Co-Administration of Anticancer Candidate MK-2206 Enhances the Efficacy of BCG Vaccine Against Mycobacterium tuberculosis in Mice and Guinea Pigs.

The failure of M. bovis BCG to induce long-term protection has been endowed to its inability to escape the phagolysosome, leading to mild activation of CD8+ mediated T cell response. Induction of apoptosis in host cells plays an important role in potentiating dendritic cells-mediated priming of CD8+ T cells, a process defined as "cross-priming." Moreover, IL-10 secretion by infected cells has been reported to hamper BCG-induced immunity against Tuberculosis (TB). Previously, we have reported that apoptosis of BCG-infected macrophages and inhibition of IL-10 secretion is FOXO3 dependent, a transcription factor negatively regulated by the pro-survival activated threonine kinase, Akt. We speculate that FOXO3-mediated induction of apoptosis and abrogation of IL-10 secretion along with M. bovis BCG immunization might enhance the protection imparted by BCG. Here, we have assessed whether co-administration of a known anti-cancer Akt inhibitor, MK-2206, enhances the protective efficacy of M. bovis BCG in mice model of infection. We observed that in vitro MK-2206 treatment resulted in FOXO3 activation, enhanced BCG-induced apoptosis of macrophages and inhibition of IL-10 secretion. Co-administration of M. bovis BCG along with MK-2206 also increased apoptosis of antigen-presenting cells in draining lymph nodes of immunized mice. Further, MK-2206 administration improved BCG-induced CD4+ and CD8+ effector T cells responses and its ability to induce both effector and central memory T cells. Finally, we show that co-administration of MK-2206 enhanced the protection imparted by M. bovis BCG against Mtb in aerosol infected mice and guinea pigs. Taken together, we provide evidence that MK-2206-mediated activation of FOXO3 potentiates BCG-induced immunity and imparts protection against Mtb through enhanced innate immune response.

The Phenolic compound Kaempferol overcomes 5-fluorouracil resistance in human resistant LS174 colon cancer cells.

Resistance to 5-Fluorouracil chemotherapy is a major cause of therapeutic failure in colon cancer cure. Development of combined therapies constitutes an effective strategy to inhibit cancer cells and prevent the emergence of drug resistance. For this purpose, we investigated the anti-tumoral effect of thirteen phenolic compounds, from the Tunisian quince Cydonia oblonga Miller, alone or combined to 5-FU, on the human 5-FU-resistant LS174-R colon cancer cells in comparison to parental cells. Our results showed that only Kaempferol was able to chemo-sensitize 5-FU-resistant LS174-R cells. This phenolic compound combined with 5-FU exerted synergistic inhibitory effect on cell viability. This combination enhanced the apoptosis and induced cell cycle arrest of both chemo-resistant and sensitive cells through impacting the expression levels of different cellular effectors. Kaempferol also blocked the production of reactive oxygen species (ROS) and modulated the expression of JAK/STAT3, MAPK, PI3K/AKT and NF-κB. In silico docking analysis suggested that the potent anti-tumoral effect of Kaempferol, compared to its two analogs (Kaempferol 3-O-glucoside and Kampferol 3-O-rutinoside), can be explained by the absence of glucosyl groups. Overall, our data propose Kaempferol as a potential chemotherapeutic agent to be used alone or in combination with 5-FU to overcome colon cancer drug resistance.

FOXO3 Transcription Factor Regulates IL-10 Expression in Mycobacteria-Infected Macrophages, Tuning Their Polarization and the Subsequent Adaptive Immune Response.

Alveolar Macrophages play a key role in the development of a robust adaptive immune response against the agent of Tuberculosis (TB), Mycobacterium tuberculosis (M.tb). However, macrophage response is often hampered by the production of IL-10, a potent suppressor of the host immune response. The secretion of IL-10 correlates with TB pathogenesis and persistence in host tissues. Concordantly, inhibition of IL-10 signaling, during BCG vaccination, confers higher protection against M.tb through a sustained Th1 and Th17 responses. Therefore, uncovering host effectors, underlying mycobacteria-induced expression of IL-10, may be beneficial toward the development of IL-10-blocking tools to be used either as adjuvants in preventive vaccination or as adjunct during standard treatment of TB. Here, we investigated the role of FOXO3 transcription factor in mycobacteria-induced secretion of IL-10. We observed that PI3K/Akt/FOXO3 axis regulates IL-10 expression in human macrophages. Knocking down of FOXO3 expression resulted in an increase of IL-10 production in BCG-infected macrophages. The gene reporter assay further confirmed the transcriptional regulation of IL-10 by FOXO3. In silico analysis identified four Forkhead binding motifs on the human IL-10 promoter, from which the typical FOXO3 one at position -203 was the major target as assessed by mutagenesis and CHIP binding assays. Further, we also observed a decrease in gene expression levels of the M1 typical markers (i.e., CD80 and CD86) in SiFOXO3-transfected macrophages while activation of FOXO3 led to the increase in the expression of CD86, MHCI, and MHCII. Finally, co-culture of human lymphocytes with siFOXO3-transfected macrophages, loaded with mycobacterial antigens, showed decreased expression of Th1/Th17 specific markers and a simultaneous increase in expression of IL-4 and IL-10. Taken together, we report for the first time that FOXO3 modulates IL-10 secretion in mycobacteria-infected macrophage, driving their polarization and the subsequent adaptive immune response. This work proposes FOXO3 as a potential target for the development of host-directed strategies for better treatment or prevention of TB.

Ultrasensitive detection of hazardous reactive oxygen species using flexible organic transistors with polyphenol-embedded conjugated polymer sensing layers.

Here we report that superoxide, one of the hazardous reactive oxygen species (ROS), can be quickly detected by flexible organic field-effect transistors (OFETs) with the polyphenol-embedded conjugated polymer micro-channels. Rutin, one of the abundant polyphenols found in a variety of plants, was employed as a sensing molecule and embedded in the poly(3-hexylthiophene) (P3HT) matrix. The rutin-embedded P3HT layers showed randomly distributed micro-domains, which became bigger as the rutin content increased. The best transistor performance was achieved at the rutin content of 10 wt%, while the OFETs exhibited proper and controllable transistor performances even in the phosphate buffer solutions. The sensing test revealed that the present OFET sensors could stably detect superoxide using very small amount (<10 µl) of samples at extremely low concentrations (500 pM), while they exhibited outstanding stability and durability upon repeated detection and storage-reuse tests. Finally, the present flexible OFET sensors could deliver confident sensing results for the detection of superoxide generated from the mouse RAW264.7 macrophages.

Helix aspersa maxima mucus exhibits antimelanogenic and antitumoral effects against melanoma cells.

Snail secretion is currently revolutionizing the world of cosmetics and human skin care. The efficacy of snail secretion in wounds healing has been proven both in vitro and by clinical studies. However, the potential anti-tumor effect of snail secretion was poorly investigated. In this report, our in vitro study showed that Helix aspersa maxima species snail slime (SS) could not only treat melanogenesis but also endowed with anti-tumoral activity against human melanoma cells. Indeed, SS reduced melanin content and tyrosinase activity on B16F10 cells with IC50 values of 288 µg/mL and 286 µg/mL, respectively, without altering cell viability. This effect was also observed, at a lesser extent, on human melanoma IGR-39 and SK-MEL-28 cell lines. On another hand, SS specifically inhibited the viability of IGR-39 and SK-MEL-28 cells associated to an apoptotic effect highlighted by PARP cleavage. It is worth to note that SS did not affect the viability of B16F10 cells and non tumorigenic HaCaT cells. Interestingly, this extract was found to inhibit migration and invasion of both human melanoma cells through reducing the expression of Matrix metalloproteinase MMP2. Snail slime also exerted a high inhibitory effect on IGR-39 cell adhesion through blocking the function of α2ß1 (45%), αvß3 (38%) integrins and by reducing the expression levels of αv and ß1 integrins. The presented results shed light on the potential anti-melanoma effect of SS and support its use against skin diseases.

Mertensene, a Halogenated Monoterpene, Induces G2/M Cell Cycle Arrest and Caspase Dependent Apoptosis of Human Colon Adenocarcinoma HT29 Cell Line through the Modulation of ERK-1/-2, AKT and NF-κB Signaling.

Conventional treatment of advanced colorectal cancer is associated with tumor resistance and toxicity towards normal tissues. Therefore, development of effective anticancer therapeutic alternatives is still urgently required. Nowadays, marine secondary metabolites have been extensively investigated due to the fact that they frequently exhibit anti-tumor properties. However, little attention has been given to terpenoids isolated from seaweeds. In this study, we isolated the halogenated monoterpene mertensene from the red alga Pterocladiella capillacea (S.G. Gmelin) Santelices and Hommersand and we highlight its inhibitory effect on the viability of two human colorectal adenocarcinoma cell lines HT29 and LS174. Interestingly, exposure of HT29 cells to different concentrations of mertensene correlated with the activation of MAPK ERK-1/-2, Akt and NF-κB pathways. Moreover, mertensene-induced G2/M cell cycle arrest was associated with a decrease in the phosphorylated forms of the anti-tumor transcription factor p53, retinoblastoma protein (Rb), cdc2 and chkp2. Indeed, a reduction of the cellular level of cyclin-dependent kinases CDK2 and CDK4 was observed in mertensene-treated cells. We also demonstrated that mertensene triggers a caspase-dependent apoptosis in HT29 cancer cells characterized by the activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). Besides, the level of death receptor-associated protein TRADD increased significantly in a concentration-dependent manner. Taken together, these results demonstrate the potential of mertensene as a drug candidate for the treatment of colon cancer.

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5ß1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc.

Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2 Pathways and the Abrogation of VEGF Secretion.

Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK1/2, Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

Asteraceae Artemisia campestris and Artemisia herba-alba Essential Oils Trigger Apoptosis and Cell Cycle Arrest in Leishmania infantum Promastigotes.

We report the chemical composition and anti-Leishmania and antioxidant activity of Artemisia campestris L. and Artemisia herba-alba Asso. essential oils (EOs). Our results showed that these extracts exhibit different antioxidant activities according to the used assay. The radical scavenging effects determined by DPPH assay were of IC50 = 3.3 mg/mL and IC50 = 9.1 mg/mL for Artemisia campestris and Artemisia herba-alba essential oils, respectively. However, antioxidant effects of both essential oils, determined by ferric-reducing antioxidant power (FRAP) assay, were in the same range (2.3 and 2.97 mg eq EDTA/g EO, resp.), while the Artemisia herba-alba essential oil showed highest chelating activity of Fe2+ ions (27.48 mM Fe2+). Interestingly, we showed that both EOs possess dose-dependent activity against Leishmania infantum promastigotes with IC50 values of 68 µg/mL and 44 µg/mL for A. herba-alba and A. campestris, respectively. We reported, for the first time, that antileishmanial activity of both EOs was mediated by cell apoptosis induction and cell cycle arrest at the sub-G0/G1 phase. All our results showed that EOs from A. herba-alba and A. campestris plants are promising candidates as anti-Leishmania medicinal products.

Two distinct conformational states of Mycobacterium tuberculosis virulent factor early secreted antigenic target 6 kDa are behind the discrepancy around its biological functions.

Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.

Quince peel polyphenolic extract blocks human colon adenocarcinoma LS174 cell growth and potentiates 5-fluorouracil efficacy.

BACKGROUND: Development of alternative cancer-specific drugs would be of paramount importance to overcome toxicity toward normal tissues and tumor resistance. Here, we investigated the potential anti-tumoral effect of peel (Peph) and pulp polyphenolic extracts from the Tunisian quince Cydonia oblonga Miller on both no-tumorigenic cells NIH 3T3 Fibroblasts and HEK 293 cells and human colon adenocarcinoma LS174 cells. METHODS: Cell proliferation and cytotoxicity were measured with MTT and LDH assays respectively. Cell cycle distribution and the apoptosis levels were assessed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were determined using the fluorescent probe CM-H2DCFDA. Western blot was used to further characterize cell death and analyze the signaling pathways affected by Peph treatment. The expression level of VEGF-A was evaluated by real time quantitative PCR and further verified by quantifying the secreted cytokines by enzyme-linked immunosorbent assay. RESULTS: We found that Peph extract displayed the highest anti-proliferative effect specifically on LS174 cells. However, each Peph phenolic compound alone did not exhibit any anti-proliferative activity, suggesting a synergistic effect of phenolic molecules. Such effect was associated with a cell cycle arrest in the G1/S phase, a caspase-independent apoptosis and an increase of the ROS production. Peph extract inhibited the pro-survival signaling pathway NFκB and suppressed the expression of various cellular markers known to be involved in cell cycling (cyclin D1) and angiogenesis (Vascular Endothelial Growth Factor, VEGF). Interestingly, the combination Peph extract and 5-FU exerted synergistic inhibitory effect on cell viability. CONCLUSION: These data propose the quince Peph extract as a promising cost effective non toxic drug to employ alone or in combination with conventional anti-colorectal cancer. Moreover, quince rich regimen may prevent the development and the progress of colon cancer.

Forkhead box O3 (FOXO3) transcription factor mediates apoptosis in BCG-infected macrophages.

Enhanced apoptosis of BCG-infected macrophages has been shown to induce stronger dendritic cell-mediated cross-priming of T cells, leading to higher protection against tuberculosis (TB). Uncovering host effectors underlying BCG-induced apoptosis may then prove useful to improve BCG efficacy through priming macrophage apoptosis. Her we report that BCG-mediated apoptosis of human macrophages relies on FOXO3 transcription factor activation. BCG induced a significant apoptosis of THP1 (TDMs) and human monocytes (MDMs)-derived macrophages when a high moi was used, as shown by annexin V/7-AAD staining. BCG-induced apoptosis was associated with dephosphorylation of the prosurvival activated threonine kinase (Akt) and its target FOXO3. Cell fractionation and immunofluorescence microscopy showed translocation of FOXO3 to the nucleus in BCG-infected cells, concomitantly with an increase of FOXO3 transcriptional activity. Moreover, FOXO3 expression knock-down by small interfering RNA (siRNA) partially inhibited the BCG-induced apoptosis. Finally, real-time quantitative PCR (qRT-PCR) analysis of the expression profile of BCG-infected macrophages showed an upregulation of two pro-apoptotic targets of FOXO3, NOXA and p53 upregulated modulator of apoptosis (PUMA). Our results thus indicate that FOXO3 plays an important role in BCG-induced apoptosis of human macrophages and may represent a potential target to improve vaccine efficacy through enhanced apoptosis-mediated cross-priming of T cells.