YAP Inhibition by Resveratrol via Activation of AMPK Enhances the Sensitivity of Pancreatic Cancer Cells to Gemcitabine

Resveratrol, a natural polyphenol present in most plants, inhibits the growth of numerous cancers both in vitro and in vivo. Aberrant expression of YAP has been reported to activate multiple growth-regulatory pathways and confer anti-apoptotic abilities to many cancer cells. However, the role of resveratrol in YES-activated protein (YAP) expression and that of YAP in pancreatic cancer cells’ response to gemcitabine resistance remain elusive. In this study, we found that resveratrol suppressed the proliferation and cloning ability and induced the apoptosis of pancreatic cancer cells. These multiple biological effects might result from the activation of AMP-activation protein kinase (AMPK) (Thr172) and, thus, the induction of YAP cytoplasmic retention, Ser127 phosphorylation, and the inhibition of YAP transcriptional activity by resveratrol. YAP silencing by siRNA or resveratrol enhanced the sensitivity of gemcitabine in pancreatic cancer cells. Taken together, these findings demonstrate that resveratrol could increase the sensitivity of pancreatic cancer cells to gemcitabine by inhibiting YAP expression. More importantly, our work reveals that resveratrol is a potential anticancer agent for the treatment of pancreatic cancer, and YAP may serve as a promising target for sensitizing pancreatic cancer cells to chemotherapy.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant and lethal tumors, with an overall five-year survival rate less than 7% [1]. Over recent decades, the prognosis of patients with this malignancy has not improved due to aggressive local invasion, metastases, and resistance to chemotherapy [2]. Currently, surgical resection is the only opportunity for curing pancreatic cancer at an early stage. Unfortunately, only approximately 20% of patients are eligible for surgical resection at the time of diagnosis, with most patents losing the opportunity for radical surgery. At present, gemcitabine is the first-line chemotherapeutic agent for pancreatic cancer patients. However, a low response rate to gemcitabine is common in the clinic, and less than 20% of patients experience the ideal effects of gemcitabine [3]. In recent years, FOLFIRINOX (oxaliplatin, irinotecan, fluorouracil, and leucovorin) has become the recommended frontline chemotherapeutic regimen for metastatic pancreatic cancer patients [4]. However, the seriously adverse reaction and acquired drug resistance dialyzed heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere containing 5% CO 2 at 37 • C.

Cell Viability Assay
Cancer cell lines (Panc-1, BxPC-3) were plated into 96-well plates at a density of 5000 cells/well and treated with various concentrations (0, 25, 50, 100, and 200 µM) of resveratrol and various concentrations (0, 1, 2, 5, 10, and 20 µM) of gemcitabine for designated lengths of time (24,48, and 72 h). After being transfected with siRNA for 48 h, cells were plated in 96-well plates at a density of 5000 cells/well and treated with 2 µM gemcitabine for 72 h. Cell viability was assessed by the MTT assay. Ten microliters of 5 mg/mL MTT was added into each well after media were removed and incubated at 37 • C for 4 h. Then, 100 µL DMSO was added to each well, and the optical density (OD) was measured at 490 nm on a multifunction microplate reader (POLARstar OPTIMA; BMG, Offenburg, Germany). The proliferation inhibition rate was calculated according to the following equation: Proliferation inhibition rate = (1 − OD sample/OD control) × 100%.

Apoptosis Assay
Cell apoptosis was assessed by flow cytometry with an Annexin V-FITC/7-AAD apoptosis detection kit from Becton, Dickinson and Company (BD) (Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Briefly, cancer cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well, after being starved overnight, and each treatment was applied for 48 h. Then, cells were trypsinized, washed with phosphate buffered saline (PBS) and stained with Annexin V and 7-AAD. The percentage of apoptotic cells was quantified by flow cytometry using a FACSCalibur (BD Biosciences, San Diego, CA, USA) instrument. The total apoptosis rate was calculated by summing the rate of populations stained with Annexin V-FITC+/7-AAD-(early apoptotic cells) and Annexin V-FITC+/7-AAD+ (late apoptotic cells).

Immunofluorescence Staining
Cells were fixed in 4% formaldehyde diluted in phosphate buffered saline (PBS) for 15 min, permeabilized with 0.3% Triton X-100, treated with blocking buffer (5% BSA in PBS), and then incubated overnight with the primary antibody at 4 • C. Cells were then incubated with the Red conjugated secondary antibody from Jackson Immunoresearch Laboratories (West Grove, PA, USA) for 1 h at room temperature. Slides were mounted and examined using a Zeiss Instruments confocal microscope.

Gene Silencing by Small Interfering RNA
Loss-of-function analysis was performed using siRNAs targeting AMPK and YAP, which were purchased from GenePharm (Shanghai, China). The siRNA sequences are provided in Supplementary Materials Table S2. Each siRNA (100 nM) was transfected into pancreatic cancer cells using Lipofectamine 2000 according to the manufacturer's instructions. The knockdown of each target gene was confirmed by Western blot analysis. The cells were used for subsequent experiments 48 h after transfection.

Western Blot Analysis
Total proteins were extracted by RIPA lysis buffer (Beyotime, Guangzhou, China), and the concentration of proteins was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instruction. The proteins were then subjected to SDS-PAGE using a 10% polyacrylamide gel with a 5% stacking gel. The proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk in Tris-buffered saline-Tween (TBS-T) for 2 h and then incubated with the primary antibodies (listed in Supplementary Materials Table S1) at 4 • C overnight. Then, the membranes were incubated with a secondary antibody (diluted 1:10,000) for 2 h at room temperature. Chemiluminescence detection of bound antibodies was performed using an enhanced chemiluminescence (ECL) PLUS system and a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).

Real-Time PCR
Total RNA was extracted using the Fastgen1000 RNA isolation system (Fastgen, Shanghai, China) according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the Prime Script RT reagent kit (TaKaRa, Dalian, China). Real-time PCR was used to quantitatively examine the expression of YAP, CTGF and CYR61 at the mRNA level. Real-time PCR was conducted according to a previous report [25]. The PCR primer sequences for YAP, CTGF, CYR61, and β-actin are shown in Supplementary Materials Table S3. The expression of each target gene was determined using β-actin as the normalization control. Relative gene expression was calculated using the 2 −∆∆Ct method [26].

Colony Formation Assay
One thousand cells were seeded into a 35-mm petri dish and allowed to adhere overnight. The next day, a different treatment was applied to the dishes for 24 h, after which the medium was replaced with drug-free medium. Cells were further cultured for two weeks to allow colonies to form. At the indicated time point, colonies were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet solution, rinsed, and then imaged. The number of colonies larger than 0.5 mm in diameter was counted using a microscope (Nikon Eclipse Ti-S, Tokyo, Japan) at a magnification of 400×.

Statistical Analysis
Each experiment was performed at least three times. Data are presented as means ± standard deviation. Differences were evaluated using Student's t-test, with p < 0.05 considered to be statistically significant.

Resveratrol Inhibits the Proliferation of Pancreatic Cancer Cells
First, we examined the effects of resveratrol on the viability of cancer cells. Pancreatic cancer cells Panc-1 and BxPC-3 were treated with increasing doses of resveratrol (0, 25, 50, 100, and 200 µM). At the indicated time points (24,48, and 72 h), the cell viability was assessed by the MTT assay. As shown in Figure 1, resveratrol decreased the growth of cancer cell lines in a dose-and time-dependent manner. The 50% inhibitory concentration (IC50) for both BxPC-3 and Panc-1 cells was approximately 50 µM resveratrol, which exhibited no cytotoxic effects on the BxPC-3 and Panc-1 cells. These results were in accord with our previous results. Therefore, cells were treated with 50 µM resveratrol in subsequent experiments.

Resveratrol Inhibits Clone Formation and Induces Apoptosis of Pancreatic Cancer Cells
To address the underlying mechanism governing the inhibitory effect of resveratrol (Res) on pancreatic cancer cell viability, we measured Res-induced apoptosis in BxPc-3 and Panc-1 cells by flow cytometry. The flow cytometric analyses were conducted after Panc-1 and BxPC-3 cells were treated with or without resveratrol (50 µM) for 48 h. As shown in Figure 2A,B, treatment of cancer cells with resveratrol caused an increase in the apoptotic population compared with that of the untreated control cells. Next, we detected the effect of resveratrol on the clone formation ability of cancer cells Panc-1 and BxPC-3. As shown in Figure 2C,D, treatment with 50 µM resveratrol markedly decreased the number of colonies compared with the untreated control cells. These results demonstrate that Res has a potent effect against clone formation and induces apoptosis of cancer cells.

Resveratrol Inhibits YAP Expression of Pancreatic Cancer Cells
Increasing evidence has suggested that overexpression of YAP plays a key role in cancer cell survival and progression [27]. In particular, YAP can be phosphorylated at Ser127 and forms a more stable complex with the 14-3-3 proteins; therefore, it is retained in the cytoplasm and subject to degradation [28]. To determine whether resveratrol affects the YAP expression of cancer cells, Panc-1 and BxPC-3 cells were treated with resveratrol (0, 25, 50, and 100 µM) for 24 h. The protein expression of YAP and p-YAP (Ser127) in the pancreatic cancer cells exposed to resveratrol was evaluated by Western blot analysis. As shown in Figure 3A,B, resveratrol treatment up-regulated the level of p-YAP (Ser127), and the total level of YAP was significantly inhibited by resveratrol in a dose-dependent manner. Connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) are two YAP-mediated downstream effectors that play an important role in tumor progression [29,30]. We therefore examined the expression of YAP, CTGF and CYR61 in response to treatment with resveratrol. The results showed that the mRNA levels of YAP, CTGF, and CYR61 were downregulated upon treatment with resveratrol (50 µM) ( Figure 3C,D). Additionally, the nuclear translocation of YAP was decreased due to the effect of resveratrol, as demonstrated by immunofluorescence ( Figure 3E). Together, these data indicate that resveratrol inhibits YAP expression of cancer cells via YAP phosphorylation at Ser127.

Resveratrol Inhibits YAP Expression of Pancreatic Cancer Cells
Increasing evidence has suggested that overexpression of YAP plays a key role in cancer cell survival and progression [27]. In particular, YAP can be phosphorylated at Ser127 and forms a more stable complex with the 14-3-3 proteins; therefore, it is retained in the cytoplasm and subject to degradation [28]. To determine whether resveratrol affects the YAP expression of cancer cells, Panc-1 and BxPC-3 cells were treated with resveratrol (0, 25, 50, and 100 μM) for 24 h. The protein expression of YAP and p-YAP (Ser127) in the pancreatic cancer cells exposed to resveratrol was evaluated by Western blot analysis. As shown in Figure 3A,B, resveratrol treatment up-regulated the level of p-YAP (Ser127), and the total level of YAP was significantly inhibited by resveratrol in a dosedependent manner. Connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) are two YAP-mediated downstream effectors that play an important role in tumor progression [29,30]. We therefore examined the expression of YAP, CTGF and CYR61 in response to treatment with resveratrol. The results showed that the mRNA levels of YAP, CTGF, and CYR61 were downregulated upon treatment with resveratrol (50 μM) ( Figure 3C,D). Additionally, the nuclear translocation of YAP was decreased due to the effect of resveratrol, as demonstrated by immunofluorescence ( Figure 3E). Together, these data indicate that resveratrol inhibits YAP expression of cancer cells via YAP phosphorylation at Ser127.

Knockdown of AMPK Rescues Resveratrol Induced Suppression of YAP in Pancreatic Cancer Cells
Previous studies have established that resveratrol can activate the AMPK pathway [31]. The activation of AMPK leads to the suppression of YAP expression [32]. Based on the abovementioned promising findings, we speculated that the effect of resveratrol on cancer cell YAP inhibition may be mediated by AMPK signaling. To test this hypothesis, we further examined the effect of resveratrol on the activity of AMPK signaling. Immunoblotting results revealed that the phosphorylation level of AMPK (p-AMPK) in pancreatic cancer cells was significantly increased in response to resveratrol treatment in a dose-dependent manner ( Figure 4A,B). To verify that resveratrol-inhibited YAP expression in cancer cells is mediated by AMPK signaling, siRNA technology was developed to knock down AMPK expression. We found that knocking down AMPK expression alone did not affect the expression of YAP or the phosphorylation level of p-YAP in Panc-1 and BxPC-3 cells (Figure 4C,D). However, resveratrol induced the activation of p-AMPK and p-YAP, and inhibition of YAP was restored by AMPK knockdown (Figure 4C,D). Additionally, the immunofluorescence results indicated that the nuclear translocation and total level of YAP was inhibited by resveratrol and that this inhibition effect was restored by AMPK knockdown ( Figure 4E). Together, these data suggest that AMPK signaling is involved in resveratrol-suppressed YAP expression in pancreatic cancer cells.

Knockdown of YAP Increased Gemcitabine Sensitivity in Pancreatic Cancer Cells
First, we used the MTT assay to examine the effects of gemcitabine on the proliferation of the Panc-1 and BxPC-3 cell lines. As shown in Figure 5A,B, we found that BxPC-3 was sensitive whereas Panc-1 was resistant to gemcitabine, in accord with previous findings [33]. Therefore, we used Panc-1 in further experiments. To evaluate the effects of YAP on cell survival and resistance to chemotherapy, we treated Panc-1 cells, which express high levels of YAP natively and are resistant to gemcitabine, with 2 µM gemcitabine in the presence of either siControl or siYAP and confirmed the silencing of YAP in the cells using Western blot analysis ( Figure 5H). MTT assay results showed that the proliferation capacity was lower in siYAP cells than in siControl cells after treating them with gemcitabine ( Figure 5C). Furthermore, silencing of YAP increased the apoptotic response to treatment with gemcitabine (2 µM) in Panc-1 cells ( Figure 5D,E). The clone ability was significantly decreased after silencing YAP, and siYAP enhanced the gemcitabine inhibition effect on clone ability ( Figure 5F,G). Together, these data suggest that YAP silencing enhances the sensitivity of gemcitabine in gemcitabine-resistant pancreatic cancer cells.

Knockdown of AMPK Rescues Resveratrol Induced Suppression of YAP in Pancreatic Cancer Cells
Previous studies have established that resveratrol can activate the AMPK pathway [31]. The activation of AMPK leads to the suppression of YAP expression [32]. Based on the abovementioned promising findings, we speculated that the effect of resveratrol on cancer cell YAP inhibition may be mediated by AMPK signaling. To test this hypothesis, we further examined the effect of resveratrol on the activity of AMPK signaling. Immunoblotting results revealed that the phosphorylation level of AMPK (p-AMPK) in pancreatic cancer cells was significantly increased in response to resveratrol treatment in a dose-dependent manner ( Figure 4A,B). To verify that resveratrol-inhibited YAP expression in cancer cells is mediated by AMPK signaling, siRNA technology was developed to knock down AMPK expression. We found that knocking down AMPK expression alone did not affect the expression of YAP or the phosphorylation level of p-YAP in Panc-1 and BxPC-3 cells ( Figure  4C,D). However, resveratrol induced the activation of p-AMPK and p-YAP, and inhibition of YAP was restored by AMPK knockdown (Figure 4C,D). Additionally, the immunofluorescence results indicated that the nuclear translocation and total level of YAP was inhibited by resveratrol and that this inhibition effect was restored by AMPK knockdown ( Figure 4E). Together, these data suggest that AMPK signaling is involved in resveratrol-suppressed YAP expression in pancreatic cancer cells.

Knockdown of YAP Increased Gemcitabine Sensitivity in Pancreatic Cancer Cells
First, we used the MTT assay to examine the effects of gemcitabine on the proliferation of the Panc-1 and BxPC-3 cell lines. As shown in Figure 5A,B, we found that BxPC-3 was sensitive whereas Panc-1 was resistant to gemcitabine, in accord with previous findings [33]. Therefore, we used Panc-1 in further experiments. To evaluate the effects of YAP on cell survival and resistance to chemotherapy, we treated Panc-1 cells, which express high levels of YAP natively and are resistant to gemcitabine, with 2 μM gemcitabine in the presence of either siControl or siYAP and confirmed the silencing of YAP in the cells using Western blot analysis ( Figure 5H). MTT assay results showed that the proliferation capacity was lower in siYAP cells than in siControl cells after treating them with gemcitabine ( Figure 5C). Furthermore, silencing of YAP increased the apoptotic response to treatment with gemcitabine (2 μM) in Panc-1 cells ( Figure 5D,E). The clone ability was significantly decreased after silencing YAP, and siYAP enhanced the gemcitabine inhibition effect on clone ability ( Figure 5F,G). Together, these data suggest that YAP silencing enhances the sensitivity of gemcitabine in gemcitabine-resistant pancreatic cancer cells.

Inhibition of YAP Activity by Resveratrol Enhanced the Sensitivity of Pancreatic Cancer Cells to Gemcitabine
To determine whether resveratrol increases the susceptibility of Panc-1 cells to gemcitabine, we treated Panc-1 cells with 50 μM resveratrol and 2 μM gemcitabine. The MTT assay results showed that the proliferation capacity was significantly lower in the resveratrol and gemcitabine therapy group than in the resveratrol alone or gemcitabine alone group ( Figure 6A). The protein expression of YAP and p-YAP (Ser127) in Panc-1 exposed to resveratrol and gemcitabine was evaluated by Western blot analysis. As shown in Figure 6B, resveratrol treatment up-regulated the level of p-YAP (Ser127), and the total level of YAP was significantly inhibited by resveratrol. However, gemcitabine had no effect on YAP expression. Next, we measured the apoptosis rate in Panc-1 cells by flow cytometry. Flow cytometric analyses were conducted after Panc-1 cells were treated with or without resveratrol (50 μM) and gemcitabine (2 μM) for 48 h. As shown in Figure 6C,D, treatment of cancer cells with resveratrol caused an increase in apoptotic population compared with the untreated control cells, but almost all cells underwent apoptosis in the resveratrol and gemcitabine combined therapy group. Next, we detected the clone formation ability of Panc-1 cancer cells after treatment with resveratrol and gemcitabine. As shown in Figure 6E,F, treatment with resveratrol and gemcitabine markedly decreased the number of colonies compared with the number measured for the gemcitabine or resveratrol alone cells. Taken together, these results demonstrate that resveratrol has

Inhibition of YAP Activity by Resveratrol Enhanced the Sensitivity of Pancreatic Cancer Cells to Gemcitabine
To determine whether resveratrol increases the susceptibility of Panc-1 cells to gemcitabine, we treated Panc-1 cells with 50 µM resveratrol and 2 µM gemcitabine. The MTT assay results showed that the proliferation capacity was significantly lower in the resveratrol and gemcitabine therapy group than in the resveratrol alone or gemcitabine alone group ( Figure 6A). The protein expression of YAP and p-YAP (Ser127) in Panc-1 exposed to resveratrol and gemcitabine was evaluated by Western blot analysis. As shown in Figure 6B, resveratrol treatment up-regulated the level of p-YAP (Ser127), and the total level of YAP was significantly inhibited by resveratrol. However, gemcitabine had no effect on YAP expression. Next, we measured the apoptosis rate in Panc-1 cells by flow cytometry. Flow cytometric analyses were conducted after Panc-1 cells were treated with or without resveratrol (50 µM) and gemcitabine (2 µM) for 48 h. As shown in Figure 6C,D, treatment of cancer cells with resveratrol caused an increase in apoptotic population compared with the untreated control cells, but almost all cells underwent apoptosis in the resveratrol and gemcitabine combined therapy group. Next, we detected the clone formation ability of Panc-1 cancer cells after treatment with resveratrol and gemcitabine. As shown in Figure 6E,F, treatment with resveratrol and gemcitabine markedly decreased the number of colonies compared with the number measured for the gemcitabine or resveratrol alone cells. Taken together, these results demonstrate that resveratrol has a potent effect in enhancing the sensitivity of pancreatic cancer cells to gemcitabine by inhibiting YAP expression.

Discussion
In this study, we observed that resveratrol inhibited pancreatic cancer cell proliferation and clone formation and induced cell apoptosis, which was accompanied by the activation of p-AMPK and p-YAP and a decreased level of YAP in BxPc-3 and Panc-1 cells. Moreover, the results suggest that AMPK signaling is essential in resveratrol-suppressed YAP expression in pancreatic cancer cells. Furthermore, we found that YAP silencing enhances the sensitivity of gemcitabine in gemcitabineresistant Panc-1 cancer cells. Resveratrol has a synergistic effect with gemcitabine in Panc-1 cells by inhibiting YAP expression. Our findings suggest that resveratrol is a potential drug for adjuvant therapy or a complementary alternative medicine for the management of pancreatic cancers via activation of AMPK and inhibition of YAP.
Despite great advances in modern medicine over the past several years, pancreatic cancer is still associated with an extremely high mortality rate [1]. Currently, gemcitabine [3] and FOLFIRINOX [4] are considered first-line drugs for treating pancreatic cancer, but their efficacy is still low because of

Discussion
In this study, we observed that resveratrol inhibited pancreatic cancer cell proliferation and clone formation and induced cell apoptosis, which was accompanied by the activation of p-AMPK and p-YAP and a decreased level of YAP in BxPc-3 and Panc-1 cells. Moreover, the results suggest that AMPK signaling is essential in resveratrol-suppressed YAP expression in pancreatic cancer cells. Furthermore, we found that YAP silencing enhances the sensitivity of gemcitabine in gemcitabine-resistant Panc-1 cancer cells. Resveratrol has a synergistic effect with gemcitabine in Panc-1 cells by inhibiting YAP expression. Our findings suggest that resveratrol is a potential drug for adjuvant therapy or a complementary alternative medicine for the management of pancreatic cancers via activation of AMPK and inhibition of YAP.
Despite great advances in modern medicine over the past several years, pancreatic cancer is still associated with an extremely high mortality rate [1]. Currently, gemcitabine [3] and FOLFIRINOX [4] are considered first-line drugs for treating pancreatic cancer, but their efficacy is still low because of a seriously adverse reaction and because acquired drug resistance limits their cytotoxic efficacy. Therefore, we must find a novel target for enhancing current chemotherapy to improve the outcomes of patients with pancreatic cancer.
Resveratrol is a natural polyphenolic phytoalexin that is widely found in plants and in traditional Chinese medicines [16,17]. Resveratrol has been shown to directly inhibit the proliferation and viability of human pancreatic cancer cells in vitro in a dose-and time-dependent manner [22]. Accordingly, our results demonstrated that resveratrol inhibited cell proliferation and clone formation and induced cell apoptosis. Interestingly, we found that Panc-1 and BxPC-3 cells respond differently to gemcitabine (BxPC-3 being more sensitive). But their biochemical and functional response to resveratrol appears to be the same. As we know, BxPC-3 and Panc-1 cells have many differences such as K-ras mutation types (BxPC-3 is a K-ras wild cell while Panc-1 is a K-ras mutant cell) [34] and epithelial-mesenchymal transition (EMT) regulated genes (E-cadherin and Zeb-1) [35] which could affect gemcitabine sensitivity in pancreatic cancer cells. Our previous reports have shown that resveratrol could inhibit the EMT of pancreatic cancer cells via suppression of the PI3K/Akt/NF-kappaB pathway in Panc-1 and BxPC-3 cells [22]. Furthermore, resveratrol could also inhibit pancreatic cancer stem cell characteristics in human and Kras (G12D) transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition [36]. However, the specific mechanism needs to be further explored.
The AMPK system is traditionally considered a sensor of cellular energy status and a regulator of metabolism [37]. Recent studies have provided novel evidence that AMPK may function as a suppressor of cell proliferation. Indeed, activation of AMPK has been shown to benefit a variety of malignant tumors by inhibiting the proliferation of tumor cells [38,39]. In this respect, our results demonstrated that resveratrol could activate AMPK (Thr172) and suppress the proliferation of pancreatic cancer cells, indicating the anti-proliferative action of AMPK in pancreatic cancer cells. The effect of AMPK on cell proliferation appears to be mediated through multiple mechanisms, mainly the regulation of cell cycle progression, inhibition of protein synthesis, and de novo fatty acid biosynthesis [40,41]. Previous reports have suggested that AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition [42]. Accordingly, our results demonstrated that resveratrol induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation via activation of AMPK. Knockdown of AMPK rescues resveratrol-induced suppression of YAP in pancreatic cancer cells. Some reports have suggested that glucose starvation and energy stress could result in phosphorylation of YAP and contribute to its inactivation [43]. Harris et al. [44] demonstrated that resveratrol could inhibit glycogen synthesis, which may serve as the underlying mechanism in the inhibition of YAP. However, Jung-Soon Mo [32] and his colleagues also found that AMPK inhibits YAP activity through phosphorylation of serine 94. Whether resveratrol could induce YAP phosphorylation at serine 94 need further investigation.
As an oncogene, YAP is abundantly expressed in many types of cancers [15,27], and tremendous progress has been made toward our understanding of the roles of YAP in tumorigenesis, size control, and stem cell renewal and differentiation [45,46]. However, YAP's function in chemotherapeutic drug response is largely unknown in pancreatic cancer cells. Ciamporcero et al. [47] demonstrated that YAP overexpression protected, whereas YAP knockdown sensitized, urothelial cell carcinoma (UCC) cells to chemotherapy and radiation effects by increasing the accumulation of DNA damage and apoptosis. Verteporfin, a pharmacological YAP inhibitor, could inhibit tumor cell proliferation and restore sensitivity to cisplatin. In our study, we found that the chemotherapeutic sensitivity of pancreatic cancer to gemcitabine can be increased by YAP inhibition in vitro. YAP may participate in regulating the chemosensitivity of pancreatic cancer to chemotherapy. As a dietary and synthetic agent that is a pharmacological YAP inhibitor, resveratrol may exhibit greater efficacy and lower toxicity for the prevention and treatment of pancreatic cancer. However, its more specific mechanisms and safety require further investigation.

Conclusions
In conclusion, the present study demonstrated that resveratrol suppressed the proliferation and cloning ability and induced the apoptosis of pancreatic cancer cells. These multiple biological effects might result from the activation of AMPK (Thr172), thus, inducing YAP cytoplasmic retention and S127 phosphorylation, and inhibiting YAP transcriptional activity by resveratrol. YAP silencing by siRNA or resveratrol could enhance the sensitivity of gemcitabine in pancreatic cancer cells. These results suggest that resveratrol is a potential anticancer agent for the treatment of pancreatic cancer. However, whether other mechanisms are involved in the anti-tumor effects of resveratrol and its safety in humans warrant further study.