Inhibition of YAP/TAZ pathway contributes to the cytotoxicity of silibinin in MCF-7 and MDA-MB-231 human breast cancer cells.

Breast cancer is one of the most common cancers threatening women's health. Our previous study found that silibinin induced the death of MCF-7 and MDA-MB-231 human breast cancer cells. We noticed that silibinin-induced cell damage was accompanied by morphological changes, including the increased cell aspect ratio (cell length/width) and decreased cell area. Besides, the cytoskeleton is also destroyed in cells treated with silibinin. YAP/TAZ, a mechanical signal sensor interacted with extracellular pressure, cell adhesion area and cytoskeleton, is also closely associated with cell survival, proliferation and migration. Thus, the involvement of YAP/TAZ in the cytotoxicity of silibinin in breast cancer cells has attracted our interests. Excitingly, we find that silibinin inhibits the nuclear translocation of YAP/TAZ in MCF-7 and MDA-MB-231 cells, and reduces the mRNA expressions of YAP/TAZ target genes, ACVR1, MnSOD and ANKRD. More importantly, expression of YAP1 gene is negatively correlated with the survival of the patients with breast cancers. Molecular docking analysis reveals high probabilities for binding of silibinin to the proteins in the YAP pathways. DARTS and CETSA results confirm the binding abilities of silibinin to YAP and LATS. Inhibiting YAP pathway either by addition of verteporfin, an inhibitor of YAP/TAZ-TEAD, or by transfection of si-RNAs targeting YAP or TAZ further enhances silibinin-induced cell damage. While enhancing YAP activity by silencing LATS1/2 or overexpressing YAPS127/397A, an active form of YAP, attenuates silibinin-induced cell damage. These findings demonstrate that inhibition of the YAP/TAZ pathway contributes to cytotoxicity of silibinin in breast cancers, shedding lights on YAP/TAZ-targeted cancer therapies.

Gelatin but not type I collagen promotes bacteria phagocytosis in PMA-treated U937 human lymphoma cells.

PURPOSE: Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. MATERIALS AND METHODS: Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. RESULTS: Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. CONCLUSION: This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy.

Silibinin ameliorates STING-mediated neuroinflammation via downregulation of ferroptotic damage in a sporadic Alzheimer's disease model.

Ferroptosis, an iron-dependent cell death, is caused by lipid peroxidation. Noteworthily, accumulation of iron and lipid peroxidation are found in the proximity of the neuritic plaque, a hallmark of Alzheimer's disease (AD), but the relationship between ferroptosis and neuroinflammation in AD is unclear. Silibinin, extracted from the Silybum marianum, is possibly developed as an agent for AD treatment from its neuroprotective effect, but the effect of silibinin on sporadic AD that accounts for more than 95% of AD remains unclear. To determine whether silibinin alleviates the pathogenesis of sporadic AD and investigate the underlying mechanisms, STZ-treated HT22 murine hippocampal neurons and intracerebroventricular injection of streptozotocin (ICV-STZ) rats, a sporadic AD model, were used in this study. Results show that silibinin not only promotes survival of STZ-treated HT22 cells, but also ameliorates the cognitive impairment and anxiety/depression-like behavior of ICV-STZ rats. We here demonstrate that silibinin evidently inhibits the protein level of p53 as well as upregulates the protein level of cystine/glutamate antiporter SLC7A11 and ferroptosis inhibitor GPX4, but not p21, leading to the protection against STZ-induced ferroptotic damage. Immunofluorescent staining also shows that accumulation of lipid peroxidation induced by ferroptotic damage leads to increased fluorescence of 8-oxo-deoxyguanosine (8-OHDG), a maker of oxidized DNA. The oxidized DNA then leaks to the cytoplasm and upregulates the expression of the stimulator of interferon gene (STING), which triggers the production of IFN-ß and other inflammatory cascades including NF-κB/TNFα and NLRP3/caspase 1/IL-1ß. However, the treatment with silibinin blocks the above pathological changes. Moreover, in HT22 cells with/without STZ treatment, GPX4-knockdown increases the protein level of STING, indicating that the ferroptotic damage leads to the activation of STING signaling pathway. These results imply that silibinin exerts neuroprotective effect on an STZ-induced sporadic AD model by downregulating ferroptotic damage and thus the downstream STING-mediated neuroinflammation.

Increased mitochondrial fission induces NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis in UVB-irradiated immortalized human keratinocyte HaCaT cells.

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N-acetyl-l-cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury.

Impaired mitophagy causes mitochondrial DNA leakage and STING activation in ultraviolet B-irradiated human keratinocytes HaCaT.

Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNß, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages.

Silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in liver cell lines.

Alcoholic liver disease has become one of the main causes of liver injury, and its prevention and cure are important medical tasks. Silibinin, a natural flavonoid glycoside, is a conventional hepatic protectant. This study elucidates the modulation of ferroptosis in silibinin's protective effects on ethanol- or acetaldehyde-induced liver cell damage by using human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells. Our results show that ferroptosis is induced in the cells treated with ethanol or acetaldehyde, as evidenced by the increased ROS stress and iron level. Silibinin resolves the oxidative stress and reduces iron level. Ferroptosis induced by ethanol- or acetaldehyde involving nuclear receptor co-activator 4 (NCOA4)-dependent autophagic degradation of ferritin, a protein for storing iron is rescued by silibinin. PINK1 and Parkin-mediated mitophagy is arrested in ethanol- or acetaldehyde-treated cells but reversed by silibinin. Ferritin degradation and ROS level are further increased when PINK1 or Parkin is silenced in the cells treated with ethanol or acetaldehyde. Collectively, our study reveals that silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines, HepG2 and HL7702 cells, providing new therapeutic strategies for alcoholic liver injury.

Protective effects of silibinin against ethanol- or acetaldehyde-caused damage in liver cell lines involve the repression of mitochondrial fission.

Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis.

IGF-1R/YAP signaling pathway is involved in collagen V-induced insulin biosynthesis and secretion in rat islet INS-1 cells.

PURPOSE: Type V collagen (collagen V) is one of the important components of extracellular matrix (ECM) in pancreas. We previously reported that pre-coating collagen V on the culture dishes enhanced insulin production in INS-1 rat pancreatic ß cells. In this study, we investigate the underlying mechanism. RESULTS: Insulin biosynthesis and secretion are both increased in INS-1 cells cultured on collagen V-coated dishes, accompanied by the reduced nuclear translocation of Yes-associated protein (YAP), a transcriptional co-activator. YAP, the downstream effector of Hippo signaling pathway, plays an important role in the development and function of pancreas. Inhibition of YAP activation by verteporfin further up-regulates insulin biosynthesis and secretion. Silencing large tumor suppressor (LATS), a core component of Hippo pathway which inhibits activity of YAP by phosphorylation, by siRNA transfection inhibits both insulin biosynthesis and secretion. In the present study, the protein level of insulin-like growth factor 1 receptor (IGF-1 R), detected as the upstream molecule of YAP, is reduced in the INS-1 cells cultured on the dishes coated with collagen V. The silencing of IGF-1 R by siRNA transfection further enhances insulin biosynthesis and secretion. IGF-1 treatment reduces collagen V-induced up-regulation of insulin biosynthesis and secretion, accompanying the increased nuclear YAP. CONCLUSION: Inhibition of IGF-1 R/YAP signal pathway is involved in collagen V-induced insulin biosynthesis and secretion in INS-1 cells.

Epithelial-Mesenchymal Transition Induced in Cancer Cells by Adhesion to Type I Collagen.

The epithelial-mesenchymal transition (EMT) is an important biological process that is physiologically observed during development, wound healing, and cancer invasion. During EMT induction, cancer cells lose their epithelial properties owing to various tumor microenvironmental factors and begin to exhibit mesenchymal properties, such as loss of apical-basal polarity, weakened intercellular adhesion, and promotion of single cell migration. Several factors, including growth factor stimulation and adhesion to type I collagen (Col-I), induce EMT in cancer cells. Cells adhere to Col-I via specific receptors and induce EMT by activating outside-in signals. In vivo, Col-I molecules often form fibrils, which then assemble into supramolecular structures (gel form). Col-I also self-assembles in vitro under physiological conditions. Notably, Col-I can be used as a culture substrate in both gel and non-gel forms, and the gel formation state of Col-I affects cell fate. Although EMT can be induced in both forms of Col-I, the effects of gel formation on EMT induction remain unclear and somewhat inconsistent. Therefore, this study reviews the relationship between Col-I gel-forming states and EMT induction in cancer cells.

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 ERß 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. ERß 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 ERß diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERß were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERß 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 ERß pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage.

DNA damage-triggered activation of cGAS-STING pathway induces apoptosis in human keratinocyte HaCaT cells.

Exposure to ultraviolet B (UVB) from sunlight causes DNA damage, serious cellular inflammation and aging, and even cell death in the skin, commonly known as sunburn, leading to cutaneous tissue disorders. DNA damage can be sensed as a danger-associated molecular pattern (DAMP) by the innate immune system. It has not been studied, however, whether cGAS-STING activation is involved in the apoptosis induced by UVB irradiation or by cisplatin treatment. Here we report the findings that within hours of DNA damages keratinocytes show an innate immune response, which involves the activation of cGAS-STING; a cytosolic DNA receptor, cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase), cyclic GMP-AMP (cGAMP) synthase, and DNA sensing adaptor, STING (protein stimulator of interferon genes). Either UVB irradiation or cisplatin treatment can cause DNA damages, releasing fragmented DNA from nucleus and/or mitochondria. Roles of cGAS-STING were examined in the HaCaT cells with DNA damages caused by UVB irradiation or cisplatin treatment. Silencing STING by siRNA rescued HaCaT cells from UVB or cisplatin-induced apoptosis. NF-κB, one of the major downstream components of STING pathway, which usually regulates the classical STING apoptotic pathway, was translocated to nucleus in the HaCaT cells irradiated with UVB. This translocation was attenuated by STING silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-induced apoptosis. cGAS-STING-mediated production of IFNß was induced by nuclear translocation of interferon regulatory factor 3 (IRF3). UVB irradiation inceased the nuclear translocation of IRF3, accompanied by enhanced expression level of IFNß mRNA. The nuclear translocation of IRF3 and expression of IFNß mRNA were attenuated by STING silencing. Treatment with MRT67307, an inhibitor of TBK1-IRF3-IFNß pathway, blocked UVB-induced apoptosis. Therefore, we conclude that NF-κB pathway and IFNß pathway residing in the downstream of STING are resposible for apoptosis of UVB-irradiated or cisplatin-treated HaCaT cells.

Silibinin-induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells.

We reported previously that higher doses (150-250 µM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying apoptosis of double-positive breast cancer cell line MCF-7 cells and triple-negative breast cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to mitophagy, 2) Whether mitophagy positively contributes to apoptosis and 3) Whether estrogen receptor-positive (ER+) MCF-7 cells and estrogen receptor-negative (ER-) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of mitophagy as well as autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of mitophagy by Pink1 or Parkin knockdown increased silibinin-induced apoptosis of these cells, respectively, suggesting that the mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of apoptosis of cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced mitophagy and apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERß are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231.

Type I collagen inhibits adipogenic differentiation via YAP activation in vitro.

Extracellular matrix (ECM) has a marked influence on adipose tissue development. Adipose tissue formation is initiated with proliferation of preadipocytes and migration before undergoing further differentiation into mature adipocytes. Previous studies showed that collagen I (col I) provides a good substratum for 3T3-L1 preadipocytes to grow and migrate. However, it remains unclear whether and how col I regulates adipogenic differentiation of preadipocytes. This study reports that lipid accumulation, representing in vitro adipogenesis of the 3T3-L1 preadipocytes or the mouse primary adipocyte precursor cells derived from subcutaneous adipose tissue in the inguinal region is inhibited by the culture on col I, owing to downregulation of adipogenic factors. Previous study shows that col I enhances 3T3-L1 cell migration via stimulating the nuclear translocation of yes-associated protein (YAP). In this study, we report that downregulation of YAP is associated with in vitro adipogenesis of preadipocytes as well as with in vivo adipose tissue of high-fat diet fed mice. Increased expression of YAP in the cells cultured on col I-coated dishes is correlated with repression of adipogenic differentiation processes. The inactivation of YAP using YAP inhibitor, verteporfin, or YAP small-interfering RNA enhanced adipogenic differentiation and reversed the inhibitory effect of col I. Activation of YAP either by the transfection of YAP plasmid or the silence of large tumor suppressor 1 (LATS1), an inhibitory kinase of YAP, inhibited adipogenic differentiation. The results indicate that col I inhibits adipogenic differentiation via YAP activation in vitro.

Type I collagen promotes the migration and myogenic differentiation of C2C12 myoblasts via the release of interleukin-6 mediated by FAK/NF-κB p65 activation.

Skeletal muscle regeneration is a complicated process, requiring the proliferation, migration and differentiation of myoblasts whose processes are highly regulated by the extracellular matrix (ECM) surrounding the muscle tissues in vivo. However, the effects of respective ECM components on the regulation of myoblast behaviors are unknown. In this study, we report on the effect of collagen I, a major ECM component in muscle tissue and a popular food supplement, on mouse C2C12 myoblast proliferation, migration and differentiation as well as the underlying mechanisms. Collagen I (col 1) enhances the migration and myogenic differentiation of C2C12 cells, but has no effect on cell proliferation. Col I significantly promotes the production and release of interleukin-6 via nuclear translocation of nuclear factor κB (NF-κB) p65. The release of IL-6 plays a critical role in the col I-enhanced migration and differentiation of C2C12 cells. Furthermore, col I increases phosphorylation of focal adhesion kinase (FAK) that is involved in the nuclear translocation of NF-κB p65. Collectively, col I enhances the migration and differentiation of C2C12 cells through IL-6 release induced by FAK/NF-κB p65 activation.

Silibinin inhibits migration and invasion of breast cancer MDA-MB-231 cells through induction of mitochondrial fusion.

Human triple negative breast cancer cells, MDA-MB-231, show typical epithelial to mesenchymal transition associated with cancer progression. Mitochondria play a major role in cancer progression, including metastasis. Changes in mitochondrial architecture affect cellular migration, autophagy and apoptosis. Silibinin is reported to have anti-breast cancer effect. We here report that silibinin at lower concentrations (30-90 µM) inhibits epithelial to mesenchymal transition (EMT) of MDA-MB-231, by increasing the expression of epithelial marker, E-cadherin, and decreasing the expression of mesenchymal markers, N-cadherin and vimentin. Besides, silibinin inhibition of cell migration is associated with reduction in the protein expression of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) and paxillin. In addition, silibinin treatment increases mitochondrial fusion through down-regulating the expression of mitochondrial fission-associated protein dynamin-related protein 1 (DRP1) and up-regulating the expression of mitochondrial fusion-associated proteins, optic atrophy 1, mitofusin 1 and mitofusin 2. Silibinin perturbed mitochondrial biogenesis via down-regulating the levels of mitochondrial biogenesis regulators including mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator (PGC1) and nuclear respiratory factor (NRF2). Moreover, DRP1 knockdown or silibinin inhibited cell migration, and MFN1&2 knockdown restored it. Mitochondrial fusion contributes to silibinin's negative effect on cell migration. Silibinin decreased reactive oxygen species (ROS) generation, leading to inhibition of the NLRP3 inflammasome activation. In addition, knockdown of mitofusin 1&2 (MFN 1&2) relieved silibinin-induced inhibition of NLRP3 inflammasome activation. Repression of ROS contributes to the inhibition of the expression of NLRP3, caspase-1 and IL-ß proteins as well as of cell migration. Taken together, our study provides evidence that silibinin impairs mitochondrial dynamics and biogenesis, resulting in reduced migration and invasion of the MDA-MB-231 breast cancer cells.

Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels.

BACKGROUND: Extracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well. AIM: This study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs). METHODS: MTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. RESULTS: MPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1ß, TNFα and prostaglandin E2 (PGE2) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB. CONCLUSION: Col I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels.

Silibinin-induced autophagy mediated by PPARα-sirt1-AMPK pathway participated in the regulation of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes.

Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.

Type I collagen-induced YAP nuclear expression promotes primary cilia growth and contributes to cell migration in confluent mouse embryo fibroblast 3T3-L1 cells.

The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibitor verteporfin repressed the growth of primary cilia as well as the expressions of ciliogenesis-associated proteins in confluent 3T3-L1 cells cultured on Col I-coated plate. Moreover, knockdown of either YAP or IFT88, one of the ciliogenesis-associated proteins, reversed the migration of confluent 3T3-L1 cells promoted by Col I-coating. In conclusion, activation of YAP pathway by Col I-coating of culture plate for confluent 3T3-L1 cells is positively associated with the primary cilia growth, which eventually results in promoted migration.

Reactive oxygen species are responsible for the cell aggregation and production of pro-inflammatory mediators in phorbol ester (PMA)-treated U937 cells on gelatin-coated dishes through upregulation of autophagy.

Purpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.

Gelatin promotes cell aggregation and pro-inflammatory cytokine production in PMA-stimulated U937 cells by augmenting endocytosis-autophagy pathway.

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.