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Abstract Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle [...]

Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX-2) specific inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. The effects of chebulagic acid (CA), a benzopyran tannin from Terminalia chebula having COX-2/5-LOX dual inhibitory properties, on the sensitivity of doxorubicin (Dox) in human hepatocellular carcinoma cell line HepG2 were studied in the present investigation. CA increased the accumulation of Dox in a concentration dependant manner and also enhanced the cytotoxicity of Dox in HepG2 cells by 20 folds. Quantitation of interaction by calculating Combination Index (CI) showed a strong synergistic interaction between CA and Dox in terms of cell growth inhibition. Calculation of dose reduction index (DRI) for CA-Dox combinations also showed a significant decrease in the dosage of Dox in the presence of CA. The induction of MDR1 expression by PGE(2), a metabolite of COX-2, and its downregulation by COX-2 knockdown or CA implies that the enhanced sensitivity of HepG2 cells to doxorubicin by CA is mediated by the downregulation of MDR1 expression, via COX-2-dependent mechanism. Further studies reveal the inactivation of signal transduction pathways involving Akt, ERK, JNK and p38 and the transcription factor NF-κB in the CA induced down regulation of MDR1. The present study shows the efficacy of CA to overcome MDR-1 mediated drug resistance in HepG2 cells through COX-2 dependant modulation of MDR-1.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.