability assay (CCK8)

Wound healing assay

Migration/Invasion assay

Apoptosis assay

Caspase-3/9 activity assay

Cellular immunofluorescence (IF)

Gene knockdown using siRNA and transient transfection

Preparation of total cell extracts and western blot analysis

Statistical analysis

Results

DHM reduced cell migration and invasion in SKOV3 and A2780 cells

Fig 1. DHM reduces cell migration and invasion.

DHM induced cell apoptosis in A2780 and SKOV3 cells

Fig 2. DHM induces apoptosis in SKOV3 and A2780 cells.

DHM downregulated GRASP65 expression in SKOV3 and A2780 cells

Fig 3. DHM downregulates GRASP65 expression in SKOV3 and A2780 cells.

DHM-induced caspase-3 activation was crucial for suppression of GRASP65 expression in SKOV3 and A2780 cells

Fig 4. Caspase-3 activation is crucial for suppression of GRASP65 expression in SKOV3 and A2780 cells.

Effects of GRASP65 on DHM-induced cell apoptosis in A2780 cells

Fig 5. Effects of GRASP65 on DHM-induced apoptosis in A2780 cells.

Effects of GRASP65 on DHM-mediated cell viability and migration in A2780 cells

Fig 6. Effects of GRASP65 on DHM-induced cell viability and migration in A2780 cells.

JNK/ERK pathway participated in DHM-mediated apoptosis in A2780 cells

Fig 7. ERK/JNK pathway participated in DHM-mediated apoptosis in A2780 cells.

Suppression of the ERK/JNK pathway attenuated DHM-induced apoptosis in A2780 cells

Fig 8. Suppression of the ERK/JNK pathway attenuated DHM-induced apoptosis in A2780 Cells.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Yi-Hsien Hsieh

Roles

Author response to Decision Letter 0

Decision Letter 1

Yi-Hsien Hsieh

Roles

Acceptance letter

Yi-Hsien Hsieh

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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