Silibinin Treatment Research Articles

Quantitative proteomics analysis based on tandem mass tag labeling coupled with labeling coupled with liquid chromatography-tandem mass spectrometry discovers the effect of silibinin on non-alcoholic fatty liver disease in mice

ABSTRACT In recent years, the beneficial effects of silibinin (SIL) on nonalcoholic fatty liver disease (NAFLD) have attracted widespread attention. We tried to study the intervention effect of SIL on NAFLD, and explore the potential mechanisms and targets of SIL on NAFLD improvement. Thirty-three male C57BL6/J mice were divided into three groups, and, respectively, fed a normal diet (ND), a high-fat diet (HFD) or a HFD given SIL treatment (HFD+SIL). Biochemical indexes and histopathological changes of mice in each group were detected. In addition, quantitative proteomics analysis based on tandem mass tag (TMT) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis was performed on protein changes in the livers. SIL could reduce the weight of mice, reduce liver lipid deposition, and improve glucose metabolism. Through comparison among the three experimental groups, a total of 30 overlapping proteins were found. These identified proteins were closely linked to liver lipid metabolism and energy homeostasis. Moreover, some drug targets were found, namely perilipin-2, phosphatidate phosphatase LPIN1, farnesyl pyrophosphate synthase, and glutathione S-transferase A1. In conclusions, high-fat diet increases the expressions of proteins implicated in lipid synthesis and transport in the liver, which can result in disorders of liver lipid metabolism. SIL can decrease liver lipid deposition and increase insulin sensitivity by regulating the expressions of these proteins. It not only improves the disorder of lipid metabolism in vivo, but also improves the disorder of glucose metabolism.

Silibinin improves L-cell mass and function through an estrogen receptor-mediated antioxidative mechanism

BackgroundSilibinin, a major component of milk thistle extract silymarin, promotes hypoglycemia by activating estrogen receptor (ER) α and β-mediated pathways in pancreatic β-cells. Glucagon-like peptide-1 (GLP-1) is the enteroendocrine peptide produced in L-cells, and it controls glucose homeostasis through multiple pathways. The effect of silibinin on L-cell mass and function is still unknown. PurposeThe protective effect of silibinin on palmitate (PA)-treated intestinal L-cell line GLUTag cells and the SHRSP•Z-Leprfa/Izm-Dmcr (SP•ZF) diabetic rat model was investigated in current study. MethodsAfter pre-incubation with 50 μM silibinin for 4 h, GLUTag cells were treated with 0.125 mM PA. MTT, Annexin V/PI apoptosis, Hoechst 33342 staining, western blot, DCFH-DA, GLP-1 ELISA, qRT-PCR and immunofluorescence analyses were undertaken to determine ER-dependent protection of silibinin against PA-induced cellular damage. The differential protein expression of GLUTag cells under different treatments was examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The SP•ZF diabetic rat model was chosen for in vivo study. After 4 weeks of gastric gavage with 100 or 300 mg kg−1 of silibinin, the physiological indexes of the rats were measured. Cells expressing GLP-1, 8‑hydroxy-2′-deoxyguanosine (8-OHdG), ERα, and/or ERβ in duodenum tissues were detected by immunofluorescence. ResultsThe current study showed that the GLUTag cells preincubated with silibinin activated the transcription factor nuclear erythroid-2 like factor-2 (Nrf2)-antioxidant pathway, reduced reactive oxygen species (ROS) generation, and improved cell survival and GLP-1 content, while the antioxidative effect of silibinin was blocked by the selective ERα antagonist MPP or ERβ antagonist PHTPP in GLUTag cells. Our proteomics data further revealed that ERα or β inactivation reduced glutathione peroxide and proteins associated with endocytosis and reproduction, thus at least partially reversing the protective effect of silibinin. SP•ZF rats received silibinin treatment showed increased serum GLP-1 content and improved glucose homeostasis. Furthermore, silibinin upregulated ERα and β levels and reduced the level of 8-OHdG in GLP-1-positive cells. ConclusionsOur study showed that silibinin improved L-cell mass and function through an ER-mediated antioxidant pathway, and the proteomics analysis revealed for the first time the differential regulation of proteins by PA and silibinin in GLUTag cells.

Silibinin Can Promote Bone Regeneration of Selenium Hydrogel by Reducing the Oxidative Stress Pathway in Ovariectomized Rats.

Osteoporosis-related bone defects are a major public health concern. Considering poor effects of a singular pharmacological treatment, many have sought combination therapies, including local treatment combined with systemic intervention. Based on recent evidence that selenium and silibinin increase bone formation and bone mineral density, it is hypothesized that systemic administration with silibinin plus local treatment with selenium may have an additive effect on bone regeneration in an OVX rat model with bone defects. To verify this hypothesis, 3-month-old ovariectomized Sprague- Dawley rats (n = 10/gp) were intraperitoneally with a dose of 50mg/kg silibinin with selenium hydrogel scaffolds implanted into femoral metaphysis bone defect. Moreover, the MC3T3-E1 cells were co-cultured with selenium and silibinin, and observed any change of cell viability, ROS, and osteogenic activity. Experiment results show that the cell mineralization and osteogenic activity of silibinin plus selenium (SSe) group is enormously higher than the control (Con) group and selenium (Se) group, while ROS appears to be immensely reduced. Osteogenic protein expressions such as SIRT1, SOD2, RUNX-2 and OC of SSe group are significantly higher than Con group and Se group. Micro-CT and Histological analysis evaluation display that group SSe, compared with Con group and Se group, presents the strongest effect on bone regeneration, bone mineralization and higher expression of SIRT1 and SOD2. RT-qPCR analysis indicates that SSe group manifests increased SIRT1, SOD1, SOD2 and CAT than the Con group and Se group (p < 0.05). Our current study demonstrates that systemic administration with SIL plus local treatment with Se is a scheme for rapid repair of femoral condylar defects, and these effects may be achieved via reducing the oxidative stress pathway.

Silibinin inhibits the migration, invasion and epithelial-mesenchymal transition of prostate cancer by activating the autophagic degradation of YAP.

Silibinin (SB), a flavonoid extracted from milk thistle seeds, has been found to exert antitumor effects in numerous tumor types. Our previous study reported that SB had anti-metastatic effects in prostate cancer (PCa). However, the exact underlying molecular mechanisms remain to be determined. The present study aimed to investigate the effects of SB on the migration, invasion and epithelial-mesenchymal transition (EMT) of castration-resistant PCa (CRPC) cells using wound healing, Transwell assays, and western blotting. The results revealed that SB treatment significantly inhibited the migration and invasion of CRPC cell lines. Moreover, SB was confirmed to activate autophagy, as determined using LC3 conversion, LC3 turnover and LC3 puncta assays. Further mechanistic studies indicated that the expression levels of Yes-associated protein (YAP) were downregulated in an autophagy-dependent manner after SB treatment. In addition, the SB-induced autophagic degradation of YAP was associated with the anti-metastatic effects of SB in CRPC. In conclusion, the findings of the present study suggested that SB might inhibit the migration, invasion and EMT of PCa cells by regulating the autophagic degradation of YAP, thus representing a potential novel treatment strategy for metastatic CRPC.

Physicochemical Properties of Silibinin-Phosphatidylcholine Complex and its Implications for Drug Formulations

Silibinin, an active constituent of silymarin possesses several medicinal properties. Although, the poor water solubility and less bioavailability restrict the clinical utility of silibinin, the complex of silibinin and phosphatidylcholine manifested outstanding biological activities in vivo. Thereby, the complex of silibinin was prepared with phosphatidylcholine (1:2, w/w) for better solubility and therapeutic efficacy and their physicochemical properties were analyzed. Thermal analysis, nuclear magnetic resonance and Fourier transform infrared spectroscopy data revealed that complex of silibinin with phosphatidylcholine improved binding efficiency of silibinin with non-covalent interactions. Kinetics of in vitro drug release with high performance liquid chromatography analysis showed that complex of silibinin-phosphatidylcholine released maximum 30 %-32 % silibinin into phosphate-buffered saline between 3 h to 6 h at pH 3 and 7 at 37°. Release profile of silibinin from silibinin-phosphatidylcholine complex in Swiss albino mice suggested that the complex increased silibinin concentration three times more in plasma samples of mice than individual silibinin treatment after 10 d. Furthermore, no significant changes were observed in body weight and diet consumption of mice throughout the study. Therefore, the study suggested that complex of silibinin-phosphatidylcholine improves the solubility and bioavailability of silibinin, which explains better efficacy and clinical potential of silibinin in its complex form against various disorders including cancer.

Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.

Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells.

Anxiolytic effects of silibinin and naringenin on zebrafish model: A preclinical study.

OBJECTIVE:Stress exacerbates the pathophysiology of major neurodegenerative disorders. In this study, the zebrafish (Danio rerio), the frequently used model for experimental studies of stress and other central nervous system disorders, was used to evaluate the anxiolytic potential of flavonoids, namely silibinin and naringenin on alleviating acute stress-induced anxiety.MATERIALS AND METHODS:A molecular docking study with Molegro Virtual Docker software was done to assess the binding potential of flavonoids on serotonin and dopamine receptors. To determine the bioactivity and investigate the toxicity of the flavonoids, silibinin, and naringenin, brine shrimp lethality assay, and an acute toxicity study was conducted according to Organization for Economic Co-operation and Development (OECD) Test Guideline 203. The effect of silibinin and naringenin was assessed using behavioral tasks such as the novel tank assay and the light-dark test on the zebrafish model of acute stress.RESULTS:Molecular docking studies showed a higher affinity of silibinin and naringenin for the serotonin and dopamine receptors. In comparison to the LC50 value, 13.15 μg/ml of the reference standard potassium dichromate, silibinin, and naringenin yielded higher LC50 values, 34.10 μg/ml and 91.33 μg/ml, respectively. The LC50 value of silibinin and naringenin was observed to be >100 mg/l from the acute toxicity study on adult zebrafish. After transferring to a novel tank, silibinin and naringenin-treated zebrafish groups were found to explore the upper level of the tank, similar to standard drugs, and spent a long time in the upper level of the tank compared to the control group (p < 0.01). Both silibinin and naringenin treatment group spent increased amounts of time in the tank's illuminated part in contrast to that of the dark side as evidenced by the number of zebrafish entering or remaining in the illuminated part of the tank through the light-dark test. Silibinin and naringenin treated groups were found to spend increased time in the light side significantly on the day 15th of evaluation as compared to the control group (p < 0.05 and p < 0.001, respectively).CONCLUSION:The flavonoids, silibinin, and naringenin were found to mitigate acute stress-induced anxiety, owing to their anxiolytic properties in the zebrafish model and may be explored as the potential therapeutic agents for treating anxiety.

Protective Effects of Silibinin on Helicobacter pylori-induced Gastritis: NF-κB and STAT3 as Potential Targets.

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.

Oral Administration of Silibinin Ameliorates Cognitive Deficits of Parkinson's Disease Mouse Model by Restoring Mitochondrial Disorders in Hippocampus.

Besides motor disorder, cognitive dysfunction is also common in Parkinson's disease (PD). Essentially no causal therapy for cognitive dysfunction of PD exists at present. In this study, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD was used to analyze the neuroprotective potential of orally administered silibinin, a proverbial hepatoprotective flavonoid derived from the herb milk thistle (Silybum marianum). Results demonstrated that silibinin administration significantly attenuated MPTP-induced cognitive impairment in behavioral tests. Nissl staining results showed that MPTP injection significantly increases the loss of neurons in the hippocampus. However, these mice were protected by oral administration of silibinin, accompanying reduction in the cell apoptosis in the hippocampus. The hippocampal aggregates of α-synuclein (α-syn) appeared in MPTP-injected mice, but were significantly decreased by silibinin treatment. MPTP injection induced oxidative stress, as evidenced by increased malondialdehyde (MDA) and decreased superoxide dismutase (SOD). The oxidative stress was alleviated by silibinin treatment. Mitochondrial disorder including the decline of mitochondrial membrane potential (MMP) was another signature in the hippocampus of MPTP-treated mice, accompanying increased mitochondrial fission and decreased fusion. Silibinin administration restored these mitochondrial disorders, as expected for the protection against MPTP injury. These findings suggest that silibinin has a potential to be further developed as a therapeutic candidate for cognitive dysfunction in PD.

Autophagy and glycolysis independently attenuate silibinin-induced apoptosis in human hepatocarcinoma HepG2 and Hep3B cells.

The mechanism of cytotoxicity of silibinin on two human hepatocellular carcinoma (HCC) cell lines, HepG2 (p53 wild-type) and Hep3B cells (p53 null), is examined in relation with the induction of autophagy and phosphorylation of AMP-activated protein kinase (p-AMPK). Levels of apoptosis in relation to the levels of autophagy and those of glycolysis-related proteins, glucose transporter 1/4 (Glut1/4) and hexokinase-II (HK2), in HepG2 and Hep3B cells were examined. Silibinin-induced apoptosis was incomplete for HCC cell death in that up-regulated autophagy and/or reduced level of glycolysis, which are induced by silibinin treatment, antagonized silibinin-induced apoptosis. Inhibition of autophagy with 3-methyl adenine (3MA) or blocking of AMP-activated protein kinase (AMPK) activation with Compound C (CC) enhanced silibinin-induced apoptosis. The results confirm that AMPK involved in autophagy as well as in glycolysis remaining with silibinin is responsible for attenuation of silibinin-induced apoptosis. Blocking of AMPK or autophagy contributes to the enhancement of silibinin's cytotoxicity to HepG2 and Hep3B cells. This study shows that incomplete apoptosis of HCC by silibinin treatment becomes complete by repression of autophagy and/or glycolysis.

MDM2 induces EMT via the B‑Raf signaling pathway through 14‑3‑3.

MDM2 proto-oncogene, E3 ubiquitin protein ligase (MDM2) is a well-known oncogene and has been reported to be closely associated with epithelial-to-mesenchymal transition (EMT). The present study first demonstrated that the expression levels of MDM2 were markedly increased in TGF-β-induced EMT using quantitative PCR and western blotting. In addition, MDM2 was demonstrated to be associated with pathological grade in clinical glioma samples by immunohistochemical staining. Furthermore, overexpression of MDM2 promoted EMT in glioma, lung cancer and breast cancer cell lines using a scratch wound migration assay. Subsequently, the present study explored the mechanism by which MDM2 promoted EMT and revealed that MDM2 induced EMT by upregulating EMT-related transcription factors via activation of the B-Raf signaling pathway through tyrosine 3-monooxygenase activation protein ε using RNA sequencing and western blotting. This mechanism depended on the p53 gene. Furthermore, in vivo experiments and the colony formation experiment demonstrated that MDM2 could promote tumor progression and induce EMT via the B-Raf signaling pathway. Since EMT contributes to increased drug resistance in tumor cells, the present study also explored the relationship between MDM2 and drug sensitivity using an MTT assay, and identified that MDM2 promoted cell insensitivity to silibinin treatment in an EMT-dependent manner. This finding is crucial for the development of cancer therapies and can also provide novel research avenues for future biological and clinical studies.

Monocytes from preeclamptic women previously treated with silibinin attenuate oxidative stress in human endothelial cells

ABSTRACT Objective: To investigate whether the supernatant from monocytes of preeclamptic and normotensive pregnant women, cultured in vitro with silibinin, can modulate oxidative stress in HUVEC. Methods: Concentrations of IL-1β, IL-10, and TNF-α in monocyte culture supernatants were determined by ELISA. HUVEC and their supernatant cultures were employed for determination of NO, nitrite and nitrate, lipid peroxidation, and hemeoxygenase-1 (HO-1). Results: HUVEC treatment with supernatant of preeclamptic monocytes cultured with silibinin produced increased levels of nitrite, reduced lipid peroxidation, and increased HO-1. Conclusion: Supernatant of monocytes from preeclamptic women induce oxidative stress in HUVEC which can be reduced by silibinin treatment. Abbreviations: DAF-FMTM, Diaminofluorescein-FM; EDTA, Ethylenediaminetetraacetic acid; HO-1, heme oxygenase-1; HPLC, high-performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; MDA, malondialdehyde; NO, nitric oxide; NT, normotensive; PE, preeclampsia; ROS, reactive oxygen species; Sb, silibinin.

Silibinin treatment protects human skin cells from UVB injury through upregulation of estrogen receptors

Ultraviolet B (UVB) from the sunlight is a major environmental cause for human skin damages, inducing cell death, inflammation, senescence and even carcinogenesis. The natural flavonoid silibinin, clinically used as liver protectant, has protective effects against UVB-caused skin injury in vivo and in vitro. Silibinin is often classified as a phytoestrogen, because it modulates the activation of estrogen receptors (ERs). However, whether silibinin's estrogenic effect contributes to the skin protection against UVB injury remains to be elucidated. The issue was explored in this study by using the human foreskin dermal fibroblasts (HFF) and human non-malignant immortalized keratinocytes (HaCaT). In HFF, pre-treatment with silibinin rescued UVB-irradiated cells from apoptosis. Interestingly, silibinin increased the whole cellular and nuclear levels of ERα and ERβ in UVB-irradiated cells. Activation of ERs by treatment with estradiol elevated the cell survival and reduced apoptosis in UVB-treated cells. ERα agonist increased cell survival, while its antagonist decreased it. ERβ agonist also increased cell survival, but the antagonist had no effect on cell survival. Transfection of the cells with the small interfering RNAs (si-RNAs) to ERα or ERβ diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERβ were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERβ by specific antagonists and si-RNAs, respectively, reduced cell survival in UVB-treated HaCaT cells regardless of silibinin treatment. Taken together, it is summarized that silibinin up-regulates both ERα and ERβ pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage.

Lrp6 Genotype affects Individual Susceptibility to Nonalcoholic Fatty Liver Disease and Silibinin Therapeutic Response via Wnt/β-catenin-Cyp2e1 Signaling.

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious threat to human health worldwide, with a high genetic susceptibility. Rs2302685, a functional germline variant of LRP6, has been recently found to associate with NAFLD risk. This study was aimed to clarify the underlying mechanism associated with rs2302685 risk and its impact on pharmacotherapy in treatment of NAFLD.Methods: Venous blood samples were collected from NAFLD and non-NAFLD patients for SNP genotyping by using mass spectrometry. The Lrp6-floxdel mouse (Lrp6(+/-)) was generated to model the partial function associated with human rs2302685. The liver injury and therapeutic effects of silibinin were compared between Lrp6(+/-) and Lrp6(+/+) mice received a methionine-choline deficient (MCD) diet or normal diet. The effect of Lrp6 functional alteration on Wnt/β-catenin-Cyp2e1 signaling activities was evaluated by a series of cellular and molecular assays.Results: The T allele of LRP6 rs2302685 was confirmed to associate with a higher risk of NAFLD in human subjects. The carriers of rs2302685 had reduced level of AST and ALT as compared with the noncarriers. The Lrp6(+/-) mice exhibited a less severe liver injury induced by MCD but a reduced response to the treatment of silibinin in comparison to the Lrp6(+/+) mice, suggesting Lrp6 as a target of silibinin. Wnt/β-catenin-Cyp2e1 signaling together with ROS generation could be exacerbated by the overexpression of Lrp6, while decreased in response to Lrp6 siRNA or silibinin treatment under NAFLD modeling.Conclusions: The Lrp6 function affects individual susceptibility to NAFLD and the therapeutic effect of silibinin through the Wnt/β-catenin-Cyp2e1 signaling pathway. The present work has provided an underlying mechanism for human individual susceptibility to NAFLD associated with Lrp6 polymorphisms as well as a rationale for the effective use of silibinin in NAFLD patients.

The effects of Silibinin on nitrosative stress and matrix metalloproteinase -9 expression in Balb/c mice with breast cancer tumor

Background: Among the factors associated with cancer are the nitrosative stress and increased expression of some Matrix Metalloproteinase (MMP). Silibinin has an anti-tumor effect, therefore, the present study evaluates the effects of slibinin on nitrosative stress indices and Matrix Metalloproteinase -9 (MMP-9) expression in the animal models of breast cancer. Methods: In this study, 48 Balb/c mice were divided into 6 groups (each group contains 8 mouse): healthy ( control and cancer ) and treatment groups: treatment groups included cancerous mice which receiving 20 mg, 40 mg and 80 mg of silibinin for 3 weeks. In order to induce cancer, 4T1 cell line was used. After enrolling serum and breast tumor samples, NO level and MMP-9 expression in breast tumor biopsy were evaluated. Data were analyzed using one-way ANOVA, Kruskal-Wallis and T-test (p &lt;0.05). Results: The results revealed that the use 80 mg of silibinin resulted in significantly decreased in NO level in cancerous mice(170.88±15.42μM/ml) compared with untreated cancerous mice(190.38±21.19 μM/ml) (p=0.05). Furthermore, the use of three dose of silibinin led to a non-significant reduction of MMP-9 in the treatment group compared to untreated cancerous mice. Conclusion: Silibinin treatment has been shown to improve nitrosative stress in breast cancer mice.

Hypoxia-inducible Factor-1α Suppression in Ovarian Clear-cell Carcinoma Cells by Silibinin Administration.

Advanced ovarian clear-cell carcinoma (CCC) fails to respond to standard chemotherapy, and has a poor prognosis. Since hypoxia-inducible factor-1 (HIF-1) stimulates various genes involved in cancer, we aimed to examine the efficacy of silibinin, an active component of milk thistle belonging to Asteraceae, in suppressing HIF-1 activity, and elucidate the underlying mechanism in human CCC cell lines. Human ovarian CCC cell lines HAC-2, OVISE, and RMG-1 were treated with 500 μM silibinin for 4 h under normoxic and hypoxic conditions. Using DNA microarray, we analysed genes whose expression modulated more than 2-fold in response to hypoxia, whereas HIF-1α expression was measured using ELISA. Silibinin treatment decreased HIF-1α protein in all cell lines, and eIF4E2 and RPS6 mRNA in HAC-2 and RMG-1 cells. Silibinin suppressed HIF-1α protein under hypoxic conditions in CCC cell lines and could be a potential anti-cancer drug.

Natural flavonoid silibinin promotes the migration and myogenic differentiation of murine C2C12 myoblasts via modulation of ROS generation and down-regulation of estrogen receptor α expression.

Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50μM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration.

Silibinin Triggers the Mitochondrial Pathway of Apoptosis in Human Oral Squamous Carcinoma Cells.

Background:Silibinin, a natural polyphenolic flavonoid present in seed extracts of milk thistle (Silybum marianum). It has been shown to interact with various cancer-related cell signalling pathways in preclinical models, demonstrating promising anticancer effects in vitro and in vivo. Materials and Methods:The cytotoxic effect of silibinin was evaluated in human oral squamous carcinoma (SCC-25) cells by MTT assay. The apoptosis-related morphological changes were investigated by AO/EB dual staining. The cytochrome c, caspases-3, and -9, B-cell lymphoma-2 (Bcl-2), and B-cell associated X protein (Bax) gene expressions were analysed by PCR. Results:We have shown that silibinin treatment for 24 h in SCC-25 cells induced cytotoxicity in a concentration-dependent manner. The cytotoxic potential was due to the induction of apoptosis via the release of mitochondrial cytochrome c into the cytosol and subsequent activation of caspases-3 and -9. Dual staining assay was further confirmed the induction of early apoptosis upon silibinin treatment. Conclusion:The results from this study show that silibinin can be considered as a promising drug candidate for the treatment of oral squamous cell carcinoma.

Silibinin induces metabolic crisis in triple‐negative breast cancer cells by modulating EGFR‐MYC‐TXNIP axis: potential therapeutic implications

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment modalities and poor prognosis. Metabolic reprogramming in cancer is considered a hallmark of therapeutic relevance. Here, we report disruption of metabolic reprogramming in TNBC cells by silibinin via modulation of EGFR-MYC-TXNIP signaling. Metabolic assays combined with LC-MS-based metabolomics revealed inhibition of glycolysis and other key biosynthetic pathways by silibinin, to induce metabolic catastrophe in TNBC cells. Silibinin-induced metabolic suppression resulted in decreased cell biomass, proliferation, and stem cell properties. Mechanistically, we identify EGFR-MYC-TXNIP as an important regulator of TNBC metabolism and mediator of inhibitory effects of silibinin. Highlighting the clinical relevance of our observations, the analysis of METABRIC dataset revealed deregulation of EGFR-MYC-TXNIP axis in TNBC and association of EGFRhigh -MYChigh -TXNIPlow signature with aggressive glycolytic metabolism and poor disease-specific and metastasis-free survival. Importantly, combination treatment of silibinin or 2-deoxyglucose (glycolysis inhibitor) with paclitaxel synergistically inhibited proliferation of TNBC cells. Together, our results highlight the importance of EGFR-MYC-TXNIP axis in regulating TNBC metabolism, demonstrate the anti-TNBC activity of silibinin, and argue in favor of targeting metabolic vulnerabilities of TNBC, at least in combination with mainstay chemotherapeutic drugs, to effectively treat TNBC patients.

Silibinin treatment results in reducing OPA1&MFN1 genes expression in a rat model hepatic ischemia-reperfusion.

The mitochondrial damage has a pivotal role in triggering apoptosis and cell death. This study assessed the effect of silibinin on optical atrophy-1 (OPA1) and mitofusin-1 (MFN1) gene expression in liver tissue during hepatic warm ischemia-reperfusion (IR). Four groups of rats, eight rats each were designed: Vehicle: the rats received normal saline and encountered to laparotomy, Sili: silibinin (60 mg/kg) was administered to animals, IR: the rats received the normal saline and insulted by liver IR procedure, and IR + Sili: silibinin was injected to rats. All groups were subjected to the same process of injection of the solvent or silibinin (30 min before laparotomy or ischemia and immediately after the reperfusion), intraperitoneally (IP). After 3 h of reperfusion, blood and liver tissue samples were collected for future examinations. Our results showed no significant differences between the Vehicle and Sili groups in all assessed parameters. In IR + Sili, the increased serum levels of AST and ALT in comparison with the control group were markedly reduced by silibinin treatment. Silibinin lowered the elevated expression of OPA1 and MFN1 mRNAs in the IR group. Histology revealed silibinin could decline tissue degeneration compared to the IR group. Electron microscopy of control and silibinin groups showed no fusion of mitochondria and tissue degradation both of which were observed in the IR group. The extent of tissue destruction and mitochondrial fusion decreased significantly with silibinin treatment. Silibinin has a protective effect on liver cells against IR induced injuries by preserving mitochondrial membrane.