Exploring host epigenetic enzymes as targeted therapies for visceral leishmaniasis: in silico design and in vitro efficacy of KDM6B and ASH1L inhibitors.

In order to combat various infectious diseases, the utilization of host-directed therapies as an alternative to chemotherapy has gained a lot of attention in the recent past, since it bypasses the existing limitations of conventional therapies. The use of host epigenetic enzymes like histone lysine methyltransferases and lysine demethylases as potential drug targets has successfully been employed for controlling various inflammatory diseases like rheumatoid arthritis and acute leukemia. In our earlier study, we have already shown that the functional knockdown of KDM6B and ASH1L in the experimental model of visceral leishmaniasis has resulted in a significant reduction of organ parasite burden. Herein, we performed a high throughput virtual screening against KDM6B and ASH1L using > 53,000 compounds that were obtained from the Maybridge library and PubChem Database, followed by molecular docking to evaluate their docking score/Glide Gscore. Based on their docking scores, the selected inhibitors were later assessed for their in vitro anti-leishmanial efficacy. Out of all inhibitors designed against KDM6B and ASH1L, HTS09796, GSK-J4 and AS-99 particularly showed promising in vitro activity with IC50 < 5 µM against both extracellular promastigote and intracellular amastigote forms of L. donovani. In vitro drug interaction studies of these inhibitors further demonstrated their synergistic interaction with amphotericin-B and miltefosine. However, GSK-J4 makes an exception by displaying an in different mode of interaction with miltefosine. Collectively, our in silico and in vitro studies acted as a platform to identify the applicability of these inhibitors targeted against KDM6B and ASH1L for anti-leishmanial therapy.

Understanding the Crosstalk Between Epigenetics and Immunometabolism to Combat Cancer.

The interaction between metabolic and epigenetic events shapes metabolic adaptations of cancer cells and also helps rewire the proliferation and activity of surrounding immune cells in the tumor microenvironment (TME). Recent studies indicate that the TME imposes metabolic constraints on immune cells, inducing them to attain a tolerogenic state, incompetent of mounting effective tumor eradication. Owing to extensive mutations acquired over repeated cell divisions, tumor cells selectively accumulate metabolites that regulate the activity of key epigenetic enzymes to mediate activation/suppression of genes associated with T-cell function and macrophage polarization. Further, multiple modulators connecting epigenetic and metabolic pathways help dictate the preferential induction of cytokines and expression of lineage-specifying genes associated with immunosuppressive T-cell differentiation.In this chapter, we attempt to discuss the mechanisms underpinning the metabolic and epigenetic interplay in immune cells of the TME and how modulating these events can boost the application of existing anticancer immunotherapy.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Leishmania donovani Subverts Host Immune Response by Epigenetic Reprogramming of Macrophage M(Lipopolysaccharides + IFN-γ)/M(IL-10) Polarization.

Reciprocal changes in histone lysine methylation/demethylation of M(LPS + IFN-γ)/M(IL-10) genes is one of the factors that direct macrophage polarization and contribute to host defense/susceptibility toward infection. Although, histone lysine methyltransferases and lysine demethylases orchestrate these events, their role remains elusive in visceral leishmaniasis, a disease associated with macrophage M(IL-10) polarization. In this study, we observed that L. donovani induced the expression of histone lysine methyltransferases Ash1l, Smyd2, and Ezh2 and histone lysine demethylases Kdm5b and Kdm6b in J774 macrophages and BALB/c mice. Chromatin immunoprecipitation analysis revealed that L. donovani facilitated H3K36 dimethylation at TNF-α promoter by Smyd2 and H3K27 trimethylation at inducible NO synthase promoter by Ezh2 to suppress their expression in macrophages. Furthermore, infection-induced Kdm5b and Kdm6b modulated H3K4 and H3K27 trimethylation at IL-12, TNF-α, and arginase-1 promoters, respectively, whereas H3K4 trimethylation by Ash1l at IL-10 promoter induced its expression. Analysis of transductional events revealed that HIF-1α upregulated Kdm5b and Kdm6b expression, whereas Ash1l and Ezh2 expression were induced by transcription factor MeCP2. Additionally, Smyd2 was induced by c-Myc in infected macrophages. Knockdown of Ash1l, Ezh2, Kdm5b, and Kdm6b by specific small interfering RNA and Vivo-Morpholino, as well as inhibition of Smyd2 by its specific inhibitor, AZ505, led to increased protective proinflammatory response and inhibited amastigote multiplication in infected J774 macrophages and BALB/c mice, respectively. Collectively, our findings demonstrate that L. donovani exploits specific histone lysine methyltransferases/demethylases to redirect epigenetic programming of M(LPS + IFN-γ)/M(IL-10) genes for its successful establishment within the host.

Differential Induction of SOCS Isoforms by Leishmania donovani Impairs Macrophage-T Cell Cross-Talk and Host Defense.

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow-derived macrophages (BMMфs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells, respectively, whereas the SOCS1 level remains consistently high in BMMфs and SOCS3 expression is pronounced and long lasting in T cells. Consequently, this inhibits STAT1-mediated IL-12 induction in macrophages & STAT4-mediated IFN-γ synthesis in T cells. Mechanistically, PI3K/Akt-mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMфs. Additionally, L. donovani activates IDO/kynurenine/AHR signaling in BMMфs to maintain prolonged SOCS1 expression. Later, PGE2, secreted from infected BMMфs induces cAMP-PKA pathway by binding to the EP2/EP4 receptor of CD4+ T cells, leading to SP1, CREB, and GATA1 activation and SOCS3 expression. Small interfering RNA-mediated silencing of SOCS1 and SOCS3 in macrophage and T cells, respectively, restored IL-12 and IFN-γ cytokine levels and BMMф-T cell interaction. Vivo morpholino-mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden significantly in L. donovani-infected BALB/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage-T cell cross-talk and preserve its own niche.

Leishmania donovani Exploits Tollip, a Multitasking Protein, To Impair TLR/IL-1R Signaling for Its Survival in the Host.

IL-1R/TLR signaling plays a significant role in sensing harmful foreign pathogens and mounting effective innate and adaptive immune responses. However, the precise mechanism by which Leishmania donovani, an obligate intramacrophagic pathogen, breaches IL-1R/TLR signaling and host-protective immunity remains obscure. In this study, we report the novel biphasic role of Toll-interacting protein (Tollip), a negative regulator of the IL-1R/TLR pathway, in the disease progression of experimental visceral leishmaniasis. We observed that during early hours of infection, L. donovani induced phosphorylation of IRAK-1, resulting in the release of Tollip from the IL-1R-associated kinase (IRAK)-1 complex in J774 macrophages, which then acted as an endocytic adaptor on cell surface IL-1R1 and promoted its lysosomal degradation. In the later stage, Tollip shuttled back to IRAK-1, thereby inhibiting IRAK-1 phosphorylation in association with IRAK-M to neutralize downstream TLR signaling in infected macrophages. Moreover, during late infection, L. donovani enhanced nuclear translocation and recruitment of transcription factors early growth response protein 2, NF erythroid 2-related factor 2, and Ahr on Tollip promoter for its induction. Small interfering RNA-mediated silencing of Tollip in infected macrophages significantly enhanced NF-κB activation and induced host-defensive IL-12 and TNF-α synthesis, thereby reducing amastigote multiplication. Likewise, abrogation of Tollip in L. donovani-infected BALB/c mice resulted in STAT-1-, IRF-1-, and NF-κB-mediated upregulation of host-protective cytokines and reduced organ parasite burden, thereby implicating its role in disease aggravation. Taken together, we conclude that L. donovani exploited the multitasking function of Tollip for its own establishment through downregulating IL-1R1/TLR signaling in macrophages.

Ammonium trichloro [1,2-ethanediolato-O,O']-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway.

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4ß7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Mahanine exerts in vitro and in vivo antileishmanial activity by modulation of redox homeostasis.

Earlier we have established a carbazole alkaloid (mahanine) isolated from an Indian edible medicinal plant as an anticancer agent with minimal effect on normal cells. Here we report for the first time that mahanine-treated drug resistant and sensitive virulent Leishmania donovani promastigotes underwent apoptosis through phosphatidylserine externalization, DNA fragmentation and cell cycle arrest. An early induction of reactive oxygen species (ROS) suggests that the mahanine-induced apoptosis was mediated by oxidative stress. Additionally, mahanine-treated Leishmania-infected macrophages exhibited anti-amastigote activity by nitric oxide (NO)/ROS generation along with suppression of uncoupling protein 2 and Th1-biased cytokines response through modulating STAT pathway. Moreover, we have demonstrated the interaction of a few antioxidant enzymes present in parasite with mahanine through molecular modeling. Reduced genetic and protein level expression of one such enzyme namely ascorbate peroxidase was also observed in mahanine-treated promastigotes. Furthermore, oral administration of mahanine in acute murine model exhibited almost complete reduction of parasite burden, upregulation of NO/iNOS/ROS/IL-12 and T cell proliferation. Taken together, we have established a new function of mahanine as a potent antileishmanial molecule, capable of inducing ROS and exploit antioxidant enzymes in parasite along with modulation of host's immune response which could be developed as an inexpensive and nontoxic therapeutics either alone or in combination.

15d-Prostaglandin J2 induced reactive oxygen species-mediated apoptosis during experimental visceral leishmaniasis.

UNLABELLED: 15-Deoxy-delta (12,14)-prostaglandin J2 (15d-PgJ2) is a potent bioactive lipid mediator, known to possess several roles in cell regulation and differentiation along with antimicrobial efficacy against different bacterial and viral infections. In the present study, we investigated the therapeutic efficacy and mechanism of action of 15d-PgJ2 in vitro in Leishmania donovani promastigotes and infected J774 macrophages, and in vivo in Balb/c mice/golden hamster model of experimental visceral leishmaniasis. 15d-PgJ2 effectively killed L. donovani promastigotes and amastigotes in vitro with IC50 of 104.6 and 80.09 nM, respectively. At 2 mg/kg (mice) and 4 mg/kg (hamster) doses, 15d-PgJ2 decreased >90 % spleen and liver parasite burden. It significantly reduced interleukin (IL)-10 and transforming growth factor (TGF)-ß synthesis in infected macrophages and splenocytes. 15d-PgJ2 induced reactive oxygen species (ROS)-dependent apoptosis of promastigotes by triggering phosphatidyl serine externalization, mitochondrial membrane damage and inducing caspase-like activity. In vitro drug interaction studies revealed an indifference to the synergistic association of 15d-PgJ2 with Miltefosine and Amphotericin-B (Amp-B). Moreover, when combined with sub-curative doses of Miltefosine and Amphotericin-B, 15d-PgJ2 resulted in >95 % parasite removal. Our results suggested that 15d-PgJ2 induces mitochondria-dependent apoptosis of L. donovani and is a good therapeutic candidate for adjunct therapy against experimental visceral leishmaniasis. KEY MESSAGE: 15d-PgJ2 effectively eliminated both promastigotes and amastigotes form of L. donovani. 15d-PgJ2 decreased parasite burden from infected mice and hamsters with reduced Th2 cytokines. 15d-PgJ2 induced ROS-mediated mitochondrial apoptosis of L. donovani promastigotes. 15d-PgJ2 is a good therapeutic candidate for adjunct therapy with Miltefosine and Amp-B.

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.

Protection against filarial infection by 45-49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction.

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators' release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24-48.64 kDa), F11 (33.44-38.44 kDa) and F12 (28.44-33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1ß, IL-10, TGF-ß release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1ß significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.

A novel 2,6-diformyl-4-methylphenol based chemosensor for Zn(II) ions by ratiometric displacement of Cd(II) ions and its application for cell imaging on human melanoma cancer cells.

A new chelating ligand [4-methyl-2,6-bis-(pyridin-2-yl-hydrazonomethyl)-phenol] (1) was prepared by the condensation of 2-hydrazinylpyridine with 2,6-diformyl-p-cresol. Compound 1 exhibits weak fluorescence due to intramolecular photoinduced electron transfer (PET). The sensor (1) demonstrates Zn(2+)-specific emission enhancement due to the "PET off" process through a 1:1 binding mode with the metal ion. The fluorescence quantum yield of chemosensor 1 is only 0.020, and it increases more than 14-fold (0.280) in the presence of one equivalent of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to remain either unchanged or weakened except for Cd(2+). The new sensor showed 'naked-eye' detection of Zn(2+) ions: a color change of the solution from colorless to yellow. Ratiometric displacement of Cd(2+) ions from the complex by Zn(2+) ions supports the formation of a more stable sensor­Zn(2+) complex over the sensor­Cd(2+) complex. The experimental findings have been correlated with theoretical results using the B3LYP functional and 6-31G (d, p), LANL2DZ basis set for Cd(2+) (2) and Zn(2+) (3) complexes, respectively, by the Density Functional Theory (DFT) method. Moreover, the ability of probe 1 to sense Zn(2+) within human melanoma cancer cells has been explored, and the Zn(2+)-probing process in living cells was found to be reversible with zinc chelator solution of N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or EDTA.

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 ß-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-1ß, 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.

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-ß 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.

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.

18ß-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 18ß-glycyrrhetinic acid (GRA) a pentacyclic triterpenoid of the ß-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.

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.

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 TGFß-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-ß 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.

MAPK-directed phosphatases preferentially regulate pro- and anti-inflammatory cytokines in experimental visceral leishmaniasis: involvement of distinct protein kinase C isoforms.

The role of phosphatases in the impairment of MAPK signaling, which is directly responsible for Leishmania-induced macrophage dysfunction, is still poorly understood. Gene expression profiling revealed that Leishmania donovani infection markedly up-regulated the expression of three phosphatases: MKP1, MKP3, and PP2A. Inhibition of these phosphatases prior to infection points toward preferential induction of the Th2 response through deactivation of p38 by MKP1. On the other hand, MKP3 and PP2A might play significant roles in the inhibition of iNOS expression through deactivation of ERK1/2. Among various PKC isoforms, PKCzeta was associated with induction of MKP3 and PP2A in infected macrophages, whereas PKCepsilon was correlated with MKP1 induction. Inhibition of phosphatases in L. donovani-infected BALB/c mice shifted the cytokine balance in favor of the host by inducing TNF-alpha and iNOS expression. This was validated by cystatin, an immunomodulator and curing agent for experimental visceral leishmaniasis, which showed that inhibition of MKPs and PP2A activity may be necessary for a favorable T cell response and suppression of organ parasite burden. This study, for the first time, suggests the possibility of the involvement of MAPK-directed phosphatases in the establishment of L. donovani infection.