ABSTRACT
Baicalein is a flavonoid (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran4-one) and an active principle in Scutellaria baicalensis. The present study was performed to investigate the mechanisms underlying the anti-fibrotic effects of baicalein with a focus on Rho kinase (ROCK) inhibition. The effect of baicalein on ROCK activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The underlying mechanisms of baicalein were examined using angiotensin II-stimulated H9c2 cells. Rho kinase (ROCK1 and ROCK2) studies using IMAP-TR-FRET showed that baicalein possesses potent ROCK inhibitory activity with IC50 values of 6.55 and 2.82 µM, respectively. Pretreatment with baicalein (for 2 h) concentration-dependently decreased the angiotensin II-induced phosphorylation of myosin phosphatase (MYPT) and myosin light chain (MLC). Furthermore, baicalein also concentration-dependently suppressed actin stress fiber formation in angiotensin II-stimulated H9c2 cells. These results suggest that baicalein potently inhibits ROCK and that by so doing it modulates actin stress fiber formation. These anti-fibrotic effects of baicalein explain, at least in part, its pharmacology and mode of action.
Subject(s)
Actins/metabolism , Angiotensin II/pharmacology , Flavanones/pharmacology , Plant Extracts/pharmacology , Scutellaria baicalensis/chemistry , Stress Fibers/drug effects , rho-Associated Kinases/antagonists & inhibitors , Animals , Cell Line , Dose-Response Relationship, Drug , Fibrosis/metabolism , Fibrosis/prevention & control , Flavanones/therapeutic use , Inhibitory Concentration 50 , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Phosphorylation , Phytotherapy , Plant Extracts/therapeutic use , Rats , Stress Fibers/metabolism , Stress Fibers/pathology , Vasoconstrictor Agents/pharmacologyABSTRACT
PURPOSE: To test whether tanshinones inhibit prostate cancer (PCa) growth at least in part through inhibiting androgen receptor (AR) signaling. METHODS: We evaluated cell growth, survival and AR signaling parameters of PCa cells after exposure to tanshinones in in vitro models. We also tested the in vivo inhibitory efficacy of tanshinone IIA (TIIA) against LNCaP xenograft model in athymic nude mice. RESULTS: For androgen-dependent LNCaP cells, a colony growth assay showed strong inhibitory potency following the order of TIIA≈cryptotanshinone>tanshinone I, being 10-30 folds higher than Casodex (racemic). TIIA inhibited growth of LNCaP cells more than several androgen-independent PCa cell lines. All 3 tested tanshinones were devoid of AR agonist activity under castrate condition. Mechanistically, tanshinones inhibited AR nuclear translocation within 2 h, decreased protein and mRNA abundance of AR and its target prostate-specific antigen within 12 h, and stimulated proteosomal degradation of AR. Oral administration of TIIA (25 mg/kg, once daily) retarded LNCaP xenograft growth and down-regulated tumor AR abundance in athymic nude mice. CONCLUSION: AR targeting action of tanshinones was distinct from Casodex and contributed to prostate cancer growth suppression in vitro and in vivo.
Subject(s)
Abietanes/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cell Proliferation/drug effects , Drugs, Chinese Herbal/pharmacology , Phenanthrenes/pharmacology , Prostatic Neoplasms/drug therapy , Receptors, Androgen/drug effects , Salvia miltiorrhiza , Signal Transduction/drug effects , Abietanes/isolation & purification , Active Transport, Cell Nucleus , Androgen Antagonists/pharmacology , Anilides/pharmacology , Animals , Antineoplastic Agents, Hormonal/pharmacology , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/isolation & purification , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Nitriles/pharmacology , Phenanthrenes/isolation & purification , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Salvia miltiorrhiza/chemistry , Time Factors , Tosyl Compounds/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Our group previously reported that tanshinone IIA induced apoptosis via a mitochondria dependent pathway in LNCaP prostate cancer cells. In the present study, the roles of androgen receptor (AR) and p53 signaling pathways were investigated in tanshinone IIA-induced G1 arrest in LNCaP cells. Tanshinone IIA significantly inhibited the growth and proliferation of LNCaP cells by colony formation and BrdU incorporation assays, respectively. Tanshinone IIA induced cell cycle arrest at G1 phase and down-regulated cyclin D1, CDK2 and CDK4. Furthermore, tanshinone IIA activated the phosphorylation of p53 at Ser 15 residue and its downstream p21 and p27. Additionally, tanshinone IIA suppressed the expression of AR and prostate specific antigen (PSA). Conversely, silencing p53 using its specific siRNA reversed cyclin D1 expression inhibited by tanshinone IIA. However, knockdown of AR had no effect on the p53/p21/p27 signaling pathway activated by tanshinone IIA in LNCaP cells. In AR siRNA-transfected cells, tanshinone IIA did not cause cell cycle arrest and reduce cyclin D1, implying that AR is essential to induce G1 arrest by tanshinone IIA in LNCaP cells. Taken together, the findings suggest that tanshinone IIA induces G1 arrest via activation of p53 signaling and inhibition of AR in LNCaP cells.
Subject(s)
Abietanes/pharmacology , Androgen Receptor Antagonists/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , G1 Phase Cell Cycle Checkpoints/drug effects , Receptors, Androgen/drug effects , Tumor Suppressor Protein p53/drug effects , Abietanes/chemistry , Abietanes/isolation & purification , Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cell Proliferation/drug effects , Down-Regulation/drug effects , Humans , Male , Plant Roots/chemistry , Prostate/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , RNA, Small Interfering , Receptors, Androgen/metabolism , Salvia miltiorrhiza/chemistry , Signal Transduction/drug effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables that exerts anti-oxidant, anti-inflammatory, anti-cancer and radio-sensitizing activities. Nonetheless, the mechanism responsible for SFN-induced cell death is not fully understood. In the present study, anti-cancer mechanism of SFN was elucidated in LNCaP prostate cancer cells. RESULTS: SFN exerted cytotoxicity and increased TUNEL positive cells in a concentration-dependent manner in LNCaP cells. Proteomics study revealed that levels of nine proteins including tubulin ß-2, phosphoglucomutase-3 (PGM3), melanoma-derived leucine zipper containing extra-nuclear factor, activin A type I receptor precursor, smoothelin-A, KIA0073, hypothetical protein LOC57691 and two unnamed proteins were changed over 8 folds in SFN treated LNCaP cells compared to untreated control. We have further confirmed that SFN reduced PGM3 expression with western blotting and showed that PGM3 siRNA enhanced cytotoxicity demonstrated by cell morphology and TUNEL assays in LNCaP cells. CONCLUSION: Taken together, these findings suggest that PGM3 plays a role in mediating SFN-induced cell death in LNCaP cells, and is a potential molecular therapeutic target for prostate cancer.
ABSTRACT
Tanshinone IIA (Tan IIA; 14,16-epoxy-20-nor-5(10),6,8,13,15-abietapentaene-11,12-dione), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been reported to posses anti-angiogenic, anti-oxidant, anti-inflammatory and apoptotic activities. However, the cancer growth inhibitory/cytocidal effects and molecular mechanisms in prostate cancer cells have not been well studied. In the present study, we demonstrate that Tan IIA significantly decreased the viable cell number of LNCaP (phosphate and tensin homolog (PTEN) mutant, high AKT, wild type p53) prostate cancer cells more sensitively than against the PC-3 (PTEN null, high AKT, p53 null) prostate cancer cells. Tan IIA significantly increased TdT-mediated dUTP nick-end labeling (TUNEL) positive index and sub-G1 DNA contents of treated cells, consistent with apoptosis. Tan IIA treatment led to cleavage activation of pro-caspases-9 and 3, but not pro-caspase-8, and cleavage of poly (ADP ribose) polymerase (PARP), a caspase-3 substrate. Additionally, Tan IIA treatment induced cytochrome c release from the mitochondria into the cytosol and reduced mitochondrial membrane potential and suppressed the expression of mitochondria protective Bcl-2 family protein Mcl-1(L). Tan IIA reduced the expression of phosphoinositide 3-kinase (PI3K) p85 subunit, and the phosphorylation of AKT and mammalian target of rapamycin (mTOR) in a concentration-dependent manner. Moreover, the combination of Tan IIA and LY294002, a specific PI3K inhibitor, enhanced PARP cleavage of LNCaP and PC-3, but not in MDA-MB-231 breast cancer cells which do not contain detectable active AKT. The findings suggest that Tan IIA-induced apoptosis involves mitochondria intrinsic caspase activation cascade and an inhibition of the PI3K/AKT survival pathway.
Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Abietanes/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Prostatic Neoplasms/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Salvia miltiorrhiza/chemistry , Abietanes/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Caspases/metabolism , Cell Line, Tumor , DNA/metabolism , Dose-Response Relationship, Drug , Enzymes/metabolism , Humans , Male , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/physiology , Phosphorylation , Phytotherapy , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Plant Roots , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Signal Transduction/drug effectsABSTRACT
No abstract available.
