Reduction Of Mitochondrial Potential Research Articles

Phototoxic Reactions Inducted by Hydrochlorothiazide and Furosemide in Normal Skin Cells-In Vitro Studies on Melanocytes and Fibroblasts.

Hypertension is known to be a multifactorial disease associated with abnormalities in neuroendocrine, metabolic, and hemodynamic systems. Poorly controlled hypertension causes more than one in eight premature deaths worldwide. Hydrochlorothiazide (HCT) and furosemide (FUR), being first-line drugs in the treatment of hypertension, are among others the most frequently prescribed drugs in the world. Currently, many pharmacoepidemiological data associate the use of these diuretics with an increased risk of adverse phototoxic reactions that may induce the development of melanoma and non-melanoma skin cancers. In this study, the cytotoxic and phototoxic potential of HCT and FUR against skin cells varied by melanin pigment content was assessed for the first time. The results showed that both drugs reduced the number of metabolically active normal skin cells in a dose-dependent manner. UVA irradiation significantly increased the cytotoxicity of HCT towards fibroblasts by approximately 40% and melanocytes by almost 20% compared to unirradiated cells. In the case of skin cells exposed to FUR and UVA radiation, an increase in cytotoxicity by approximately 30% for fibroblasts and 10% for melanocytes was observed. Simultaneous exposure of melanocytes and fibroblasts to HCT or FUR and UVAR caused a decrease in cell viability, and number, which was confirmed by microscopic assessment of morphology. The phototoxic effect of HCT and FUR was associated with the disturbance of redox homeostasis confirming the oxidative stress as a mechanism of phototoxic reaction. UVA-irradiated drugs increased the generation of ROS by 10-150%, and oxidized intracellular thiols. A reduction in mitochondrial potential of almost 80% in melanocytes exposed to HCT and UVAR and 60% in fibroblasts was found due to oxidative stress occurrence. In addition, HCT and FUR have been shown to disrupt the cell cycle of normal skin cells. Finally, it can be concluded that HCT is the drug with a stronger phototoxic effect, and fibroblasts turn out to be more sensitive cells to the phototoxic effect of tested drugs.

Phytochemical Profiling of Microalgae Euglena tuba and Its Anticancer Activity in Dalton's Lymphoma Cells.

Natural phytochemicals are considered safe to use as therapeutic agents. There is a growing trend toward exploring anticancer effects of crude algal extracts or their active ingredients. Euglena tuba, a microalga, contains excellent antioxidant potential. However, the anticancer property of E. tuba has not been explored. This study investigates the chemical profiling as well as antitumor property of methanolic extract of E. tuba (ETME) against Dalton's lymphoma (DL) cells. E. tuba, procured from northern part of India, was extracted in 70% methanol, dried at room temperature, and stored at -20 ∘C for future use. A freshly prepared aqueous solution of ETME of different concentrations was employed into each experiment. The ETME mediated anti-tumor response in Dalton's lymphoma was evaluated in the inbred populations of BALB/c (H2d) strain of mice of either sex at 8-12 weeks of age. The cytotoxicity of ETME in cancer cells, effects on morphology of cell and nucleus, alteration in the mitochondrial membrane potential, and level of expression of proapoptotic proteins (Bcl-2, cyt C, Bax and p53) were done using known procedures. The ETME contained high content of total alkaloids (96.02 ± 3.30 mg/100 mg), flavonoids (15.77 ± 2.38 mg/100 mg), carbohydrate (12.71 ± 0.59 mg/100 mg), ascorbic acid (12.48 ± 2.59 mg/100 mg), and phenolics (0.94 ± 0.05 mg/100 mg). Gas chromatography-mass spectrometry (GC-MS) analysis indicated the presence of 23 phytochemicals with known anticancer properties. DL cells treated with ETME exhibited significant and concentration dependent cytotoxicity. Florescent microscopy and flow cytometry of ETME treated DL cells indicated significant repair in cellular morphology and decreased mitochondrial potential, respectively. Western blot analysis displayed up-regulation of proapoptotic proteins (Bax, Cyt-c, p53) and down regulation of anti-apoptotic protein (Bcl2) in DL cells treated with ETME. The findings of this study clearly indicated that the anticancer property of ETME was mediated via reduction in mitochondrial potential and induction of apoptotic mechanism. Further studies are warranted to explore the anticancer activities of active ingredients present in this microalga of pharmaceutical importance.

Exposure to benzo(a)pyrene suppresses mitophagy via ANT1-PINK1-Parkin pathway in ovarian corpus luteum during early pregnancy

Exposure to benzo (a)pyrene (BaP) has been confirmed to interfere with embryo implantation. As the primary organ of progesterone synthesis during early pregnancy, the ovarian corpus luteum (CL) is essential for embryo implantation and pregnancy maintenance. We previously demonstrated that BaP impaired luteal function, but the molecular mechanism remains unclear. In CL cells, mitochondria are the main sites of progesterone synthesis. Mitophagy, a particular type of autophagy, regulates mitochondrial quality by degrading damaged mitochondria and ensuring the homeostasis of cell physiology. Therefore, the present study investigated the effects and the potential molecular mechanisms of BaP on ovarian mitophagy during early pregnancy. We found that BaP and its metabolite, BPDE, inhibited autophagy and PINK1/Parkin-mediated mitophagy in the pregnant ovaries and luteinized granulosa cell, KGN. Notably, adenine nucleotide translocator 1 (ANT1), a crucial mediator of PINK1-dependent mitophagy, was suppressed by BaP and BPDE both in vivo and in vitro. The inhibition of ANT1 leads to the decrease in the PINK1 bound to the outer membrane of mitochondria and consequently reduces recruitment of Parkin to the mitochondria, which is required for the subsequent clearance of mitochondria. Meanwhile, exposure to BPDE also damaged mitochondrial function, causing the reduction in mitochondrial potential and ATP production. Overexpression of ANT1 in KGN cells partially relieved the inhibition of mitophagy caused by BPDE, restored mitochondrial function and expression of hormone synthesis-associated genes. Collectively, our study firstly clarified that BaP and BPDE suppress mitophagy of CL cells via the ANT1-PINK1-Parkin pathway, which provides a new insight to explore the detailed mechanism of the BaP-induced ovarian toxicity.

The inflammation influence on pathological remodeling of pulmonary arteries in monocrotaline-induced pulmonary hypertension in rats

Abstract Aim To investigate the inflammation role on pathological remodeling of pulmonary arteries (PA) in monocrotaline-induced pulmonary hypertension (mPAH) in rats with joint assessment serum and tissue inflammatory biomarkers and the morphological arteries changes. Methods The mPAH was induced by a subcutaneous monocrotaline injection (60 mg/kg) in male rats and control group received a single saline solution. Baseline and every 2, 4, 6, 8 weeks after the serum concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10) were measured by enzyme-linked immunosorbent assay; matrix metalloproteinase-9 (MMP-9), interleukin-1β (IL-1β), collagen type 1 and 3, smooth muscle actin α (SMA-α) in lung tissue were investigated immunohistochemically and quantitative measurements of intima and media thickness were done by planimetry. The functional activity neutrophil changes measured by chemiluminescence and fluorescent methods. Results The IL-10 increased after 2 weeks of mPAH (5,9 vs 0,6 pg/ml, p<0,05) vs control and then it decreased to initial values by 8 weeks (0,06 vs 0,62 pg/ml, p>0,05). The increasing IL-6 (30,3 vs 0,01 pg/ml, p<0,05) and the maximum expression of IL-1β in the lung tissue (0,119 vs 0,099 index of expression (IE), p<0,05) we observed 4 weeks after mPAH. The SMA-α (29,4 vs 40,2 IE, p<0,05) and MMP-9 (1,6 vs 0,8 IE, p<0,05) expression level significantly raised 4 weeks later vs control and remained in a high level until 8 week. A significant increase of type 1 collagen expression was observed at all phases of the experiment, and high level type 3 collagen expression was observed from 4 to 8 weeks (7,0 vs 10,4 IE, p<0,05). The histological characteristics of remodeling these were a thickening of the media (30,6 vs 56,0 μm, p<0,05) and the subintimal layer (1,6 vs 10,9 μm, p<0,05) of the PA. 2 weeks after mPAH cell priming occurred which manifested by modification ROS generation systems, decrease NADPH oxidase activity, increase of myeloperoxidase secretion (MPO), enhance of unbound cytosolic calcium ions, mitochondrial potential reduction. From 4 to 8 weeks an increase NADPH oxidase activity and MPO secretion was revealed into the extracellular environment which leads to overproduction of hypochlorous acid. This functional activity reprogramming of circulating neutrophils indicate associated with time-development of mPAH. Conclusion The inflammation is the most important mediator of pathological remodeling processes in mPA. The monocrotaline launches a neutrophil reaction at an early stage of PAH with changes their functional activity which leads to immune cells recruitment into the lung tissue, producing inflammation and proliferation biomarkers. The hyperplasia of smooth muscle cells and reconstruction of the extracellular matrix are the result of this process and leads to increase intima and media thickness. The high MMP-9, SMA-α, IL-6 activity in 6–8 weeks reflects maintenance local inflammatory potential. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Basic and applied sciences - medicine, subprogram “Diagnostics and therapy of diseases” on the assignment “To establish the molecular-cellular mechanisms of the development of irreversible remodeling of pulmonary vessels in pulmonary arterial hypertension in an experiment in vivo.”

Endocytosis and the Participation of Glycosaminoglycans Are Important to the Mechanism of Cell Death Induced by β-Hairpin Antimicrobial Peptides.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for β-hairpin peptides.

A Novel Peptide Ameliorates TNFα- and LPS-Induced Endothelia Dysfunction in Preeclampsia.

To investigate the protective effects of the novel peptide antiendothelial dysfunction peptide in preeclampsia (AEDPPE) on tumor necrosis factor α (TNFα)- and lipopolysaccharide (LPS)-induced injury in the vascular endothelium in preeclampsia. The effects of AEDPPE on TNFα-induced vascular endothelial injury were assessed by enzyme-linked immunosorbent assay, quantitative real-time PCR, mitochondrial membrane potential assay, Cell Counting Kit-8 assay, THP-1 monocyte-human umbilical vein endothelial cell (HUVEC) adhesion assay, endothelial tube-forming assay, transcriptomic analysis, preeclamptic symptom analysis, and histological analysis in preeclampsia-like rat models induced by LPS. AEDPPE alleviated the upregulation of antiangiogenic factors including soluble fms-like tyrosine kinase-1, endothelin-1, and tissue plasminogen activator and attenuated the reduction in mitochondrial potential induced by TNFα in HUVECs. In addition, AEDPPE treatment counteracted the decrease in tube formation and decreased the numbers of THP-1 monocytes attached to HUVECs caused by TNFα. Mechanistically, cytokine-cytokine receptor interactions enriched many genes and the TNF signaling pathway may be involved in this phenomenon. Moreover, cotreatment with LPS and AEDPPE significantly reversed the preeclampsia-like phenotype including hypertension and proteinuria and improved the functions of the kidney and placenta. AEDPPE effectively ameliorated the vascular endothelial injury induced by TNFα and LPS in preeclampsia. We suggest that AEDPPE may be a novel therapeutic candidate for preeclampsia treatment. These findings demonstrate that AEDPPE may play an effective role in ameliorating vascular endothelial dysfunction and be a potential therapeutic agent for preeclampsia.

In vitro vascular toxicity assessment of NitDOX, a novel NO-releasing doxorubicin

The conjugation of doxorubicin (DOX) with nitric oxide (NO)-releasing groups gave rise to novel anthracyclines, such as nitrooxy-DOX (NitDOX), capable to overcome multidrug resistance. The widely described anthracycline cardiovascular toxicity, however, might limit their clinical use. This study aimed to investigate NitDOX-induced effects, as potential hazard, on vascular smooth muscle A7r5 and endothelial EA.hy926 cell viability, on the mechanical activity of freshly and cultured rat aorta rings, as well as on Cav1.2 channels of A7r5 cells. DOX was used as a reference compound.Although an increase in intracellular radicals and a reduction in mitochondrial potential occurred upon treatment with both drugs, A7r5 and EA.hy926 cells proved to be more sensitive to DOX than to NitDOX. Both compounds promoted comparable effects in A7r5 cells, whereas NitDOX was less active than DOX in inducing DNA damage and in eliciting apoptotic-mediated cell death revealed as an increase in sub-diploid-, DAPI- and annexin V-positive- EA.hy926 cell percentage. Moreover, in EA.hy926 cells, NitDOX doubled basal NO content, while preincubation with the NO-scavenger PTIO increased NitDOX-induced cytotoxicity.DOX exhibited a negligible contracturing effect in endothelium-intact rings, while NitDOX induced a significant ODQ-sensible, vasodilation in endothelium-denuded rings. In arteries cultured with both drugs for 7 days, NitDOX prevented either phenylephrine- or KCl-induced contraction at a concentration 10-fold higher than that of DOX. These results demonstrate that NitDOX displays a more favourable vascular toxicity profile than DOX. Taking into account its greater efficacy against drug-resistant cells, NitDOX is worth of further investigations in preclinical and clinical settings.

Cigarette smoke extract increases mitochondrial membrane permeability through activation of adenine nucleotide translocator (ANT) in lung epithelial cells

Cigarette smoke is one of major risk factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). It is generally believed that cigarette smoke induces mitochondrial damage in the alveolar epithelial cells to contribute to COPD. However, the exact molecular mechanism remains unknown for the mitochondrial damage. In this study, cigarette smoke extract (CSE) was found to induce the mitochondrial membrane permeability (MMP), which promoted proton leakage leading to the reduction in mitochondrial potential and ATP production. ANT in the mitochondrial inner membrane was activated by CSE for the alteration of MMP. The activation was observed without an alteration in the protein level of ANT. Inhibition of the ANT activity with ADP or bongkrekic acid prevented the MMP alteration and potential drop upon CSE exposure. The ANT activation was observed with a rise in ROS production, inhibition of the mitochondrial respiration, decrease in the complex III protein and rise in mitophagy activity. The results suggest that ANT may mediate the toxic effect of cigarette smoke on mitochondria and control of ANT activity is a potential strategy in intervention of the toxicity.

MiR-31 Modulates Liver Cancer HepG2 Cell Apoptosis and Invasion via ROCK1/F-Actin Pathways.

PurposeLiver cancer is one of the most common malignant tumor in the world. miR-31 is downregulated in liver cancer and associated with tumor growth and metastasis. However, the underlying mechanism remains unclear.MethodsCellular apoptosis was detected via MTT, TUNEL assay, LDH release and Annexin V/PI flow-cytometry analysis. Cellular migration and invasion were measured by the Transwell chamber assay. Mitochondrial functions were evaluated via mitochondrial membrane potential JC-1 staining and mPTP opening assessment. The mitophagy activity was examined via Western blots.ResultsIn the present study, our results confirm that miR-31 promotes apoptosis and inhibits proliferation and metastasis in liver cancer HepG2 cells. In vitro, miR-31 promotes HepG2 cell apoptosis through the mitochondrial pathway as indicated by mitochondrial potential reduction, increased mPTP opening time, cty-c release and imbalance of pro- and anti-apoptotic proteins. Furthermore, miR-31 reduces the energy generation by inhibiting mitochondrial respiratory function. At last, it is demonstrated that miR-31 triggers the mitochondrial damage via ROCK1/F-actin pathway. Inhibiting the ROCK1/F-actin pathway abolishes the effects of miR-31 mimic on mitochondrial injury, apoptosis, proliferation arrest and migration inhibition.ConclusionOur results reveal that miR-31 can inhibit HepG2 cell survival and metastasis by activating the ROCK1/F-actin pathway.

Mst1 deletion reduces septic cardiomyopathy via activating Parkin‐related mitophagy

Cardiomyocyte function and viability are highly modulated by mammalian Ste20-like kinase 1 (Mst1)-Hippo pathway and mitochondria. Mitophagy, a kind of mitochondrial autophagy, is a protective program to attenuate mitochondrial damage. However, the relationship between Mst1 and mitophagy in septic cardiomyopathy has not been explored. In the present study, Mst1 knockout mice were used in alipopolysaccharide (LPS)-induced septic cardiomyopathy model. Mitophagy activity was measured via immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay. Pathway blocker and small interfering RNA were used to perform the loss-of-function assay. The results demonstrated that Mst1 was rapidly increased in response to LPS stress. Knockout of Mst1 attenuated LPS-mediated inflammation damage, reduced cardiomyocyte death, and improved cardiac function. At the molecular levels, LPS treatment activated mitochondrial damage, such as mitochondrial respiratory dysfunction, mitochondrial potential reduction, mitochondrial ATP depletion, and caspase family activation. Interestingly, in response to mitochondrial damage, Mst1 deletion activated mitophagy which attenuated LPS-mediated mitochondrial damage. However, inhibition of mitophagy via inhibiting parkin mitophagy abolished the protective influences of Mst1 deletion on mitochondrial homeostasis and cardiomyocyte viability. Overall, our results demonstrated that septic cardiomyopathy is linked to Mst1 upregulation which is followed by a drop in the protective mitophagy.

Liraglutide protects renal mesangial cells against hyperglycemia‑mediated mitochondrial apoptosis by activating the ERK‑Yap signaling pathway and upregulating Sirt3 expression.

Diabetic nephropathy results from hyperglycemia‑mediated renal glomerular cell death via mitochondrial apoptosis. There is an emerging requirement for novel approaches with mitochondrial protective effects that alleviate the hyperglycemia‑induced loss of functional cells during diabetic renal damage. Liraglutide, a type of glucagon‑like peptide‑1 agonist, has been suggested to inhibit the progression of obesity and hyperglycemia. However, the contributions and mechanism of action of liraglutide on hyperglycemia‑mediated cell mitochondrial apoptosis in diabetic kidneys have not been illustrated. The present study demonstrated that liraglutide may protect human renal mesangial cells (HRMCs) against hyperglycemia‑induced cell death by inhibiting mitochondrial apoptosis. Liraglutide administration also maintained HRMC viability and promoted HRMC proliferation within a high glucose stress environment. Functional studies demonstrated that hyperglycemia triggered mitochondrial dysfunction, including mitochondrial potential reduction, mitochondrial permeability transition pore opening, reactive oxygen species overproduction and the activation of the mitochondrial apoptotic pathway. However, liraglutide treatment preserved mitochondrial function and prevented activation of mitochondrial apoptosis by upregulating sirtuin 3 (Sirt3) expression. Deletion of Sirt3 abrogated the protective effects of liraglutide on mitochondrial homeostasis following high glucose challenge. In addition, molecular analysis confirmed that liraglutide upregulated Sirt3 via activating the extracellular signal‑regulated kinase‑Yes‑associated protein (ERK‑Yap) signaling pathway. Inhibition of the ERK‑Yap axis negated the action of liraglutide on Sirt3 activation, leading to mitochondrial injury and HRMC apoptosis. Taken together, the present study illustrated that liraglutide protected renal mesangial cells from hyperglycemia‑mediated mitochondrial apoptosis by upregulating Sirt3 expression and activation of the ERK‑Yap signaling pathway.

Mst1 regulates non-small cell lung cancer A549 cell apoptosis by inducing mitochondrial damage via ROCK1/F‑actin pathways.

Mammalian STE20-like kinase 1 (Mst1) is well recognized as a major tumor suppressor in cancer development, growth, metabolic reprogramming, metastasis, cell death and recurrence. However, the roles of Mst1 in non-small cell lung cancer (NSCLC) A549 cell phenotypic alterations remain to be elucidated. The present study aimed to explore the functional role and underlying mechanisms of Mst1 with regards to A549 cell proliferation, migration and apoptosis; this study focused on mitochondrial homeostasis and Rho-associated coiled-coil containing protein kinase 1 (ROCK1)/F-actin pathways. The results demonstrated that Mst1 was downregulated in A549 cells compared with in a normal pulmonary epithelial cell line. Subsequently, overexpression of Mst1 in A549 cells reduced cell viability and promoted cell apoptosis. Furthermore, overexpression of Mst1 suppressed A549 cell proliferation and migration. At the molecular level, the reintroduction of Mst1 in A549 cells led to activation of mitochondrial apoptosis, as evidenced by a reduction in mitochondrial potential, overproduction of ROS, cytochrome c release from the mitochondria into the nucleus, and upregulation of pro-apoptotic protein expression. In addition, Mst1 overexpression was closely associated with impaired mitochondrial respiratory function and suppressed cellular energy metabolism. Functional studies illustrated that Mst1 overexpression activated ROCK1/F-actin pathways, which highly regulate mitochondrial function. Inhibition of ROCK1/F-actin pathways in A549 cells sustained mitochondrial homeostasis, alleviated caspase-9-dependent mitochondrial apoptosis, enhanced cancer cell migration and increased cell proliferation. In conclusion, these data firmly established the regulatory role of Mst1 in NSCLC A549 cell survival via the modulation of ROCK1/F-actin pathways, which may provide opportunities for novel treatment modalities in clinical practice.

Sirt3 attenuates post-infarction cardiac injury via inhibiting mitochondrial fission and normalization of AMPK-Drp1 pathways

Mitochondrial damage is involved in the pathogenesis of post-infarction cardiac injury. However, the upstream regulators of mitochondrial damage have not yet been identified. The aim of our study is to explore the role of Sirt3 in post-infarction cardiac injury with a particular focus on mitochondrial fission and AMPK-Drp1 pathways. Our results indicated that Sirt3 was downregulated in the progression of post-infarction cardiac injury. Overexpression of Sirt3 attenuated cardiac fibrosis, sustained myocardial function, inhibited the inflammatory response, and reduced cardiomyocyte death. Functional studies illustrated that chronic post-infarction cardiac injury was characterized by increased mitochondrial fission, which triggered mitochondrial oxidative stress, metabolic disorders, mitochondrial potential reduction and caspase-9 apoptosis in cardiomyocytes. However, Sirt3 overexpression attenuated mitochondrial fission and thus preserved mitochondrial homeostasis and cardiomyocyte viability. Furthermore, our results confirmed that Sirt3 repressed mitochondrial fission via normalizing AMPK-Drp1 pathways. Inhibition of AMPK activity re-activated Drp1 and thus abrogated the inhibitory effect of Sirt3 on mitochondrial fission. Altogether, our results indicate that Sirt3 enhancement could be an effective approach to retard the development of post-infarction cardiac injury via disrupting mitochondrial fission and normalizing the AMPK-Drp1 axis.

NR4A1 Promotes Cerebral Ischemia Reperfusion Injury by Repressing Mfn2-Mediated Mitophagy and Inactivating the MAPK-ERK-CREB Signaling Pathway.

Mitochondrial dysfunction has been acknowledged as the key pathogenic mechanism in cerebral ischemia-reperfusion (IR) injury. Mitophagy is the protective system used to sustain mitochondrial homeostasis. However, the upstream regulator of mitophagy in response to brain IR injury is not completely understood. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been found to be associated with mitochondrial protection in a number of diseases. The aim of our study is to explore the functional role of NR4A1 in cerebral IR injury, with a particular focus on its influence on mitophagy. Wild-type mice and NR4A1-knockout mice were used to generate cerebral IR injury in vivo. Mitochondrial function and mitophagy were detected via immunofluorescence assays and western blotting. Cellular apoptosis was determined via MTT assays, caspase-3 activity and western blotting. Our data revealed that NR4A1 was significantly increased in the reperfused brain tissues. Genetic ablation of NR4A1 reduced the cerebral infarction area and repressed neuronal apoptosis. The functional study demonstrated that NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, evoked cellular oxidative stress, interrupted ATP generation, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-mediated mitophagy. Knockdown of NR4A1 elevated Mfn2 expression and therefore reversed mitophagic activity, sending a prosurvival signal for mitochondria in the setting of cerebral IR injury. Further, we demonstrated that NR4A1 modulated Mfn2 expression via the MAPK-ERK-CREB signaling pathway. Blockade of the ERK pathway could abrogate the permissive effect of NR4A1 deletion on mitophagic activation, contributing to neuronal mitochondrial apoptosis. Overall, our results demonstrate that the pathogenesis of cerebral IR injury is closely associated with a drop in protective mitophagy due to increased NR4A1 through the MAPK-ERK-CREB signaling pathway.

Bidesmosidic betulin saponin bearing L-rhamnopyranoside moieties induces apoptosis and inhibition of lung cancer cells growth in vitro and in vivo.

Betulin has a wide range of biological and pharmacological properties with its anticancer activity attracting most of the attention as it offers a possible alternative treatment to chemotherapy. However, betulin’s in vivo biological effectiveness is limited by its poor solubility. As such, we synthesized polar glycosylated derivatives to increase its hydrosolubility and enhance its pharmacological properties. Among these synthesized compounds, 28-O-α-l-rhamnopyranosylbetulin 3β-O-α-l-rhamnopyranoside (Bi-L-RhamBet) was assessed for its cytotoxic effects against a suite of lung cancer cell lines. We also investigated its mechanism of action using an A549 lung cancer cell line. Our results showed that Bi-L-RhamBet exhibited potent cytotoxic activity toward lung cancer cell lines including A549, NCI-H2087, NCI-H522, NCI-H1993 NCI-H1755, and LLC1 having IC50 values ranging from 2.9 to 5.9 μM. Moreover, Bi-L-RhamBet (50 mg/kg) significantly inhibited tumor growth with a treatment-to-control ratio (T/C) of 0.54 and a tumor growth inhibition rate of 46% at day 18 (p < 0.05). Microscopic observations of A549 cells, double stained with acridine orange and ethidium bromide, showed apoptotic features. Bi-L-RhamBet induced activation of pro-apoptotic caspases 8, 9, and 3/7 as well as causing DNA fragmentation. Moreover, a marked increase in mitochondrial ROS (mROS) was coupled with a reduction of mitochondrial potential. Interestingly, the presence of mitochondrial electron transport chain (ETC) inhibitors, including rotenone, malonate, and antimycin A, reduced mROS production, and the activation of caspases suggesting that Bi-L-RhamBet disturbs the ETC. Finally, dichloroacetate, a pyruvate dehydrogenase kinase inhibitor potentiated the cytotoxicity of Bi-L-RhamBet against A549 cells. Taken together, these data suggest that Bi-L-RhamBet can induce apoptotic cell death via disturbance of mitochondrial electron transfer chain, reduced ROS production, and decreased membrane potential.

Effects of melatonin on fatty liver disease: The role of NR4A1/DNA-PKcs/p53 pathway, mitochondrial fission, and mitophagy.

Mitochondrial dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) through poorly defined mechanisms. Melatonin supplementation has been found to protect liver function in diabetes and obesity. Here, we intensively explored the role and mechanism of melatonin in the development of NAFLD. We demonstrated that the onset of diet-induced NAFLD greatly caused NR4A1 upregulation in hepatocytes, leading to the activation of DNA-PKcs and p53. On the one hand, p53 aided Drp1 migration in the mitochondria and consequently drove mitochondrial fission. On the other hand, p53 repressed Bnip3 transcription and expression, resulting in mitophagy arrest. The excessive fission and deficient mitophagy dramatically mediated mitochondrial dysfunction, including extensive mPTP opening, reduction in mitochondrial potential, oxidative stress, calcium overload, mitochondrial respiratory collapse, and ATP shortage. However, genetic deletion of NR4A1 or DNA-PKcs could definitively reverse NAFLD progression and the mitochondrial dysfunction. Similarly, melatonin supplementation could robustly reduce the damage to liver and mitochondrial structure and function in NAFLD. Mechanistically, melatonin halted fission but recovered mitophagy via blockade of NR4A1/DNA-PKcs/p53 pathway, finally improving mitochondrial and liver function in the setting of NAFLD. Our results identify NR4A1/DNA-PKcs/p53 pathway as the novel molecular mechanism underlying the pathogenesis of NAFLD via regulation of Drp1-mediated mitochondrial fission and Bnip3-related mitophagy. Meanwhile, we also confirm that melatonin has the ability to cut off the NR4A1/DNA-PKcs/p53 pathway, which confers a protective advantage to hepatocytes and mitochondria. The manipulation of NR4A1/DNA-PKcs/p53 pathway by melatonin highlights a new entry point for treating NAFLD.

Neo-tanshinlactone D-ring modified novel analogues induce apoptosis in human breast cancer cell via DNA damage

Neo-tanshinlactone (NTL) a natural product is known for its specificity and selectivity towards the breast cancer cells. By NTL D-ring modification approach, 13 new analogues were synthesized (1A–1M). Among them 1J showed the best anticancer activity in MCF-7 (ER+, PR+/−, HER2−), SKBR3 (ER−, PR−, HER2+) and MDA-MB-231 (ER−, PR−, HER2−) cells lines with IC50 value 11.98nM, 23.71nM, and 62.91nM respectively. 1J showed minor grove binding interaction with DNA at AT-rich region and induced DNA double strand breaks (DDSBs). This had triggered several key molecular events involving, activation of ATM, Chk2 and p53, reduction in mitochondrial potential (Δψm) leading to caspase-3 and PARP cleavage mediated apoptosis. These results along with other biochemical studies strongly suggest that novel NTL analogue 1J caused DNA cleavage mediated apoptosis in the breast cancer cells and this may serve as potential lead for future breast cancer treatment.

Inflammation-related pro-apoptotic activity of exopolysaccharides isolated from Lactococcus lactis subsp. lactis

Exopolysaccharides (EPS) have attracted attention recently for possible use in suppressing early stage breast cancer. In this study, a mannan EPS produced by Lactococcus lactis subsp. lactis was found to affect the production of inflammatory cytokines. EPS (300 μg/ml) can significantly enhance tumour necrosis factor alpha and inducible NO synthase release in MCF-7 cells compared to control cells in a concentration-dependent manner. Also the intracellular calcium level was found to increase with the concentration of EPS. After EPS-treatment, a significant reduction in mitochondrial potential was observed, as was nuclear condensation and cell shrinkage. These results may be helpful in further understanding the anti-tumour properties of lactic acid bacteria.

Antioxidant, anticancer activities and mechanistic studies of the flavone glycoside diosmin and its oxidovanadium(IV) complex. Interactions with bovine serum albumin

The natural antioxidant flavonoid diosmin, found in citric fruits, showed low antioxidant properties among other flavonoids due to its structural characteristics and low cytotoxicity against lung (A549) and breast (T47D, SKBR3 and MDAMB231) cancer cell lines. The anticancer behavior has been improved by the metal complex generated with the flavonoid and the oxidovanadium(IV) ion. This new complex, [VO(dios)(OH)3]Na5·6H2O (VOdios), has been synthesized and characterized both in solid and solution states. The interaction of the metal ion through the sugar moiety of diosmin precluded the improvement of the antioxidant effects. However, the cell-killing effects tested in human lung A549 and breast T47D, SKBR3 and MDAMB231 cancer cell lines, were enhanced by complexation. The anti-proliferative effects on the human lung cancer cell line were accompanied by cellular ROS generation and an increase in cytoplasm condensation. The breast cancer cell lines did not produce caspase3/7 activation, mitochondrial potential reduction and ROS generation. Therefore, a non-apoptotic form of cell death in a caspase- and oxidative stress-independent manner has been proposed. The protein binding ability has been monitored by the quenching of tryptophan emission in the presence of the compounds using bovine serum albumin (BSA) as a model protein. Both compounds could be distributed and transported in vivo and the complex displayed stronger binding affinity and higher contributions to the hydrogen bond and van der Waals forces.

Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.

A study using cultured human annulus cells and human annular tissue. To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo. Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis. High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis. Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P < 0.00001 by analysis of variance). A significant negative correlation for mitochondrial potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P < 0.0001). When exposed to the K ionophore valinomycin at sublethal levels to induce apoptosis, a significant reduction in mitochondrial potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1). Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration. N/A.