Andrographolide causes apoptosis via inactivation of STAT3 and Akt and potentiates antitumor activity of gemcitabine in pancreatic cancer

Toxicology Letters 222 (2013) 23–35

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Andrographolide causes apoptosis via inactivation of STAT3 and Akt and potentiates antitumor activity of gemcitabine in pancreatic cancer Guo-Qing Bao, Bai-Yong Shen, Chun-Peng Pan, Ya-Jing Zhang, Min-Min Shi, Cheng-Hong Peng ∗ Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of medicine (SJTU-SM), Shanghai 200025, PR China

h i g h l i g h t s • ANDRO causes apoptosis and cell cycle arrest in pancreatic cancer cells. • Combining ANDRO with GEM exhibits synergistic anti-pancreatic cancer effect. • The anti-pancreatic cancer effect of ANDRO is due to inactivation of STAT3 and Akt.

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Article history: Received 3 May 2013 Received in revised form 21 June 2013 Accepted 27 June 2013 Available online 8 July 2013 Keywords: Pancreatic cancer Andrographolide Gemcitabine STAT3 Akt

a b s t r a c t Gemcitabine is a first-line drug utilised in the chemotherapy of pancreatic cancer; however, this drug induces chemo-resistance and toxicity to normal tissue during treatment. Here, we firstly report that andrographolide (ANDRO) alone not only has anti-pancreatic cancer activity, but it also potentiates the anti-tumour activity of gemcitabine. Treatment with ANDRO alone inhibits proliferation of the pancreatic cancer cell lines in a dose- and time-dependent manner in vitro. Interestingly, ANDRO induces cell cycle arrest and apoptosis of pancreatic cancer cells by inhibiting STAT3 and Akt activation, upregulating the expression of p21WAF1 and Bax, and downregulating the expression of cyclinD1, cyclinE, survivin, X-IAP and Bcl-2. Additionally, ANDRO combined with gemcitabine significantly induce stronger cell cycle arrest and more obvious apoptosis than each single treatment. The mechanistic study demonstrates that this synergistic effect is also dependent on the inhibition of STAT3 and Akt activations which subsequently regulates the pathways involved in the apoptosis and cell cycle arrest. Furthermore, both ANDRO alone and the combination treatments exhibit efficacious anti-tumour activity in vivo. Overall, our results provide solid evidence supporting that ANDRO alone or its combination with gemcitabine is a potential chemotherapeutic approach for treating human pancreatic cancer in clinical practice. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Pancreatic cancer is a lethal malignancy with extremely poor prognosis, and it is currently the fourth leading cause of cancerrelated deaths in the United States and other industrialised countries (Vincent et al., 2011; Bardeesy and DePinho, 2002). According to NIH-National Cancer Institute statistics, it is estimated that 43,920 new cases of pancreatic cancer were diagnosed in the United States in 2012; among them, 37,390 would die of this disease (Siegel et al., 2012). The dismal prognosis of pancreatic cancer, with a median survival of 6 months and overall 5-year survival rate of <6%, is attributable to delayed

∗ Corresponding author. Department of General Surgery, Ruijin Hospital, Shanghai JiaoTong University School of medicine (SJTU-SM), 197 Ruijin Er Road, Shanghai 200025, China. Tel.: +86 021 64370045x680503, fax: +86 021 64370045x680503. E-mail address: [email protected] (C.-H. Peng). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.06.241

diagnosis and insensitivity to most therapeutic strategies, including chemotherapy, radiotherapy, and immunotherapy (Vincent et al., 2011). The pyrimidine nucleoside analogue gemcitabine, which inhibits DNA synthesis and cell replication, has been the first-line treatment for advanced and metastatic pancreatic cancer since the late 1990s (Burris et al., 1997); however, the clinical response rate remains less than 10%, triggering only a marginal survival advantage. Moreover, prolonged exposure to the drug leads to acquired chemo-resistance and notorious toxicities to normal cells, such as gastrointestinal toxicity, renal damage and bone marrow suppression (Vincent et al., 2011; Bardeesy and DePinho, 2002). Many combinations using gemcitabine as a backbone were designed and tested in clinical trials. Unfortunately, none of them is confirmed to be superior to gemcitabine monotherapy, except the combination of the EGFR inhibitor, erlotinib, plus gemcitabine (Moore et al., 2007). Therefore, new effective treatment strategies are urgently needed.

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In recent years, naturally occurring compounds screened from traditional medicinal plants with strong antitumour activities and few adverse side effects have gained great attention due to their roles in the inactivation of core survival signalling cascades within cancer (Mann, 2002). Strikingly, some phytochemicals among them exert synergism with routine clinical chemotherapy drugs, such as gemcitabine (Thoennissen et al., 2009; Banerjee et al., 2009; Wang et al., 2010). Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone, has been extracted and purified as the principal bioactive chemical ingredient from the herb Andrographis paniculata, which has been widely used as a traditional folklore herbal remedy in China, India, Malaysia and Thailand for hundreds of years for the therapy of various illnesses including bacterial dysentery, flu, diarrhoea and upper respiratory tract infections (Lim et al., 2012). ANDRO is well tolerated and largely free of any adverse side effects (Lim et al., 2012). Accumulating evidence reveals that ANDRO exerts a number of potent bioactivities such as antibiotic (Shen et al., 2002), hepatoprotective (Trivedi et al., 2007) and immunomodulatory (Iruretagoyena et al., 2005) activities. In the context of cancer, this active compound has been reported to possess strong anticancer activity in a variety of human cancer cell lines derived from hepatoma (Ji et al., 2011), colorectal carcinoma (Shi et al., 2008), breast cancer (Kumar et al., 2012), glioblastoma (Li et al., 2012), prostate cancer (Chun et al., 2010), non-small cell lung cancer(Lin et al., 2011; Lee et al., 2010), lymphoma (Yang et al., 2010) and epidermoid carcinoma (Tan et al., 2010). The mechanisms of action for its antitumour effect have been reported to be associated with cell cycle arrest (Li et al., 2007, 2012; Jada et al., 2008; Shi et al., 2008), apoptosis induction (Yang et al., 2010; Ji et al., 2011; Jada et al., 2008; Zhou et al., 2006), antiangiogenesis (Sheeja et al., 2007), suppression of migration(Shi et al., 2009; Kumar et al., 2012; Jiang et al., 2007; Lee et al., 2010), increased sensitisation to radiation and anticancer drugs (Zhou et al., 2010; Yang et al., 2009), empowered cytotoxicity of immune cells (Sheeja and Kuttan, 2007) and anti-cancer stem cell activity (Gunn et al., 2011). These features have rendered ANDRO as a promising chemotherapeutic agent against various types of cancers. However, the effect of ANDRO in pancreatic cancers remains elusive, especially with regard to its roles in combined chemotherapy with gemcitabine. Recently, many new insights into the core signalling pathways in pancreatic cancer have been made, including the signal transducer and activator of transcription-3 (STAT3) (Scholz et al., 2003; Corcoran et al., 2011) and protein kinase B (Akt) pathways (Nicholson and Anderson, 2002; Kagawa et al., 2012). These pathways are often constitutively activated in subsets of human pancreatic cancer tissues and cell lines (Thoennissen et al., 2009; Kagawa et al., 2012; Jamieson et al., 2011; Scholz et al., 2003; Corcoran et al., 2011; Jones et al., 2008). Furthermore, specific inhibitors of STAT3 (Lin et al., 2010; Sun et al., 2005) and/or AKT (Ahmad et al., 2012; Simon et al., 2009) reduce the growth of pancreatic cancer cell lines, and the combination with clinical standard chemotherapy causes synergic effects (Kagawa et al., 2012; Thoennissen et al., 2009). Previous studies have reported that ANDRO suppressed prostate cancer cell growth by inhibition of the IL-6/STAT3 pathway (Chun et al., 2010), and inhibition of the JAK/STAT3 pathway by ANDRO also increased the sensitivity of cancer cells to doxorubicin (Zhou et al., 2010). In addition, ANDRO inhibits the PI3 K/Akt signalling pathway to mediate proliferation suppression and cell cycle arrest in human glioblastoma cells (Li et al., 2012), and down-regulation of the PI3 K/Akt pathway by ANDRO also plays an important role in the inhibition of the migration and invasion of human non-small cell lung cancer A549 cells (Lee et al., 2010). Therefore, in the context of pancreatic cancer, we hypothesised that ANDRO might be a novel chemotherapy agent, possibly due to inactivation of the STAT3 pathway and/or Akt

pathway, resulting in inactivation of multiple downstream survival factors. 2. Materials and methods 2.1. Reagents and antibodies Andrographolide (with >98% purity) was purchased from Sigma–Aldrich (St. Louis, MO, USA) and dissolved in DMSO (Sigma) as a 100 mM stock solution and stored at −20 ◦ C in the dark. Control experiments used the same volume of DMSO (final concentration <0.1% in all experiments). Gemcitabine (Sigma) was reconstituted as a 1 mM stock solution in sterile PBS and stored at 4 ◦ C on the day of use. The antibodies against Bcl-2, Bax, Survivin, X-IAP, Cleaved Casepase-3, Casepase-9, PARP, CyclinD1, CyclinE, p21WAF1 , Cytochrome c, Akt, p-Akt (Ser473), Src, p-Src(Tyr419), Jak2, p-Jak2(Tyr1007/1008), STAT3, p-STAT3 (Tyr705), p53, Ki-67, ␤-actin, GADPH, and COX IV were purchased from Cell Signalling (Beverly, MA, USA). Horseradish peroxidise-conjugated anti-mouse secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). 2.2. Cell lines and cell culture Human pancreatic cancer cell lines AsPC-1, Panc-1, BxPC-3, SW1990, and Capan-1 and the human normal liver cell line L02 were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences, Shanghai, China. AsPC1, BxPC-3 and L-02 cells were cultured in RPMI1640, Panc-1and SW1990 cells were cultured in DMEM, and Capan-1 was cultured in IMDM. All media were supplemented with foetal bovine serum (10%), 100 units/ml penicillin, and 10 mg/ml streptomycin. Cells were maintained at 37 ◦ C in an humidified 5% CO2 incubator. 2.3. CCK-8 assay Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Tokyo, Japan) is based on highly water-soluble tetrazolium salt WST-8 instead of MTT, which is similar to MTT assay to evaluate the cell viability. Five thousand cells per well with 200 ␮l of medium were seeded into 96-well plates. Following the various incubations, CCK-8 solution (10 ␮l) was added to each well, followed by incubation for 2 h at 37 ◦ C, after which the absorbance was read at a wavelength of 490 nm. Medium containing 10% CCK-8 was used as a control. All experiments were repeated in triplicate. 2.4. Crystal violet viability assay Cancer cells (Panc-1, AsPc-1) were treated as designated before being reseeded in six-well plates (5000 cells/well) with fresh medium. After 2 weeks, the surviving clones were stained with crystal violet solution (0.5% crystal violet, 20% methanol) for 1 h. After the removal of the crystal violet solution, plates were washed three times and left to dry at 37 ◦ C. Photos were taken using a digital camera, and the relative staining intensities were quantified using ImageJ software. The experiments were repeated thrice. 2.5. Flow cytometric analysis of apoptosis An Annexin V/PI (Roche) kit was used according to the manufacturer’s instructions to detect apoptosis of the cancer cells (AsPc-1, Panc-1) by flow cytometry. Briefly, cells were seeded in 6-well plates. Then, the floating cells and adherent cells were harvested and counted after treatment as designated. 1 × 105 cells were resuspended in 100 ␮l binding buffer, 5 ␮l of Annexin V and 5 ␮l of PI were added, and the solution was incubated in the dark for 15 min at

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room temperature following flow cytometry to determine the apoptosis rate (%) with a cytometer (Beckman Coulter, CA, USA). The AnnexinV-positive cells were defined as apoptotic cells, including late apoptotic cells (the Annexin V-positive/PI-positive) and early apoptotic cells (the AnnexinV-positive/PI-negative). The experiments were repeated three times, and each sample was assayed in duplicate. 2.6. Cell cycle analysis After treatment as designated, AsPc-1 and Panc-1 cells were harvested by trypsinisation, washed with ice-cold PBS and fixed with ice-cold 70% ethanol overnight at 4 ◦ C. After centrifugation, the ethanol was removed. About 106 cells were re-suspended in PBS containing PI (50 ␮g/ml, Sigma) and ribonuclease A (50 ␮g/ml, Sigma) for 30 min in the dark. The percentage of cells at G0/G1, S, or G2/M phase was analysed by flow cytometry using FACScaliber (Becton Dickinson, San Jose, CA, USA). MultiCycle software (Phoenix Flow Systems, San Diego, CA, USA) is used to quantify cycle phase percentages in the flow cytometry. The experiments were repeated three times. 2.7. Protein extraction and western blot analysis 5 × 105 cells were disrupted by sonication in lysis buffer (cell signalling) in the presence of phosphatise and protease inhibitors (Sigma) and homogenised. An equal amount of protein (50 ␮g) was run on a 10% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane and blotted with mouse monoclonal antibody against Bcl-2, Bax, Survivin, X-IAP, Cleaved Casepase-3, Casepase-9, PARP, CyclinD1, CyclinE, p21WAF1 , p53, Akt, p-Akt (Ser473), Jak2, p-Jak2(Tyr1007/1008), Src, pSrc(Tyr419), STAT3, or p-STAT3 (Tyr705). Then, the membrane was incubated with horseradish peroxidise-conjugated goat antimouse antibody. The immune complexes were visualised with ECL Western blotting Detection Reagent (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. ␤-actin or GAPDH was simultaneously used as an internal control. 2.8. Analysis of cytochrome c release Mitochondrial fractions were prepared using a mitochondria isolation kit for cultured cells from Pierce (Pierce, Rockford, IL, USA) following the manufacturer’s instructions. The mitochondrial pellet was re-suspended in sample buffer for SDS-gel electrophoresis and analysed by western blotting for Cytochrome c antibodies (cell signalling). COX IV (cell signalling) was used as an internal control for the mitochondrial fraction. 2.9. Animal experimental design 4-week-old male nude BALB/c mice (weight 12–14 g) were purchased from Shanghai Slac Animal Centre (Shanghai, China). AsPC-1 cells were cultured, harvested, washed with PBS and re-suspended in PBS. Tumours were established by subcutaneous injection of 4 × 106 AsPC-1 cells into the flanks of mice. Tumour size was monitored with callipers measuring tumour length (L) and width (W) every 3 days, and the volume was calculated according to the formula: V = (length × width2 ) × ␲/6. When the tumours had grown for 2 weeks, reaching about 100 mm3 , the mice were randomly divided into four groups (4 per cohort): (a) untreated control (DMSO was dissolved in 200 ␮l PBS, thrice weekly by i.p. injection); (b) gemcitabine (80 mg/kg, thrice weekly by i.p. injection); (c) ANDRO (15 mg/kg, thrice weekly by i.p. injection); and (d) ANDRO and gemcitabine, following the same schedule of individual drugs. Tumour size and mouse weight were monitored every three days.

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Therapy was continued for 2 weeks. A week following the last treatment dose, all the mice were euthanised and tumours were excised neatly and weighted. One part of the tumour tissue was fixed in 10% neutral-buffered formalin, and another part was frozen in liquid nitrogen. The animal experiment was approved by the Animal Care and Use Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine, China, and conducted according to the guidelines of the Laboratory Animal Centre of Ruijin Hospital, Shanghai Jiaotong University School of Medicine. 2.10. Quantitation of Ki-67 proliferation index Microwave antigen retrieval was applied to unmask the interfering epitope. Formalin-fixed, paraffin-embedded tumour sections (5 ␮m) were rinsed with PBS and blocked with 3% hydrogen peroxide for 3 min and 5% bovine serum albumin for 2 h. They were then incubated with an anti-Ki-67 antibody overnight. Diaminobenzidine (DAB) and CoCl2 were used to develop the stain (Sigma-Aldrich, Shanghai, China). The sections were counterstained with haematoxylin and mounted. Tumour cells positive for the Ki-67 antigen were identified by intranuclear DAB staining (brown) and counted in 10 randomly selected ×400 high-power fields under microscopy. The Ki-67 proliferation index was counted by the following formula: the number of Ki-67-positive cells/total number of nucleated cells × 100%. 2.11. In situ detection of apoptotic cells Tumour sections were stained with the TUNEL agent (Roche, Shanghai, China), and the TUNEL-positive cells were counted in 10 randomly selected ×400 high-power fields under microscopy. The apoptosis index was measured according to the following formula: the number of apoptotic cells/the total cell count × 100%. 2.12. Statistical analysis All in vitro experiments were repeated three times to confirm the results. Dates were expressed as mean values ± standard deviation, and were analysed using Student’s t-test or One-way analysis of variance (ANOVA). A value of less than 0.05 (P < 0.05) was considered statistically significant. 3. Results 3.1. ANDRO suppresses proliferation of pancreatic cancer cells To examine the effects of ANDRO on the viability of a panel of five pancreatic cancer cell lines (AsPC-1, Panc-1, BxPC-3, SW1990, and Capan-1) and the normal human liver cell line L02, a dose escalation experiment was conducted via CCK-8 assay. Cells were incubated with increasing concentrations of ANDRO (0–100 ␮mol/l) for 0, 12, 24, 48, and 72 h. As shown in Fig. 1A, ANDRO inhibited the proliferation of all five pancreatic cancer cell lines in a dose- and time-dependent manner. In this test, no differences were detected between 0.1% DMSO and the negative control (data not shown). There was strong cytostatic effects observed on AsPC-1, Panc-1, BxPC-3, SW1990 and Capan-1 cells after 48 h treatment with 25 ␮mol/l ANDRO although different pancreatic cancer cells exhibited different sensitivities to ANDRO. The experiment demonstrates that ANDRO efficiently inhibits cell proliferation of pancreatic cancer cells as a single agent. In contrast, ANDRO had a minimal effect on the normal human liver cell line L-02 with equivalent concentrations (Fig. 1A). An anti-proliferative effect of ANDRO on AsPC-1 and Panc-1 cells was further confirmed by crystal violet assay. As shown in Fig. 1B,

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Fig. 1. ANDRO inhibited the cell proliferation of pancreatic cancer cells and had no cytotoxic effect in the non-tumorigenic epithelial cell line L-02. (A) Cell growth curve of pancreatic cancer cells (AsPC-1, Panc-1, BxPC-3, SW1990, and Capan-1) and normal human liver cell lines L-02. These cells were treated with doses of 0, 5, 10, 25, 50, 100 ␮mol/l ANDRO for 0, 12, 24, 48, 72 h as determined by CCK8 assay. (B) Cells were treated with doses of 0, 5, 25, 50 ␮mol/l ANDRO for 48 h before reseeded in six-well plates (5000 cells/well), respectively. After 2 weeks, the survival clones were stained by 0.5% crystal violet for 1 h and photos were taken using digital camera. The relative signal intensity was evaluated by ImageJ software (“*”P < 0.05, versus control. “**”P < 0.01, versus control). Data are representative of values from at least three independent experiments.

with ascending concentration of ANDRO, the colony formation ability of AsPC-1 and Panc-1 cells was strongly inhibited, especially when cells were treated with 50 ␮mol/l ANDRO. These results were consistent with the CCK-8 assay. Our results indicate that ANDRO suppresses the proliferation of pancreatic cancer cells. 3.2. ANDRO induces apoptosis and causes cell cycle arrest To assess whether the loss of cell viability could in part be due to apoptosis, the apoptotic efficacy of ANDRO was evaluated by

FACS analysis using the AsPC-1 and Panc-1 pancreatic cancer cell lines. As shown in Fig. 2A and B, the compound augmented pancreatic cancer cell apoptosis in a time- and dose-dependent manner. The apoptosis rate of AsPC-1 and Panc-1 cells induced by exposure to ANDRO (25 ␮mol/l; 48 h or 50 ␮mol/l; 24 h) were significantly different (P < 0.01). Nevertheless, no obvious discrepancies were detected when L-02 cells were treated by ANDRO at a concentration of 25 ␮mol/l for 0, 12, 24 or 48 h (Fig. 2B). These data confirmed ANDRO’s minimal cytostatic effects in normal cells. The effect of ANDRO on the cell cycle progression was also examined by flow cytometry. Fig. 2 C shows that an exposure to 10 ␮mol/l

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Fig. 2. ANDRO led to cell cycle arrest and increased levels of apoptosis of pancreatic cancer cells, and had no apoptotic effect in L-02 cells. (A) Apoptosis was measured by flow cytometric detection via Annexin-V-FITC and propidium iodide labelling of 105 pancreatic cancer cells (AsPC-1, and Panc-1) after treatment with doses of 0, 10, 25, 50 ␮mol/l ANDRO for 24 h. (B) Apoptosis was measured by flow cytometric detection via Annexin-V-FITC and propidium iodide labelling of 105 pancreatic cancer cells (AsPC-1, and Panc-1) and 105 L-02 cells after treatment with 25 ␮mol/l ANDRO for 0, 12, 24, 48 h. (C) Cell cycle progression of AsPC-1 and Panc-1 was analysed using flow cytometry after exposure to doses of 0, 10, 25, 50 ␮mol/l ANDRO for 24 h. (“*”P < 0.05, versus control. “**”P < 0.01, versus control). These figures are from representative experiments carried out at least three times.

ANDRO for 24 h caused a slight but significant increase in cell frequency at G2/M, but treatment with higher concentrations (25 or 50 ␮mol/l) for 24 h led to an apparent accumulation of the cells in the G0/G1 phase. Distribution of the G0/G1 phase cells in the population increased from 34.1% ± 2.4 to 65.9 ± 5.1% in Aspc-1 cells and from 23.8 ± 3.3% to 60.2 ± 5.0% in Panc-1 cells in a dose-dependent manner. Simultaneously, the compound treatment of pancreatic

cancer cells from both cell lines led to a dose-dependent decrease in the percentage of cells present in S phase. When increasing ANDRO concentration, we found that contraction of cell cycle profiles along the X-axis. ANDRO may affect chromatin condensation to make difficult PI intercalation to cause this phenomenon. Our data suggest that ANDRO induces cycle arrest and apoptosis of pancreatic cancer cells.

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3.3. ANDRO regulates the expression of proteins involved in apoptotic pathways and cell cycle regulation To further understand the molecular basis of ANDRO-induced apoptosis and cell cycle arrest, we detected the proteins associated with apoptosis and cell-cycle progression by western blot analysis using AsPC-1 and Panc-1 cells after exposure to increasing concentrations of ANDRO for 48 h. As shown in Fig. 3A, the expression of the pro-apoptotic protein Bax was significantly induced in a concentration-dependent manner. Simultaneously, Bcl-2 expression was markedly inhibited. Therefore, the ratio of Bax/Bcl-2 was dramatically up-regulated, which is a critical determinant of apoptosis. Furthermore, the antiapoptotic molecules Survivin and X-IAP were also examined. They were down-regulated in a dose-dependent manner after exposure to ANDRO. The cleaved active components of PARP, caspase-3, and caspase9 markedly increased in a dose-dependent manner following treatment with ANDRO (Fig. 3A). An upstream event in the activation of the caspase cascade is the release of cytochrome c from mitochondria. As shown in Fig. 3A, ANDRO caused the release of cytochrome c, indicating that ANDRO-induced apoptosis in AsPC-1 and Panc-1 cells is mediated at least in part by the mitochondrial pathway. The expression of cyclin D1, cyclin E, and p21WAF1 , which are associated with cell cycle regulation, were also measured by western-blot. Cyclin D1 and cyclin E were downregulated by 50 ␮mol/l ANDRO exposure for 48 h in AsPC-1 cells. We also obtained an analogous result in Panc-1 cells. Cyclin-dependent kinase inhibitor p21WAF1 , which acts as negative cell cycle regulator, was up-regulated significantly with exposure to ANDRO in the two pancreatic cancer cells. These results reveal that induction of the cell cycle arrest of AsPC-1 and Panc-1 cells exposed to ANDRO is partly associated with down-regulation of cyclin D1, cyclin E and up-regulation of p21WAF1 . 3.4. ANDRO inhibits activation of STAT3 and AKT in human pancreatic cancer cells In view of the important role of STAT3 activation in cell growth, proliferation, and survival in many human cancers (Sun et al., 2005; Lin et al., 2010), including pancreatic cancer (Thoennissen et al., 2009; Lin et al., 2010; Scholz et al., 2003; Corcoran et al., 2011), and STAT3 could be activated by Src (an oncogenic kinase) (Al Zaid Siddiquee and Turkson, 2008), we tested the ability of ANDRO to modulate the expression of these proteins. We exposed AsPC-1 and Panc-1 cells to ANDRO (25 ␮mol/l) for various times and assessed the levels of phosphorylated-Src (P-Src), Src, phosphorylated-STAT3 (P-STAT3) and STAT3 by western blotting. Src and STAT3 were found to be constitutively active, and ANDRO effectively downregulated P-Src and P-STAT3 levels (Fig. 3B). Interleukin-6 is known to activate Jak2/STAT3 signalling pathway (Al Zaid Siddiquee and Turkson, 2008). Therefore, we determined whether ANDRO affects Jak2/STAT3 activation induced by IL-6. IL-6 (100 ng/ml) induced phospho-Jak2 and phospho-STAT3 in AsPC-1 and Panc-1 cells in a time-dependent manner (Fig. 3C), and treatment with ANDRO (25 ␮mol/l; 48 h) led to a dramatic suppression of IL-6-induced phosphorylation of Jak2 and STAT3 (Fig. 3C). These results suggest that ANDRO can suppress both constitutive and IL6-induced STAT3 activation by dual inhibition of Src/STAT3 and Jak2/STAT3 pathways. Activation of AKT also plays a major role in cancer cell survival (Nicholson and Anderson, 2002; Ahmad et al., 2012), including pancreatic cancer cells (Simon et al., 2009). Hence, we examined whether ANDRO modulated the activation of AKT in pancreatic cancer cells. AKT was proved to be constitutively active in

AsPC-1 and Panc-1 cells, and ANDRO suppressed these constitutive phosphorylated-AKT levels in a time-dependent manner, with maximum inhibition observed at 48 h (Fig. 3B). 3.5. ANDRO potentiates the anti-proliferative effects of gemcitabine In light of the anti-pancreatic cancer activity of ANDRO alone, we examined whether combined therapy with gemcitabine, which is known to be a standard agent in pancreatic cancer patients, could have combinatory effects. The effect of gemcitabine on the viability of AsPC-1 and Panc-1 cells was determined by CCK-8 assay (data not shown). Cells were treated with ANDRO, chosen suboptimal doses of gemcitabine, or both drugs in combination, and cell viability was determined (Fig. 4A). We found co-treatment with ANDRO (10 ␮mol/l; 48 h) and gemcitabine (200 nmol/l or 400 nmol/l; 48 h) in AsPC-1 cells caused more significant suppression of cell viability than monotherapy of either drug, all with statistical significance (P < 0.05, Fig. 4A). The results were similar in Panc-1 cells treated with ANDRO (10 ␮mol/l; 48 h) and gemcitabine (500 nmol/l or 1000 nmol/l; 48 h). Moreover, for evaluating the synergistic efficacies, the values of CDI (coefficient of drug interaction) were determined as described by Wang et al. (Wang et al., 2012). CDI is calculated as follows: CDI = (observed fraction value/expected fraction value). The observed fraction = (mean value of experiment)/(mean value of control). The expected fraction of combination = (mean fraction of ANDRO) × (mean fraction of gemcitabine). CDI < 1 indicates synergism, CDI > 1 indicates antagonism, and CDI = 1 indicates a simple additive effect. The values of CDI were 0.86 and 0.70 in ASPC-1 cells when treated with ANDRO (10 ␮mol/l; 48 h) and gemcitabine (200 nmol/l or 400 nmol/l; 48 h). The CDI values were 0.96 and 0.95 in Panc-1 cells treated with ANDRO (10 ␮mol/l; 48 h) and gemcitabine (500 nmol/l or 1000 nmol/l; 48 h). The results indicate the two drugs have a synergistic activity in AsPC-1 cells and an additive effect in Panc-1 cells. We also performed a crystal violet assay to verify the combinatory effects of the two drugs using AsPC-1 and Panc-1 cells treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine(400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or both drugs in combination for 48 h. The relative signal intensity of combination treatment was decreased dramatically more than that of either agent alone (P < 0.05, Fig. 4B). These data were consistent with the aforementioned CCK-8 assay. 3.6. ANDRO augments the effects of apoptosis and cell cycle arrest induced by gemcitabine We next investigated whether enhanced cytostatic effects by co-treatment of ANDRO and gemcitabine was mediated by apoptosis. For these studies, cells were treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination, stained with AnnexinV/PI, and subjected to flow cytometry to determine the apoptosis rate. As shown in Fig. 5A, the apoptotic rate induced by the combination treatment was 46.93 ± 2.25% in AsPC-1 cells, while monotherapy with gemcitabine or ANDRO achieved only 16.13 ± 1.09% and 22.67 ± 1.73%, respectively (both with P < 0.05, versus monotherapy). In Panc-1 cells, co-treatment with ANDRO and gemcitabine caused the apoptotic index to rise to 45.37 ± 3.72%, but either alone achieved only 15.07 ± 0.99% and 21.10 ± 2.06%, respectively (both with P < 0.05, versus monotherapy; Fig. 5A). In addition, we evaluated the apoptotic effect of the combination of ANDRO with gemcitabine on L-02 cells using flow cytometry. As shown in Fig. 5A, combination therapy did not cause

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Fig. 3. ANDRO regulated the expression of proteins involved in apoptotic pathways and cell cycle regulation and suppressed activation of STAT3 and AKT in human pancreatic cancer cells. (A) Western blot depicted alterations in apoptosis and cell cycle progression related proteins in lysates prepared from AsPC-1 and Panc-1 cells after treatment with various doses of ANDRO for 48 h. (B) Western blot showed changes in Src, P-Src, STAT3, P-STAT3, AKT, and P-AKT in lysates prepared from AsPC-1 and Panc-1 cells after exposure to 25 ␮mol/l ANDRO for 0, 12, 24, 48 h. (C) Western blot indicated alterations of Jak2, P-Jak2, Stat3 and P-Stat3 in lysates prepared from AsPC-1 and Panc-1 cells after incubated with or without ANDRO (25 ␮mol/l) for 48 h in the absence of serum and then Interleukin-6 (100 ng/ml) was added for the indicated times. Densitometry analysis of the blot was carried out using the ImageJ software. (“*”P < 0.05, versus control. “#”P < 0.05, versus IL-6/15 min. “and”P < 0.05, versus IL-6/30 min). Data are representative of values from at least three independent experiments.

any obvious apoptotic effect relative to either agent alone or the control, indicating negligible normal human cell toxicity of ANDRO combined with gemcitabine. To elucidate the specific effect of combination treatment on cell cycle progression, we analysed the cell cycle distribution of AsPC-1 and Panc-1 cells treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. As shown in Fig. 5B, combination therapy

stimulated greater G0 –G1 arrest of AsPC-1 and Panc-1 cells compared to either drug alone (both with P < 0.05). Relative to gemcitabine treatment alone, the distribution of the G0 –G1 phase cells in the population increased from 64.1 ± 3.3% to 80.2 ± 4.6% in AsPC1 cells and from 70.7 ± 3.1% to 87.8 ± 3.1% in Panc-1 cells. These results suggest that enhanced cell cycle G0 -G1 arrest and promoted apoptosis involve the synergistic or additive anti-proliferative effects of co-treatment with ANDRO and gemcitabine.

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Fig. 4. ANDRO potentiated the anti-proliferative effects of gemcitabine. (A) AsPC-1 and Panc-1 Cells were treated with ANDRO (10 ␮mol/l; 48 h), chosen suboptimal doses of gemcitabine (48 h), or their combination, and cell viability was determined using the CCK8 assay. (B) Cells were treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination before being reseeded in six-well plates (5000 cells/well). After 2 weeks, the surviving clones were stained with 0.5% crystal violet for 1 h and photos were taken using digital camera. The relative signal intensity was evaluated by Image J software. (“*”P < 0.05, versus control. “**”P < 0.01, versus control. “* #” P < 0.05, versus monotherapy). Data are representative of values from at least three independent experiments.

3.7. Combination therapy of ANDRO and gemcitabine modulates the expression of proteins associated with apoptosis and cell cycle arrest

3.8. Combination therapy causes enhanced inhibition of activated STAT3 and suppression of activated Akt in human pancreatic cancer cells

To determine the molecular mechanism of combinationinduced apoptosis and cell cycle arrest, we determined the proteins involved in apoptosis and cell-cycle progression by western blot analysis using Panc-1 and AsPC-1 cells treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. As shown in Fig. 6A, co-treatment in AsPC-1 and Panc-1 cells showed a comparatively stronger band of cleaved PARP, caspase-3, and caspase-9, although these bands were visible in cells treated with the single agents. The pro-apoptotic protein Bax in combined treatment was significantly upregulated compared to monotherapy. Simultaneously, the anti-apoptotic member Bcl-2 was markedly inhibited relative to either agent alone. Therefore, the ratio of Bax/Bcl-2 was dramatically upregulated, which is crucial to apoptosis. Other prosurvival molecules such as Survivin and X-IAP were determined and showed more significant downregulation with combination treatment (Fig. 6A). Down regulation of cyclin D1 and cyclin E was more obvious in combination treatment compared to either single agent therapy in the two pancreatic cancer cells. These results could partly explain the synergistic or additive effect of the two drugs.

To further understand the sensitisation mechanism, we also examined phosphorylated-STAT3 and phosphorylated-Akt in pancreatic cancer cells (AsPC-1 and Panc-1) after treated with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. As shown in Fig. 6B, downregulation of P-STAT3 was more obvious in combination treatment compared to either single agent therapy in AsPC-1 and Panc-1 cells. The activation of Akt was inhibited after treatment with ANDRO, but not by gemcitabine (Fig. 6B). Combined ANDRO with gemcitabine led to significant inhibition of P-Akt compared to gemcitabine alone. Our results partially explained the synergistic or additive effect of the two drugs, as both P-STAT3 and P-Akt are related to chemo-resistance in pancreatic cancer (Thoennissen et al., 2009; Kagawa et al., 2012). 3.9. ANDRO decreases the growth of AsPC-1 human pancreatic tumour xenografts and enhances the in vivo therapeutic effect of gemcitabine Building on these results, we examined the effects of ANDRO and gemcitabine, either alone or in combination, on

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Fig. 5. ANDRO augmented the effects of gemcitabine on apoptosis and cell cycle arrest in human pancreatic cancer cells. (A) Apoptosis was measured by flow cytometric detection via Annexin-V-FITC and propidium iodide labelling of 105 pancreatic cancer cells (AsPC-1, and Panc-1) and 105 L-02 cells after treatment with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1 and L-02; 48 h), or their combination. (B) Cell cycle progression of AsPC-1 and Panc-1 was analszed using flow cytometry after exposure to ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. (“*”P < 0.05, versus control. “**”P < 0.01, versus control. “*#” P < 0.05, versus monotherapy). Data are representative of values from at least three independent experiments.

the growth of human pancreatic tumour xenografts in vivo. As shown in Fig. 7A, the tumour volume in the control group reached 1590.4 ± 237.7 mm3 . In contrast, the tumour volume in ANDRO group was significantly (P < 0.05) lower, reaching only 937.5 ± 140.2 mm3 , suggesting the efficacy of ANDRO in the suppression of pancreatic cancer cells in vivo. Gemcitabine also significantly (P < 0.05) decreased tumour volume growth (1069.5 ± 164.3 mm3 ) compared with the control. However, the tumour volume in the ANDRO plus gemcitabine group was not only greatly significantly lower than the control group (P < 0.01), but also lower than either group using a single agent (P < 0.05), reaching only 587.6 ± 99.32 mm3 . In addition, as shown in Fig. 7B, the average tumour weight in the ANDRO group decreased compared to the control group, to 879.8 ± 80.7 mg from 1489.0 ± 97.1 mg (P < 0.01). The combination of ANDRO and gemcitabine also significantly reduced the tumour weight to 482.5 ± 51.9 mg as compared to 965.3 ± 65.6 mg in those treated with gemcitabine alone (P < 0.05). Fig. 7C shows the fractional tumours excised from each treatment group. We also evaluated the changes in body weight during the therapy in the four groups. As shown in Fig. 7D, the body weight of the ANDRO group was not lower than the control group. Furthermore, no significant weight loss was observed in the combination therapy group as compared to the single agent group or the control group, suggesting that good therapeutic effect was gained without significant addition of toxicity. Ki-67 (a common marker of cell proliferation) and TUNEL (an indicator of apoptosis) were further detected in tumour sections prepared from the above tumours. As shown in Fig. 7E, monotherapy with either ANDRO or gemcitabine significantly decreased the

expression of Ki-67 compared with the control (P < 0.05). Furthermore, the combination of the two drugs significantly reduced the expression of Ki-67 not only more than the control (P < 0.01), but also more than either group treated with a single agent (P < 0.05). As shown in Fig. 7F, the ANDRO group showed a higher apoptosis index than the control group (P < 0.05), while the combination group significantly up-regulated the apoptosis index to levels higher than both the control group (P < 0.01) and either single agent alone (P < 0.05). Our results demonstrate that ANDRO could effectively decrease the growth of human pancreatic tumour xenografts and enhance the in vivo therapeutic effect of gemcitabine. 4. Discussion The ominous prognosis of pancreatic cancer is due to its tendency for late presentation, aggressive local invasion, early metastases, and poor response to chemotherapy (Vincent et al., 2011). Since 1997, gemcitabine has been the first-line therapy for patients with locally advanced and metastatic pancreatic cancer (Burris et al., 1997). Unfortunately, gemcitabine treatment results in an objective tumour response rate of less than 10%, which is partly attributable to intrinsic and acquired drug resistance. Although much effort has been made in combination therapy with other agents, the overall results have not been encouraging (Moore et al., 2007). Therefore, investigation of new drugs for pancreatic cancer is urgent. Plant-derived natural products occupy an important position in the area of cancer chemotherapy. Andrographolide, the principal bioactive chemical constituent of Andrographis paniculata Nees,

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Fig. 6. Combination therapy of ANDRO with gemcitabine modulated the expression of proteins associated with apoptosis and cell cycle G1 arrest and also significantly suppressed activation of STAT3 and Akt in human pancreatic cancer cells. (A) Western blot showed alterations in apoptosis and cell cycle progression related-proteins in lysates prepared from AsPC-1 and Panc-1 cells after treatment with ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. (B) Western blot indicated changes to STAT3, P-STAT3, Akt, and P-Akt in lysates prepared from AsPC-1 and Panc-1 cells after exposure to ANDRO (10 ␮mol/l; 48 h), gemcitabine (400 nmol/l for AsPC-1 and 1000 nmol/l for Panc-1; 48 h), or their combination. Densitometry analysis of the blot was carried out using the ImageJ software. (“*”P < 0.05, versus control. “* #” P < 0.05, versus monotherapy). Data are representative of values from at least three independent experiments.

draws our attention because of its potent anti-tumour activities in many kinds of cancers (Lim et al., 2012). In the present study, we provide strong evidence that ANDRO inhibits the growth of pancreatic cancer cells and augments the anti-tumour activity of gemcitabine in vitro and in vivo. Loss of cell proliferation and/or induction of apoptosis are two key mechanisms by which cytotoxic drugs kill cancer cells (Gordaliza, 2007; Mann, 2002). In the present study, we demonstrated that ANDRO dramatically inhibited pancreatic cancer cell proliferation using both the short-term CCK8 assay and the longterm colony formation assay. Furthermore, we showed that ANDRO significantly enhanced apoptosis in a dose- and time-dependent manner, which was associated with upregulation of caspase family members 3 and 9 and subsequent cleavage of the PARP protein. It has been reported that ANDRO induces cancer cell apoptosis via a mitochondria pathway by modulation of Bcl-2 family members (Zhou et al., 2006). In the present study, we also showed that ANDRO effectively downregulated the anti-apoptotic protein Bcl2 and upregulated the pro-apoptotic factor Bax. This modulation led to the release of cytochrome c from mitochondria and initiated subsequent apoptosis. These results are consistent with the data of Dey et al. (Dey et al., 2013), indicating increased apoptosis induced by ANDRO and its derivatives via mitochondrial apoptotic pathway in MiaPaCa-2 cells. Survivin and XIAP, which are predominantly over-expressed in pancreatic cancer cells and are associated with poor prognosis (Jamieson et al., 2011; Tamm et al., 2000;

Lee et al., 2005), were also inhibited by ANDRO treatment in our study. In the spectrum of cell cycle progression, ANDRO caused cell cycle arrest in pancreatic cancer cells, which was associated with downregulation of cyclin D1 and cyclin E and upregulation of p21WAF1 . P21WAF1 not only mediates cell cycle arrest dependent of p53 but may also be responsible for the anti-proliferative effects independent of p53 (Zeng and el-Deiry, 1996; Warfel and El-Deiry, 2013). ANDRO is able to upregulate the expression of p21WAF1 independent of p53 as both AsPC-1 and Panc-1 cells have p53 mutations and our data demonstrated that ANDRO could downregulate the expression of the mutant p53 in a dose-dependent manner in Panc-1 cells (Fig. S1). This is relevant because p53 mutations have been found in 81.1% of pancreatic cancer tissues (Oshima et al., 2013). Apoptosis-related proteins (Bcl-2, Bax, and survivin) and cell cycle regulation proteins (p21WAF1 and cyclin D1) are specific target genes that are transcriptionally regulated by activated STAT3 (Darnell, 1997; Scholz et al., 2003) and/or Akt (Nicholson and Anderson, 2002). In the present study, we showed that ANDRO suppressed both constitutive and IL-6-induced STAT3 activation by dual inhibition of Src/STAT3 and Jak2/STAT3 pathways. In addition, ANDRO suppressed constitutive phosphorylated-AKT levels in a time-dependent manner, which may be related to inactivation of Src (Frame, 2002). It is conceivable that ANDRO inhibits proliferation and induces apoptosis via inactivation of STAT3 and Akt, as well as subsequent modulation of their down-stream gene products.

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Fig. 7. ANDRO decreased the growth of AsPC-1 human pancreatic tumour xenografts and enhanced the in vivo therapeutic effect of gemcitabine. (A) Tumour volume was estimated using callipers and calculated using the formula: V = (length × width2 ) ×␲/6 at indicated time points. (B) Date represent the mean tumour weight of each group. (C) Illustration showing the tumours excised from each treatment group. (D) The change in body weight during the treatment with ANDRO, gemcitabine or their combination. (E) Analysis of proliferation marker Ki-67 by immunohistochemistry. The proliferation index of tumour cells was measured by Ki-67-positive cell percentage counted in 10 randomly selected × 400 high-power fields under microscopy. (F) Analysis of apoptotic markers by in situ TUNEL assay. TUNEL-positive cells were counted in 10 randomly selected × 400 high-power fields under microscopy to measure the apoptotic index. (“*”P < 0.05, versus control. “**”P < 0.01, versus control. “*#” P < 0.05, versus monotherapy).

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. toxlet.2013.06.241. Combining some new phytochemicals with the standard chemotherapeutic drug gemcitabine is a promising exploration for treatment of pancreatic cancer (Wang et al., 2010; Banerjee et al., 2009; Thoennissen et al., 2009). Therefore, we evaluated whether ANDRO and gemcitabine had combinatory effects, and our results demonstrated that their co-treatment exhibited synergistic or additive proliferation suppression, enhanced cell cycle arrest and promoted apoptosis compared to either single application. Furthermore, combined treatment significantly downregulated cyclin D1, cyclin E, Bcl-2, Survivin and X-IAP and up-regulated Bax and p21WAF1 . Pro-apoptotic Bax expression has also been shown to be involved in the synergistic effect of ANDRO and 5-fluorouacil on SMMC-7721 cells via enhanced apoptosis through a caspase-8 dependent mitochondrial pathway (Yang et al., 2009). Activation of STAT3 has been shown to confer resistance to chemotherapyinduced apoptosis in pancreatic cancer cells (Thoennissen et al., 2009; Nagaraj et al., 2011). ANDRO has been shown to enhance the chemo-sensitivity of cancer cells to doxorubicin via inhibition of the JAK/STAT3 pathway (Zhou et al., 2010). In the present study, we found that downregulation of p-STAT3 was more obvious in combination treatment compared to either single agent therapy. In addition, combined ANDRO with gemcitabine led to significant inhibition of p-Akt compared to gemcitabine alone, and activated Akt has been shown to be crucial for gemcitabine resistance in pancreatic cancer cells (Kagawa et al., 2012). Thus, via enhanced inhibition of activated STAT3 and suppression of activated Akt, ANDRO potentiates the anti-tumour activity of gemcitabine in pancreatic cancer cells. Recently, a global genomic analysis proved pancreatic cancer to be highly heterogenous, containing many genetic alterations (average of 63) that affect a core set of 12 signalling pathways (Jones et al., 2008). Therefore, targeting these complex and overlapping signalling pathways, rather than just the products of a single gene, should be a more promising direction for treatment in pancreatic cancer (Nagaraj et al., 2011). A strong argument can be made that by targeting STAT3 and Akt pathways concurrently, ANDRO may be more efficacious than if it has only one single target. In addition, ANDRO with dual specificity may have the extra advantage of being less likely to induce drug resistance. Our in vitro results were also recapitulated in an in vivo subcutaneous pancreatic cancer model. In vivo, we found that ANDRO alone had potential anti-pancreatic cancer effects, but that the combination of ANDRO with gemcitabine exhibited the strongest anti-tumour efficacy. These results draw a parallel with increased apoptosis as documented by increased TUNEL staining, and reduced proliferation as documented by Ki-67 immunoreactivity. Furthermore, the chemotherapeutic approaches did not cause any obvious addition of toxicity in terms of progressive weight loss. In conclusion, we show for the first time that ANDRO, a multitargeted agent, not only has profound anti-pancreatic cancer activity when used alone, but also potentiates the anti-tumour activity of gemcitabine in vitro and in vivo against human pancreatic cancer cells. The underlying mechanisms may be, at least in part, due to inactivation of STAT3 and Akt and modulation of their down-stream gene products to initiate cell cycle arrest and increased levels of apoptosis. Given the low toxicity of ANDRO, we believe that ANDRO alone or combined with gemcitabine may hold great promise for development as a novel chemotherapeutic approach in human pancreatic cancer in the future, and we suggest that a clinical trial be performed.

Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported in part by Grants from the Shanghai Commission of Science and Technology (11JC1407801) and Shanghai JiaoTong University School of medicine (2012073). We thank Dr. Yuan Fang for critical reading of the manuscript. References Ahmad, K., Biersack, B., Li, Y., Bao, B., Kong, D., Schobert, R., Padhye, S.B., Sarkar, F.H., 2012. Deregulation of PI3K/Akt/mTOR signaling pathways by isoflavones and its implication in cancer treatment. Anticancer Agents in Medical Chemistry [Epub ahead of print]. Al Zaid Siddiquee, K., Turkson, J., 2008. STAT3 as a target for inducing apoptosis in solid and hematological tumors. Cell Research 18, 254–267. Banerjee, S., Kaseb, A.O., Wang, Z., Kong, D., Mohammad, M., Padhye, S., Sarkar, F.H., Mohammad, R.M., 2009. Antitumor activity of gemcitabine and oxaliplatin is augmented by thymoquinone in pancreatic cancer. 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