Susanta Kar

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.

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.

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.

Successful Therapy of Visceral Leishmaniasis With Curdlan Involves T-Helper 17 Cytokines

The aim of this study was to evaluate and characterize the therapeutic potential of curdlan, a naturally occurring β-glucan immunomodulator, against visceral leishmaniasis, a fatal parasitic disease. Curdlan eliminated the liver and spleen parasite burden in a 45-day BALB/c mouse model of visceral leishmaniasis at a dosage of 10 mg/kg/day as determined by Giemsa-stained organ impression smears. Curdlan was associated with production of the disease-resolving T-helper (Th) 1 and Th17-inducing cytokines interleukin (IL)-6, IL-1β, and IL-23, as well as with production of Th17 cytokines IL-17 and IL-22, as determined by enzyme-linked immunosorbent assay (ELISA) and real time polymerase chain reaction (RT-PCR). Reversal of curdlan-mediated protection by anti-IL-17 and anti-IL-23 monoclonal antibodies showed the importance of Th17 cytokines. Significantly decreased production of both IL-17 and IL-22 by mice that received anti-IL-23 antibody suggested the essential role of IL-23 in Th17 differentiation. Although administration of recombinant IL-17 or IL-23 caused significant suppression of the organ parasite burden, with marked generation of interferon γ and nitric oxide (NO), effects were much faster for IL-17. These results documented that although both IL-23 and IL-17 play major roles in the antileishmanial effect of curdlan, the effect of IL-23 may occur indirectly, through the induction of IL-17 production.

Hesperetin Induces Apoptosis in Breast Carcinoma by Triggering Accumulation of ROS and Activation of ASK1/JNK Pathway

Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF‐7 cells in a concentration‐ and time‐dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF‐10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl‐serine externalization, DNA fragmentation, caspase‐7 activation, and PARP cleavage. Apoptosis was associated with caspase‐9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl‐2 ratio. Pre‐treatment with caspase‐9 specific inhibitor (Z‐LEHD‐fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow‐cytometric analysis revealed triggering of ROS in a time‐dependent manner. Pre‐treatment with ROS scavenger N‐acetylcysteine (NAC) and glutathione markedly abrogated hesperetin‐mediated apoptosis whereas carbonyl cyanide m‐chlorophenylhydrazone (CCCP) pretreatment along with DHR123‐based flow‐cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre‐treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin‐mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin‐mediated apoptosis suggesting that hesperetin‐mediated apoptosis of MCF‐7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA‐MB‐231 cells via intrinsic pathway via activation of caspase ‐9 and ‐3 and increase in Bax:Bcl‐2 ratio. J. Cell. Physiol. 230: 1729–1739, 2015.

Fucoidan cures infection with both antimony-susceptible and -resistant strains of Leishmania donovani through Th1 response and macrophage-derived oxidants

OBJECTIVES The aim of this study was to evaluate and characterize the antileishmanial efficacy of fucoidan, a polyanionic sulphated polysaccharide from brown algae, in experimental infections of BALB/c mice with antimony-susceptible (AG83) and -resistant (GE18ER) Leishmania donovani. METHODS The effect of fucoidan was assessed against intracellular parasites in cultured macrophages and in suppressing splenic and liver parasite burdens in a BALB/c mouse model of visceral leishmaniasis by microscopic evaluation of surviving intracellular amastigotes stained with Giemsa. To evaluate the type of immunological responses, real-time PCR and ELISA were performed for various Th1 and Th2 cytokines in both in vitro and in vivo infected conditions. To determine the effector mechanism, reactive oxygen species (ROS) and NO were measured in fucoidan-treated animals by H(2)DCFDA-based fluorometric analysis and Griess reaction, respectively. RESULTS In addition to having appreciable inhibitory effect on amastigote multiplication within macrophages (>93% inhibition at 50 μg/mL), complete elimination of liver and spleen parasite burden was achieved by fucoidan at a dose of 200 mg/kg/day given orally, 3 times weekly, in a 6-week mouse model of both antimony-susceptible and -resistant strains. This curative effect is associated with switching of T cell differentiation from Th2 to Th1 mode. Further, splenocytes of fucoidan-treated infected (AG83 and GE18FR) mice generated significantly enhanced levels of superoxide and NO. Not only was this treatment curative when administered orally 15 days post-infection, but it also imparted resistance to reinfection. CONCLUSIONS These results suggest the effectiveness of fucoidan as potent immunomodulator for controlling both antimony-susceptible and -resistant visceral leishmaniasis.

Cystatin cures visceral leishmaniasis by NF-κB-mediated proinflammatory response through co-ordination of TLR/MyD88 signaling with p105-Tpl2-ERK pathway.

Cystatin could completely cure experimental visceral leishmaniasis by switching the differentiation of Th2 cells to Th1 type, as well as upregulating NO, and activation of NF‐κB played a major role in these processes. Analysis of upstream signaling events revealed that TLR 2/4‐mediated MyD88‐dependent participation of IL‐1R‐activated kinase (IRAK)1, TNF receptor‐associated factor (TRAF)6 and TGFβ‐activated kinase (TAK)1 is essential to induce cystatin‐mediated IκB kinase (IKK)/NF‐κB activation in macrophages. Cystatin plus IFN‐γ activated the IKK complex to induce phosphorylation‐mediated degradation of p105, the physiological partner and inhibitor of the MEK kinase, tumor progression locus 2 (Tpl‐2). Consequently, Tpl‐2 was liberated from p105, thereby stimulating activation of the MEK/ERK MAPK cascade. Cystatin plus IFN‐γ‐induced IKK‐β post‐transcriptionally modified p65/RelA subunit of NF‐κB by dual phosphorylation in infected phagocytic cells. IKK induced the phosphorylation of p65 directly on Ser‐536 residue whereas phosphorylation on Ser 276 residue was by sequential activation of Tpl‐2/MEK/ERK/MSK1. Collectively, the present study indicates that cystatin plus IFN‐γ‐induced MyD88 signaling may bifurcate at the level of IKK, leading to a divergent pathway regulating NF‐κB activation by IκBα phosphorylation and by p65 transactivation through Tpl‐2/MEK/ERK/MSK1.

Combination of liposomal CpG oligodeoxynucleotide 2006 and miltefosine induces strong cell-mediated immunity during experimental visceral leishmaniasis.

Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.

Expression of IL-10-triggered STAT3-dependent IL-4Rα is required for induction of arginase 1 in visceral leishmaniasis

Although enhanced macrophage‐specific arginase activity is directly related to increased parasite burden in cutaneous leishmaniasis (CL), the regulation and precise role of arginase in the disease outcome of visceral leishmaniasis (VL) has yet to be explored. As in CL, BALB/c mice infected with Leishmania donovani showed increased levels of arginase in acute infection. Arginase 1 is the major isoform associated with infection and while the IL‐4‐induced arginase pathway is operative in CL, IL‐10 plays a crucial role in modulating arginase activity in VL, although a synergism with IL‐4 is required. IL‐10, in combination with IL‐4, regulated both in vivo and ex vivo arginase 1 induction in a STAT6 and C/EBPβ‐dependent fashion. Further investigation toward the cause of such synergism suggests that induction of a STAT3‐dependent IL‐10‐mediated cascade in VL triggers the expression and surface localization of the IL‐4 receptor alpha (IL‐4Rα) which, in turn, enhances IL‐4 responsiveness toward STAT6 and C/EBPβ‐dependent signaling for arginase 1. This could also offer a mechanistic explanation for the fact that, in spite of the low level of IL‐4 in VL, enhanced IL‐4‐Rα expression by IL‐10 might markedly amplify IL‐4‐mediated arginase 1 signaling and provide a possible mechanism for synergistic induction of arginase 1.