Piu Saha

Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-κB pathway

The crude ethanol extract of Piper betle leaf is reported to possess anti-inflammatory activity which has been suggested to be mediated by allylpyrocatechol (APC). In the present study, we have demonstrated the anti-inflammatory effects of APC (10 mg/kg, p.o.) in an animal model of inflammation. To investigate the mechanism(s) of this anti-inflammatory activity, we examined its effects on the lipopolysaccaride (LPS)-induced production of NO and PGE(2) in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE(2) in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-kappaB (NF-kappaB) appears to play a central role in transcriptional regulation of these proteins, we investigated the effects of APC on this transcription factor. APC inhibited LPS induced nuclear factor-kappaB (NF-kappaB) activation, by preventing degradation of the inhibitor kappaB (IkappaB). Taken together, our data indicates that APC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2 and IL-12 p40 through down regulation of the NF-kappaB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.

Efficacy of artemisinin in experimental visceral leishmaniasis

Visceral leishmaniasis (VL), caused by the protozoan Leishmania sp., affects 500000 people annually, with the Indian subcontinent contributing a significant proportion of these cases. Emerging refractoriness to conventional antimony therapy has emphasised the need for safer yet effective antileishmanial drugs. Artemisinin, a widely used antimalarial, demonstrated anti-promastigote activity and the 50% inhibitory concentration (IC(50)) ranged from 100 microM to 120 microM irrespective of Leishmania species studied. Leishmania donovani-infected macrophages demonstrated decreased production of nitrite as well as mRNA expression of inducible nitric oxide synthase, which was normalised by artemisinin, indicating that it exerted both a direct parasiticidal activity as well as inducing a host protective response. Furthermore, in a BALB/c model of VL, orally administered artemisinin (10mg/kg and 25mg/kg body weight) effectively reduced both splenic weight and parasite burden, which was accompanied by a restoration of Th1 cytokines (interferon-gamma and interleukin-2). Taken together, these findings have delineated the therapeutic potential of artemisinin in experimental VL.

Monitoring of intracellular nitric oxide in leishmaniasis: Its applicability in patients with visceral leishmaniasis

Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5‐diaminofluorescein diacetate (DAF‐2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S‐nitroso‐N‐acetyl‐penicillamine (SNAP)] and a NO scavenger [2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide, C‐PTIO]. Using 40 μM DAF‐2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti‐leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF‐2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF‐2DA, being 2 μM. Following parasitization, levels of NO decreased which was normalized following treatment with anti‐leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti‐leishmanial compounds.

Immunomodulation by chemotherapeutic agents against Leishmaniasis.

Leishmaniasis is caused by protozoan parasites of the genus Leishmania and causes a wide spectrum of clinical manifestations ranging from self-healing cutaneous lesions to the fatal visceral form. The use of pentavalent antimony, the mainstay of therapy of Leishmaniasis is now limited by its toxicity and alarming increase in unresponsiveness, especially in the Indian subcontinent. Furthermore, other anti-leishmanial drugs are unaffordable in many affected countries and as vaccination based approaches have not yet proved to be effective, chemotherapy remains the only alternative, emphasizing the need for identifying novel drug targets. In this review, we have described the different host immune signaling pathways that could be considered as potential drug targets for Leishmania chemotherapy.

Berberine chloride causes a caspase-independent, apoptotic-like death in Leishmania donovani promastigotes.

Berberine chloride, a quarternary isoquinoline alkaloid, is a promising anti-leishmanial compound, IC50 being 7.1 µM in L. donovani promastigotes. This leishmanicidal activity was initiated by its pro-oxidant effect, evidenced by enhanced generation of reactive oxygen intermediates that was accompanied by depletion of thiols; pre-incubation in N-acetyl cysteine, attenuated its cell viability, corroborating that generation of free radicals triggered its parasiticidal activity. Externalization of phosphatidylserine and elevation of intracellular calcium preceded depolarization of the mitochondrial membrane potential, which translated into an increase in the sub G0/G1 population and was accompanied by DNA laddering, hallmarks of apoptosis. Berberine chloride failed to induce caspase activity and anti-leishmanial activity in the presence of a pan caspase inhibitor, Z-Val-Ala-DL-Asp (methoxy)-fluoromethylketone remained unchanged, which indicated that the apoptosis was caspase independent. Collectively, the data indicates that Berberine chloride triggers an apoptosis-like death following enhanced generation of reactive oxygen species, thus meriting further pharmacological investigations.

Berberine Chloride Mediates Its Anti-Leishmanial Activity via Differential Regulation of the Mitogen Activated Protein Kinase Pathway in Macrophages

Background A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages. Principal Findings The IC50 of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC50 being 2.8 fold lower than its IC50 in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10. Conclusions Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.

Malabaricone-A Induces A Redox Imbalance That Mediates Apoptosis in U937 Cell Line

Background The ‘two-faced’ character of reactive oxygen species (ROS) plays an important role in cancer biology by acting both as secondary messengers in intracellular signaling cascades and sustaining the oncogenic phenotype of cancer cells, while on the other hand, it triggers an oxidative assault that causes a redox imbalance translating into an apoptotic cell death. Principal Findings Using a tetrazolium [{3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium] based cell viability assay, we evaluated the cytotoxicity of a plant derived diarylnonanoid, malabaricone-A on leukemic cell lines U937 and MOLT-3. This cytotoxicity hinged on its ability to cause a redox imbalance via its ability to increase ROS, measured by flow cytometry using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate and by decreasing glutathione peroxidase activity. This redox imbalance mediated apoptosis was evident by an increase in cytosolic [Ca2+], externalization of phosphatidyl serine as also depolarization of the mitochondrial membrane potential as measured by flow cytometry. There was concomitant peroxidation of cardiolipin, release of free cytochrome c to cytosol along with activation of caspases 9, 8 and 3. This led to cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase that caused DNA damage as proved by labeling with 4′,6-diamidino-2-phenylindole (DAPI); furthermore, terminal deoxy ribonucleotide transferase catalysed incorporation of deoxy uridine triphosphate confirmed DNA nicking and was accompanied by arrest of cell cycle progression. Conclusions Taken together, compounds like MAL-A having pro-oxidant activity mediate their cytotoxicity in leukemic cells via induction of oxidative stress triggering a caspase dependent apoptosis.

Reprogramming of TAM toward proimmunogenic type through regulation of MAP kinases using a redox-active copper chelate

TAMs, present in the tumor microenvironment, play an immunosuppressive role, leading to tumor progression and metastasis. Recently, numerous attempts have been made to switch immunosuppressive TAMs into an immunostimulatory type. Previously, we showed that a copper chelate, viz., copper N‐(2‐hydroxy acetophenone) glycinate [CuNG], can reprogram TAMs toward the proimmunogenic type to mount an antitumor immune response, but the underlying molecular mechanisms of skewing TAMs toward the proimmunogenic type remain elusive. Herein, we tried to explore the signaling mechanisms responsible for the reprogramming of TAMs. We observed that CuNG‐induced ROS generation triggers activation of two MAPKs, i.e., p38MAPK and ERK1/2, and also causes up‐regulation of intracellular glutathione. Furthermore, activation of p38 MAPK up‐regulated the initial IL‐12 production and the activation of ERK1/2 in tandem with GSH, found responsible for IFN‐γ production by TAMs. This IFN‐γ, in turn, prolonged IL‐12 production and down‐regulated TGF‐β production and thus, plays the decisive role in CuNG‐mediated reprogramming of regulatory cytokine production by TAMs. Our work highlights that ROS‐mediated activation of MAPKs can convert suppressive macrophages toward the proimmunogenic type. Thus, the present study opens the possibility of targeting TAMs by the use of redox‐active compounds for designing a novel, therapeutic strategy against cancer.

The antioxidant activity of allylpyrocatechol is mediated via decreased generation of free radicals along with escalation of antioxidant mechanisms.

Allylpyrocatechol (APC) is responsible for the antiinflammatory activity exhibited by the methanolic extract of leaves of Piper betle. As antiinflammatory compounds may display antioxidant properties and vice versa, we investigated the antioxidant effect of APC. APC effectively reduced phorbol‐myristate‐acetate‐induced generation of reactive oxygen species and superoxide in murine peritoneal macrophages as well as inhibited Escherichia‐coli‐induced phagocytic activity of macrophages. Furthermore, pBluescript SK+ plasmid DNA damage induced by addition of sodium ascorbate was attenuated by APC as it inhibited transformation of the supercoiled form to a relaxed form. In addition, APC increased the enzymatic (catalase) and nonenzymatic (GSH) antioxidant components of murine macrophages. Taken together, APC exhibited an antioxidant activity which was mediated both via decreased generation of free radicals along with increase in cellular antioxidants. Copyright

Iron enhances generation of free radicals by Artemisinin causing a caspase-independent, apoptotic death in Leishmania donovani promastigotes

Abstract An increasing incidence of unresponsiveness to antimonials in Leishmaniasis has led to identification of plant-derived anti-leishmanial compounds like Artemisinin. Since iron-mediated generation of free radicals sustains the anti-malarial activity of Artemisinin, this study investigated whether similar mechanisms accounted for its activity in Leishmania promastigotes. Artemisinin effectively disrupted the redox potential via an increased generation of free radicals along with a decrease in levels of non-protein thiols. Attenuation of its IC50 by a free radical scavenger N-acetyl l-cysteine and an iron chelator desferoxamine established the pivotal role of free radicals and of the potentiating effect of iron. An enhanced Fluo-4 fluorescence reflected Artemisinin-induced mobilization of intracellular calcium, which triggered apoptosis. However, the absence of any detectable caspase activity indicated that the leishmanicidal activity of Artemisinin is mediated by an iron-dependent generation of reactive intermediates, terminating in a caspase-independent, apoptotic mode of cell death.