Nisin induces apoptosis in cervical cancer cells via reactive oxygen species generation and mitochondrial membrane potential changes

Nisin, an antimicrobial peptide produced by Lactococcus lactis, is widely used as a safe food preservative and has been recently attracting the attention of many researchers as a potential anticancer agent. The cytotoxicity of nisin against HeLa, OVCAR-3, SK-OV-3, and HUVEC cells was evaluated using MTT assay. The apoptotic effect of nisin was identified by Annexin-V/propidium iodide assay, and then it was further confirmed by western blotting analysis, mitochondrial membrane potential (ΔΨm) analysis, and reactive oxygen species (ROS) assay. The MTT assay showed concentration-dependent cytotoxicity of nisin towards cancer cell lines, with the IC50 values of 11.5-23 µM, but less toxicity against normal endothelial cells. Furthermore, treatment of cervical cancer cells with 12 µM nisin significantly (P<0.05) increased the Bax/Bcl-2 ratio (4.9-fold), reduced ΔΨm (70%), and elevated ROS levels (1.7-fold). These findings indicated that nisin might have anticancer and apoptogenic activities through mitochondrial dysfunction and oxidative stress damage in cervical cancer cells.

Is the Mitochondrial Membrane Potential (∆Ψ) Correctly Assessed? Intracellular and Intramitochondrial Modifications of the ∆Ψ Probe, Rhodamine 123

The mitochondrial membrane potential (∆Ψ) is the driving force providing the electrical component of the total transmembrane potential of hydrogen ions generated by proton pumps, which is utilized by the ATP synthase. The role of ∆Ψ is not limited to its role in bioenergetics since it takes part in other important intracellular processes, which leads to the mandatory requirement of the homeostasis of ∆Ψ. Conventionally, ∆Ψ in living cells is estimated by the fluorescence of probes such as rhodamine 123, tetramethylrodamine, etc. However, when assessing the fluorescence, the possibility of the intracellular/intramitochondrial modification of the rhodamine molecule is not taken into account. Such changes were revealed in this work, in which a comparison of normal (astrocytic) and tumor (glioma) cells was conducted. Fluorescent microscopy, flow cytometry, and mass spectrometry revealed significant modifications of rhodamine molecules developing over time, which were prevented by amiodarone apparently due to blocking the release of xenobiotics from the cell and their transformation with the participation of cytochrome P450. Obviously, an important role in these processes is played by the increased retention of rhodamines in tumor cells. Our data require careful evaluation of mitochondrial ∆Ψ potential based on the assessment of the fluorescence of the mitochondrial probe.

Sesquiterpene from Polygonum Barbatum Disrupt Mitochondrial Membrane Potential to Induce Apoptosis and Inhibits Metastasis by Downregulating Matrix Metalloproteinase and Osteopontin in NCI-H460 Cells

Background: Globally, lung cancer accounts for 18% of cancer-associated mortalities. Among the subtypes, non-small-cell lung cancer (NSCLC) is the most prevalent one. The increased resistance and poor survival rates, signifies disease aggressiveness and thus require a search for an alternative anticancer molecule. Hypothesis/Purpose: Earlier, the isolated sesquiterpene i.e. compound 1 ((E)-Methyl 6-acetoxy-7-methoxy-1-(2-methylpropylidene)-1H-indene-3-carboxylate) from P. barbatum was isolated, characterized by us and reported for preliminary anticancer activity. Considering its potent activity, this study was designed to explore the underlying molecular mechanism of apoptosis and metastasis against NCI-H460 cells. Method: The molecular mechanism of compound 1 inducing apoptosis and inhibiting metastasis was elucidated by analyzing mitochondrial membrane potential, DNA fragmentation, clonogenic assay, invasion assay and expression of apoptotic (Caspases 3, 6, 8, 9 and BAK) and metastatic markers (MMP 2, 9 and osteopontin). Results: Compound 1 significantly inhibited cell proliferation and induced apoptosis via intrinsic route i.e. the mitochondrial pathway by disrupting mitochondrial membrane potential. The enhanced expression of caspases 6, 9, BAK and HRK with downregulation of Bcl-2L1 and Ki67 further confirmed the involvement of the intrinsic apoptotic pathway. Moreover, compound 1 restricted the invasive nature of NCI-H460 cells evinced by reduced cell invasion in Boyden chamber invasion assay and downregulating the expression of metastatic markers i.e. matrix metalloproteinase 2 / 9 and VEGF. It was also found that it blocks osteopontin by negatively regulating its expression; a marker protein in cancer management. Conclusion: Conclusively, this sesquiterpene exhibited potent anticancer and anti-metastatic activity and can be explored further as possible pharmacophores

Paris Polyphylla Monomer PP-26 Induces Mitochondrial Pathway-Mediated Cell Apoptosis by Inhibiting the PI3K/Akt Pathway in Human Gastric Cancer Cells

Background: Paris polyphylla is a traditional Chinese medicinal herb that has been used as a haemostatic, antimicrobial and anticancer agent. Gastric cancer (GC) is a global health problem, with more than 1 million people newly diagnosed with gastric cancer worldwide each year.Methods: The MTT and colony formation assay were used to test the anti-proliferative effects of PP-26 on MGC-803 and BGC-823 cells. Flow cytometry assays, Hoechst 33258 staining assay and Caspase inhibitor Z-VAD-FMK were used to test apoptosis. JC-1 staining used to measure changes in mitochondrial membrane potential and western blot analysis were used to test apoptotic and PI3K/Akt pathway related proteins.Results: PP-26 had a dose-dependent inhibitory effect on the proliferation of MGC-803 and BGC-823 cells, but had no obvious anti-proliferative effect on normal liver LO2 cells and normal embryonic kidney HEK-293 cells. Additionally, PP-26 induced typical apoptotic morphological changes, such as nuclear pyknosis, nuclear cracking and apoptotic bodies. Moreover, PP-26 induced a decrease in mitochondrial membrane potential. And PP-26 modulated the expression of Bcl-xL, Mcl-1, Bax, caspase-9/-3 and PARP proteins and induced cell apoptosis through the mitochondrial apoptotic pathway. Next, using an irreversible general caspase inhibitor (Z-VAD-FMK), we confirmed the activation of the mitochondrial apoptotic pathway induced by PP-26. Furthermore, PP-26 inhibited the phosphorylation of Akt and GSK-3β. The inhibition of Akt protein activated the mitochondrial apoptotic pathway.Conclusion: Collectively, these results indicated that PP-26 inhibited the proliferation of MGC-803 and BGC-823 cells by inhibiting the Akt signalling pathway and activating the mitochondrial apoptotic pathway.

Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia, and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation-perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single-pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm, and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone-induced and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.

Flurochloridone Induced Abnormal Spermatogenesis by Damaging Testicular Sertoli Cells in Mice

BackgroundFlurochloridone (FLC), a selective herbicide used on a global scale, has been reported to have male reproductive toxicity which evidence is limited and the mechanism is still unclear. The present study was conducted to systematically explore the male reproductive toxicity of FLC, including sperm quality, spermatogenesis process, toxicity targets and possible mechanisms. MethodsMale C57BL/6 mice aged 6-7 weeks received gavage administration of FLC (365/730 mg/kg body weight) for 28 consecutive days. Then the tissue and sperm of mice were collected for analysis. We measured the coefficient of male reproductive organs, and analyzed sperm concentration, motility, malformation rate and mitochondrial membrane potential. Spermatocyte immunofluorescence staining was performed to analyze meiosis processes. At the same time, we performed pathological staining on the testis and epididymis tissue, and performed TUNEL staining, immunohistochemical analysis and ultrastructural observation on the testicular tissue.ResultsThe results showed that FLC caused mice testicular weight reduction, dysfunction and architectural damage, but no significant adverse effect was found in epididymis. The exposure interfered with the proliferation of spermatogonia and the process of meiosis, affecting sperm concentration, motility, kinematic parameters, morphology and mitochondrial membrane potential, leading to sperm quality decline. Furthermore, mitochondrial damage and apoptosis of testicular Sertoli cells were observed in mice treated with FLC. ConclusionWe found that FLC has significant adverse effects on spermatogonia proliferation and meiosis. Meanwhile, apoptosis and mitochondrial damage may be the potential mechanism of Sertoli cell damage. Our study demonstrated that FLC could induce testicular Sertoli cell damage, leading to abnormal spermatogenesis which resulted in sperm quality decline and provided a methodological reference for related studies.

GSK-3β Inhibitor SB216763 Resists Oxidative Stress-Induced Apoptosis in Nucleus Pulposus Cell

Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this process remain unclear. Increasing evidence indicates that GSK-3β is related to cell apoptosis induced by oxidative stress. In this study, we explored whether GSK-3β inhibition protects human NP cell against apoptosis under oxidative stress. Immunofluorescence staining was used to show the expression of GSK-3β in human NP cells (NPCs). Flow cytometry, mitochondrial staining and western blot were used to detect apoptosis of treated NPCs, changes of mitochondrial membrane potential and the expression of mitochondrial apoptosis-related proteins using GSK-3β specific inhibitor SB216763. Coprecipitation was used to demonstrate the interaction between GSK-3β and Bcl-2 in an GSK-3β knockdown in vitro model. We delineated the protective effect of GSK-3β specific inhibitor SB216763 on human NP cell apoptosis induced by oxidative stress in vitro. Further, we showed SB216763 exert the protective effect by preservation of the mitochondrial membrane potential and inhibition of caspase 3/7 activity during oxidative injury. The detailed mechanism underlying the antiapoptotic effect of GSK-3β inhibition was also studied by analyzing mitochondrial apoptosis pathway in vitro. We concluded that the GSK-3β inhibitor SB216763 protected mitochondrial membrane potential to delay nucleus pulposus cell apoptosis by inhibiting the interaction between GSK-3β and Bcl-2 and subsequently reducing Cyto-C release and caspase-3 activation. Together, inhibition of GSK-3β using SB216763 in NP may be a favorable therapeutic strategy to slow intervertebral disc degeneration.

Association Between Plasma Redox State/Mitochondria Function and a Flu-Like Syndrome/COVID-19 in the Elderly Admitted to a Long-Term Care Unit

Background/Aims: It is widely known that the imbalance between reactive oxygen species (ROS)/antioxidants and mitochondrial function could play a pivotal role in aging and in the physiopathology of viral infections. Here, we correlated the plasma oxidants/antioxidants levels of the elderly admitted to a long-term care (LTC) unit with clinical data in relation to flu-like disease/COVID-19. Moreover, in vitro we examined the effects of plasma on cell viability, ROS release and mitochondrial function.Materials and Methods: In 60 patients admitted to LTC unit for at least 1 year at moderate or high care load, demographic and clinical variables were taken. Blood samples were collected for the evaluations of oxidants/antioxidants, as thiobarbituric acid reactive substances, 8-hydroxy-2-deoxyguanosine, 8-isoprostanes, superoxide dismutase activity, glutathione, and vitamin D. In vitro, human umbilical vascular endothelial cells (HUVEC) were used to examine the effects of plasma on viability, ROS release and mitochondrial membrane potential.Results: The results obtained showed that the redox state of the elderly was quite balanced; mitochondrial membrane potential of HUVEC was reduced by about 20%, only. Also, the correlation analysis evidenced the association between mitochondrial function and the patients’ outcomes. Interestingly, lower levels of mitochondrial membrane potential were found in the elderly who had symptoms suggestive of COVID-19 or with a confirmed diagnosis of COVID-19.Conclusion: The results of this study highlight the importance of mitochondrial function in the tendency to get a flu-like syndrome like COVID-19 in the elderly admitted to LTC unit. This information could have clinical implications for the management of old population.

UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.