Pijush K. Das

Curcumin, the major component of food flavour turmeric, reduces mucosal injury in trinitrobenzene sulphonic acid-induced colitis.

Inflammmatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leucocyte infiltration and upregulation of proinflammatory cytokines. In this study, we have investigated the protective effects of curcumin, an anti‐inflammatory and antioxidant food derivative, on 2,4,6‐ trinitrobenzene sulphonic acid‐induced colitis in mice, a model for IBD. Intestinal lesions (judged by macroscopic and histological score) were associated with neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa), increased serine protease activity (may be involved in the degradation of colonic tissue) and high levels of malondialdehyde (an indicator of lipid peroxidation). Dose–response studies revealed that pretreatment of mice with curcumin (50 mg kg−1 daily i.g. for 10 days) significantly ameliorated the appearance of diarrhoea and the disruption of colonic architecture. Higher doses (100 and 300 mg kg−1) had comparable effects. In curcumin‐pretreated mice, there was a significant reduction in the degree of both neutrophil infiltration (measured as decrease in myeloperoxidase activity) and lipid peroxidation (measured as decrease in malondialdehyde activity) in the inflamed colon as well as decreased serine protease activity. Curcumin also reduced the levels of nitric oxide (NO) and O2− associated with the favourable expression of Th1 and Th2 cytokines and inducible NO synthase. Consistent with these observations, nuclear factor‐κB activation in colonic mucosa was suppressed in the curcumin‐treated mice. These findings suggest that curcumin or diferuloylmethane, a major component of the food flavour turmeric, exerts beneficial effects in experimental colitis and may, therefore, be useful in the treatment of IBD.

Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4+ T cell differentiation. A 6-fold increase in NO production was observed with 0.5 μM chicken cystatin, a natural cysteine protease inhibitor, in IFN-γ-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-γ. In contrast to the case with promastigotes, cystatin and IFN-γ inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-γ or anti-TNF-α Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-γ treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4+ differentiation from Th2 to Th1, indicative of long-term resistance.

Carnosic acid modulates Akt/IKK/NF-κB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells

This study investigates the efficacy of carnosic acid (CA), a polyphenolic diterpene, isolated from the plant rosemary (Rosemarinus officinalis), on androgen-independent human prostate cancer PC-3 cells. CA induced anti-proliferative effects in PC-3 cells in a concentration- and time-dependent manner, which was due to apoptotic induction as evident from flow-cytometry, DNA laddering and TUNEL assay. Apoptosis was associated with the activation of caspase-8, -9, -3 and -7, increase in Bax:Bcl-2 ratio, release of cytochrome-c and decrease in expression of inhibitor of apoptosis (IAP) family of proteins. Apoptosis was attenuated upon pretreatment with specific inhibitors of caspase-8 (Z-IETD-fmk) and caspase-9 (Z-LEHD-fmk) suggesting the involvement of both intrinsic and extrinsic apoptotic cascades. Further, apoptosis resulted from the inhibition of IKK/NF-κB pathway as evident from decreased DNA binding activity, nuclear translocation of p50 and p65 and IκBα phosphorylation. The down-regulation of IKK/NF-κB was associated with inhibition of Akt phosphorylation and its kinase activity with a concomitant increase in the serine/threonine protein phosphatase 2A (PP2A) activity. Pharmacologic inhibition of PP2A by okadaic acid and calyculin A, significantly reversed CA-mediated apoptotic events in PC-3 cells indicating that CA induced apoptosis by activation of PP2A through modulation of Akt/IKK/NF-κB pathway. In addition, CA induced apoptosis in another androgen refractory prostate cancer DU145 cells via intrinsic pathway as evidenced from the activation of caspase 3, cleavage of PARP, increase in Bax:Bcl-2 ratio and cytochrome-c release. Carnosic acid, therefore, may have the potential for use in the prevention and/or treatment of prostate cancer.

Targeting of liposomes towards different cell types of rat liver through the involvement of liposomal surface glycosides

Studies on the uptake of liposomes by isolated cell types of rat liver after in vivo administration reveal that hepatocytes are three times more efficient than nonparenchymal cells in taking up liposomes having β-galactoside on their surface whereas α-mannoside liposomes are taken up preferentially by nonparenchymal cells. Nonsugar liposomes are taken up by both cells. Glycoside-containing liposomes are also cleared from the circulation at a faster rate than nonsugar liposomes. Asialofetuin and mannan inhibit both the clearance and the uptake by isolated cells of β-Gal and α-Man liposomes, respectively. These findings show that surface β-galactoside and α-mannoside can mediate selective targeting of liposomes toward parenchymal and nonparenchymal cells, respectively, of rat liver.

Protection from experimental colitis by theaflavin-3,3′-digallate correlates with inhibition of IKK and NF-κB activation

Inflammatory bowel disease (IBD) is associated with activation of nuclear factor κ B (NF‐κB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin‐3,3′‐digallate (TFDG), the most potent anti‐oxidant polyphenol of black tea, down‐regulates NF‐κB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines.

Uncoupling Protein 2 Negatively Regulates Mitochondrial Reactive Oxygen Species Generation and Induces Phosphatase-Mediated Anti-Inflammatory Response in Experimental Visceral Leishmaniasis

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.

Synergistic Effect of Interferon-γ and Mannosylated Liposome-Incorporated Doxorubicin in the Therapy of Experimental Visceral Leishmaniasis

Active targeting of doxorubicin to macrophages was studied by incorporating it in mannose-coated liposomes by use of visceral leishmaniasis in BALB/c mice as the model macrophage disease. Mannosylated liposomal doxorubicin was more effective than liposomal doxorubicin or free doxorubicin. Because leishmaniasis is accompanied by immunosuppression, immunostimulation by interferon (IFN)-gamma was evaluated to act synergistically with mannosylated liposomal doxorubicin therapy. Combination chemotherapy with a suboptimal dose of IFN-gamma resulted in possibly complete elimination of spleen parasite burden. Analysis of mRNA levels of infected spleen cells suggested that targeted drug treatment together with IFN-gamma, in addition to greatly reducing parasite numbers, resulted in reduced levels of interleukin (IL)-4 but increased levels of IL-12 and inducible nitric oxide synthase. Such combination chemotherapy may provide a promising alternative for the cure of leishmaniasis, with a plausible conversion of antiparasitic T cell response from a Th2 to Th1 pattern indicative of long-term resistance.

18β-glycyrrhetinic acid induces apoptosis through modulation of Akt/FOXO3a/Bim pathway in human breast cancer MCF-7 cells.

Triterpenes found in plants display a multitude of biological activities, including anti‐tumor properties. The present study investigates the effect of 18β‐glycyrrhetinic acid (GRA) a pentacyclic triterpenoid of the β‐amyrin type, isolated from the root of Licorice (Glycyrrhizza glabra) on human breast cancer cells, MCF‐7. GRA showed potent inhibitory effects on MCF‐7 proliferation in a concentration‐ and time‐dependent manner without affecting immortalized normal mammary epithelial cell line (MCF‐10A). Growth inhibition of MCF‐7 cells by GRA occurred through apoptosis, as evident from phosphatidyl serine externalization and DNA fragmentation. Apoptosis was primarily mediated through mitochondrial death cascade as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase‐9. GRA induced an increase in Bax:Bcl‐2 ratio along with a significant increase in the protein level of the BH3 protein Bim. SiRNA‐mediated knock down of Bim markedly attenuated GRA‐mediated apoptosis. Profiling of transcriptional regulators of Bim revealed a role of Forkhead box O 3a transcription factor (FOXO3a) as judged by increased expression and nuclear translocation of FOXO3a. Silencing of FOXO3a resulted in marked attenuation in the expression of Bim as well as protection against GRA‐mediated apoptosis. Furthermore, GRA‐induced activation and nuclear localization of FOXO3a was associated with a reduced activity of Akt kinase. These results suggest that GRA induces apoptosis in human breast carcinoma MCF‐7 cells via caspase activation and modulation of Akt/FOXO3a pathway. J. Cell. Physiol. 227: 1923–1931, 2012.

Leishmania donovani Exploits Host Deubiquitinating Enzyme A20, a Negative Regulator of TLR Signaling, To Subvert Host Immune Response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK–NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK–TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-β synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.

18β-Glycyrrhetinic Acid Triggers Curative Th1 Response and Nitric Oxide Up-Regulation in Experimental Visceral Leishmaniasis Associated with the Activation of NF-κB

The efficacy of 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpene belonging to the β-amyrin series of plant origin, was evaluated in experimental visceral leishmaniasis. GRA is reported to have antitumor and immunoregulatory activities, which may be attributable in part to the induction of NO. Indeed, an 11-fold increase in NO production was observed with 20 μM GRA in mouse peritoneal macrophages infected with Leishmania donovani promastigotes. In addition to having appreciable inhibitory effects on amastigote multiplication within macrophages (IC50, 4.6 μg/ml), complete elimination of liver and spleen parasite burden was achieved by GRA at a dose of 50 mg/kg/day, given three times, 5 days apart, in a 45-day mouse model of visceral leishmaniasis. GRA treatment resulted in reduced levels of IL-10 and IL-4, but increased levels of IL-12, IFN-γ, TNF-α, and inducible NO synthase, reflecting a switch of CD4+ differentiation from Th2 to Th1. This treatment is likely to activate immunity, thereby imparting resistance to reinfection. GRA induced NF-κB migration into the nucleus of parasite-infected cells and caused a diminishing presence of IκB in the cytoplasm. The lower level of cytoplasmic IκBα in GRA-treated cells resulted from increased phosphorylation of IκBα and higher activity of IκB kinase (IKK). Additional experiments demonstrated that GRA does not directly affect IKK activity. These results suggest that GRA exerts its effects at some level upstream of IKK in the signaling pathway and induces the production of proinflammatory mediators through a mechanism that, at least in part, involves induction of NF-κB activation.