Sophoridine induces apoptosis and S phase arrest via ROS-dependent JNK and ERK activation in human pancreatic cancer cells

Sophoridine was kindly provided by National Institute for the Control of Pharmaceutical and Biological Products. Its purity was at least 95% as determined by HPLC analysis. Rhodamine 123, Hoechst 33,342 and Cycloheximide (CHX) were obtained from Sigma-Aldrich (MO, USA). Annexin V/PI apoptosis kit and Cell Counting Kit-8 (CCK-8) were relatively purchased from Invitrogen (CA, USA) or Dojindo Laboratories (Japan). DC protein assay kits were purchased from Bio-Rad, and the enhanced chemiluminescence plus system was purchased from Amersham Pharmacia Biotech. The antibodies against Bax, Bad, Bcl-XL, Bcl-2, cleaved-caspase 3 (Asp175), PARP cyt C and GAPDH were purchased from Cell Signaling Technology (MA, USA). ERK, JUNK and p-38 antibodies were purchased from Santa Cruz. Antibodies against Cyclin A, CDK2 and Cyclin D1 were purchased from Epitomics (CA, USA). PCNA antibody was obtained from Abcam (Cambridge, UK).

The cell lines Miapaca-2, PANC-1 and HPDE were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Miapaca-2 cells were maintained in RPMI-1640 (Gibco, NY, USA) containing with 100 U/mL penicillin-streptomycin (Hyclone, UT, USA) and 10% fetal bovine serum (Gibco). PANC-1 cells were cultured in DMEM medium (Gibco) containing 10% FBS. HPDE cells were cultured in K-SFM medium (Gibco) containing 10% FBS and 1% epidermal growth factor. All cell lines were maintained at cell culture incubator with 37 °C and 5% CO2. The details of other cell lines are available in Additional file 1.

Cell viability was measured with CCK-8 kit, followed the manufacturer. Briefly, cancer cells seeded in 96-well plates were either treated with Sophoridine at serial concentrations for 48 h, or were treated for various time points (0, 24, 48, or 72 h). After treatment for indicated time, CCK- 8 solution was added and incubated with cancer cells for 4 h. The percentages of cell survival was measured by SpectraMax190 microplate reader (Molecular Devices) based on the absorbance.

Propidium iodide (PI) staining assay was used to analyze the cell cycle distribution. After exposed to different concentrations of Sophoridine for 48 h, cancer cells were harvested and fixed with 70% ethanol, followed by centrifugation (3000 rpm, 5 min), incubation with 100 mg/mL RNase in PBS for 30 min at 37 °C, and then staining with 50 mg/mL PI in PBS. The cell cycle distribution were analyzed by a Cell Lab Quanta SC flow cytometer (Beckman Coulter, USA). The Annexin V–FITC Apoptosis Detection Kit (BioVision) was used for the apoptosis analysis. Cells (5 × 10⁵) were exposed to different concentrations of Sophoridine for 48 h. After resuspended in 500 ml binding buffer, cells were incubated with Annexin V– fluorescein isothiocyanate (FITC; 5 ml) and PI (5 ml). After 30-min incubation, cells were analyzed by fluorescence-activated cell sorting (FACS) by flow cytometer (Becton Dickinson). Annexin V–FITC–stained only cells which indicated early apoptosis and cells with Annexin V–FITC- and PI double positive signals were combined for analysis.

Five hundred cancer cells per well were seeded into 6-well plates, and then treated with Sophoridine at different concentrations for 48 h. After treatment, the cells were allowed to form cell colonies for another 7 days. Then cell colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. After 3 times washing and air-dried, the stained colonies were counted and photographed under microscope (Leica, Germany).

Hoechst 33,342 was used to identify the apoptotic cells based on the morphological changes in the nuclear assembly as previously described [16].

After 20 μM Sophoridine cells were stained with JC-1, which can indicates the change of mitochondrial membrane potential. The wavelengths of 490/540 nm was used. The mitochondrial membrane potential was indicated by the ratio between 590 nm and 540 nm (red signal and green signal, relatively).

Miapaca-2 and PANC-1 cancer cells were treated with Sophoridine at different concentrations for 48 h, and then whole-cell lysates were prepared for western blot analysis as previously described [17].

The peroxide-sensitive fluorescent probe DCFH-DA was used to measure the intracellular ROS levels. Briefly, cells were suspended in 1 mM DCFH-DA at 37 °C for 30 min. After incubation, cells were washed twice with PBS and re-suspended in PBS. ROS accumulation was detected with Calibur flow cytometry system at the wavelength 488/538 nm.

BALB/c homozygous (nu/nu) nude mice (6 weeks, 18 g) were purchased from Shanghai SLAC Laboratory Animal (China). All animal experiments were followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. The mice were maintained in pathogen-free environment for one week after arrival. 2 × 10⁶ Miapca-2 cells in 100 μl PBS were inoculated in the right flank of nude mice. One week later, mice were randomly divided into 3 groups (7 mice/group). 3 groups received relatively an intraperitoneally (i.p.) injection of PBS as control, 20 mg/kg of Sophoridine, or 40 mg/kg of Sophoridine daily. After 3 weeks treatment, mice were sacrificed to weigh the tumors.

Tumors were excised to 4-mm sections, fixed with formalin and embedded with paraffin. Slides were stained with antibodies against phospho-ERK, JNK, cleaved-caspase-3 and PCNA, then after washing stained with secondary antibody and visualized by the ChemMate EnVision Kit. The stained sections were analyzed under microscope at a magnification of × 400. Some sections were stained with H&E for the histological analysis.

Sophoridine is a monomeric alkaloid extracted from sophora alopecuroides L (Fig. 1a). Modern pharmacologic evaluations have established that this herb medicine has potent cytotoxicity to cancer cells. We conducted a cell-based screening, examining the effects of Sophoridine on the cell viability of various tumor cells and normal cells (Fig. 1b, Additional file 2: Table S1). Eleven human cancer cell lines and 8 normal cell lines were exposed to various concentrations of Sophoridine (0–500 μmol/L) for 48 h. Cell viability was determined by the CCK-8 assay. Sophoridine exhibited remarkable inhibition effects to the growth of human pancreatic, gastric, liver, colon, gallbladder, and prostate carcinoma cells with IC50 values of about 20 μmol/L to 200 μmol/L. Among them, pancreatic cancer cells were the most sensitive cell lines to the cytotoxic effects of Sophoridine. Normal human pancreatic ductal epithelial cell (HPDE) and normal human bronchial epithelial cells (BEAS-2B) incubated with Sophoridine for 48 h, exhibited less sensitivity, indicating that Sophoridine selectively kills cancer cells.

To evaluate the cytotoxic effects of Sophoridine on pancreatic cancer cells, two human pancreatic cancer cell lines (Miapaca-2 and PANC-1) and pancreatic ductal epithelial cell line (HPDE) were treated with Sophoridine at different concentrations (0, 10, 20, 40, 80, or 100 μM) for different lengths of time (24 h, 48 h, or 72 h). The CCK-8 assay showed that Sophoridine exhibited both concentration-dependent and time-dependent killing effects on multiple pancreatic cancer cell lines, but had no significant effect on HPDE cells (Fig. 2a-d), indicating that Sophoridine selectively kill cancer cells but not normal cells. Furthermore, we adopted the doses 0, 10, 20 and 40 μM of Sophoridine based on the initial cytotoxicity results and the concentrations used in the following colony formation assays (Fig. 2e). Quantitative analysis further revealed that colony numbers decreased with increased Sophoridine dosage (Fig. 2f). These data suggests that Sophoridine inhibits pancreatic cancer cell growth in both dose-dependent and time-dependent manners.

In order to examine whether Sophoridine inhibits cell growth via cell cycle disturbance, cell cycle distribution and related cell cycle checkpoint factors were analyzed. We found that 20 μM Sophoridine treatment for 48 h to cancer cells leads to accumulated population in the S phase (Fig. 3a). Compared with the control, Sophoridine treatment increases S phase cell population from 26.23% (control) to 38.67% in Miapaca-2 cells and from 29.56% (control) to 39.16% in PANC-1 cells, about a 1.5-fold and a 1.3-fold increase, respectively. Additionally, Sophoridine treatment decreases the expression of Cyclin A, CDK2 and Cyclin D1 (Fig. 3b). In sum, S phase arrest contributes to the antiproliferative effects of Sophoridine in both cells and more significantly in Miapca-2 cells.

Next, the apoptotic effects of Sophoridine in pancreatic cancer cells were tested. Annexin V-FITC/PI double stainings were used to make a quantitative measurement of apoptosis. Twenty μM Sophoridine treatment for 48 h induces 10.65 ± 2.91% and 15.34 ± 2.36% apoptosis in Miapca-2 and PANC-1 cells, respectively (Fig. 4a). In addition, apoptotic cells indicated by nuclear condensation and fragmentation can be visualized by DAPI staining (Fig. 4b). We further tested the changes of the mitochondrial membrane potential (ΔΨm), bcl-2 family proteins, cytochrome c release, proteolytic cleavage of procaspase and the general caspase inhibitor (z-VAD-fmk) rescue assay. The loss of ΔΨm, measured by JC-1, can be measured after cells were exposed to 20 μM Sophoridine in both cell lines, especially in Miapaca-2 cells (Fig. 4c). To further test whether Sophoridine-induced cell apoptosis was caspase dependent, z-VAD-fmk, a pan caspase inhibitor, was used. As indicated in Fig. 4d and Additional file 3: Figure S1, the addition of z-VAD-fmk reduced the apoptotic population induced by Sophoridine from 10.89 to 3.73% in Miapaca-2 cells and from 14.79 to 2.32% in PANC-1 cells. The proto-oncoprotein Bcl-2 is a powerful antagonist of the mitochondrial pathway of apoptosis initiated by a variety of extra- and intracellular stresses. Therefore, we examined whether Sophoridine could alter the balance between proapoptotic Bax and antiapoptotic Bcl-2 proteins at the mitochondrial membrane. West blotting analysis showed that treatment of Miapca-2 and PANC-1 cells with 20 μM. Sophoridine significantly increased bad and bax levels, and decreased bcl-2 and bcl-xl levels in contrast, with a significant increase in Bax/Bcl-2 ratio. In consistent, cytochrome c was released, and cleavages of procaspase-3 and PARP were also observed (Fig. 4e). In addition, we found Sophoridine could upregulate ER stress mediators PERK and IRE but have no effect on ATF6 (Additional file 4: Figure S2). These results indicated that the activation of intrinsic apoptosis pathway was induced by Sophoridine.

MAPK signaling pathway has been shown to play essential roles in the regulation of a wide variety of cellular processes, including cell growth, cell cycle regulation, migration, differentiation, development, and apoptosis [18]. To further study the underlying mechanisms of Sophoridine anti-tumor effects, the activations of ERK, JNK and p38 MAP kinases were evaluated in both cell lines. West blotting showed that the phosphorylation levels of ERK and JNK kinases were significantly increased after Sophoridine treatment, but the phosphorylation level of p38 was little affected (Fig. 5a). Pre-treatment with 20 μM PD98059 or SP600125 almost completely blocked the phosphorylation of ERK or JNK induced by Sophoridine (Fig. 5b). Interestingly, as shown in Fig. 5c and d and Additional file 5: Figure S3, only the ERK inhibitor PD98059 had a reversible effects on the S phase cell cycle arrest induced by Sophoridine and the JNK inhibitor SP600125 significantly decreased cell apoptosis in response to Sophoridine. Consistent with the results of flow cytometry, western blot revealed that PD98059 treatment had an apparently opposite effects compared with Sophoridine on the S transition-related protein levels, but it did not significantly affect the apoptosis-related proteins. In contrast, SP600125 decreased the Sophoridine-induced apoptosis proteins, but it did not affect S phase transition-related proteins (Fig. 5e and f). These data indicated that the effects of apoptosis and S phase arrest in Miapaca-2 cells induced by Sophoridine were mainly mediated by activation of the JNKand the ERK signaling pathway.

Since ROS generation is associated with the loss of mitochondrial membrane potential [19], we measured the levels of ROS in Miapaca-2 cells treated with Sophoridine. As shown in Fig. 6a, the levels of ROS in cells after Sophoridine treatment were increased in a time-dependent manner. Next, to test whether the increased ROS generation may have a role in Sophoridine-induced cell cycle arrest or apoptosis, we pretreated the cells with the antioxidant N-acetylcysteine (NAC) 1 h before 48-h Sophoridine treatment. The results showed that pretreatment with NAC leaded to a significant inhibition of the Sophoridine-induced cell cycle arrest and apoptosis (Fig. 6b and c and Additional file 6: Figure S4). In addition, pivotal proteins associated with the cell cycle transition and apoptosis were further test to validate the role of ROS in Sophoridine’s antineoplastic effects. Western blotting analysis revealed that NAC restored the expression of Cyclin A and Cyclin D1 (Fig. 6d). Similarly, NAC decreased the cleavage of caspase-3 and PARP, and the expression of bax, and increased the Bcl-2 expression (Fig. 6e). Moreover, the activation of ERK and JNK kinases in Miapaca-2 cells treated with Sophoridine can be attenuated by NAC (Fig. 6f). These data suggest that Sophoridine-induced ROS generation activates ERK and JNK kinases, which trigger cell cycle arrest and mitochondrial apoptotic pathways in pancreatic cancer cells.

To further validate Sophoridine can inhibit tumor growth in vivo, 2 × 10⁶ Miapaca-2 cells were subcutaneously inoculated in Balb/c nude mice. Sophoridine treatment was administrated from the 7th day at 20 or 40 mg/kg intraperitoneally for 21 days. We found that Sophoridine showed significant inhibitory effects on tumor volume (Fig. 7a and b). The mass of tumors under 20 or 40 mg/kg Sophoridine treatment was significantly less than that of the control group (Fig. 7c). However, there was no significant changes on body weight under Sophoridine treatment (Fig. 7d). Furthermore, the activation of ERK and JNK in xenograft tumor tissues was tested by immunohistochemistry and immunoblotting. It showed that both ERK and JNK were activated in Sophoridine-treated xenograft tumor tissues (Fig. 7e and Additional file 7: Figure S5). In addition, IHC analysis of proliferating cell nuclear antigen (PCNA), cleaved caspase-3 and tunel staining showed significantly fewer proliferative cells, and more apoptotic cells in Sophoridine-treated tumors (Fig. 7e). The liver and kidney biochemical functions were monitored, and there were no obvious difference between control and Sophoridine treatment group (Fig. 7f). The liver and kidney from control and Sophoridine group were stained with H&E to further evaluate the toxicity after treatment. The histological structure of liver and kidney were observed and compared microscopically, and there were almost no histological changes after Sophoridine treatment (Fig. 7g). These results suggest that Sophoridine was an effective agent that can inhibit the growth of xenograft pancreatic tumors in vivo with well-tolerated toxicity.