Dual Effects of Silibinin on Human Pancreatic Cancer Cells

Background Silibinin is a avonoid with antihepatotoxic properties, and exhibits pleiotropic anticancer effects. However, the molecular mechanisms responsible for its anticancer actions in pancreatic cancer cells, and the effects on such cells and normal pancreatic cells, remain unclear. The objective of this study was to determine the effect of silibinin on human pancreatic cancer cells and normal ductal cells. Methods Human pancreatic cancer cells (MIA PaCa-2 and PANC-1) and normal ductal cells (hTERT-HPNE) were cultured with 0–400 μM silibinin for 48 h. Thereafter, the proliferation, invasion, apoptosis, and signaling pathways of the pancreatic cells were evaluated. Results Silibinin signicantly inhibited the proliferation, invasion, and spheroid formation of human pancreatic cancer cells in vitro in a dose-dependent manner (p < 0.05). It also induced apoptosis in a dose-dependent manner. Western blot analysis showed that silibinin downregulated extracellular signaling-regulated kinase (ERK) and serine/threonine protein kinase (AKT) in human pancreatic cancer cells. It also upregulated microtubule associated protein 1 light chain 3 β (LC3B) and cleaved caspase-3 via c-Jun N-terminal kinases (JNK) signaling. On the other hand, silibinin increased the mRNA and protein levels of c-Jun, Twist-related protein 1, and Snail. It also decreased exogenous p53 levels, but increased endogenous c-Jun protein levels in human pancreatic cancer cells. However, silibinin did not affect cell viability and endogenous c-Jun levels in pancreatic normal ductal cells. It increased exogenous p53 levels, but decreased stemness-related gene expression in pancreatic normal ductal cells. Silibinin increased Ki-67 levels in pancreatic cancer cells, but decreased them in pancreatic normal ductal cells. Conclusion Silibinin not only exerted anticancer effects by inhibiting AKT–ERK and JNK signaling, but also upregulated cancer stemness–related genes in human pancreatic cancer cells. These results suggest that silibinin should be used as a therapeutic agent for human pancreatic cancer with caution.


Introduction
Pancreatic cancer is a highly malignant disease that is characterized by locally advanced, unresectable disease, or metastasis at the time of diagnosis. Despite signi cant advances in surgery and chemotherapy, the ve-year survival rate of patients with pancreatic cancer has not signi cantly improved beyond <5% [1][2][3]. In particular, no curative treatment options exist for patients with advanced cancer. Resistance of pancreatic cancer cells to chemotherapeutic agents is a major problem in oncology [4].
Therefore, new treatment options that can overcome the chemotherapy resistance of pancreatic cancer cells are urgently needed.
Silybum marianum or milk thistle, from which silibinin is extracted, is an annual or biannual plant of the Asteraceae family. Originally native to Southern Europe and Asia, the plant is now found throughout the world. The medicinal part of the plant is its ripe seed [5,6]. It has been used to treat liver diseases (cirrhosis, jaundice, and hepatitis) and gallbladder disease. It has been claimed to protect the liver against poisons [7]. Silibinin is a hepatoprotective antioxidant that can stabilize and protect the membrane lipids of hepatocytes [8]. It can inhibit peroxidase and lipoxygenase [9,10]. Previous studies have shown that silibinin exerts anticancer effects on colon cancer, liver cancer, and breast cancer [11][12][13][14]. The role of silibinin in pancreatic cancer cells is as follows. Firstly, silibinin induces apoptosis of pancreatic cancer cells through a multi-signal pathway. Silibinin induces apoptosis by activating c-Jun N-terminal kinases /Stress-activated protein kinases (JNK/SAPK) signaling in SW1990, a human pancreatic cancer cell line [15,16]. Furthermore, silibinin induces apoptosis by downregulating the glucagon-like peptide-1 receptor and protein kinase A (GLP-1R/PKA) signal pathway in cells of INS-1, a rat insulinoma cell line, with amylin [17]. Secondly, silibinin reduces the tumor volume and inhibits weight loss in nude mice with BxPC-3 and PANC-1(kind of pancreatic ductal adenocarcinoma cell lines) tumor xenografts [18].
The molecular mechanisms involved in the anticancer action of silibinin are complex. Moreover, the targets or molecular mechanisms in human pancreatic cancer cells are the least studied compared with those in other cancers.
Cancer stem cells (CSCs) are cells that possess the ability to self-renew and differentiate into various types of mature cells. Such cells are rare in cancer. However, they play an important role in cancer homeostasis, metastasis, resistance to therapy, and subsequent tumor recurrence [19,20]. Pancreatic CSCs are associated with poor prognosis, tumor recurrence and metastasis, and epithelial mesenchymal transition [21]. c-Jun protein is an oncogenic transcription factor that is encoded by the Jun gene in humans, and undergoes a combination Activator protein 1 (AP-1) early response transcription with c-Fos [22]. It is involved in AKT-ERK and JNK/SAPK signaling [23]. c-Jun is an important signal for cell divisions. The G1 phase of the cell cycle arrests when c-Jun is de cient in cells. This implies that c-Jun regulates cell cycle progression and apoptosis [24]. c-Jun is required for tumor development stages, such as cell proliferation, angiogenesis, and metastasis [25,26].
Snail is zinc-nger protein that is encoded by the Snail gene in humans. It represses E-cadherin for the promotion of epithelial mesenchymal transition during embryonic development [33]. Snail causes the recurrence of human breast cancer by repressing E-cadherin and upregulating epithelial mesenchymal transition [34].
p53 is a known tumor suppressor gene in various organisms. Mutant p53 is associated with the drugresistant property of pancreatic cancer [35].
Prior studies have only shown the positive effects of silibinin on human pancreatic cancer cells, and there are no data on the effect of silibinin on human normal pancreatic cells.
The objective of this study was to investigate the molecular mechanism of silibinin and the responses of human pancreatic cells to silibinin treatment.

Flow cytometry analysis
Apoptosis assay MIA PaCa-2, PANC-1, and hTERT-HPNE cells were seeded in six-well cell culture plates at a density of 1 × 10 5 cells/well. After overnight culture, the cells were treated with various concentrations (0-400 μM) of silibinin for 24 h; then, the cells were harvested using 0.005 M trypsin-EDTA, and washed thrice with PBS.
The cells were then stained with Annexin V Apoptosis Detection Kit I (51-66211E; BD Pharmingen ™ , USA).
The stained cells were acquired and analyzed using Canto II (BD Pharmingen ™ ) and FlowJo software (Tree Star Inc., USA).

Western blot analysis
Proteins were extracted from silibinin (200 µM/mL)-treated pancreatic cells (the cells were cultured to 70% con uence in 60 mm dishes) using a radioimmunoprecipitation assay buffer (Sigma R0278, Sigma-Aldrich Co. LLC, USA) containing a protease inhibitor (#p8340, Sigma-Aldrich) and a phosphatase inhibitor (#p2850, Sigma-Aldrich). The proteins were separated using 10% SDS-polyacrylamide gel electrophoresis, and blotted onto PVDF membranes (Millipore Corporation, Billerica, MA, USA). These membranes were blocked with 5% (w/v) skim milk in TBS-T (20 mM Tris, pH 7.6, 136 mM NaCl containing 0.1% (v/v) Tween-20) at 25℃ for 1 h. After washing thrice with TBS-T, the membranes were incubated overnight with diluted primary antibodies (all antibodies were from Cell Signaling Technology Inc., USA; antibody:TBS-T = 1:1,000) at 4 °C. After washing thrice with TBS-T for 10 min each, the membranes were incubated with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies. ECL SuperSignal chemiluminescent substrate (Millipore Corporation, Billerica, MA, USA) was used to develop the membrane. Protein bands were then visualized using a LAS 3000 Imaging System (FujiFilm, R&D Systems, Minneapolis, MN, USA).

Invasion assay
Cell invasion was carried out overnight using a transwell lter chamber (8.0-μm pores) coated with 1% gelatin/DMEM, followed by drying at RT. Pancreatic cancer cells were harvested, washed once with the growth culture medium, and seeded on the upper chamber at 2 × 10 5 cells in 120 μL 0.2% BSA medium.
Cells that invaded the bottom surface of the upper chamber were xed with 70% ethanol and stained with Diff-Quik solution (Sysmex, Kobe, Japan), according to the manufacturer's protocol. Non-invasive cells on the top surface were wiped off with cotton balls and stained. Cells on the bottom surface were counted in ve selected elds (each 0.5 mm 2 ), using a hematocytometer under a light microscope at ×400 magni cation. The results were expressed as means ± SE of the number of cells per eld from three individual experiments.

RNA isolation
Total RNA was extracted using TRIzol (Takara, Japan). Brie y, 1 mL TRIzol solution was added into each well, and the suspensions were transferred to 1.5 mL tubes. After adding 200 μL chloroform (Sigma-Aldrich) and vortex mixing for 15 s, the mixtures were centrifuged at 4℃ and 6,000 × g (grams) for 20 min. The supernatants were then collected, mixed with equal amounts of isopropyl alcohol (MERCK), and centrifuged at 4℃ and 6,000 × g for 20 min. The pellets were washed with 1 mL 70% ethyl alcohol (MERCK) and centrifuged at 4℃ and 6,000 × g for 5 min. After removing the remaining ethyl alcohol, the RNA pellets were air-dried at RT. They were then resuspended in 50 µL diethyl pyrocarbonate water.
7. Real-time polymerase chain reaction (PCR) Total RNAs were converted to cDNAs using a reverse transcription system (Promega Corporation, WI 53711-5399, USA). Real-time PCR was performed with an Applied Biosystems StepOnePlus ™ (Thermo Fisher Scienti c, MA, USA) Real-Time PCR system, according to the manufacturer's protocol.

Statistical analysis
The two-tailed paired t-test was used to assess statistical signi cance appropriately. The statistical signi cance of differences between data sets was determined using the paired t-test and one-way ANOVA test. All the reported p values are two-sided; p ≤ 0.05 was considered statistically signi cant. The Statistical Package for the IBM SPSS Statistics 23 software (SPSS Statistics Inc., Chicago, IL, USA) was used for all statistical analyses.

Silibinin induces apoptosis of pancreatic cancer cell lines
Cells were stained with AnnexinV-FITC and propidium iodide (PI), and analyzed using ow cytometry. The binding of uorescently labeled Annexin V to externalized phosphatidylserine was also determined using ow cytometric analysis to quantify early apoptotic cells. PI uptake was measured to assess cells in the late stage of apoptosis, or cells that sustained direct plasma membrane damage. Figure 1 shows the ow cytometric plots obtained with AnnexinV-FITC PI assay after 24 h exposure to different concentrations (0-400 μM) of silibinin. After cell exposure to silibinin, the number of early and late apoptotic or directly damaged MIA PaCa-2 and PANC-1 cells signi cantly (p < 0.001) increased. However, silibinin did not affect the cell viability of hTERT-HPNE cells (p < 0.001) (Fig. 1).

Silibinin decreases phosphorylation of ERK-SAPK/JNK signaling proteins in human pancreatic cancer cell lines
Silibinin can modulate the phosphorylation of mitogen-activated protein kinase (MAPK) cascade signaling proteins in human pancreatic cancer cells [9]. The effect of silibinin on the MAPK signal pathway, which is essential for cell survival and growth during development and carcinogenesis, was examined. Silibinin treatment signi cantly decreased the expression levels of phosphorylated ERK 1/2 and phosphorylated p42/44 MAPK ( Figs. 2A and 2B, and 2D and 2E, respectively). As crosstalk between signal transduction pathways is common, we examined whether silibinin prevented the phosphorylation of p-ERK or SAPK/JNK in human pancreatic cancer cells. Silibinin treatment decreased the phosphorylation levels of AKT and SAPK/JNK. However, it did not alter the levels of total ERK 1/2, SAPK/JNK, p42/44-MAPK, and AKT ( Figs. 2A and 2B, and 2D and 2E, respectively). Furthermore, silibinin did not affect phosphorylation, or the total levels of AKT and SAPK/JNK in hTERT-HPNE cells (Figs. 2C and 2F).

Silibinin increases autophagy and apoptosis signaling in pancreatic cancer cell lines
Silibinin-induced apoptosis is associated with the modulation of apoptotic regulatory proteins in human pancreatic cancer cells [13][14][15]. The expression levels of cleaved caspase-3 and LC3B signi cantly increased in MIA PaCa-2 and PANC-1 cells following treatment with 200 µM silibinin (Figs. 3A and 3B, and 3D and 3E, respectively). However, silibinin did not affect the levels of cleaved caspase-3 and LC3B in hTERT-HPNE cells (Figs. 3C and 3F).

Silibinin decreases spheroid formation in pancreatic cancer cell lines
Cancer cells can be cultured in suspension to form spheres in a serum-free replacement medium. To test whether human pancreatic cancer cell lines could form spheres, MIA PaCa-2 and PANC-1 cells were cultured in a suspension culture system. Silibinin decreased the number of colonies after 14 days of incubation. We also observed a smaller number of colonies in silibinin-treated cultures relative to that in the control, for each cell line (p < 0.05). On average, silibinin exposure decreased the colony size of each cell line (Fig. 4B).

Silibinin decreases invasion and migration of pancreatic cancer cell lines
To determine whether silibinin in uenced the biological activity of the human pancreatic cancer cells used, we performed an invasion assay. After measuring six random squares of a 0.5 mm × 0.5 mm microscope eld, we found that the numbers of invading MIA PaCa-2 cells after treatment with 100 and 200 µM/mL silibinin were 239 ± 3.5 and 240 ± 15.1, respectively, whereas that for the negative control was 286 ± 7.3 (p = 0.007) (Fig. 4C). The numbers of invading PANC-1 cells after treatment with 100 and 200 µM/mL silibinin were 66 ± 4.24 and 35 ± 4.24, respectively, whereas that for the negative control was 112 ± 2.12 (p = 0.001) (Fig. 4C). Furthermore, silibinin treatment of MIA PaCa-2 and PANC-1 cells decreased migration, compared to that in negative control cells (Fig. 4A).

Silibinin increases expression of stemness-related signal pathway in pancreatic cancer cell lines
Silibinin increased the expression levels of TWIST1, Snail, and c-Jun mRNAs in pancreatic cancer cells, compared to those in the control (Figs. 5A and 5B) (p < 0.05). However, it decreased the expression levels of TWIST1, Snail, and c-Jun in pancreatic normal ductal cells (hTERT-HPNE) (Fig. 5C), compared with those in the control (p < 0.05). The TWIST1, Snail, and c-Jun mRNA expression levels were signi cantly higher in silibinin-treated cancer cells than in the negative control cells (p < 0.05) (Fig. 5D). However, the TWIST1, Snail, and c-Jun mRNA expression levels in silibinin-treated normal cells were lower than those in the negative control cells (p < 0.05) (Fig. 5D).

Silibinin decreases wild-type p53 protein levels and increases c-JUN protein levels in pancreatic cancer cell lines
Silibinin decreased the p53 protein levels in overexpressed wild-type p53 protein pancreatic cancer cell lines (Figs. 6A and 6B), however, increased those in overexpressed wild-type p53 protein pancreatic normal ductal cells (hTERT-HPNE) (Fig. 6C). In addition, silibinin increased c-Jun protein levels in pancreatic cancer cell lines (Figs. 6A and 6B), but decreased those in pancreatic normal ductal cells (hTERT-HPNE) (Fig. 6C).

Silibinin increases expression of Ki-67 in pancreatic adenocarcinoma cell lines
Ki-67 is a protein encoded by the MKI67 gene, and is used as a marker of proliferation. It is overexpressed in the G1, S, G2, and M phases of cell cycles, but is not expressed in the G0 phase [36]. Silibinin increased the expression of Ki-67 by 16.7% and 23.1% in the human pancreatic adenocarcinoma cell lines, MIA PaCa-2 and PANC-1, respectively. On the other hand, silibinin decreased the expression of Ki-67 by 7.3% in the pancreatic normal ductal cell line, hTERT-HPNE (p = 0.0001) (Figs. 7A and 7B).

Discussion
The present study demonstrated the following ndings: 1) silibinin does not affect cell viability in pancreatic normal ductal cells, but reduces cell viability, migration, invasion, and spheroid formation by downregulating AKT-ERK signaling in pancreatic adenocarcinoma cell lines; 2) silibinin decreases stemness-related signals, including the expression of c-Jun, TWIST1, and Snail, in pancreatic normal ductal cells, but increases those in pancreatic adenocarcinoma cell lines; 3) although silibinin increases wild-type p53 levels in pancreatic normal ductal cells, silibinin reduces those in pancreatic adenocarcinoma cell lines.
In addition, a limitation of these previous studies is that they focused less on the signal pathway of cell death in pancreatic cancer than that in other cancers, and only the positive effects of silibinin on human pancreatic cancer cells were reported. Furthermore, no studies have been conducted on the effects of silibinin on human pancreatic normal ductal cells. Our study has shown the effect of silibinin on pancreatic cancer cell lines as well as pancreatic normal ductal cell lines. The results of our study show that the effect of silibinin is broader than that indicated by other studies; it is a potent anticancer agent, but also has the potential to cause cancer recurrence. A recent study suggested that silibinin could induce cell death possibly via a bcl-2-sensitive apoptotic pathway in a human pancreatic adenocarcinoma cell line (SW1990) [13]. In the present study as well, 0-400 µM/mL silibinin induced the apoptotic cell death of pancreatic adenocarcinoma cell lines (MIA PaCa-2 and PANC-1), based on ow cytometer analysis (Fig. 1). Western blot analysis showed that silibinin downregulated the levels of AKT-ERK and SAPK/JNK in human pancreatic adenocarcinoma cells (Fig. 2). ERK 1/2-SAPK/JNK signaling is known to regulate cellular activities, including gene expression, mitosis, movement, metabolism, and programmed death [40]. Therefore, the results of the present study demonstrate that silibinin causes cell death through the p-AKT and p-SAPK/JN-ERK pathway (Fig. 2). The present study has also demonstrated the additional anticancer effects of silibinin; it reduced cell migration, invasion, and spheroid formation by downregulating p-AKT and p-SAPK/JNK-ERK signaling in pancreatic adenocarcinoma cell lines. However, silibinin did not downregulate p-AKT and p-SAPK/JNK-ERK signaling in hTERT-HPNE cells (Fig. 4). Our data show that cell death was induced by a decrease in c-Jun-related AKT-ERK and JNK/SAPK signaling, but this mechanism does not explain the increase in c-Jun levels. This study did not reveal the mechanism for the increase in c-Jun levels. However, we speculate that the increase in c-Jun levels involves a molecular mechanism other than AKT-ERK and JNK/SAPK signaling, because this mechanism is complex [41]. A previous study has shown that CSCs can self-renew and differentiate into mature cells [42]. They also play important roles in cancer homeostasis, metastasis, resistance to therapy, and subsequent tumor recurrence [43]. Pancreatic CSCs are highly resistant to a majority of chemotherapeutic agents [44].
It is unknown whether silibinin affects pancreatic cancer stemness. The present study demonstrated that after exposure to silibinin, the expression levels of cancer stemness-related genes increase in human pancreatic cancer cells and other organ cancer cells, such as HepG2 (hepatocarcinoma cell line) and HCT116 (colon adenocarcinoma cell line); conversely, they decrease in human normal pancreatic ductal cells (hTERT-HPNE) and normal kidney cells (293T) (Fig. 5). These results suggest that the effects of silibinin on pancreatic cancer cells and pancreatic normal ductal cells differ.
p53 is a tumor suppressor protein encoded by the Tp53 gene. p53 is an important cell-cycle inhibitor that prevents cancer in multicellular organisms. A mutation in the p53 protein cannot kill damaged cells. However, other healthy cells can break down such damaged cells. If a mutation of the p53 gene does not allow the p53 protein to function properly, cells with the damaged DNA will progress to cell division. These damaged cells can continue to divide. They will not be able to recognize the damaged DNA, resulting in more mutations. Such mutations cause cancer [45][46][47][48][49]. p53 protein overexpression is a common genetic alteration, and occurs very early in the development of pancreatic cancer [50][51]. The results of the present study indicate that silibinin-induced increases in stemness-related signals may be mediated by mutations in the p53 protein. In normal pancreatic ductal cells (hTERT-HPNE) that overexpress wild-type p53, silibinin increases the p53 protein levels. However, it decreases the p53 protein levels in p53-mutated cells such as MIA PaCa-2 and PANC-1 cells that overexpress wild-type p53 (Fig. 6). These results suggest that mutations in p53 may in uence the effect of silibinin. However, the present study did not demonstrate that silibinin increases the expression of stemness-related genes, such as those of c-Jun, TWIST1, and Snail, in wild-type p53 pancreatic adenocarcinoma cells. Further studies are necessary to investigate the relationship between wild-type p53 pancreatic adenocarcinoma cells (such as SW1990) and silibinin.
Silibinin not only exerts anticancer effects by inhibiting AKT-ERK and JNK, but also increases the expression of cancer stemness-related genes in human pancreatic adenocarcinoma cells. However, silibinin does not have an adverse effect on pancreatic normal ductal cells.

Conclusions
In conclusion, pancreatic cancer cells susceptible to silibinin undergo cell death; on the other hand, if pancreatic cancer cells become resistant to silibinin and survive, it is possible that they will engage in activity similar to that of CSCs. These results suggest that when silibinin is used as a therapeutic agent for human pancreatic cancer, caution should be exercised. Analysis and interpretation of data; discussion and data review.