Gorla V. Reddy

Alteration of mitochondrial membrane potential by Spirulina platensis C‐phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular‐carcinoma cell line HepG2

C‐PC (C‐phycocyanin) is a water‐soluble biliprotein from the filamentous cyanobacterium Spirulina platensis with potent antioxidant, anti‐inflammatory and anticancerous properties. In the present study, the effect of C‐PC was tested on the proliferation of doxorubicin‐sensitive (S‐HepG2) and ‐resistant (R‐HepG2) HCC (hepatocellular carcinoma) cell lines. These studies indicate a 50% decrease in the proliferation of S‐ and R‐HepG2 cells treated with 40 and 50 μM C‐PC for 24 h respectively. C‐PC also enhanced the sensitivity of R‐HepG2 cells to doxorubicin. R‐HepG2 cells treated with C‐PC showed typical apoptotic features such as membrane blebbing and DNA fragmentation. Flow‐cytometric analysis of R‐HepG2 cells treated with 10, 25 and 50 μM C‐PC for 24 h showed 18.8, 39.72 and 65.64% cells in sub‐G0/G1‐phase respectively. Cytochrome c release, decrease in membrane potential, caspase 3 activation and PARP [poly(ADP‐ribose) polymerase] cleavage were observed in C‐PC‐treated R‐HepG2 cells. These studies also showed down‐regulation of the anti‐apoptotic protein Bcl‐2 and up‐regulation of the pro‐apoptotic Bax (Bcl2‐associated X‐protein) protein in the R‐HepG2 cells treated with C‐PC. The present study thus demonstrates that C‐PC induces apoptosis in R‐HepG2 cells and its potential as an anti‐HCC agent.

Eicosanoids in inflammation and cancer: the role of COX-2.

Eicosanoids, a family of oxygenated metabolites of eicosapolyenoic fatty acids, such as arachidonic acid, formed via the lipoxygenase, cyclooxygenase (COX) and epoxygenase pathways, play an important role in the regulation of various pathophysiological processes, including inflammation and cancer. COX-2, the inducible isoform of COX, has emerged as the key enzyme regulating inflammation, and promises to play a considerable role in cancer. Although NSAIDs have been in use for centuries, the COX-2 selective inhibitors – coxibs – have emerged as potent anti-inflammatory drugs with fewer gastric side effects. As COX-2 plays a major role in neoplastic transformation and cancer growth, by downregulating apoptosis and promoting angiogenesis, invasion and metastasis, coxibs have a potential role in the prevention and treatment of cancer. Recent studies indicate their possible application in overcoming drug resistance by downregulating the expression of MDR-1. However, the cardiac side effects of some of the coxibs have limited their application in treating various inflammatory disorders and warrant the development of COX-2 inhibitors without side effects. This review will focus on the role of COX-2 in inflammation and cancer, with an emphasis on novel approaches to the development of COX-2 inhibitors without side effects.

Selective inhibition of cyclooxygenase‐2 (COX‐2) by 1α,25‐dihydroxy‐16‐ene‐23‐yne‐vitamin D3, a less calcemic vitamin D analog

Inducible cyclooxygenase‐2 (COX‐2) has been implicated to play a role in inflammation and carcinogenesis and selective COX‐2 inhibitors have been considered as anti‐inflammatory and cancer chemopreventive agents. 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1α,25(OH)2D3 and one of its less calcemic synthetic analogs, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 on the activity of both COX‐1 and COX‐2 in an in vitro enzyme assay. Preliminary data indicated that both 1α,25(OH)2D3 and 1α,25(OH)2‐16‐ene‐23‐yne‐D3 inhibited selectively the activity of COX‐2 with no effect on the activity of COX‐1. Out of the two compounds, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1α,25(OH)2‐16‐ene‐23‐yne‐D3 only. 1α,25(OH)2‐16‐ene‐23‐yne‐D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX‐2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin‐2 (IL‐2). Furthermore, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX‐2, iNOS, and IL‐2 in the tissues of the same air pouch. In both cases, 1α,25(OH)2‐16‐ene‐23‐yne‐D3 did not show any effect on the expression of COX‐1. In summary, our results indicate that 1α,25(OH)2‐16‐ene‐23‐yne‐D3, a less calcemic vitamin D analog, exhibits potent anti‐inflammatory effects and is a selective COX‐2 inhibitor. J. Cell. Biochem. 104: 1832–1842, 2008.

Anti-leukemic effects of gallic acid on human leukemia K562 cells: downregulation of COX-2, inhibition of BCR/ABL kinase and NF-κB inactivation.

Gallic acid (GA) induces apoptosis in various cancer cell lines. In this study, we investigated the apoptotic activity induced by GA on chronic myeloid leukemia (CML) cell line-K562 and the underlying mechanism. GA reduced the viability of K562 cells in a dose and time dependent manner. GA led to G0/G1 phase arrest in K562 cells by promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. Further studies indicated apoptosis with impaired mitochondrial function as a result of deranged Bcl-2/Bax ratio, leakage of cytochrome c and PARP cleavage along with DNA fragmentation and by up-regulating the expression of caspase-3. GA also activated the protein expressions of fatty acid synthase ligand and caspase-8. GA is more effective in imatinib resistant-K562 (IR-K562) cells (IC50 4 μM) than on K562 cells (IC50 33 μM). GA inhibited cyclooxygenase-2 (COX-2) in K562 as well as IR-K562 cells appears to be COX-2 involved in the suppression of growth. Interestingly, GA also inhibited BCR/ABL tyrosine kinase and NF-κB. In conclusion, GA induced apoptosis in K562 cells involves death receptor and mitochondrial-mediated pathways by inhibiting BCR/ABL kinase, NF-κB activity and COX-2.

C-Phycocyanin inhibits MDR1 through reactive oxygen species and cyclooxygenase-2 mediated pathways in human hepatocellular carcinoma cell line

The effects of C-Phycocyanin (C-PC), a biliprotein from Spirulina platensis on the regulation of multidrug resistance-1 (MDR1), a poly glycoprotein in human hepatocarcinoma cell line, HepG2 were reported. The results revealed that a significant down regulation of MDR1 expression in C-PC treated HepG2 cells was through reactive oxygen species and cyclooxygenase-2 (COX-2) mediated pathways. C-PC in a concentration dependent manner increased the accumulation of doxorubicin in HepG2 cells and enhanced sensitivity of the cells to doxorubicin by 5 folds. The induction of MDR1 expression by PGE₂ and its down regulation by C-PC and DPI (Diphenylene iodonium, NADPH oxidase inhibitor) or by COX-2 knockdown suggest that the enhanced sensitivity of HepG2 cells to doxorubicin by C-PC is mediated by the down regulation of MDR1 expression. Further studies reveal the involvement of NF-κB and AP-1 in the C-PC induced down regulation of MDR1. Also the inactivation of the signal transduction pathways involving Akt, ERK, JNK and p38 by C-PC was observed. The present study thus demonstrates the efficacy of C-PC in overcoming the MDR1 mediated drug resistance in HepG2 cells by the down regulation of reactive oxygen species and COX-2 pathways via the involvement of NF-κB and AP-1.

Inhibition of 12-LOX and COX-2 reduces the proliferation of human epidermoid carcinoma cells (A431) by modulating the ERK and PI3K-Akt signalling pathways.

Abstract:  Eicosanoids, the oxygenated metabolites of arachidonic acid (AA), mediate a variety of human diseases, such as cancer, inflammation and arthritis. To evaluate the role of eicosanoids in epidermoid carcinoma, the expression of AA metabolizing enzymes, such as lipoxygenases (LOXs) and cyclooxygenases (COXs), was analysed in a human epidermoid carcinoma cell line (A431). These studies revealed overexpression of 12‐R‐LOX and COX‐2 in A431 cells. Baicalein (a 12‐LOX inhibitor) and celecoxib (a COX‐2 inhibitor) significantly reduced thymidine incorporation, whereas 12‐(R)‐HETE and 12‐(S)‐HETE (12‐LOX metabolites) and PGE2 (COX‐2 metabolite) significantly enhanced thymidine incorporation, suggesting a role for these enzymes in the regulation of A431 cell proliferation. Further studies on the mechanism of cell death by baicalein and celecoxib revealed that the induction of apoptosis in A431 cells was associated with reduction in the Bcl‐2/Bax ratio, release of cytochrome c, activation of caspase‐3 and PARP cleavage. The apoptosis induced by baicalein and celecoxib was mediated by down regulation of ERK and PI3K‐Akt pathways. Further, 12‐(R)‐HETE, 12‐(S)‐HETE and PGE2 upregulated the p‐ERK and p‐Akt levels, suggesting the involvement of ERK and Akt pathways in the 12‐LOX‐ and COX‐2‐mediated regulation of growth in A431 cells. Our findings suggest that 12‐R‐LOX and COX‐2 play a critical role in the regulation of growth in epidermoid carcinoma and that their inhibitors may be of potential therapeutic importance.

15-Lipoxygenase metabolites of α-linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome

The ratio of ω-6 to ω-3 polyunsaturated fatty acids (PUFAs) appears to be critical in the regulation of various pathophysiological processes and to maintain cellular homeostasis. While a high proportion of dietary intake of ω-6 PUFAs is associated with various inflammatory disorders, higher intake of ω-3 PUFAs is known to offer protection. It is now well established that beneficial effects of ω-3 PUFAs are mediated in part by their oxygenated metabolites mainly via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. However, the down-stream signaling pathways that are involved in these anti-inflammatory effects of ω-3 PUFAs have not been elucidated. The present study evaluates the effects of 15-LOX metabolites of α-linolenic acid (ALA, ω-3 PUFA) on lipopolysaccharide (LPS) induced inflammation in RAW 264.7 cells and peritoneal macrophages. Further, the effect of these metabolites on the survival of BALB/c mice in LPS mediated septic shock and also polymicrobial sepsis in Cecal Ligation and Puncture (CLP) mouse model was studied. These studies reveal the anti-inflammatory effects of 13-(S)-hydroperoxyoctadecatrienoic acid [13-(S)-HPOTrE] and 13-(S)-hydroxyoctadecatrienoic acid [13-(S)-HOTrE] by inactivating NLRP3 inflammasome complex through the PPAR-γ pathway. Additionally, both metabolites also deactivated autophagy and induced apoptosis. In mediating all these effects 13-(S)-HPOTrE was more potent than 13-(S)-HOTrE.

Chebulagic acid synergizes the cytotoxicity of doxorubicin in human hepatocellular carcinoma through COX-2 dependant modulation of MDR-1.

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.

Effects of (15S)‐hydroperoxyeicosatetraenoic acid and (15S)‐hydroxyeicosatetraenoic acid on the acute‐ lymphoblastic‐leukaemia cell line Jurkat: activation of the Fas‐mediated death pathway

The antiproliferative effects of 15‐LOX (15‐lipoxygenase) metabolites of arachidonic acid {(15S)‐HPETE [(15S)‐hydroperoxyeicosatetraenoic acid] and (15S)‐HETE [(15S)‐hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T‐cell leukaemia cell line Jurkat. (15S)‐HPETE, the hydroperoxy metabolite of 15‐LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC50 value of 10 μM. The hydroxy metabolite of 15‐LOX, (15S)‐HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC50 value of 40 μM. The cells exposed to 10 μM (15S)‐HPETE for 3 h or to 40 μM (15S)‐HETE for 6 h showed increased expression of Fas ligand and FADD (Fas‐associated death domain), caspase 8 activation, Bid (BH3‐interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase‐induced ROS generation activates the Fas‐mediated death pathway.

Evaluation of Anti-Inflammatory Properties of Isoorientin Isolated from Tubers of Pueraria tuberosa

Inflammation is the major causative factor of different diseases such as cardiovascular disease, diabetes, obesity, osteoporosis, rheumatoid arthritis, inflammatory bowel disease, and cancer. Anti-inflammatory drugs are often the first step of treatment in many of these diseases. The present study is aimed at evaluating the anti-inflammatory properties of isoorientin, a selective cyclooxygenase-2 (COX-2) inhibitor isolated from the tubers of Pueraria tuberosa, in vitro on mouse macrophage cell line (RAW 264.7) and in vivo on mouse paw edema and air pouch models of inflammation. Isoorientin reduced inflammation in RAW 264.7 cell line in vitro and carrageenan induced inflammatory animal model systems in vivo. Cellular infiltration into pouch tissue was reduced in isoorientin treated mice compared to carrageenan treated mice. Isoorientin treated RAW 264.7 cells and animals showed reduced expression of inflammatory proteins like COX-2, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-lipoxygenase (5-LOX), and interleukin 1-β (IL-1-β) both in vitro and in vivo. The antioxidant enzyme levels of catalase and GST were markedly increased in isoorientin treated mice compared to carrageenan treated mice. These results suggest that isoorientin, a selective inhibitor of COX-2, not only exerts anti-inflammatory effects in LPS induced RAW cells and carrageenan induced inflammatory model systems but also exhibits potent antioxidant properties.