Anti-CDK1-a monoclonal antibody, clone 4F2.2

Background Hedgehog signaling pathway (Hh) is abnormally stimulated in colon cancer. Evidence suggests the therapeutic effectiveness of andrographolide against several cancers. This study attempts to delineate the effect of andrographolide on Hh signaling pathway in colon cancer HCT-116 cells.Methods:Effects of andrographolide were studied on HCT-116 cells by evaluating cytotoxicity by MTT assay, morphology assessment, trypan blue exclusion, and colony formation assay; migratory potential by scratch assay; apoptosis by DAPI, Hoechst staining, FITC-Annexin V assay, and caspases activation; mitochondrial membrane potential (Delta psi m) by Mito Tracker and Rhodamine 123. Intracellular ROS by DCFH-DA staining. Cell cycle regulation by flow cytometry. Expression of BAX, BAD, BCL2, Cyclin B1, CDK1, Smo, and Gli1 by qRT-PCR. Interaction between andrographolide and Smo protein by in-silico molecular docking.Results:Andrographolide induced antiproliferative effect on HCT-116 cells in a dose-dependent and time-dependent manner. It also induced apoptosis and anti-migratory effect in HCT-116 cells. In combination with 5FU, andrographolide exhibited synergistic effect. It Induced G2/M phase arrest through downregulating CDK1 and Cyclin B1. Andrographolide also inhibited Hh signaling by downregulating Smo and Gli1 in HCT-116 cells. It showed high affinity toward Smo protein in-silico.Conclusion:Andrographolide repressed the colon cancer cell growth via inhibiting Hh signaling pathway.

Ethnopharmacological relevance: Crateva adansonii DC (Capparaceae) is a shrub used to treat tumors in Cameroon. In our previous reports, a Crateva adansonii dichloromethane-methanol (DCM/MeOH) extract was shown to prevent chemically induced tumors in Wistar rats. Aim of study: To determine the bioactive principle of Crateva adansonii extract and to elucidate its underlying mechanism. Materials and methods: An activity-guided fractionation was realized using MTT assay. To investigate if the bioactive compound daucosterol (CA2) accounted for the previously observed anticancer effects of the C. adansonii extract, it was tested on cell growth, cell proliferation, cell cycle, cell death mechanism and cell migration. In addition, cell cycle- and apoptosis-regulating proteins were assessed by Western blotting. Results: Daucosterol (CA2), a steroid saponin, was identified as major anticancer principle of the C. adansonii extract. Daucosterol significantly inhibited LNCaP, DU145 and PC3 prostate carcinoma cell growth and proliferation at the optimal concentration of 1 mu g/mL. It also significantly increased the number of late apoptotic (DU145) and apoptotic (PC3) cells. The number of cells in S phase increased in DU145, while the number of G0/G1 cells decreased. Cell cycle proteins (cdk1, pcdk1, cyclin A and B) were down-regulated in DU145 and PC3 cells, whereas only cdk2 was down-regulated in PC3 cells. Moreover, the anti-apoptotic Akt, pAKT and Bcl-2 proteins were down-regulated, while the pro-apoptotic protein Bax was up-regulated. CA2 induced anti-metastatic effects by decreasing chemotaxis and cell migration, while it increased cell adhesion to fibronectin and collagen matrix. Conclusion: These results suggest that daucosterol is the major active principle responsible at least in part for the anticancer effect of the extract of Crateva adansonii.