Melatonin Synergizes with Sorafenib to Suppress Pancreatic Cancer via Melatonin Receptor and PDGFR-β/STAT3 Pathway

Pancreatic ductal adenocarcinoma (PDAC), the fourth-most frequent cause of human cancer-related deaths, is extremely aggressive with a high risk of metastasis, recurrence, and the overall poorest prognosis [1, 2]. Although surgical resection is currently the main therapeutic strategy for PDAC, consequent outcomes are not outstanding [3]. Moreover, conventional chemotherapies including gemcitabine and its combinations with other therapeutic agents have often failed owing to weak responsiveness and the incidence of side effects [4]. These observations have led us to develop a more effective and safer therapeutic approach toward this malignant tumor.

The multi–kinase inhibitor sorafenib (Bay 43-9006, Nexavar) inhibits various signaling pathways including RAF/MEK/ERK cascades, resulting in the suppression of cell proliferation and survival [5]. It also exhibits anti-angiogenic effect through inhibition of various target receptors including VEGFR and PDGFR-β [5]. In addition, it inhibits DNA synthesis and induces tumor cell death in various human cancers [6]. Sorafenib alone and its combination with other drugs has shown potent anti-proliferative and pro-apoptotic efficacy in PDAC cells [7, 8]. Notably, it increases apoptotic cell death through the mitochondrial-dependent intrinsic pathway, which is associated with cytochrome c release from the mitochondria into the cytoplasm [9]. Despite these promising preclinical findings, sorafenib have shown modest results or incomplete therapeutic outcomes in clinical trials due to limited efficacy and drug resistance, as well as countless side effects [10, 11]. Hence, new chemotherapeutic strategies are needed to increase the efficacy of sorafenib and reduce its toxicity. Numerous evidences support combination therapies in cancer, using anticancer chemicals or various supplements [12, 13]. Supplements such as vitamins (C, E, and K) and hormones (estrogen and insulin) have exhibited beneficial anticancer effects when used as a single agent or in combination with conventional anticancer drugs [8, 13]. For example, sorafenib combined with hymecromone, a dietary supplement, was shown to be highly effective in controlling renal cell carcinoma [14].

Melatonin (N-acetyl-5-methoxytryptamine), a commonly taken supplement used as a sleep aid, is a controller of the human circadian rhythm [15]. As a major product of the pineal gland, melatonin is known to regulate immunological function, exert antioxidant properties, and protect against organ injury [16, 17]. It has also been proposed to act as an oncostatic agent against several types of cancer [18, 19]. In addition, combinations of melatonin with various potent anticancer agents, such as cisplatin, 5-fluorouracil (5-FU), and doxorubicin, exhibited strong anticancer effects from in vitro and in vivo studies [20-22]. Accordingly, melatonin is considered as a potential candidate to enhance the anticancer activity of chemotherapeutic agents. In PDAC, melatonin is known to stimulate pro-apoptotic activity through regulation of Bcl-2 family proteins, caspase-3 and caspase-9 in vitro [23]. Moreover, melatonin promoted apoptosis by inducing the intrinsic mitochondrial pathway in rat PDAC cells [24]. Although accumulating evidences support the anti-cancer effect of melatonin, the specific molecular mechanism by which melatonin inhibits tumor is unclear, and uncertainty regarding combinational treatment of melatonin and anticancer drugs remains.

Recently, combined treatment of sorafenib and melatonin was reported to exert synergistic anticancer activity via the JNK/c-Jun pathway in hepatocellular carcinoma [25]. However, these agents have never been tested to treat PDAC. Given the enhancing effect of melatonin on sorafenib efficacy in other cancer types, we sought to determine whether these two agents act synergistically against PDAC and investigated the underlying mechanism of action.

MIAPaCa-2 and PANC-1 human PDAC cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37°C in a humidified atmosphere composed of 95% air and 5% CO₂. Sorafenib (LC Laboratories, Woburn, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM before use. Melatonin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 2 M before use (final DMSO concentration, < 0.1%).

Cell viability was assessed using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cells were plated at a density of 6x10³ cells per well in 96-well plates and incubated for 24 h. The medium was then removed, and cells were treated with different concentrations of sorafenib (1-10 µM) alone or with melatonin (0.1, 1, or 2 mM) for 48 h. Next, 20 µl of an MTT solution (2 mg/mL) was added to each well, and the plate was incubated at 37°C for an additional 4 h. The formazan crystals that formed in the cells were dissolved in DMSO (200 µl per well) with constant shaking for 30 min. Absorbance of the solution was then measured with a microplate reader at 540 nm. This assay was conducted in triplicate, and a combination index for sorafenib and melatonin was calculated using CompuSyn v1.0 (Biosoft, Cambridge, USA) according to the method of Chou and Talalay, where CI < 1, CI = 1, and CI > 1 indicate synergistic, additive and antagonistic effects, respectively [26].

After incubating for 24 h with sorafenib (10 µM) and/or melatonin (2 mM), cells were fixed with an acetic acid:ethanol (1: 2) solution for 5 min at -20°C, DNA fragmentation was detected using a terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay, as described by the manufacturer of the ApopTag Peroxidase In Situ Apoptosis Detection (Kit#S7100; Merck Millipore, Temecula, CA, USA).

MIAPaCa-2 cells were plated on 18-mm glass coverslips and grown to approximately 70% confluence. The cells were treated with sorafenib and melatonin, alone and in combination for 24 h, then permeabilized, blocked and incubated with anti-Annexin V primary antibody (Abcam, Cambridge, USA) at 4°C overnight. The cells were incubated with fluorescence-conjugated anti-mouse secondary antibody for 1 h and then stained with DAPI (4’,6-diamidino-2-phenylindole) solution (NucBlue; Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark at room temperature. The cells were mounted with DABCO (Sigma-Aldrich, St. Louis, MO, USA) and viewed under a confocal laser-scanning microscope (Fluo View 1000; Olympus, Tokyo, Japan).

Total cellular proteins were extracted with sterile RIPA lysis buffer (Biosesang, Korea) consisting of 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, and 2 mM EDTA supplemented with Xpert Protease & Phosphatase Inhibitor Cocktail (GenDEPOT, Barker, TX, USA) solution (100X) prior to use. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were incubated first with diluted primary antibodies overnight with gentle shaking at 4°C, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies. Thereafter, membranes were washed three times in PBS containing 0.1% Tween-20 (PBST), and antigen-antibody complexes were visualized using enhanced chemiluminescence reagents (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA, USA). Antibodies against Survivin, Bcl-2, PARP, phosphorylated CRAF (p-CRAF), p-MEK, MEK, p-ERK, ERK, p-PDGFR-β, p-STAT3, STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA), and XIAP, CRAF, SOD-2, PDGFR-β from Santa Cruz Biotechnology (Dallas, TX, USA), VDAC-1 from Abcam (Cambridge, USA), p-DAPK1 from GeneTex (Irvine, CA, USA ), and β-actin from Sigma-Aldrich (St. Louis, MO, USA). Secondary goat anti-rabbit, goat anti-mouse, and donkey anti-goat antibodies were purchased from Santa Cruz Biotechnology.

MIAPaCa-2 cells were plated on 18-mm glass coverslips and grown to approximately 70% confluence. The cells were treated with sorafenib and melatonin, alone and in combination for 12 h, then permeabilized, and incubated with 500 nM MitoTracker Red probe (Molecular Probes Inc., Eugene, OR, USA) for 30 min at 37°C. Next, the cells were washed twice with PBS, fixed in an acetic acid:ethanol (1: 2) solution for 10 min at -20°C, and then incubated overnight at 4°C with an anti-cytochrome c antibody (1: 50; Santa Cruz Biotechnologies, Dallas, TX, USA). On the following day, the cells were washed twice with PBS and incubated with fluorescence-labeled anti-mouse secondary antibody (1: 100; Dianova, Hamburg, Germany) for 1 h at room temperature. The cells were also stained with DAPI to visualize nuclei. The slides were then washed twice with PBS, mounted with DABCO (Sigma-Aldrich, St. Louis, MO, USA), and viewed under a confocal laser-scanning microscope (Fluo View 1000; Olympus).

MIAPaCa-2 cells were plated on 18-mm glass coverslips and grown to approximately 70% confluence. The cells were then treated with sorafenib and melatonin, alone and in combination for 16 h, followed by incubation with JC-1 solution (Cayman Chemical, Ann Arbor, MI, USA) for 20 min at 37°C in the dark. After staining with DAPI to visualize nuclei, slides were washed twice with PBS, mounted with DABCO (Sigma-Aldrich, St. Louis, MO, USA), and viewed by confocal laser-scanning microscopy (Fluo View 1000; Olympus).

MIAPaCa-2 cells were plated on 18-mm glass coverslips, grown to approximately 70% confluence, and treated with sorafenib and melatonin, alone and in combination for 24 h. Cells were then permeabilized with 0.5% Triton X-100, blocked with 0.25% goat and 0.25% horse serum for 1 h, and incubated first with an anti-cleaved caspase-3 antibody (1: 20; Cell Signaling Technology, Danvers, MA, USA) overnight and then with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated anti-rabbit secondary antibody (1: 50; Dianova, Hamburg, Germany) for 1 h. Slides were stained by DAPI to visualize nuclei, washed twice with PBS, and mounted with DABCO (Sigma-Aldrich, St. Louis, MO, USA) for confocal laser-scanning microscopy (Fluo View 1000; Olympus).

MIAPaCa-2 cells (5 × 10⁵) were plated in 100-mm culture dishes at ∼70% confluence and incubated at 37°C for 24 h. The next day, cells were treated with sorafenib and melatonin, alone and in combination. Floating and adherent cells were collected and fixed overnight in a cold 70% ethanol at 4°C. After washing with PBS, the cells were stained with 50 µg/mL propidium iodide and 100 µg/mL RNase A for 30 min in the dark at 37°C, and then analyzed by fluorescence-activated cell sorting (FACS) using a FACS Calibur^TM flow cytometer (Becton Dickinson, San Jose, CA, USA) to determine the percentage of cells in the sub-G₁ (apoptotic) phase of the cell cycle. All experiments were performed in triplicate.

Detached cells were washed with cold DPBS, resuspended in hypotonic buffer (5 mM Tris, 5 mM KCl, 1.5 mM MgCl₂, 0.1 mM EGTA, and 1 mM DTT), and homogenized using a glass-Teflon tissue grinder (40–50 strokes). A nuclear fraction (pellet) and a post-nuclear fraction (supernatant) were prepared by centrifugation at 600 × g for 10 min at 4°C. A mitochondrial fraction (pellet) and cytosolic fraction (supernatant) were obtained by centrifugation of the post-nuclear fraction at 7000 × g for 10 min at 4°C. For purification of mitochondria, the pellet was resuspended in a hypotonic buffer and centrifuged for 10 min at 7000 × g. This washing step was repeated two times. Finally, the mitochondrial pellet was resuspended in RIPA buffer.

Cell lysates were prepared by washing cells twice in cold 1× PBS and incubating in NP-40 lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 1x protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) for 30 min at 4°C with gentle rocking. Lysates were cleared by microcentrifugation at 13, 000 r.p.m for 30 min at 4°C. For co-immunoprecipitation, equal amounts of cell lysates were incubated with primary antibodies overnight on an end-over-end tube rotator at 4°C. Pre-washed protein G agarose beads (Santa Cruz Biotechnology) were then added, and tubes were gently rocked at 4°C for an additional 6 h. Complexes were washed twice with cold lysis buffer and examined by immunoblot analysis.

Heterodimerization of the serine/threonine kinases BRAF and CRAF was measured using an in situ proximity ligation assay (PLA). In this assay, a pair of oligonucleotide-labeled secondary antibodies (PLA probes) generates a signal only when the two antibodies are bound in close proximity, allowing the detection of protein–protein interaction in situ. MIAPaCa-2 cells were plated on 18-mm glass coverslips and allowed to adhere overnight at 37°C. Cells were treated with sorafenib and melatonin, alone and in combination for 6 h, then fixed with an acetic acid:ethanol (1: 2) solution for 5 min at -20°C, and then BRAF– CRAF heterodimerization was visualized by PLA using anti-CRAF and anti-BRAF antibodies.

The relative levels of tyrosine phosphorylation of 49 distinct receptor tyrosine kinases (RTKs) were determined using a Human Phospho-RTK Assay kit (R&D Systems, Minneapolis, MN, USA). MIAPaCa-2 cells were treated with sorafenib and melatonin, alone and in combination for 6 h, and protein lysates were prepared as described above. After blocking for 1 h using Array Buffer 1, the arrays were incubated with 800 µg of protein lysates overnight at 4°C, then washed and incubated with an HRP-conjugated anti-phospho-tyrosine detection antibody (1: 5000). The membranes were developed using a Chemi Reagent Mix provided by the manufacturer, and the expression levels were densitometrically quantified using ImageJ software.

Relative phosphorylation levels of 43 distinct kinases were determined using a Human Phospho-Kinase Array kit (R&D Systems). MIAPaCa-2 cells were treated with sorafenib and melatonin, alone and in combination for 6 h, and then lysed as described above. After blocking for 1 h using Array Buffer 1, membranes were incubated with 500 µg of protein lysates overnight at 4°C, then washed and incubated with a streptavidin-HRP detection antibody (1: 5000). Membranes were developed using ECL Western blotting detection reagents (Chemi reagent A and Chemi reagent B) provided by the manufacturer, and the expression levels were densitometrically quantified using ImageJ software.

Xenograft models were prepared by inoculating 5 × 10⁶ MIAPaCa-2 cells suspended in PBS (200 µL per mouse) in the flank of 6-wk-old male BALB/c nude mice (Orient Bio, Korea). After tumors reached approximately 100 mm³ in size, mice were randomly divided into four groups: group 1, vehicle (control); group 2, sorafenib only (10 mg/kg); group 3, melatonin only (40 mg/kg); group 4, combined sorafenib and melatonin, and were intraperitoneally administered once daily for 27 days. Tumor size was measured every 2 days, and tumor volume was calculated using the formula, 0.5 × length × width². All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Inha University Medical School (INHA IACUC).

Paraffin-embedded sections of tumors were blocked with normal goat or horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h, and incubated at 4°C overnight in 1: 50 dilutions of primary antibodies against cleaved caspase-3 and p-STAT3 (Cell Signaling Technology); proliferating cell nuclear antigen (PCNA; Abcam); and p-PDGFR-β (Santa Cruz Biotechnology). Sections were then incubated with biotinylated secondary antibodies (1: 100) for 1 h. Immunoreactive proteins were visualized by first applying an avidin–biotin peroxidase complex solution (ABC kit; Vector Laboratories, Burlingame, CA, USA), then washing in PBS and developing with a diaminobenzidine tetrahydrochloride (DAB) substrate for 15 min with subsequent hematoxylin counterstaining. At least three random fields in each section were examined at a magnification of 200×.

Data are expressed as means ± standard deviation (S.D.). The significant differences among and between means was evaluated using analysis of variance (ANOVA) or unpaired Student's t-test, as appropriate. P-values < 0.05 were considered statistically significant. Statistical analyses were performed using SPSS software for the MS Windows operating system (version 10.0; SPSS, Chicago, IL, USA).

We evaluated anticancer activity of sorafenib and melatonin in human PDAC cells using a cell viability assay. Cells were treated with different concentrations of sorafenib (1, 5, 10 µM), melatonin (0.1, 1, or 2 mM), or both for 48 h. Compared to treatment with either agent alone, co-treatment with both agents caused greater inhibition of cell growth (Fig. 1A). To identify synergistic effects of sorafenib and melatonin, we calculated combination index (CI) values using CompuSyn v1.0 (Biosoft). As shown in Fig. 1B, sorafenib and melatonin produced significant synergistic effects, with CI values < 1 for the combination of 10 mµ sorafenib and 2 mM melatonin in MIAPaCa-2 (CI = 0.053) and PANC-1 (CI = 0.828) cells.

Next, we examined whether the combination of sorafenib (10 µM) and melatonin (2 mM) induced apoptosis in MIAPaCa-2 cells. The combined treatment induced large numbers of TUNEL-positive cells compared with single treatment with either agent, which produced few apoptotic cells (Fig. 2A). Annexin-V staining assay, performed in parallel with TUNEL assays to measure DNA damage, showed that the combined treatment induced a higher level of apoptosis than either agent alone (Fig. 2B). And cell cycle analyses showed that the combined treatment also produced a significant increase in the sub-G1, apoptotic population (∼2.8-fold) compared with either agent alone group (Fig. 2C).

Based on the apoptosisinducing effects of combination treatment, we next assessed whether the combined treatment altered the mitochondrial membrane potential in MIAPaCa-2 cells, using the membrane potentialsensitive fluorescent dye JC-1. In control cells, JC-1 accumulated in mitochondria as aggregates that emitted a red fluorescence in live cells, whereas the monomeric form which emitted a green fluorescence was prevalent in the cytoplasm of dead cells (Fig. 3A). As shown in Fig. 3A, combined treatment induced marked changes in mitochondrial membrane potential, as evidenced by a clear decrease in red fluorescence or increase in green fluorescence in most cells. Because changes in mitochondrial membrane potential are known to cause the release of cytochrome c from the inner membrane of the mitochondria into the cytoplasm, thereby initiating the intrinsic apoptosis pathway, we also examined cytochrome c staining. As expected, the combined treatment significantly increased the release of cytochrome c, with a concomitant decrease in the co-localization of cytochrome c and mitochondria (Fig. 3B).

To further confirm that the combined treatment induces apoptosis through a mitochondria-mediated pathway, we analyzed expression of mitochondrial proteins that regulate apoptosis. Upon treatment with sorafenib or melatonin, the anti-apoptotic factors, the expression of Bcl-2, Survivin, Mcl-1, and XIAP were suppressed, whereas the proapoptotic factor PARP exhibited greater cleavage in the combined treatment group than either single agent treatment group (Fig. 3D), resulting in an increase in cleaved caspase-3, a dominant player in apoptosis (Fig. 3C). Collectively, our results strongly suggest that the synergistic effects of combined treatment on apoptosis are mediated by the mitochondrial pathway in PDAC cells.

To investigate the mechanisms by which the combination of sorafenib and melatonin caused anticancer activity in PDAC, we first tested the effect on the RAF/MEK/ERK pathway, the target of the antiproliferative activity of sorafenib [27]. Unexpectedly, both sorafenib alone and in combination with melatonin activated rather than inhibited the RAF/MEK/ ERK pathway, as evidenced by increased levels of p-CRAF, p-MEK, and p-ERK (Fig. 4A). Similar results were obtained in other cancer cells harboring KRAS mutant as shown in Fig. 4B, consistent with the contradictory effects of sorafenib on the RAF/MEK/ERK pathway in PDAC cells containing KRAS mutants. In addition to critical cytoplasmic functions, CRAF in mitochondria is also essential for the regulation of mitochondrial shape and cellular distribution [28]. In addition, co-localization of CRAF with p-DAPK (death-associated protein kinase) in the mitochondria facilitates mitochondrial remodeling [28]. Indeed, recent studies on renal cell carcinoma have shown that combined treatment of sorafenib and another RAF inhibitor induces anticancer effects by targeting mitochondrial CRAF [29]. Therefore, we isolated mitochondria and examined mitochondrial CRAF as well as DAPK phosphorylation. However, contrary to expectation, neither sorafenib nor combined treatment decreased the mitochondrial activity of CRAF or DAPK (Fig. 4C).

Because RAF dimerization is a critical determinant of the effects of RAF inhibitors [30], we next investigated whether the combination of sorafenib and melatonin inhibited RAF dimerization in PDAC cells. Co-immunoprecipitation analyses showed that neither combination treatment nor sorafenib alone reduced BRAF-CRAF heterodimerization, but instead increased it, which was further validated by in situ PLA assays (Fig. 4D).

Collectively, these results indicate that the effects of combined treatment of sorafenib and melatonin are not attributable to actions on RAS/RAF/MEK signaling, mitochondrial RAF, or RAF dimerization, suggesting the involvement of other signaling pathways. To identify anticancer mechanisms that are regulated by sorafenib and melatonin in PDAC, we investigated the activation (phosphorylation) status of 49 different RTKs by performing phospho-RTK analyses. Among tested RTKs, PDGFR-β showed the most significant decrease in phosphorylation in response to treatment with sorafenib alone or together with melatonin (Fig. 5A). In addition, we performed a phospho-kinase analysis to determine which other kinases are regulated by sorafenib and its combination. Notably, among the 43 different kinases tested in this assay, p-STAT3 was the most significantly down-regulated by combined treatment (Fig. 5B). These data were validated by Western blotting analysis and immunoprecipitation, which showed that combined treatment also decreased levels of p-PDGFR-β and p-STAT3 and intracellular interaction in both MIAPaCa-2 and PANC-1 cell lines (Fig. 5C and D). Furthermore, combined treatment reduced nuclear translocation of p-STAT3 compared with treatment with either agent alone, as observed by immunofluorescence staining (Fig. 5D). These results suggest that the antiproliferative and proapoptotic effects of the combined treatment may be mediated by the PDGFR-β/STAT3 pathway in PDAC cells.

The physiological activity of melatonin are regulated through its communication with two membrane receptors, melatonin receptor type 1A (MT1) and 1B (MT2), which are coupled to the guanine nucleotide binding proteins (G proteins) [31]. In various types of cancer, melatonin is known to have anti-tumorigenic properties and loss of MT could represent decreased sensitivity to the anti-proliferative properties of melatonin [32, 33]. Thus, we measured expression of melatonin receptors in PDAC cells. As shown in Fig. 6A, the expression of MT1 and MT2 was higher in PDAC cells than in normal cells. Especially, the MT2 expression was significantly elevated in all PDAC cells compared with normal cells; human umbilical endothelial cells (HUVEC) and normal pancreatic epithelial cells (HPNE). Based on the evidences that melatonin inhibited activation of STAT-3 and MT was associated with activation of STAT-3 [34, 35], we next identified that melatonin inhibited MT-mediated STAT3 activation in PDAC cells. For this experiment, PDAC cells were treated with melatonin in the presence or absence of luzindole, a MT1/MT2 antagonist. As expected, the decrease of STAT3 expression by melatonin was completely rescued by MT inhibition of luzindole, suggesting that down-regulation of STAT3 by melatonin in combination treatment is mediated via MT as well as PDGFR-β (Fig. 6B).

To test the effect of combined treatment on tumor growth in vivo, we used a MIAPaCa-2 xenograft model. As shown in Fig. 7, tumor growth in mice treated with melatonin or sorafenib alone was only slightly delayed compared with the control group. In contrast, combination treatment resulted in tremendous antitumor effects. In addition, no significant changes in body weight were observed, indicating that the combination treatment regimen possesses a good safety profile, even with multiple administrations. To further confirm that the combination effectively inhibits tumor growth by inducing apoptosis, we investigated the expression level of cleaved caspase-3 and determined the number of TUNEL-positive cells in pancreatic tumor tissues. The combined treatment increased cleaved caspase-3 expression and the number of TUNEL-positive cells compared with the control and single-treatment groups. Moreover, combination treatment significantly attenuated intratumoral expression of p-PDGFR-β and p-STAT3 (Fig. 7B).

PDAC is an aggressive malignancy, reflecting its extensive local invasion, subsequent development of metastasis, and resistance to chemo- and radiotherapy. Although gemcitabine has been proposed as a standard chemotherapy, the median survival with this treatment paradigm in advanced PDAC is only ∼5.4 months. With the failure of single-agent chemotherapy, combination therapy has progressively gained research attention. However, previous studies have demonstrated that gemcitabine combined with common anticancer drugs yields poor responses or limited therapeutic effects with prominent side effects [4]. To date, a number of research groups have reported that combination chemotherapy with dietary supplements may lead to beneficial effects in cancer treatment, producing better therapeutic efficacy with fewer side effects [8, 13, 14]. In addition, the combination of sorafenib with other agents has also shown noteworthy results in preclinical studies [14, 36]. However, studies of sorafenib in PDAC, alone or in combination with other agents, are limited.

Given that melatonin appears to exert anticancer effects in different types of cancer cells [36, 37], we hypothesized that the combination of sorafenib and melatonin might achieve synergistic effects in pancreatic cells, and further investigated its mechanism of action. We found that combined treatment significantly inhibited the growth of PDAC cells compared with treatment with either agent alone. As shown in CI values, simultaneous treatment with sorafenib (10 µM) and melatonin (2 mM) showed the highest synergistic effects in PDAC cells. Since each sorafenib or melatonin have been reported to induce apoptosis [8, 21], we also investigated whether the combination treatment causes a synergic apoptotic effect. TUNEL staining showed that the combination induced significant apoptosis, as evidenced by increased TUNEL-positive nuclear fragmentation. In addition, an Annexin-V assay revealed the induction of DNA damage after the combined treatment. These results were further confirmed by an assessment of cell-cycle distribution, which revealed a ∼2-fold increase in the sub-G1, apoptotic cell population.

In general, apoptosis is classified into two different pathways: the extrinsic pathway, mediated by death receptors, and the intrinsic pathway, mediated by mitochondrial factors. In case of the intrinsic apoptotic pathway, a loss of mitochondrial membrane potential, permeabilization of the outer mitochondrial membrane, and consequent release of apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF) are characterized. There are also increasing reports that the combination of sorafenib with other agents triggers the intrinsic mitochondrial apoptotic pathway [9]. We previously reported that the combination of sorafenib and a PI3K inhibitor induces mitochondria-mediated apoptosis by decreasing mitochondrial membrane potential and releasing cytosolic cytochrome c in PDAC cells [38]. Here, we extended this analysis by investigating the involvement of mitochondrial membrane potential in the effects of combined treatment on apoptosis by testing changes in membrane potential and immunofluorescence staining for cytochrome c. We found that the combined treatment synergistically reduced mitochondrial membrane potential and significantly increased cytochrome c. To confirm these results, we investigated the expression of the Bcl-2 family members Bcl-2, Survivin, Mcl-1 and XIAP, as well as inhibitory apoptosis proteins (IAPs) that contribute to mitochondria-mediated apoptosis [39]. These analyses showed that combined treatment significantly decreased the expression of Bcl-2, Survivin, Mcl-1 and XIAP, while increasing cleaved caspase-3 and cleaved PARP levels in PDAC cells. Collectively, our findings demonstrate that the synergistic apoptotic effects of the combined treatment may be regulated by the mitochondrial-mediated apoptotic pathway in PDAC cells.

Sorafenib has been approved as first-line chemotherapy for the clinical management of primary hepatic cellular carcinoma and kidney cancer, and its major target has been identified as RAF, which is involved in the RAF/MEK/ERK signaling cascade [7, 40]. Because the RAF/MEK/ERK pathway is commonly activated in PDAC owing to a high frequency of KRAS mutations, we examined common elements of the RAF/MEK/ERK pathway in both whole-cell lysates and mitochondria after the combined treatment. Notably, it has been shown that mitochondrial p-CRAF, an important modulator of mitochondria structural remodeling, is down-regulated by RAF inhibitors including sorafenib, in renal cell carcinoma [29]. However, we found that neither sorafenib alone nor its combination with melatonin negatively regulated the RAF/MEK/ERK pathway, as evidenced by an increase rather than a decrease in both cytosolic and mitochondrial p-CRAF. This activation of RAF/MEK/ ERK signaling in PDAC is thought to reflect the presence of KRAS mutants in these cancer cells. These results are supported by a study by Holderfield et al, which showed that RAF inhibitors including sorafenib, are less potent inhibitors of RAF/MEK/ERK signaling in cells with activating RAS mutations [41]. Accordingly, we suggest that the antiproliferative and proapoptotic effects of sorafenib alone or combination with melatonin are independent of the RAF/MEK/ERK pathway in PDAC cells (Fig. 4B). Accumulating evidences suggest that RAF inhibitors promote dimerization of RAF in cells expressing mutated KRAS, leading to reconstitution of the MAPK signaling pathway [30, 42]. On the other hand, it is well known that KRAS is the most frequently mutated gene in PDAC (up to ∼95%), but there are few reports of the occurrence of RAF dimerization, and there is still no evidence that sorafenib affects RAF dimerization in PDACs. Hence, we investigated whether effects of melatonin and sorafenib in combination involve inhibition of RAF dimerization in PDAC. Contrary to expectation, we found that sorafenib alone increased RAF dimerization in PDAC cells, and its combination with melatonin did not block RAF dimerization (Fig. 4D). These results indicate that the anticancer effect of the combined treatment is not related to inhibition of RAS/RAF/MEK signaling, mitochondria RAF, or RAF dimerization.

In addition to the RAF/MEK/ERK pathway, sorafenib is known to target various RTKs, including PDGFR-β, VEGFR, c-Kit, and Flt-3 [5]. Thus, to further elucidate the detailed molecular mechanisms underlying the anticancer activity of the combined treatment, we used phospho-RTKs and phospho-kinases array kits, which are representative factors involved in cancer signaling pathways. Among RTKs examined, PDGFR-β was the most significantly decreased by either sorafenib alone or together with melatonin. Additionally, several other effectors including STAT family members and PI3K/AKT, are known to be targets of sorafenib [25, 43, 44], and constitutive STAT3 activation has been shown to be critical for cancer initiation, progression, invasion, and motility of carcinoma cells [45]. In addition, blockade of constitutively STAT3 pathway has inhibited growth of human PDAC [46].

In line with these observations, our analysis of phospho-kinases revealed that the combined treatment regimen decreased p-STAT3 levels. Notably, PDGFR-β is known to induce STAT3 phosphorylation, and activated STAT3 leads to cell transformation [47, 48]. Consequently, elevated STAT3 signaling inhibits apoptosis and supports cancer survival, producing a phenotype similar to chemo-resistance [49]. In the current study, sorafenib alone suppressed p-PDGFR-β and p-STAT3 levels as well as nuclear translocation of STAT3, and the combination treatment completely blocked the PDGFR-β/STAT3 signaling pathway (Fig. 5 and 6). Considering the previous study that sorafenib suppresses STAT3 signaling associated with growth arrest and causes apoptosis [50], it seems that melatonin improves the anticancer activity of sorafenib through PDGFR-β/STAT3 pathway, and not the RAF/ MEK/ERK signaling pathway [49].

Melatonin has been reported to bind and activate two distinct receptor types, MT1 and MT2 [51]. MT1 and MT2 are expressed in various organs and neoplastic cells such as cancer [52]. In numbers of studies, melatonin has shown anti-cancer properties, and loss of MT expression has decreased sensitivity to the anti-proliferative properties of melatonin [32, 33]. Indeed, overexpression of MT1 melatonin receptor enhanced anti-proliferative effect of melatonin in human breast cancer cells [51]. Also, MT is linked to the modulation of STAT3 [53, 54] and inhibition of MT expression has been reported to down-regulate activation of STAT-3 [55]. In addition, melatonin has shown to inhibit tumorigenicity via STAT3 signaling [35]. Considering significant role between MT and STAT3, we expected that melatonin bound with MT in PDAC cells, which inhibited expression of p-STAT-3. Interestingly, we observed that MT1/MT2 was highly expressed in five PDAC cells compared with normal cells, and down-regulation of STAT3 by melatonin was recovered via MT inhibition by MT1/MT2 antagonist. These results imply that melatonin enhances combination effect with sorafenib by inhibition of STAT-3 via MT and PDGFR-β.

A trend similar to in vitro results was confirmed in a mouse xenograft model. Here, sorafenib treatment alone caused negligible inhibition of tumor growth, whereas combination treatment of sorafenib and melatonin exerted potent antitumor effects that surpassed those of either single treatment, confirming the sensitizing effect of melatonin on pancreatic xenografts (Fig. 7A). Moreover, combination treatment showed a good safety profile, similar to either sorafenib or melatonin alone group. Notably, the potent anticancer and proapoptotic effects induced by combination treatment were associated with inhibition of PDGFR-β/STAT3 signaling in tumor tissue (Fig. 7B).

Our data show that melatonin in combination with sorafenib synergistically inhibited the growth of PDAC cells and induced mitochondrial-mediated apoptosis through inhibition of the PDGFR-β/STAT3 pathway and MT-mediated STAT-3. Our findings provide new insight into potential therapeutic strategies in PDAC and lay the foundation for possible future clinical regimens.

This research was supported by the National Research Foundation (NRF) Grant (2015R1A2A1A10054108, 2014M3C1A3051476), and the Korea Health Technology R&D Project (HI15C0554), Ministry of Health and Welfare, Republic of Korea.

No conflict of interests exists.

Zhenghuan Fang and Kyung Hee Jung contributed equally to this work.