Membrane Potential Generation Research Articles

Low-intensity ultrasound (LIUS) differentially modulates mitochondrial reactive oxygen species (mtROS) generation by three different chemicals in PC12 cells

We have previously shown that low-intensity ultrasound (LIUS) can modulate mitochondrial complex I activity and the generation of mitochondrial reactive oxygen species (mtROS) in PC12 cells. This study investigated the mechanism of LIUS by comparing its effect on mitochondrial dysfunction by three different pathways. LIUS was shown to reverse the effects of rotenone, a Q-site blocker, on the complex I inhibition, mtROS generation, and drop of mitochondrial membrane potential (Δψm). In contrast, common antioxidants, N-acetyl cysteine (NAC), and uric acid (UA) blocked rotenone-induced mtROS generation and Δψm drop without recovering the complex I activity, which suggested that Δψm drop is correlated with mtROS generation rather than complex I inhibition itself. Ionomycin, an ionophore for Ca2+, and L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, induced mtROS generation and Δψm drop without inhibiting complex I activity via different mechanisms. LIUS showed no effect on ionomycin-induced Δψm drop but showed partial inhibition on the other effects of ionomycin and BSO. These results suggest that LIUS might have redundant mechanisms but acted mainly on the complex I activity thereby modulating mtROS and Δψm levels. LIUS appeared to act on the Q-module of complex I because it showed no inhibitory effect on Zn2+, an inhibitor of the proton transporting P-module of complex I. Interestingly, pretreatment of LIUS for up to an hour in advance blocked the rotenone effect as efficiently as the co-treatment. Further studies are needed to reveal the exact mechanism of LIUS to inhibit complex I activity.

Ginsenoside RG1 augments doxorubicin-induced apoptotic cell death in MDA-MB-231 breast cancer cell lines.

This study determined the chemosensitizing potential of ginsenoside Rg1 in triple-negative MDA-MB-231 breast cancer cell lines. Ginsenoside Rg1 (10 µM) treated breast cancer cells were exposed to 8 nM of doxorubicin, and the chemosensitizing potential was measured by cell-based assays. Ginsenoside Rg1 (10 µM) treatment lowered the doxorubicin IC50 value to 0.01 nM. Furthermore, the ginsenoside pretreatment augments doxorubicin-mediated reactive oxygen species (ROS) generation and subsequent alterations of mitochondrial membrane potential in MDA-MB-231 cell lines. The alkaline comet assay results illustrated an increased % tail DNA during ginsenoside Rg1 plus doxorubicin treatment than doxorubicin alone treatment. In addition, the number of apoptotic cells was also increased in ginsenoside Rg1 plus doxorubicin-treated cells. Furthermore, the polymerase chain reaction array results illustrate activation of mitogen-activated protein kinase (MAPK) gene expression (AKT, ERK, and MAPK) during doxorubicin alone treatment and it has been attenuated by ginsenoside Rg1 pretreatment. Moreover, ginsenoside Rg1 treatment before doxorubicin activates the DNA damage response elements (ATM, H2AX, RAD51, and XRCC1) and subsequent apoptosis-related gene expression (p21, TP53. APAF1, Bax, CASP3, and CASP9) patterns in MDA-MB-231 cell lines. The ginsenoside Rg1 plus doxorubicin combination shows less cytotoxicity and ROS generation in MDA10A normal breast cancer cell lines. Therefore, the present results support the chemosensitizing property of ginsenoside Rg1 in triple-negative breast cancer cell lines.

Mechanisms of abemaciclib, a CDK4/6 inhibitor, induced apoptotic cell death in prostate cancer cells in vitro

The therapeutic effects of abemaciclib (ABE), an inhibitor of cyclin- dependent kinases (CDK) 4/6, on the proliferation of two types of prostate cancer (PC) cells were revealed. In this study, in vitro cytotoxic and apoptotic effects of ABE on metastatic castration-resistant prostate cancer (mCRPC) androgen receptor (AR) negative PC-3 and AR mutant LNCaP PC cells were analyzed with WST-1, Annexin V, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential, RT-PCR, western blot, and apoptosis protein array. ABE considerably inhibited the growth of PC cells in a dose-dependent manner (p<0.01) and caused significant apoptotic cell death through the suppression of CDK4/6-Cyclin D complex, ROS generation and depolarization of mitochondria membrane potential. However, PC-3 cells were more sensitive to ABE than LNCaP cells. Furthermore, the expression levels of several pro-apoptotic and cell cycle regulatory proteins were upregulated by ABE in especially PC-3 cells with the downregulation of apoptotic inhibitor proteins. Our results suggest that ABE inhibits PC cell growth and promotes apoptosis and thus ABE treatment may be a promising treatment strategy in especially mCRPC. Further preclinical and clinical studies should be performed to clarify the clinical use of ABE for the treatment of PC.

The involvement of TRPV4 on the hypoxia-induced oxidative neurotoxicity and apoptosis in a neuronal cell line: Protective role of melatonin

The hypoxia (HYPX)-mediated excessive generation of mitochondrial free reactive oxygen species (mROS) and the overload Ca2+ influx via the inhibition of TRPV4 are controlled by the treatment of antioxidants. However, the molecular mechanisms underlying melatonin (MLT)'s neuroprotection remains elusive. We investigated the role of MLT via modulation of TRPV4 on oxidative neurodegeneration and death in SH-SY5Y neuronal cells. The SH-SY5Y cells were divided into five groups as follows: control, MLT (1 mM for 2 h), HYPX (200 μM CoCl2 for 24 h), HYPX + MLT, and HYPX + TRPV4 blockers (ruthenium red-1 μM for 30 min). The HYPX caused to the increase of TRPV4 current density and overload Ca2+ influx with an increase of mitochondrial membrane potential and mROS generation. The changes were not observed in the absence of TRPV4. When HYPX exposure and TRPV4 agonist (GSK1016790A)-induced TRPV4 activity were inhibited by the treatment of ruthenium red or MLT, the increase of mROS, lipid peroxidation, apoptosis, Zn2+ concentrations, TRPV4, caspase -3, caspase -9, Bax, and Bcl-2 expressions were restored via upregulation of reduced glutathione, glutathione peroxidase, and total antioxidant status. The levels of apoptosis and cell death in the cells were enriched with increases of caspase -3 and -9 activations, although they were decreased by MLT treatment. In conclusion, the treatment of MLT modulates HYPX-mediated mROS, apoptosis, and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting HYPX-mediated neurological diseases associated with the increase of mROS, Ca2+, and Zn2+ concentration.

Trypanosoma brucei Tim50 Possesses PAP Activity and Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity.

ABSTRACTTrypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of the translocase of the mitochondrial inner membrane 50 (TbTim50). It has a pair of characteristic phosphatase signature motifs, DXDX(T/V). Here, we demonstrated that, besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. Mutations of D242 and D244, but not of D345and D347, to alanine abolished these activities. In silico structural homology models identified the putative binding interfaces that may accommodate different phosphosubstrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential (ΔΨ). TbTim50 knockdown (KD) also reduced the population of G2/M phase and increased that of G1 phase cells; inhibited segregation and caused overreplication of kinetoplast DNA (kDNA), and reduced BF cell growth. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed with upregulation of expression of a few stumpy marker genes. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice. Proteomics analysis showed reductions in levels of the translation factors, flagellar transport proteins, and many proteasomal subunits, including those of the mitochondrial heat shock locus ATPase (HslVU), which is known to play a role in regulation of kinetoplast DNA (kDNA) replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Together, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and the cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis.

Induction of mitochondrial-dependent apoptosis by essential oil of Toona sinensis root through Akt, mTOR and NF-κB signalling pathways in human renal cell carcinoma cells.

Natural products have long been considered as a kind of complementary medicine. In this study, we investigate the apoptotic effect of essential oils of Toona sinensis roots (TSR) on human clear cell renal cell carcinomas (ccRCC). The sesquiterpene content of TSR essential oil was determined via GC/MS analysis. TSR decreased ccRCC cell viabilities, inducing ROS generation and reduction of the mitochondrial membrane potential. Moreover, TSR inhibited Bcl-2 and Hsp90 expression but increased PARP-1 cleavage and cytochrome c release. Akt, mTOR and NF-κB phosphorylation and HIF-α expression were all inhibited, which likely contributed to the anti-proliferative and anti-adhesive effects of TSR.

Proteasome inhibition induces macrophage apoptosis via mitochondrial dysfunction.

Dysfunction of the ubiquitin-proteasome system has been linked to the pathogenesis of a variety of diseases. Proteasome inhibition not only exerts antitumor effects but also affects inflammatory signaling pathways. MG132, a proteasome inhibitor, has been shown to induce tumor cell apoptosis. However, its role in the induction of macrophage apoptosis remains unknown. In our study, we investigated the mechanism of the proapoptotic effects of MG132 in macrophages. Our data showed that MG132 treatment induced mitochondrial reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential in macrophages. We found that proteasome inhibition induced a significant increase in the apoptosis rate, as evidenced by cleavage of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenyl-phosphonium chloride (Mito-TEMPO) attenuated MG132-induced apoptosis. In conclusion, proteasome inhibition by MG132 can induce macrophage apoptosis by promoting the production of ROS and mitochondrial dysfunction.

P–048 Effects of bisphenol S and bisphenol F on human spermatozoa: an in vitro study

Abstract Study question Are plasticizers bisphenol S (BPS) and bisphenol F (BPF) safer alternatives to bisphenol A (BPA) for human sperm function? Summary answer Unlike BPA, the analogues BPS and BPF do not significantly affect human sperm mitochondrial functions, motility and viability What is known already The widespread distribution of BPA, along with its reputation to be an endocrine disruptor has generated concerns about possible adverse effects for human health, thus prompting the European Food Safety Authority and the Food and Drug Administration to ban the use of this chemical in many plastic products. Following such restrictions, several substitutes have been developed, with BPS and BPF representing the main replacements to BPA. While it has been demonstrated that BPA promotes oxidative damages in spermatozoa from different species, including human, the possible effects exerted by BPS and BPF on human sperm, have not yet been investigated. Study design, size, duration We explored the effect of 4 h in vitro exposure to scalar concentrations of BPS and BPF (from 10 to 400 μM), and to 400 μM BPA on sperm motility, viability, mitochondrial membrane potential (Δm) and mitochondrial generation of reactive oxygen species (ROS). In a set of experiments, the effect of a combination of both BPF (400 μM) and BPS (400 μM) on ΔΨm and mitochondrial ROS generation was also assessed. Participants/materials, setting, methods Sperm Δm was analyzed by flow cytometry with the fluorescent lipophilic cationic dye JC–1. Flow cytometric assessment of mitochondrial generation of ROS was carried out using the lipid soluble cation MitoSOX red (MSR). Sperm motility and viability were evaluated by Computer-Aided Semen Analysis (CASA) and eosin assay, respectively. Main results and the role of chance The exposure to scalar concentration of BPS did not significantly affect sperm motility and viability with respect to untreated controls. A lower, albeit not significant, sperm motility was registered in samples exposed to the highest concentrations of BPF (300 μM and 400 μM). As expected, 400 μM BPA produced a complete sperm immobilization along with a dramatically loss in sperm viability. No significant differences were observed in sperm Δm and ROS generation after exposure to scalar concentration of BPS compared to untreated controls and the trend towards lower Δm and higher mitochondrial ROS generation at the highest concentrations of BPF did not reach statistical significance. On the contrary, after 4 h exposure to 400 μM BPA a significant lower Δm and higher mitochondrial ROS generation were observed. Finally, the exposure to a combination of BPF and BPS at high concentrations (400 μM) did not significantly affect sperm Δm, or mitochondrial ROS generation, when compared to 400 μM BPA, used as positive control. Limitations, reasons for caution: The present study only evaluated BPS and BPF effects, but in daily-life people are exposed to several plasticizers containing different bisphenols at different concentrations. Therefore, adverse effects of synergic exposure to BPA analogues other than BPS and BPF, alone or in combination with BPA, cannot be ruled out. Wider implications of the findings: The analogues BPS and BPF, alone or in combination, appeared to be safer alternatives to BPA on sperm biology as they exert a neutral effect on sperm motility, viability, and mitochondrial functions even at high concentrations. These results could be useful to identify more secure plasticizer components. Trial registration number Not applicable

P28-8 Paclitaxel inhibits the proliferation of PC9 cells through cell cycle arrest and apoptosis

Paclitaxel is used as a first-line and subsequent therapy for the treatment of various cancers. However, the function and mechanisms of paclitaxel in non-small cell lung cancer (NSCLC) remain unknown. Here, we investigated paclitaxel in vitro for the molecular mechanism of its anti-cancer activity in human PC9 (NSCLC, exon 19 deletion mutation) cells. PC9 cells were treated with paclitaxel and then evaluated with cell viability assay, Giemsa staining, DAPI staining, flow cytometry, SA-beta-gal staining, and western blot analysis. Our results revealed that paclitaxel markedly decreased the viability of PC9 cells and induced cell morphological changes. The apoptotic effects of paclitaxel were observed via increased levels of cleaved caspase-3 and cleaved PARP, as well as ROS generation and loss of mitochondrial membrane potential (MMP). Moreover, paclitaxel-induced DNA damage that triggered the death receptor apoptotic pathway. Additionally, this study revealed that paclitaxel effectively suppressed the EGFR/PI3K/AKT/mTOR signaling pathway. Paclitaxel markedly induced cell cycle arrest at the G1 phase through up-regulation of p21WAF1/CIP1 and downregulation of cyclin E1/cdk2 complex. In light of these results, paclitaxel may be a promising agent for anti-cancer therapy against human NSCLC.

Phenolic acid metabolites of polyphenols act as inductors for hormesis in C. elegans

IntroductionAging represents a major risk factors for metabolic diseases, such as diabetes, obesity, or neurodegeneration. Polyphenols and their metabolites, especially simple phenolic acids, gained growing attention as a preventive strategy against age-related, non-communicable diseases, due to their hormetic potential. Using Caenorhabditis elegans (C. elegans) we investigate the effect of protocatechuic, gallic, and vanillic acid on mitochondrial function, health parameters, and the induction of potential hormetic pathways. MethodsLifespan, heat-stress resistance and chemotaxis of C. elegans strain P X 627, a specific model for aging, were assessed in 2-day and 10-day old nematodes. Mitochondrial membrane potential (ΔΨm) and ATP generation were measured. mRNA expression levels of longevity and energy metabolism-related genes were determined using qRT-PCR. ResultsAll phenolic acids were able to significantly increase the nematodes lifespan, heat-stress resistance and chemotaxis at micromolar concentrations. While ΔΨm was only affected by age, vanillic acid (VA) significantly decreased ATP concentrations in aged nematodes. Longevity pathways, were activated by all phenolic acids, while VA also induced glycolytic activity and response to cold. ConclusionWhile life- and health span parameters are positively affected by the investigated phenolic acids, the concentrations applied were unable to affect mitochondrial performance. Therefore we suggest a hormetic mode of action, especially by activation of the sirtuin-pathway.

NAD+ deficiency and mitochondrial dysfunction in granulosa cells of women with polycystic ovary syndrome‡.

Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine disorder characterized by ovulation dysfunction, androgen excess, ovarian polycystic changes, insulin resistance, and infertility. Although underlying mechanisms for PCOS are still unknown, inflammation and mitochondrial dysfunction in granulosa cells (GCs) of PCOS patients have been reported. Here, we found that Nicotinamide Adenine Dinucleotide (NAD+) levels in GCs of PCOS patients was significantly decreased when compared with controls. Also, we found that higher expression of inflammation factors, increased reactive oxygen species (ROS) accumulation, lower adenosine triphosphate (ATP) generation, and decreased mitochondrial membrane potential, as well as abnormal mitochondrial dynamics in GCs of PCOS patients. In addition, the NAD+ levels were decreased after activation of inflammation in human granulosa-like tumor cell line (KGN) treated by Lipopolysaccharide (LPS). However, supplementation of nicotinamide riboside (NR), a NAD+ precursor, could largely restore the NAD+ content, reduce ROS levels and improve mitochondrial function demonstrated by increased mitochondrial membrane potential and ATP generation in LPS-treated KGN cells. Our data suggested that inflammation decreased NAD+ levels in GCs of PCOS patients, while supplementation of NR could restore NAD+ levels and alleviated mitochondrial dysfunction in GCs of PCOS patients.

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba3 from Thermus thermophilus

Cytochrome ba3 from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba3. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.

The need for reconsideration of a mechanism of membrane potential generation using Ling's adsorption theory.

Membrane theory attributes the mechanism of generation of membrane potential to transmembrane ion transport, and is typified by the Goldman-Hodgkin-Katz equation (GHK eq.). Despite broad acceptance of the GHK eq. in physiology, it seems unable to explain some characteristics of the membrane potential. The long-underrated Ling's adsorption theory (LA theory) is another mechanism for membrane potential generation. The LA theory attributes the generation mechanism of the membrane potential to an ion adsorption-desorption process. Although the LA theory has not been seriously considered up until today, there are no serious defects in it as a membrane potential generation mechanism. In this work, the authors explain problematic facets of membrane theory from the view of the GHK eq. We propose an alternative concept based on the LA theory that addresses problematic issues with membrane theory. Consequently, an ion adsorption-desorption process could be a genuine mechanism of membrane potential generation as predicted by the LA theory.

What can S-shaped potential profiles tell us about the mechanism of membrane potential generation?

Membrane theory attributes the generation mechanism of the membrane potential to transmembrane ion transport, while Cheng's ISE (Ion selective electrode) mechanism attributes the ISE potential generation to ion adsorption on to the ISE surface. Although the membrane potential generation mechanism is different from the ISE potential generation mechanism, both the membrane potential and the ISE potential exhibit quite similar characteristics. For instance, both become indifferent to the variation of the ion concentration in both the high and the low ion concentration environment. Our experimental and theoretical investigations suggest that such a characteristic membrane potential behavior could be explained by the ion adsorption mechanism called Ling's adsorption theory (LA theory) instead of by membrane theory. If the membrane potential generation mechanism is explained by the LA theory, then the significant similarity between the membrane potential and the ISE potential is understandable, since both the LA theory and Cheng's ISE mechanism rely on the ion adsorption process. Although the LA theory is not acknowledged as the mechanism for the membrane potential generation in the mainstream physiology community, it does not have any serious defect in principleas a membrane potential generation mechanism. Hence, it is worth investigating if the current membrane potential generation mechanism needs reevaluation in light of evidence presented here. We conclude that the LA theory is a quite plausible membrane potential generation mechanism, suggesting that it may contribute to it.

The msaABCR Operon Regulates Persister Formation by Modulating Energy Metabolism in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that causes chronic, systemic infections, and the recalcitrance of these infections is mainly due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient, antibiotic tolerance accompanied by a transient halt in growth. However, upon cessation of antibiotic treatment, a resumption in growth of persister cells causes recurrence of infections and treatment failure. Previously, we reported the involvement of msaABCR in several important staphylococcal phenotypes, including the formation of persister cells. Additionally, observations of the regulation of several metabolic genes by the msaABCR operon in transcriptomics and proteomics analyses have suggested its role in the metabolic activities of S. aureus. Given the importance of metabolism in persister formation as our starting point, in this study we demonstrated how the msaABCR operon regulates energy metabolism and subsequent antibiotic tolerance. We showed that deletion of the msaABCR operon results in increased tricarboxylic acid (TCA) cycle activity, accompanied by increased cellular ATP content and higher NADH content in S. aureus cells. We also showed that msaABCR (through MsaB) represses the ccpE and ndh2 genes, thereby regulating TCA cycle activity and the generation of membrane potential, respectively. Together, the observations from this study led to the conclusion that msaABCR operon deletion induces a metabolically hyperactive state, leading to decreased persister formation in S. aureus.

Mechanisms of microRNA‑142 in mitochondrial autophagy and hippocampal damage in a rat model of epilepsy.

Researchers have confirmed the microRNA (miRNA/miR)-epilepsy association in rodent models of human epilepsy via a comprehensive database. However, the mechanisms of miR-142 in epilepsy have not been extensively studied. In the present study, a rat model of epilepsy was first established by an injection of lithium chloride-pilocarpine and the successful establishment of the model was verified via electroencephalogram monitoring. The levels of miR-142, phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1), marker proteins of mitochondrial autophagy, and apoptosis-related proteins were measured. Additionally, the pathological changes in the hippocampus, the ultrastructure of the mitochondria, and degeneration and the apoptosis of neurons were observed using different staining methods. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the hippocampus, mitochondrial membrane potential (MTP) and reactive oxygen species (ROS) generation were detected. Furthermore, the targeting association between miR-142 and PINK1 was predicted and verified. Consequently, apoptosis increased, and mitochondrial autophagy decreased, in the hippocampus of epileptic rats. Following miR-142 inhibition, the epileptic rats exhibited an increased Bax expression, a decreased Bcl-2 expression, upregulated marker protein levels of mitochondrial autophagy, a reduced MDA content, an enhanced SOD activity, an increased MTP and decreased ROS generation. PINK1 is a target gene of miR-142, and its overexpression protected against hippocampal damage. Taken together, the results of the present study demonstrated that miR-142 inhibition promotes mitochondrial autophagy and reduces hippocampal damage in epileptic rats by targeting PINK1. These findings may provide useful information for the treatment of epilepsy.

Berberine-photodynamic therapy sensitizes melanoma cells to cisplatin-induced apoptosis through ROS-mediated P38 MAPK pathways

The clinical use of cisplatin are limited due to its drug resistance. Thus, it is urgent to find effective combination therapy that sensitizes tumor cells to this drug. The combined chemo-photodynamic therapy could increase anti-tumor efficacy while also reduce the side effects of cisplatin. Berberine is an isoquinoline alkaloid, which has been reported to show high photosensitizing activity.In this study, we have examined the effect of a combination of cisplatin and berberine-PDT in cisplatin-resistant melanoma cells. The cytotoxic effects of berberine-PDT alone or in combination with cisplatin were tested by MTT assays. We then examined the subcellular localization of berberine with confocal fluorescence microscopy. The percentage of apoptotic cells, the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation assessed using flow cytometry analysis. Western blotting used in this study to determine the expression levels of MAPK signaling pathways and apoptosis-related proteins. Experimental data revealed that the mode of cell death is the caspase-dependent mitochondrial apoptotic pathways. Excessive accumulation of ROS played a key role in this process, which is confirmed by alleviation of cytotoxicity upon pretreatment with NAC. Furthermore, we found that the combined treatment activated MAPK signaling pathway. The inhibition of p38 MAPK by pretreating with SB203580 block the combined treatment-induced apoptotic cell death.In conclusion, berberine-PDT could be used as a chemo-sensitizer by promoting cell death through activation of a ROS/p38/caspase cascade.

Synthesis and biological activities of novel mitochondria-targeted artemisinin ester derivatives

A series of novel artemisinin ester derivatives were designed and synthesized for targeting mitochondria. Cytotoxicity against SMMC-7721, HepG2, OVCAR3, A549 and J82 cancer cell lines was evaluated. Compound 2c (IC50 = 3.0 μM) was the most potent anti-proliferative molecule against the OVCAR3 cells with low cytotoxicity in normal HUVEC cells. The mechanism of action of compound 2c was further investigated by analyzing cell apoptosis, mitochondrial membrane potential (Δψm) and intracellular ROS generation. The results indicated that compound 2c targeted mitochondria and induced cell apoptosis. ROS and heme attributed to the cytotoxicity and cell apoptosis of compound 2c. These promising findings indicated the compound 2c could serve as a great candidate against ovarian cancer for further investigation.

Cinnamide derived pyrimidine-benzimidazole hybrids as tubulin inhibitors: Synthesis, in silico and cell growth inhibition studies

An approach in modern medicinal chemistry to discover novel bioactive compounds is by mimicking diverse complementary pharmacophores. In extension of this strategy, a new class of piperazine-linked cinnamide derivatives of benzimidazole-pyrimidine hybrids have been designed and synthesized. Their in vitro cytotoxicity profiles were explored on selected human cancer cell lines. Specifically, structural comparison of target hybrids with tubulin-DAMA-colchicine and tubulin-nocodazole complexes has exposed a deep position of benzimidazole ring into the αT5 loop. All the synthesized compounds were demonstrated modest to interesting cytotoxicity against different cancer cell lines. The utmost cytotoxicity has shown with an amine linker of benzimidazole-pyrimidine series, with specificity toward A549 (lung cancer) cell line. The most potent compound in this series was 18i, which inhibited cancer cell growth at micromolar concentrations ranging 2.21–7.29 µM. Flow cytometry studies disclosed that 18i inhibited the cells in G2/M phase of cell cycle. The potent antitumor activity of 18i resulted from enhanced microtubule disruption at a similar level as nocodazole on β-tubulin antibody, explored using immunofluorescence staining. The most active compound 18i also inhibited tubulin polymerization with an IC50 of 5.72 ± 0.51 µM. In vitro biological analysis of 18i presented apoptosis induction on A549 cells with triggering of ROS generation and loss of mitochondrial membrane potential, resulting in DNA injury. In addition, 18i displayed impairment in cellular migration and inhibited the colony formation. Notably, the safety profile of most potent compound 18i was revealed by screening against normal human pulmonary epithelial cells (L132: IC50: 69.25 ± 5.95 μM). The detailed binding interactions of 18i with tubulin was investigated by employing molecular docking, superimposition and free energy analyses. Thus remarks made in this study established that pyrimidine-benzimidazole hybrids as a new class of tubulin polymerization inhibitors with significant anticancer activity.

IGF-1 Alleviates Mitochondrial Apoptosis through the GSK3β/NF-κB/NLRP3 Signaling Pathway in LPS-Treated PC-12 Cells.

Inflammation contributes to mitochondrial dysfunction and neuronal apoptosis. The aim of this study was to determine whether insulin-like growth factor-1 (IGF-1) alleviates mitochondrial apoptosis in lipopolysaccharide (LPS)-treated PC-12 cells, and to further explore the mechanism involved.Prepared PC-12 cells were treated with IGF-1, Mdivi-1 (DRP1 blocker), LY294002 (PI3K blocker), betulinic acid (NF-κB activator) or their combinations. Mitochondrial membrane potential and ATP generation were then measured to assess mitochondrial function. The rate of apoptosis was determined using flow cytometry. The expression of several apoptosis proteins (i.e. Bax, cleaved caspase-9 and cleaved caspase-3) and signaling proteins (i.e. p-GSK3β, NF-κB and NLRP3) was measured using western blotting.Compared with the control cells, the LPS-treated cells showed evidence of mitochondrial dysfunction, increased apoptosis and upregulation of apoptosis proteins, which were significantly alleviated by Mdivi-1. These findings indicate that neuronal apoptosis was activated partly through the mitochondrial pathway. IGF-1 treatment inhibited mitochondrial apoptosis in a dose-dependent manner in the LPS-treated cells. The reagent also increased the expression of p-GSK3β and decreased the expression of NF-κB and NLRP3. Both LY294002 and betulinic acid reversed the protective effect of IGF-1. In addition, LY294002 affected the expression of the three signaling proteins, while betulinic acid only affected the expression of NF-κB and NLRP3.These findings indicated a GSK3β/NF-κB/NLRP3 signaling pathway was existed and was involved in the protective mechanism of IGF-1. In conclusion, IGF-1 alleviated mitochondrial apoptosis through GSK3β/NF-κB/NLRP3 signaling pathway in LPS-treated PC-12 cells.