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Hao He,
Ling-He Zang,
Yong-Sheng Feng,
Jian Wang,
Wei-Wei Liu,
Li-Xia Chen,
Ning Kang, Shin-Ichi Tashiro,
Satoshi Onodera,
Feng Qiu,
Takashi Ikejima
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ABSTRACT: Physalin A (1) is a withanolide isolated from Physalis alkekengi var. franchetii. In this study, the selective growth inhibitory effects on tumor cells induced by 1 were screened, and the mechanism was investigated on 1-induced growth inhibition, including apoptosis and autophagy, in human fibrosarcoma HT1080 cells. Apoptosis induced by 1 in HT1080 cells was associated with up-regulation of caspase-3 and caspase-8 expression. However, there were no significant changes in caspase-9, Bid, Bax, and Bcl-2 expression, indicating that 1-induced apoptosis in HT1080 cells occurs mainly through activation of the death receptor-associated extrinsic apoptotic pathways. Autophagy induced by 1 was found to antagonize apoptosis in HT1080 cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor 3-methyladenine (3MA). Loss of beclin 1 (as an autophagic regulator) function led to similar results to 3MA. However, 1 did not show inhibitory effects on normal human cells (human peripheral blood mononuclear cells). Taken together, these results suggest that 1 may be a promising agent for the treatment of cancer.
Journal of Natural Products 05/2013; · 3.13 Impact Factor
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Weiwei Liu,
Wuxiyar Otkur,
Lingzhi Li,
Qiong Wang,
Hao He,
Yuanchao Ye,
Yuning Zhang,
Toshihiko Hayashi, Shin-Ichi Tashiro,
Satoshi Onodera,
Takashi Ikejima
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ABSTRACT: Ultraviolet B (UVB) in the sun light is a major cause of skin damage, which accompanies complex alterations in irradiated skin cells, including DNA lesions, oxidative stress, inflammation and caspase activation. The protection against UVB damage requires multiple interruptions such as repair of the DNA lesions, scavenging of the reactive oxygen species (ROS), repression of the inflammation and others. Silibinin is suggested as an anti-UVB reagent, but the underlying mechanisms have not been fully elucidated. In this study, we found a role of autophagy in the anti-UVB effect of silibinin in A431 cells. Autophagy was reduced after UVB-irradiation while restored by silibinin through the suppression of the IGF-1R signalling pathway. The protective effect of silibinin in UVB-irradiated A431 cells was further enhanced by pre-treatment with an autophagy inducer, rapamycin, while it was reversed by an autophagy inhibitor, wortmannin, indicating that elevated autophagy contributed to the cell survival. Consistently, cell apoptosis was augmented by siRNAs targeting Beclin 1 and Atg5, supporting the hypothesis that autophagy induced by silibinin plays a protective role against UVB-induced epidermal apoptosis.
Journal of photochemistry and photobiology. B, Biology 04/2013; 123C:23-31. · 1.87 Impact Factor
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ABSTRACT: Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Our previous study demonstrated that PAB induced mitotic catastrophe in L929 cells and indicated that only a small percentage (12%) of the cells undergoing mitotic catastrophe displayed an apoptotic phenotype after PAB treatment for 72 h. In this study, we found that a minority of the cells undergoing mitotic catastrophe ended in apoptosis, and a majority of them entered a period of senescence. Further data confirmed that PAB induced autophagy, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in L929 cells. Subsequently, we found that autophagy inhibitors significantly delayed the senescence process, indicating that autophagy facilitated senescence. Moreover, ROS scavenger significantly decreased the autophagic level and improved mitochondrial function. Additionally, autophagy inhibitors effectively reduced ROS levels and ameliorated mitochondrial function. In conclusion, autophagy promoted senescence via enhancement of ROS generation and mitochondrial dysfunction in PAB-treated L929 cells.
Journal of Pharmacological Sciences 02/2013; · 2.08 Impact Factor
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Weiwei Liu,
Wuxiyar Otkur,
Lingzhi Li,
Qiong Wang,
Hao He,
Linghe Zang,
Toshihiko Hayashi, Shin-Ichi Tashiro,
Satoshi Onodera,
Mingyu Xia,
Takashi Ikejima
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ABSTRACT: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
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ABSTRACT: Apoptosis in murine dermal cells is retarded by ultraviolet B (UVB) irradiation-induced autophagic intervention while simultaneously epidermal cells commit apoptosis, during which inflammatory cytokines released from the lost epidermal cells promote immune responses of dermal inflammatory cells, forming morphological symptoms of acute cutaneous diseases. Autophagy is involved in prevention or provocation of apoptosis of dermal or epidermal cells of UVB-irradiated mice via modulation of intracellular metabolism, intervening the balance between cell death and survival in dermis and epidermis. p53 expressed in immune system affects autophagy function through activating or inactivating genes encoding apoptotic factors and inflammatory cytokines. Silibinin protects dermal and epidermal cells of UVB irradiated skin against abnormally autophagy-mediated apoptosis adjustments. In this study, how UVB irradiation intervenes autophagy in dermal and epidermal cells as well as how silibinin protects UVB irradiated skin through physiological recovering of autophagy function in dermis and epidermis are focused and elucidated preliminarily. Silibinin treatment (50 mg/kg/day for 4 days) reversed dermal and epidermal autophagy levels from UVB irradiation-induced improper autophagy intervention, repaired the balance between cell survival and death in dermis and epidermis, and protected skin against damage through mediation of p53 activation in dermal and epidermal cells.
Journal of Asian natural products research 02/2013; 15(2):117-29. · 0.61 Impact Factor
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ABSTRACT: We reported previously that phagocytosis of apoptotic cells by U937 cells was enhanced by the treatment with oridonin that showed high activity to induce the generation of reactive oxygen species (ROS) in many cells. ROS, important signaling molecules, are involved in the immune defenses, cell repair and proliferation. In this study, oridonin caused modest amount of ROS generation in U937 cells, with hydrogen peroxide (H(2)O(2)) and hydroxyl free radical (OH) as the major types. Meanwhile, H(2)O(2) and OH were positive regulators involved in oridonin-enhanced engulfment of apoptotic cells through down-regulating mitochondrial membrane potential (MMP) and inducing autophagy. The ROS-mediated phagocytosis was independent of cellular adenosine triphosphate (ATP) levels. H(2)O(2) and OH generation also activated phosphatidylinositol 3-kinases-Akt (PI3K-Akt) and phospholipase C γ-protein kinase C(PLC γ)-Ras-Raf-ERK signaling pathways, which were essential for oridonin-induced engulfment of apoptotic cells. Phagocytosis, the loss of MMP, autophagy and the activated signaling pathways were all suppressed by ROS scavenger N-acetyl-l-cysteine (NAC), H(2)O(2)scavenger catalase or OH scavenger glutathione (GSH). However, superoxide anion (O(2)-) and its scavenger superoxide dismutase (SOD) did not significantly affect these oridonin-induced biological processes.
International immunopharmacology 01/2013; · 2.21 Impact Factor
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ABSTRACT: Previous study proved that norcantharidin (NCTD) could exert its anticancer activity in a variety of malignant cell lines, including human cervical carcinoma HeLa cells. In this study, we found that NCTD-activated p38 mitogen-activated protein kinase (p38 MAPK)-nuclear transcription factor kappa B (NF-κB) signaling pathway induced mitochondrial apoptotic pathway activation and G2/M cell cycle arrest in HeLa cells. NCTD-induced mitochondria-associated apoptosis was concomitant with the collapse of mitochondrial membrane potential (ΔΨ(m)), translocation of Bax, down-regulation of Bcl-2 expression, and release of cytochrome c. NCTD-led G2/M cell-cycle arrest was associated with the up-regulated p21 and p-cdc25c expression and the down-regulated cyclin B and cdc2 expression. Treatment of the cells with p38 inhibitor SB203580 and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) showed that p38 functioned upstream of NF-κB, while augmented apoptosis and cell cycle arrest were induced in response to NCTD with NF-κB activation. Intriguingly, NF-κB had a negative feedback regulatory effect on p38 activation. Moreover, NCTD-induced apoptosis and cell cycle arrest were significantly blocked by SB203580 and PDTC but not by pifithrin-α (p53 inhibitor). Therefore, p38-NF-κB induced mitochondrial apoptotic pathway and G2/M cell cycle arrest in NCTD-treated HeLa cells.
Journal of Asian natural products research 11/2012; 14(11):1008-19. · 0.61 Impact Factor
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ABSTRACT: Abstract Oridonin, a diterpenoid compound, extracted and purified from Rabdosia rubescen has been reported to have cytotoxic effect on tumour cells through apoptosis, and tyrosine kinase pathways are involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478 was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant human epidermoid carcinoma A431 cells. Inhibition of EGFRaugmented oridonin-induced A431 cell apoptosis, while the changes of expression of downstream proteins, Bcl-2, Bcl-xL, Bax, cytochrome c, pro-caspase-3, Fas, FADD and pro-caspase-8 suggested that both the intrinsic and extrinsic apoptotic pathways are involved in these processes. Pretreatment with AG1478 aggravated oridonin-induced loss of mitochondrial membrane potential (MMP) and increased ROS generation in A431 cells, while a ROS scavenger, N-acetylcysteine (NAC) completely reversed oridonin- and AG1478-induced ROS generation and apoptosis. Therefore, AG1478 augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478 decreased the expression of downstream MAPK proteins ERK, JNK and P38 and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38 inhibitors, only JNK inhibitor SP600125 effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonised apoptosis and exerted a protective effect in A431 cells, and both AG1478 and SP600125 decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis and this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonising apoptosis and ROS generation.
Free radical research 08/2012; 46(11):1393-405. · 2.22 Impact Factor
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ABSTRACT: 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.
Free radical research 08/2012; 46(11):1346-60. · 2.22 Impact Factor
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ABSTRACT: To investigate the role of nitric oxide (NO) in oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells and the underlying molecular mechanisms.
Cell viability was measured using MTT assay. Intracellular NO level, SubG(1) cell ratio and autophagy cell ratios were analyzed with flow cytometry after diaminofluorescein-2 diacetate (DAF-2DA), propidium iodide (PI) and monodansylcadaverine (MDC) staining, respectively. Protein expression was examined using Western blot analysis.
Exposure of L929 cells to oridonin (50 μmol/L) for 24 h led to intracellular NO production. Pretreatment with NOS inhibitor 1400w or L-NAME inhibited oridonin-induced apoptosis and autophagy in L929 cells. The pretreatment decreased the apoptosis-related protein Bax translocation and cytochrome c release, increased Bcl-2 level, reversed the autophagy-associated protein Beclin 1 increase and conversion of LC3 I to LC3 II. Furthermore, pretreatment with NO scavenger DTT completely inhibited oridonin-induced apoptosis and autophagy in L929 cells. In addition, oridonin (50 μmol/L) activated ERK and p53 in L929 cells, and the interruption of ERK and p53 activation by PD 98059, pifithrin-α, or ERK siRNA decreased oridonin-induced apoptosis and autophagy. The inhibition of NO production reduced oridonin-induced ERK and p53 activation, and NO production was down-regulated by blocking ERK and p53 activation.
NO played a pivotal role in oridonin-induced apoptosis and autophagy in L929 cells. Taken together with our previous finding that ERK contributes to p53 activation, it appears that NO, ERK, and p53 form a positive feedback loop. Consequently, we suggest that oridonin-induced apoptosis and autophagy are modulated by the NO-ERK-p53 molecular signaling mechanism in L929 cells.
Acta Pharmacologica Sinica 07/2012; 33(8):1055-61. · 1.95 Impact Factor
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ABSTRACT: Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.
Free radical research 06/2012; 46(10):1207-19. · 2.22 Impact Factor
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ABSTRACT: The pharmacological activity of SU11274 is primarily due to its inhibition of hepotocyte growth factor receptor (c-Met) kinase overexpression. In this study, we demonstrated that the pathway involved in SU11274-induced autophagy was presumably through inhibition of c-Met and its down-stream pathways, including phosphatidylinositol 3-kinases – Akt (PI3K–Akt) and the growth factor receptor bound protein-2 / son of sevenless – Ras – p38 MAPK (Grb2/SOS–Ras–p38) pathway. SU11274 time-dependently induced the generation of superoxide anion (O2(•−)) and hydrogen peroxide (H2O2). There is a negative feedback loop between reactive oxygen species (ROS) induction and SU11274. Then, we investigated the role of ROS in protecting cells against SU11274-induced autophagic cell death in A549 cells. O2(•−) and H2O2 generation activated c-Met–PI3K–Akt and c-Met–Grb2/SOS–Ras–p38 signaling pathways, which were suppressed by O2(•−) scavenger superoxide dismutase (SOD) and H2O2 scavenger catalase. In conclusion, O2(•−) and H2O2 evoked cell resistance to SU11274 via activating c-Met–PI3K–Akt and c-Met–Grb2/SOS–Ras–p38 pathways in A549 cells. SU11274 also induced ROS generation in Caenorhabditis elegans.
Journal of Pharmacological Sciences 05/2012; 119(2):150-9. · 2.08 Impact Factor
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ABSTRACT: 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.
Free radical research 05/2012; 46(9):1082-92. · 2.22 Impact Factor
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ABSTRACT: Skin inflammation induced by ultraviolet B (UVB) radiation is characterized by migration and chemotaxis of inflammatory cells, epidermic thickening and erythema. Apoptosis and autophagy of epidermal and dermal cells are involved in its development through the adjustment of balance between cell survival and death. In this study, the role of balance between cell survival and apoptosis in dermis and epidermis in UVB-induced skin inflammation and the effect of autophagy on the balance were elucidated, and the protective mechanism of silibinin was investigated through the examination of the influence of autophagy activation or inhibition on erythema, migration, and chemotaxis of inflammatory cells as well as apoptosis adjustments. In UVB-irradiated controls, dermal apoptosis was retarded and the survival of inflammatory cells was promoted through the up-regulation of dermal autophagic level; epidermal apoptosis was increased through the down-regulation of epidermal autophagic level, causing migration and chemotaxis of neutrophils and mast cells as well as skin erythema. In silibinin-treated group (50 mg/kg/day for 4 days), dermal apoptosis was increased through inhibiting dermal autophagy; improper adjustment of epidermal apoptosis was attenuated through promoting epidermal autophagy, presenting dual effects on the balance between autophagy and apoptosis of epidermal and dermal cells and the protection.
Journal of Asian natural products research 05/2012; 14(7):688-99. · 0.61 Impact Factor
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ABSTRACT: SU11274, a small molecule inhibitor of c-Met, was reported to induce apoptosis in human non-small-cell lung cancer (NSCLC) cells. However, SU11274-mediated autophagy in NSCLC cells has rarely been reported. The aim of this study was to elucidate the molecular mechanisms mediating SU11274-induced autophagy in NSCLC A549 cells. Here we reported that SU11274-induced autophagy was accompanied with an increase in the conversion of LC3-I to LC3-II and up-regulation of Beclin-1 expression. Subsequently, we also found that small interfering RNA against c-Met induced A549 cell autophagy while promotion of c-Met by hepatocyte growth factor (HGF) suppressed A549 cell autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) suppressed SU11274-induced cell death, suggesting that SU11274-induced autophagy caused cell death. Further study showed that ERK and p53 were activated after SU11274 treatment. Interruption of ERK and p53 activities decreased SU11274-induced autophagy, and blocking of ERK by the specific inhibitor PD98059 suppressed SU11274-induced p53 activation. Moreover, ERK activation upregulated Beclin-1 expression through induction of Bcl-2 phosphorylation, but p53 did not induce Bcl-2 phosphorylation. In conclusion, inhibition of c-Met induced autophagic cell death, which was associated with ERK-p53 activation and ERK-mediated Bcl-2 phosphorylation in A549 cells.
Journal of Pharmacological Sciences 03/2012; 118(4):423-32. · 2.08 Impact Factor
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ABSTRACT: Improper adjustments of autophagy and silent information regulator 1 (Sirt-1) expression were reported to be closely associated with metabolic disorders. In this study, we examined the roles of Sirt-1 and autophagy in streptozotocin-induced diabetes mellitus, assessed the relationship between autophagy and Sirt-1, and investigated the protective mechanism of silibinin. Diabetes was induced in 6-week-old mice by intravenous injection of streptozotocin (150 mg/kg/day, for 2 weeks). In the treatment groups, silibinin (50 mg/kg/day, intramuscular injection, for 8 weeks) or inhibitors (50 mg/kg/day, subcutaneous injection, for 8 weeks) were given. Diabetic control animals received vehicle for the same time. Compared with diabetic controls, silibinin or autophagy inhibitor, 3-methyladenine, treated mice showed decreased levels of glycosylated hemoglobin A1C (P < 0.01), serum triglyceride (P < 0.01), cholesterol (P < 0.01), blood glucose (P < 0.05), autophagy (P < 0.05), and apoptosis ratio (P < 0.05) of pancreatic β-cells. Systemic administration of silibinin reversed streptozotocin-induced downregulation of Sirt-1 expression. Sirt-1 may play a role in regulating the physiological level of autophagy and is associated with loss of pancreatic β-cells and metabolic biochemical disorders. Through promoting Sirt-1 expression and recovering autophagy physiologically, silibinin may reverse hyperglycemia and repair damaged pancreatic β-cells.
Journal of Asian natural products research 03/2012; 14(5):413-23. · 0.61 Impact Factor
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ABSTRACT: Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Previous studies have demonstrated that PAB arrests cells in G2/M phase in several cancer cell lines without significantly perturbing the G2/M transition-associated proteins. CylinB1, a marker for mitotic phase arrest, was up-regulated in cells treated with PAB. Therefore, we investigated whether PAB affects cell cycle progression at the mitotic phase. The mitotic index increased during a 24h treatment with PAB, suggesting that PAB arrested cell cycle progression at mitosis. In addition, after a prolonged mitotic arrest, the cells underwent mitotic catastrophe. After an extended treatment with PAB (longer than 24h), the protein levels of cylinB1 and cdc2 significantly decreased in both nuclear and cytosolic extracts. According to these results, we concluded that mitotic slippage could be due to the inactivation of the cylinB1-cdc2 complex resulting from prolonged treatment with PAB. The cells undergoing mitotic catastrophe died via apoptosis.
European journal of pharmacology 03/2012; 683(1-3):16-26. · 2.59 Impact Factor
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ABSTRACT: 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.
Free radical research 03/2012; 46(3):310-9. · 2.22 Impact Factor
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ABSTRACT: Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.
Biological & Pharmaceutical Bulletin 01/2012; 35(12):2148-59. · 1.66 Impact Factor
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ABSTRACT: The aim of this study was to elucidate the molecular mechanisms mediating hepatocyte growth factor (HGF)-induced protection against oridonin-induced apoptosis in A549 cells. Oridonin induced decrease in Bcl-2/Bax ratio and activation of caspase-3, while these processes were reversed by HGF, suggesting that HGF played an anti-apoptotic role in oridonin-induced A549 cell death. HGF-induced protective effect was partially attributed to the activation of nuclear factor (NF)-κB and cyclooxygenase 2 (COX-2), since the protective effect was abolished by inhibition of NF-κB or interruption of COX-2. Then the activated COX-2 could prevent cells from initiating the apoptotic response by promoting prostaglandin E₂ (PGE₂) release. Activation of NF-κB-COX-2 by HGF-treatment triggered the increase in Bcl-2/Bax ratio, inhibition of procaspase-3 cleavage, promotion of Ca²⁺-independent intracellular phospholipase A2 (iPLA2) expression and augmentation of PGE₂ release, leading to antagonizing oridonin-induced cell death in A549 cells. HGF-induced cell survival in response to oridonin administration was associated with the activation of c-Met-NF-κB-COX-2 and c-Met-Bcl-2-caspase-3 signaling pathways. iPLA2, downstream effector of caspase-3, also participated in these processes.
Biological & Pharmaceutical Bulletin 01/2012; 35(7):1150-8. · 1.66 Impact Factor
