TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor ß-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

Phosphoprotein patterns predict trametinib responsiveness and optimal trametinib sensitisation strategies in melanoma.

Malignant melanoma is a highly aggressive form of skin cancer responsible for the majority of skin cancer-related deaths. Recent insight into the heterogeneous nature of melanoma suggests more personalised treatments may be necessary to overcome drug resistance and improve patient care. To this end, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified. In this study, we applied multiplex phosphoproteomic profiling across a panel of 24 melanoma cell lines with different disease-relevant mutations, to predict responsiveness to MEK inhibitor trametinib. Supported by multivariate statistical analysis and multidimensional pattern recognition algorithms, the responsiveness of individual cell lines to trametinib could be predicted with high accuracy (83% correct predictions), independent of mutation status. We also successfully employed this approach to case specifically predict whether individual melanoma cell lines could be sensitised to trametinib. Our predictions identified that combining MEK inhibition with selective targeting of c-JUN and/or FAK, using siRNA-based depletion or pharmacological inhibitors, sensitised resistant cell lines and significantly enhanced treatment efficacy. Our study indicates that multiplex proteomic analyses coupled with pattern recognition approaches could assist in personalising trametinib-based treatment decisions in the future.

Anti-Mycobacterial Activity of Tamoxifen Against Drug-Resistant and Intra-Macrophage Mycobacterium tuberculosis.

Recently, it has become a struggle to treat tuberculosis with the current commercial antituberculosis drugs because of the increasing emergence of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis. We evaluated here the antimycobacterial activity of tamoxifen, known as a synthetic anti-estrogen, against eight drug-sensitive or resistant strains of Mycobacterium tuberculosis (TB), and the active intracellular killing of tamoxifen on TB in macrophages. The results showed that tamoxifen had antituberculosis activity against drug-sensitive strains (MIC, 3.125-6.25 µg/ml) as well as drug-resistant strains (MIC, 6.25 to 12.5 µg/ml). In addition, tamoxifen profoundly decreased the number of intracellular TB in macrophages in a dose-dependent manner.

Ursolic Acid Activates Intracellular Killing Effect of Macrophages During Mycobacterium tuberculosis Infection.

Tuberculosis is one of the most threatening infectious diseases to public health all over the world, for which Mycobacterium tuberculosis (MTB) is the etiological agent of pathogenesis. Ursolic acid (UA) has immunomodulatory function and exhibits antimycobacterial activity. However, the intracellular killing effect of UA has yet to be elucidated. The aim of this study was to evaluate the intracellular killing effect of UA during mycobacterial infection. The intracellular killing activity of UA was evaluated in the macrophage cell line THP-1 by the MGIT 960 system as well as by CFU count. The production of reactive oxygen species (ROS) and the level of nitric oxide (NO) were measured using DCF-DA and Griess reagent, respectively. Phagocytosis was observed by a fluorescence-based staining method, and the colony forming units were enumerated on 7H11 agar medium following infection. In addition, MRP8 mRNA expression was measured by qRT-PCR. UA significantly decreased the number of intracellular Mycobacterium through generation of ROS and NO. In addition, it profoundly activated the phagocytosis process of THP-1 cells during MTB-infection. Furthermore, our data demonstrated that UA activated the phagocytosis process in human monocyte cells through MRP8 induction. These data suggest that UA firmly contributes to the intracellular killing effect of macrophages during mycobacterial infection.

Cytoprotective effect of kaempferol on paraquat-exposed BEAS-2B cells via modulating expression of MUC5AC.

Mucins are highly glycosylated secretary proteins produced by most epithelial cells. Hypersecretion of mucins is one of the prominent symptoms of several airway diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. Paraquat (PQ), a common herbicide, has been associated with pulmonary damage and is a potent reactive oxygen species (ROS) producer. However, until now the role of PQ on mucin overproduction has not been studied. The aim of this study is to explore how kaempferol (KM), a widely used dietary flavonoid, affects the protection of human PQ-exposed bronchial epithelium BEAS-2B cells by suppressing Mucin gene expression via nuclear factor-kappa B (NF-κB). We observed that PQ generates intracellular ROS, and also induces lipid peroxidation in BEAS-2B cells. Additionally, we found that PQ effectively induces the expression of the MUC5AC gene; however, co-treatment of PQ with KM drastically reduces its expression. Furthermore, we observed that PQ activates NF-κB, while co-treatment with KM occludes its nuclear translocation, and additionally KM repressed the PQ phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in BEAS-2B cells. Based on our data, we believe that KM can suppress the over-expression of the MUC5AC gene. This would contribute to the protection of PQ cytotoxicity to exposed BEAS-2B cells, and allow further study toward a better understanding of ROS-associated diseases.

Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 µg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 µg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 µg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression.

Naringenin exerts cytoprotective effect against paraquat-induced toxicity in human bronchial epithelial BEAS-2B cells through NRF2 activation.

We have previously shown that paraquat (PQ)-induced oxidative stress causes dramatic damage in various human cell lines. Naringenin (NG) is an active flavanone, which has been reported to have beneficial bioactivities, including antioxidative, anti-inflammatory, and antitumorigenic activities, with a relatively low toxicity to normal cells. In this study, we intended to assess the cytoprotective effect of NG against PQ-induced toxicity in the human bronchial epithelial BEAS-2B cell line. Co-treatment with NG in PQ-treated BEAS-2B cells can reduce PQ-induced cellular toxicity. NG can also decrease the generation of intracellular ROS caused by PQ treatment. We also observed that treatment with NG in PQ-exposed BEAS-2B cells can significantly induce the expression of antioxidant-related genes, including GPX2, GPX3, GPX5, and GPX7. NG co-treatment can also activate the NRF2 transcription factor and promote its nuclear translocation. In addition, NG co-treatment can induce the expression of NRF2-downstream target genes such as that of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). A small interfering RNA study revealed that the knockdown of NRF2 can abrogate NG-mediated protection of the cells from PQ-induced cellular toxicity. We propose that NG effectively alleviates PQ-induced cytotoxicity in human bronchial epithelial BEAS-2B cells through the NRF2-regulated antioxidant defense pathway, and NG might be a good therapeutic candidate molecule in oxidative stress-related diseases.

Cytoprotective effect of bioactive sea buckthorn extract on paraquat-exposed A549 cells via induction of Nrf2 and its downstream genes.

The extract of sea buckthorn (SBT) [Hippophae rhamnoides L. (Elaeagnaceae)], is used as a food supplement and traditional medicine in numerous countries. This study investigated the protective effects of the functional extract of SBT against paraquat (PQ)-induced toxicity via antioxidant mechanisms in A549 cells. The methanol extract of SBT (25-200 µg/ml) was used to protect cells against PQ (200 µM)-induced cell death. A viability assay was conducted using 3-(4,5-dimethylthioazol-2-ly)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase (LDH). Total intracellular reactive oxygen species (ROS) were measured and plotted. For validation of the SBT-induced expression of nuclear factor-E2-related factor 2 (Nrf2) and its target genes, western blot analysis and qPCR were performed. The present study showed that pretreatment of A549 cells with SBT extract significantly attenuated PQ (200 µM)-induced cellular toxicity. The maximum cytoprotective effect was identified using 200 µg/ml SBT extract; it began 24 h following exposure and was sustained up to 120 h (P<0.05). SBT extract significantly reduced LDH activity by 35.63% and ROS levels by 30.90% (P<0.05). Pretreatment with SBT extract activated Nrf2 mRNA and protein expression and its nuclear translocation. The SBT extract effectively induced Nrf2 target genes, such as NAD(P)H dehydrogenase quinone 1, glutathione peroxidase 1, glutathione reductase and catalase following treatment with PQ. Based on these results, it was hypothesized that SBT extract may be used as a potential therapeutic agent for the treatment of various oxidative stress-related diseases.

Cytoprotective effect of alpha-lipoic acid on paraquat-exposed human bronchial epithelial cells via activation of nuclear factor erythroid related factor-2 pathway.

Alpha-lipoic acid (LA), a metabolic antioxidant, is a natural compound and its biological function has been well studied in various human diseases. The present study was designed to investigate the cytoprotective effect and the molecular mechanisms of LA in paraquat (PQ)-induced oxidative stress injury using BEAS-2B human bronchial epithelial cells. LA co-treatment prevented PQ-induced BEAS-2B cell death. LA also prevented PQ-induced increases in total reactive oxygen species (ROS), lactate dehydrogenase (LDH) and malondialdehyde (MDA). LA also increased the expression of detoxifying phase II enzyme encoding genes and antioxidant genes including HO-1, NQO1, CAT, GPX3 and GPX4, resulting in the attenuation of the decreases of antioxidants during PQ-induced oxidative stress. Nuclear factor erythroid related factor 2 (Nrf2) was induced by LA. Additionally, translocation of Nrf2 from the cytoplasm to the nucleus was promoted by LA treatment. While LA was responsible for the upregulation of Nrf2, it also activated and up-regulated the downstream proteins heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1 (NQO1). The data collectively suggest that the beneficial effect of LA involving the activation of cytoprotective antioxidant genes make LA a potential candidate in the prevention of PQ-induced oxidative stress-related bronchial cell death, pending clinically relevant studies.

Antioxidant effect of silymarin on paraquat-induced human lung adenocarcinoma A549 cell line.

Paraquat (PQ) is not only widely used as a potent herbicide but also causes severe fatality to humans around the world due to accidental or intentional ingestion. Silymarin is a well-known phytochemical whose multi-functional effects in humans include anti-oxidant, anti-inflammatory and anti-cancer activities. The efficacy of silymarin in protecting against PQ-induced cytotoxicity is unknown. This study investigated the potential role of silymarin against PQ-induced oxidative stress on human A549 adenocarcinoma cell line. Colorimetric-based viability assay, determination of reactive oxygen species, cell damage assay based on lactate dehydrogense retention, anti-oxidant enzyme assay, Western blot and quantitative reverse transcription-polymerase chain reaction analyses were done. Our data revealed that silymarin could dramatically prevent cell toxicity, and reduce the LDH retention induced by PQ on A549 cell line. Silymarin acted as a potent cytoprotective effector through the effective induction of anti-oxidant related genes, including Nrf2, NQO1 and HO-1, in the presence of PQ. The induction of the Nrf2, HO-1 and NQO1 genes was first evident after 3h. The data indicate the potential of silymarin in alleviating PQ intoxication.