An updated review at molecular pharmacological level for the mechanism of anti-tumor, antioxidant and immunoregulatory action of silibinin / 药学学报

Silibinin, from milk thistle (Silybum marianum), is a flavonolignan with anti-oxidative and anti-inflammatory properties. It has been therapeutically used for the treatment of hepatic diseases in China, Germany and Japan. Recently, increasing evidences prove that silibinin is also a potent antitumor agent, and the major anti-tumor mechanism for silibinin is the prominent inhibition of the activities of receptor tyrosine kinases (RTKs) and their downstream signal molecules in a variety of tumor cell lines, such as epidermal growth factor receptor 1 (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) signaling pathways. Meanwhile, the evidences that silibinin selectively scavenges hydroxyl free radical (*OH) and specifically inhibits the action of nuclear factor kappaB (NF-kappaB) provide more complicated explanations for its antioxidant and anti-inflammatory effects. Some new findings such as that silibinin attenuating the cognitive deficits induced by amyloid beta protein (Abeta) peptide through its antioxidative and anti-inflammatory properties is valuable to broad the medical prospect of silibinin. In this review, we discuss the molecular pharmacological mechanisms of silibinin, focusing on its inhibition of tyrosine kinases, actions of antioxidation, free radical scavenging, immunoregulation and anti-inflammation.

An updated review at molecular pharmacological level for the mechanism of anti-tumor, antioxidant and immunoregulatory action of silibinin / 药学学报

Silibinin, from milk thistle (Silybum marianum), is a flavonolignan with anti-oxidative and anti-inflammatory properties. It has been therapeutically used for the treatment of hepatic diseases in China, Germany and Japan. Recently, increasing evidences prove that silibinin is also a potent antitumor agent, and the major anti-tumor mechanism for silibinin is the prominent inhibition of the activities of receptor tyrosine kinases (RTKs) and their downstream signal molecules in a variety of tumor cell lines, such as epidermal growth factor receptor 1 (EGFR) and insulin-like growth factor 1 receptor (IGF-1R) signaling pathways. Meanwhile, the evidences that silibinin selectively scavenges hydroxyl free radical (*OH) and specifically inhibits the action of nuclear factor kappaB (NF-kappaB) provide more complicated explanations for its antioxidant and anti-inflammatory effects. Some new findings such as that silibinin attenuating the cognitive deficits induced by amyloid beta protein (Abeta) peptide through its antioxidative and anti-inflammatory properties is valuable to broad the medical prospect of silibinin. In this review, we discuss the molecular pharmacological mechanisms of silibinin, focusing on its inhibition of tyrosine kinases, actions of antioxidation, free radical scavenging, immunoregulation and anti-inflammation.

Regulatory effects of Shc-related phosphotyrosine adaptor proteins on aging / 药学学报

Aging-related oxidative stress and free radical theory has become accepted increasingly as explaination, at least in part of the aging process. In murine models of aging, a genetic deficiency of the p66(Shc) (66-kilodalton isoform of Shc gene products) gene, which encodes a phosphotyrosine signal adapter protein, extends life span by 30%, and confers resistance to oxidative stress. Upon oxidative stress, p66(Shc) is phosphorylated at Ser36, contributing to inactivation of the forkhead-type transcription factors (FKHR/ FoxO1), which regulates the gene expression of cellular antioxidants. The p66(Shc) has a direct connection with the life span related signaling, which is conserved evolutionarily. Shc is basically not expressed in mature neurons of the adult brain and spinal cord. Instead, two Shc homologues, Sck/ShcB and N-Shc/ ShcC, which have been proved to be effective on oxidative stress and aging, are expressed in neural system. The expression of Shc-related genes is affected in the aging process, which may be relevant to cellular dysfunction, stress response and/or cognitive decline during aging.

Mechanism of downregulation of apoptosis by autophagy induced by oridonin in HeLa cells / 药学学报

To study the mechanism of downregulation of apoptosis by autophagy induced by oridonin in HeLa cells, the cell viability was measured by MTT method. DNA fragmentation was assayed by agarose gel electrophoresis. Autophagic and apoptotic ratio was determined by flowcytometric analysis. Protein expression was detected by Western blotting analysis. Oridonin induced both apoptosis and autophagy in HeLa cells. Apoptosis was upregulated by introduction of the inhibitor of autophagy, 3-methyladenine (3-MA). Addition of oridonin increased Bax/Bcl-2 expression ratio and cytochrome c, whereas the expression of SIRT-1 was decreased, and 3-MA pre-application enhanced these changes. Oridonin-induced autophagy antagonized apoptosis in HeLa cells through mitochondrial pathway.

Protective effect of silibinin against isoproterenol-induced injury to cardiac myocytes and its mechanism / 药学学报

Silibinin is a polyphenolic flavanoid derived from fruits and seeds of milk thistle (Silybum marianum). To investigate the effect and mechanism of silibinin on beta-isoproterenol-induced rat neonatal cardiac myocytes injury, the viability, the activation of lactate dehydrogenase (LDH) and the content of maleic dialdehyde (MDA) were chosen for measuring the degree of cardiac myocytes injury. Superoxide dismutase (SOD) activity, mitochondrial membrane potential (deltapsi) detected by flow cytometric analysis, and Western blotting analysis were applied to determine the related proteins. Silibinin protected isoproterenol-treated rat cardiac myocytes from death and significantly decreased LDH release and MDA production. Silibinin increased superoxide dismutase (SOD) activity, and increased mitochondrial membrane potential (deltapsi). Furthermore, the release of pro-apoptotic cytochrome c from mitochondria was reduced by silibinin. Silibinin increased the expression of anti-apoptotic Bcl-2 family protein Bcl-2, and up-regulation of SIRT1 inhibited the translocation of Bax from cytoplasm to mitochondria, which caused mitochondrial dysfunction and cell injury. Silibinin protects cardiac myocytes against isoproterenol-induced injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members.

Involvement of JNK-initiated p53 accumulation and phosphorylation of p53 in pseudolaric acid B induced cell death

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.

Effect of protein kinase C on human melanoma A375-S2 cell death induced by evodiamine / 药学学报

<p><b>AIM</b>To study the role of PKC in evodiamine-induced A375-S2 cell death.</p><p><b>METHODS</b>Ratio of apoptosis induced by evodiamine was determined by TUNEL assay. MTT assay was carried out to assess cytotoxic effect of evodiamine. The influence on expression of ERK, phospho-ERK and Bcl-2 was detected by Western blotting analysis.</p><p><b>RESULTS</b>TUNEL assay indicated that apoptosis was the type of A375-S2 cell death induced by evodiamine treatment for 24 h. Both staurosporine (inhibitor of PKC) and PD98059 (inhibitor of ERK) cooperated with evodiamine to further induce A375-S2 cell death. Evodiamine inhibited PKC activity, down-regulated the expression of ERK, phospho-ERK and Bcl-2, and staurosporine was capable of augmenting these effects induced by evodiamine.</p><p><b>CONCLUSION</b>PKC lies upstream and exhibits regulatory effect on ERK and Bcl-2 in evodiamine-induced cell death.</p>

Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin.</p><p><b>METHODS</b>The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4',6'-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.</p><p><b>RESULTS</b>Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.</p><p><b>CONCLUSION</b>Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.</p>

Oridonin induced U937 cell apoptosis through ERK pathway / 中国中药杂志

<p><b>OBJECTIVE</b>To study the mechanisms of oridonin-induced U937 cell apoptosis, and to examine the role of ERK MAPK.</p><p><b>METHOD</b>MTT, Hoechst 33258 staining, DNA agarose gel electrophoresis and Western blot analysis were used.</p><p><b>RESULT</b>Oridonin inhibited U937 cell growth in a time- and dose-dependent manner. Apoptotic bodies were found with Hoechst 33258 staining after treatment with 27 micromol x L(-1) oridonin. Simultaneously, ERK phosphorylation was significant. ERK inhibitor PD98059 partially blocked the growth-inhibitory effect as well as DNA fragmentation. The expression of antiapoptotic mitochondrial protein Bcl-XL decreased time-dependently, and that of proapoptotic protein Bax increased. However, PD98059 reversed the effect of oridonin on Bcl-XL and Bax.</p><p><b>CONCLUSION</b>Oridonin induces U937 cell apoptosis through activation of ERK and alteration of the ratio of Bax/Bcl-XL.</p>

Mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells.</p><p><b>METHODS</b>MTT assay, fluorescence microscopy, DNA agarose gel electrophoresis, flow cytometry and Western blot analysis were carried out to assess the morphological and biochemical changes of A375-S2 cells after capsaicin treatment.</p><p><b>RESULTS</b>Capsaicin induced A375-S2 cell death in a time- and dose-dependent manner. Sub-diploid peak was seen at 24 h after 250 micromol/L capsaicin treatment, and apoptotic bodies and DNA ladder were observed at 36 h after capsaicin treatment. The expression of inhibitor of caspase activated DNase (ICAD) was reduced with the lapse of time.</p><p><b>CONCLUSION</b>Capsaicin induces A375-S2 cell apoptosis and down-regulation of ICAD contributes to this process.</p>

Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.</p><p><b>METHODS</b>We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.</p><p><b>RESULTS</b>The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.</p><p><b>CONCLUSION</b>These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.</p>

IL-1beta Acts in Synergy with Endogenous IL-1beta in A375-S2 Human Melanoma Cell Apoptosis Through Mitochondrial Pathway

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.

Mechanism of dracorhodin perchlorate-induced Hela cell apoptosis / 药学学报

<p><b>AIM</b>To study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis.</p><p><b>METHODS</b>Cell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis.</p><p><b>RESULTS</b>Dracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules.</p><p><b>CONCLUSION</b>Dracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.</p>

Pseudolaric acid B induces apoptosis via activation of c-Jun N-terminal kinase and caspase-3 in HeLa cells

Pseudolaric acid B was isolated from Pseudolarix kaempferi Gordon (Pinaceae) and was evaluated for the anti-cancer effect in HeLa cells. We observed that pseudolaric acid B inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. HeLa cells treated with pseudolaric acid B showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. JNK inhibitor, SP600125, markedly inhibited pseudolaric acid B-induced cell death. In addition, Bcl-2 expression was down-regulated while Bax protein level was up-regulated. Caspase-3 inhibitor, z-DEVD-fmk, partially blocked pseudolaric acid B-induced cell death, and the expression of two classical caspase substrates, PARP and ICAD, were both decreased in a time- dependent manner, indicative of downstream caspase activation.

Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.

Pseudolaric acid B induced apoptosis and mitotic arrest circumventing Fas receptor pathway in MCF-7 cells / 中国药理学通报

Aim To study the mechanisms of Pseudolaric acid B(PAB)-induced MCF-7 cell apoptosis and mitotic arrest.Methods MTT assay was performed to assess the cell growth inhibition,contrast phase microscope was used to observe cellular morphologic alteration,and the change of DNA was detected by fluorescent microscopy.The distribution of cell cycle was determined by flow cytometric analysis of propidium iodide staining,and the protein expression was examined by Western blot analysis.Results PAB inhibited MCF-7 cell growth in a dose-and time-dependent manner.4 ?mol?L-1 PAB induced DNA condensation at 24 h.PAB cleaved PARP in a time-dependent manner.At 36 h,PAB up-regulated the expression of cdc 2 and nuclear cyclin B1.Fas antagonistic antibody UB2 had no effect on apoptosis,but agonistic antibody CH11 enhanced the apoptosis induced by PAB.UB2 exerted no effect on cell cycle arrest,and CH11 had the same action as UB2 except for reducing the mitotic arrest through enhancing apoptotic subdiploid peak.Conclusion PAB inhibited MCF-7 cell growth through mitotic arrest and apoptosis.Apoptosis and mitotic arrest were independent of Fas pathway.

Evodiamine induces A375-S2 cell death through two different pathways / 药学学报

<p><b>AIM</b>To study the mechanism of evodiamine-induced cell death of A375-S2.</p><p><b>METHODS</b>The changes in cell morphology were observed by invert microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. The effects of evodiamine on apoptosis and cell cycle were studied by flow cytometric analysis.</p><p><b>RESULTS</b>Evodiamine was shown to markedly inhibit the growth of A375-S2 cells in dose- and time-dependent manners. At the early stage, evodiamine activated caspase cascades, which unexpectedly did not induce typical DNA fragmentation. At later stage, caspase inhibitors failed to block A375-S2 cell death induced by evodiamine. Evodiamine-induced cell death was shown to be not directly associated with cell cycle arrest.</p><p><b>CONCLUSION</b>At the early stage, evodiamine initiates caspase-dependent and a typical apoptosis pathway in A375-S2 cells, but later it induces cell death through caspase-independent pathway which might be necrosis.</p>

Studies on evodiamine induced HeLa cell apoptosis / 药学学报

<p><b>AIM</b>To study the mechanism of evodiamine-induced growth inhibition of HeLa cells.</p><p><b>METHODS</b>HeLa cells viability and the effect of caspase inhibitors on evodiamine-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.</p><p><b>RESULTS</b>Evodiamine was found to inhibit HeLa cell growth in dose- and time-dependent manners. Caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk) was shown to partially inhibit evodiamine-induced apoptosis. However, caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize evodiamine induced cell death.</p><p><b>CONCLUSION</b>Evodiamine suppresses the growth of HeLa cells in vitro by apoptosis. Evodiamine-induced apoptosis is partially dependent on caspase-3 pathway in HeLa cells. Other apoptotic pathways might be also related to the induction of apoptosis by evodiamine.</p>

Regulation of ERK MAPK in evodiamine-induced A375-S2 cell death / 中国病理生理杂志

AIM: To compare the cytotoxic effect of evodiamine with chemotherapy drugs on A375-S2 cells, and to examine the relationship between the effects of PKC and ERK on evodiamine-induced cell death. METHODS: MTT assay and Western blot analysis were applied. RESULTS: Compared to actinomycin D, cisplatin and 5-FU, evodiamine showed less cytotoxic effects on A375-S2 cells, but it induced more significant inhibition of proliferation in A375-S2 cells incubated with evodiamine for 24 h, followed by continuous culture in drug-free medium. The activation of PKC induced by 10 ?g?L -1 PMA partially blocked evodiamine-induced cell death, which was reversed by PKC and ERK inhibitors. Moreover, evodiamine down-regulated the expressions of ERK and phosphorylated ERK. CONCLUSION: Evodiamine has a strong inhibitory influence on proliferation of A375-S2 cells, even after removal of evodiamine. Evodiamine blocks the protective role of ERK to A375-S2 cells through the downregulation of ERK and phosphorylated ERK expression. [

Human interleukin-1? induces A375-S2 human melanoma apoptosis through caspase pathway / 中国病理生理杂志

AIM: To study the molecular biological mechanism and signal transduction pathway of interleukin-1? (IL-1?)-induced apoptosis in A375-S2 melanoma cells. METHODS: Photomicrocropy showed typical apoptotic changes. The cytotoxic effect of IL-1? in vitro and influences of caspases in this effect were measured by MTT assay. The cytotoxicity of cells was assessed by LDH-based assay. Degradation of DNA was detected by agarose gel electrophoresis. RESULTS: The inhibitory effect of IL-1? on A375-S2 cell growth was in a dose and time-dependent manner, and cell death rate reached more than 90% at 72 h after treatment with 10~(-9)mol/L IL-1?. The inhibitors of caspase-family, -1, -3, -8, -9, and -10, partially blocked cell death at early stage. LDH assay showed that major IL-1?-induced cell death was apoptosis, and in a dose and time-dependent manner. Typical apoptotic DNA ladder was observed in agarose gel electrophoresis. CONCLUSION: IL-1? induced apoptosis in melanoma A375-S2 cells by activating caspase pathway. [