cyclin-d1 and andrographolide

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

Topics: 1-Naphthylisothiocyanate; Animals; Bile Ducts; Cell Proliferation; Cholestasis, Intrahepatic; Cyclin D1; Diterpenes; Gene Expression Regulation; Hepatic Stellate Cells; Liver; Male; NF-kappa B; Rats

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.. AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.. SRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Diterpenes; DNA Fragmentation; Down-Regulation; Guinea Pigs; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Our aim was to explore anti-cell proliferative and anti-angiogenic potential of andrographolide by analyzing the expression pattern of cell proliferative (PCNA, Cyclin D1) and angiogenic (VEGF) markers during 7, 12-dimethylbenz(a)anthracene (DMBA) induced hamster buccal pouch carcinogenesis. DMBA painting three times a week for 14 weeks in the buccal pouch of golden Syrian hamsters resulted in oral tumors which were histopathologically diagnosed as well differentiated squamous cell carcinoma. Immunohistochemical (PCNA, VEGF) and RT-PCR (Cyclin D1) studies revealed over expression of PCNA, VEGF and Cyclin D1 in the buccal mucosa of hamsters treated with DMBA alone. Oral administration of andrographolide at a dose of 50 mg/kg bw to hamsters treated with DMBA not only suppressed the histological abnormalities but also down regulated the expression of PCNA, VEGF and Cyclin D1. The results of the present study suggest that andrographolide suppressed tumor formation in the buccal mucosa of hamsters treated with DMBA through its anti-cell proliferative and anti-angiogenic potential.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Angiogenesis Inhibitors; Animals; Anticarcinogenic Agents; Apoptosis; Carcinogens; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Cricetinae; Cyclin D1; Diterpenes; Immunoenzyme Techniques; Male; Mesocricetus; Mouth Mucosa; Mouth Neoplasms; Neoplasms, Experimental; Neovascularization, Pathologic; Proliferating Cell Nuclear Antigen; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

The NF-κB family of transcription factors is essential for promoting cell proliferation and preventing cell apoptosis. We have previously shown that Andrographolide (Andro) isolated from an herbal plant, Andrographis paniculata, covalently modifies reduced cysteine(62) in the oligonucleotide binding pocket of p50 for inhibition of NF-κB activation. Here we report that Andro, but not its inactive structural analog 4H-Andro, potently suppressed squamous cell carcinogenesis induced by 7,12-dimethyl-1,2-benzanthracene (DMBA) in the hamster model of cheek buccal pouch. Compared with 4H-Andro, Andro reduced phosphorylation of p65 (Ser536) and IκBα (Ser32/36) for inhibiting aberrant NF-κB activation, suppressed c-Myc and cyclin D1 expression and attenuated neoplastic cell proliferation, promoted cancerous cell apoptosis, and mitigated tumor-induced angiogenesis. Consistently, Andro retarded growth, decreased proliferation, and promoted apoptosis of Tb cells, a human tongue squamous cell carcinoma cell line, in time- and dose-dependent manners, with concomitant reduction of the expression of NF-κB targeting molecules in vitro. Our results thus demonstrate that NF-κB activation plays important roles in the pathogenesis of chemically induced squamous cell carcinoma. By inhibition of aberrant NF-κB activation, Andro treats chemically induced oral squamous cell carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Cheek; Cricetinae; Cyclin D1; Diterpenes; Dose-Response Relationship, Drug; eIF-2 Kinase; Humans; I-kappa B Kinase; Mouth Neoplasms; Neovascularization, Pathologic; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-myc