Karnati R. Roy

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.

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.

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

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.

A Lipid-Modified Estrogen Derivative that Treats Breast Cancer Independent of Estrogen Receptor Expression through Simultaneous Induction of Autophagy and Apoptosis

It is a challenge to develop a universal single drug that can treat breast cancer at single- or multiple-stage complications, yet remains nontoxic to normal cells. The challenge is even greater when breast cancer–specific, estrogen-based drugs are being developed that cannot act against multistaged breast cancer complications owing to the cells differential estrogen receptor (ER) expression status and their possession of drug-resistant and metastatic phenotypes. We report here the development of a first cationic lipid-conjugated estrogenic derivative (ESC8) that kills breast cancer cells independent of their ER expression status. This ESC8 molecule apparently is nontoxic to normal breast epithelial cells, as well as to other noncancer cells. ESC8 induces apoptosis through an intrinsic pathway in ER-negative MDA-MB-231 cells. In addition, ESC8 treatment induces autophagy in these cells by interfering with the mTOR activity. This is the first example of an estrogen structure–based molecule that coinduces apoptosis and autophagy in breast cancer cells. Further in vivo study confirms the role of this molecule in tumor regression. Together, our results open new perspective of breast cancer chemotherapy through a single agent, which could provide the therapeutic benefit across all stages of breast cancer. Mol Cancer Res; 9(3); 364–74. ©2011 AACR.

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.

C‐Phycocyanin ameliorates 2‐acetylaminofluorene induced oxidative stress and MDR1 expression in the liver of albino mice

Aim:  To study the effect of C‐Phycocyanin (C‐PC), a biliprotein isolated from Spirulina platensis, on 2‐acetylaminofluorene (2‐AAF) induced oxidative stress and MDR1 expression in the liver of albino mice.

Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4

S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α‐operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell‐free system of rabbit reticulocytes. The translation‐blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.—Yadaiah, M., Sudhamalla, B., Rao, P. N., Roy, K. R., Ramakrishna, D., Hussain Syed, G., Ramaiah, K. V. A., Bhuyan, A. K. Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4. FASEB J. 27, 803–810 (2013). www.fasebj.org

Na+-Stimulated Na+/H+ Exchange and an Unfavorable Ca2+ Homeostasis Initiate the Cycloxygenase-2 Inhibitors-Induced Apoptotic Signals in Colonic Epithelial Cells During the Early Stage of Colon Carcinogenesis

Evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cycloxygenase (COX) and production of the proinflammatory prostaglandin, PGE2, and thus prevent carcinogenesis in the colon. Indeed, one of the specific COX-2 inhibitors, celecoxib, had been accepted by the US FDA for the treatment of familial adenomatous polyposis. However, the molecular mechanism of such inhibition is not clear, although apoptosis appears to be the dominant antiproliferative end effect. The present study delineates the intracellular ionic milieu in the colonocytes that could generate strong apoptotic signals where DMH-induced carcinogenesis was studied in the initiation stage in rats and its regression with the COX inhibitors. While DMH treatment produced a significant elevation in the Na+/H+ exchanger activity and resultant proton efflux, this was reversed by the NSAIDs, particularly so with celecoxib and etoricoxib compared to aspirin. Similarly, the intracellular pH was changed, with more alkalosis noted in DMH, which was reversed by NSAIDs. Also, an intracellular Ca2+ build up was noted by Fura 2 AM, which was also supported by a reduced Ca2+ ATPase and an enhanced inward movement of Ca2+. Further, mitochondrial dysfunction-related cyt C release, increased DNA ladder formation, activation of caspase-3, and cleavage product of poly (ADP-ribose) polymerase (PARP) were not seen in DMH but well noted in NSAIDs. Our results indicate that NSAIDs can induce apoptosis through a change in the colonic Na+/H+ exchange, intracellular pH, and an unfavorable Ca2+ homeostasis.