Loss Of Mitochondrial Transmembrane Potential Research Articles

Telomerase inhibitors induce mitochondrial oxidation and DNA damage-dependent cell death rescued by Bcl-2/Bcl-xL

BackgroundReactivation of telomerase is a hallmark of cancer and the majority of cancers over-express telomerase. Telomerase-dependent telomere length maintenance confers immortality to cancer cells. However, telomere length-independent cell survival functions of telomerase also play a critical role in tumorigenesis. Multiple telomerase inhibitors have been developed as therapeutics and include anti-sense oligonucleotides, telomerase RNA component targeting agents, chemical inhibitors of telomerase, small molecule inhibitors of hTERT, and telomerase vaccine. In general, telomerase inhibitors affect cell proliferation and survival of cells depending on the telomere length reduction, culminating in replicative senescence or cell death by crisis. However, most telomerase inhibitors kill cancer cells prior to significant reduction in telomere length, suggesting telomere length independent role of telomerase in early telomere dysfunction-dependent cell death. MethodsIn this study, we explored the mechanism of cell death induced by three prominent telomerase inhibitors utilizing a series of genetically encoded sensor cells including redox and DNA damage sensor cells. ResultsWe report that telomerase inhibitors induce early cell cycle inhibition, followed by redox alterations at cytosol and mitochondria. Massive mitochondrial oxidation and DNA damage induce classical cell death involving mitochondrial transmembrane potential loss and mitochondrial permeabilization. Real-time imaging of the progression of mitochondrial oxidation revealed that treated cells undergo a biphasic mitochondrial redox alteration during telomerase inhibition, emphasizing the potential role of telomerase in the redox regulation at mitochondria. Additionally, silencing of hTERT confirmed its predominant role in maintaining mitochondrial redox homeostasis. Interestingly, the study also demonstrated that anti-apoptotic Bcl-2 family proteins still confer protection against cell death induced by telomerase inhibitors. ConclusionThe study demonstrates that redox alterations and DNA damage contribute to early cell death by telomerase inhibitors and anti-apoptotic Bcl-2 family proteins confer protection from cell death by their ability to safeguard mitochondria from oxidation damage.

(2E)-1-(2,4,6-Trimethoxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one, a Chalcone Derivative, Promotes Apoptosis by Suppressing RAS-ERK and AKT/FOXO3a Pathways in Hepatocellular Carcinoma Cells.

Liver cancer is an extremely common cancer with the highest mortality rate and poor prognosis. Owing to their low systemic toxicity and few side effects, natural compounds may provide better therapeutic effects for patients. (2E)-1-(2,4,6-trimethoxyphenyl)-3-(4-chlorophenyl)prop-2-en-1-one (TMOCC), a chalcone derivative, exhibits cytotoxicity towards many tumor cells. However, the anticancer mechanism of TMOCC has not been elucidated in human hepatocellular carcinoma (HCC). Cell Counting Kit-8 and colony formation assays were used to evaluate the effects of TMOCC on viability and proliferation. Mitochondrial transmembrane potential and flow cytometry assays were used to detect apoptosis. The expression levels of proteins related to apoptosis, the RAS-ERK and AKT/FOXO3a signaling pathways were assessed using western blot. Potential targets of TMOCC were detected using molecular docking analysis. TMOCC inhibited viability and proliferation, and induced the loss of mitochondrial transmembrane potential, apoptosis and DNA double-strand breaks in both HCC cells. The RAS-ERK and AKT/FOXO3a signaling pathways were suppressed by TMOCC. Finally, ERK1, PARP-1, and BAX were identified as potential targets of TMOCC. Taken together, our results show that TMOCC promotes apoptosis by suppressing the RAS-ERK and AKT/FOXO3a signaling pathways. TMOCC may be a potential multi-target compound that is effective against liver cancer.

Effect of a Novel Dihydroartemisinin Dimer Containing Nitrogen Atoms SM 1044 on Apoptosis of Human Leukemia Cell Line NB4-R1

To investigate the effect of a water-soluble novel dihydroartemisinin dimer containing nitrogen atoms SM 1044 on the apoptosis of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) NB4-R1 cells and its potential mechanism. The effects of SM 1044 on cell apoptosis, mitochondrial transmembrane potential, and the level of reactive oxygen species (ROS) were assessed by flow cytometry. Expressions of apoptosis-related proteins were determined by Western blot. The effects of SM 1044 on MAPK (ERK, JNK) signaling pathway, PML/RARα fusion protein, and expressions of apoptosis-related proteins were detected by Western blot. SM 1044 could significantly induce apoptosis and the loss of mitochondrial transmembrane potential in NB4-R1 cells, and activate apoptosis-related proteins caspase-3, caspase-8, caspase-9 and poly (ADP-ribose) polymerase (PARP). SM 1044 could also induce NB4-R1 cells to produce ROS. Western blot showed that SM 1044 activated the phosphorylation of MAPK (ERK, JNK) signaling pathway and down-regulated the expression of PML/RARα fusion protein. SM 1044 can induce apoptosis of ATRA resistant APL NB4-R1 cells, which may be related to ROS/ERK and ROS/JNK signaling pathway, and can also induce by down-regulating PML/RARα fusion protein.

Naringenin Induces HepG2 Cell Apoptosis via ROS-Mediated JAK-2/STAT-3 Signaling Pathways.

Hepatocarcinoma is one of the most prevalent digestive system tumors worldwide and lacks effective therapy. Recently, naringenin has been isolated from some citrus fruits, and its anticancer effects have been tested. However, the molecular mechanisms of naringenin and the potential implications of oxidative stress in naringenin-induced cytotoxicity in HepG2 cells remain elusive. Based on the above, the present study examined the effect of naringenin on the cytotoxic and anticancer mechanisms of HepG2 cells. Naringenin-induced HepG2 cell apoptosis was confirmed via the accumulation of the sub-G1 cell population, phosphatidylserine exposure, mitochondrial transmembrane potential loss, DNA fragmentation, caspase-3 activation, and caspase-9 activation. Furthermore, naringenin enhanced cytotoxic effects on HepG2 cells and triggered intracellular reactive oxygen species; the signaling pathways of JAK-2/STAT-3 were inhibited, and caspase-3 was activated to advance cell apoptosis. These results suggest that naringenin plays an important role in inducing apoptosis in HepG2 cells and that naringenin may be a promising candidate for cancer therapy.

A novel benzothiazole derivative induces apoptosis via the mitochondrial intrinsic pathway producing antitumor activity in colorectal cancer.

Background: Colorectal cancer (CRC) is one of the most common malignancies causing the third highest mortality rate in the world. It is particularly urgent to explore effective therapeutic strategies to overcome this disease. We identified a novel benzothiazole derivative (BTD) that may serve as a potentially effective agent against CRC. Method: MTT assays, cell colony formation assays, EdU staining assays, flow cytometry, RNA-seq, Western blotting, and migration and invasion assays were used to examine the effects of BTD on cell proliferation, apoptosis, metastasis, and the cell cycle. The antitumor activity of BTD in vivo was investigated in a CT26 tumor-bearing mouse model. Immunohistochemistry (IHC) was performed to examine the protein expression in mouse tumors. Hematology, biochemical analysis, and H&E staining were used to assess the biosafety of BTD. Results: We observed that BTD suppressed cell proliferation and metastasis and promoted the apoptosis of tumor cells in vitro. Treatment with BTD at a tolerable dose significantly reduced tumor growth in CT26-tumor-bearing mice and appeared to be safe. Treatment of BTD induced apoptosis by increasing the generation of reactive oxygen species (ROS) and evoking the loss of mitochondrial transmembrane potential. Overall, BTD suppressed cell proliferation and metastasis, and induced apoptosis of colorectal tumor cells through the ROS-mitochondria-mediated apoptotic pathway. The preliminary proof of the antitumor activity and relative safety of BTD were validated in a mouse model. Conclusion: Our findings suggest that BTD could serve as a potentially safe and effective candidate for CRC treatment.

Polypeptide antibiotic actinomycin D induces Mcl-1 uncanonical downregulation in lung cancer cell apoptosis

AimsActinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation. Main methodsHuman adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles. Key findingsWe found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis. SignificanceAll of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.

Anticancer polypyrrole-polyethylenimine drug-free nanozyme for precise B-cell lymphoma therapy

As an alternative strategy for cancer treatment, the combination of cancer nanomedicine and immunotherapy is promising with regard to efficacy and safety; however, precise modulation of the activation of antitumor immunity remains challenging. Therefore, the aim of the present study was to describe an intelligent nanocomposite polymer immunomodulator, drug-free polypyrrole-polyethyleneimine nanozyme (PPY-PEI NZ), which responds to the B-cell lymphoma tumor microenvironment, for precision cancer immunotherapy. Earlier engulfment of PPY-PEI NZs in an endocytosis-dependent manner resulted in rapid binding in four different types of B-cell lymphoma cells. The PPY-PEI NZ effectively suppressed B cell colony-like growth in vitro accompanied by cytotoxicity via apoptosis induction. During PPY-PEI NZ-induced cell death, mitochondrial swelling, loss of mitochondrial transmembrane potential (MTP), downregulation of antiapoptotic proteins, and caspase-dependent apoptosis were observed. Deregulated AKT and ERK signaling contributed to glycogen synthase kinase-3-regulated cell apoptosis following deregulation of Mcl-1 and MTP loss. Additionally, PPY-PEI NZs induced lysosomal membrane permeabilization while inhibiting endosomal acidification, partly protecting cells from lysosomal apoptosis. PPY-PEI NZs selectively bound and eliminated exogenous malignant B cells in a mixed culture system with healthy leukocytes ex vivo. While PPY-PEI NZs showed no cytotoxicity in wild-type mice, they provided long-term and efficient inhibition of the growth of B-cell lymphoma-driven nodules in a subcutaneous xenograft model. This study explores a potential PPY-PEI NZ-based anticancer agent against B-cell lymphoma.

Low-Molecular-Weight β-1,3-1,6-Glucan Derived from Aureobasidium pullulans Exhibits Anticancer Activity by Inducing Apoptosis in Colorectal Cancer Cells.

β-glucan, a plant polysaccharide, mainly exists in plant cell walls of oats, barley, and wheat. It is attracting attention due to its high potential for use as functional foods and pharmaceuticals. We have previously reported that low-molecular-weight Aureobasidium pullulans-fermented β-D-glucan (LMW-AP-FBG) could inhibit inflammatory responses by inhibiting mitogen-activated protein kinases and nuclear factor-κB signaling pathways. Bases on previous results, the objective of the present study was to investigate the therapeutic potential of LMW-AP-FBG in BALB/c mice intracutaneously transplanted with CT-26 colon cancer cells onto their backs. Daily intraperitoneal injections of LMW-AP-FBG (5 mg/kg) for two weeks significantly suppressed tumor growth in mice bearing CT-26 tumors by reducing tumor proliferation and inducing apoptosis as compared to phosphate buffer-treated control mice. In addition, LMW-AP-FBG treatment reduced the viability of CT-26 cells in a dose-dependent manner by inducing apoptosis with loss of mitochondrial transmembrane potential and increased activated caspases. Taken together, LMW-AP-FBG exhibits anticancer properties both in vivo and in vitro.

Polysaccharides from Annonamuricata leaves protect against cisplatin‑induced cytotoxicity in macrophages by alleviating mitochondrial dysfunction.

Cisplatin is a prominent chemotherapeutic agent that can induce significant damage to normal cells. Therefore, it is important to develop agents that protect normal cells without influencing the chemotherapeutic effect of cisplatin. The present study was conducted to explore the protective effects of Annonamuricata leaf polysaccharides (ALPS) against cisplatin‑induced toxicity in macrophages. Apoptosis was assessed in macrophages and lung cancer cells to investigate the cytoprotective effect of ALPS, their effect on the production of cisplatin‑induced reactive oxygen species (ROS) and the loss of the mitochondrial transmembrane potential (MTP). Cisplatin, when used alone or in combination with ALPS, showed significant toxicity against A549 and H460 lung cancer cells. However, cisplatin‑induced cytotoxicity was suppressed by cotreatment of RAW 264.7 macrophages with ALPS. ALPS significantly inhibited the upregulation of Bax, cytosolic cytochrome c and caspases‑3, ‑8 and ‑9. Moreover, ALPS resulted in the cleavage of PARP and downregulation of Bcl‑2 levels in a concentration‑dependent manner, which ultimately led to a reduction in the apoptotic and necrotic populations of cisplatin‑treated RAW 264.7 macrophages. The suppression of the apoptotic signaling pathways was mediated through the reduction of ROS and MTP loss in cisplatin‑treated RAW 264.7 macrophages. In addition, ALPS alleviated cell damage by suppressing the mitochondrial apoptotic pathways in cisplatin‑treated bone marrow‑derived macrophages. Together, these findings suggested that ALPS may alleviate the toxic side effects of chemotherapeutic agents and act as a potential candidate for use as an effective adjuvant therapy.

Benzimidazole based bis-carboxamide derivatives as promising cytotoxic agents: Design, synthesis, in silico and tubulin polymerization inhibition

A new series of carboxamide-bearing benzimidazole derivatives have been reported for their cytotoxicity profile (in vitro) on selected human tumour cells like A549, HCT116, SK-Mel-28, and MCF-7. Among the derivatives, the utmost cytotoxicity was shown by compounds 10g and 10m, with specificity toward SK-Mel-28 skin cancer cells displaying potent IC50 values (0.41–51.21 μM). The compounds 10g and 10m unveiled to be safe on testing them against human non-cancerous lung epithelial cells (BEAS: IC50: >100 μM). The annexin V binding assay and JC-1 were implemented to evaluate the scope of apoptosis and loss of mitochondrial transmembrane potential in SK-Mel-28 cell lines. The G2/M phase arrest in the cell cycle examination was observed by active molecule 10m dose-dependently. Target-based findings suggested tubulin polymerization inhibition by 10m (IC50 of 2.36 ± 0.20 μM) and its efficacious binding with DNA. The detailed binding interactions of compounds 10g and 10m with tubulin and DNA was examined by docking studies on PDB ID: 3E22 and DNA hexamer (PDB ID: 1NAB) respectively.

Apoptin mediates mitophagy and endogenous apoptosis by regulating the level of ROS in hepatocellular carcinoma

BackgroundApoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes.MethodsUsing crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing.ResultsThe results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy.ConclusionThe above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells.Graphical abstractF4GajfVYHPvFTjahqy1NffVideo abstract

Microcystin-leucine arginine exhibits adverse effects on human aortic vascular smooth muscle cells in vitro

Microcystin-leucine arginine (MC-LR) is a kind of toxin produced by cyanobacteria, which can do harm to human and livestock health. MC-LR can easily enter tissues and organs through the blood circulation and accumulate in certain target organs. Vessels are prone to contact with MC-LR during growth and development. Previous study had demonstrated that MC-LR had potential vascular toxicity. However, it is not clear whether MC-LR has adverse effects on vascular smooth muscle cells. In this study, we evaluated the cytotoxicity of MC-LR exposure (0.01, 0.05, 0.1, 0.5, and 1 μM) on human aortic vascular smooth muscle cells (HAVSMCs) in vitro. The data showed that MC-LR exposure inhibited the HAVSMC proliferation and migration, induced HAVSMC apoptosis, cytoskeleton destruction, S-phase arrest, mitochondrial transmembrane potential (MMP) loss, and reactive oxygen species (ROS) production. In addition, MC-LR exposure resulted in the imbalance between oxidants and antioxidants, increased the caspase-3 and caspase-9 activities, and down-regulated the gene expressions (integrin β1, Rho, ROCK, MLC). Taken together, MC-LR could induce the generation of ROS in HAVSMCs, leading to apoptosis by the mitochondrial signaling pathway. MC-LR could also induce cytoskeletal disruption by integrin-mediated FAK/ROCK signaling pathway, leading to cell cycle arrest and the inhibition of HAVSMCs proliferation and migration. The current findings facilitate an understanding of the mechanism of MC-LR toxicity involved in angiocardiopathy.

Evaluation of antimicrobial, anticancer potential and Flippase induced leakage in model membrane of Centella asiatica fabricated MgONPs.

The use of chemically synthesized nanoparticles and crude plant extracts as antimicrobial -anticancer agents have many limitations. In this study, we have used Centella asiatica extract (CaE) having relatively less explored but tremendous medicinal properties, as reducing and stabilizing agents to green synthesize magnesium oxide nanoparticles (MgONPs) using magnesium nitrate. In comparison to the bulk material, capabilities of Ca-MgONPs as an improved antibacterial, antifungal, and anticancer agent in human prostatic carcinoma cells (PC3), as well as membranolytic capability in model cell membrane, were studied. The phyto-functionalized Ca-MgONPs were characterized using UV-Visible spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Energy Dispersive X-Ray Spectroscopy (EDX), X-ray Diffraction (XRD), Fourier Transform Infra-Red Spectroscopy (FT-IR) and Atomic Force Microscopy (AFM). Observation of characteristic peaks by spectroscopic and microscopic analysis confirmed the synthesis of Ca-MgONPs. The Ca-MgONPs showed broad spectrum of bactericidal activity against both gram-positive and gram-negative bacteria and fungicidal activity against two species of the Candida fungus. The Ca-MgONPs also exhibited dose-dependent and selective inhibition of proliferating PC3 cells with IC50 of 123.65±4.82μg/mL at 24h, however, without having any cytotoxicity toward non-cancerous HEK293 cells. Further studies aimed at understanding the probable mechanism of toxicity of Ca-MgONPs in PC3 cells, the results indicated a significant reduction in cell migration capacities, increment in cytosolic ROS, loss of mitochondrial transmembrane potential, DNA damage and S-phase cell cycle arrest. Ca-MgONPs also induced pore formation in a synthetic large unilamellar vesicle. Thus, Ca-MgONPs might be useful in the effective management of several human pathogens of concern and some more cancer types.

Pyrroloquinoline quinone ameliorates renal fibrosis in diabetic nephropathy by inhibiting the pyroptosis pathway in C57BL/6 mice and human kidney 2 cells.

Diabetic nephropathy (DN), which is characterized by renal fibrosis, is a major complication of diabetes, a disease that afflicted more than 460 million people worldwide in 2019. Pyroptosis is an essential signaling pathway in DN-related injuries, such as renal fibrosis. Pyrroloquinoline quinone (PQQ) is a naturally occurring bioactive compound that protects human kidney 2 (HK-2) cells from oxidative stress-induced damage caused by high glucose concentrations. However, the nature and underlying mechanism of the effect of PQQ on DN-related renal fibrosis remains unclear. In this study, we evaluated whether PQQ has potential protective effects against renal fibrosis due to DN by establishing type 1 diabetes in mice via streptozotocin treatment and then inhibiting their pyroptosis signaling pathway. We found that compared to control mice, the area of renal fibrosis and injury were significantly increased in diabetic mice, and this was accompanied by increased levels of expression of collagen Ⅰ and transforming growth factor-β1; increased concentrations of the inflammatory cytokines, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α; and activation of the pyroptosis pathway components nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, IL-1β, and IL-18. All of these changes were reversed by PQQ treatment. Analogously, we treated cultured HK-2 cells with a high concentration of glucose (35mmol/L), which caused these cells to exhibit significantly increased concentrations of reactive oxygen species (ROS), phosphorylated (p)-nuclear factor kappa B (NF-κB), p-IkappaB, NLRP3, caspase-1, IL-1β, and IL-18, and the loss of mitochondrial transmembrane potential. However, PQQ treatment significantly blunted these effects. In conclusion, in this study we demonstrated that PQQ attenuates renal fibrosis by alleviating mitochondrial dysfunction, reducing ROS production, and inhibiting the activation of the NF-κB/pyroptosis pathway under conditions of DN and hyperglycemia.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

Chemoprevention of NCI-H322 Lung Cancer Cells Proliferation and Tumor Regression in Murine Ascites Model by Dietary Flavonoid Quercetin

Prunus cerasus L (Sour cherries) contain diverse secondary metabolites which exhibit various biological activities, including anticancer, antioxidant, and anti-inflammatory properties. The present study aimed to determine the anticancer efficacy of four compounds, quercetin, daidzin, rutin, and chlorogenic acid, isolated from Prunus cerasus fruit. The antiproliferative activity of four cherry isolates was determined against five different cancer cell lines (NCI-H322, A549, THP-1, MCF-7, and PC-3) by Tetrazolium bromide assay, followed by apoptosis Cell cycle analyses, mitochondrial membrane potential, cell migration test, and in vivo Ehrlich Ascites Carcinoma studies using potent bioactive lead. The cytotoxicity profile of the four molecules demonstrated that quercetin induced significant cell growth inhibition in all cancer cell lines with paramount 79% cytotoxicity against NCI-H322 lung cancer cells (IC50 value 24μM). Incubation of NCI-H322 cells with quercetin showed a concentration-dependent increase in hypo-diploid sub G0/G1 DNA fraction, exhibited consequential changes in nuclear morphology, and caused mitochondrial transmembrane potential loss of 60.3% augmented at 30 µM. Pertaining to in vivo potency, quercetin manifested 89% tumor inhibition at 50 mg/kg body weight in EAC-bearing mice. The current studies raise the potential usefulness of quercetin in chemoprevention against lung cancer cells and support its empirical use as a promising nutraceutical agent.

Glia Maturation Factor Beta as a Novel Biomarker and Therapeutic Target for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. The novel sensitive biomarkers and therapeutic targets are urgently needed for the early diagnosis of HCC and improvement of clinical outcomes. Glia maturation factor-β (GMFB) is a growth and differentiation factor for both glia and neurons and has been found to be tightly involved in inflammation and neurodegeneration conditions. In our study, the expression level of GMFB was significantly up-regulated in patients with HCC and positively co-expression with tumor node metastases (TNM) stage and histopathological grade of HCC. The high expression level of GMFB was remarkably associated with poor overall survival, which mainly occurred in males rather than females. Multivariate analysis revealed GMFB to be an independent prognostic factor for overall survival in patients with HCC. Results of Gene Ontology (GO) and KEGG pathways analysis showed that down-regulation of pathways related to protein translation and mitochondria function were enriched. Protein-protein interaction analysis revealed the central role of mitochondria protein in HCC. The downregulation of genes involved in glycolysis and gluconeogenesis was observed among the co-expression genes of GMFB. Knockdown of GMFB in Hep3B significantly inhibited proliferation, migration, and invasion of Hep3B cells, and also downregulated the expression levels of some of metal matrix proteinase (MMP), increased mtDNA copy number and loss of mitochondrial transmembrane potential. GMFB influences the malignancy rate of HCC possibly through regulation of the expression of MMPs, mtDNA function and glycolysis. We proposed that GMFB was a promising HCC diagnostic and prognostic biomarker and therapeutic target in HCC.

Epoxiconazole profoundly alters rat brain and properties of neural stem cells

Epoxiconazole (EPX), a widely used fungicide for domestic, medical, and industrial applications, could cause neurodegenerative diseases. However, the underling mechanism of neurotoxicity is not well understood. This study aimed to investigate the possible toxic outcomes of Epoxiconzole, a triazole fungicide, on the brain of adult rats in vivo, and in vitro on neural stem cells derived from the subventricular zone of newborn Wistar rats. Our results revealed that oral exposure to EPX at these concentrations (8, 24, 40, 56 mg/kg bw representing respectively NOEL (no observed effect level), NOEL × 3, NOEL × 5, and NOEL × 7) for 28 days caused a considerable generation of oxidative stress in adult rat brain tissue. Furthermore, a significant augmentation in lipid peroxidation and protein oxidation has been found. Moreover, it induced an elevation of DNA fragmentation as assessed by the Comet assay. Indeed, EPX administration impaired activities of antioxidant enzymes and inhibited AChE activity. Concomitantly, this pesticide produced histological alterations in the brain of adult rats. Regarding the embryonic neural stem cells, we demonstrated that the treatment by EPX reduced the viability of cells with an IC50 of 10 μM. It also provoked the reduction of cell proliferation, and EPX triggered arrest in G1/S phase. The neurosphere formation and self-renewal capacity was reduced and associated with decreased differentiation. Moreover, EPX induced cytoskeleton disruption as evidenced by immunocytochemical analysis. Our findings also showed that EPX induced apoptosis as evidenced by a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspase-3. In addition, EPX promoted ROS production in neural stem cells. Interestingly, the pretreatment of neural stem cells with the N-acetylcysteine (ROS scavenger) attenuated EPX-induced cell death, disruption of neural stem cells properties, ROS generation and apoptosis. Thus, the use of this hazardous material should be restricted and carefully regulated.

Protective effects of FK 506 against haemorrhagic shock‐induced microvascular hyperpermeability

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from the intravascular space to the interstitium, is a devastating clinical concern in haemorrhagic shock (HS), sepsis, burn and so forth. Previous studies have shown that HS-induced microvascular hyperpermeability is associated with activation of the mitochondria-mediated 'intrinsic' apoptotic signalling cascade and caspase-3 mediated disruption of the endothelial cell barrier. In this study, our objective was to test if FK506, an immunomodulator that is also known to protect mitochondria, would protect barrier functions and decrease vascular hyperpermeability following HS by acting on this pathway. FK506 (25µM) was given 10 minutes before the shock period in a rat model of HS. The HS model was a non-traumatic/fixed pressure model of hypovolemic shock developed by withdrawing blood to reduce the mean arterial pressure to 40mm Hg for 60minutes. The mesenteric post-capillary venules were monitored for changes in permeability using intravital microscopic imaging. The changes in mitochondrial transmembrane potential (MTP) were determined using the cationic dye 5,5',6,6' tetrachoro-1,1',3,3' tetraethyl benzimidazolyl carbocyanine iodide (JC-1), that was superfused on the mesenteric vasculature followed by intravital imaging. The mesenteric caspase-3 activity was measured fluorometrically. Haemorrhagic shock induced a significant increase in hyperpermeability compared to the sham-control group and FK506 treatment decreased HS-induced hyperpermeability significantly (P<.05). FK506 dampened HS-induced loss of MTP and elevation of caspase-3 activity significantly (P<.05). FK506 has protective effects against HS-induced microvascular hyperpermeability. The maintenance of the MTP and protection against caspase-3 mediated endothelial cell barrier disruption are possible mechanisms by which FK506 attenuates HS-induced hyperpermeability. FK506, currently used in clinical settings as an immunomodulator, needs to be explored further for its therapeutic usefulness against HS-induced vascular hyperpermeability and associated complications.

Tingenone and 22-hydroxytingenone target oxidative stress through downregulation of thioredoxin, leading to DNA double-strand break and JNK/p38-mediated apoptosis in acute myeloid leukemia HL-60 cells

Acute myeloid leukemia (AML) is the most lethal form of leukemia. Standard anti-AML treatment remains almost unchanged for decades. Tingenone (TG) and 22-hydroxytingenone (22-HTG) are quinonemethide triterpenes found in the Amazonian plant Salacia impressifolia (Celastraceae), with cytotoxic properties in different histological types of cancer cells. In the present work, we investigated the anti-AML action mechanism of TG and 22-HTG in the AML HL-60 cell line. Both compounds exhibited potent cytotoxicity in a panel of cancer cell lines. Mechanistic studies found that TG and 22-HTG reduced cell growth and caused the externalization of phosphatidylserine, the fragmentation of internucleosomal DNA and the loss of mitochondrial transmembrane potential in HL-60 cells. In addition, pre-incubation with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, prevented TG- and 22-HTG-induced apoptosis, indicating cell death by apoptosis via a caspase-dependent pathway. The analysis of the RNA transcripts of several genes indicated the interruption of the cellular antioxidant system, including the downregulation of thioredoxin, as a target for TG and 22-HTG. The application of N-acetyl-cysteine, an antioxidant, completely prevented apoptosis induced by TG and 22-HTG, indicating activation of the apoptosis pathway mediated by oxidative stress. Moreover, TG and 22-HTG induced DNA double-strand break and phosphorylation of JNK2 (T183/Y185) and p38α (T180/Y182), and co-incubation with SP 600125 (JNK/SAPK inhibitor) and PD 169316 (p38 MAPK inhibitor) partially prevented apoptosis induced by TG and 22-HTG. Together, these data indicate that TG and 22-HTG are new candidate for anti-AML therapy targeting thioredoxin.