Microtubule-based chemotherapeutics, primarily Taxane-derived agents are still used as the major live-saving agents, yet have several side effects including serious loss of immune cells, bone density etc. which lowers the quality of life. This imposes the need to understand the effects of these agents on Mesenchymal Stem Cells (MSCs) in details. In this work we demonstrate that Taxol and Nocodazole affects the endogenous expression of TRPV1, a non-selective cation channel in MSCs. These agents also affect the status of polymerized Actin as well as Tyrosinated-tubulin, basal cytosolic Ca2+ and mitochondrial membrane potential (ΔΨm). Notably, pharmacological modulation of TRPV1 by Capsaicin or Capsazepine can also alter the above-mentioned parameters in a context-dependent manner. We suggest that endogenous expression of TRPV1 and pharmacological modulation of TRPV1 can be utilized to rescue some of these parameters effectively. These findings may have significance in the treatments and strategies with Microtubule-based chemotherapeutics and stem-cell based therapy.

Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC.

With the introduction of nanosecond (ns) pulses, it was suggested that such pulses could be used to permeabilize intracellular membranes, including the mitochondrial membrane. The results presented thus far, however, are not conclusive. Interestingly, the effect of longer microsecond (μs) pulses on changes in mitochondria has never been investigated. We, therefore, investigated the changes in mitochondrial membrane permeability through changes in mitochondrial membrane potential (MMP) in CHO and H9c2 cells after electroporation with 4 ns, 200 ns, and 100 μs pulses. In the range of reversible electroporation, the decrease in MMP generally depended on the cell line. In CHO, ns pulses decreased MMP at lower electroporation intensities than μs. In H9c2, ns and μs were equally effective. In the range of irreversible electroporation, MMP decreased even further, regardless of pulse duration and cell type. The analysis at different time points showed that the changes in MMP within the first hour after pulse treatment are dynamic. Our results on the efficacy of ns pulses are consistent with published data, but with this study we show that μs pulses cause similar changes in MMP as ns pulses, demonstrating that electroporation affects MMP regardless of pulse duration. At the same time, however, differences in MMP changes were observed between different cell lines, indicating some dependence of MMP changes on cell type.

BACKGROUND: Adenomyosis is a common gynecological disease, characterized by overgrowth of endometrial glands and stroma in the myometrium, however its exact pathophysiology still remains uncertain. Emerging evidence has demonstrated the elevated level of arginase 2 (ARG2) in endometriosis and adenomyosis. This study aimed to determine whether ARG2 involved in mitochondrial function and epithelial to mesenchymal transition (EMT) in adenomyosis and its potential underlying mechanisms.
METHODS: RNA interference was used to inhibit ARG2 gene, and then Cell Counting Kit (CCK-8) assay and flow cytometery were performed to detect the cell proliferation capacity, cell cycle, and apoptosis progression, respectively. The mouse adenomyosis model was established and RT-PCR, Western blot analysis, mitochondrial membrane potential (Δψm) detection and mPTP opening evaluation were conducted.
RESULTS: Silencing ARG2 effectively down-regulated its expression at the mRNA and protein levels in endometrial cells, leading to decreased enzyme activity and inhibition of cell viability. Additionally, ARG2 knockdown induced G0/G1 cell cycle arrest, promoted apoptosis, and modulated the expression of cell cycle- and apoptosis-related regulators. Notably, the interference with ARG2 induces apoptosis by mitochondrial dysfunction, ROS production, ATP depletion, decreasing the Bcl-2/Bax ratio, releasing Cytochrome c, and increasing the expression of Caspase-9/-3 and PARP. In vivo study in a mouse model of adenomyosis demonstrated also elevated levels of ARG2 and EMT markers, while siARG2 treatment reversed EMT and modulated inflammatory cytokines. Furthermore, ARG2 knockdown was found to modulate the NF-κB and Wnt/β-catenin signaling pathways in mouse adenomyosis.
CONCLUSIONS: Consequently, ARG2 silencing could induce apoptosis through a mitochondria-dependent pathway mediated by ROS, and G0/G1 cell cycle arrest via suppressing NF-κB and Wnt/β-catenin signaling pathways in Ishikawa cells. These findings collectively suggest that ARG2 plays a crucial role in the pathogenesis of adenomyosis and may serve as a potential target for therapeutic intervention.

OBJECTIVE: To investigate the effect of chrysophanol, a phytochemical derived from Radix et Rhizoma Rhei on HepG2 liver cancer cells.
METHODS: HepG2 cell line was treated with different concentrations chrysophanol (0-100 μmol/L) for 24 h. The cell counting kit 8 assay was employed to assess cell viability. Intracellular calcium levels were examined using Fluo-4 AM and Mag-fluo-4 AM staining, followed by flow cytometry analysis. Mitochondrial membrane potential was measured with JC-1 assay kit. Additionally, the expressions of key proteins such as p-JNK, Bax, cytochrome c (Cyt C), cleaved caspase-3 (cCaspase-3), and caspase-8 were analyzed by Western blot. The inhibitory effects of chrysophanol on the invasion of cells were determined using a Transwell assay. Analysis of invasiveness was conducted by wound healing assay.
RESULTS: Chrysophanol significantly reduced the proliferation of HepG2 liver cancer cells by affecting intracellular calcium distribution, diminishing mitochondrial membrane potential, and enhancing the expressions of p-JNK, Bax, Cyt C, cCaspase-3, and caspase-8 in the groups treated with 75 or 100 μmol/L chrysophanol compared to the control group (P<0.05). Additionally, 75 and 100 μmol/L chrysophanol exhibited inhibitory effects on cell migration and wound healing.
CONCLUSIONS: Chrysophanol demonstrates potential against HepG2 liver cancer cells, suggesting its potential use as a therapeutic agent for liver cancer treatment.

BACKGROUND: As an emerging myocardial ablation technique, the mechanism of nanosecond pulse electric field (nsPEF) ablation is currently less studied. Mitochondria are one of the important membrane structure organelles in cells, participating in numerous life activities within the cell. This study aimed to explore the morphological changes of mitochondria in living cells following nsPEF treatment.
METHODS: Myocardial cells were treated with a self-made solid-state LTD high-voltage nanosecond pulse generator with a pulse width of 100 ns for 80 times. The changes in mitochondrial membrane potential and cell apoptosis in rat myocardial cells after nsPEFs were investigated using JC-1 assay kit, apoptosis double staining assay kit, and mitochondrial fluorescence probe.
RESULTS: The results showed that after nsPEF treatment, the mitochondrial membrane potential decreased, apoptosis increased, and the average mitochondrial area decreased from 0.48 µm2 in live myocardial cells to 0.16 µm2. The average circumference ranges from 3.17 µm dropped to 1.60 µm. The shape factor decreased from 1.92 to 1.41. The aspect ratio has decreased from 2.16 to 1.59. nsPEF treatment induces changes in the morphology of myocardial cell mitochondria.
CONCLUSIONS: Based on the results of mitochondrial membrane potential and apoptosis, it can be inferred that under this equipment and parameter conditions, nsPEF treatment first causes changes in mitochondrial morphology, and then initiates the mitochondrial apoptosis pathway, which may provide experimental basis for investigating the potential mechanism of nsPEF ablation of myocardial cells.

This study investigated the effects of ergosterol peroxide(EP) on the proliferation and apoptosis of MCF-7 breast cancer cells, explored its possible mechanisms of action, and verified the effects and mechanisms by in vitro experiments. Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI) networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects. The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation, and the colony formation assay was used to assess the cell cloning ability. Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis, mitochondrial membrane potential and reactive oxygen species(ROS) levels. Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), caspase-7, cleaved caspase-7, phosphatidylinositol 3-kinase(PI3K), and se-rine/threonine kinase B(AKT) in MCF-7 cells treated with EP. The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer; the results of Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway, PI3K-AKT signaling pathway, cellular senescence signaling pathway, and viral carcinogenesis pathway; and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group, exhibiting a time-and concentration-dependent trend, and EP can inhibit colony formation of MCF-7 breast cancer cells. Treatment with 10, 20, and 40 μmol·L~(-1) EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, and a downregulation of p-PI3K, p-AKT, and Bcl-2 protein expression in MCF-7 cells. Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.

This study aims to investigate the effect of ergosterol peroxide(EP) on the apoptosis of human hepatocellular carcinoma and its mechanism of action. The cell viability of HepG2 and SK-Hep-1 cells with 0(blank control), 2.5, 5, 10, 20, 40, and 80 μmol·L~(-1) of EP after 24, 48, and 72 h of action was detected by using CCK-8 assay, and the half inhibitory concentrations(IC_(50)) at 24, 48, and 72 h were calculated. Formal experiments were performed to detect the effect of EP on intracellular reactive oxygen species(ROS) using DCFH-DA staining, the effect of EP on intracellular mitochondrial membrane potential using JC-1 staining, the number of apoptotic cells using Annexin V-FITC/PI double-staining after HepG2 cells were co-cultured with 0(blank control), 10, 20, 40 μmol·L~(-1) EP for 48 h. The effects of EP at different concentrations on apoptotic morphology were detected using AO/EB staining. The effects of different concentrations of EP on the protein expression of mitochondrial apoptosis pathway-related proteins B cell lymphoma 2(Bcl-2), cytochrome C(Cyt-C), Bcl-2-related X protein(Bax), caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were examined by using Western blot. The results showed that different concentrations of EP could inhibit the proliferation of hepatocellular carcinoma with concentration-and time-dependent trends. Compared with the blank control group, the ROS level in the EP-treated group increased significantly(P<0.05). The mitochondrial membrane potential decreased significantly(P<0.05). The total apoptosis rate increased significantly(P<0.05). The expression of Bcl-2 protein was significantly down-regulated, and the expression of Cyt-C, Bax, cleaved caspase-9, and cleaved caspase-3 were significantly up-regulated(P<0.05). In summary, EP may inhibit the proliferation of hepatocellular carcinoma by modulating the mitochondria-mediated apoptosis pathway and induce apoptosis.

BACKGROUND: Besides adenine triphosphate (ATP) production for sustaining motility, the mitochondria of sperm also host other critical cellular functions during germ cell development and fertilization including calcium homeostasis, generation of reactive oxygen species (ROS), apoptosis, and in some cases steroid hormone biosynthesis. Normal mitochondrial membrane potential with optimal mitochondrial performance is essential for sperm motility, capacitation, acrosome reaction, and DNA integrity.
RESULTS: Defects in the sperm mitochondrial function can severely harm the fertility potential of males. The role of sperm mitochondria in fertilization and its final fate after fertilization is still controversial. Here, we review the current knowledge on human sperm mitochondria characteristics and their physiological and pathological conditions, paying special attention to improvements in assistant reproductive technology and available treatments to ameliorate male infertility.
CONCLUSIONS: Although mitochondrial variants associated with male infertility have potential clinical use, research is limited. Further understanding is needed to determine how these characteristics lead to adverse pregnancy outcomes and affect male fertility potential.

UNASSIGNED: This study investigated potential predictive models associated with natural killer (NK) cell mitochondrial membrane potential (MMP or ΔΨm) in predicting death among critically ill patients with COVID-19.
UNASSIGNED: We included 97 patients with COVID-19 of different severities attending Peking Union Medical College Hospital from December 2022 to January 2023. Patients were divided into three groups according to oxygen and mechanical ventilation use during specimen collection and were followed for survival and death at 3 months. The lymphocyte subpopulation MMP was detected via flow cytometry. We constructed a joint diagnostic model by integrating identified key indicators and generating receiver operating curves (ROCs) and evaluated its predictive performance for mortality risk in critically ill patients.
UNASSIGNED: The NK-cell MMP median fluorescence intensity (MFI) was significantly lower in critically ill patients who died from COVID-19 (p<0.0001) and significantly and positively correlated with D-dimer content in critically ill patients (r=0.56, p=0.0023). The random forest model suggested that fibrinogen levels and NK-cell MMP MFI were the most important indicators. Integrating the above predictive models for the ROC yielded an area under the curve of 0.94.
UNASSIGNED: This study revealed the potential of combining NK-cell MMP with key clinical indicators (D-dimer and fibrinogen levels) to predict death among critically ill patients with COVID-19, which may help in early risk stratification of critically ill patients and improve patient care and clinical outcomes.