Atmospheric Oxygen Tension Slows Myoblast Proliferation via Mitochondrial Activation

Background

Mitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ∼20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O₂) relative to more physiological oxygen (5% O₂).

Methodology/Principal Findings

Murine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.

Conclusions/Significance

These results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.     

Serum withdrawal-induced accumulation of phosphoinositide 3-kinase lipids in differentiating 3T3-L6 myoblasts: distinct roles for Ship2 and PTEN

Phosphoinositide 3-kinase (PI3K) activation and synthesis of phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃) lipids mediate growth factor signaling that leads to cell proliferation, migration, and survival. PI3K-dependent activation of Akt is critical for myoblast differentiation induced by serum withdrawal, suggesting that in these cells PI3K signaling is activated in an unconventional manner. Here we investigate the mechanisms by which PI3K signaling and Akt are regulated during myogenesis. We report that PI-3,4-P₂ and PI-3,4,5-P₃ accumulated in the plasma membranes of serum-starved 3T3-L6 myoblasts due to de novo synthesis and increased lipid stability. Surprisingly, only newly synthesized lipids were capable of activating Akt. Knockdown of the lipid phosphatase PTEN moderately increased PI3K lipids but significantly increased Akt phosphorylation and promoted myoblast differentiation. Knockdown of the lipid phosphatase Ship2, on the other hand, dramatically increased the steady-state levels of PI-3,4,5-P₃ but did not affect Akt phosphorylation and increased apoptotic cell death. Together, these results reveal the existence of two distinct pools of PI3K lipids in differentiating 3T3-L6 myoblasts: a pool of nascent lipids that is mainly dephosphorylated by PTEN and is capable of activating Akt and promoting myoblast differentiation and a stable pool that is dephosphorylated by Ship2 and is unable to activate Akt.     

Downregulation of hTERT: An Important As2O3 Induced Mechanism of Apoptosis in Myelodysplastic Syndrome

Two myelodysplastic syndrome (MDS) celllines, MUTZ-1 and SKM-1 cells, were used to study the effect of arsenic trioxide (As₂O₃) on hematological malignant cells. As₂O₃ induced this two cell lines apoptosis via activation of caspase-3/8 and cleavage of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. As₂O₃ reduced NF-κB activity, which was important for inducing MUTZ-1 and SKM-1 cells apoptosis. As₂O₃ also inhibited the activities of hTERT in MUTZ-1 and SKM-1 cells. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), had no effect on caspase-8 activation, although PDTC did inhibit MUTZ-1 and SKM-1 cells proliferation. Incubation of MUTZ-1 cells with a caspase-8 inhibitor failed to block As₂O₃-induced inhibition of NF-κB activity. Our findings suggest that As₂O₃ may induce apoptosis in MUTZ-1 and SKM-1 cells by two independent pathways: first, by activation of caspase-3/8 and PARP; and second, by inhibition of NF-κB activity, which results in downregulation of hTERT expression. We conclude that hTERT and NF-κB are important molecular targets in As₂O₃-induced apoptosis.     

Arsenic trioxide induces G2/M arrest in hepatocellular carcinoma cells by increasing the tumor suppressor PTEN expression

Arsenic trioxide (As₂O₃), an effective agent against acute promyelocytic leukemia, has been reported to inhibit the viability of solid tumors cell lines recently. The detailed molecular mechanism underlying the As₂O₃‐induced inactivation of the cdc2 and possible functional role of PTEN in the observed G2/M arrest has yet to be elucidated. Here, we assessed the role of PTEN in regulation of As₂O₃‐mediated G2/M cell cycle arrest in Hepatocellular carcinoma cell lines (HepG2 and SMMC7721). After 24 h following treatment, As₂O₃ induced a concentration‐dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by As₂O₃ is associated with decreased cdc2 protein and increased phospho‐cdc2(Tyr15). As₂O₃ treatment increased Wee1 levels and decreased phospho‐Wee1(642). Moreover, As₂O₃ substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in As₂O₃‐treated cells increased phospho‐Wee1(Ser642) while decreased phospho‐cdc2(Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by As₂O₃ involved upregulation of PTEN. J. Cell. Biochem. 113: 3528–3535, 2012. © 2012 Wiley Periodicals, Inc.     

Choline Plasmalogens Isolated from Swine Liver Inhibit Hepatoma Cell Proliferation Associated with Caveolin-1/Akt Signaling

Plasmalogens play multiple roles in the structures of biological membranes, cell membrane lipid homeostasis and human diseases. We report the isolation and identification of choline plasmalogens (ChoPlas) from swine liver by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC)/MS. The growth and viability of hepatoma cells (CBRH7919, HepG2 and SMMC7721) was determined following ChoPlas treatment comparing with that of human normal immortal cell lines (HL7702). Result indicated that ChoPlas inhibited hepatoma cell proliferation with an optimal concentration and time of 25 μmol/L and 24 h. To better understand the mechanism of the ChoPlas-induced inhibition of hepatoma cell proliferation, Caveolin-1 and PI3K/Akt pathway signals, including total Akt, phospho-Akt(pAkt) and Bcl-2 expression in CBRH7919 cells, were determined by western blot. ChoPlas treatment increased Caveolin-1 expression and reduced the expression of phospho-Akt (pAkt) and Bcl-2, downstream targets of the PI3K/Akt pathway. Further cell cycle analysis showed that ChoPlas treatment induced G₁ and G₁/S phase transition cell cycle arrest. The expression of essential cell cycle regulatory proteins involved in the G₁ and G₁/S phase transitions, cyclin D, CDK4, cyclin E and CDK2, were also analyzed by western blot. ChoPlas reduced CDK4, cyclin E and CDK2 expression. Taken together, the results indicate that swine liver-derived natural ChoPlas inhibits hepatoma cell proliferation associated with Caveolin-1 and PI3K/Akt signals.     

Near Infrared Imaging of EGFR of Oral Squamous Cell Carcinoma in Mice Administered Arsenic Trioxide

Background

The effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.

Material and Methods

A431 OSCC in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As₂O₃ for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As₂O₃ treatment. Next, mice with pharynx-implanted A431 cells received As₂O₃ i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.

Results

In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As₂O₃ dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As₂O₃ treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As₂O₃ treatment in a dose responsive pattern.

Conclusion

This study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As₂O₃ treatment.     

Arsenic Trioxide and Resveratrol Show Synergistic Anti-Leukemia Activity and Neutralized Cardiotoxicity

Cardiotoxicity is an aggravating side effect of many clinical antineoplastic agents such as arsenic trioxide (As₂O₃), which is the first-line treatment for acute promyelocytic leukemia (APL). Clinically, drug combination strategies are widely applied for complex disease management. Here, an optimized, cardiac-friendly therapeutic strategy for APL was investigated using a combination of As₂O₃ and genistein or resveratrol. Potential combinations were explored with respect to their effects on mitochondrial membrane potential, reactive oxygen species, superoxide dismutase activity, autophagy, and apoptosis in both NB4 cells and neonatal rat left ventricular myocytes. All experiments consistently suggested that 5 µM resveratrol remarkably alleviates As₂O₃-induced cardiotoxicity. To achieve an equivalent effect, a 10-fold dosage of genistein was required, thus highlighting the dose advantage of resveratrol, as poor bioavailability is a common concern for its clinical application. Co-administration of resveratrol substantially amplified the anticancer effect of As₂O₃ in NB4 cells. Furthermore, resveratrol exacerbated oxidative stress, mitochondrial damage, and apoptosis, thereby reflecting its full range of synergism with As₂O₃. Addition of 5 µM resveratrol to the single drug formula of As₂O₃ also further increased the expression of LC3, a marker of cellular autophagy activity, indicating an involvement of autophagy-mediated tumor cell death in the synergistic action. Our results suggest a possible application of an As₂O₃ and resveratrol combination to treat APL in order to achieve superior therapeutics effects and prevent cardiotoxicity.     

Construction of a BALB/c-Nu Mouse Model of Invasive Bladder Carcinoma and Preliminary Studies on the Treatment of Bladder Tumors through Internal Iliac Arterial Infusion of Albumin-Bound Arsenic Trioxide (As2O3)

To establish a BALB/c-nu mouse model of invasive bladder carcinoma and to investigate the feasibility, efficacy, and side effects of treating the mouse xenografts with internal iliac arterial infusion of albumin-bound arsenic trioxide (As₂O₃). Bladder tumors were established by intravesicular injection. Color Doppler were used to monitor tumor growth. Albumin-bound As₂O₃ and bovine serum albumin (BSA) nanoparticles were synthesized by cross-linking. BALB/c-nu mice were randomly divided into four treatment groups: 1) normal saline, 2) BSA nanoparticles, 3) As₂O₃ injections, and 4) albumin-bound As₂O₃. In an attempt to replicate the treatment of bladder cancer in humans using internal iliac arterial infusion, the drugs were injected into the mouse abdominal aorta. Tumor xenografts were established successfully. Mice treated with As₂O₃ injections and with albumin-bound As₂O₃ had significantly smaller bladders (36.59% and 37.82% smaller, respectively) than mice given normal saline injections (P < 0.01). Mice receiving As₂O₃ injections had lower white blood cell (WBC) and platelet counts compared with mice receiving normal saline injections only (P < 0.05). However, mice treated with albumin-bound As₂O₃ did not experience a significant decrease in WBC or platelet counts compared with control mice. A model of intra-arterial bladder cancer treatment was successfully established in BALB/c-nu mice. In this model, albumin-bound As₂O₃ appeared to be an effective method for treating bladder tumors, with less severe hematologic side effects compared with As₂O₃ alone. The infusion of albumin-bound As₂O₃ through the internal iliac artery is a promising method of bladder cancer therapy.     

Arsenic exposure alters activity behaviour of key nitrogen assimilatory enzymes in growing rice plants

Rice seedlings when grown in sand cultures for 5–20 days under 25 and 50 M As₂O₃ in the medium showed a marked decline in growth when compared to controls. Increased absorption of arsenic from the medium, against the concentration gradient was observed. Greater localization of absorbed arsenic was noted in roots than in shoots. Rice plants grown for 20 days with 50 mol l^–1 arsenic in the medium accumulated upto 370 mol arsenic kg^–1 dry weight in roots. Increasing levels of As₂O₃ in situ caused a marked decline in the activities of the nitrate assimilatory enzymes nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS), whereas an increase in the activities of alanine and aspartate aminotransferases was observed. The activities of aminating (NADH-GDH) and deaminating (NAD⁺-GDH) glutamate dehydrogenases increased at moderately toxic level (25 M) of As₂O₃ whereas a higher As level of 50 M was inhibitory to the enzymes. Addition of 1 M proline in the reaction medium caused significant restoration in As-led loss of NR and GS activities. NR and GS extracted from arsenic exposed seedlings showed higher K _m values compared to the enzymes extracted from control-grown seedlings, whereas GDHs extracted from As-stressed seedlings showed a decrease in K _m. Results suggest that inhibition in the activities of N assimilatory enzymes accompanied with decreased affinity of the enzymes towards their substrates would eventually lead to a marked suppression of N assimilation and impaired growth of rice seedlings in As polluted environment.     

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

Background

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca²⁺ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H₂O₂-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.

Results

Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H₂O₂ exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H₂O₂-induced injury in H9c2 cells. H₂O₂ exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca²⁺ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H₂O₂-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H₂O₂-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.

Conclusions

Collectively, these findings suggest that EGCG blunts the H₂O₂-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.     

Opposite effect of ERK1/2 and JNK on p53-independent p21 WAF1/CIP1 activation involved in the arsenic trioxide-induced human epidermoid carcinoma A431 cellular cytotoxicity

Summary While arsenic trioxide (As₂O₃) is an infamous carcinogen, it is also an effective chemotherapeutic agent for acute promyelocytic leukemia and some solid tumors. In human epidermoid carcinoma A431 cells, we found that As₂O₃ induced cell death in time- and dose-dependent manners. Similarly, dependent regulation of the p21 ^WAF1/CIP1 (p21) promoter, mRNA synthesis, and resultant protein expression was also observed. Additionally, transfection of a small interfering RNA of p21 could block the As₂O₃-induced cell growth arrest. The As₂O₃-induced p21 activation was attenuated by inhibitors of EGFR and MEK in a dose-dependent manner. Using a reporter assay, we demonstrated the involvement of the EGFR-Ras-Raf-ERK1/2 pathway in the promoter activation. In contrast, JNK inhibitor enhanced the As₂O₃-induced p21 activation, also in a dose-dependent fashion. Over-expression of a dominant negative JNK plasmid likewise also enhanced this activation. Furthermore, MEK inhibitor attenuated the anti-tumor effect of As₂O₃. In contrast, in combination with JNK inhibitor and As₂O₃ enhanced cellular cytotoxicity. Therefore, we conclude that in A431 cells the ERK1/2 and JNK pathways might differentially contribute to As₂O₃-induced p21 expression and then due to cellular cytotoxicity.     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

2-Methoxyestradiol Induces Mitotic Arrest,Apoptosis, and Synergistic Cytotoxicity with Arsenic Trioxide in Human Urothelial Carcinoma Cells

2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As₂O₃), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As₂O₃ provides a novel implication in clinical treatment of UC.     

miR-152 regulates the proliferation and differentiation of C2C12 myoblasts by targeting <Emphasis Type="Italic">E2F3</Emphasis>

The development of skeletal muscle is a complex process involving the proliferation, differentiation, apoptosis, and changing of muscle fiber types in myoblasts. Many reports have described the involvement of microRNAs in the myogenesis of myoblasts. In this study, we found that the expression of miR-152 was gradually down-regulated during myoblast proliferation, but gradually up-regulated during the differentiation of myoblasts. Transfection with miR-152 mimics restrained cell proliferation and decreased the expression levels of cyclin E, CDK4, and cyclin D1, but promoted myotube formation and significantly increased the mRNA expression levels of MyHC, MyoD, MRF4, and MyoG in C2C12 myoblasts. However, treatment with miR-152 inhibitors promoted cell proliferation and restrained differentiation. Moreover, over-expression of miR-152 significantly decreased E2F3 production in C2C12 myoblasts. A luciferase assay confirmed that miR-152 could bind to the 3′ UTR of E2F3. In conclusion, this study showed that miR-152 inhibited proliferation and promoted myoblast differentiation by targeting E2F3.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Ellagic acid mitigates arsenic‐trioxide‐induced mitochondrial dysfunction and cytotoxicity in SH‐SY5Y cells

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As₂O₃)‐induced toxicity in SH‐SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH‐SY5Y cells with EA (10 and 20 μM) for 60 min followed by exposure to 2 μM As₂O₃ protected the SH‐SY5Y cells against the harmful effects of the second. Also, EA pre‐treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As₂O₃ alone. EA supplementation also inhibited As₂O₃‐induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As₂O₃‐induced mitochondria‐mediated neuronal toxicity under in vitro system.