Silibinin protects rat pancreatic ß-cell through up-regulation of estrogen receptors' signaling against amylin- or Aß1-42 -induced reactive oxygen species/reactive nitrogen species generation.

Amylin and amyloid-ß (Aß) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic ß-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and ß (ERα and ERß) and generation of ROS/RNS in amylin/Aß1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aß1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aß1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aß1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aß1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aß1-42 -treated INS-1 cells.

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.

Activated toll-like receptor 4 is involved in oridonin-induced phagocytosis via promotion of migration and autophagy-lysosome pathway in RAW264.7 macrophages.

In our previous study, we demonstrated that oridonin enhances phagocytosis of apoptotic bodies by macrophage-like cells by inducing autophagy. However, the direct sensor of autophagy and the key event controlling phagocytosis remains unknown. Herein, we showed that Toll-like receptor 4 (TLR4), known to mediate immune responses, was activated by oridonin. Activated TLR4 contributes to phagocytosis of apoptotic cells by RAW264.7 macrophages. Indeed, inhibition or small interfering RNA (siRNA) silencing of TLR4 significantly attenuated oridonin-induced phagocytosis. Inhibition of TLR4 also decreased the level of autophagy and its associated proteins, Beclin-1 and light chain 3 (LC3), suggesting that activated TLR4 is involved in activation of autophagy. LPS-induced activation of TLR4 promoted phagocytosis and autophagy progression. Activation of TLR4 accompany increase in activities of lysosome acid phosphatase and cathepsin B as well as in up-regulation of lysosomal-associated membrane protein (LAMP 1 and 2) levels. Furthermore, TLR4 in association with translocation to cytoplasm leads to macrophage motility or migration through increased plasticity of skeleton and/or membrane structure. These results suggest that oridonin-induced phagocytosis of apoptotic bodies by macrophages is TLR4 signal pathway-mediated, via activation of the autophagy-lysosome pathway as well as increase of cell 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.

Silibinin ameliorates amylin-induced pancreatic ß-cell apoptosis partly via upregulation of GLP-1R/PKA pathway.

The objective was to investigate the mechanism of the protective effect of silibinin on amylin/Aß1-42-induced INS-1 cell apoptosis, with special reference to the roles of glucagon-like peptide-1 receptor (GLP-1R) and protein kinase A (PKA). The effects of silibinin on apoptosis, insulin secretion, GLP-1R, and PKA expression in the INS-1 cells treated with amylin/Aß1-42 were examined. INS-1 cells exposed to amylin showed increased TUNEL-positive ratio, reduced expression of GLP-1R and PKA. GLP-1R antagonists or PKA inhibitor enhanced the expression of apoptosis-associated proteins and TUNEL-positive ratio. Silibinin exerted antiapoptotic effect on and upregulation of GLP-1R and PKA. However, Aß1-42-induced INS-1 cell apoptosis, GLP-1R, and PKA expressions were not changed. Our results indicate that down-regulation of GLP-1R and PKA contributes to INS-1 cell apoptosis induced with amylin. Silibinin protects INS-1 cells from amylin-induced apoptosis through activation of GLP-1R/PKA signaling. Silibinin's inhibition of the toxic effects of Aß1-42 is independent of GLP-1R/PKA pathway.

Persistent IKKα phosphorylation induced apoptosis in UVB and Poly I:C co-treated HaCaT cells plausibly through pro-apoptotic p73 and abrogation of IκBα.

Toll-like receptor 3 (TLR3), a member of pattern recognition receptors, is reported to initiate skin inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. Recently, we have discovered the NF-κB pathway activated by TLR3 is involved in apoptosis of UVB-Poly I:C-treated HaCaT cells. The real culprit for apoptosis has not been precisely identified since the system of NF-κB pathway is complex. In this study, we silenced main transcriptional factors in NF-κB family, RelA, RelB and c-Rel, but to our surprise the results show that none of them participate in apoptosis induction in UVB-Poly I:C-treated HaCaT cells. Therefore, we moved to investigate the apoptosis-associated molecules in the upstream of NF-κB pathway. We firstly checked the expression of IκBα, an NF-κB inhibitor. UVB (4.8 mJ/cm2) and Poly I:C (0.3 µg/mL) co-treatment decreased IκBα expression level in a time-dependent manner. Silencing IκBα with siRNA further enhanced UVB-Poly I:C-induced cell death. We then investigated IκB kinase (IKK) complex that contributes to the degradation of IκBα. IKK is composed of IKKα, IKKß and NEMO. Treatment with IKK-16, an IKKα/ß inhibitor, significantly diminished UVB-Poly I:C-induced IκBα degradation and thus apoptosis. Silencing either IKKα or NEMO but not IKKß with corresponding siRNA inhibited apoptosis. Tumor repressor p73, a homologue of p53, is reported to mediate IKKα-induced apoptosis in DNA damage response. Silencing p73 reduced cell apoptosis in UVB-Poly I:C-treated HaCaT cells. In summary, UVB and Poly I:C co-treatment activates IKKα and NEMO, which diminishes anti-apoptotic IκBα, resulting in enhancement of apoptosis through p73. The findings partially clarify the possible molecular mechanism of pro-apoptotic NF-κB pathway activated by TLR3 in the fate of UVB-irradiated epidermis.

L-A03, a dihydroartemisinin derivative, promotes apoptotic cell death of human breast cancer MCF-7 cells by targeting c-Jun N-terminal kinase.

L-A03 is a dihydroartemisinin derivative and exerts distinct anti-tumor activity in vitro. Previous studies showed that induction of autophagy and deficiency in nitric oxide (NO) generation contributed to apoptotic cell death in L-A03-treated MCF-7 cells. However, the detailed mechanism is still unclear. In this study, the role of mitogen-activated protein kinases (MAPKs) in this apoptotic process was investigated. L-A03 (7.5-30 µM) selectively inhibited the activation of c-Jun N-terminal protein kinase (JNK) with no significant effect on extracellular signal related kinase (ERK) and p38. In addition, the possible mechanism of interaction between JNK and L-A03 was also investigated by molecular docking. In the presence of SP600125, a specific JNK inhibitor, induction of autophagy and apoptosis with L-A03 at 15 µM were elevated, but NO generation was attenuated, indicating that JNK inactivation is essential for apoptotic cell death. Interestingly, autophagy induction and NO generation did not affect the activation of JNK, demonstrating that JNK is upstream to autophagy and NO. Taken together, L-A03-induced JNK inactivation enhances autophagic and apoptotic cell death, but represses the generation of NO. This study provides a new insight on the mechanism of L-A03-induced cell death by targeting JNK.

Sub-lethal ultraviolet B irradiation and Poly I:C treatment synergistically induced apoptosis of HaCaT cells through NF-κB pathway.

Ultraviolet B (UVB) irradiation exerts multiple effects on skin cells, inducing apoptosis, senescence and carcinogenesis. Toll-like receptor 3, a member of pattern recognition receptors, is reported to initiate inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. It has not been studied, however, whether apoptosis induction in UVB irradiation is attributed to TLR3 activation. Here, we report on the pro-apoptotic role of TLR3 in UVB-irradiated epidermal cells. Poly I:C, an analogue of dsRNA that activates TLR3, was used in combination with sub-lethal UVB (4.8 mJ/cm2) irradiation for investigating the effects of TLR3 activation on human immortalized keratinocyte HaCaT cells. Although sub-lethal dose of either Poly I:C or UVB alone did not induce cell death, UVB-Poly I:C co-treatment synergistically induced cell death by activation of caspase-3 and cleavages of ICAD and PARP, with apoptotic features when stained with Annexin V/PI or Hoechst 33342. Treatment with pan-caspase inhibitor, Z-VAD, attenuated UVB-Poly I:C-induced cell death. Silencing TLR3 by siRNA rescued HaCaT cells from UVB-Poly I:C-induced apoptosis. NF-κB, a major downstream component of TLR3 pathway, that usually negatively regulates the classical TLR3 apoptotic pathway, was analyzed by western blotting and immunofluorescence confocal microscopy. The results indicate to our surprise that NF-κB is translocated to nucleus in the cells co-treated with UVB-Poly I:C. The nuclear translocation of NF-κB is attenuated by TLR3 silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-Poly I:C-induced apoptosis. Therefore, we conclude that NF-κB pathway plays a cytotoxic role in UVB-Poly I:C-treated HaCaT cells, mediating TLR3-related apoptosis.

Involvement of estrogen receptors in silibinin protection of pancreatic ß-cells from TNFα- or IL-1ß-induced cytotoxicity.

Silibinin is a polyphenolic flavonoid that exhibits anticarcinogenic, anti-inflammatory and cytoprotective effects. The effect of silibinin on pancreatic islet ß-cell is yet largely unknown in spite of well documented-hepatoprotective effects. Protecting the functional insulin-producing ß-cells in the pancreas is a major therapeutic challenge in the patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). This study reports the effect of silibinin on the rat pancreatic ß-cell line, INS-1, damaged with pro-inflammatory cytokine, TNFα or IL-1ß. Exposure to TNFα or IL-1ß for 48 h caused INS-1 cells to reduce the production of insulin as well as cell viability. These actions of TNFα or IL-1ß are associated with suppression of the expression of estrogen receptors (ERs). Further study revealed that silibinin protected the suppression in the expression of both ERα and ERß that were involved in insulin synthesis and release, respectively. Furthermore, evidence is obtained that silibinin may impede the loss of critical INS-1 cells by promoting autophagy and preventing apoptosis. Direct cytoprotective effects of silibinin on INS-1 cells suggest that silibinin may be therapeutically beneficial for diabetes.

Salicylate induces reactive oxygen species and reduces ultraviolet C susceptibility in Staphylococcus aureus.

This study demonstrates that growth of Staphylococcus aureus in the presence of salicylate reduces ultraviolet C (UVC)-induced cell death and increases the generation of reactive oxygen species (ROS). In addition, compounds that scavenge ROS (N-acetylcysteine, glutathione, catalase and superoxide dismutase) reverse the increased UVC survival induced by growth in the presence of salicylate, while ROS donors (tert-butylhydroperoxide, H2O2 and NaClO) enhance survival of salicylate challenged cultures. Collectively, these findings suggest that ROS production induced by growth in the presence of salicylate protects S. aureus from UVC-induced cell death.

Estrogen Receptors Are Involved in the Neuroprotective Effect of Silibinin in Aß1-42-Treated Rats.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by a cascade of pathologic changes. A widely discussed theory indicates that amyloid ß (Aß) peptides are the causative agents of AD. Silibinin, a flavonoid derived from milk thistle, is well known for its hepato-protective activities and we have reported the neuroprotective effects of silibinin. In this study, we investigated the role of estrogen receptors (ERs) in silibinin's neuroprotective effect on Aß1-42-injected rats. Results of Morris water maze and novel object-recognition tests demonstrated that silibinin significantly attenuated Aß1-42-induced memory impairment. Silibinin attenuated ERs and PI3K-Akt pathways, as well as modulated mitogen-activated protein kinases in the hippocampus of Aß1-42-injected rats. Taken together, silibinin is a potential candidate in the treatment of Alzheimer's disease.

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.

Phorbol ester (PMA)-treated U937 cells cultured on type I collagen-coated dish express a lower production of pro-inflammatory cytokines through lowered ROS levels in parallel with cell aggregate formation.

The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1ß, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1ß, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Blocking the utilization of glucose induces the switch from senescence to apoptosis in pseudolaric acid B-treated human lung cancer cells in vitro.

Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, exerts anti-tumor effects in several cancer cell lines. Our previous study showed that PAB mainly induced senescence via p53-p21 activation rather than apoptosis in suppression of the growth of human lung cancer A549 cells (p53 wild-type). In p53-null human lung cancer H1299 cells, however, PAB caused apoptosis without senescence. In this study we investigated what mechanism was responsible for the switch from senescence to apoptosis in PAB-treated human lung cancer cell lines. Senescent cells were examined by SA-ß-gal staining. Glucose uptake and the apoptosis ratio were assessed using a FACScan flow cytometer. Commercial assay kits were used to measure the levels of ATP and lactate. Transfection of siRNA was used to knockdown the expression of p53 or p21. Western blot analysis was applied to measure the protein expression levels. In p53 wild-type A549 cells, PAB (20 µmol/L) caused senescence, and time-dependently increased glucose utilization; knockdown of p53 or p21 significantly decreased the uptake and metabolism of glucose but elevated PAB-induced apoptosis. Inhibition of glucose utilization using a glycolytic inhibitor 2-DG (1 mmol/L) significantly enhanced apoptosis induction. Similar results were observed in another p53 wild-type H460 cells treated with PAB. Opposite results were found in p53-null H1299 cells, where PAB time-dependently decreased glucose utilization, and induced only apoptosis. Our results demonstrate that PAB-induced senescence is associated with enhanced glucose utilization, and lower glucose utilization might contribute to apoptosis induction. Thus, blocking glucose utilization contributes to the switch from senescence to apoptosis, and p53 plays an important role in this process.

Timosaponin AIII induces apoptosis and autophagy in human melanoma A375-S2 cells.

Timosaponin AIII (AIII), a steroidal saponin isolated from Anemarrhena asphodeloides Bge. Our study showed that AIII induced both apoptosis and autophagy, and autophagy inhibited apoptosis in A375S2 cells. Furtherly, this study was carried out to investigate what kind of cytokines plays an important role in this process. The results revealed that AIII induced apoptosis through activating c-Jun N-terminal protein kinase (JNK) or extracellular signal related kinase (ERK) signaling pathway and generating NO. However, JNK or ERK inhibited autophagy, while NO had no effect on autophagy. Therefore, JNK, ERK or NO regulates two programmed death processes in different ways. AIII did not show obvious cytotoxic effect on human peripheral blood mononuclear cells, which indicated that AIII has less side effects on normal cells, and could be considered as a leading compound for developing novel anticancer drug.

Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in regulating cell survival and death. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with anti-tumor activities, but it was found to induce cytoprotective ROS/RNS in human breast cancer MCF-7 cells. Furthermore, treatment with silibinin down-regulates ERα expression in MCF-7 cells, and inducing both autophagy and apoptosis. In this study we explored the relationship between ER-associated pathways and RNS/ROS in MCF-7 cells. We also investigated the molecular mechanisms underlying the reciprocal regulation between ROS/RNS levels and autophagy in the death signaling pathways in silibinin-treated MCF-7 cells. Silibinin (100-300 µmol/L) dose-dependently increased ROS/RNS generation in MCF-7 cells (with high expression of ERα and low expression of ERß) and MDA-MB-231 cells (with low expression of ERα and high expression of ERß). Scavenging ROS/RNS significantly enhanced silibinin-induced death of MCF-7 cells, but not MDA-MB231 cells. Pharmacological activation or blockade of ERα in MCF-7 cells significantly enhanced or decreased, respectively, silibinin-induced ROS/RNS generation, whereas activation or block of ERß had no effect. In silibinin-treated MCF-7 cells, exposure to the ROS/RNS donators decreased the autophagic levels, whereas inhibition of autophagy with 3-MA significantly increased ROS/RNS levels. We further showed that increases in ROS/RNS generation, ERα activation or autophagy down-regulation had protective roles in silibinin-treated MCF-7 cells. Under a condition of ERα activation, scavenging ROS/RNS or stimulating autophagy enhanced the cytotoxicity of silibinin. These results demonstrate the existence of two conflicting pathways in silibinin-induced death of MCF-7 cells: one involves the down-regulation of ERα and thereby augmenting the pro-apoptotic autophagy downstream, leading to cell death; the other involves the up-regulation of pro-survival ROS/RNS; and that the generation of ROS/RNS and autophagy form a negative feedback loop whose balance is regulated by ERα.

Collagen gel protects L929 cells from TNFα-induced death by activating NF-κB.

PURPOSE: Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel. MATERIAL AND METHODS: We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy. RESULTS: L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed. CONCLUSION: NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro.

AIM: Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, has shown to exert anti-tumor effects via inducing cell cycle arrest followed by apoptosis in several cancer cell lines. Here we reported that PAB induced a mitotic catastrophe in human lung cancer A549 cells, which resulted in senescence without apoptosis or necrosis. METHODS: Three human lung cancer cell lines (A549, H460 and H1299 cells) were examined. Cell growth inhibition was assessed with MTT assay. Cell cycle distribution was determined using a flow cytometer. Cell nuclear morphology was observed under a fluorescence microscope. Senescent cells were detected using SA-ß-Gal staining. Apoptotic and senescent protein expression was examined using Western blot analysis. The expression of p53 and p21 in the cells was downregulated by siRNAs. RESULTS: Treatment with PAB (5-80 µmol/L) inhibited the growth of A549 cells in dose- and time-dependent manners. Prolonged treatment with PAB (20 µmol/L) caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2, which eventually resulted in cell senescence between days 3 and 4 without cell death (apoptosis or necrosis). Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells (p53 wild). Furthermore, PAB-induced senescence was also observed in human lung cancer H460 cells (p53 wild), but not in human lung cancer H1299 cells (p53 null). CONCLUSION: The anti-tumor action of PAB against human lung cancer A549 cells in vitro involves the induction of senescence through activation of the p53 pathway.