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Two new series of betulin derivatives with semicarbazone ( 7a - g ) or thiosemicarbazone ( 8a - g ) groups at the C-28 position were synthesized. All compounds were evaluated for their in vitro cytotoxicities in human hepatocellular carcinoma cells (HepG2), human breast carcinoma cells (MCF-7), human lung carcinoma cells (A549), human colorectal cells (HCT-116) and normal human gastric epithelial cells (GES-1). Among these compounds, 8f displayed the most potent cytotoxicity with an IC 50 value of 5.86 ± 0.61 μM against MCF-7 cells. Furthermore, the preliminary mechanism studies in MCF-7 cells showed that compound 8f could trigger the intracellular mitochondrial-mediated apoptosis pathway by losing MMP level, which was related with the upregulation of Bax, P53 and cytochrome c expression; the downregulation of Bcl-2 expression; activation of the expression levels of caspase-3, caspase-9, cleaved caspase-3 and cleaved caspase-9; and an increase in the amounts of intracellular reactive oxygen species. These results indicated that compound 8f may be used as a valuable skeleton structure for developing novel antitumor agents.

Background/aims: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear., Methods: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis., Results: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine., Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Euphorbia fischeriana Steud is a traditional Chinese Medicine that is known to possess a variety of anticarcinogenic properties. However, the bioactive constituents in Euphorbia fischeriana Steud and molecular mechanisms underlying this action in cancer treatment remain poorly understood. The present study investigated the chemotherapy activity and molecular targets of Ethyl gallate, which is identified as the major constituent extracted from the roots of Euphorbia fischeriana Steud in breast cancer cell lines in vitro. The results showed Ethyl gallate obviously decreased cell proliferation in MDA-MB-231 and MCF-7 cells in a dose- and time-dependent manner. Highly invasive MDA-MB-231 cells were found to be highly sensitive to treatment. Furthermore, significantly decreased metastatic potential of highly metastatic MDA-MB‑231 cells by Ethyl gallate was identified via the inhibition of cell motility using invasion and migration through a polyethylene terephthalate membrane. Ethyl gallate treatment decreased the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 by the downregulation of mRNA levels using RT-PCR, enzymes that are critical to tumor invasion. Treatment with Ethyl gallate decreased phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-κB (NF-κB) activation in MDA-MB-231 cells. These results indicate that Ethyl gallate suppresses proliferation and invasion in human breast cancer cells by modulating the PI3K/Akt pathway, which may contribute to inhibiting their downstream targets such as NF-κB p-65, Bcl-2/Bax, and mRNA levels of MMP-2 and MMP-9 in breast cancer cells. Thus, the present study shed new light on Ethyl gallate, an important bioactive constituent of Euphorbia fischeriana Steud, in human breast cancer treatment. The findings may provide basal theories for wide therapeutic application in human breast cancer.

The phosphoinositol-3-kinase (PI3K)/Akt signal transduction pathway is critically important for tumor cell growth, proliferation and apoptosis. Apoptosis activation has been reported to be a good target in cancer therapies. In this study, we have found that jolkinolide B (JB), a diterpenoid from the traditional Chinese medicinal herb Euphorbia fischeriana Steud, strongly inhibited the expression of the PI3K p85 subunit and the phosphorylation of Akt. Furthermore, we evaluated the effects of JB on the proliferation and apoptosis of MDA-MB-231 human breast cancer cells. Our results show significant induction of apoptosis in MDA-MB-231 cells incubated with JB. This effect was enhanced by combination with LY294002. In addition, treatment with JB could induce downregulation of the Bcl-2/Bax ratio, and subsequent promotion of mitochondrial release of cytochrome c and activation of caspase-3. Taken together, JB-induced apoptosis of MDA-MB-231 cells occurs through the mitochondrial pathway. Further, the PI3K/Akt signaling cascade plays a role in the induction of apoptosis in JB-treated cells. These observations suggest that JB may have therapeutic applications in the treatment of cancer.

Apoptosis is a widely accepted component of the pathogenesis of Parkinson's disease (PD), a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. In this study, we investigated the neuroprotective effects of puerarin and possible mechanisms by which puerarin acts against MPP(+)-induced toxicity in rat pheochromocytoma PC12 cells. PC12 cells exposed to MPP(+) (500 μM) significantly decreased the viability of PC12 cells when examined by MTT assay, DNA ELISA assay, and Annexin V assays, which was prevented by puerarin in a dose-dependent manner. PC12 cells exposed to MPP(+) (500 μM) elicited phosphorylation of MKK7, c-Jun-NH(2)-terminal kinase (JNK), and c-Jun which followed by the increase in cytochrome c levels, and which was prevented by puerarin. Moreover, puerarin inhibited the activation of caspase-9 and caspase-3 in MPP(+)-exposed PC12 cells. Whereas, the neuroprotective effect of puerarin against MPP(+) insults can be blocked by SP600125 (inhibitor of JNK). Taken together, these results suggest that puerarin protected PC12 cells against MPP(+)-induced neurotoxicity through the inhibition of the JNK signaling pathways. Therefore, puerarin has the possible beneficial effects in PD by attenuating MPP(+)-induced toxicity., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Elevated levels of β-amyloid (Aβ) in the brains being a hallmark of Alzheimer's disease have been believed to play a critical role in the cognitive dysfunction that occurs in Alzheimer's disease. Recent evidence suggests that Aβ induces neuronal apoptosis in the brain and in primary neuronal cultures. In this study, we investigated the effects of puerarin, a phytoestrogen isolated from Pueraria lobata, on cognitive function and neuronal apoptosis in the intrahippocampal injection of Aβ rats and its mechanism of action. The results show the intrahippocampal injection of Aβ induced a spatial memory deficit, apoptosis, and caspase-9 activation in hippocampal neurons. Puerarin treatment ameliorated Aβ(1-42)-induced cognitive impairment and reversed the increase of apoptosis in the hippocampus. The attenuation is associated with the activation of Akt and phosphorylation of Bad. These results suggest that puerarin may be an anti-Alzheimer's disease candidate drug to suppress both Alzheimer's disease-related neuronal cell apoptosis and dysfunction of the memory system., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Neurodegenerative disorders, such as Alzheimer's disease (AD), is associated with the loss of neuronal cells, and it has been suggested that apoptosis is a crucial pathway in neuronal loss in AD patients. Recent evidence suggests that amyloid beta peptide (Abeta) induces neuronal apoptosis in the brain and in primary neuronal cultures. In this study, we investigated the impact of beta-asarone against the apoptosis induced by Abeta in rat hippocampus. The results showed that intrahippocampal injections of Abeta (1-42) caused apoptosis in rat hippocampus. Oral administration of beta-asarone (12.5, 25, or 50 mg/kg) for 28 d reverse the increase in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus tissue. Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in AD. Therefore, we investigated nuclear translocation of apoptosis induction factors. Our results showed that beta-asarone afforded a beneficial inhibition on both mRNA and protein expression of Bad, Bax, and cleavage of caspases 9 in rat hippocampus following intrahippocampal injections of Abeta (1-42). Our further investigation revealed that ASK1, p-MKK7, and p-c-Jun were significantly decreased after beta-asarone treatment, implicating that the modulation of ASK1/c-JNK-mediated intracellular signaling cascades might be involved in therapeutic effect of beta-asarone against Abeta toxicity. Taken together, these results suggest that beta-asarone may be a potential candidate for development as a therapeutic agent for AD.

Elevated levels of beta-amyloid (Abeta) in the brains being a hallmark of Alzheimer's disease (AD) have been believed to play a critical role in the cognitive dysfunction that occurs in AD. Recent evidence suggests that Abeta induces neuronal apoptosis in the brain and in primary neuronal cultures. In this study, we investigated the effects of beta-asarone, the major ingredient of Acorus Tatarinowii Schott, on cognitive function and neuronal apoptosis in Abeta hippocampus injection rats and its mechanism of action. The results show that the Abeta (1-42) injection caused impairments in spatial reference memory in a Morris water maze task and apoptosis in hippocampus. Oral administration of beta-asarone with three different dose (12.5, 25, or 50 mg/kg) for 28 d ameliorated Abeta (1-42)-induced cognitive impairment and reversed the increase of apoptosis in the hippocampus. Abeta-induced c-Jun N-terminal kinase (JNK) results in phosphorylation, subsequent down-regulation of Bcl-2 and Bcl-w expression, and caspase-3 activation. Beta-asarone attenuate Abeta (1-42)-induced neuronal apoptosis in hippocampus by reversal down-regulation of Bcl-2, Bcl-w, caspase-3 activation, and JNK phosphorylation. These results suggest that beta-asarone may be a potential candidate for development as a therapeutic agent to manage cognitive impairment associated with conditions such as Alzheimer's disease.

Abstract Background/Aims: Resveratrol (Res), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. This study was undertaken to investigate whether Res can protect the heart suffering from hypertrophy injuries induced by isoproterenol (ISO), and whether the protective effect is mediated by endoplasmic reticulum (ER) stress. Methods: Cardiomyocytes were randomly assigned to the control group, ISO group (100 nM ISO for 48 h), Res + ISO group (50 μM Res and 100 nM ISO for 48 h) and Res group (50 μM Res for 48h only). Hypertrophy was estimated by measuring the cell surface area and the atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using Hoechst 33258 staining and transmission electron microscopy. Protein expression of ER stress and apoptosis factors was analyzed using Western Blot analysis. Results: Res effectively suppress the cardiomyocytes hypertrophy and apoptosis induced by ISO, characterized by the reduction of the myocardial cell surface area, the ANP gene expression, the LDH and MDA leakage amount and the rate of cell apoptosis, while decrease of the protein expression of GRP78, GRP94 and CHOP, and reverse the expression of Bcl-2 and Bax. Conclusion: In summary, Res treatment effectively suppressed myocardial hypertrophy and apoptosis at least partially via inhibiting ER stress. [ABSTRACT FROM AUTHOR]

Background/Aim: Polyamines (putrescine, spermidine and spermine) play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS) in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO). Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO). Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP) gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM) treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) level in the culture medium. In addition, DFMO (0.5 mM) down regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM). Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Epidemiological data have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer''s disease (AD). Phytoestrogens have been proposed as potential alternatives to ERT. The aim of the present study was to assess the neuroprotective effects of puerarin, a phytoestrogen isolated from Pueraria lobata, against the toxicity of beta-amyloid (Aβ) in relation to the mitochondria-mediated cell death process, and to elucidate the role the activation of Akt and modulation of the pro- and antiapoptotic proteins in puerarin-induced neuroprotection. The present study shows that puerarin afforded protection against Aβ-induced toxicity through inhibiting apoptosis in PC12 cells. This result was also confirmed by the activated caspase-3 assay. P-Akt, Bcl-2 and p-Bad expression increased after pretreatment with puerarin in PC12 cells exposed to Aβ25–35, whereas Bax expression and cytochrome c release decreased. Interestingly, these effects of puerarin against Aβ25–35 insult were abolished by wortmannin, an inhibitor of PI3K phosphorylation. These findings suggest that puerarin prevent Aβ-induced neurotoxicity through inhibiting neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD. [Copyright &y& Elsevier]

Abstract: Neurodegenerative brain disorders such as Alzheimer''s disease have been well investigated. However, significant methods for the treatment of the promotion and progression of Alzheimer''s disease are unavailable to date. Apoptosis is a crucial pathway in neuronal loss in Alzheimer''s disease patients. Thus, the suppression of apoptosis may be an effective therapeutic strategy for Alzheimer''s disease. In this study, we evaluated the effect of β-asarone on β-amyloid (Aβ)-induced toxicity in cultured PC12 cells. Our data show significant induction of apoptosis in PC12 cells incubated with Aβ peptide, and this effect was reduced by β-asarone. Beta-asarone reduced Aβ-induced JNK activation. In addition, β-asarone attenuates Aβ-induced down-regulation of Bcl-w and Bcl-xL in a JNK-dependent manner, and subsequent inhibition mitochondrial release of cytochrome c and activation of caspase-3. Together, these findings indicate that Aβ-induced apoptosis of PC12 cells proceeds through mitochondrial pathway. Further, the JNK signaling cascade plays a role in regulating the anti-apoptotic effects of β-asarone. Thus, our results indicate that β-asarone might be a potentially therapeutic compound for Alzheimer''s disease. [Copyright &y& Elsevier]

Euphorbia fischeriana S. (E. fischeriana) is a classic Chinese herb with toxicity that is mainly used for cancer treatment and in insect repellent, anti-inflammatory and anti-edema applications (Liu et al., 2001). 12-Deoxyphorbol13-palmitate (DP), a tetracyclic diterpene monomer compound, was extracted from the roots of E. fischeriana by our research groups. Previous studies found that DP could inhibit the proliferation of leukemia cells in vitro. However, the underlying mechanism of DP in leukemia is unknown. Hence, DP's pharmacological effect on leukemia cells was investigated in this study. DP was obtained from the Natural Medicine Chemistry Laboratory of Qiqihaer Medical University. In vitro, K562 cells and HL60 cells were incubated with DP or DP combined with LY294002 at different concentrations. Cell proliferation and apoptosis were detected by the relevant experimental methods. In vivo, nude mouse xenograft models were established by injecting K562 cells. DP was intraperitoneally administered to observe the influence on the growth of transplanted tumors. Gene detection and immunoblot analysis were performed to validate the mechanisms. The cell counting kit-8 (CCK-8) assay proved that DP inhibited the growth of K562 and HL60 cells in a time- or dose-dependent manner. At 12 h, DP could induce apoptosis by Annexin V-FITC/propidium iodide (PI) dual labeling, loss of mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS), acridine orange/ethidium bromide (AO/EB) staining and transmission electron microscopy (TEM) observation in K562 or HL60 cells. Furthermore, in an assay of gene and protein expression, we found that DP could downregulate the gene and protein expression levels of Bcl-2, upregulate the gene and protein expression levels of Bax and Bim, and downregulate the protein expression levels of PI3k, p-Akt, and p-FoxO3a. Moreover, the effects of DP on proliferation and apoptosis in K562 cells were enhanced by LY294002. Then, we tested the antitumor effects of DP in vivo. Nude mouse xenograft models were established by subcutaneously injecting K562 cells. We found that tumor volume was significantly decreased in DP-treated xenograft nude mice. Morphologic changes, apoptosis degree, and related gene and protein expression levels in transplanted tumor tissue of DP-treated nude mice were assessed by different experimental methods. The in vivo and in vitro experimental results indicated that DP might inhibit the proliferation and induce the apoptosis of leukemia cells, which might be a result of suppressing the PI3k/Akt signaling pathways. The in vivo and in vitro experimental results indicated that DP might inhibit the proliferation and induce the apoptosis of leukemia cells, which might be a result of suppressing the PI3k/Akt signaling pathways. [Display omitted] [ABSTRACT FROM AUTHOR]