Intracellular Caspase-3 Activity Research Articles

Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells.

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.

The Effect of κ-Carrageenan on Porcine Sperm Cryo-Survival.

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen-thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress.

Visual monitoring of cisplatin-regulated caspase-3 activity in living cells based on a reduced graphene oxide-loaded fluorescent probe.

Cisplatin (DDP) is a potent chemotherapeutic drug, which can regulate tumor cell apoptosis by up-regulating caspase-3 activity. Thus, monitoring caspase-3 activity in breast cancer cells can directly illustrate the efficiency of DDP treatment. In this study, by using reduced graphene oxide (rGO) as a quencher of a fluorescence labeled peptide, we developed an "off to on" method to monitor the effect of DDP on caspase-3 in breast cancer cells. In this method, the rGO quenched fluorescence with an ultra-high level of efficiency. Caspase-3 hydrolyzed the polypeptide probe, generating two segments of different lengths. The release of a short segment marked with fluorophores led to the recovery of the fluorescence signal (Ex/Em = 450/521 nm). Under the optimal conditions, the linear range of caspase-3 was 0.4-7 U mL-1 and the limit of detection was 0.33 U mL-1. The upregulating effect of DDP on intracellular caspase-3 activity was visualized with the "off to on" method and flow cytometry assay showed that caspase-3 activity increased along with the apoptosis rate of tumor cells. The above results show the practical application of the method for evaluating the efficacy of drugs against cancer cells.

Andrew David Hamilton Wyllie. 24 January 1944—26 May 2022

Andrew Wyllie graduated from the University of Aberdeen, becoming an academic pathologist in Aberdeen, Edinburgh and Cambridge. He was the co-discoverer of apoptotic cell death, having observed single cells dying following carcinogen exposure. Together with Alastair Currie and John Kerr, he realized the profound importance of this novel mode of cell death that showed a distinctive series of morphological changes, which he first described as a new cell death process. Wyllie and Currie introduced the term ‘apoptosis’ for this cell death process in a seminal paper in 1972. Another landmark discovery was of chromatin fragmentation in apoptosis, due to activation of an endogenous endonuclease that caused internucleosomal DNA cleavage (‘chromatin laddering’), which was the first biochemical mechanism of apoptosis described. He further characterized chromatin fragmentation in the 1980s, followed by investigations of cell surface changes to produce ‘eat-me’ signals to trigger rapid phagocytosis of the apoptotic cells and bodies, intracellular calcium ion signalling, caspase activation and other mechanisms of apoptosis. His cancer research helped identify the location of APC and generated his demonstration that apoptosis was regulated by oncogenes MYC and RAS and by tumour suppressor genes, such as TP53 . He showed how apoptosis occurred in response to DNA damage and was a key process influencing both carcinogenesis and tumour growth. Andrew made a major scientific observation that changed the understanding of how cells die in health and disease, although it took time for the scientific establishment to understand its fundamental importance. Andrew Wyllie is widely known as the ‘Father of Apoptosis’.

Anticancer effect of umbelliferone on MKN-45 and MIA PaCa-2 cell lines

In this study, the anticancer activity of umbelliferone (7-hydroxycoumarin-UMB) was investigated in MKN-45 human gastric cancer and MIA PaCa-2 human pancreatic cancer cells. The cytotoxic effect of UMB on MKN-45 and MIA PaCa-2 cells was determined by WST-8 cell viability assay; the effect on colony formation and migration potential by colony forming assay and wound healing/cell migration assay. Apoptotic effect of UMB was determined by measuring the change in mitochondrial membrane potentials, reactive oxygen species levels, and Caspase-3 activities in cells. Anticancer drugs cisplatin and gemcitabine were used as positive controls in experiments, and NIH/Swiss 3 T3 mouse embryonic fibroblast cells were used as a healthy cell group.The results of this study showed that umbelliferone had a significant cytotoxic effect in MKN-45 and MIA PaCa-2 cells, especially after 72 h treatment, while its cytotoxic effect in NIH/3 T3 cells was low. Furthermore, UMB reduces significantly the potential of cells to colonize and migrate; it has been determined that it causes apoptosis by decreasing the mitochondrial membrane potential, increasing intracellular ROS levels and Caspase-3 activity. UMB was found to have more anticancer effect on MIA PaCa-2 cells compared to MKN-45 cells. This showed that UMB has a cell-selective effect.

Roles of DNA Target in Cancer Cell-Selective Cytotoxicity by Dicopper Complexes with DNA Target/Ligand Conjugates.

The DNA target/ligand conjugates (HLX, X = Pn and Mn, n = 1-3) were synthesized where various lengths of -CONH(CH2CH2O)nCH2CH2NHCO- linkers with a 9-phenanthrenyl (P) or methyl (M) terminal as DNA targets replace the methyl group of 2,6-di(amide-tether cyclen)-p-cresol ligand (HL). DNA binding, DNA cleavage, cellular uptake, and cytotoxicity of [Cu2(μ-OH)(LX)](ClO4)2 (1X) are examined and compared with those of [Cu2(μ-OH)(L)](ClO4)2 (1) to clarify roles of DNA targets. Upon reaction of 1X with H2O2, μ-1,1-O2H complexes are formed for DNA cleavage. 1P1, 1P2, and 1P3 are 22-, 11-, 3-fold more active for conversion of Form II to III in the cleavage of supercoiled plasmid DNA with H2O2 than 1, where the short P-linker may fix a dicopper moiety within a small number of base pairs to facilitate DNA double-strand breaks (dsb). This enhances the proapoptotic activity of 1P1, 1P2, and 1P3, which are 30-, 12-, and 9.9-fold cytotoxic against HeLa cells than 1. DNA dsb and cytotoxicity are 44% correlated in 1P1-3 but 5% in 1M1-3, suggesting specific DNA binding of P-linkers and nonspecific binding of M-linkers in biological cells. 1P1-3 exert cancer cell-selective cytotoxicity against lung and pancreas cancer and normal cells where the short P-linker enhances the selectivity, but 1M1-3 do not. Intracellular visualization, apoptosis assay, and caspase activity assay clarify mitochondrial apoptosis caused by 1P1-3. The highest cancer cell selectivity of 1P1 may be enabled by the short P-linker promoting dsb of mitochondrial DNA with H2O2 increased by mitochondrial dysfunction in cancer cells.

Effect of the dinitrosyl iron complex with N-ethylthiourea on ROS and NO intracellular levels and caspase activity in HeLa tumor cells

Effect of the dinitrosyl iron complex with N-ethylthiourea on ROS and NO intracellular levels and caspase activity in HeLa tumor cells

Green rooibos extract attenuates high glucose induced oxidative stress in a human derived (HepG2) liver cell line

• Oxidative stress biomarkers are attenuated by REE in a glucotoxic HepG2 cell model. • REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose. • REE activates NRF2 antioxidant pathway in HepG2 cells exposed to high glucose. The exposure of hepatocytes to high concentrations of glucose results in a glucotoxic environment and ensuing hepatocellular injury, often due to oxidative stress. The popularly consumed herbal tea, Rooibos ( Aspalathus linearis ), demonstrates pleiotropic pharmacological and antioxidant activities. Our study investigated the molecular events regulating the antioxidant effects of a Rooibos ethanolic extract (REE) in a glucotoxic HepG2 liver cell model. It is hypothesized that REE enhances antioxidant enzyme profiles of HepG2 cells exposed to high glucose to attenuate glucotoxic damage. Glucotoxicity was induced by treating the cells with 25 mM glucose. For this study, colorimetric, luminometric and western blot analyses were used to determine the potential of the REE to reduce biomarkers associated with oxidative damage and its potential to enhance the intracellular antioxidant defense system. The REE was found to be hepatoprotective as evidenced by reduced levels of oxidative damage biomarkers (MDA and protein carbonylation) and enhanced antioxidant capacity under glucotoxic conditions as evidenced by elevated GSH (reduced form). We also observed decreased intracellular hydrogen peroxide levels and caspase activity in the REE treated cells and further showed that the REE enhanced SOD2, CAT, and GPx1 protein expression. These effects were dependent on the transcriptional activity of NRF2. Collectively, our results show that the REE enhances the cellular antioxidant defense capacity and may provide a relevant supportive therapeutic option for treating diabetic complications associated with oxidative stress.

Antioxidant Activity of Fucoidan Modified with Gallic Acid Using the Redox Method.

Antioxidant compounds decrease the amount of intracellular reactive oxygen species (ROS) and, consequently, reduce the deleterious effects of ROS in osteoblasts. Here, we modified a 21 kDa fucoidan (FucA) with gallic acid (GA) using the redox method, to potentiate its antioxidant/protective capacity on pre-osteoblast-like cells (MC3T3) against oxidative stress. The 20 kDa FucA-GA contains 37 ± 3.0 mg GA per gram of FucA. FucA-GA was the most efficient antioxidant agent in terms of total antioxidant capacity (2.5 times), reducing power (five times), copper chelation (three times), and superoxide radical scavenging (2 times). Exposure of MC3T3 cells to H2O2 increased ROS levels and activated caspase-3 along with caspase-9. In addition, the cell viability decreased approximately 80%. FucA-GA also provided the most effective protection against oxidative damage caused by H2O2. Treatment with FucA-GA (1.0 mg/mL) increased cell viability (~80%) and decreased intracellular ROS (100%) and caspase activation (~80%). In addition, Fuc-GA (0.1 mg/mL) abolished H2O2-induced oxidative stress in zebra fish embryos. Overall, FucA-GA protected MC3T3 cells from oxidative stress and could represent a possible adjuvant for the treatment of bone fragility by counteracting oxidative phenomena.

Platelets Inhibit Methicillin-Resistant Staphylococcus aureus by Inducing Hydroxyl Radical-Mediated Apoptosis-Like Cell Death.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most common drug-resistant bacteria and poses a significant threat to human health. Due to the emergence of multidrug resistance, limited drugs are available for the treatment of MRSA infections. In recent years, platelets have been reported to play important roles in inflammation and immune responses, in addition to their functions in blood hemostasis and clotting. We and other researchers have previously reported that platelets can inhibit Staphylococcus aureus growth. However, it remained unclear whether platelets have the same antibacterial effect on drug-resistant strains. In this study, we hypothesized that platelets may also inhibit the growth of MRSA; the results confirmed that platelets significantly inhibited the growth of MRSA in vitro. In a murine model of MRSA infection, we found that a platelet transfusion alleviated the symptoms of MRSA infection; in contrast, depletion of platelets aggravated infective symptoms. Moreover, we observed an overproduction of hydroxyl radicals in MRSA following platelet treatment, which induced apoptosis-like death of MRSA. Our findings demonstrate that platelets can inhibit MRSA growth by promoting the overproduction of hydroxyl radicals and inducing apoptosis-like death.IMPORTANCE The widespread use of antibiotics has led to the emergence of drug-resistant bacteria, particularly multidrug-resistant bacteria. MRSA is the most common drug-resistant bacterium that causes suppurative infections in humans. As only a limited number of drugs are available to treat the infections caused by drug-resistant pathogens, it is imperative to develop novel and effective biological agents for treating MRSA infections. This is the first study to show that platelets can inhibit MRSA growth in vitro and in vivo. Our results revealed that platelets enhanced the production of hydroxyl radicals in MRSA, which induced a series of apoptosis hallmarks in MRSA, including DNA fragmentation, chromosome condensation, phosphatidylserine exposure, membrane potential depolarization, and increased intracellular caspase activity. These findings may further our understanding of platelet function.

The Granger Causal Effects of Canady Helios Cold Plasma on the Inhibition of Breast Cancer Cell Proliferation

Cold atmospheric plasma (CAP) has become a promising tool for modern medicine. With its recent applications in oncology, regenerative medicine, and immunotherapy, CAP can be used for a myriad of different clinical treatments. When using CAP specifically for the treatment of tumors, it is known to elicit an oxidative response within malignant cancer cells, inducing cell cycle arrest and apoptosis. In this study, data of intracellular reactive oxygen species (ROS), caspase activity, Ki-67 expression, and cell cycle activity in the G1 phase were acquired to determine the causal relationships these intermediates have with cell proliferation and death after Canady Helios Cold Plasma (CHCP) treatment. The data were derived from four different subtypes of breast cancer cell lines: BT-474, MCF-7, MDA-MB-231, and SK-BR-3. Data transformation techniques were conducted on the time-series data for the input into the causal model code. The models were created on the basis of Granger causality principles. Our results demonstrated that there was a Granger causal relationship among all potentially causal variables (ROS, caspase, Ki-67, and G1 activity) and cell proliferation after 5 min CHCP treatment; however, not all variables were causal for the 3 min models. This same pattern did not exist for cell death models, which tested all potentially causal variables (ROS, Ki-67, and G1 activity) vs. caspase activity. All models were validated through a variety of statistical tests and forecasting accuracy metrics. A pseudo data set with defined causal links was also created to test R’s ability in picking up known causal relationships. These models, while nonexhaustive, elucidated the effects cold plasma has on cell activity regulators. Research in causal modeling is needed to help verify the exact mechanism of cold plasma for the ultimate optimization of its application in the treatment of cancers.

Experimental study on thioredoxin redox inhibitor 1-methylpropyl 2-imidazolyl disulfide promoting apoptosis of multiple myeloma cells in vitro and in vivo.

To explore the in vitro and in vivo experimental study of thioredoxin-1(Trx1) inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) promoting multiple myeloma H929 cell apoptosis, investigate the relationship between the inhibitory effect of PX-12 on H929 cells and reactive oxygen species (ROS). Inhibition of PX-12 on H929 cells in relation to reactive oxygen species (ROS), cell cycle, and apoptosis were assessed by flow cytometry. ELISA kit, IVIS Imaging, Hematoxylin and eosin (H&E) staining and immunohistochemical staining assessment were applied to assess the anti-myeloma effect in the SCID mice model established by H929EL cells. PX-12 inhibited proliferation of H929 cells performed time and dose dependent style. Furthermore, it significantly induced a G2/M phase arrest of the cell cycle in H929 cells. It also increased intracellular ROS and caspase-3 activity in H929 cells indicating that cells have undergone apoptosis. There was an almost 3-5-fold decrease in tumor viability measured by the Living-Imaging system after 21 and 28 days after PX-12 injection compared with the control group. Importantly, PX-12 caused significant decrease in expression of Kappa chain in vivo assessed by immunohistochemical staining. The results suggest that PX-12 may be a potential strategy for the treatment of MM, and the inhibition of TRX-1 in the treatment of myeloma deserves further research.

Red Blood Cell Abnormalities as the Mirror of SARS-CoV-2 Disease Severity: A Pilot Study.

PurposeUnraveling the pathophysiology of COVID-19 disease is of crucial importance for designing treatment. The purpose of this study is to investigate the effects of the disease on erythrocytes (RBCs) and to correlate the findings with disease severity.Materials and MethodsHospitalized patients (n = 36) with COVID-19 and control group of healthy volunteers (n = 18) were included in the study. Demographic data, clinical, laboratory and chest Computed Tomography (CT) findings at time of admission were recorded. Laboratory measurements included: Hemoglobin (H b), indirect billirubin, LDH, D-Dimers, and plasma free hemoglobin (plasma free-Hb). On RBCs were performed: osmotic fragility (MCF), Free-Hb after mechanical stress (Free-Hb-MECH), intracellular RBC concentration of calcium ions (iCa2+), intracellular ROS (iROS), G6PD, intracellular active caspase-3 (RBC-caspase-3), IgG immunoglobulins (RBC-IgGs), which are bound on RBCs’ senescent neo-antigen proteins and RBC surface phosphatidylserine (RBC-PS).ResultsThe percentage of males was 50 and 66% and the mean age was 65.16 ± 14.24 and 66.33 ± 13.48 years among patients and controls respectively (mean ± SD, p = 0.78). Upon admission patients’ PO2/FiO2 ratio was 305.92 ± 76.75 and distribution of infiltration extend on chest CT was: 0–25% (N = 19), 25–50%: (N = 7), and 50–75% (N = 9). Elevated hemolysis markers (LDH and plasma free-Hb) were observed in patients compared to the control group. Patients’ RBCs were more sensitive to mechanical stress, and exhibited significantly elevated apoptotic markers (iCa2+, RBC-PS). Plasma free Hb levels correlated with the extend of pulmonary infiltrates on chest CT in COVID-19 patients. Surprisingly, patients’ RBC-iROS were decreased, a finding possibly related with the increased G6PDH levels in this group, suggesting a possible compensatory mechanism against the virus. This compensatory mechanism seemed to be attenuated as pulmonary infiltrates on chest CT deteriorated. Furthermore, RBC-IgGs correlated with the severity of pulmonary CT imaging features as well as the abnormality of lung function, which are both associated with increased disease severity. Lastly, patients’ D-Dimers correlated with RBC surface phosphatidylserine, implying a possible contribution of the red blood cells in the thrombotic diathesis associated with the SARS-CoV-2 disease.ConclusionThis pilot study suggests that SARS-CoV-2 infection has an effect on red blood cells and there seems to be an association between RBC markers and disease severity in these patients.

Exposure to zinc oxide nanoparticles (ZnO-NPs) induces cardiovascular toxicity and exacerbates pathogenesis – Role of oxidative stress and MAPK signaling

The precise toxico-pathogenic effects of zinc oxide nanoparticles (ZnO-NPs) on the cardiovascular system under normal and cardiovascular disease (CVD) risk factor milieu are unclear. In this study, we have investigated the dose-dependent effects of ZnO-NPs on developing chicken embryo and cell culture (H9c2 cardiomyoblast, HUVEC and aortic VSMC) models. In addition, the potentiation effect of ZnO-NPs on simulated risk factor conditions was evaluated using; 1. Reactive oxygen species (ROS) induced cardiac remodeling, 2. Angiotensin-II induced cardiac hypertrophy, 3. TNF-α induced HUVEC cell death and 4. Inorganic phosphate (Pi) induced aortic VSMC calcification models. The observed results illustrates that ZnO-NPs exposure down regulates vascular development and elevates oxidative stress in heart tissue. At the cellular level, ZnO-NPs exposure reduced the cell viability and increased the intracellular ROS generation, lipid peroxidation and caspase-3 activity in a dose-dependent manner in all three cell types. In addition, ZnO-NPs exposure significantly suppressed the endothelial nitric oxide (NO) generation, cardiac Ca2+ - ATPase activity and enhanced the cardiac mitochondrial swelling. Moreover, inhibition of p38 MAPK and JNK signaling pathways influence the cytotoxicity. Overall, ZnO-NPs exposure affects the cardiovascular system under normal conditions and it exacerbates the cardiovascular pathogenesis under selected risk factor milieu.

Characterization and function analysis of Epinephelus coioides Hsp40 response to Vibrio alginolyticus and SGIV infection

Heat shock protein 40 (Hsp40), a member of Heat shock proteins (Hsps) family, plays a crucial role in regulation of cell proliferation, survival and apoptosis in mammals. In this study, Hsp40, EcHsp40, was identified from Epinephelus coioides, an economically important marine-cultured fish in China and Southeast Asian counties. The full length of EcHsp40 was 2236 bp in length containing a 1026 bp open reading frame (ORF) encoding 341 amino acids, with a molecular mass of 37.88 kDa and a theoretical pI of 9.09. EcHsp40 has two conserved domains DnaJ and DnaJ_C. EcHsp40 mRNA was detected in all tissues examined, and the expression was significantly up-regulated response to challenged with Vibrio alginolyticus or Singapore grouper iridovirus (SGIV), one of the important pathogens of marine fish. EcHsp40 was distributed in both the cytoplasm and nucleus, over-expression of EcHsp40 can inhibit the activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), significantly promote SGIV-induced apoptosis, intracellular caspase-3 activity and viral replication, suggesting that the EcHsp40 may play an important role in pathogenic stimulation.

In Situ Imaging of Cellular Reactive Oxygen Species and Caspase-3 Activity Using a Multifunctional Theranostic Probe for Cancer Diagnosis and Therapy.

In this work, a multifunctional theranostic nanoprobe (Au-Ag-HM) was skillfully designed for simultaneous imaging of intracellular reactive oxygen species (ROS) and caspase-3 activity. The Au-Ag-HM was fabricated by coloading of silver nanoparticles (AgNPs) and hematoporphyrin monomethyl ether (HMME) to Au nanoflowers (AuNFs). When Au-Ag-HM was devoured by cancer cells, HepG2 cells were used as the model, and under laser irradiation, the photogenerated intracellular ROS by the photosensitizer HMME would induce the apoptosis of cancer cells. Meanwhile, the intracellular ROS triggered the oxidative etching of AgNPs on Au-Ag-HM, which led to a tremendous localized surface plasmon resonance response and scattering color changes in Au-Ag-HM, allowing in situ dark-field imaging of the ROS level in cancer cells. On the other hand, the ROS-induced activation of cellular caspase-3, which cleaved the C-peptide-containing caspase-3-specific recognition sequence (DEVD) and allowed HMME to release from the nanoprobe, resulted in a significant fluorescence recovery related to caspase-3 activity. Both photogenerated ROS and enhanced caspase-3 activity contributed to the synergistic effect of laser-mediated chemotherapy and photodynamic therapy. Therefore, the as-prepared theranostic probe could be used for simultaneous detection of cellular ROS and caspase-3 activity, distinguishing between tumor cells and normal cells, inducing the apoptosis of cancer cells, and providing a new method for diagnosis and therapy of cancer.

Vincamine, a safe natural alkaloid, represents a novel anticancer agent

Vincamine, a well-known plant alkaloid, has been used as a dietary supplement and as a peripheral vasodilator to combat aging in humans. In this study, for the very first time, we demonstrated that vincamine can function as an anticancer agent in a human alveolar basal epithelial cell line A549 (IC50 = 309.7 μM). The anticancer potential of vincamine in A549 cells was assessed by molecular assays to determine cell viability, generation of intracellular ROS, nuclear condensation, caspase-3 activity and inhibition, and change in mitochondrial membrane potential (ΔΨm). In silico studies predicted that the anti-proliferative potential of vincamine is enhanced by its interaction with the apoptotic protein caspase-3, and that this interaction is driven by two hydrogen bonds and has a high free energy of binding (−5.64 kcal/mol) with an estimated association constant (Ka) of 73.67 μM. We found that vincamine stimulated caspase-3-dependent apoptosis and lowered mitochondrial membrane potential, which ultimately led to cytochrome C release. Vincamine was also found to quench hydroxyl free radicals and deplete iron ions in cancer cells. As a dietary supplement, vincamine is almost non-toxic in BEAS-2B and 3T3-L1 cells. Therefore, we propose that vincamine represents a safe anticancer agent in lung cancer cells. Its role in other cancers has yet to be explored.

Single-cell dynamics of pannexin-1-facilitated programmed ATP loss during apoptosis.

ATP is essential for all living cells. However, how dead cells lose ATP has not been well investigated. In this study, we developed new FRET biosensors for dual imaging of intracellular ATP level and caspase-3 activity in single apoptotic cultured human cells. We show that the cytosolic ATP level starts to decrease immediately after the activation of caspase-3, and this process is completed typically within 2 hr. The ATP decrease was facilitated by caspase-dependent cleavage of the plasma membrane channel pannexin-1, indicating that the intracellular decrease of the apoptotic cell is a 'programmed' process. Apoptotic cells deficient of pannexin-1 sustained the ability to produce ATP through glycolysis and to consume ATP, and did not stop wasting glucose much longer period than normal apoptotic cells. Thus, the pannexin-1 plays a role in arresting the metabolic activity of dead apoptotic cells, most likely through facilitating the loss of intracellular ATP.

Hypoxic preconditioning combined with curcumin promotes cell survival and mitochondrial quality of bone marrow mesenchymal stem cells, and accelerates cutaneous wound healing via PGC-1α/SIRT3/HIF-1α signaling

Restrained survival and function of relocated bone marrow mesenchymal stem cells (BMSCs) is a major impediment to BMSCs-mediated tissue repair. Accumulating evidences have indicated that hypoxic preconditioning of BMSCs could enhance BMSCs’ adaptability after transplantation and thus improve their therapeutic properties. Curcumin, a natural dietary product, is known to exert profound protective effects on various cellular processes. Here we showed that mild hypoxic preconditioning combined with curcumin significantly increased cell survival, enriched more cells in G2/M and S phase, and improved mitochondrial function in BMSCs. Meanwhile, hypoxic preconditioning combined with curcumin altered mitochondrial cristae shape and strongly inhibited mitochondrial cytochrome c release, which consequently suppressed an apoptosis signal as revealed by reduced caspase-3 cleavage in BMSCs. Moreover, hypoxic preconditioning remarkably promoted mitochondrial quality via increasing mitochondrial fusion and elevating the activity of oxidative phosphorylation (OXPHOS) and mitochondrial complex Ⅰ enzyme in BMSCs, which were in accordance with the up-regulated expression of OPA1, PINK1 and Parkin. At the mechanistic level, the destabilization of HIF-1α and the up-regulated expression of PGC-1α and SIRT3 synergistically contributed to the protective effects of hypoxic preconditioning combined with curcumin in BMSCs. The proteasome inhibitor MG132 stabilized HIF-1a expression, but not PGC-1α or SIRT3, and dramatically restrained BMSCs survival under hypoxia combined with curcumin condition. MG132 also increased mitochondrial superoxide and intracellular hydrogen peroxide (H2O2) production and caspase-3 activation in hypoxia combined with curcumin-treated BMSCs. Furthermore, knockdown of SIRT3 and PGC-1α by RNAi both led to caspase-3 activation in BMSCs after hypoxia and curcumin treatment. Notably, SIRT3 RNAi suppressed OXPHOS activity, while PGC-1α RNAi triggered mitochondrial superoxide and intracellular H2O2 production in hypoxia combined with curcumin-treated BMSCs. Finally, we showed that hypoxia combined with curcumin-treated BMSCs accelerated the cutaneous wound healing process in a mice wound model. Overall, this study suggests that hypoxic preconditioning combined with curcumin could serve as an attractive strategy for facilitating BMSCs-mediated tissue repair, and further sheds new light on the rich repertoire of PGC-1α/SIRT3/HIF-1α signaling involved in the regulation of mitochondrial quality and function for cellular adaption to hypoxia.

Neodymium YAG laser chemical vapor deposition growth of luminescent Mo2S3 nanocrystals using bulk MoS2 and its structural, optical properties and caspase-mediated apoptosis in THP-1 monocytic cells

Metal-sulphur phase-based semiconductor materials are often investigated in the field of nanotechnology. Herein, we describe the synthetic route to obtain Mo2S3 nanocrystals from MoS2 via a laser-furnace technique. These nanocrystals have been tested for cytotoxicity against the monocytic leukemic THP-1 cell line. The synthetic process caused a phase change from MoS2 to Mo2S3 at a temperature of 1100 °C and a pressure of 400 Torr. Powder X-ray diffraction analysis confirmed the crystallinity and structure and X-ray photoelectron spectroscopy confirmed the chemical composition and oxidation states of the Mo2S3 nanocrystals. The morphology of the Mo2S3 has been observed by using field emission scanning electron microscopy and high-resolution transmission electron microscopy. The in vitro cytotoxicity evaluation of the Mo2S3 nanocrystals was determined in the THP-1 cell line. The cytotoxic potential of the Mo2S3 against THP-1 cells was assessed by the WST-1 assay and intracellular caspase activities. The caspase activities were significantly elevated indicating the initiation of both the intrinsic and extrinsic apoptotic pathways when compared to untreated cells. Field emission scanning electron microscopy revealed that the Mo2S3 NCs compromised the cell membrane integrity resulting in a loss of THP-1 cell viability and apoptosis. Thus, Mo2S3 is an aqueous nanocrystal which shows promising cytotoxicity effect against THP-1 with good photoluminescent properties.