Induction of cancer-specific cytotoxicity towards human prostate and skin cells using quercetin and ultrasound

Bioflavonoids, such as quercetin, have recently emerged as a new class of chemotherapeutic drugs for the treatment of various cancer types, but are marred by their low potency and poor selectivity. We report that a short application of low-frequency ultrasound selectively sensitises prostate and skin cancer cells against quercetin. Pretreatment of cells with ultrasound (20 kHz, 2 W cm−2, 60 s) selectively induced cytotoxicity in skin and prostate cancer cells, while having minimal effect on corresponding normal cell lines. About 90% of the viable skin cancer cell population was lost within 48 h after ultrasound-quercetin (50 μM) treatment. Ultrasound reduced the LC50 of quercetin for skin cancer cells by almost 80-fold, while showing no effect on LC50 for nonmalignant skin cells

Evidence for Oxidative Stress and Defective Antioxidant Response in Guinea Pigs with Tuberculosis

The development of granulomatous inflammation with caseous necrosis is an important but poorly understood manifestation of tuberculosis in humans and some animal models. In this study we measured the byproducts of oxidative stress in granulomatous lesions as well as the systemic antioxidant capacity of BCG vaccinated and non-vaccinated guinea pigs experimentally infected with Mycobacterium tuberculosis. In non-vaccinated guinea pigs, oxidative stress was evident within 2 weeks of infection as measured by a decrease in the serum total antioxidant capacity and blood glutathione levels accompanied by an increase in malondialdehyde, a byproduct of lipid peroxidation, within lesions. Despite a decrease in total and reduced blood glutathione concentrations, there was an increase in lesion glutathione by immunohistochemistry in response to localized oxidative stress. In addition there was an increase in the expression of the host transcription factor nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates several protein and non-proteins antioxidants, including glutathione. Despite the increase in cytoplasmic expression of Nrf2, immunohistochemical staining revealed a defect in Nrf2 nuclear translocation within granulomatous lesions as well as a decrease in the expression of the Nrf2-regulated antioxidant protein NQO1. Treating M. tuberculosis–infected guinea pigs with the antioxidant drug N-acetyl cysteine (NAC) partially restored blood glutathione concentrations and the serum total antioxidant capacity. Treatment with NAC also decreased spleen bacterial counts, as well as decreased the lung and spleen lesion burden and the severity of lesion necrosis. These data suggest that the progressive oxidative stress during experimental tuberculosis in guinea pigs is due in part to a defect in host antioxidant defenses, which, we show here, can be partially restored with antioxidant treatment. These data suggest that the therapeutic strategies that reduce oxidant-mediated tissue damage may be beneficial as an adjunct therapy in the treatment and prevention of tuberculosis in humans

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

Background: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaMbinding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems

PE-PGRS33 contributes to Mycobacterium tuberculosis entry in macrophages through interaction with TLR2

PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140-260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2