Apoptotic and antioxidant effects in HCT-116 colorectal carcinoma cells by a spiro-acridine compound, AMTAC-06.

BACKGROUND: Acridine compounds have been described as promising anticancer agents. Previous studies showed that (E)-1'-((4-chlorobenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-06), a spiro-acridine compound, has antitumor activity on Ehrlich tumor and low toxicity. Herein, we investigated its antitumor effect against human cells in vitro. METHODS: MTT assay was used to assess cytotoxicity of AMTAC-06 (3.125-200 µM) against tumor and non-tumor cells, and the half-maximal inhibitory concentration (IC50) and the selectivity index (SI) were calculated. The effects on the cell cycle (propidium iodide-PI-staining), apoptosis (Annexin V-FITC/PI double staining by flow cytometry), and production of reactive oxygen species, ROS (DCFH assay) were also evaluated. Statistical analysis was achieved using ANOVA followed by Tukey's post-test. RESULTS: AMTAC-06 showed higher cytotoxicity against colorectal carcinoma HCT-116 cells (IC50: 12.62 µM). The SI showed that AMTAC-06 was more selective for HCT-116 cells (HaCaT SI: 1.41; PBMC SI: 0.62) than doxorubicin (HaCaT SI: 0.10; PBMC SI: 0.01). AMTAC-06 (15 and 30 µM) induced an increase in the sub-G1 peak (p < 0.000001) and cell cycle arrest in S phase (p = 0.003547). Moreover, treatment with this compound (15 and 30 µM) resulted in increased early (p < 0.000001) and late apoptotic cells (p < 0.000001). In addition, there was a reduction on ROS production (p < 0.000001). CONCLUSIONS: AMTAC-06 presents anticancer activity against HCT-116 cells by regulating the cell cycle, inducing apoptosis and an antioxidant action.

Characterization of Regulatory T Cells in Patients Infected by Leishmania Infantum.

High IL-10 levels are pivotal to parasite survival in visceral leishmaniasis (VL). Antigenic stimuli induce IL-10 expression and release of adenosine by CD39/CD73. Due their intrinsic ability to express IL-10 and produce adenosine from extracellular ATP, we evaluated the IL-10, CD39, and CD73 expression by Regulatory T cells (Treg) correlated with VL pathology. Using flow cytometry, Treg cells was analyzed in peripheral blood samples from VL patients (in the presence and absence of Leishmania infantum soluble antigen (SLA)) and healthy individuals (negative endemic control-NEC group), without any treatment. Additionally, IL-10 levels in leukocytes culture supernatant were measured in all groups by ELISA assay. VL patients presented more Treg frequency than NEC group, independently of stimulation. ELISA results demonstrated that SLA induced higher IL-10 expression in the VL group. However, the NEC group had a higher Treg IL-10+ compared to the VL group without stimulation and SLA restored the IL-10 in Treg. Additionally, an increase in Treg CD73+ in the VL group independently of stimuli compared to that in the NEC group was observed. We suggest that Treg are not the main source of IL-10, while the CD73 pathway may be an attempt to modulate the exacerbation of immune response in VL disease.

CD45RO+ T Cells and T Cell Activation in the Long-Lasting Immunity after Leishmania infantum Infection.

Manifestations of Leishmania infantum infection range from asymptomatic to symptomatic visceral leishmaniasis (VL). People with symptomatic VL (sVL) have suppressed immune responses against Leishmania antigens that are reversed after clinical cure. The intradermal leishmanin skin test (LST) is negative during sVL, but it becomes positive after treatment. The aim of this study was to compare T cell responses in individuals with sVL, recovered VL (RecVL), and endemic controls. Endemic controls were household contacts of a VL case and they were grouped by their LST results, either positive (LST+) or negative (LST-). Mononuclear cells were studied ex vivo or after stimulation with soluble Leishmania antigens (SLA); cell surface markers and cytokines were determined. T cells, ex vivo, from individuals with sVL and from LST+ individuals presented a higher activation for CD4+ and CD8+ cells expressing CD69. However, lymphocytes from sVL stimulated with SLA had lower percentages of CD4+ and CD8+ cells expressing CD69 and CD8+ cells expressing CD25, with no release of interferon-γ or tumor necrosis factor. sVL subjects had lower percentage of memory cells (CD4+ CD45RO+), ex vivo, without SLA stimulation than RecVL, LST+, or LST- (P = 0.0022). However, individuals with sVL had fewer regulatory cells after SLA stimulation (CD4+ CD25HIGH, P = 0.04 and CD4+ FOXP3+, P = 0.02) than RecVL. The decrease in specific memory and activated CD4+ and CD8+ cells, as in response to Leishmania antigens, could explain, in part, the immune impairment during sVL. Finally, protective T cell responses are long lasting because both RecVL or LST+ individuals maintain a specific protective response to Leishmania years after the primary infection.

Transcriptional changes that characterize the immune reactions of leprosy.

BACKGROUND: Leprosy morbidity is increased by 2 pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL). METHODS: To discover host factors related to immune reactions, global transcriptional profiles of peripheral blood mononuclear cells were compared between 11 RR, 11 ENL, and 19 matched control patients, with confirmation by quantitative polymerase chain reaction. Encoded proteins were investigated in skin biopsy specimens by means of immunohistochemistry. RESULTS: There were 275 genes differentially expressed in RR and 517 differentially expressed in ENL on the microarray. Pathway analysis showed immunity-related pathways represented in RR and ENL transcriptional profiles, with the "complement and coagulation" pathway common to both. Interferon γ was identified as a significant upstream regulator of the expression changes for RR and ENL. Immunohistochemical staining of skin lesions showed increased C1q in both RR and ENL. CONCLUSIONS: These data suggest a previously underrecognized role for complement in the pathogenesis of both RR and ENL, and we propose new hypotheses for reaction pathogenesis.