Pterostilbene Enhances Cytotoxicity and Chemosensitivity in Human Pancreatic Cancer Cells

Gemcitabine (GEM) drug resistance causes high mortality rates and poor outcomes in pancreatic ductal adenocarcinoma (PDAC) patients. Receptor for advanced glycation end products (RAGE) involvement in the GEM resistance process has been demonstrated. Therefore, finding a safe and effective way to inhibit receptors for RAGE-initiated GEM resistance is urgent. Pterostilbene (PTE), a natural methoxylated analogue derived from resveratrol and found in grapes and blueberries, has diverse bioactivities, such as antioxidative, anti-inflammatory, and anticancer qualities. The overall research objective was to determine the potential of PTE to enhance tumor cytotoxicity and chemosensitivity in PDAC cells. Our results have demonstrated that PTE induced S-phase cell cycle arrest, apoptosis, and autophagic cell death and inhibited multidrug resistance protein 1 (MDR1) expression by downregulating RAGE/PI3K/Akt signaling in both MIA PaCa-2 and MIA PaCa-2 GEMR cells (GEM-resistant cells). Remarkably, convincing evidence was established by RAGE small interfering RNA transfection. Taken together, our study demonstrated that PTE promoted chemosensitivity by inhibiting cell proliferation and MDR1 expression via the RAGE/PI3K/Akt axis in PDAC cells. The observations in these experiments indicate that PTE may play a crucial role in MDR1 modulation for PDAC treatment.


Introduction
Over 90% of pancreatic ductal adenocarcinomas (PDACs) are exocrine pancreatic cancer, which is a highly malignant tumor that with the growing cause of cancer-related mortality worldwide. In the early stages of PDAC, the signs or symptoms are rarely noticeable, and patients often have local invasion or distant metastasis at diagnosis. Unfortunately, less than 15%-20% of stage I-II PDAC patients are resectable due to most PDAC patients have carcinoma in-situ intravasation, perineural invasion, and distant metastasis at diagnosis [1]. The poor prognosis and outcomes of PDAC patients lead to a low five-year survival rate (6%) and a high incidence [2]. A report by the American Association for Cancer Research (AACR) estimated that PDAC may grow into the second leading cause of cancer-associated deaths by 2030 [3]. Thus, PDAC is a silent disease that remains a therapeutic challenge.
Gemcitabine (GEM), a deoxycytidine nucleoside analogue for cancer DNA synthesis obstruction, is the most common chemotherapeutic drug for PDAC treatment. However, approximately 75% of

Cell Cycle Measurement
The cells were incubated with different concentrations of PTE for 72 h. The cell cycle was measured by propidium iodide (PI) staining and analyzed by FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) [17].

RAGE siRNA Transfection
The procedure was performed as reported previously [17]. Briefly, cells at a density of 1 × 10 5 cells/mL were cultured on 6-well plates overnight and then incubated with 25 nM non-targeting control siRNA or RAGE siRNA for 24 h. The knockdown efficiency was verified by western blot analysis.

Statistical Analysis
Experimental results were repeated in three independent operations and quantified as the mean ± standard deviation (SD). Data were analyzed by Student's t test using SPSS 16.0 statistical software (IBM Corporation, Armonk, NY, USA).

PTE Induced S-Phase Cell Cycle Arrest in PDAC Cell Lines
A stable GEM-tolerant MIA PaCa-2 GEMR cell line that can resist 0.5 µM GEM-induced cytotoxicity was established ( Figure 1A,B). To assess the cytotoxicity effect triggered by PTE in both MIA PaCa-2 and MIA PaCa-2 GEMR cells. Cells were treated with PTE at different concentrations (0, 5, 10, 25, 50, and 75 µM) for 48 or 72 h. Cell proliferation suppressed by PTE treatment in a time-and dose-response manner was observed by MTT analysis ( Figure 1C, D). The IC 50 values of PTE in MIA PaCa-2 and MIA PaCa-2 GEMR cells were 41.8 and 42.0 µM (72 h), respectively. The spindle-shaped morphology and loss of viability by PTE treatment for 72 h compared with untreated cells are shown in Figure 1E. In addition, the detailed role of PTE on cell proliferation was validated by cell cycle analysis. Cell cycle analysis with propidium iodide (PI) staining showed that S-phase arrest was induced in PTE-treated MIA PaCa-2 cells compared to untreated cells in a dose-dependent manner ( Figure 2A). Similar results were observed in GEM-resistant cells ( Figure 2B), indicating that cell proliferation inhibition was induced via PTE-induced S-phase cell cycle arrest in both cell types.  . P values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control. . p values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control. . p values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control.

PTE Triggered Apoptotic and Autophagic Cell Death in PDAC Cell Lines
A recent report found that PTE induces cell cycle arrest-mediated apoptotic progression in ovarian cancer [20]. Regarding the anticancer characteristics of PTE, our study demonstrated that PTE treatment degraded Bcl-xL and elevated Bax protein expression in a dose-dependent manner in both cell lines ( Figure 3). Our research also focused on PTE in autophagic cell death modulation. As shown in Figure 4A-D, PTE significantly enhanced Atg5, Beclin-1, and LC3-II protein expression in MIA PaCa-2 cells. Moreover, the protein levels of Atg5 and Beclin-1 were increased by PTE treatment in a dose-dependent manner but did not reach statistical significance in MIA PaCa-2 GEMR cells ( Figure 4E-G). Nevertheless, LC3-II significantly increased in MIA PaCa-2 GEMR cells subjected to 75 µM PTE treatment compared to the untreated cells ( Figure 4E,H). These results suggested that PTE induced apoptosis and autophagy in parental and GEM-resistant PDAC cells. . P values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control.

PTE Triggered Apoptotic and Autophagic Cell Death in PDAC Cell Lines
A recent report found that PTE induces cell cycle arrest-mediated apoptotic progression in ovarian cancer [20]. Regarding the anticancer characteristics of PTE, our study demonstrated that PTE treatment degraded Bcl-xL and elevated Bax protein expression in a dose-dependent manner in both cell lines ( Figure 3). Our research also focused on PTE in autophagic cell death modulation. As shown in Figure 4A-D, PTE significantly enhanced Atg5, Beclin-1, and LC3-II protein expression in MIA PaCa-2 cells. Moreover, the protein levels of Atg5 and Beclin-1 were increased by PTE treatment in a dose-dependent manner but did not reach statistical significance in MIA PaCa-2 GEMR cells ( Figure  4E-G). Nevertheless, LC3-II significantly increased in MIA PaCa-2 GEMR cells subjected to 75 μM PTE treatment compared to the untreated cells ( Figure 4E,H). These results suggested that PTE induced apoptosis and autophagy in parental and GEM-resistant PDAC cells. . p values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control. Effect of pterostilbene on apoptosis-related protein expression in MIA PaCa-2 cells and MIA PaCa-2 GEMR cells. Cells were treated with 50 and 75 μM pterostilbene for 72 h, and the expression levels of Bax and Bcl-xL protein in (A) MIA PaCa-2 and (E) MIA PaCa-2 GEMR cells were analyzed by western blotting. The relative expression levels of (B,F) Bax, (C,G) Bcl-xL, and (D,H) the ratio of Bax/Bcl-xL are expressed as the mean ± SD (n = 3). P values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control.  . palues were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control.

Autophagy Induction Was Required for PI3K/Akt Signaling Pathway Inhibition
Activation of the PI3K/Akt cascade is highly related to carcinogenesis, tumor progression, and chemoresistance. Targeting the PI3K/Akt signaling pathway is an ongoing effort in several clinical trials [21,22]. Currently, our evidence shows that quercetin treatment or RAGE silencing significantly increases apoptosis, autophagy, and chemosensitivity by suppressing the PI3K/Akt axis in PDAC [17]. Here, our results showed that 75 µM PTE treatment significantly inhibited the phosphorylation activities of PI3K and Akt proteins in both cell lines ( Figure 5).

Autophagy Induction Was Required for PI3K/Akt Signaling Pathway Inhibition
Activation of the PI3K/Akt cascade is highly related to carcinogenesis, tumor progression, and chemoresistance. Targeting the PI3K/Akt signaling pathway is an ongoing effort in several clinical trials [21,22]. Currently, our evidence shows that quercetin treatment or RAGE silencing significantly increases apoptosis, autophagy, and chemosensitivity by suppressing the PI3K/Akt axis in PDAC [17]. Here, our results showed that 75 μM PTE treatment significantly inhibited the phosphorylation activities of PI3K and Akt proteins in both cell lines ( Figure 5).  . p values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated control.

Higher RAGE and MDR1 Protein Levels Were Found in GEM-Resistant PDAC Cells
High RAGE expression is correlated with chemotherapy resistance in PDAC patients [23,24]. Although the underlying molecular mechanism is still unclear, our current evidence demonstrates that abundant RAGE expression in MIA PaCa-2 GEMR cells consequently enhances GEM chemoresistance [17]. Similarly, higher RAGE protein expression was observed in GEM-resistant cells compared to MIA PaCa-2 cells in this study ( Figure 6A). In addition, a high level of MDR1 protein expression was found in MIA PaCa-2 GEMR cells ( Figure 6B). Apoptosis and autophagy are both key mediators in chemotherapy-induced cancer cell death [25,26]. RAGE and MDR1 protein expression, as well as cell proliferation were assessed to validate the effect of PTE on chemosensitivity in PDAC. RAGE expression was suppressed in PTE-treated MIA PaCa-2 cells compared to untreated cells ( Figure 6C). In addition, PTE treatment significantly repressed RAGE protein levels in a dose-dependent manner in MIA PaCa-2 GEMR cells ( Figure 6D). Interestingly, MDR1 protein expression was reduced by 75 µM PTE treatment in both cell lines ( Figure 6E,F).

MDR1 Expression Was Upregulated by the RAGE-Initiated PI3K/Akt Signaling Pathway
We next examined whether MDR1 protein expression was regulated by the RAGE-initiated PI3K/Akt signaling pathway. Knocking down RAGE expression significantly reduced PI3K and Akt phosphorylation levels and consequently inhibited MDR1 expression in MIA PaCa-2 cells (Figure 7).

Chemosensitivity Induced by PTE Treatment
To examine whether chemosensitivity was enhanced by PTE incubation, the inhibitory effect of PTE combined with GEM on cell proliferation was assessed by MTT assay. In Figure 9A, treatment with 0.5 µM GEM dramatically prohibited cell proliferation in MIA PaCa-2 cells compared to untreated cells. Moreover, cell proliferation was inhibited 50 and 75 µM PTE-pretreated MIA PaCa-2 cells ( Figure 9A). Not surprisingly, GEM treatment led to extensive cell death in 50 and 75 µM PTE-pretreated MIA PaCa-2 cells ( Figure 9A). Remarkably, treatment with PTE combined with GEM significantly reduced cell viability compared with that in GEM-treated MIA PaCa-2 cells ( Figure 9A). Chemosensitivity was also evaluated in PTE-pretreated GEM-resistant cells. As anticipated, the cell viability of MIA PaCa-2 GEMR cells was not influenced by GEM treatment ( Figure 9B). Cell proliferation was inhibited in PTE-treated cells compared to untreated MIA PaCa-2 GEMR cells in a dose-dependent manner ( Figure 9B). In addition, PTE combined with GEM notably enhanced MIA PaCa-2 GEMR cell cytotoxicity compared to that with GEM treatment alone or PTE pretreatment alone ( Figure 9B).

Chemosensitivity Induced by PTE Treatment
To examine whether chemosensitivity was enhanced by PTE incubation, the inhibitory effect of PTE combined with GEM on cell proliferation was assessed by MTT assay. In Figure 9A, treatment with 0.5 μM GEM dramatically prohibited cell proliferation in MIA PaCa-2 cells compared to untreated cells. Moreover, cell proliferation was inhibited 50 and 75 μM PTE-pretreated MIA PaCa-2 cells ( Figure 9A). Not surprisingly, GEM treatment led to extensive cell death in 50 and 75 μM PTEpretreated MIA PaCa-2 cells ( Figure 9A). Remarkably, treatment with PTE combined with GEM significantly reduced cell viability compared with that in GEM-treated MIA PaCa-2 cells ( Figure 9A). Chemosensitivity was also evaluated in PTE-pretreated GEM-resistant cells. As anticipated, the cell viability of MIA PaCa-2 GEMR cells was not influenced by GEM treatment ( Figure 9B). Cell proliferation was inhibited in PTE-treated cells compared to untreated MIA PaCa-2 GEMR cells in a dose-dependent manner ( Figure 9B). In addition, PTE combined with GEM notably enhanced MIA PaCa-2 GEMR cell cytotoxicity compared to that with GEM treatment alone or PTE pretreatment alone ( Figure 9B). . P values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with untreated control. The values were considered statistically significant at †p < 0.05 and † † †p < 0.001, compared with the gemcitabine group. The values were considered statistically significant at ‡p < 0.05, ‡ ‡p < 0.01 and ‡ ‡ ‡p < 0.001, compared with the pterostilbene group. The results are expressed as the mean ± SD (n = 3). p values were considered statistically significant when *p < 0.05, **p < 0.01, and ***p < 0.001 compared with untreated control. The values were considered statistically significant at †p < 0.05 and † † †p < 0.001, compared with the gemcitabine group. The values were considered statistically significant at ‡p < 0.05, ‡ ‡p < 0.01 and ‡ ‡ ‡p < 0.001, compared with the pterostilbene group.

Discussion
PDAC is a very aggressive form of cancer, and patients have a five-year survival rate lower than 6% because GEM resistance develops within weeks after chemotherapy initiation. Unfortunately, approximately 75% of PDAC patients have GEM resistance, which has a long-lasting effect on global disease burden [4]. Therefore, finding novel effective preventative strategies is urgently required. Cancer chemoprevention with dietary phytochemicals has been discovered because of their high antitumor capacity and limited side effects [27]. PTE, a bioactive component of various Vaccinium berries, has been shown to have a tumor suppression effect in colorectal cancer, lung cancer, and human melanomas [28][29][30]. Likewise, our previous reports demonstrated that PTE restricts breast cancer cell motility, invasion, and metastasis via modulation of the tumor microenvironment and epithelial-mesenchymal transition (EMT)-associated signaling pathways [14,31]. In the present study, we found that the chemopreventive effect of PTE occurs through inducing apoptosis and autophagy in both MIA PaCa-2 cells and MIA PaCa-2 GEMR cells.
Some studies indicate that cell cycle arrest in cancer cells can eventually induce apoptosis [32,33]. Enhancement of cell cycle arrest is a successful strategy for attenuating of cancer development and progression. Our results showed that 25-75 µM PTE exhibited a powerful cytotoxic effect that not only resulted in S-phase cell cycle arrest, but also increased apoptosis-associated protein expression in a dose-dependent manner in both PDAC cell lines. Consistently, other reports also found that PTE triggers S-phase cell cycle arrest and apoptosis in colon cancer cells and diffuse large B-cell lymphoma cells (DLBCL) [34,35]. Next, our research results were in agreement with previous reports demonstrating that PTE treatment induces autophagy and inhibits tumor progression in human bladder cancer and hepatocellular carcinoma cells [13,36]. For example, PTE treatment significantly increases autophagy-associated protein LC3-II and Bcl-xl (negative regulator of apoptosis) levels through AKT/mTOR/p70S6K axis regulation in T24 human bladder cancer cells [13]. In the present study, similar promoting effects on autophagy and apoptosis were observed in PTE-treated cells. However, another study showed that PTE inhibits human hepatocellular carcinoma cell growth by apoptosis-independent signaling [36]. Lifting endoplasmic reticulum (ER) stress accelerates PTE-induced autophagy in human hepatocellular carcinoma cells, indicating that tumor-specific cytotoxicity responses such as cell cycle arrest, ER stress, apoptosis, and autophagy may be elicited by PTE treatment.
Although PTE-induced antioxidant-, anti-inflammatory-and tumor cytotoxicity-related mechanisms have been established, the underlying mechanism of PTE-mediated chemosensitivity enhancement in PDAC is still unclear. A previous report showed that PTE induces apoptosis and autophagy in chemoresistant lung cancer cells and bladder cancer cells, which is initiated by the suppression of AKT signaling [13,15]. Additionally, PTE induces autophagy-and apoptosis-related cell death and inhibits MDR1 expression through Akt-Ser473 phosphorylation in cisplatin-resistant human oral cancer CAL 27 cells [37]. In agreement with this, our results showed that PTE triggered cell cycle arrest and apoptosis-and autophagy-mediated cytotoxicity and inhibited MDR1 expression by regulating the RAGE/PI3K/Akt axis. Although several natural compounds are used in combined therapies against pancreatic cancer such as catechins, resveratrol, and curcumin [38][39][40]. However, few available publications showed that natural compounds directly target MDR1 expression and promote chemosensitivity. Our results support the idea that PTE is an MDR1 modulator that increases GEM-induced tumor cell death in PDAC.
Gemcitabine (GEM) drug resistance causes high mortality rates and poor outcomes in PDAC patients. RAGE has been demonstrated to be involved in the GEM resistance process. Therefore, finding a safe and effective way to inhibit RAGE-initiated GEM resistance is urgent. Pterostilbene (PTE), a natural methoxylated analogue derived from resveratrol and found in grapes and blueberries, has diverse bioactivities such as antioxidative, anti-inflammatory, and anticancer characteristics. Based on our results, it was demonstrated that PTE induced S-phase cell cycle arrest and apoptotic and autophagic cell death and inhibited MDR1 expression by inhibiting the RAGE/PI3K/Akt axis, which consequently enhanced chemosensitivity in both MIA PaCa-2 cells. Remarkably, convincing evidence was established by RAGE small interfering RNA transfection.

Conclusions
Taken together, our study demonstrates that PTE promotes chemosensitivity by inhibiting cell proliferation and MDR1 expression via the RAGE/PI3K/Akt signaling pathway in PDAC cells. The observations in these experiments indicate that PTE may play a pivotal role in MDR1 modulation for PDAC chemoprevention. Funding: This research work was supported in part by the grant MOST 108-2320-B-005-001-from the Ministry of Science and Technology, Taiwan.

Conflicts of Interest:
The authors declare no conflict of interest.