Piu Saha

Publications of Piu Saha

• The Antioxidant Activity of Allylpyrocatechol is Mediated via Decreased Generation of Free Radicals Along With Escalation of Antioxidant Mechanisms.

  Authors: Debjani Sarkar, Sunanda Kundu, Soumita De, Chellaram Hariharan, Piu Saha, Alak Manna, Subrata Chattopadhyay, Mitali Chatterjee

  Phytotherapy research : PTR. 05/2012;

  Allylpyrocatechol (APC) is responsible for the antiinflammatory activity exhibited by the methanolic extract of leaves of Piper betle. As antiinflammatory compounds may display antioxidant propertiesAllylpyrocatechol (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 © 2012 John Wiley & Sons, Ltd.

• Targets for immunochemotherapy in leishmaniasis.

  Authors: Debanjan Mukhopadhyay, Piu Saha, Mitali Chatterjee

  Expert review of anti-infective therapy. 03/2012; 10(3):261-4.

• Malabaricone-a induces a redox imbalance that mediates apoptosis in U937 cell line.

  Authors: Alak Manna, Piu Saha, Avijit Sarkar, Debanjan Mukhopadhyay, Ajay K Bauri, Deepak Kumar, Padma Das, Subrata Chattopadhyay, Mitali Chatterjee

  PloS one. 01/2012; 7(5):e36938.

  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 theThe '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. 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 [Ca(2+)], 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. 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.

• Immunomodulation by chemotherapeutic agents against Leishmaniasis.

  Authors: Piu Saha, Debanjan Mukhopadhyay, Mitali Chatterjee

  International immunopharmacology. 08/2011; 11(11):1668-79.

  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.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.

• Monitoring of intracellular nitric oxide in leishmaniasis: its applicability in patients with visceral leishmaniasis.

  Authors: Avijit Sarkar, Piu Saha, Goutam Mandal, Debanjan Mukhopadhyay, Susmita Roy, Sunny Kumar Singh, Sukhen Das, R P Goswami, Bibhuti Saha, Deepak Kumar, Padma Das, Mitali Chatterjee

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 01/2011; 79(1):35-45.

  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. AsNitric 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.

• Berberine chloride mediates its anti-leishmanial activity via differential regulation of the mitogen activated protein kinase pathway in macrophages.

  Authors: Piu Saha, Surajit Bhattacharjee, Avijit Sarkar, Alak Manna, Subrata Majumder, Mitali Chatterjee

  PloS one. 01/2011; 6(4):e18467.

  A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. InA 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. The IC(50) 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 IC(50) being 2.8 fold lower than its IC(50) 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. Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.

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

  Authors: Rupashree Sen, Piu Saha, Avijit Sarkar, Sudipto Ganguly, Mitali Chatterjee

  Free radical research. 11/2010; 44(11):1289-95.

  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 ofAn 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.

• Efficacy of artemisinin in experimental visceral leishmaniasis.

  Authors: Rupashree Sen, Sudipto Ganguly, Piu Saha, Mitali Chatterjee

  International journal of antimicrobial agents. 07/2010; 36(1):43-9.

  Visceral leishmaniasis (VL), caused by the protozoan Leishmania sp., affects 500000 people annually, with the Indian subcontinent contributing a significant proportion of these cases. EmergingVisceral 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.

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

  Authors: Piu Saha, Rupashree Sen, Chellaram Hariharan, Deepak Kumar, Padma Das, Mitali Chatterjee

  Free radical research. 09/2009;

  Berberine chloride, a quarternary isoquinoline alkaloid, is a promising anti-leishmanial compound, IC(50) being 7.1 microM in L. donovani promastigotes. This leishmanicidal activity was initiated byBerberine chloride, a quarternary isoquinoline alkaloid, is a promising anti-leishmanial compound, IC(50) being 7.1 microM 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 G(0)/G(1) 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.

• Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

  Authors: Goutam Mandal, Avijit Sarkar, Piu Saha, Neeloo Singh, Shyam Sundar, Mitali Chatterjee

  Indian journal of biochemistry & biophysics. 03/2009; 46(1):86-92.

  The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated anThe recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

• Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress.

  Authors: Samiran Bandyopadhyay, Sudipto Ganguly, Goutam Mandal, Rupashree Sen, Piu Saha, Monoj Kumar Ghosh, Mihir Sarkar, Mitali Chatterjee

  Research in veterinary science. 02/2009;

  In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The presentIn Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC(50) being 13.8+/-1.11mug/mL. The effect of S-EtOH (1mug/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate (H(2)DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55+/-0.03 to 47.32+/-2.25 (p<0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90+/-0.72muM to 18.79+/-0.32muM (p<0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95+/-0.48 to 33.34+/-1.66 (p<0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5+/-49.44 to 407.4+/-12.61 (p<0.01). Additionally, S-EtOH (14mug/mL, 24h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.

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

  Authors: Debjani Sarkar, Piu Saha, Sunita Gamre, Surajit Bhattacharjee, Chellaram Hariharan, Sudipto Ganguly, Rupashree Sen, Goutam Mandal, Subrata Chattopadhyay, Subrata Majumdar, Mitali Chatterjee

  International immunopharmacology. 10/2008; 8(9):1264-71.

  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 haveThe 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.

• An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis.

  Authors: Avijit Sarkar, Rupashree Sen, Piu Saha, Sudipto Ganguly, Goutam Mandal, Mitali Chatterjee

  Parasitology research. 05/2008; 102(6):1249-55.

  An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally knownAn unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

• Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes.

  Authors: Rupashree Sen, Samiran Bandyopadhyay, Avijit Dutta, Goutam Mandal, Sudipto Ganguly, Piu Saha, Mitali Chatterjee

  Journal of medical microbiology. 10/2007; 56(Pt 9):1213-8.

  A major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, aA major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an established anti-malarial compound that showed anti-leishmanial activity in both promastigotes and amastigotes, with IC(50) values of 160 and 22 microM, respectively, and, importantly, was accompanied by a high safety index (>22-fold). The leishmanicidal activity of artemisinin was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, in situ labelling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. Taken together, these data indicate that artemisinin has promising anti-leishmanial activity that is mediated by programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

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

  Authors: Debjani Sarkar, Piu Saha, Sunita Gamre, Surajit Bhattacharjee, Chellaram Hariharan, Sudipto Ganguly, Rupashree Sen, Goutam Mandal, Subrata Chattopadhyay, Subrata Majumdar, Mitali Chatterjee

  International Immunopharmacology.

  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 haveThe 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 PGE2 in a murine macrophage cell line, RAW 264.7. APC inhibited production of NO and PGE2 in a dose dependent manner as also decreased mRNA expression of iNOS, COX-2, IL-12p40 and TNF-alpha. Since nuclear factor-κB (NF-κB) 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-κB) activation, by preventing degradation of the inhibitor kappaB (IκB). 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-κB pathway, indicating that APC may have therapeutic potential in inflammation associated disorders.