Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC(50) of approximately 80-100 and 40-60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.     

P53-mediated GSH depletion enhanced the cytotoxicity of NO in silibinin-treated human cervical carcinoma HeLa cells

Silibinin is an active constituent extracted from the blessed milk thistle (Silybum marianum). In a previous study, we demonstrated that silibinin treatment induced the generation of reactive nitrogen species (RNS), which were associated with reactive oxygen species (ROS), and caused apoptosis and autophagy in HeLa cells. Another study reported that silibinin treatment attenuated the apoptotic effect of sodium nitroprusside (SNP) by generating ROS in rat pheochromocytoma PC12 cells [ 1 ]. To clarify the relationship between RNS and nitric oxide (NO) in HeLa cells, we chose SNP as a NO donor to inhibit the cell viability. We found that silibinin treatment did not reduce the cytotoxicity of NO by reducing the ROS-induced RNS levels; conversely, silibinin treatment enhanced the cytotoxicity of NO. Pre-treatment with the NO scavenger PTIO preserved the viability of SNP- or silibinin-treated cells. Buthionine sulfoximine (BSO) treatment was also used to deplete the level of glutathione (GSH) and subsequently enhance the cytotoxicity of NO. Pre-treatment with BSO enhanced the SNP-induced reduction of cell viability but had no such effects in the silibinin-treated cells. These results led us to investigate whether silibinin treatment could induce the depletion of GSH. JNK and p53 have been shown to mediate the depletion of GSH [ 2 , 3 ], and we previously demonstrated the existence of a ROS-JNK-p53 cycle in silibinin-treated HeLa cells [ 4 ]. Thus, we speculated that p53 also plays a crucial role in the silibinin-induced GSH depletion. To elucidate the role of p53 in this process, A431 cells were used because they are naturally devoid of a functional p53 (p53His273 mutation). To our surprise, silibinin treatment did not lower the GSH level in A431 cells but rather elevated the GSH level. Unlike the ROS level, the NO level was still up-regulated by silibinin treatment in A431 cells. Cumulatively, these findings support the idea that the silibinin-induced GSH depletion, which is mediated by p53, enhances the cytotoxicity of NO in HeLa cells.     

Nitric oxide (•NO) generation but not ROS plays a major role in silibinin-induced autophagic and apoptotic death in human epidermoid carcinoma A431 cells

Abstract

Silibinin, a major active constituent of silymarin, is clinically used as a hepatoprotectant, and in recent years, it has been used for the treatment of cancer in China. Because the mechanism of silibinin action on cancer cells was still unclear, we investigated the contribution of silibinin to the induction of apoptosis and autophagy via generation of reactive oxygen species (ROS) and nitric oxide (?NO) in human epidermoid carcinoma A431 cells. Silibinin inhibited the cell growth in a dose‐and time-dependent manner. Obvious autophagy was observed after treatment with different doses of silibinin. At a high dose (400 μM), silibinin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. Loss of mitochondrial membrane potential by silibinin led to mitochondrial dysfunction and decreased ROS levels, suggesting that silibinin might act as an antioxidant in this process. Furthermore, silibinin induced ?NO generation in a time‐and dose-dependent manner. The ?NO scavenger PTIO could effectively clear ?NO and exerted a minor cell protection effect through partial inhibition of silibinin-induced apoptosis and autophagy.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Abstract

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Silibinin Inhibits Glioma Cell Proliferation via Ca<Superscript>2+</Superscript>/ROS/MAPK-Dependent Mechanism In Vitro and Glioma Tumor Growth In Vivo

Anticancer activity of silibinin, a flavonoid, has been demonstrated in various cancer cell types. However, the underlying mechanism and in vivo efficacy in glioma were not elucidated. The present study was undertaken to determine the effect of silibinin on glioma cell proliferation in vitro and to examine whether silibinin inhibits tumor growth in vivo. Silibinin resulted in inhibition of proliferation in a dose- and time-dependent manner, which was largely attributed to cell death. Silibinin induced a transient increase in intracellular Ca²⁺ followed by an increase in reactive oxygen species (ROS) generation. The silibinin-induced cell death was prevented by EGTA, calpain inhibitor and antioxidants (N-acetylcysteine and Trolox). Western blot analysis showed that silibinin also induced ROS-dependent activation of extracellular signal-regulated kinase, p38 kinase, and c-Jun N-terminal kinase. Inhibitors of these kinases prevented the silibinin-induced cell death. Silibinin caused caspase activation and the silibinin-induced cell death was prevented by caspase inhibitors. Glioma cell migration was also decreased by silibinin treatment. Oral administration of silibinin in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in Ki-67 positive cells, an increase in TUNEL-positive cells, and caspase activation. These results indicate that silibinin induces a caspase-dependent cell death via Ca²⁺/ROS/MAPK-mediated pathway in vitro and inhibits glioma growth in vivo. These data suggest that silibinin may serve as a potential therapeutic agent for malignant human gliomas.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Silibinin induces the generation of nitric oxide in human breast cancer MCF-7 cells

Abstract

Increasing research has concentrated on the anti-tumour efficacy of silibinin, a flavonolignan that is clinically used as an hepatoprotectant. However, previous work has found that silibinin-induced apoptosis is accompanied by protective superoxide (O₂??) generation in MCF-7 cells. This study further reports the formation of reactive nitrogen species (RNS) in the same system. It finds that silibinin induces nitric oxide (?NO) generation in a time- and concentration-dependent manner. Moreover, the results support that there exists an inter-regulation pattern between RNS and reactive oxygen species (ROS) generation. In addition, silibinin is also found to induce RNS and ROS generation in the isolated populations of mouse peripheral blood mononuclear cells (PBMCs) and a simple in vivo model of Caenorhabditis elegans.     

Silibinin induced apoptosis of human epidermal cancer A431 cells by promoting mitochondrial NOS

The antitumor effects of silibinin are of increasing interest, though its mechanism is not yet clear. The goal of this study was to clarify the mechanism of silibinin-induced cell death in the A431 human epidermoid carcinoma cell line. We used a cell viability assay, flow cytometry, nitric oxide (NO) assay, and western blotting to examine relationships between silibinin, NO generation and apoptosis in A431 cells. Silibinin inhibited A431 cell growth in a dose-dependent manner, inducing mitochondrial damage, and apoptosis at a high dose. At the same time, high dose silibinin increased NO levels in A431 cells and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) attenuated silibinin-induced cell growth inhibition. By western blotting, silibinin caused increased eNOS phosphorylation in the mitochondria. The AMP-activated protein kinase inhibitor compound C significantly decreased p-eNOS expression, while blocking eNOS did not affect p-AMPK levels, suggested that AMPK acted upstream of eNOS. This study showed that silibinin increased NO levels in A431 cells by activating the AMPK–eNOS pathway, leading to mitochondrial dysfunction and apoptosis. In this mechanism of action, mitochondrial eNOS played an important role. The results provided new understanding of the functions of intracellular NO.     

Silibinin induces protective superoxide generation in human breast cancer MCF-7 cells

Abstract

The pharmacological activity of polyphenolic silibinin from milk thistle (Silybum marianum) is primarily due to its antioxidant property. However, this study found that silibinin promoted sustained superoxide (O₂^·–) production that was specifically scavenged by exogenous superoxide dismutase (SOD) in MCF-7 cells, while the activity of endogenous SOD was not changed by silibinin. Previous work proved that silibinin induced MCF-7 cell apoptosis through mitochondrial pathway and this study further proved that O₂^·– generation induced by silibinin was also related to mitochondria. It was found that respiratory chain complexes I, II and III were all involved in silibinin-induced O₂^·– generation. Moreover, it was found that silibinin-induced O₂^·– had protective effect, as exogenous SOD markedly enhanced silibinin-induced apoptosis.     

Silibinin ameliorates Aβ<Subscript>25-35</Subscript>-induced memory deficits in rats by modulating autophagy and attenuating neuroinflammation as well as oxidative stress

Alzheimer’s disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid β (Aβ)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aβ_25-35-injected rats. Results demonstrated that silibinin significantly attenuated Aβ_25-35-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aβ_25-35-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aβ_25-35-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.     

Silibinin-induced endoplasmic reticulum stress and mitochondrial dysfunction suppress growth of endometriotic lesions

Silibinin is a flavonolignan extracted from milk thistle, which has been used for treating liver disorders, various cancers, and gynecological diseases. However, attempts for treating endometriosis with silibinin are lacking. In this study, we observed that silibinin exerts antiproliferative and apoptotic effects on human endometriotic cell lines VK2/E6E7 and End1/E6E7. We also identified that silibinin-induced oxidative stress and lipid peroxidation in human endometriotic cells. Moreover, we observed upregulation of calcium concentration in the cytosol and mitochondrial matrix, which resulted in mitochondrial dysfunction. Furthermore, induction of endoplasmic reticulum stress signals with rapid mitogen-activated protein kinase (MAPK) pathway signaling resulted in apoptosis of both cells. Using an animal model mimicking the retrograde menstruation hypothesis, we verified the effects of silibinin on reducing endometriotic lesions by inhibiting the expression of inflammatory cytokines in mice. Silibinin might be used as a novel therapeutic agent or supplement for inhibiting progression of endometriosis in vitro and in vivo.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

Background

In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.

Results

We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.

Conclusion

In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.     

Galantamine inhibits β‐amyloid‐induced cytostatic autophagy in PC12 cells through decreasing ROS production

Objectives

Alzheimer's disease (AD) is one of the most prevalent brain diseases among the elderly, majority of which is caused by abnormal deposition of amyloid beta‐peptide (Aβ). Galantamine, currently the first‐line drug in treatment of AD, has been shown to diminish Aβ‐induced neurotoxicity and exert favourable neuroprotective effects, but the detail mechanisms remain unclear.

Materials and methods

Effects of galantamine on Aβ‐induced cytotoxicity were checked by MTT, clone formation and apoptosis assays. The protein variations and reactive oxygen species (ROS) production were measured by western blotting analysis and dichloro‐dihydro‐fluorescein diacetate assay, respectively.

Results

Galantamine reversed Aβ‐induced cell growth inhibition and apoptosis in neuron cells PC12. Aβ activated the entire autophagy flux and accumulation of autophagosomes, and the inhibition of autophagy decreased the protein level of cleaved‐caspase‐3 and Aβ‐induced cytotoxicity. Meanwhile, galantamine suppressed Aβ‐mediated autophagy flux and accumulation of autophagosomes. Moreover, Aβ upregulated ROS accumulation, while ROS scavengers N‐acetyl‐l ‐cysteine impaired Aβ‐mediated autophagy. Further investigation showed that galantamine downregulated NOX4 expression to inhibit Aβ‐mediated ROS accumulation and autophagy.

Conclusions

Galantamine inhibits Aβ‐induced cytostatic autophagy through decreasing ROS accumulation, providing new insights into deep understanding of AD progression and molecular basis of galantamine in neuroprotection.

     

Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.     

Protective Effect of Silibinin on Learning and Memory Impairment in LPS-Treated Rats via ROS–BDNF–TrkB Pathway

Silibinin, a flavonoid derived from the herb milk thistle (Silybum marianum), has been used as a hepato-protectant in the clinical treatment of liver disease. In the present study, the effect of silibinin on lipopolysaccharide (LPS)-induced neuroinflammatory impairment in rats is investigated. Injection of LPS into lateral ventricle caused learning and memory impairment. Rats were treated with silibinin to see the effect in comparison with resveratrol as a positive control. Y-maze and Morris water maze tests showed that silibinin significantly attenuated memory damage caused by LPS treatment. At the molecular analysis, the levels of IL-1β and of IL-4 in the hippocampus were decreased and enhanced, respectively, by the treatment with silibinin. NF-κB expression was attenuated by silibinin treatment. Furthermore, generation of total reactive oxygen species (ROS) in the hippocampus was elevated in silibinin-treated groups, and so were the expressions of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB). At the same time, LPS-induced reduction of neurons in hippocampus was reversed by silibinin. In conclusion, silibinin ameliorated the impairment of learning and memory of LPS-injection rats, possibly due to the activation of ROS–BDNF–TrkB pathway in the hippocampus as well as the suppression of inflammatory response. This study gives an insight on the beneficial consequences of ROS in central nervous system. Silibinin might be a potential candidate drug for neurodegenerative diseases.     

Atmospheric Pressure Room Temperature Plasma Jets Facilitate Oxidative and Nitrative Stress and Lead to Endoplasmic Reticulum Stress Dependent Apoptosis in HepG2 Cells

Atmospheric pressure room temperature plasma jets (APRTP-Js) that can emit a mixture of different active species have recently found entry in various medical applications. Apoptosis is a key event in APRTP-Js-induced cellular toxicity, but the exact biological mechanisms underlying remain elusive. Here, we explored the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in APRTP-Js-induced apoptosis using in vitro model of HepG2 cells. We found that APRTP-Js facilitated the accumulation of ROS and RNS in cells, which resulted in the compromised cellular antioxidant defense system, as evidenced by the inactivation of cellular antioxidants including glutathione (GSH), superoxide dismutase (SOD) and catalase. Nitrotyrosine and protein carbonyl content analysis indicated that APRTP-Js treatment caused nitrative and oxidative injury of cells. Meanwhile, intracellular calcium homeostasis was disturbed along with the alteration in the expressions of GRP78, CHOP and pro-caspase12. These effects accumulated and eventually culminated into the cellular dysfunction and endoplasmic reticulum stress (ER stress)-mediated apoptosis. The apoptosis could be markedly attenuated by N-acetylcysteine (NAC, a free radical scavenger), which confirmed the involvement of oxidative and nitrative stress in the process leading to HepG2 cell apoptosis by APRTP-Js treatment.