The Dual Effects of Silibinin on Human Pancreatic Cells

Research Article

Austin J Cancer Clin Res. 2021; 8(2): 1092.

The Dual Effects of Silibinin on Human Pancreatic Cells

Su-Mi L¹*, Gil-Woo L²*, Seon-Young P¹, Hosouk J¹, Eun-Ae C¹, Hyun-Soo K¹, Sung-Kyu C¹, Jong-Sun R¹ and Chang Hwan P*¹

¹Department of Internal Medicine, Division of Gastroenterology and Hepatology, Chonnam National University Medical School, Gwangju, South Korea

²Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, South Korea

*Corresponding author: Chang-Hwan P, Department of Internal Medicine, Chonnam National University Medical School42, Jaebongro, Dong-ku, Gwangju, 61469, South Korea

Received: July 06, 2021; Accepted: July 24, 2021; Published: July 31, 2021


Objective: Silibinin is a flavonoid 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 significantly inhibited the proliferation, invasion, and spheroid formation of human pancreatic cancer cells in vitro in a dosedependent 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 AKTERK 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.

Keywords: Silibinin; Pancreatic cancer cells; Anti-cancer drug; Pancreatic normal cells


Pancreatic cancer is a highly malignant disease that is characterized by locally advanced, unresectable disease, or metastasis at the time of diagnosis. Despite significant advances in surgery and chemotherapy, the five-year survival rate of patients with pancreatic cancer has not significantly improved beyond <5% [1-3]. 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 [11-14]. 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 selfrenew 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 deficient 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].

Twist-related protein 1 (TWIST1) is a transcription factor encoded by the TWIST1 gene in humans [27,28]. TWIST1 acts as an oncogene in several cancers. It cooperates with N-Myc, and plays an essential role in cancer metastasis, angiogenesis, and epithelial mesenchymal transition [29-32].

Snail is zinc-finger 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 drug-resistant 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.

Materials and Methods

Cell lines and culture

MIA PaCa-2 and PANC-1 are p53-mutated pancreatic cancer cell lines that are derived from human pancreatic carcinoma [18]. hTERT-HPNE is a human pancreatic normal ductal cell line. These pancreatic cancer cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (GIBCO Invitrogen Inc., USA) containing 4.5 mg/L glucose, 100 mg/L streptomycin, and 2 mM L-glutamine supplemented with 10% Fetal Bovine Serum (FBS) (GIBCO Invitrogen Inc.). hTERT-HPNE (ATCC® CRL-4023™, USA) cells were grown in DMEM containing 1.0 mg/L glucose (low glucose), 100 mg/L streptomycin, and 2 mM L-glutamine supplemented with 10% FBS and 10 ng/mL human epidermal growth factor (hEGF) (MERCK, USA). The cells were sub-cultured following enzymatic digestion using trypsin-EDTA solution (GIBCO Invitrogen Inc.), and were maintained under a humidified atmosphere at 37°C in a 5% CO2 incubator (Sanyo, Japan).

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 1×105 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).

Ki-67 analysis: Cells were stained with Ghost dye (BV510; #59863, CST, USA) and washed thrice with PBS. For intracellular staining, cells were stained using a FoxP3/transcription factor staining buffer set (00-5523; eBioscience, USA) and APC-conjugated anti-human Ki-67 (350514; Biolegend, USA).

Western blot analysis

Proteins were extracted from silibinin (200 μM/mL)-treated pancreatic cells (the cells were cultured to 70% confluence 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 25ºC for 1h. 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 4ºC. 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).

Spheroid formation assay

Cells were seeded in six-well plates at a density of 1×10³ cells/well, and cultured in F-12 DMEM (GIBCO Invitrogen Inc.) containing 10 ng/mL human recombinant basic Fibroblast Growth Factor (bFGF) (R&D Systems) and 10 ng/mL hEGF (R&D Systems) with 1×N2 supplement (GIBCO Invitrogen Inc.). The cells were incubated at 37ºC under a humidified atmosphere containing 5% CO2. Spheroids were confirmed after 14 days.

Invasion assay

Cell invasion was carried out overnight using a transwell filter 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 2×105 cells in 120 μL 0.2% BSA medium. Then, 400 μL 0.2% BSA medium containing 20 μg/mL human plasma fibronectin (Calbiochem, La Jolla, CA, USA) was loaded into the lower chamber. The transwell apparatus was incubated at 37ºC for 24 h. Cells that invaded the bottom surface of the upper chamber were fixed 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 five selected fields (each 0.5 mm²), using a hematocytometer under a light microscope at ×400 magnification. The results were expressed as means ± SE of the number of cells per field from three individual experiments.

RNA isolation

Total RNA was extracted using TRIzol (Takara, Japan). Briefly, 1mL 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 4ºC and 6,000 × g (grams) for 20 min. The supernatants were then collected, mixed with equal amounts of isopropyl alcohol (MERCK), and centrifuged at 4ºC and 6,000 × g for 20 min. The pellets were washed with 1 mL 70% ethyl alcohol (MERCK) and centrifuged at 4ºC 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.

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 Scientific, MA, USA) Real-Time PCR system, according to the manufacturer’s protocol. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH: HS02786624-g1) was used as a control, and the del/del threshold cycles ( 2-ΔΔCT CTs) values were calculated for stem markers (TWIST1: HS 01675818_s1, c-Jun: HS01103582_s1, and Snail: HS00195591_m1). The probes used were Thermo Fisher Taqman® Assay probes (Thermo Fisher Scientific).

Wild-type p53 gene transfection

Cells were seeded in six-well plates and incubated at 37ºC overnight. The cells were then transfected with pcDNA3/wild-type p53 (plasmid #69003, Addgene, MA 02472, USA) expression plasmid vector, using FuGENE®6 (Cat.#E2693, Promega Corporation) reagent, according to the manufacturer’s protocol.

Statistical analysis

The two-tailed paired t-test was used to assess statistical significance appropriately. The statistical significance of differences between data sets was determined using the paired t-test and oneway ANOVA test. All the reported p values are two-sided; p≤0.05 was considered statistically significant. 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 flow cytometry. The binding of fluorescently labeled Annexin V to externalized phosphatidylserine was also determined using flow 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 flow cytometric plots obtained with AnnexinVFITC PI assay after 24h 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 significantly (p<0.001) increased. However, silibinin did not affect the cell viability of hTERT-HPNE cells (p<0.001) (Figure 1).