Thymoquinone Pretreatment Overcomes the Insensitivity and Potentiates the Antitumor Effect of Gemcitabine Through Abrogation of Notch1, PI3K/Akt/mTOR Regulated Signaling Pathways in Pancreatic Cancer

Abstract

Background

The gemcitabine-insensitivity remains the main challenge for pancreatic cancer treatment. Thymoquinone, the predominant bioactive ingredient of Nigella sativa, has been shown to possess promising anti-cancer and chemo-sensitizing effects on pancreatic cancer, however, its meticulous mechanism is still indistinct.

Aim

The objective of the present study was to investigate the potency of thymoquinone in combination with gemcitabine in inducing apoptosis and preventing the development of gemcitabine-insensitivity in pancreatic cancer cells.

Methods

The anti-tumor effects of thymoquinone and gemcitabine were analyzed via evaluation of alterations of cell viability, tumor weight, apoptosis-related proteins, caspase-3, -9 activities and NF-κB DNA binding activity in pancreatic cancer cells in vitro and PANC-1 cells orthotopic xenograft in vivo.

Results

Thymoquinone pretreatment following gemcitabine treatment synergistically caused an increase in pancreatic cancer cells apoptosis and tumor growth inhibition both in vitro and in vivo. The novel combinational regimen also contributes to alterations of multiple molecular signaling targets, such as the suppression of Notch1, NICD accompanying with up-regulation of PTEN, the inactivation of Akt/mTOR/S6 signaling pathways, and the suppression of phosphorylation and nuclear translocation of p65 induced by TNF-α. Thymoquinone pretreatment and gemcitabine also induced down-regulation of anti-apoptotic Bcl-2, Bcl-xL, XIAP and up-regulation and activation of pro-apoptotic molecules including Caspase-3, Caspase-9, Bax and increased release of cytochrome c.

Conclusions

This novel modality of thymoquinone pretreatment can enhance the anti-cancer activity of gemcitabine and may be a promising option in the treatment of pancreatic cancer.

Introduction

Despite increasing treatment options, the 5-year survival of patients with pancreatic cancer remains dismal [1]. Currently, gemcitabine-based chemotherapy is the most common choice for patients with inoperable pancreatic cancers. Resistance to chemotherapeutic agents, either natural or acquired, is a major factor that limits the efficacy to cure pancreatic cancer patients [2]. Multiple factors contribute to the intrinsic and acquired chemoresistance of human pancreatic cancers to conventional chemotherapeutic agents, such as reduction of intracellular chemotherapeutic drugs, increase of DNA damage impairment, as well as emergence and progression of epithelial mesenchymal transition [3, 4]. Previous studies also reported that several cell signaling pathways vital for the chemoresistance in pancreatic cancer have also been identified, such as the notable Notch1, PI3K/Akt, Hedgehog, and NF-κB pathways [58].

One advantage of combination treatment is the sensitization of cancer cells to gemcitabine [9, 10]. Moreover, in the present study, we employed combination therapy and the use of a naturally occurring thymoquinone to aim multiple molecular targets in preventing the gemcitabine-insensitivity and inducing more apoptotic deaths in pancreatic cancer cells, besides, reducing the therapeutic dose of gemcitabine [11, 12]. Thymoquinone is the main bioactive constituent derived from the volatile oil of the black seed (Nigella sativa). Previous studies revealed that co-treatment of thymoquinone potentiated the apoptotic effect of gemcitabine or oxaliplatin and enhanced the effect of gemcitabine or oxaliplatin in suppressing the growth of pancreatic cancer cell xenograft tumor in nude mice [12]. Likewise, a combination of thymoquinone and cisplatin synergistically blocked the proliferation and invasion of cisplatin-resistant lung cancer cells [13]. Several molecular signaling pathways have been reported to be responsible for the chemo-sensitizing effect of thymoquinone, such as the abrogation of NF-κB activity and Akt activation, decreased mitochondrial membrane potential, increased Bax/Bcl-2 ratio, and up-regulated expression of p53 and p21 proteins [1113].

Hence, in the present study, we investigated whether thymoquinone can significantly overcome the gemcitabine-insensitivity and argument for the apoptotic effect of gemcitabine in in vitro pancreatic cancer PANC-1, AsPC-1 and BxPC-3 cell lines and in an in vivo xenograft mouse model. In addition, the underlying mechanism of action of thymoquinone, especially with respect to alterations of Notch1, PTEN, Akt/mTOR/S6 and NF-κB mediated pathways, was assessed.

Methods

Antibodies and Reagents

Antibodies were obtained from the following commercial sources: anti-rabbit Notch1, β-actin, and pho-p65 (Ser 536) (Santa Cruz Biotechnology, USA); anti-rabbit Bcl-2, Bcl-xL, Bax, caspase-3, caspase-9, NICD, Akt, pho-Akt (Ser 473), mTOR, pho-mTOR (Ser 2448), S6, pho-S6 (Ser 235/236), cytochrome c and p65 antibody (Cell Signaling Technology, USA); anti-porin-2 was obtained from Novus Biological. Goat anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 555 Conjugate) was the secondary antibody (Cell Signaling Technology, USA). Horseradish peroxidase-labeled secondary antibodies and chemiluminescence reagents were obtained from Pierce, Thermo Scientific (Rockford, USA). Thymoquinone (Sigma Aldrich, UK) was dissolved in DMSO (Sigma Aldrich, UK) to make 20 mmol/L stock solution. Gemcitabine was purchased from Lilly France. Tumor necrosis factor-α (TNF-α) was purchased from sigma (Sigma Aldrich, UK); 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI) was obtained from Roche (Roche, Japan).

Cell Culture

The human pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell lines were maintained in continuous logarithmic growth in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY) supplemented with 10 % fetal bovine serum, 100 U/ml of penicillin sodium, and 100 μg/ml of streptomycin sulfate in a humidified incubator containing 5 % CO2 in air at 37 °C.

Evaluation of Thymoquinone and/or Gemcitabine Cytotoxicity

Cells (5 × 103 cells/well) seeded in 96-well culture plates were cultured overnight and the medium was replaced with fresh medium containing different concentrations of thymoquinone and/or gemcitabine. After 48 h incubation, cell viabilities were determined by WST-8 dye at 450 nm according to the optimized manufacturer’s recommendations (Dojindo, Japan).

Protein Extraction and Western Blotting

Treated cells were harvested and lysed in lysis buffer containing 50 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 150 mmol/L NaCl, 1 % Triton X-100, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, protease and phosphatase inhibitors cocktail (Roche, Japan).

Cytosolic and mitochondrial protein extracts were isolated using the cytochrome c Releasing Apoptosis Assay Kit (Millipore, Germany) following the manufacturer’s recommendation. The nuclear protein extracts were isolated using the Nuclear Extraction Kit from Active Motif (Carlsbad, USA). Proteins (40 μg per sample) were separated on SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Pall, NY). The membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies bound to immunoreactive bands were visualized by chemiluminescence detection system (Millipore) through incubation with horseradish peroxidase conjugated secondary antibodies. The porin-2, actin and PARP proteins were considered as markers of mitochondrial, cytosolic and nuclear proteins, respectively.

Caspase-3 and Caspase-9 Activity Assay

The activities of caspase-3, 9 were measured using Caspase-3 and Caspase-9 Colorimetric Assay Kits (R&D system). The cellular lysates (50 μl) were transferred to 96-well plates. The lysates were co-cultured for 4 h with 50 μl 2 × reaction buffer and 5 μl caspase-3 or caspase-9 substrate, respectively, then the activities were quantified by microplate reader at 405 nm.

Flow Cytometric Analysis of Cell Cycle

Cell cycle distribution was measured as previously described [14]. Treated cells were collected and washed with PBS and then fixed with 70 % ice-cold ethanol overnight at −20 °C. The cells were centrifuged, incubated with propidium iodide (50 μg/ml) and RNase A (100 μg /ml) for 40 min at 37 °C. The stained cells were determined by a FAC Scan flow cytometer, and the Cell Quest Histogram analysis program was used to analyze the percentages of cells in pre-G1, G0/G2, S, and G2/M phases.

Immunofluorescence Assay

Cells were seeded onto 20-mm sterile circular microscope coverslips at 1 × 105 cells/ml and cultured overnight to adhere to the slides. Cells pretreated with thymoquinone for 24 h were exposed to TNF-a for an additional 4 h. After washing twice with sterile phosphate buffered saline (PBS), cells were fixed in preheated 3.7 % paraformaldehyde (PFA) (Sigma Aldrich, UK) for 15 min and permeated with 0.4 % Trion-X 100 for 10 min. Nonspecific binding sites were blocked for 60 min with 0.1 % Tween-20/5 % BSA/PBS solution. For staining, coverslips were incubated with anti-p65 antibody overnight and visualized using Alexa Fluor® 555 Conjugate) secondary antibody. Nuclei were stained with 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI, 1 μg/ml). Images were required using upright Olympus fluorescence microscope.

NF-κB DNA-Binding Assay

Nuclear extracts of pancreatic tumor samples were prepared as described above. Nuclear NF-κB activity was determined using ELISA-based TransAM® NF-κB Kit (Active, Motif, Carlsbad, USA) following the manufacturer’s recommendations. Briefly, nuclear proteins (5 μg/well) were loaded into a 96-well plate, which was immobilized with oligonucleotide containing NF-κB consensus-binding sites (5′-GGGACTTTCC-3′). The active forms of NF-κB in the nuclear extracts were specifically bound to the oligonucleotide and detected colorimetrically. The NF-κB activities were determined by reading absorbance at 450 nm with an optional reference wavelength of 655 nm.

Animal Experiments and Orthotopic Implantation of Pancreatic Cancer Cells

Six-week old female BALB/c nude mice were purchased from Beijing HFK Bioscience Co., Ltd. All in vivo studies were conducted according to the Chinese national guidelines for the care and use of laboratory animals. All mice were randomized into four treatment groups: control group; gemcitabine treated group, 50 mg/kg given intraperitoneally three times per week; thymoquinone treated group, 1 mg/mouse given intragastrically daily; a combination of gemcitabine and thymoquinone treated group. The procedure of orthotopic implantation was operated as previously described [15]. Untreated pancreatic cancer PANC-1 cells were harvested from sub-confluent cultures and re-suspended in PBS containing 0.1 % Matrigel (BD Biosciences). PANC-1 cells (1 × 106 cells/50 μl) were injected into the pancreatic parenchyma. All mice were sacrificed on day 35, the primary pancreatic tumors were isolated and weighed. Tumors were divided in half, one part of which was lysed for western blotting assay, while the other was prepared for NF-κB DNA assay and caspase-3 activity assay.

Statistical Analysis

For all cell culture experiments, mean values ± SD of at least triplicate experimental points were calculated. Statistical significance between values of different experimental conditions was analyzed using Student’s t test. P < 0.01 was considered to indicate a statistically significant difference.

Results

Thymoquinone Potently Reduces Viabilities of Pancreatic Cancer Cells

To determine the effect of thymoquinone and gemcitabine alone on the cell viability of pancreatic cancer cells in vitro, PANC-1, AsPC-1 and BxPC-3 cells were initially treated with increasing concentrations of either thymoquinone (0–50 μmol/L) or gemcitabine (0–200 μmol/L) (Fig. 1a, b). Thymoquinone treatment on PANC-1, AsPC-1 and BxPC-3 cells resulted in an IC50 of 25.12 ± 0.67, 21.9 ± 0.37 and 10.48 ± 0.15 μmol/L, respectively, indicating enhanced cytotoxicity. Gemcitabine treatment of PANC-1, AsPC-1 and BxPC-3 cells produced an IC50 of 49.96 ± 2.34, 28.1 ± 1.32 and 9.05 ± 0.36 μmol/L, respectively. Thymoquinone (0–50 μmol/L) showed less cytotoxic effect on normal cells.

Fig. 1
figure1

Thymoquinone dose-dependently induced clear growth inhibition and elevated apoptosis of pancreatic cancer cells. Cell viability assays of PANC-1, AsPC-1 and BxPC-3 cells treated with hymoquinone (a) and gemcitabine (b). Points represent mean ± SD, n = 4, P < 0.01. c Caspase-3 and caspase-9 (d) activity assays of PANC-1, AsPC-1 and BxPC-3 cells exposed to increasing concentrations of thymoquinone. Data are presented as mean ± SD, n = 3, P < 0.01. e The pro- and anti-apoptotic protein expressions of PANC-1, AsPC-1 and BxPC-3 cells exposed to thymoquinone were analyzed by western blotting assay. f The band intensities were quantitated. Data are presented as mean ± SD, n = 3, P < 0.01

Thymoquinone Dose-Dependently Induced Apoptosis of Pancreatic Cancer Cells

To clarify the molecular basis of thymoquinone induced apoptosis, the PANC-1, AsPC-1 and BxPC-3 pancreatic cancer cells were treated with varying concentrations of thymoquinone (Fig. 1e, f). Thymoquinone dose-dependently induced clearly down-regulations of anti-apoptotic proteins Bcl-2 and Bcl-xL, and up-regulations of pro-apoptotic protein Bax, indicating the apoptosis-promoting effect of thymoquinone is partly attributed to up-regulating Bax/Bcl-2 protein ratio. The other NF-κB regulated molecules XIAP and survivin were also significantly inhibited in thymoquinone-treated cells (Fig. 1e, f).

Thymoquinone treatment also dose-dependently caused increasing release of cytochrome c from the mitochondria of PANC-1, AsPC-1 and BxPC-3 cells. In a sequential manner, the cytochrome c released into cytosol recruits and activates the caspase families that are the executioners of apoptosis. The western blotting analysis revealed that the cleaved active components of caspase-3 and caspase-9 were significantly elevated due to the thymoquinone treatment (Fig. 1e, f). In parallel with immunoblotting analysis, relative caspase-3 and caspase-9 activities of all three pancreatic cancer cells were also noticeably up-regulated along with the increasing concentrations of thymoquinone (Fig. 1c, d).

Thymoquinone Sensitizes Pancreatic Cancer Cells to Gemcitabine

The subsequent studies were undertaken to determine whether thymoquinone pretreatment could enhance the susceptibility of pancreatic cancer cells to gemcitabine. PANC-1, AsPC-1 and BxPC-3 cells were pretreated with thymoquinone (an IC50 of 25, 21 and 10 μmol/L, respectively, for 48 h) and followed by 24 h incubation of varying concentrations of gemcitabine (0–50 μmol/L), which was defined as thymoquinone pretreatment (Fig. 2a). Meanwhile, combination treatments were carried out by varying gemcitabine (0–50 μmol/L) in the presence of thymoquinone (an IC50 of 25, 21 and 10 μmol/L for PANC-1, AsPC-1 and BxPC-3 cells, respectively) for 48 h (Fig. 2a). As shown in Fig. 2a, thymoquinone pretreatment resulted in a clearly leftward lift of the concentration response curve compared to the combination treatment, indicating thymoquinone pretreatment could sensitize pancreatic cancer cells to gemcitabine. According to the results, the dosage of 100 nmol/L gemcitabine was selected for the thymoquinone pretreatment, which caused significant cell death.

Fig. 2
figure2

Apoptotic patterns of pancreatic cancer cells treated with thymoquinone and/or gemcitabine. a PANC-1, AsPC-1, BxPC-3 cells were treated with combination of thymoquinone (25, 21, 10 μmol/L, respectively) and varying concentrations of gemcitabine (0–50 μmol/L) for 48 h, or pretreated with thymoquinone (25, 21, 10 μmol/L, respectively, 48 h) following gemcitabine treatment (0–50 μmol/L, for 24 h), and the relative cell viabilities were determined by WST-8 assay. Data were presented as mean ± SD, n = 3, P < 0.01. b–e PANC-1, AsPC-1, BxPC-3 cells were then treated with thymoquinone (IC50 of 25, 21, 10 μmol/L, respectively, for 48 h), or gemcitabine (IC50 of 50, 28, 9 μmol/L, respectively, for 48 h), or pretreated with thymoquinone (25, 21, 10 μmol/L, respectively, for 48 h) following gemcitabine treatment (100 nmol/L, for 24 h). b Caspase-9 and caspase-3 (c) activities in PANC-1, AsPC-1 and BxPC-3 cells were determined by colorimetric assay. Data were presented as mean ± SD, n = 3. Asterisk P < 0.01 versus untreated control; inverted triangle < 0.01 versus gemcitabine-treated group. d Pro- and anti-apoptotic molecules in PANC-1, AsPC-1 and BxPC-3 cells were determined using western blotting. Porin-2 and actin were represented as the markers of mitochondrial and cytosolic proteins, respectively. e The band intensities were quantitated. Data are presented as mean ± SD, = 3, < 0.01

Thymoquinone Pretreatment Augments Apoptosis Induced by Gemcitabine in Pancreatic Cancer Cells

To explore the molecular evidence of chemosensitization and obvious apoptotic effect induced by thymoquinone pretreatment, the statuses of cytochrome c and cleaved caspase-3, caspase-9 and several other apoptosis related molecules were determined using western blotting. Thymoquinone or gemcitabine alone treated PANC-1 (IC50 of thymoquinone 25 μmol/L and gemcitabine 50 μmol/L), AsPC-1 (IC50 of thymoquinone 21 μmol/L and gemcitabine 28 μmol/L) and BxPC-3 (IC50 of thymoquinone 10 μmol/L and gemcitabine 9 μmol/L) cells exhibited significantly elevated release of cytochrome c from mitochondria compared with the untreated cells. However, PANC-1, AsPC-1 and BxPC-3 cells pretreated with thymoquinone (IC50 of thymoquinone 25, 21, 10 μmol/L, respectively, for 48 h) was incubated with 100 nmol/L gemcitabine for an additional 24 h. Thymoquinone pretreatment further markedly aggrandized the release of cytochrome c. Increased release of cytochrome c triggers the recruitment and activation of caspase families. Thymoquinone pretreatment significantly aggrandized and exhibited comparatively strong bands for cleaved caspase-3 and caspase-9 (Fig. 2d, e). Additionally, relative caspase-3 and caspase-9 activities were determined with colorimetric assay and significantly increased in PANC-1, AsPC-1 and BxPC-3 cells treated with thymoquinone pretreatment compared to other treatment (Fig. 2b, c).

Moreover, compared to gemcitabine or thymoquinone treatment alone, the pro-apoptotic Bax protein was significantly up-regulated and the other anti-apoptotic Bcl-2, Bcl-xL and survivin proteins were significantly down-regulated in PANC-1, AsPC-1 and BxPC-3 cells exposed to thymoquinone pretreatment (Fig. 2d, e). Overall, the significantly elevated Bax/Bcl-2 ratio of thymoquinone pretreatment was consistent with the increased release of cytochrome c and activation of caspase families, indicating that thymoquinone pretreatment indeed synergizes the pro-apoptotic effect of gemcitabine.

Effect of Thymoquinone Pretreatment on Cell Cycle Distribution

The effects of thymoquinone and/or gemcitabine on PANC-1, AsPC-1 and BxPC-3 cell cycle were analyzed (Fig. 3). Untreated cells showed a relatively normal pattern, with most cells in the S phase, a lower G0–G1 phase peak of the cell cycle. Thymoquinone or gemcitabine treated PANC-1, AsPC-1 and BxPC-3 cells, as indicated above, exhibited an increased percentage of G0–G1 phase arrest (P < 0.01) (Fig. 3). Interestingly, thymoquinone pretreatment augmented the G0–G1 phase arrest compared to cells treated with gemcitabine or thymoquinone alone (P < 0.01) (Fig. 3). These findings clearly manifested that thymoquinone pretreatment ruled over arrest of cells at G0–G1 phase in progression of the mitosis.

Fig. 3
figure3

Thymoquinone induces the G1 phase arrest of the cell cycle in pancreatic cancer cells. Representative plots reveal the distribution of the cell cycle of PANC-1, AsPC-1 and BxPC-3 pancreatic cancer cells were determined using flow cytometry after the following treatment: thymoquinone (IC50 of 25, 21, 10 μmol/L, respectively, for 48 h); gemcitabine (IC50 of 50, 28, 9 μmol/L, respectively, for 48 h); pretreated with thymoquinone (25, 21, 10 μmol/L, respectively, for 48 h) following gemcitabine treatment (100 nmol/L, 24 h). Each individual experiment was carried out in triplicate, < 0.01

Thymoquinone Whittled Down the Up-Regulation of Notch1 Signaling Pathway Induced by Gemcitabine

As is now well known, Notch1 and PTEN are the most important molecules, and activated Notch1 decreased PTEN protein levels which predicts the poorer prognosis and enhanced chemoresistance [16]. Hence, the present study was carried out to determine whether thymoquinone affected the expression of Notch1 and PTEN in PANC-1, AsPC-1 and BxPC-3 cancer cells. As shown in Fig. 4, gemcitabine treatment alone, in concentrations as indicated, induced the significant up-regulation of Notch1, especially elevated expression of NICD, which is the intracellular portion (active form) of Notch1. Interestingly, thymoquinone pretreatment, in concentrations as indicated, dramatically whittled down the up-regulation of Notch1 and NICD induced by gemcitabine stimulation (Fig. 4). Moreover, PTEN, one of the most frequently lost tumor suppressors in multiple human malignancies, was clearly down-regulated by gemcitabine in PANC-1, AsPC-1 and BxPC-3 cells. However, thymoquinone pretreatment could restore PTEN protein which was inhibited by gemcitabine (Fig. 4). Rationally, the results suggested that deviant Notch1, NICD, and PTEN directly or indirectly contributed to thymoquinone-induced apoptosis or chemosensitization of pancreatic cancer cells.

Fig. 4
figure4

PANC-1, AsPC-1 and BxPC-3 cells were treated with thymoquinone (IC50 of 25, 21, 10 μmol/L, respectively, for 48 h) or gemcitabine (IC50 of 50, 28, 9 μmol/L, respectively, for 48 h) or pretreated with thymoquinone (25, 21, 10 μmol/L, respectively, for 48 h) following gemcitabine treatment (100 nmol/L, 24 h). a The protein expression profiles of Notch1, NICD, PTEN and pho-Akt (Ser 473), pho-mTOR (Ser 2448) and pho-S6 (Ser 235/236) were determined along with the total protein expression in PANC-1, AsPC-1 and BxPC-3 cells. b Densitometric analysis of NICD, Notch1, PTEN and pho-Akt, pho-mTOR, pho-S6 in western blotting. Data are presented as mean ± SD, n = 3, P < 0.01

Thymoquinone Interferes with the Akt Transduction Pathway Through Inhibition of Downstream mTOR and S6 Protein

The PI3K/Akt signaling pathway is involved in all survival and chemoresistance signaling modulation in pancreatic cancer cells [17]. The potential attenuation of Akt pathway of PANC-1, AsPC-1 and BxPC-3 cells exposed to thymoquinone were determined using western blotting assays. Interestingly, slightly increased expression of pho-Akt (Ser 473) was found in all cells treated with gemcitabine as indicated above. However, thymoquinone alone and pre-treatment introduced a significant reduction of phosphorylation of Akt (Ser 473) in PANC-1, AsPC-1 and BxPC-3 cells, as compared with that of untreated cells as well as gemcitabine treated cells (Fig. 4). The total pan-Akt protein expression remained unaffected in all treatment conditions. Simultaneously, the levels of Akt downstream molecules including pho-mTOR (Ser 2448) and pho-S6 (Ser 235/236) ribosomal protein were assessed. Thymoquinone associated treatments also inhibited the phosphorylation of mTOR and S6 compared to untreated cells without affecting the non-phosphorylated forms. Most notably, thymoquinone pretreatment clearly attenuated the up-regulated phosphorylation of mTOR, S 6 and upstream Akt due to the gemcitabine (Fig. 4). Taken together, these findings indicated that regulation of the Akt/mTOR/S6 pathway is intimately involved with thymoquinone pretreatment-induced apoptosis and growth inhibition in pancreatic cancer cells.

Thymoquinone Represses TNF-α Induced Phosphorylation and Nuclear Translocation of NF-κB

NF-κB acts as a pivotal transcription factor in the survival and proliferation of various human tumors, including the pancreatic cancers [6]. To elucidate the effect of thymoquinone on the NF-κB signal pathway regulated by TNF-α, we first determined the optimum time of exposure to TNF-α required to induce the maximized phosphorylation of NF-κB in PANC-1 cells. TNF-α time-dependently activated the maximal phosphorylation of p65 at 30 min and resulted in the obvious nuclear translocation of p65 at 60 min, thus exposure to 0.1 nmol/L TNF-α for 60 min was applied to the subsequent experiments (Fig. 5a). PANC-1, AsPC-1 and BxPC-3 cells pretreated with thymoquinone (IC50 of thymoquinone 25, 21, 10 μmol/L, respectively, for 12 h) were exposed to 0.1 nmol/L TNF-α for an additional 60 min, and the nuclear extracts were incubated with antibodies against the p65 and pho-p65 (Ser 536). Thymoquinone pretreatment significantly suppressed the phosphorylation and nuclear translocation of p65 induced by TNF-α (Fig. 5b–d). Immunofluorescence analysis also confirmed that thymoquinone pretreatment markedly restrained p65 from translocating to nucleus and phosphorylation induced by TNF-α (Fig. 5e).

Fig. 5
figure5

Thymoquinone suppresses TNF-α induced nuclear translocation and activation of p65. a PANC-1 cells treated with 0.1 nmol/L TNF-α for different times, and the optimum time of exposure to TNF-α required to induce the maximum phosphorylation fo p65 at Ser 536 site was determined. b PANC-1 and c AsPC-1, BxPC-3 cells pretreated with thymoquinone (IC50 of 25, 21, 10, respectively) for 4 h were followed by stimulation with 0.1 nmol/L TNF-α for 60 min, and the alterations of phosphorylated p65 (Ser 536) and p65 located in nucleus were determined by western blotting. d Densimetric analysis of pho-p65 (Ser 536) and p65 protein levels in western blotting. Data are presented as mean ± SD, n = 3, P < 0.01. e Immunofluorescence analysis indicated that thymoquinone restrained p65 from phosphorylation and nuclear translocation induced by TNF-α. The nucleus was stained by DAPI. All experiments were performed in triplicate

Thymoquinone Enhances In Vivo Antitumor Effect of Gemcitabine on PANC-1 Orthotopic Tumor

Based on the in vitro results, which strongly support better killing of pancreatic cancer cells when pretreated with thymoquinone, we further assessed the therapeutic potencies of thymoquinone and gemcitabine either alone or in combination in nude mice bearing pancreatic orthotopic implantation. The dosages of thymoquinone (1.0 mg/mouse/day, i.g.) and gemcitabine (50 mg/kg, ×3/week-2 cycles, i.p.) were selected and administrated as depicted in Fig. 6a. The average weight of isolated pancreatic tumor tissues in mice exposed to thymoquinone or gemcitabine treatment alone were significantly lighter than untreated control mice (0.69 vs. 2.14 g, 1.75 vs. 2.14 g, respectively; P < 0.01) (Fig. 6b). However, the combination treatment of thymoquinone and gemcitabine resulted in noticeably 81.7 and 85 % reduction in tumor weight compared with that of gemcitabine treated or control mice, respectively (0.32 vs. 1.75 g, 0.32 vs. 2.14 g; P < 0.01) (Fig. 6b). There was no severe toxicity as evaluated by weight loss, anorexia or diarrhea in all experimental mice, indicating that thymoquinone cause no serious toxic and side effects.

Fig. 6
figure6

Anti-tumor effect of thymoquinone and/or gemcitabine in PANC-1 pancreatic cancer orthotopic xenograft in nude mice. a Schematic representation of in vivo experimental design and treatment schedule. Nude mice bearing PANC-1 xenograft were randomized into four groups: Group I was given 0.1 % DMSO/PBS as vehicle control; Group II was given gemcitabine (50 mg/kg, ×3/week, i.p.); Group III was given thymoquinone (1 mg/mouse, i.g. daily) for 3 weeks; Group IV was given gemcitabine and thymoquinone for 3 weeks as described above. b Isolated pancreatic tumor weights of different group mice were measured after the end of treatment. Data are presented as mean ± SD, n = 5, P < 0.01. c The alterations of relative caspase-3 activity in mice were determined by colorimetric assay. Columns indicate the relative caspase-3 activity against the control group; bars SD, = 3, P < 0.01. d Determination of NF-κB activation of isolated tumor samples of using ELISA assay kit. Columns indicate relative NF-κB DNA binding activity; bars SD, = 3, < 0.01. e Pro- and anti-apoptotic protein expression profiles and phosphorylation of p65 using western blotting assays. f Densitometric analysis of pro- and anti-apoptotic protein levels in western blotting. Data are presented as mean ± SD, n = 3, < 0.01. g The alterations of PTEN, Notch1 and phosphorylation of Akt (Ser 473), mTOR (Ser 2448) and S6 (Ser 235/236) in tumor tissues of mice received different treatments. h Densitometric analysis of NICD, Notch1, PTEN, pho-Akt, pho-mTOR and pho-S6 protein levels in western blotting. Data are presented as mean ± SD, = 3, P < 0.01. Asterisk indicates P < 0.01 versus control group I; inverted triangle P < 0.01 versus gemcitabine treated group II

Thymoquinone Inhibits Constitutive NF-κB Activation and Downstream Proteins Expression In Vivo

Consistent with the in vitro experiments, the constitutive NF-κB activation in pancreatic cancer tissues was determined by ELISA-based Trans AM NF-κB assay kit. As clearly shown in Fig. 6d, the NF-κB activity of tumor tissue was moderately down-regulated by thymoquinone treatment alone, but mice treated with thymoquinone in combination with gemcitabine revealed considerable reduction of NF-κB DNA binding activity compared to either control or gemcitabine treated mice (P < 0.01). The inhibitory effect of thymoquinone on NF-κB was also evaluated by western blotting, and the results clearly depicted that the constitutive phosphorylation of p65 was substantially reduced in nuclear extracts from tumor samples of mice treated with a combination of thymoquinone and gemcitabine (Fig. 6e,f). In concert with our in vitro research, these findings supplement the hypothesis that the inactivation of NF-κB is one of the molecular mechanisms by which thymoquinone potentiates gemcitabine induced anti-tumor activity in vivo. Additionally, western blotting analysis revealed down-regulation of several NF-κB regulated molecules such as Bcl-2, Bcl-xL and survivin proteins and significant up-regulation of caspase-3 activity in tumor tissues of mice treated with a combination of thymoquinone and gemcitabine, providing evidence to apoptosis within tumors in vivo (Fig. 6c, e, f).

Thymoquinone Blocks the Activation of Notch1 and Restored PTEN Expression in PANC-1 Orthotopic Tumor

Protein extracts of primary pancreatic tumor tissues was subjected to determine the molecular mechanisms using western blotting assays. As compared to control mice, gemcitabine treatment alone resulted in significant up-regulation of Notch1, especially the active form NICD, accompanied with the inhibition of PTEN. However, thymoquinone treatment inhibited the expression of Notch1 and NICD and up-regulated PTEN level which was associated with the lighter of the tumor tissues and increased pro-apoptotic proteins (Fig. 6g–h). These findings confirmed the hypothesis that the tumor growth inhibition and apoptosis promoting effect of thymoquinone was partly attributed to the inactivation of Notch1-mediated pathway and up-regulation of PTEN.

Thymoquinone Blocks Akt-Mediated Signaling Pathway in PANC-1 Orthotopic Tumor

Western blotting assays revealed that gemcitabine treatment alone significantly introduced up-regulation of phosphorylation of Akt (Ser 473), mTOR (Ser 2248) and S6 (Ser 235/236) protein in PANC-1 cells growing orthotopically in nude mice. However, the phosphorylated status of Akt, mTOR and S6 proteins were distinctly down-regulated in mice treated with thymoquinone in the presence of gemcitabine or not. Meanwhile, there was no negligible or less change in the expression level of total Akt, mTOR and S6, which did not vary significantly among mice in all the four groups (Fig. 6g–h). In general, these findings suggested that Notch1/PTEN and Akt/mTOR/S6 regulatory pathways play pivotal roles in the mechanism by which thymoquinone induced apoptosis and prevented gemcitabine-insensitivity of pancreatic cancer.

Discussion

Induction of apoptotic cell death and loss of cell viability are two major mechanisms by which conventional chemotherapeutic agents kill cancer cells. Unfortunately, conventional therapies with gemcitabine-based chemotherapeutic agents have limited impacts on the control of pancreatic cancers owing to the dose-limiting toxicity to normal tissues and increased acquisition of chemoresistance [2, 18]. Thus, new chemotherapeutic strategies need to be put in place in order to develop novel chemo-sensitizing and/or chemotherapeutic agents that could improve the drug-resistant conditions. Recently, increasing evidence encourages the use of naturally occurring innocuous dietary agents to prevent the chemoresistance and argument the apoptotic effect of conventional cancer chemotherapeutic agents [19]. In this regard, thymoquinone gained our attention because survival of normal cells was not affected by thymoquinone that was cytotoxic to various human cancer cells [20, 21]. In the present study, thymoquinone pretreatment significantly potentiated the apoptotic and growth inhibition effects of gemcitabine in pancreatic cancer cells both in vivo and in vitro, indicating the promising chemo-sensitizing effects of thymoquinone.

Increasing reports have documented that the excessive activation of the Notch1 signaling pathway contributes to the aggravated aggressiveness and chemoresistance of pancreatic cancers to gemcitabine [8, 22, 23]. In the present study, Notch1 and active form NICD were noticeably activated in pancreatic cancer cells and orthotopic xenograft under gemcitabine treatment, along with the reduction of apoptotic cell death. However, compared to gemcitabine treatment alone, thymoquinone pretreatment significantly suppressed the Notch1 and inhibited the cell survival and tumor growth pancreatic cancer cells and orthotopic xenograft. PTEN (phosphatase and tensin homologue, deleted on chromosome 10), the downstream target of Notch1, is suppressed by excess activated Notch-1 through binding CBF-1 to the PTEN promoter and is best characterized as an antagonist to the PI3K/Akt signaling pathway [3, 24]. PTEN is also frequently suppressed and inactivated in multiple human malignancies through mutation, dephosphorylation or down-regulation of protein expression to promote the chemoresistance and progression of many malignancies [25]. However, there is mounting evidence documenting that up-regulation of PTEN contributes to increased chemosensitivity in gemcitabine-resistant pancreatic cancers [16, 26]. In the present study, thymoquinone pretreatment significantly restored the PTEN suppressed by gemcitabine and conspicuously restrained the downstream activation of Akt/mTOR in pancreatic cancer both in vitro and in vivo. This finding is similar to research which documents that thymoquinone up-regulates PTEN and suppresses the activation of Akt in MCF-7/DOX doxorubicin resistant breast cancer cells [26]. These findings indicates that thymoquinone pretreatment enhances the chemosensitivity of pancreatic cancer to gemcitabine through suppression of the Notch1/PTEN pathway.

The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway plays a pivotal role in carcinogenic processes, and its anti-apoptotic properties are closely linked to the chemoresistance of human malignancies to conventional chemotherapeutic drugs [27]. The constitutive activation of this pathway is supposed to be correlated with aggravated clinical chemoresistance and poor prognosis for pancreatic cancer patients [16, 17]. mTOR is a serine/threonine kinase of the PI3K kinase family and is supposed to be a key effector of the PI3K/Akt/mTOR pathway, involved in regulating cell proliferation, survival and angiogenesis [27]. S6 ribosomal protein, downstream effector of the mTOR pathway, is critical for protein-synthesis regulation [28]. We observed the activation of Akt/mTOR and S6 ribosomal protein in pancreatic cancer cells treated with gemcitabine alone. However, we further observed thymoquinone pretreatment significantly inhibited the phosphorylation of Akt/mTOR and S6 protein induced by gemcitabine both in vitro and in vivo, along with increased cell apoptosis and tumor growth inhibition. These findings suggested that thymoquinone exhibited its promising chemo-sensitizing and apoptotic effects through suppression of the activation of PI3K/AKT/mTOR and the downstream effector S6 ribosomal protein.

Based on previous publicized findings we can hypothesize that thymoquinone, as a chemo-sensitizing drug, is destined to suppress the NF-κB DNA binding activity and induces the down-regulation of a series of downstream pro-survival proteins [11, 29]. XIAP and survivin, members of the IAP family, have been validated as therapeutic targets owing to their indulgence in malignant effects of cancer cells, especially chemoresistance, cell proliferation and angiogenesis. A prevalent mechanism by which constitutive activated NF-κB augments chemoresistance of pancreatic cancer to gemcitabine is the up-regulation of pro-survival Bcl-2 and Bcl-xL, or direct inhibition of caspases by XIAP [11]. Knockdown of survivin and XIAP with small interfering RNA or inhibitors could enhance chemosensitivity of cancer cells to conventional chemotherapeutic drugs [3032]. The present study also showed that XIAP and survivin were significantly down-regulated in pancreatic cancer cells treated with thymoquinone, and in the train of loss of cell viability and noticeable up-regulation of caspase-3 and -9 activity both in vitro and in vivo. Corollary to the hypothesis, our present study provides evidence that thymoquinone pretreatment could prevent insensitivity of pancreatic cancer to gemcitabine partly through suppression of anti-apoptotic molecules in concert with induction of mitochondrial release of cytochrome c and activation of caspases.

Additionally, in the present study, thymoquinone exhibited greater apoptotic efficacy than gemcitabine treatment alone in PANC-1, AsPC-1 and BxPC-3 cell lines as evidenced by increased G1-phase cell cycle arrest, up-regulated expression of pro-apoptotic proteins and activation of caspase-3 and caspase-9. In the mouse xenograft model, thymoquinone treatment alone was more active than gemcitabine alone without additional toxicity to the mice. These results are very encouraging and indicate that thymoquinone may be a promising therapeutic agent in the treatment of pancreatic cancer.

In general, our present study indicates that thymoquinone pretreatment synergized with the low-dose of gemcitabine to prevent the gemcitabine-insensitivity and induce apoptotic cell death in pancreatic cancer cells through abrogation of Notch1/PTEN, PI3K/Akt/mTOR, NF-κB mediated signaling pathways. These observations also corroborate with previous studies and provide an important value in the possible clinical treatment with proposed combinations, minimizing the dose of gemcitabine, and thereby decreasing the risk of gemcitabine-insensitivity and toxicity. However, it would be necessary to investigate in the future if the chemo-sensitizing effect of thymoquinone is mediated through suppression of Notch1. Nevertheless, based on the results, it is apparent that the effective approach with promising results in the novel synergistic combination of thymoquinone and gemcitabine provide confidence in support of further development of thymoquinone as an adjunct to conventional chemotherapeutics for treatment of human pancreatic cancers.

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Acknowledgments

We sincerely thank Mr. Hong Xia (Key Laboratory of Hubei Province for Digestive System Disease, Wuhan, China) for his administrative support and excellent technical assistance in this work. This study was supported by grants from the Fundamental Research Funds for the Chinese Central Universities (No. 2012302020214) and National Natural Science Foundation of China (No. 81172350).

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The authors have declared that no conflicts of interest exist.

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Correspondence to Hong-gang Yu.

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Mu, Gg., Zhang, Ll., Li, Hy. et al. Thymoquinone Pretreatment Overcomes the Insensitivity and Potentiates the Antitumor Effect of Gemcitabine Through Abrogation of Notch1, PI3K/Akt/mTOR Regulated Signaling Pathways in Pancreatic Cancer. Dig Dis Sci 60, 1067–1080 (2015). https://doi.org/10.1007/s10620-014-3394-x

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Keywords

  • Notch1
  • PTEN
  • Pancreatic cancer
  • Thymoquinone
  • Chemoresistance
  • Apoptosis