Drug repurposing: identification of SARS-CoV-2 potential inhibitors by virtual screening and pharmacokinetics strategies.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic caused global health, economic, and population loss. Variants of the coronavirus contributed to the severity of the disease and persistent rise in infections. This study aimed to identify potential drug candidates from fifteen approved antiviral drugs against SARS-CoV-2 (6LU7), SARS-CoV (5B6O), and SARS-CoV-2 spike protein (6M0J) using virtual screening and pharmacokinetics to gain insights into COVID-19 therapeutics. METHODOLOGY: We employed drug repurposing approach to analyze binding performance of fifteen clinically approved antiviral drugs against the main protease of SARS-CoV-2 (6LU7), SARS-CoV (5B6O), and SARS-CoV-2 spike proteins bound to ACE-2 receptor (6M0J), to provide an insight into the therapeutics of COVID-19. AutoDock Vina was used for docking studies. The binding affinities were calculated, and 2-3D structures of protein-ligand interactions were drawn. RESULTS: Rutin, hesperidin, and nelfinavir are clinically approved antiviral drugs with high binding affinity to proteins 6LU7, 5B6O, and 6M0J. These ligands have excellent pharmacokinetics, ensuring efficient absorption, metabolism, excretion, and digestibility. Hesperidin showed the most potent interaction with spike protein 6M0J, forming four H-bonds. Nelfinavir had a high human intestinal absorption (HIA) score of 0.93, indicating maximum absorption in the body and promising interactions with 6LU7. CONCLUSIONS: Our results indicated that rutin, hesperidin, and nelfinavir had the highest binding results against the proposed drug targets. The computational approach effectively identified SARS-CoV-2 inhibitors. COVID-19 is still a recurrent threat globally and predictive analysis using natural compounds might serve as a starting point for new drug development against SARS-CoV-2 and related viruses.

DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies.

DPP8/9 inhibition induces either pyroptotic or apoptotic cell death in hematological malignancies. We previously reported that treatment with the DPP8/9 inhibitor 1G244 resulted in apoptotic cell death in myeloma, and our current study further evaluates the mechanism of action of 1G244 in different blood cancer cell lines. Specifically, 1G244 inhibited DPP9 to induce GSDMD-mediated-pyroptosis at low concentrations and inhibited DPP8 to cause caspase-3-mediated-apoptosis at high concentrations. HCK expression is necessary to induce susceptibility to pyroptosis but does not participate in the induction of apoptosis. To further characterize this DPP8-dependent broad-spectrum apoptosis induction effect, we evaluated the potential antineoplastic role for an analog of 1G244 with higher DPP8 selectivity, tominostat (also known as 12 m). In vitro studies demonstrated that the cytotoxic effect of 1G244 at high concentrations was enhanced in tominostat. Meanwhile, in vivo work showed tominostat exhibited antitumor activity that was more effective on a cell line sensitive to 1G244, and at higher doses, it was also effective on a cell line resistant to 1G244. Importantly, the weight loss morbidity associated with increasing doses of 1G244 was not observed with tominostat. These results suggest the possible development of novel drugs with antineoplastic activity against selected hematological malignancies by refining and increasing the DPP8 selectivity of tominostat.

Isofraxidin enhances hyperthermia­induced apoptosis via redox modification in acute monocytic leukemia U937 cells.

The cell­killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat­induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre­treatment. Apoptosis was measured by Annexin V­FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT­induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase­3, caspase­8 and phosphorylated­JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD­FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT­induced apoptosis via a reactive oxygen species mediated mitochondria/caspase­dependent pathway in U937 cells.

Ginger ring compounds as an inhibitor of spike binding protein of alpha, beta, gamma and delta variants of SARS-CoV-2: An in-silico study.

The available drugs against coronavirus disease 2019 (COVOD-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are limited. This study aimed to identify ginger-derived compounds that might neutralize SARS-CoV-2 and prevent its entry into host cells. Ring compounds of ginger were screened against spike (S) protein of alpha, beta, gamma, and delta variants of SARS-CoV-2. The S protein FASTA sequence was retrieved from Global Initiative on Sharing Avian Influenza Data (GISAID) and converted into ".pdb" format using Open Babel tool. A total of 306 compounds were identified from ginger through food and phyto-databases. Out of those, 38 ring compounds were subjected to docking analysis using CB Dock online program which implies AutoDock Vina for docking. The Vina score was recorded, which reflects the affinity between ligands and receptors. Further, the Protein Ligand Interaction Profiler (PLIP) program for detecting the type of interaction between ligand-receptor was used. SwissADME was used to compute druglikeness parameters and pharmacokinetics characteristics. Furthermore, energy minimization was performed by using Swiss PDB Viewer (SPDBV) and energy after minimization was recorded. Molecular dynamic simulation was performed to find the stability of protein-ligand complex and root-mean- square deviation (RMSD) as well as root-mean-square fluctuation (RMSF) were calculated and recorded by using myPresto v5.0. Our study suggested that 17 out of 38 ring compounds of ginger were very likely to bind the S protein of SARS-CoV-2. Seventeen out of 38 ring compounds showed high affinity of binding with S protein of alpha, beta, gamma, and delta variants of SARS-CoV-2. The RMSD showed the stability of the complex was parallel to the S protein monomer. These computer-aided predictions give an insight into the possibility of ginger ring compounds as potential anti-SARS-CoV-2 worthy of in vitro investigations.

Frequency Of Ventricular Septal Rupture As A Complication Of Acute St-Elevation Myocardial Infarction In Patients Presenting To A Tertiary Care Hospital.

Background: Nearly one-third of the world's population dies from cardiovascular disorders, the majority of which are caused by stroke and coronary artery problems and 80 percent of these fatalities occur in impoverished countries. This study was conducted to determine the frequency of ventricular septal rupture in patients with acute ST-elevation myocardial infarction presenting to cardiology unit Hayatabad Medical Complex Peshawar. Methods: This was descriptive cross sectional research study at the Department of Cardiology, MTI-Hayatabad Medical Complex, Peshawar, from January to July 2020. Detailed history was obtained including duration of symptoms, co-morbidities present and occupation. A thorough clinical examination was done for signs of heart failure and ventricular septal rupture. Patients' demographics, clinical and laboratory parameters were recorded on a pro forma. All the data was analyzed statistically by using SPSS version 24.0. Results: A total of 179 patients were included in our study. Ventricular Septal Rupture (VSR), was recorded in 7 (3.9%) patients having Acute ST elevation of MI. In our study age, obesity, reperfusion therapy, location of MI and history of previous shock were observed to be non significantly (p˂0.05) associated with high incidence of Ventricular Septal Rupture in patients having Acute ST elevation of MI. Conclusion: According to our findings, individuals with PI-VSR have a significant risk of acute-phase death. Furthermore, a significant incidence of acute-phase fatalities has been related to female gender and severe cardiac failure upon admission.

Overexpression of D-dopachrome tautomerase increases ultraviolet B irradiation-induced skin tumorigenesis in mice.

Ultraviolet irradiation (UV) exposure is the leading factor underlying the development of skin malignancies. D-dopachrome tautomerase (D-DT), a functional homolog of macrophage migration inhibitory factor (MIF), has functional similarities to MIF. However, its role, unlike the role of MIF in photocarcinogenesis, is unknown. We therefore explored the role of D-DT in photocarcinogenesis by developing D-DT transgenic (D-DT Tg) mice and provided a research model for future studies targeting D-DT. Chronic UVB exposure accelerated tumor development in D-DT Tg mice compared with wild-type (WT) mice, with a higher incidence of tumors observed in D-DT Tg mice than in WT mice. In D-DT Tg irradiated mouse keratinocytes, the p53, PUMA, and Bax expression was lower than that in WT mice. These results indicate that D-DT Tg overexpression confers prevention against UVB-induced apoptosis in keratinocytes. Taken together, these findings support D-DT as a functionally important cytokine in photocarcinogenesis and potential therapeutic target for the prevention of photocarcinogenesis.

Berberine induces anti-atopic dermatitis effects through the downregulation of cutaneous EIF3F and MALT1 in NC/Nga mice with atopy-like dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with severe pruritus. Berberine, a naturally occurring isoquinoline alkaloid, has anti-inflammatory effects. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In this study, NC/Nga mice with atopy-like dermatitis (dermatitis mice), fibroblast and mast cells were used. In dermatitis mice, intermittent oral administrations of berberine 3 times a week for 12 days inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the expression of cutaneous eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also attenuated IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip® microarray showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The siRNAs for them inhibited the expression of MIF and IL-4 in antigen-stimulated mast cells. These results suggest that berberine improves AD-like symptoms through the inhibition of the eotaxin and pro-inflammatory cytokine expression and the related inflammatory cell recruitment. It is also suggested that the downregulation of EIF3F and MALT1 by berberine is involved in suppressing the cytokine expression. Taken together, berberine or berberine-containing crude drugs are expected to contribute to the improvement of AD symptoms.

Small size gold nanoparticles enhance apoptosis-induced by cold atmospheric plasma via depletion of intracellular GSH and modification of oxidative stress.

Gold nanoparticles (Au-NPs) have attracted attention as a promising sensitizer owing to their high atomic number (Z), and because they are considered fully multifunctional, they are preferred over other metal nanoparticles. Cold atmospheric plasma (CAP) has also recently gained attention, especially for cancer treatment, by inducing apoptosis through the formation of reactive oxygen species (ROS). In this study, the activity of different sized Au-NPs with helium-based CAP (He-CAP) was analyzed, and the underlying mechanism was investigated. Treating cells with only small Au-NPs (2 nm) significantly enhanced He-CAP-induced apoptosis. In comparison, 40 nm and 100 nm Au-NPs failed to enhance cell death. Mechanistically, the synergistic enhancement was due to 2 nm Au-NPs-induced decrease in intracellular glutathione, which led to the generation of intracellular ROS. He-CAP markedly induced ROS generation in an aqueous medium; however, treatment with He-CAP alone did not induce intracellular ROS formation. In contrast, the combined treatment significantly enhanced the intracellular formation of superoxide (O2• -) and hydroxyl radical (•OH). These findings indicate the potential therapeutic use of Au-NPs in combination with CAP and further clarify the role of Au-NPs in He-CAP-aided therapies.

The regulation of protein kinase casein kinase II by apigenin is involved in the inhibition of ultraviolet B-induced macrophage migration inhibitory factor-mediated hyperpigmentation.

Ultraviolet (UV) radiation elicits melanogenesis and pigmentation in the skin. Apigenin (4',5,7-trihydroxyflavone [AGN]) is a plant flavone contained in various herbs, fruits, and vegetables. We herein investigated antimelanogenic properties of AGN and the molecular mechanisms of the action of AGN. In UVB-treated mice, AGN inhibited cutaneous hyperpigmentation and macrophage migration inhibitory factor (MIF) expression as a melanogenesis-related key factor. In mouse keratinocytes, AGN inhibited the expression of MIF and also the related factors (e.g., stem cell factor and proteinase-activated receptor 2) induced by MIF. In addition to ellagic acid as a casein kinase II (CK2) inhibitor, AGN suppressed CK2 enzymatic activity and UVB-induced CK2 expression and subsequent phosphorylation of IκB and MIF expression. These results suggest that AGN inhibits UVB-induced hyperpigmentation through the regulation of CK2-mediated MIF expression in keratinocytes.

DPP8 is a novel therapeutic target for multiple myeloma.

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that are ideal therapeutic targets in human diseases. Indeed, DPP4 inhibitors are widely used in clinical practice as anti-diabetic agents. In this paper, we show that DPP4 inhibitors also induced cell death in multiple human myeloma cells. Among five DPP4 inhibitors, only two of them, vildagliptin and saxagliptin, exhibited apparent cytotoxic effects on myeloma cell lines, without any difference in suppression of DPP4 activity. As these two DPP4 inhibitors are known to have off-target effects against DPP8/9, we employed the specific DPP8/9 inhibitor 1G244. 1G244 demonstrated anti-myeloma effects on several cell lines and CD138+ cells from patients as well as in murine xenograft model. Through siRNA silencing approach, we further confirmed that DPP8 but not DPP9 is a key molecule in inducing cell death induced by DPP8/9 inhibition. In fact, the expression of DPP8 in CD38+ cells from myeloma patients was higher than that of healthy volunteers. DPP8/9 inhibition induced apoptosis, as evidenced by activated form of PARP, caspases-3 and was suppressed by the pan-caspase inhibitor Z-VAD-FMK. Taken together, these results indicate that DPP8 is a novel therapeutic target for myeloma treatment.

Combination of 5-aminosalicylic acid and hyperthermia synergistically enhances apoptotic cell death in HSC-3 cells due to intracellular nitric oxide/peroxynitrite generation.

The repurposing of existing FDA-approved non-cancer drugs is a potential source of new treatment options for cancer patients. An anti-inflammatory drug, 5-aminosalicylic acid (5-ASA), has been clinically used to treat inflammatory bowel disease. Hyperthermia (HT) is widely applicable addendum therapy with the existing cancer treatment modalities. Here, we addressed how 5-ASA combined with HT induces lethal effects in human oral squamous cell carcinoma (OSCC) HSC-3 cells. We found that 5-ASA/HT combination significantly inhibited the viability of HSC-3 cells, while cytotoxic effects in primary human dermal fibroblast cells were minor. Apoptotic endpoints were significantly increased by the 5-ASA/HT combined treatment, as evidenced by presence of Annexin V-FITC/PI positive cells, loss of MMP, Bcl-2/Bax ratio alteration, and increased Fas, cleaved Bid, and caspase expression. Interestingly, the enhancement of apoptosis was reversed in the presence of ON/ONOO- scavengers. These findings indicate that the combination treatment enhances apoptosis via ON/ONOO- mediated ER stress-Ca2+-mitochondria signaling and caspase-dependent apoptotic pathways. Our findings provide novel evidence that the combination of 5-ASA and HT is a promising approach for the enhancement of apoptosis; it may serve as an effective strategy for treating human OSCC.

Differential response of immortalized human amnion mesenchymal and epithelial cells against oxidative stress.

Cells are equipped with various antioxidant defense factors to antagonize insults from reactive oxygen species (ROS), thus the antioxidant capacity has been characterized by a variety of cellular responses during the pathophysiological processes. Amniotic cells have been extensively applied in clinical practice for burn treatment, corneal repair, and tissue regeneration. However, the antioxidative properties of amniotic cells have not yet been fully understood. Therefore, the current study was aimed to observe the response of amniotic cells against ROS stimuli, and to investigate the underlying molecular mechanisms. The immortalized human amniotic mesenchymal cells (iHAMs) and immortalized human amniotic epithelial cells (iHAEs) were used. The human skin fibroblast (HSF) was used as a control cell line. Changes in intracellular ROS generation, cell viability, and cellular morphology were investigated to reveal the response of amniotic cells against oxidative stresses induced by x-rays and hydrogen peroxide. In addition, expression of apoptosis-related proteins and response to antioxidative stress was also examined. The intracellular ROS level and cell apoptosis in iHAMs was remarkably increased. iHAEs showed relatively high resistance to ROS stimulation, which can be attributed to the high SOD2 expression and up-regulation of Nrf2, HO-1 after x-rays exposure. In contrast, iHAMs were found sensitive to oxidative damage. Expression of caspase-3, caspase-8 and BAX was increased, whereas down-regulation of Bcl-xL, Nrf2, HO-1, and TrxR-1. Taken together, findings have highlighted the characterization of response of amniotic derived epithelial and mesenchymal cells to oxidative stress. In physiological processes, iHAMs may play an important role to maintain the homeostasis of the pregnancy environment. However, under oxidative stimulations, iHAEs provides protection against oxidative damage in amnion tissue.

Roles of intracellular and extracellular ROS formation in apoptosis induced by cold atmospheric helium plasma and X-irradiation in the presence of sulfasalazine.

Sulfasalazine (SSZ) is a well-known anti-inflammatory drug and also an inhibitor of the cystine-glutamate antiporter that is known to reduce intracellular glutathione (GSH) level and increase cellular oxidative stress, indicating its anti-tumor potential. However, the combination of SSZ with other physical modalities remains unexplored. Here, the effects of SSZ on cold atmospheric helium plasma (He-CAP), which produces approximately 24 x higher concentration of hydroxyl radicals (. OH) compared to X-irradiation (IR) in aqueous solution, and on IR-induced apoptosis in human leukemia Molt-4 cells were studied to elucidate the mechanism of apoptosis enhancement. Both the Annexin V-FITC/PI and DNA fragmentation assay revealed that pre-treatment of cells with SSZ significantly enhanced He-CAP and IR-induced apoptosis. Similar enhancement was observed during the loss of mitochondrial membrane potential, intracellular Ca2+ ions, and mitochondria- and endoplasmic reticulum-related proteins. The concentration of intracellular reactive oxygen species (ROS) was much higher in He-CAP treated cells than in X-irradiated cells. On the other hand, strong enhancement of Fas expression and caspase-8 and -3 activities were only observed in X-irradiated cells. It might be possible that the higher concentration of intracellular and extracellular ROS suppressed caspase activities and Fas expression in He-CAP-treated cells. Notably, pretreating the cells with an antioxidant N-acetyl-L-cysteine (NAC) dramatically decreased apoptosis in cells treated by He-CAP, but not by IR. These results suggest that IR-induced apoptosis is due to specific and effective ROS distribution since intracellular ROS formation is marginal and the high production of ROS inside and outside of cells plays unique roles in He-CAP induced apoptosis. We conclude that our data provides efficacy and mechanistic insights for SSZ, which might be helpful for establishing SSZ as a future sensitizer in He-CAP or IR therapy for cancer.

Inflammatory cytokine-mediated induction of serine racemase in atopic dermatitis.

Serine racemase (SR) is an enzyme that catalyses the synthesis of d-serine, an endogenous coagonist for N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild-type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune-reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d-serine in stratum corneum in AD patients and healthy controls using a tape-stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d-serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor -α or macrophage migration inhibitory factor. Taken together, these findings suggest that d-serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.

Cold atmospheric helium plasma causes synergistic enhancement in cell death with hyperthermia and an additive enhancement with radiation.

Cold atmospheric plasmas (CAPs) have been proposed as a novel therapeutic method for its anti-cancer potential. However, its biological effects in combination with other physical modalities remain elusive. Therefore, this study examined the effects of cold atmospheric helium plasma (He-CAP) in combination with hyperthermia (HT) 42 °C or radiation 5 Gy. Synergistic enhancement in the cell death with HT and an additive enhancement with radiation were observed following He-CAP treatment. The synergistic effects were accompanied by increased intracellular reactive oxygen species (ROS) production. Hydrogen peroxide (H2O2) and superoxide (O2•-) generation was increased immediately after He-CAP treatment, but fails to initiate cell death process. Interestingly, at late hour's He-CAP-induced O2•- generation subsides, however the combined treatment showed sustained increased intracellular O2•- level, and enhanced cell death than either treatment alone. He-CAP caused marked induction of ROS in the aqueous medium, but He-CAP-induced ROS seems insufficient or not completely incorporated intra-cellularly to activate cell death machinery. The observed synergistic effects were due to the HT effects on membrane fluidity which facilitate the incorporation of He-CAP-induced ROS into the cells, thus results in the enhanced cancer cell death following combined treatment. These findings would be helpful when establishing a therapeutic strategy for CAP in combination with HT or radiation.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in Molt-4 cells.

PURPOSE: Transforming growth factor-ß-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line. MATERIALS AND METHODS: Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity. RESULTS: Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis. CONCLUSION: Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.

Role of macrophage migration inhibitory factor in heat-induced apoptosis in keratinocytes.

In human skin, keratinocytes are constantly challenged by adverse influences, such as hot and cold temperatures; however, the effects of heat on apoptosis induction in keratinocytes are not well understood. Macrophage migration inhibitory factor (MIF) is a potent cytokine that overcomes p53 function by suppressing its transcriptional activity. Here, we evaluated the effects of MIF on hyperthermia (HT)-induced apoptosis in MIF-deficient [knockout (KO)] and MIF-transgenic (Tg) mouse keratinocytes. Cells were exposed to HT at 44°C, and increased apoptosis was observed in MIF-KO and wild-type (WT) cells compared with MIF-Tg cells. To determine the mechanism, MIF-mediated changes in the cellular p53 level and its effects on p53-dependent death signaling (Bax and p21) and JNK signaling (p-JNK, JNK, p-Bad, and Bad) were investigated. MIF-Tg cells exhibited substantially decreased levels of p53 after HT treatment compared with WT and MIF-KO cells. In addition, HT treatment caused decreased expression of p-JNK and p-Bad in MIF-Tg cells; however, no such changes were observed in MIF-KO and WT cells. These results showed that the activation of JNK (p-JNK and p-Bad) and p53 may be involved in HT-induced apoptosis in keratinocytes and that enhanced endogenous MIF expression suppressed apoptosis.-Yoshihisa, Y., Rehman, M. U., Kondo, T., Shimizu, T. Role of macrophage migration inhibitory factor in heat-induced apoptosis in keratinocytes.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in A549 cells.

KRAS mutant lung cancers have long been considered as untreatable with drugs. Transforming growth factor-ß-activated kinase 1 (TAK1) appears to play an anti-apoptotic role in response to multiple stresses and has been reported to be a responsive kinase that regulates cell survival in KRAS-dependent cells. In this study, in order to find a useful approach to treat KRAS mutant lung cancer, we focused on the combined effects of 5Z-7-oxozeaenol, a TAK1 inhibitor, with hyperthermia (HT) in KRAS mutant lung cancer cell line A549. Annexin V-FITC/PI assay, cell cycle analysis, and colony formation assay revealed a significant enhancement in apoptosis induced by HT treatment, when the cells were pre-incubated with 5Z-7-oxozeaenol in a dose-dependent manner. The enhanced apoptosis by 5Z-7-oxozeaenol was accompanied by a significant increase in reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (MMP). In addition, western blot showed that 5Z-7-oxozeaenol enhanced HT-induced expressions of cleaved caspase-3, cleaved caspase-8, and HSP70 and decreased HT-induced expressions of Bcl-2, p-p38, p-JNK, and LC3. Moreover, 5Z-7-oxozeaenol pre-treatment resulted in a marked elevation of intracellular calcium level which might be associated with endoplasmic reticulum (ER) stress-related pathway. Taken together, our data provides further insights of the mechanism of action of 5Z-7-oxozeaenol and HT treatment, and their potential application as a novel approache to treat patients with KRAS mutant lung cancer.

Helium-based cold atmospheric plasma-induced reactive oxygen species-mediated apoptotic pathway attenuated by platinum nanoparticles.

Plasma is generated by ionizing gas molecules. Helium (He)-based cold atmospheric plasma (CAP) was generated using a high-voltage power supply with low-frequency excitation (60 Hz at 7 kV) and He flow at 2 l/min. Platinum nanoparticles (Pt-NPs) are potent antioxidants due to their unique ability to scavenge superoxides and peroxides. These features make them useful for the protection against oxidative stress-associated pathologies. Here, the effects of Pt-NPs on He-CAP-induced apoptosis and the underlying mechanism were examined in human lymphoma U937 cells. Apoptosis was measured after cells were exposed to He-CAP in the presence or absence of Pt-NPs. The effects of combined treatment were determined by observing the changes in intracellular reactive oxygen species (ROS) and both mitochondrial and Fas dependent pathway. The results indicate that Pt-NPs substantially scavenge He-CAP-induced superoxides and peroxides and inhibit all the pathways involved in apoptosis execution. This might be because of the SOD/catalase mimetic effects of Pt-NPs. These results showed that the Pt-NPs can induce He-CAP desensitization in human lymphoma U937 cells.

Comparison of free radicals formation induced by cold atmospheric plasma, ultrasound, and ionizing radiation.

Plasma medicine is increasingly recognized interdisciplinary field combining engineering, physics, biochemistry and life sciences. Plasma is classified into two categories based on the temperature applied, namely "thermal" and "non-thermal" (i.e., cold atmospheric plasma). Non-thermal or cold atmospheric plasma (CAP) is produced by applying high voltage electric field at low pressures and power. The chemical effects of cold atmospheric plasma in aqueous solution are attributed to high voltage discharge and gas flow, which is transported rapidly on the liquid surface. The argon-cold atmospheric plasma (Ar-CAP) induces efficient reactive oxygen species (ROS) in aqueous solutions without thermal decomposition. Their formation has been confirmed by electron paramagnetic resonance (EPR) spin trapping, which is reviewed here. The similarities and differences between the plasma chemistry, sonochemistry, and radiation chemistry are explained. Further, the evidence for free radical formation in the liquid phase and their role in the biological effects induced by cold atmospheric plasma, ultrasound and ionizing radiation are discussed.