J Cancer Prev 2021; 26(2): 118-127
Published online June 30, 2021
© Korean Society of Cancer Prevention
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, 2Department of Food Science and Biotechnology, College of Knowledge-based Services Engineering, Sungshin Women's University, 3Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, 4Cancer Research Institute, Seoul National University, Seoul, Korea
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More than half of the world’s populations are considered to be infected by Helicobacter pylori. It causes a chronic inflammation of the stomach, which is implicated in the pathogenesis of gastric ulcer and cancer. Silibinin, a polyphenolic flavonoid derived from milk thistle, has been known for its hepatoprotective effects, and recent studies have revealed its chemopreventive potential. In the present study, we examined the anti-inflammatory effects of silibinin in human gastric cancer MKN-1 cells and in the stomach of C57BL/6 mice infected by H. pylori. Pretreatment with silibinin attenuated the up-regulation of COX-2 and inducible nitric oxide synthase (iNOS) in H. pylori-infected MKN-1 cells and mouse stomach. In addition, the elevated translocation and DNA binding of NF- κB and STAT3 induced by H. pylori infection were inhibited by silibinin treatment. Moreover, H. pylori infection in combination with high salt diet resulted in dysplasia and hyperplasia in mouse stomach, and these pathological manifestations were substantially mitigated by silibinin administration. Taken together, these findings suggest that silibinin exerts anti-inflammatory effects against H. pylori infection through suppression of NF-κB and STAT3 and subsequently, expression of COX-2 and iNOS.
Keywords: Helicobacter pylori, Silibinin, NF-κB, STAT3, Gastritis
For treating patients with
Silibinin is a major bioactive flavonolignan isolated from milk thistle seeds (Fig. 1) . Generally, silymarin rather than silibinin has widely been known to have hepatoprotective properties . Silymarin is a mixture of silidianin, silichristine, and silibinin . Silibinin possesses strong antioxidant and anti-inflammatory effects and has chemopreventive activities as well [16-18]. In the present study, we investigated the possible inhibitory effects of silibinin on inflammation caused by
Silibinin was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) and dissolved in sterile dimethyl sulfoxide and carboxymethycellulose (CMC) for treatment to cells and mice, respectively. RPMI 1640 medium and FBS were obtained from Giboco BRL (Grand Island, NY, USA). GasPak EZ, CO2 indicator, and tryptic soy were purchased from BD Bioscience (Franklin, NJ, USA). Rabbit polyclonal antibody was the product of NeoMarkers (Fremont, CA, USA). iNOS antibody for immunohistochemical analysis was supplied from BD Bioscience. Antibodies for Western blot analysis against iNOS, p65, p50 and α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-STAT3 and Anti-P-STAT3 were obtained from Cell signaling Technology (Beverly, CA, USA). Mouse anti-lamin B was purchased from Invitrogen (Carlsbad, CA, USA). The oligonuclotides containing the NF-κB and STAT3 binding consensus sequence are: NF-κB: 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, STAT3: 5′-GAT CCT TCT GGG AAT TCC TAG ATC-3’ and the luciferase assay kit with reporter lysis buffer were obtained from Promega (Madison, WI, USA).
Human gastric carcinoma (MKN-1) cells were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and a 100 ng/mL penicillin/streptomycin/fungizone mixture and maintained at 37°C under a humidified atmosphere of 5% CO2/95% air.
After pretreatment with silibinin for 1 hour, MKN-1 cells were infected with
C57 black 6 (C57BL/6) female mice (5 weeks of age) were purchased from Orient Bio Inc. (Seongnam, Korea).
Half of the stomach tissues were placed in 1 mL of tryptic soy broth containing 10% FBS and homogenized with an UltraTurrax homogenizer. Ten-fold dilutions were made, and 100 µL aliquots of the dilutions were plated onto TSA. Colonies on the plate were counted, and bacterial counts were expressed as colony-forming units per gram of tissue. The color changing grade was determined by the Campylobacter-Like Organism (CLO) kit.
Total RNAs were isolated from MKN-1 cells using the TRIzol® reagent (Invitrogen), and one microgram of total RNAs was reverse transcribed by M-MLV reverse transcriptase (Promega) following the protocol. One microliter of cDNA was subjected to PCR amplification:
Immunocytochemistry was conducted to illustrate the nuclear translocation of NF-κB-p65 and STAT3. MKN-1 cells were placed on the eight-well chamber slide (Thermo Scientific, Rockford, IL, USA), pretreated with silibinin for 1 hour and infected by
MKN-1 cells placed in a 100 mm dish were pretreated with silibinin for 1 hour and then infected with
MKN-1 cells at a confluence of 60% to 70% were transfected with mock or NF-κB luc vector with lipofectamin 2000 reagent (Invitrogen) according to the instructions supplied by the manufacturer. After 24 hours of transfection, cells were pretreated with silibilinin for 1 hour and infected with
After infected with
After treatment, all mice were sacrificed by CO2 inhalation. These tissue blots were fixed with 10% formalin before being embedded in paraffin. Each tissue blot section (4 µm) was stained with hematoxylin and eosin.
When necessary, data were expressed as means ± SD, and statistical analysis was performed by using Gel-Pro analyzer program. Statistical significance of the obtained data was determined by conducting Student’s
The broad diversity of genes regulated by NF-κB include cytokines (e.g., interleukin [IL]-6 and TNF-α), chemokines (e.g., IL-8 and RANTES), and inducible effector enzymes (e.g., iNOS and COX-2) . We investigated whether silibinin could suppress the activation of transcriptional factors in nuclear extracts of MKN-1 cells stimulated with
Besides NF-κB, STAT3 is recognized as a major mediator of inflammation associated with tumor promotion. STAT3 is activated through phosphorylation of tyrosine 705 by diverse protein tyrosine kinases [22,23]. As in the case of NF-κB, the nuclear accumulation of the phosphorylated STAT3 elevated by
Besides cell culture experiments, we investigated the effect of silibinin on
To clarify how silibinin could relieve these abnormal gross pathologies, stomach tissues of each treatment group were subjected to Western blot analysis. Compared with the control mice, mice inoculated with
All the aforementioned results demonstrate the inhibitory effects of silibinin on
Silibinin has been reported to inhibit COX-2 and iNOS in an azoxymethane-induced mouse colon cancer model  and to down-regulate COX-2 in rat hepatocellular carcinoma . The aforementioned publications address the possibility that silibinin could inhibit
Interplay or crosstalk between NF-κB and STAT3 attracted special attention in inflammation-associated carcinogenesis . Multiple interactions between NF-κB and STAT3 could contribute to the promotion of cancer . Moreover, the interaction between p65 and unphosphorylated STAT3 is prominent in the expression of inflammatory genes . There are several possibilities that the Rel A/p65 subunit of NF-κB directly interacts with either unphosphorylated or phosphorylated STAT3 to regulate transcription of a target gene. p65 and STAT3 may work independently of each other, but provokes synergistic effects by interacting with the same promoter region of a single common target gene [40-42].
According to the findings from our present study,
In the mouse gastritis caused by
This study was supported by the Global Core Research Center (GCRC) grant (No. 2011-0030001) and BK21 FOUR Program (5120200513755) from the National Research Foundation, Republic of Korea.
No potential conflicts of interest were disclosed.
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