Search

TIM-3 functions to enforce CD8+ T cell exhaustion, a dysfunctional state associated with the tolerization of tumor microenvironment. Here we report apoptosis of IFN-γ competent TIM-3+ population of tumor-infiltrating CD8+ T cells in colon cancer. In humans suffering from colorectal cancer, TIM-3+ population is higher in cancer tissue-resident relative to peripheral blood CD8+ T cells. Both the TIM-3+ and TIM-3- cancer tissue-resident CD8+ T cells secrete IFN-γ of comparable levels, although apoptotic cells are more in TIM-3+ compared to TIM-3- population. In mouse CT26 colon tumor model, majority of tumor-infiltrating CD8+ T cells express TIM-3 and execute cytolysis function with higher effector cytokine secretion and apoptosis in TIM-3+ compared to TIM-3- population. The tumor cells secrete galectin-9, which increases apoptosis of tumor-infiltrating CD8+ T cells. Galectin-9/TIM-3 signaling blockade with anti-TIM-3 antibody reduces the apoptosis and in addition, inhibits tumor growth in mice. The blockade increases therapeutic efficacy of cyclophosphamide to treat tumor in mice as well. These results reveal a previously unexplored role of TIM-3 on tumor-infiltrating CD8+ T cells in vivo.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

Leishmaniasis remains a major health problem of the tropical and subtropical world. The visceral form causes the most fatalities if left untreated. Dramatic increases in the rates of infection and drug resistance and the non-availability of safe vaccines have highlighted the need for identification of novel and inexpensive anti-leishmanial agents. This study reports that racemoside A, a water-soluble steroidal saponin purified from the fruits of Asparagus racemosus, is a potent anti-leishmanial molecule effective against antimonial-sensitive (strain AG83) and -unresponsive (strain GE1F8R) Leishmania donovani promastigotes, with IC(50) values of 1.15 and 1.31 microg ml(-1), respectively. Incubation of promastigotes with racemoside A caused morphological alterations including cell shrinkage, an aflagellated ovoid shape and chromatin condensation. This compound exerts its leishmanicidal effect through the induction of programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide, loss of mitochondrial membrane potential culminating in cell-cycle arrest at the sub-G(0)/G(1) phase, and DNA nicking shown by deoxynucleotidyltransferase-mediated dUTP end labelling (TUNEL). Racemoside A also showed significant activity against intracellular amastigotes of AG83 and GE1F8R at a 7-8-fold lower dose, with IC(50) values of 0.17 and 0.16 microg ml(-1), respectively, and was non-toxic to murine peritoneal macrophages up to a concentration of 10 microg ml(-1). Hence, racemoside A is a potent anti-leishmanial agent that merits further pharmacological investigation.

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, 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.

Leishmaniasis constitutes a complex of diseases with clinical and epidemiological diversity and includes visceral leishmaniasis, a disease that is fatal when left untreated. In earlier studies, the authors reported that Aloe vera leaf exudate (AVL) is a potent antileishmanial agent effective in promastigotes of Leishmania braziliensis, Leishmania mexicana, Leishmania tropica, Leishmania major and Leishmania infantum and also in axenic amastigotes of Leishmania donovani. In the present study, it has been demonstrated that, in promastigotes of L. donovani (IC(50) = 110 microg ml(-1)), AVL mediates this leishmanicidal effect by triggering a programmed cell death. Incubation of promastigotes with AVL caused translocation of phosphatidylserine to the outer leaflet of the plasma membrane as measured by annexin V binding, which was accompanied by loss of mitochondrial membrane potential, release of cytochrome c into the cytosol and concomitant nuclear alterations that included chromatin condensation, deoxynucleotidyltransferase-mediated dUTP end labelling and DNA laddering. As this AVL-induced leishmanicidal effect could not be inhibited by protease inhibitors including Z-Val-Ala-dl-Asp (methoxy)-fluoromethylketone, a broad-spectrum caspase inhibitor, non-involvement of caspases and major proteases was suggested. Additionally, AVL treatment caused no increase in cytosolic Ca(2+) or generation of reactive oxygen species, indicating that although promastigote death was induced by an apoptotic-like mechanism similar to metazoan apoptosis, the pathways of induction and/or execution differed at the molecular level.

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (Treg). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in Treg infiltration and CD8+ T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of Treg against CD8+ T cells. We also found tumor growth to be diminished in mice lacking CCR5, aCCL5 receptor, where a similar decrease in both Treg cell infiltration andCD8+ T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8+ T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient Treg or to enhance their cytotoxic effects against CD8+ T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of Treg in inhibiting the antitumor responses of CD8+ T cells, in the same way as CCL5 signaling blockade. Together, our findings establishthatCCL5/CCR5signaling recruits Treg to tumors and enhances their ability to kill antitumor CD8+ T cells, thereby defining a novel mechanism of immune escape in colorectal cancer. [ABSTRACT FROM AUTHOR]