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.

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.
Amyloid beta-Peptides ; metabolism ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Antioxidants ; pharmacology ; Enzyme Activation ; Free Radical Scavengers ; pharmacology ; Humans ; Milk Thistle ; chemistry ; Molecular Structure ; NF-kappa B ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Reactive Oxygen Species ; metabolism ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, IGF Type 1 ; metabolism ; Signal Transduction ; drug effects ; Silymarin ; chemistry ; isolation & purification ; pharmacology

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.
Aging ; physiology ; Animals ; Brain ; metabolism ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Gene Deletion ; Humans ; Mice ; Neurons ; metabolism ; Oxidative Stress ; physiology ; Phosphorylation ; Shc Signaling Adaptor Proteins ; deficiency ; genetics ; metabolism ; physiology ; Signal Transduction ; physiology ; Spinal Cord ; metabolism ; Src Homology 2 Domain-Containing, Transforming Protein 1 ; Src Homology 2 Domain-Containing, Transforming Protein 2 ; Src Homology 2 Domain-Containing, Transforming Protein 3

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.
Animals ; Animals, Newborn ; Blotting, Western ; Cardiotonic Agents ; isolation & purification ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Isoproterenol ; toxicity ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Milk Thistle ; chemistry ; Mitochondria, Heart ; drug effects ; metabolism ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Silymarin ; isolation & purification ; pharmacology ; Sirtuin 1 ; Sirtuins ; metabolism ; Superoxide Dismutase ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; metabolism

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.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Autophagy ; drug effects ; Blotting, Western ; Cytochromes c ; metabolism ; Diterpenes ; isolation & purification ; pharmacology ; Diterpenes, Kaurane ; isolation & purification ; pharmacology ; Flow Cytometry ; HeLa Cells ; Humans ; Isodon ; chemistry ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Sirtuin 1 ; Sirtuins ; metabolism ; bcl-2-Associated X Protein ; metabolism

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.
Tumor Suppressor Protein p53/metabolism/*physiology ; Protein Kinase C/metabolism ; Phosphorylation ; JNK Mitogen-Activated Protein Kinases/*physiology ; Humans ; Hela Cells ; Diterpenes/*pharmacology ; DNA Fragmentation/drug effects ; Cell Death/*drug effects ; Anthracenes/pharmacology

BACKGROUND: Uterine cervix cancer is one of the diseases to damage female health and degrade quality of life. It has been a hot spot to decrease the side effects of radiotherapy and chemotherapy and anti-tumor medicine by using Chinese medicine in medical domain. In Europe, silymarin had been used to treat hepatitis. It had little toxic action and large contents in Chinese herb. Therefore, many researchers are studying its anti-tumor effects now.OBJECTIVE: To study the pharmacological mechanism of silymarin on human cervical cancer cell.DESIGN: The cells cultured in 10% serum medium served as negative control group in vitro.MATERIALS: The experiment was finished in the China-Japan Research Institute of Medical and Pharmaceutical Sciences in Shenyang Pharmaceutical University; Department of Pathological Science of Showa Medical UniversityMATERIASLS: Human cervical cancer cell (HeLa) is the preservative cell line of American ATCC in the Lab. The experiment was completed in the Cell Lab of China-Japan Research Institute of Medical and Pharmaceutical Sciences in Shenyang Pharmaceutical University from Feburary 2003 to July 2003.METHODS: HeLa cell death ratio was measured by methyl thiazol tetrazolium(MTT) assay. Cellular morphologic changes were observed by phase-contrast and fluorescence microscopy. Cell cycle distribution was analyzed by flow cytometry.MAIN OUTCOME MEASURES: The changes of cell death ratio and cell cycle distribution.RESULTS: Silymarin-induced HeLa cell death was relevant to the content of serum in medium. At the does of 80 μmol /L silymarin, the cell death ratio in serum-free group reached 85.27%. The cell death ratio in 5% and 10%serum groups was 35.53% and 7.71% respectively. Morphological changes of HeLa cells demonstrated that silymarin induced HeLa cell death through apoptotic pathway at lower concentration in serum-free condition. Flow cytometry analysis showed that silymarin induced 70. 04% cells death, most of which were in G0/G1 phase.CONCLUSION: Silymarin mainly induce G0/G1 phase HeLa cell death through apoptotic pathway in serum-free medium.

OBJECTIVETo study the mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells.

METHODSMTT 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.

RESULTSCapsaicin 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.

CONCLUSIONCapsaicin induces A375-S2 cell apoptosis and down-regulation of ICAD contributes to this process.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; biosynthesis ; Capsaicin ; pharmacology ; Caspases ; metabolism ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Melanoma ; pathology ; Signal Transduction ; Skin Neoplasms ; pathology

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.
Apoptosis/*drug effects ; Blotting, Western ; Caspase 1/metabolism ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Comparative Study ; DNA Fragmentation/drug effects ; Deoxyribonucleases/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation/drug effects ; Humans ; Interleukin-1/biosynthesis/*pharmacology/physiology ; Interleukin-6/pharmacology ; Lymphotoxin/pharmacology ; Melanoma/metabolism/pathology/physiopathology ; Mitochondria/*physiology ; Poly(ADP-ribose) Polymerases/metabolism ; Proto-Oncogene Proteins c-bcl-2/biosynthesis ; Time Factors

BACKGROUNDThe 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.

METHODSThe 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.

RESULTSShikonin 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.

CONCLUSIONShikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.

Apoptosis ; drug effects ; Caspases ; physiology ; Cell Proliferation ; drug effects ; DNA Damage ; Flavonoids ; pharmacology ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Naphthoquinones ; pharmacology ; Phosphorylation ; Tumor Suppressor Protein p53 ; analysis ; Up-Regulation