Cell Mitochondrial Membrane Research Articles

Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.

LncRNA H19 inhibits oxidative stress injury of cochlear hair cells by regulating miR-653-5p/SIRT1 axis.

Oxidative stress is one of the important mechanisms of inner ear cell damage, which can lead to age-related hearing loss (ARHL). LncRNA H19 is significantly downregulated in the cochlea of old mouse, however, the role of H19 in the development of ARHL remains unclear. In this study, we aim to investigate the expression and function of H19 in oxidative stress injury of cochlear hair cells induced by HO. RT-qPCR and western blot analysis confirms that HEI-OC1 cells stimulated with HO decreases the expressions of H19 and SIRT1, but increases the expression of miR-653-5p. Overexpression of H19 could increase cell viability, ATP level and mitochondrial membrane potential, but reduce mitochondrial ROS generation and cell apoptosis ratio in HO-stimulated HEI-OC1 cells. MiR-653-5p is a target of H19, which can bind to the 3'-UTR of SIRT1. H19 is found to regulate the expression of SIRT1 through miR-653-5p. Further experiments demonstrates that H19 regulates HEI-OC1 cell viability, ATP level, mitochondrial membrane potential, mitochondrial ROS generation, and cell apoptosis ratio via the miR-653-5p/SIRT1 axis. In conclusion, lncRNA H19 inhibits oxidative stress injury of cochlear hair cells via the miR-653-5p/SIRT1 axis.

Mechanisms of cinnamic aldehyde against myocardial ischemia/hypoxia injury in vivo and in vitro: Involvement of regulating PI3K/AKT signaling pathway

To investigate the protection of cinnamic aldehyde (CA) against myocardial ischemia/hypoxia (I/H) injury and its potential mechanisms in vivo and in vitro. Mice were pretreated with CA for 7 days, and then isoproterenol (85 mg/kg) was administered for 2 consecutive days to assess its cardioprotection. Furthermore, an in vitro myocardial I/H model was established by administering CoCl2 (600 μM) to H9c2 cells for 24 h. H9c2 cells were pretreated with CA for 12 h to assess its protection. We observed that CA improved electrocardiogram and histopathological changes and decreased creatine kinase and lactate dehydrogenase activities and oxidative stress levels. The TUNEL results showed that CA reduced the degree of apoptosis. Furthermore, CA could lead to a down-regulation of the Caspase-3 and Bax protein expressions, but an up-regulation of the Bcl-2 protein expressions. Importantly, CA increased p-PI3K and p-AKT protein expressions, indicating the activation of the PI3K/AKT signaling pathway. Moreover, treatment with CA improved the cell viability rate and mitochondrial membrane potential while markedly decreasing apoptosis and oxidative stress levels in vitro. Our results suggested that CA exerts cardioprotection on myocardial I/H injury, which possibly occurred in connection with inhibition of oxidative stress and apoptosis via activation of the PI3K/AKT signaling pathway.

Design, synthesis and biological evaluation of hydantoin derivatives as Mcl-1 selective inhibitors

As a member of Bcl-2 protein family, myeloid cell leukemia-1 (Mcl-1) plays a critical role in cell apoptosis and has become a promising anti-cancer drug target. Herein, we designed and synthesized a series of hydantoin derivatives as novel Mcl-1 inhibitors based on our previously developed lead compound. Among them, compound M23 and M24 exhibited good binding affinities against Mcl-1 with Ki values of 0.49 μM and 0.33 μM respectively. Especially, compound M23 exhibited good selectivity over Bcl-xL, whereas compound M24 possessed good selectivity over both Bcl-2 and Bcl-xL. Furthermore, we also investigated the effects of these new Mcl-1 inhibitors on cell proliferation, apoptosis and mitochondrial membrane potential, as well as the stability in plasma.

Alleviative effect of melatonin on the decrease of uterine receptivity caused by blood ammonia through ROS/NF-κB pathway in dairy cow

High concentration of blood ammonia can affect the uterus receptivity and decrease fecundity in dairy cow. Melatonin can reduce reactive oxygen species (ROS) level and has antioxidant and anti-inflammatory effects. However, it is not clear whether melatonin can alleviate ammonia-induced apoptosis of endometrial epithelial cell (EEC) and reduced uterus receptivity. The bovine EEC were treated with ammonium chloride and/or melatonin. Cell viability, apoptosis, oxidative stress and mitochondrial membrane potential were measured and the expression of apoptosis-related genes (p53, Cyt-c, Bax, Bcl-2, caspase-8, caspase-9 and caspase-3), uterus receptivity related genes (VEGF, LIF and EGF) and inflammatory factors (TLR-4, IL-6 and NF-κB) were detected. In addition, the expression of VEGF was detected after adding NF-κB inhibitor (40 μM) and IL-6 (1 ng/mL and 50 ng/mL). The results showed that ammonia significantly increased intracellular ROS level, mRNA and protein expression of Bax, p53, Cyt-c, caspase-9, caspase-8, caspase-3, TLR-4, NF-κB and IL-6, promoted cell apoptosis, while decreased mitochondrial membrane potential, the mRNA and protein expression of VEGF and EGF. Interestingly, melatonin significantly mitigated ammonia-induced changes. However, melatonin could not alleviate ammonia-induced changes of IL-6 and VEGF when NF-κB signal pathway was inhibited. The addition of IL-6 significantly reduced mRNA and protein expression of VEGF. In conclusion, ammonia induced EEC apoptosis through ROS production and activation of mitochondrial apoptosis pathway, and induced inflammatory response through TLR4/NF-κB/IL-6 pathway. Melatonin alleviated EEC apoptosis by inhibiting ROS pathway, and reduced IL-6 expression by inhibiting TLR-4/NF-κB signal pathway, which eventually improved VEGF expression and uterus receptivity in dairy cows.

Fabrication of cellulose nanocrystals as potential anticancer drug delivery systems for colorectal cancer treatment

Polysaccharide nanocrystals have great potential to be used as improved drug carriers due to their low cost, high biodegradability, and biocompatibility. This study reports the synthesis of cellulose nanocrystals (CNC) loaded with 5-fluorouracil (CNC/5FU) to evaluate their anticancer activity against colorectal cancer cells. X-ray and Fourier-transform infrared spectroscopy demonstrated that acid hydrolysis successfully degraded the amorphous cellulose to liberate the crystal regions. From transmission electron microscopy, CNC/5FU appeared as rod-like nanocrystals with an average length and width of 69.53 ± 1.14 nm and 8.13 ± 0.72 nm, respectively. The anticancer drug 5FU showed improved thermal stability after being loading onto CNC. From UV–vis spectroscopy data, the drug encapsulation efficiency in CNC/5FU was estimated to be 83.50 ± 1.52%. The drug release of CNC/5FU was higher at pH 7.4 compared to those at pH 4.2 and 1.2. From the cytotoxicity assays, CNC did not affect the viability of CCD112 colon normal cells. On the other hand, CNC/5FU exhibited anticancer effects against HCT116 and HT-29 colorectal cancer cells. The anticancer actions of CNC/5FU against HCT116 cells were then confirmed using an in vitro tumor-on-chip model and clonogenic assay. Mechanistic studies demonstrated that CNC/5FU killed the cancer cells by mainly inducing cell apoptosis and mitochondrial membrane damage. Overall, this study indicated that CNC/5FU could be a potential nanoformulation for improved drug delivery and colorectal cancer treatment.

β-Elemene induces apoptosis and autophagy in colorectal cancer cells through regulating the ROS/AMPK/mTOR pathway.

β-Elemene is an effective anti-cancer ingredient extracted from the genus Curcuma (Zingiberaceae familiy). In the present study, we demonstrated that β-elemene inhibited the proliferation of colorectal cancer cells and induced cell cycle arrest in the G2/M phase. In addition, β-elemene induced nuclear chromatin condensation and cell membrane phosphatidylserine eversion, decreased cell mitochondrial membrane potential, and promoted the cleavage of caspase-3, caspase-9 and PARP proteins, indicating apoptosis in colorectal cancer cells. At the same time, β-elemene induced autophagy response, and the treated cells showed autophagic vesicle bilayer membrane structure, which was accompanied by up-regulation of the expression of LC3B and SQSTM1. Furthermore, β-elemene increased ROS levels in colorectal cancer cells, promoted phosphorylation of AMPK protein, and inhibited mTOR protein phosphorylation. In the experiments in vivo, β-elemene inhibited the tumor size and induced apoptosis and autophagy in nude mice. In summary, β-elemene inhibited the occurrence and development of colon cancer xenografts in nude mice, and significantly induced apoptosis and autophagy in colorectal cancer cells in vitro. These effects were associated with regulation of the ROS/AMPK/mTOR signaling. We offered a molecular basis for the development of β-elemene as a promising anti-tumor drug candidate for colorectal cancer.

Dipotassium Glycyrrhizinate Against Fibrotic Effects of Histamine Depletes Intracellular Glutathione to Discomfort Mitochondrial, Inducing Cell Apoptosis/ Ferroptosis of Over-Activated Lung Fibroblast

Pulmonary fibrosis (PF) is the common outcome of a variety of chronic lung diseases and systemic diseases, and the consequence of Coronavirus Disease 2019 (COVID-19). There is currently a lack of effective treatments to reverse this disease or prevent its development. The purpose of this study is to investigate the effect of dipotassium glycyrrhizinate (DG) on lung fibroblasts and the possible mechanism of alleviating pulmonary fibrosis. Extract primary lung fibroblasts, establish cell models, and use time-lapse photography to continuously monitor the effects of dipotassium glycyrrhizinate and histamine on the morphology of lung fibroblasts. The content of glutathione in cells was detected by the fluorescence method. Cell mitochondrial membrane potential was examined, protein expressions of P53, Caspase-3, Bax and Bal-2 of lung fibroblasts were evaluated by western blot and immune cytochemistry. Our data demonstrated that dipotassium glycyrrhizinate inhibited histamine-induced cell growth/proliferation, glutathione content, mitochondrial membrane potential; also down-regulated P53 and up-regulated the ratio of Bax/Bcl-2, leading to the ferritosis/apoptosis of activated lung fibroblasts, against the fibrotic effects of histamine. Present study provided a new pharmaceutics mechanism of dipotassium glycyrrhizinate for the treatment of lung fibrosis that dipotassium glycyrrhizinate against fibrotic effects of histamine depletes intracellular glutathione to discomfort mitochondrial, inducing cell apoptosis/ferroptosis of over-activated lung fibroblast.

Composition and antifungal mechanism of essential oil from Chrysanthemum morifolium cv. Fubaiju

• Components of Chrysanthemum morifolium cv. Fubaiju essential oil (CMF-EO) were characterized. • CMF-EO showed excellent antifungal activities against six common fungus. • Antifungal targets were cell membrane, mitochondrial membrane, and DNA. • Multiple antifungal mechanisms make fungi difficult to develop drug resistance. Chrysanthemum morifolium cv. Fubaiju (CMF) has been used as a dietary herb in East Asia for centuries. The chemical composition and antifungal activity of CMF essential oil (CMF-EO) were investigated in this study. We have found that CMF-EO was a light-yellow, transparent liquid (0.61% yield, w/w) and majorly contain 31 volatile components with a total of 98.41% determined by GS and GC/MS. Camphor was the major compound (16.87%), followed by 1,8-cineole (10.62%), β -curcumene (9.51%), bornyl acetate (7.43%), α -zingiberene (6.97%), β-elemene (6.47%), α -bisabolol (4.74%), borneol (4.24%) and eugenol (4.23%). In addition, contents with more than 2% of the total CMF-EO were 1-caryophyllene (3.92%), chrysanthenyl acetate (3.29%), caryophyllene oxide (2.28%), and chamazulene (2.23%). Six fungal strains were used to study the antifungal activities and mechanisms of CMF-EO using fluorescence probe–membrane integrity assay, analysis of cytoplasmic membrane permeability, electron microscopy, monitoring of the effect of the mitochondrial membrane potential and DNA binding assay. CMF-EO effectively inhibited the growth of C. albicans , C. glabrata , C. tropicalis , S. cerevisiae , D. hansenii , and Z. parabailii with minimum inhibition concentrations of 31.25, 31.25, 62.5, 31.25, 15.625, and 15.625 μg/mL, respectively, and minimum fungicidal concentrations of 62.5, 125.0, 125.0, 62.5, 31.25, and 62.5 μg/mL, respectively. The elucidated antifungal mechanisms of CMF-EO included the destruction of cytoplasmic membrane and the damage of mitochondria and DNA. The multiple antifungal mechanisms made it difficult for fungi to develop drug resistance, and this finding might shed new light on the development of new fungal agents.

The Effect of Icariin on Human Hepatoellular Carcinomas Cell Apoptosis and Cell Cycle Arrest

Background: Natural products often have novel frameworks and unique mechanisms, It is an important way to develop new anti-tumor drugs.The paper explored the effect of Icariin on HepG2 cell apoptosis and cell cycle arrest. Material and methods: Human liver cancer HepG2 cells were studied. The biological activity of Icariin on HepG2 cells was comprehensively investigated. The mechanism was preliminarily explored from the aspects of proliferation, cell cycle, and apoptosis. Results: Icariin showed significant inhibitory effect on cell proliferation after administration, and the effect was time-and-concentration-dependent. Annexin V-PI detection showed that, after 48 hours of administration of Icariin at different concentrations, the apoptosis rate of HepG2 cells increased in a concentration-dependent manner. The results of Hoechst 33342 staining showed that, after 48 hours of intervention with Icariin at different concentrations, HepG2 cells appeared densely stained and granular fluorescence, characterizing apoptosis. In the JC-1 mitochondrial membrane potential experiment, Icariin was found to destroy cell mitochondrial membrane potential and induce HepG2 cell apoptosis. After 48 h administration of Icariin at different concentrations, Bcl-2 and survivin proteins were down-regulated, and Bax was up-regulated, both in a concentration-dependent manner. PI single staining combined with flow cytometry to detect cell cycle results showed that, Icariin can induce G2/M phase arrest of HepG2 cells, and is time-and-concentration-dependent. Western Blot detection revealed that Icariin can down-regulate the cycle-related proteins Cyclin B and CDK1 in a concentration-dependent manner, and also significantly down-regulate the expressions of p-AKT, AKT, p-ERK and ERK proteins. Conclusion: Icariin is a selectively potential active compound that can treat liver cancer. The paper provided theoretical basis and experimental support for the clinical application of Icariin in the drug treatment of liver cancer. It is necessary to further study the antitumor effect of Icariin, explore and find the target, and provide higher selectivity for the treatment of liver cancer by Icariin.

Self-activating anti-infection implant

Clinically, it is difficult to endow implants with excellent osteogenic ability and antibacterial activity simultaneously. Herein, the self-activating implants modified with hydroxyapatite (HA)/MoS2 coating are designed to prevent Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections and accelerate bone regeneration simultaneously. The electron transfer between bacteria and HA/MoS2 is triggered when bacteria contacted with the material. RNA sequencing data reveals that the expression level of anaerobic respiration–related genes is up-regulated and the expression level of aerobic respiration–related genes is down-regulated when bacteria adhere to the implants. HA/MoS2 presents a highly effective antibacterial efficacy against both S. aureus and E. coli because of bacterial respiration–activated metabolic pathway changes. Meanwhile, this coating promotes the osteoblastic differentiation of mesenchymal stem cells by altering the potentials of cell membrane and mitochondrial membrane. The proposed strategy exhibits great potential to endow implants with self-activating anti-infection performance and osteogenic ability simultaneously.

An application of p-sulfonatocalix[6]arenes to attenuate cardiotoxicity of mitoxantrone in vitro: preparation, characterization and evaluation.

In this study, p-sulfonatocalix[6]arenes (SCA6) was proposed to construct a host-guest complexation to carry mitoxantrone (MIT) to maintain its anti-proliferation effect on HepG2 cells as well as to attenuate cardiotoxicity on H9C2 cells as a nano-size drug delivery system. SCA6 binding to MIT evidenced through competitive fluorescence titration method. The complex was characterized using UV-visible, Fourier transform infrared, and proton nuclear magnetic resonance (1H-NMR) spectroscopies and differential scanning calorimetry analysis. The cytotoxicity was examined by a cell counting kit-8 assay on six cells. High content analysis, cell apoptosis and cell cycle experiments were measured to investigate the mechanism of detoxification in H9C2. The host-guest complexation was formed with a stoichiometry ratio of 1:1. Cytotoxicity study demonstrated that MIT/SCA6 complex could improve the cell viability on H9C2, MCF-7, A549, Hek293 and L02 cells and remained cytotoxicity effect on HepG2 cells. High content analysis showed that MIT/SCA6 complex could enhance the cell viability, mitochondrial mass and mitochondrial membrane potential and ameliorate the nuclear swelling on H9C2 cells. Moreover, the complex were arrested in G0/G1 phase of the cell cycle and same with MIT, while the detoxication was attributed to reducing early apoptosis. The host-guest complexation between SCA6 and MIT had the ability to attenuate cardiotoxicity and provided a potential strategy for the application of soluble calixarenes in chemotherapy.

YQFM alleviated cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway

Ethnopharmacological relevanceAs a traditional compound preparation of Chinese medicine, Yiqi Fumai lyophilized injection (YQFM) has protective effects on various cardiac diseases including cardiac hypertrophy, which is the primary cause of arrhythmia. However, the involved mechanism remains unclear. Aim of the study: This study was projected to investigate whether YQFM could prevent cardiac hypertrophy and arrhythmia concurrence. Materials and Methods: The cardiac hypertrophy rats were established by transverse aortic ligation and the H9c2 hypertrophy cardiomyocyte was induced by angiotensin II (AngII). The electrocardiogram (ECG) was conducted to estimate the arrhythmia occurrence of cardiac hypertrophy rats under isoprenaline (iso) treatment. The cardiac related indicators and histopathology were also detected. The protective effects of YQFM on H9c2 hypertrophy cardiomyocyte were determined by the cell size measurement, apoptosis detection and mitochondrial membrane potential measurement. The cardiac hypertrophy relative proteins (ANP and BNP), autophagy related factors (LC3II, p62 and Beclin-1), apoptosis related markers (p53, caspase 3, Bax and Bcl-2) and the PI3K/AKT/mTOR pathway expressions were all measured by Western blot. Results: YQFM decreased the arrhythmia occurrence and improved cardiac function in cardiac hypertrophy rats. YQFM also reduced the H9c2 cardiomyocyte size and alleviated the cardiomyocyte apoptosis induced by AngII. In addition, YQFM inhibited cell apoptosis by increasing Bcl-2/Bax ratio and decreasing caspase 3 and p53 expressions in vitro and vivo. Meanwhile, YQFM regulated the autophagy pathway by down-regulating of LC3II and Beclin-1 expressions, as well as up-regulating of p62 expression. Finally, the results showed that YQFM could activate the PI3K/AKT/mTOR pathway by enhancing the p-AKT, p-PI3K and p-mTOR expressions. Conclusion: Our results displayed that YQFM attenuated the cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway.

Benzimidazole-quinoline-based copper complexes: Exploration for their possible antitumor mechanism

In this study, we synthesised and characterised two benzimidazole-quinoline-based copper complexes, namely, [Cu(btmbq)Br]2 (1) and [Cu(btmbq)Cl]2 (2), (btmbq = 3-(1-(1H-benzotriazol-1-y-l)methyl)-6-bromo-1H-benzoimidazol-2-yl)isoquinoline). Both complexes showed strong antitumor abilities against the colon cancer cell line (HCT116) and low cytotoxicity against the normal liver cell line (L-02). The DNA binding affinity was evaluated using CD and fluorescence spectroscopy, and the Ksv and Kapp values were further quantified, revealing that the complexes bound to DNA in the intercalation mode, and caused oxidative damage to pBR322 DNA. Furthermore, complex 1 interfered with the steady-state balance of redox and Ca2+ in HCT116 cells, as well as induced cell mitochondrial membrane potential (Δψm) collapse, ATP dissipation, ultimately arrested the cell cycle in G2 phase and induced cell apoptosis. Further exploration demonstrated that the production of reactive oxygen species (ROS) might be the major contributors to the apoptotic death of HCT116 cells.

Aureoverticillactam, a Potent Antifungal Macrocyclic Lactam from Streptomyces aureoverticillatus HN6, Generates Calcium Dyshomeostasis-Induced Cell Apoptosis via the Phospholipase C Pathway in Fusarium oxysporum f. sp. cubense Race 4.

Extensive efforts have been made to discover new biofungicides of high efficiency for control of Fusarium oxysporum f. sp. cubense race 4, a catastrophic soilborne phytopathogen causing banana Fusarium wilt worldwide. We confirmed for the first time that aureoverticillactam (YY3) has potent antifungal activity against F. oxysporum f. sp. cubense race 4, with effective dose for 50% inhibition (EC50) of 20.80 μg/ml against hyphal growth and 12.62 μg/ml against spore germination. To investigate its mechanism of action, we observed the cellular ultrastructures of F. oxysporum f. sp. cubense race 4 with YY3 treatment and found that YY3 led to cell wall thinning, mitochondrial deformities, apoptotic degradation of the subcellular fractions, and entocyte leakage. Consistent with these variations, increased permeability of cell membrane and mitochondrial membrane also occurred after YY3 treatment. On the enzymatic level, the activity of mitochondrial complex III, as well as the ATP synthase, was significantly suppressed by YY3 at a concentration >12.50 μg/ml. Moreover, YY3 elevated the cytosolic Ca2+ level to promote mitochondrial reactive oxygen species (ROS) production. Cell apoptosis also occurred as expected. On the transcriptome level, key genes involved in the phosphatidylinositol signaling pathway were significantly affected, with the expression level of Plc1 increased approximately fourfold. The expression levels of two apoptotic genes, casA1 and casA2, were also significantly increased by YY3. Of note, phospholipase C activation was observed with YY3 treatment in F. oxysporum f. sp. cubense race 4. These findings indicate that YY3 exerts its antifungal activity by activating the phospholipase C calcium-dependent ROS signaling pathway, which makes it a promising biofungicide.

Cytotoxic and apoptotic effects of 1,2-diborolanes with strong donor substitutes on human cancer cells

In recent years, boron compounds have become more common as chemotherapy agents against certain types of cancers. Along with the development of boron-based therapeutic agents have come investigations into the various cancers and biochemical and molecular mechanisms affected by boron compounds and the relationships between boron compounds and chemical protection against cancer. In this preliminary study, the effects of new 1,2-N-substituted-1,2-diborolane derivatives on types of breast and liver cancers were examined for the first time. Four were found to significantly affect the cell viabilities and mitochondrial membrane potential changes in MCF-7, HepG2 and Hep3B cancer cells. Each was prepared in n-hexane at various concentrations (5, 10, 25, 50, 75 and 100 µg/mL). Human peripheral blood lymphocytes were used as control cells. Compounds 1, 2, 3a, and 3b 1,2-diborolane derivatives selectively killed cancer cells, but compound 1 was cytotoxic in a concentration-dependent manner on HepG2 and Hep3B and only at concentrations of at least 75 µg/mL on MCF-7 cells. Compound 3a exhibited cytotoxic effect on lymphocytes at 75 and 100 µgmL−1 concentrations, but compounds 1, 2 and 3b, 3c and 3d have not possessed significant cytotoxic effect on lymphocytes. Compounds 3c and 3d have not possessed significant cytotoxic effects. Mitochondrial membrane potential assay results supported these findings. Our results reveal that 1,2-diborolane derivates have high cytotoxic and apoptotic activities on human hepatocarcinoma cells and are therefore potential candidates in the development of new drugs against liver cancer.

Anti-apoptotic effect of Buchholzia coriacea Engl. stem back extracts on AsPC-1 and mechanisms of action

Ethnopharmacological relevanceBuchholzia coriacea Engl. is popularly called wonderful cola due to its wide ethnomedicinal use for the treatment of various ailments. We investigated the possible cytotoxic effect of its various fractions on human pancreatic cancer cell (AsPC-1) and also determined its mechanisms of action.Materials and methodsThe AsPC-1 cells were cultivated and separately treated with 5-fluorouracil (5-FU) or Buchholzia coriacea Engl. bark (BC) (ethanol, aqueous, chloroform or ethyl acetate extract) for 72 h. Cell viability, caspase 3 and mitochondrial membrane potential (ΔΨm) were determined in vitro after the treatment. Nitric oxide (NO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals’ scavenging property, ferric reducing power and lipid peroxidation assays were also done to examine the antioxidant effect of BC in vitro.ResultsVarious extracts of BC, especially at 2500 μg/ml and 5000 μg/ml, increased the AsPC-1 viability while 5-FU decreased it. The activity of caspase 3 was increased by 5-FU but reduced by all concentrations of various extracts of BC. Incubation of AsPC-1 with 5-FU showed the majority of cells having the monomeric form of JC-1 dye (bright green fluorescence), which indicated de-energized mitochondria. However, fluorescence photomicrograph of cells incubated with different concentrations (20, 40 and 100 μg/ml) of BC extracts (aqueous, ethanol, chloroform and ethyl acetate) showed strong JC-1 aggregation (yellow), which indicated mitochondria with intact membrane potentials. BC extracts also scavenged NO and DPPH radicals, inhibited lipid peroxidation and increased ferric reduction, though not as much as ascorbic acid.ConclusionThis study suggests that BC elicits anti-apoptotic activity in AsPC-1 by increasing cell viability, decreasing caspase 3 activity, stabilizing the ∆Ψm, and scavenging free radicals. Even though BC is used ethnomedicinally as anti-cancer agent, our findings in the present study suggest that it has pro-cancer potential in-vitro, especially on pancreatic cells. Its anti-apoptotic activity in AsPC-1 could be of clinical significance, especially to counteract the effect of apoptotic agents on pancreatic cells.

MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0–300 μM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

EVALUATION OF DOSE-DEPENDENT ACTIVITY OF BISPHENOL F ON VIABILITY PARAMETERS AND STEROIDOGENESIS IN H295R CELLS

Increasing concern over bisphenol A (BPA) as an endocrine-disrupting chemical and recent imposition of restriction on the use of BPA paved the way for entry of its analogues in the market. Bisphenol F (BPF) is one of the major analogues of commercial value. Thus, its increasing production and application make it vulnerable to human exposure. The aim of our in vitro study was to assess the potential effect of BPF on H295R cells mitochondrial activity, metabolic activity, membrane integrity, lysosomal function, and testosterone synthesis. Adrenocortical carcinoma cells were cultivated during 24 h in the presence of BPF (0.1, 0.5, 1, 10, 25, 50, 75, 100, 300, 500 μM). Exposure doses of BPF caused a significant decrease of mitochondrial activity starting from 1 μM, we observed a slight increase in mitochondrial activity at the lowest concentration (0.1 μM). Metabolic activity decreased with increasing dose of BPF - from 10 to 500 μM. A significant increase in metabolic activity was observed after cultivation with 0.1 μM BPF and a slight increase was observed after cultivation with 0.5 μM BPF. We observed a slight increase in lysosomal function and membrane integrity after cultivation with 0.1 and 1 μM, although higher exposure doses (25 - 500 μM) caused significant decrease in membrane integrity and lysosomal function. Lowest exposure dose of BPF (0.1 μM) caused a significant increase in testosterone synthesis, on the other hand, higher exposure doses (50 - 500 μM) caused significant decrease of testosterone production. The obtained results confirmed that BPF at higher concentrations caused cytotoxicity and possibly have endocrine-disrupting potential.

Acute Valproate Exposure Induces Mitochondrial Biogenesis and Autophagy with FOXO3a Modulation in SH-SY5Y Cells.

Valproic acid (VPA) is an antiepileptic drug found to induce mitochondrial dysfunction and autophagy in cancer cell lines. We treated the SH-SY5Y cell line with various concentrations of VPA (1, 5, and 10 mM). The treatment decreased cell viability, ATP production, and mitochondrial membrane potential and increased reactive oxygen species production. In addition, the mitochondrial DNA copy number increased after VPA treatment in a dose-dependent manner. Western blotting showed that the levels of mitochondrial biogenesis-related proteins (PGC-1α, TFAM, and COX4) increased, though estrogen-related receptor expression decreased after VPA treatment. Further, VPA treatment increased the total and acetylated FOXO3a protein levels. Although SIRT1 expression was decreased, SIRT3 expression was increased, which regulated FOXO3 acetylation in the mitochondria. Furthermore, VPA treatment induced autophagy via increased LC3-II levels and decreased p62 expression and mTOR phosphorylation. We suggest that VPA treatment induces mitochondrial biogenesis and autophagy via changes in FOXO3a expression and posttranslational modification in the SH-SY5Y cell line.