Cytosolic Cytochrome Research Articles

Effects of chronic heat stress on Ca2+ homeostasis, apoptosis, and protein carbonylation profiles in the breast muscle of broilers

Heat stress (HS) largely impairs the quality of broiler breast meat through protein oxidative modification. This study aimed to investigate the carbonylation pattern of Ca2+ channels and apoptotic proteins in the breast muscle of heat-stressed broilers. A total of 144 twenty-eight-day-old male Arbor Acres broilers were randomly divided into three treatment groups. The normal control (NC) group was kept at 22°C and provided with unlimited feed. The HS group was exposed to 32°C and provided with unlimited feed. The pair-fed (PF) group was kept at 22°C and given an amount of feed equivalent to that consumed by the HS group on the previous day. Results showed that broilers under HS conditions had a higher respiratory rate than those in NC and PF groups (P < 0.05). HS disrupted the morphology and structure of breast muscle fibers by decreasing the average diameters and average density of myofibers compared to the NC group (P < 0.05). HS increased the mean fluorescence intensity of the positive carbonyl signal in breast muscle compared with the NC group (P < 0.05). Besides, the pectoral Ca2+ concentration in the sarcoplasmic reticulum, cytoplasm, and mitochondria was elevated by HS when compared with the NC group (P < 0.05). In comparison to the NC and PF groups, HS increased the apoptosis rate and caspase-3 activity in the breast muscle (P < 0.05). Furthermore, HS elevated the relative protein expressions of plasma membrane Ca2+-ATPase, Na+/Ca2+ exchanger 1, and sarco/endoplasmic reticulum calcium transport ATPase 1 compared to the NC group (P < 0.05). Higher relative protein expression of μ-calpain and lower relative protein expression of cytosolic cytochrome complex were found in the HS group than the NC group (P < 0.05). HS decreased the carbonylation levels of transient receptor potential canonical 1 and inositol 1,4,5-trisphosphate receptor compared to the NC group (P < 0.05). Additionally, the carbonylation levels of cleaved caspase-3 and precursor caspase-9 were increased and decreased, respectively, by HS treatment compared to the NC group (P < 0.05). In conclusion, HS damages the myofiber based on Ca2+ dyshomeostasis and apoptosis, which are potentially associated with protein carbonylation. These results shed new light on the possible mechanism behind the development of poor meat quality in broilers due to HS.

Domperidone, a Dopamine Receptor D2 Antagonist, Induces Apoptosis by Inhibiting the ERK/STAT3-Mediated Pathway in Human Colon Cancer HCT116 Cells.

Colorectal cancer (CRC) continues to demonstrate high incidence and mortality rates, emphasizing that implementing strategic measures for prevention and treatment is crucial. Recently, the dopamine receptor D2 (DRD2), a G protein-coupled receptor, has been reported to play multiple roles in growth of tumor cells. This study investigated the anticancer potential of domperidone, a dopamine receptor D2 antagonist, in HCT116 human CRC cells. Domperidone demonstrated concentration- and time-dependent reductions in cell viability, thereby inducing apoptosis. The molecular mechanism revealed that domperidone modulated the mitochondrial pathway, decreasing mitochondrial Bcl-2 levels, elevating cytosolic cytochrome C expression, and triggering caspase- 3, -7, and -9 cleavage. Domperidone decreased in formation of β-arrestin2/MEK complex, which contributing to inhibition of ERK activation. Additionally, treatment with domperidone diminished JAK2 and STAT3 activation. Treatment of U0126, the MEK inhibitor, resulted in reduced phosphorylation of MEK, ERK, and STAT3 without alteration of JAK2 activation, indicating that domperidone targeted both MEK-ERK-STAT3 and JAK2-STAT3 signaling pathways, respectively. Immunoblot analysis revealed that domperidone also downregulated DRD2 expression. Domperidone-induced reactive oxygen species (ROS) generation and N-acetylcysteine treatment mitigated ROS levels and restored cell viability. An in vivo xenograft study verified the significant antitumor effects of domperidone. These results emphasize the multifaceted anticancer effects of domperidone, highlighting its potential as a promising therapeutic agent for human CRC.

Effect of vitamin E δ-tocotrienol and aspirin on Wnt signaling in human colon cancer stem cells and in adenoma development in APCmin/+ mice.

In this study, we evaluated the effects of vitamin E δ-tocotrienol (DT3) and aspirin on Wnt signaling in human colon cancer stem cells (CCSCs) and in the prevention of adenoma formation in APCmin/+ mice. We found that knockdown of the adenomatous polyposis coli (APC) gene led to subsequent activation of Wnt signaling in colon epithelial cells (NCM460-APCsiRNA) and induction of β-catenin and its downstream target proteins c-MYC, cyclin D1, and survivin. When aspirin and DT3 were combined, cell growth and survival were inhibited and apoptosis was induced in colon epithelial cells and in CCSCs. However, DT3 and/or aspirin had little or no effect on control normal colon epithelial cells (NCM460-NCsiRNA). The induction of apoptosis was directly related to activation of caspase 8 and cleavage of BID to truncated BID. In addition, DT3 and/or aspirin-induced apoptosis was associated with cleaved PARP, elevated levels of cytosolic cytochrome c and BAX, and depletion of anti-apoptotic protein BCl-2 in CCSCs. The combination of aspirin and DT3 inhibited the self-renewal capacity, Wnt/β-catenin receptor activity, and expression of β-catenin and its downstream targets c-MYC, cyclin D1 and survivin in CCSCs. We also found that treatment with DT3 alone or combined with aspirin significantly inhibited intestinal adenoma formation and Wnt/ β-catenin signaling and induced apoptosis, compared to vehicle, in APCmin/+ mice. Our study demonstrated a rationale for further investigation of the combination of DT3 and aspirin for colorectal cancer prevention and therapy.

Mechanistic Understanding of Dexamethasone-Mediated Protection against Remdesivir-Induced Hepatotoxicity.

Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.

Green-Synthesized Silver and Selenium Nanoparticles Using Berberine: A Comparative Assessment of In Vitro Anticancer Potential on Human Hepatocellular Carcinoma Cell Line (HepG2).

A well-known natural ingredient found in several medicinal plants, berberine (Ber), has been shown to have anticancer properties against a range of malignancies. The limited solubility and bioavailability of berberine can be addressed using Ber-loaded nanoparticles. In this study, we compared the in vitro cytotoxic effects of both Ber-loaded silver nanoparticles (Ber-AgNPs) and Ber-loaded selenium nanoparticles (Ber-SeNPs) in the human liver cancer cell line (HepG2) and mouse normal liver cells (BNL). The IC50 values in HepG2 for berberine, Ber-AgNPs, Ber-SeNPs, and cisplatin were 26.69, 1.16, 0.04, and 0.33 µg/mL, respectively. Our results show that Ber and its Ag and Se nanoparticles exerted a good antitumor effect against HepG2 cells by inducing apoptosis via upregulating p53, Bax, cytosolic cytochrome C levels, and caspase-3 activity, and the down-regulation of Bcl-2 levels. Similarly, incubation with Ber and both Ber-NPs (Ag and Se) led to a significant dose-dependent elevation in inflammatory markers' (TNF-α, NF-κB, and COX-2) levels compared to the control group. In addition, it led to the arrest of the G1 cell cycle by depleting the expression of cyclin D1 and CDK-2 mRNA. Furthermore, Ber and both Ber-NPs (Ag and Se) caused a significant dose-dependent increase in LDH activity in HepG2 cells. Furthermore, our findings offer evidence that Ber and its nanoparticles intensified oxidative stress in HepG2 cells. Furthermore, the migration rate of cells subjected to berberine and its nanoforms was notably decreased compared to that of control cells. It can be inferred that Ber nanoparticles exhibited superior anticancer efficacy against HepG2 compared to unprocessed Ber, perhaps due to their improved solubility and bioavailability. Furthermore, Ber-SeNPs exhibited greater efficacy than Ber-AgNPs, possibly as a result of the inherent anticancer characteristics of selenium.

Resistomycin Suppresses Prostate Cancer Cell Growth by Instigating Oxidative Stress, Mitochondrial Apoptosis, and Cell Cycle Arrest

Globally, prostate cancer is among the most threatening and leading causes of death in men. This study, therefore, aimed to search for an ideal antitumor strategy with high efficacy, low drug resistance, and no or few adverse effects. Resistomycin is a natural antibiotic derived from marine actinomycetes, and it possesses various biological activities. Prostate cancer cells (PC3) were treated with resistomycin (IC12.5: 0.65 or IC25: 1.3 µg/mL) or 5-fluorouracil (5-FU; IC25: 7 µg/mL) for 24 h. MTT assay and flow cytometry were utilized to assess cell viability and apoptosis. Oxidative stress, apoptotic-related markers, and cell cycle were also assessed. The results revealed that the IC50 of resistomycin and 5-FU on PC3 cells were 2.63 µg/mL and 14.44 µg/mL, respectively. Furthermore, treated cells with the high dose of resistomycin showed an increased number of apoptotic cells compared to those treated with the lower dose. Remarkable induction of reactive oxygen species generation and lactate dehydrogenase (LDH) leakage with high malondialdehyde (MDA), carbonyl protein (CP), and 8-hydroxyguanosine (8-OHdG) contents were observed in resistomycin-treated cells. In addition, marked declines in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in PC3 cells subjected to resistomycin therapy were observed. Resistomycin triggered observable cell apoptosis by increasing Bax, caspase-3, and cytosolic cytochrome c levels and decreasing Bcl-2 levels. In addition, notable downregulation of proliferating cell nuclear antigen (PCNA) and cyclin D1 was observed in resistomycin-treated cancerous cells. According to this evaluation, the antitumor potential of resistomycin, in a concentration-dependent manner, in prostate cancer cells was achieved by triggering oxidative stress, mitochondrial apoptosis, and cell cycle arrest in cancer cells. In conclusion, our investigation suggests that resistomycin can be considered a starting point for developing new chemotherapeutic agents for human prostate cancer.

Protective Effects of Heat-Killed Ruminococcus albus against β-Amyloid-Induced Apoptosis on SH-SY5Y Cells.

A high level of β-amyloid (Aβ) in the brains of patients with Alzheimer's disease (AD) generates reactive oxygen species that induce neuronal death and DNA damage. The interaction between the gut microbiota and brain health has attracted attention in recent years. Heat-killed Ruminococcus albus (hkRA) reportedly protects neurons against damage induced by oxidative stress. However, whether hkRA can inhibit Aβ-induced apoptosis and thus alleviate AD remains unclear. Hence, we aimed to evaluate the protective effects of hkRA against Aβ-induced apoptosis on the human neuroblastoma SH-SY5Y cell. HkRA treatment (108 cells/ml) significantly decreased the Aβ-induced cytotoxicity and DNA damage in the SH-SY5Y cells. It also showed a significant increase of the bax/bcl-2 ratio in the Aβ-treated SH-SY5Y cells. Moreover, hkRA treatment stimulated the expression of antioxidation-related genes HO-1, Nrf2, and PKC-δ and increased the expression of brain-derived neurotrophic factor (BDNF). Meanwhile, it significantly decreased the activity of caspase-3 and protein expression of cleaved caspase-3 in the Aβ-treated SH-SY5Y cells. Additionally, the protein levels of mitochondrial and cytosolic cytochrome c increased and decreased, respectively, in the cells. These results suggest that hkRA protects human neuroblastoma cells from Aβ-induced apoptosis and oxidative stress. Thus, hkRA may be developed into a health-promoting paraprobiotic (the inactivated microbial cells of probiotics) for patients with AD.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Tanshinone I alleviates steroid-induced osteonecrosis of femoral heads and promotes angiogenesis: in vivo and in vitro studies

BackgroundThe impaired blood supply to the bones is an important pathological feature of steroid-induced osteonecrosis of the femoral head (SIONFH). Danshen is a Chinese herb that shows therapeutic effects on SIONFH, but the effects of one of its major bioactive constituents, Tanshinone I (TsI), on SIONFH remain unknown. Here, we evaluated the effects of TsI on SIONFH, particularly focusing on its effects on angiogenesis, in in vivo and in vitro research.MethodsSIONFH was induced in Sprague–Dawley rats by an intramuscular injection of methylprednisolone (40 mg/kg) in combination with an intraperitoneal injection of lipopolysaccharide (20 μg/kg). Morphological alterations of the femoral head were observed by dual-energy X-ray absorptiometry and HE staining. Western blot, qRT-PCR, and immunohistochemical/immunofluorescence staining were used to determine gene expression.ResultsTsI (10 mg/kg) alleviated bone loss and rescued the expression of angiogenesis-related molecules (CD31, VWF, VEGF, and VEGFR2) in the femoral heads of SIONFH rats. Notably, TsI rescued the down-regulated expression of SRY-box transcription factor 11 (SOX11) in CD31+ endothelial cells in the femoral heads of SIONFH rats. In vitro studies showed that TsI preserved the dexamethasone-harmed angiogenic property (migration and tube formation) of human umbilical vein cells (EA.hy926), suppressed dexamethasone-induced cell apoptosis, reduced pro-apoptotic proteins (cytosolic cytochrome C, Bax, and caspase 3/9) and increased anti-apoptotic protein Bcl-2, whereas silencing of SOX11 reversed these beneficial effects.ConclusionsThis study demonstrates that TsI alleviates SIONFH and promotes angiogenesis by regulating SOX11 expression. Our work would provide new evidence for the application of TsI to treat SIONFH.Graphical

Dynamic changes in postmortem quality of mirror carp (Cyprinus carpio L.): Based on oxidation reaction and mitochondrial function properties

The dynamic changes in the postmortem quality of mirror carp (Cyprinus carpio L.) were investigated. With extended postmortem time, conductivity, redness, lipid oxidation, and protein oxidation all increased, while lightness, whiteness, and freshness decreased. At 4 h postmortem, the pH value reached a minimum (6.58), while the centrifugal loss and hardness reached a maximum (17.13% and 2539 g). Additionally, variations in mitochondria-related parameters during apoptosis were studied. Within 72 h postmortem, the content of reactive oxygen species initially decreased and subsequently increased; furthermore, there was a significant increase in the mitochondrial membrane permeability transition pore, membrane fluidity, and swelling (P < 0.05). Meanwhile, the cytosolic cytochrome c level decreased from 0.71 to 0.23, which indicated potential mitochondrial damage. Thus, mitochondrial dysfunction during postmortem aging can give rise to oxidation and the production of ammonia and amine compounds, which leads to flesh quality deterioration.

Gamma irradiation induces a pro-apoptotic state in longer stored platelets, without progressing to an overt apoptosis by day 7 of storage.

Although gamma-irradiation to platelet products is a standard method to prevent the risk of TA-GVHD in vulnerable recipients, it induces some proteomic and redox changes, of which irradiation-induced ROS increments may potentiate platelet mitochondrial dysfunction. However, whether these changes cause platelet apoptosis, or affect their viability during storage, is the main subject of this study. PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma-irradiation. Within 7-days storage, cytosolic and mitochondrial levels of cytochrome c and pro-apoptotic molecules of Bak and Bax were evaluated by western-blotting. Intraplatelet active caspase (using FAM-DEVD-FMK) and PS-exposure were detected by flowcytometry. Caspase activity in platelet lysate was also confirmed by immunofluorescence detection of Caspase-3/7 Substrate N-Ac-DEVD-N'-MC-R110 while platelet viability was evaluated with MTT assays. Cytosolic cytochrome c gradually increased while its mitochondrial content steadily declined during 7 days of storage. In a contrary trend, reverse patterns were observed for Bak and Bax expressions. Gamma-irradiated platelets showed higher release of mitochondrial cytochrome c that reflected by higher cytosolic cytochrome c levels on day 7 of storage. Concurrently mitochondrial pro-apoptotic Bak and Bax proteins increased on day 7 in irradiated products. However, gamma-irradiation didn't significantly increase caspase activity or PS-exposure, nor did it decrease platelet viability. Here, consistent with studies on "gamma-irradiation-induced oxidative stress", we showed that gamma-ray also increases platelet pro-apoptotic signals during storage, although not strongly enough to affect platelet viability by overt apoptosis induction. Conclusively, whether supplementing ROS scavengers or antioxidants to irradiated platelets can improve their quality during storage may be of interest for future research.

Tyrosine Kinase Inhibitors Diminish Renal Neoplasms in a Tuberous Sclerosis Model Via Induction of Apoptosis.

Tuberous sclerosis complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition because recurrence of disease occurs after treatment is discontinued. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage-specific platelet-derived growth factor receptor β (PDGFRβ) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin-induced versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, RNA sequencing, and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in tuberous sclerosis Tsc2+/- mouse models (C57BL/6J mice; N = 6; 400 mg/kg/d; oral gavage) compared with Tsc2+/- mice treated with PBS (C57BL/6J mice; N = 6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared with PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17β-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

Polysaccharides from Annonamuricata leaves protect against cisplatin‑induced cytotoxicity in macrophages by alleviating mitochondrial dysfunction.

Cisplatin is a prominent chemotherapeutic agent that can induce significant damage to normal cells. Therefore, it is important to develop agents that protect normal cells without influencing the chemotherapeutic effect of cisplatin. The present study was conducted to explore the protective effects of Annonamuricata leaf polysaccharides (ALPS) against cisplatin‑induced toxicity in macrophages. Apoptosis was assessed in macrophages and lung cancer cells to investigate the cytoprotective effect of ALPS, their effect on the production of cisplatin‑induced reactive oxygen species (ROS) and the loss of the mitochondrial transmembrane potential (MTP). Cisplatin, when used alone or in combination with ALPS, showed significant toxicity against A549 and H460 lung cancer cells. However, cisplatin‑induced cytotoxicity was suppressed by cotreatment of RAW 264.7 macrophages with ALPS. ALPS significantly inhibited the upregulation of Bax, cytosolic cytochrome c and caspases‑3, ‑8 and ‑9. Moreover, ALPS resulted in the cleavage of PARP and downregulation of Bcl‑2 levels in a concentration‑dependent manner, which ultimately led to a reduction in the apoptotic and necrotic populations of cisplatin‑treated RAW 264.7 macrophages. The suppression of the apoptotic signaling pathways was mediated through the reduction of ROS and MTP loss in cisplatin‑treated RAW 264.7 macrophages. In addition, ALPS alleviated cell damage by suppressing the mitochondrial apoptotic pathways in cisplatin‑treated bone marrow‑derived macrophages. Together, these findings suggested that ALPS may alleviate the toxic side effects of chemotherapeutic agents and act as a potential candidate for use as an effective adjuvant therapy.

Involvement of AMPKα and MAPK-ERK/-JNK Signals in Docetaxel-Induced Human Tongue Squamous Cell Carcinoma Cell Apoptosis.

Cancers of the oral cavity can develop in the anatomic area extending from the lip, gum, tongue, mouth, and to the palate. Histologically, about 85-90% of oral cavity cancers are of the type squamous cells carcinomas (SCCs). The incidence of oral tongue SCC is higher in the tongue than any other anatomic area of the oral cavity. Here, we investigated the therapeutic effects and molecular mechanisms of docetaxel, which is a paclitaxel antitumor agent, on the cell growth of a human tongue SCC-derived SAS cell line. The results showed that docetaxel (10-300 nM) induced cytotoxicity and caspase-3 activity in SAS cells. Moreover, docetaxel (100 nM) promoted the expression of apoptosis-related signaling molecules, including the cleavages of caspase-3, caspase-7, and poly (ADP-ribose) polymerase (PARP). In mitochondria, docetaxel (100 nM) decreased the mitochondrial membrane potential (MMP) and Bcl-2 mRNA and protein expression and increased cytosolic cytochrome c protein expression and Bax mRNA and protein expression. In terms of mitogen-activated protein kinase (MAPK) and adenosine monophosphate-activated protein kinase (AMPK) signaling, docetaxel increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-c-Jun N-terminal kinase (JNK), and p-AMPKα protein expression but not p-p38 protein expression. Moreover, the increase in caspase-3/-7 activity and Bax protein expression and decreased Bcl-2 protein expression and MMP depolarization observed in docetaxel-treated SAS cells could be reversed by treatment with either SP600125 (a JNK inhibitor), PD98059 (an MEK1/2 (mitogen-activated protein kinase kinase 1/2) inhibitor), or compound c (an AMPK inhibitor). The docetaxel-induced increases in p-JNK, p-ERK, and p-AMPKα protein expression could also be reversed by treatment with either SP600125, PD98059, or compound c. These results indicate that docetaxel induces human tongue SCC cell apoptosis via interdependent MAPK-JNK, MAPK-ERK1/2, and AMPKα signaling pathways. Our results show that docetaxel could possibly exert a potent pharmacological effect on human oral tongue SCC cell growth.

ATF5 is a regulator of exercise-induced mitochondrial quality control in skeletal muscle

ObjectivesThe Mitochondrial Unfolded Protein Response (UPRmt) is a compartment-specific mitochondrial quality control (MQC) mechanism that uses the transcription factor ATF5 to induce the expression of protective enzymes to restore mitochondrial function. Acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis, however, the roles of ATF5 and the UPRmt in maintaining basal mitochondrial content, function and exercise-induced MQC mechanisms in skeletal muscle are not known. MethodsATF5 KO and WT mice were examined at rest or after a bout of acute endurance exercise. We measured protein content in whole muscle, nuclear, cytosolic and mitochondrial fractions, in addition to mRNA transcript levels in whole muscle. Using isolated mitochondria, we quantified rates of oxygen consumption and ROS emission to observe the effects of the absence of ATF5 on organelle function. ResultsATF5 KO mice exhibited a larger and less functional muscle mitochondrial pool, most likely a culmination of enhanced biogenesis via increased PGC-1α expression, and attenuated mitophagy. The absence of ATF5 resulted in a reduction in antioxidant proteins and increases in mitochondrial ROS emission, cytosolic cytochrome c, and the expression of mitochondrial chaperones. KO muscle also displayed enhanced exercise-induced stress kinase signaling, but a blunted mitophagic and UPRmt gene expression response, complemented by significant increases in the basal mRNA abundance and nuclear localization of ATF4. Instead of promoting its nuclear translocation, acute exercise caused the enrichment of ATF5 in mitochondrial fractions. We also identified PGC-1α as an additional regulator of the basal expression of UPRmt genes. ConclusionThe transcription factor ATF5 retains a critical role in the maintenance of mitochondrial homeostasis and the appropriate response of muscle to acute exercise for the optimization of mitochondrial quality control.

Preservation effect of imeglimin on pancreatic β-cell mass: Noninvasive evaluation using 111In-exendin-4 SPECT/CT imaging and the perspective of mitochondrial involvements.

Progressive loss of β-cell mass (BCM) has a pernicious influence on type 2 diabetes mellitus (T2DM); evaluation of BCM has conventionally required an invasive method that provides only cross-sectional data. However, a noninvasive approach to longitudinal assessment of BCM in living subjects using an indium 111–labeled exendin-4 derivative ([Lys12(111In-BnDTPA-Ahx)]exendin-4) (111In-exendin-4) has been developed recently. Imeglimin is a novel antidiabetic agent that is reported to improve glycemic control and glucose-stimulated insulin secretion (GSIS) via augmentation of mitochondrial function. However, the influence of imeglimin on BCM is not fully understood. We have investigated the effects of imeglimin on BCM in vivo in prediabetic db/db mice using a noninvasive 111In-exendin-4 single-photon emission computed tomography/computed tomography (SPECT/CT) technique. During the 5-week study period, imeglimin treatment attenuated the progression of glucose intolerance, and imeglimin-treated mice retained greater BCM than control, which was consistent with the results of 111In-exendin-4 SPECT/CT scans. Furthermore, immunohistochemical analysis revealed reduced β-cell apoptosis in the imeglimin-treated db/db mice, and also lowered release of cytosolic cytochrome c protein in the β cells. Furthermore, electron microscopy observation and membrane potential measurement revealed improved structural integrity and membrane potential of the mitochondria of imeglimin-treated islets, respectively. These results demonstrate attenuation of progression of BCM loss in prediabetic db/db mice partly via inhibition of mitochondria-mediated apoptosis.

S9.4b Trimetallic Cu-Zn-Fe nanoparticles induced apoptosis and cell cycle arrest in multidrug-resistant Candida auris S9.4 Free oral presentations (late breaking), September 23, 2022, 4:45 PM - 6:15 PM

Background Candida species are opportunistic fungus that can cause serious infections, particularly in immunocompromised population. The number of fungal infections has increased steadily with Candida species being responsible for ˃ 70% of these instances, particularly in hospitalized patients with significant underlying conditions. Pharmacological resistance in Candida species and the advent of Candida auris have elevated candidiasis to a major public health concern. Candida auris is an emerging multidrug-resistant fungus that can cause catastrophic bloodstream infections and high fatality rates, particularly in hospitalized patients with major medical issues. Antifungal study of trimetallic nanoparticles (NPs) of various types have been studied as a therapy option for efficient and safe control of candidiasis. These NPs were highlighted for being environmentally friendly and sustainable synthetic preparative possibilities.ObjectiveThis work aimed to synthesize and characterize novel Cu-Zn-Fe trimetallic NPs and determine their in vitro antifungal activity and mechanism of antifungal action against Candida auris isolates.MethodsThe synthesis and characterization of Cu-Zn-Fe trimetallic NPs was done by standard methods. The antifungal capability of these NPs were determined by calculating minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) following CLSI recommended guidelines. Susceptibility on planktonic cells and biofilms was further confirmed by MuseTM cell count and viability assay and scanning electron microscopy (SEM) respectively. For insight antifungal mechanisms, apoptosis and cell cycle arrest were studied by exploring different apoptotic markers and MuseTM cell analyzer.ResultsCharacterizations by Fourier-transform infrared spectroscopy (FTIR), diffuse reflectance UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) determine the successful biosynthesis of Cu-Zn-Fe trimetallic NPs. Susceptibility assay confirmed the fungicidal activity of Cu-Zn-Fe NPs with MIC and MFC values of 12.5 and 25 μg/ml respectively. These results were further confirmed by viability assay reporting the cell viability of 45.5%, 13.5%, and 1.8% when C. auris cells were treated with 1/2 MIC, MIC, and 2MIC respectively. Cell cycle analysis revealed that 91.2% of healthy developing untreated control cells were in G0/G1 phase, whereas 5.2% and 3.7% of cells were in the S phase and G2/M phase, respectively. In contrast, NP-treated cells were observed to be arrested in S phase with 49.3% cells at 2MIC. To study the physiology of cell death caused by NPs, we investigated mitochondrial membrane potential (∆ψm), with live cells having stable (∆ψm) whereas treated cells showed loss of (∆ψm). Another important parameter of apoptosis in yeast cells is the release of cytochrome C from the mitochondria to the cytosol and NP-treated cells resulting in decreased mitochondrial cytochrome C and elevated cytosolic cytochrome C levels. Both results confirmed the potential test NPs in causing apoptotic cell death in C. auris.ConclusionThe trimetallic (Cu-Zn-Fe) nanoparticles displayed strong antifungal activity against C. auris, with a potential to arrest the cell cycle at S-phase, which could be linked to the DNA damage. Important yeast apoptotic markers suggested that the test NPs have a potential to cause apotosis in C. auris. All these findings suggest the potential of these trimetallic NPs to be taken to the next level of research in the development of novel antifungal medications.

Work and oxygen consumption of isolated right ventricular papillary muscle in experimental pulmonary hypertension.

Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis tested was that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular muscle preparations were subjected to 5Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption ( ) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane and monoamine oxidase and glucose 6-phosphate dehydrogenase activities and phosphatidylglycerol/cardiolipin contents were determined. Mechanical efficiency ranged from 23% to 11% in control (n=6) and from 22% to 1% in MCT-PH (n=15) and correlated with work (r2 =0.68, P<0.0001) but not with (r2 =0.004, P=0.7919). for cross-bridge cycling was proportional to work (r2 =0.56, P=0.0005). Blebbistatin-resistant (r2 =0.32, P=0.0167) and proton permeability of the mitochondrial inner membrane (r2 =0.36, P=0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination r2 =0.79, P=0.0001). Cytosolic cytochrome c correlated inversely with work (r2 =0.28, P=0.0391), but not with efficiency (r2 =0.20, P=0.0867). Glucose 6-phosphate dehydrogenase, monoamine oxidase and phosphatidylglycerol/cardiolipin increased in the right ventricular wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is a result of activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant are discussed. KEY POINTS: Mechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown. Work and oxygen consumption are determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements. Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension. Reduced work and mechanical efficiency are related to mitochondrial dysfunction. Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is a resiult of the excessive production of reactive oxygen species.

Sublethal cytochrome c release generates drug-tolerant persister cells

Drug-tolerant persister cells (persisters) evade apoptosis upon targeted and conventional cancer therapies and represent a major non-genetic barrier to effective cancer treatment. Here, we show that cells that survive treatment with pro-apoptotic BH3 mimetics display a persister phenotype that includes colonization and metastasis invivo and increased sensitivity toward ferroptosis by GPX4 inhibition. We found that sublethal mitochondrial outer membrane permeabilization (MOMP) and holocytochrome c release are key requirements for the generation of the persister phenotype. The generation of persisters is independent of apoptosome formation and caspase activation, but instead, cytosolic cytochrome c induces the activation of heme-regulated inhibitor (HRI) kinase and engagement of the integrated stress response (ISR) with the consequent synthesis of ATF4, all of which are required for the persister phenotype. Our results reveal that sublethal cytochrome c release couples sublethal MOMP to caspase-independent initiation of an ATF4-dependent, drug-tolerant persister phenotype.