Changes In Mitochondrial Potential Research Articles

Search for new biologically active compounds: in vitro studies of antitumor and antimicrobial activity of dirhodium(II,II) paddlewheel complexes.

Four neutral Rh1-Rh4 complexes of the general formula [Rh2(CH3COO)4L2], where L is an N-alkylimidazole ligand, were synthesized and characterized using various spectroscopic techniques, and in the case of Rh4 the crystal structure was confirmed. Investigation of the interactions of these complexes with HSA by fluorescence spectroscopy revealed that the binding constants Kb are moderately strong (∼104 M-1), and site-marker competition experiments showed that the complexes bind to Heme site III (subdomain IB). Competitive binding studies for CT DNA using EB and HOE showed that the complexes bind to the minor groove, which was also confirmed by viscosity experiments. Molecular docking confirmed the experimental data for HSA and CT DNA. Antimicrobial tests showed that the Rh2-Rh4 complexes exerted a strong inhibitory effect on G+ bacteria B. cereus and G- bacteria V. parahaemolyticus as well as on the yeast C. tropicalis, which showed a higher sensitivity compared to fluconazole. The cytotoxic activity of Rh1-Rh4 complexes tested on three cancer cell lines (HeLa, HCT116 and MDA-MB-231) and on healthy MRC-5 cells showed that all investigated complexes elicited more efficient cytotoxicity on all tested tumor cells than on control cells. Investigation of the mechanism of action revealed that the Rh1-Rh4 complexes inhibit cell proliferation via different mechanisms of action, namely apoptosis (increase in expression of the pro-apoptotic Bax protein and caspase-3 protein in HeLa and HCT116 cells; changes in mitochondrial potential and mitochondrial damage; release of cytochrome c from the mitochondria; cell cycle arrest in G2/M phase in both HeLa and HCT116 cells together with a decrease in the expression of cyclin A and cyclin B) and autophagy (reduction in the expression of the protein p62 in HeLa and HCT116 cells).

Isomerization and phosphorylation significantly influence the effect of beta amyloid on blood‐brain barrier endothelial cells

AbstractBackgroundThe blood‐brain barrier (BBB) is typically compromised in Alzheimer’s disease (AD): its permeability and intracellular parameters of the endothelial cells are affected. AD‐related amyloid isoforms –Aβ42, isoD7‐Aβ42 and pS8‐Aβ42, inducing amyloidogenesis in a different mode – may have peripheral origin and affect the BBB cells. This study aimed to assess this effect.MethodMouse brain endothelial cells bEnd.3 underwent incubation with 10 µM of Aβ42, isoD7‐Aβ42 and pS8‐Aβ42 for 4, 24 and 48 hours to assess nitric oxide (NO), glutathione (GSH), reactive oxygen species (ROS), mitochondrial potential, Ca2+, cell viability by flow cytometry and mitochondrial functioning by Seahorse technology. To analyze the integrity of the cellular monolayer, the expression level of tight junction proteins and permeability of bEnd.3 monolayer to fluorescent tracers were assessed by Western Blotting and transwell‐modeling, correspondingly .ResultpS8‐Aβ42 and isoD7‐Aβ42 effects on the redox status of the cells and mitochondria functioning differ from the effects of Aβ42. Even at 4 hours, Aβ42 and isoD7‐Aβ42 induce immense changes in mitochondrial potential and NO level but pS8‐Aβ42 does not. Prolonged incubation with three isoforms leads to an increase in ROS, and greater increase is observed after incubation with isoD7‐Aβ42. pS8‐Aβ42 induces ROS increase later than other isoforms. Above all, incubation with pS8‐Aβ42 results in a less pronounced NO и GSH growth compared with Aβ42 и isoD7‐Aβ42 which induce activation of mitochondrial respiration and increase in mitochondrial potential, compared with pS8‐Aβ42. IsoD7‐Aβ42 is mostly toxic to bEnd.3 cells. Although Aβ42 and isoD7‐Aβ42 significantly affect redox parameters of bEnd.3 cells, their effects on BBB permeability and tight junction protein expression are less pronounced. Our data demonstrate that inhibition of NO synthases and NMDA receptors induces changes in BBB cells’ response to Aβ peptides.ConclusionAmyloid isoforms differently affect redox parameters of BBB cells significantly modulating mitochondria functioning. Iso‐Aβ42 induces higher cytotoxicity, thus, being a more pathogenic form. Post‐translational modifications of Aβ42 alter the amyloid effect on the BBB cells. It may result in different changes in BBB functioning and its permeability under amyloid isoforms.This research was funded by Russian Science Foundation (Grant No. #19‐74‐30007).

Evaluating Tumor Hypoxia Radiosensitization Via Electron Paramagnetic Resonance Oxygen Imaging (EPROI).

Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.

Mechanistic investigation of photodynamic therapy using Visudyne in human KB carcinoma cells

Photodynamic therapy is a therapy that utilizes a specialized drug, a photosensitizer, which is not toxic by itself, but upon activation with light, is activated and leads to the production of singlet oxygen species. This production of singlet oxygen species starts a cascade that leads to cell death. Photodynamic therapy is a potential treatment for cancer patients, and it might be helpful to promote human health by providing alternative treatment to cancer. In-vitro study with Visudyne (VD) in KB carcinoma cells unveiled the mechanism of cell injury to cell death upon photodynamic therapy (PDT). In this study, we investigated real time monitoring of the production of singlet oxygen (SO), reactive oxygen species (ROS), mitochondria integrity and chromatin integrity in VD treated live cells by irradiating with a mercury lamp light source and 665–675 nm and 480–560 nm emission filter. Upon PDT we also observed cytoskeleton remodeling and cytochrome –C release using immunofluorescence techniques. We investigate the cytotoxicity of PDT and performed quantitative analysis for SO, ROS and mitochondrial potential change using colocalization analysis between VD and mitochondria.

The Effect of Xanthohumol Derivatives on Apoptosis Induction in Canine Lymphoma and Leukemia Cell Lines.

Xanthohumol is a cancer chemopreventive agent that can interfere with the initiation, promotion, and progression phase of carcinogenesis via a variety of inhibitory mechanisms. Xanthohumol was reported as an effective agent against leukemia/lymphoma cells. In the present study, we investigated the effect of xanthohumol and its natural and semisynthetic derivatives against various canine leukemia/lymphoma cell lines. Xanthohumol, three hops minor prenylflavonoids (xanthohumol C, xanthohumol D, α,β-dihydroxanthohumol) and four derivatives obtained by biotransformation (xanthohumol 4'-O-β-D-(4‴-O-methyl)-glucopyranoside) as well as by chemical modification (1″,2″-dihydroxanthohumol K, 2,3-dehydroisoxanthohumol, (Z)-6,4'-dihydroxy-4-methoxy-7-prenylaurone) were tested for their antiproliferative and pro-apoptotic activities against the following canine leukemia/lymphoma cell lines: CLBL-1 (B-cell lymphoma), CLB70 (B-cell leukemia), and GL-1 (B-cell leukemia). The compounds were tested at a final concentration range of 0.1-30 µM for 48 h. All eight of the tested flavonoids exerted concentration-dependent cytotoxicity in the selected canine lymphoma/leukemia cell lines. Three compounds markedly decreased the viability of all cell lines with IC50 in the range of 0.5 to 8 μM. Double-staining of the treated cells with AnnexinV and propidium iodide revealed that the dying cells were mostly in the late apoptosis stage. ROS production and changes in mitochondrial potential were detected. Western blot analysis showed a decreased expression of Bcl-2. Canine lymphoma and leukemia cell lines are sensitive to xanthohumol derivatives, and the compounds acted through an apoptotic cell-death mechanism. These compounds, either used alone or in combination with other therapies, may be useful for the treatment of canine leukemia/lymphoma.

Mechanistic Studies of Arene-Ruthenium(II) Complexes with Carbothioamidopyrazoles as Alternative Cancer Drugs.

Arene-ruthenium(II) complexes with carbothioamidopyrazoles at the C-2 and C-5 positions have been recognized as chemotherapeutic agent alternatives to cisplatin and its oxaliplatin analogs. The aim of this study was to continue research on the biological aspect of arene-ruthenium(II) complexes and their anticancer activity. The present paper includes an additional 12 new tumor cells, analyzed by MTT, and employs a series of extended bioassays to better understand their potential mechanism of antitumor activity. The following tests were conducted: membrane permeability studies, intramolecular reactive oxygen and nitrogen species (ROS/RNS) assays, mitochondrial potential changes, DNA analysis by comet assay using the electrophoresis method, measurement of cleaved PARP protein levels, and determination of apoptotic and necrotic cell fractions by fluorescence microscopy. Additionally, the article presents lipophilicity studies based on RP-TLC and molecular docking studies. We hope that the presented data will prove useful in practical treatment, especially for patients with cancer.

Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study

Abstract Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (>30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (< 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing >160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation. Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-06.

SOD2 in platelets: with age comes responsibility

SOD2 in platelets: with age comes responsibility

Activation of pancreatic stellate cells attenuates intracellular Ca2+ signals due to downregulation of TRPA1 and protects against cell death induced by alcohol metabolites

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis.

Physcion Induces Potential Anticancer Effects in Cervical Cancer Cells.

Background: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. Methods: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. Results: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. Conclusion: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

Recapitulating GSK3β and Tau interaction in brain injury dynamic model

IntroductionLiving systems experience various form of mechanical stimulations in daily life. Some mechanical stimulation are required for cell survival, others are capable of causing injuries. In vivo, the neuronal system is linked closely to the muscular system. As a result, we respond to pain immediately after physical injury. Proliferating neuron cells can be used to study the effect of mechanical stimulation on neurons. However, cell models developed from neurons have been lacking due to the fact that neuron cells do not or are difficult to proliferate by in vitro culture. In this study, we investigated the effect of stretching on the SH‐SY5Y cells, neuroblastoma cells that proliferate in vitro. The cells retain the dopamine‐β‐hydroxylase activity; thus, it is suitable for neuron mechanistic study.MethodThe culture conditions contain non‐stretched (control) and stretched groups. SH‐SY5Y cells were cultured on stretchable membranes, subjected to 6, 24h of cyclic stretching, 25% elongation at 1Hz.ResultsAfter 6 and 24h stretching, cells showed decreased red fluorescence for JC‐1 probe which stains the mitochondrial membrane, indicating that cells experienced oxidative stress. Results show that stretching increased b‐amyloid precursor, total Tau protein by western blot. Co‐immunoprecipitation showed that Tau formed complex with GSK3b after 6h stretching.SummaryThe observations of change in mitochondrial potential, and formation of Tau complex imply that the neuron cells were under a stress. The stress may be a kind of injury suffered from physical stimulation.Support or Funding Information108‐2320‐B‐075B‐002 ‐

The Influence of Lycopene, [6]-Gingerol, and Silymarin on the Apoptosis on U-118MG Glioblastoma Cells In Vitro Model.

Background: Lycopene, gingerol, and silymarin have a potential anticancer activity in many types of neoplasms. Healthy lifestyle and proper diet are associated with a reduced risk of cancer and other diseases. Increasingly, clinical research focuses on the mechanisms of action of natural compounds and their impact on the development of cancer. The aim of the present study was to determine the effect of lycopene, gingerol, and silymarin on apoptosis, mitochondrial potential and caspase-3/7 activity in the U118-MG cell line. Methods: Human glioblastoma cells were incubated with lycopene, [6]-gingerol, and silymarin for 24 and 48 h. Apoptosis was monitored using the Annexin V labelling, caspase-3/7 activity, and early hallmark of apoptosis were determined with mitochondrial membrane potential changes. Results: Our data showed a significant decrease in the viability glioblastoma cells U118-MG after 48 h treatment with lycopene, [6]-gingerol, and silymarin. Conclusions: Our data could confirm the stimulative effects of used compounds on apoptosis and changes in mitochondrial potential in a dose-dependent manner.

Application of a Flow-Based Hollow-Fiber Co-Culture System to Study Cellular Influences under Hyperglycemic Conditions

Elucidation of the basic mechanisms underlying human disease pathogenesis depends on the findings afforded to us through in vivo and in vitro approaches. While there are inherent limitations in any model system, 2D in vitro culture systems tend to be particularly restricted due to their static nature. Here, we adapted a flow-based hollow-fiber cartridge system to better understand the cellular influences of human retinal microvascular endothelial cells and mouse-derived neutrophils under high glucose conditions similar to those observed in diabetes. Analyses by western blot and flow cytometry indicate that pro-inflammatory molecules known to be associated with the pathogenesis of diabetic retinopathy were significantly elevated following high glucose exposure, including VEGF, ICAM-1, and ROS. Changes in mitochondrial potential were also observed. Further, we demonstrate that this innovative system allows for cross-species co-culture as well as long-term culturing conditions. This in vitro modeling system not only mimics the retinal microvasculature, it also allows for the examination of cellular interactions and mechanisms that contribute to diabetic retinopathy, a visually debilitating complication of diabetes.

Antioxidant and Neuroprotective Potential of the Brown Seaweed Bifurcaria bifurcata in an in vitro Parkinson's Disease Model.

Bifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1–F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin–Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studies.

Selectively Targeting Checkpoint Kinase By Prexasertib Had Potent Anti-Tumor Activity As Single Agent or Combined with Other Chemotherapeutic Drugs in Diffuse Large B-Cell Lymphoma, Especially Double Hit Lymphoma

Background: Relapsed/Refractory diffuse large B-cell lymphoma (DLBCL) patients had a poor prognosis, especially those relapsing within 12 months of completion therapy. Clinically, there is a need to identify and target pathways associated with acquired treatment resistance. Using rituximab-chemotherapy resistant DLBCL cell lines generated in our laboratory, we demonstrated the acquirement of rituximab resistance is associated with changes in the cell cycle distribution (S phase arrest) and expression of cell cycle regulatory proteins (cyclin B, CDC2, CDK7, Wee1, checkpoint kinase 1 [CHK1] and checkpoint kinase 2 [CHK2]). CHK1 and 2 are serine/threonine kinases that control: 1) G2/M phase transition in response of DNA damage, 2) stabilization of DNA replication fork and 3) coordinate the mitosis. Inhibition of CHKs is an attractive strategy in cancer medicine. CHK1 inhibitors are been studied in phase I clinical trials in solid tumor malignancies (i.e. breast, pancreas or lung cancer). We hypothesized that CHK inhibition by Prexasertib has effective anti-tumor activity in DLBCL pre-clinical models. Methods: We used a panel of DLBCL cell lines including rituximab sensitive (Raji, RL, DHL4, DHL6, TMD8, RIVA, and U2932), rituximab resistant (Raji 4RH and RL 4RH), and double hit DLBCL (DOH2, ROS50 and VAL). In addition, experiments were conducted in primary tumor cells isolated from lymphoma patients. Cells were exposed to prexasertib alone or in combination with chemotherapy agents. Cell viability were determined by Presto Blue assay. IC50 was calculated by Graphpad software and coefficient of synergy was calculated using the CalcuSyn software. Subsequently, cells were expose to prexasertib and changes in cell cycle distribution and apoptosis were determined by propidium iodide staining (Pi), annexing V/PI staining and by Caspase3/7 activity detection assay. Changes in mitochondrial potential were determined by DiOC6 staining. Induction of DNA double strain break was investigated by western blot probed with phosphory-H2A.X antibody. At molecular basis, downstream pathway activities ( Mcl-1, phosphor-GSK3β, phosphory-H3, and wee1) were determined by western blot. Results: Prexasertib exhibited a dose dependent anti-lymphoma activity in all all cell lines tested including DHL cell lines. The IC50 of the cells ranged from 11nM to 2.8uM at 72 hours. Additive/synergistic effects were observed by combining prexasertib and doxorubicin, etoposide, vincristine, gemcitabine and proteasome inhibitors. At the dosage of 10nM and 25nM, prexasertib induced cell cycle arrest at G2/M phase. It also induced low membrane potential, caspse3/7 activation and apoptosis in all DLBCL cell lines. Interestingly, western blot also showed CHK inhibitor at 10nM was able to reduce Mcl-1 and p-GSK 3beta. Conclusion: Our data suggests that targeting checkpoint kinase is a promising therapeutic strategy in the lymphoma, including in double hit lymphoma pre-clinical models. In vivo studies are planned. Our data supports the clinical development of prexasertib in DLBCL. (Supported by Roswell Park Cancer Institute Alliance Foundation Grant) DisclosuresNo relevant conflicts of interest to declare.

Collapse of Neuronal Energy Balance As a Basis of L-Homocysteine Neurotoxicity

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca2+ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca2+ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca2+ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca2+ overload, while only a small portion of neurons were able to compensate the intracellular Ca2+ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φmit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φmit so that an uncoupling agent FCCP did not cause any additional changes in φmit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φmit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.

New N-9- sulfonylpurine derivatives induce changes in mitochondrial function and ROS accumulation in carcinoma and leukemia cells in vitro

Our recent studies showed significant antiproliferative capacity of newly synthesized N-9-sulfonylpyrimidine derivatives on human carcinoma, lymphoma, and leukemia cells. These derivatives behave as antimetabolites exploiting cellular metabolism to induce cytotoxicity in treated tumors’ cells [1-3]. Based on previously obtained results on N-9-sulfonylpyrimidine derivatives, a new series of N-9-sulfonylpurine derivatives were synthesized and tested on biological potential. The aim of this study was to determine if measured antitumor activity of N-9-sulfonylpurine derivatives is linked with ROS accumulation and mitochondrial membrane destabilization in human cervix adenocarcinoma (HeLa), human chronic myelogenous leukemia (K562) and human Burkitt’s lymphoma (Raji) cells. Changes in the mitochondrial membrane potential (∆Ψm) of tested compounds were determined by flow cytometry, measuring fluorescence of JC-1 dye in tumor cells after 24 hours of treatment. Intracellular accumulation of ROS was determined after 1 hour in cells exposed to N-9-sulfonylpurine derivatives by flow cytometry as well. Tested derivatives induced increased accumulation of ROS and mitochondrial disruption in more than 80% of HeLa and Raji cells. The effect of tested derivatives on K562 cells is reflected by the change of mitochondrial potential in more than 70% with slightly reduced accumulation of ROS. Based on obtained results we can conclude that newly synthesized N-9-sulfonylpurine derivatives are good candidates for further antitumor studies.

Andrographolide Exhibits Anticancer Potential Against Human Colon Cancer Cells by Inducing Cell Cycle Arrest and Programmed Cell Death via Augmentation of Intracellular Reactive Oxygen Species Level

ABSTRACTAndrographolide, a diterpenoid lactone and a major constituent of Andrographis paniculata Nees, exhibits remarkable anticancer activity. However, the effect of andrographolide on colon cancer has not been completely elucidated yet. Thus, we investigated the chemopreventive potential of andrographolide in colon cancer HT-29 cells. The cytotoxic potential of andrographolide on HT-29 cells was determined by MTT assay, trypan blue exclusion assay, colony formation assay, and morphological analysis; and apoptotic property by DAPI and Hoechst staining, FITC-Annexin V assay, DNA fragmentation assay and caspase-3 activity assay. To elucidate andrographolide action, intracellular reactive oxygen species (ROS) level was determined by DCFDA dye; change in mitochondrial potential by Rhodamine123 and Mito Tracker Red CMXRos dye; and cell cycle modulatory property by flow cytometric analysis. Results of the study have shown that andrographolide decreased cell viability of HT-29 cells in a dose- and time-dependent manner. Furthermore, andrographolide induced apoptosis in HT-29 cells which seemed to be linked with augmented intracellular ROS level and disruption of mitochondrial membrane potential. Interestingly, andrographolide caused significant cell cycle arrest in G2/M phase at lower doses, but, in G0/G1 phase at higher doses. In summary, our results indicated that andrographolide exhibited antiproliferative and apoptotic properties against colon cancer HT-29 cells.

A novel series of pyrazole-platinum(II) complexes as potential anti-cancer agents that induce cell cycle arrest and apoptosis in breast cancer cells

Six novel compounds of platinum(II) with pyrazole derivatives PtPz1–PtPz6 were synthesised and characterised (PtPz1 - [Pt2N-hydroksymethyl-3,5-dimethylpyrazole4(berenil)2]Cl4; PtPz2 - [Pt23,5-dimethylpyrazole4(berenil)2]Cl4; PtPz3 - [Pt23,4-dimethylpyrazole4(berenil)2]Cl4; PtPz4 - [Pt2pyrazole4(berenil)2]Cl4; PtPz5- [Pt25-methylpyrazole4(berenil)2]Cl4; PtPz6 - [Pt2N-ethylpyrazole4(berenil)2]Cl4). The cytotoxic activity of these complexes against MCF-7 and MDA-MB-231 breast cancer cell lines was determined using the MTT assay. Evaluation of apoptosis induction was done with the Annexin V-fluorescein isothiocyanate/propidium iodide assay. In addition, using a flow cytometer, we determined the influence of test compounds on the cell cycle and caspase-3, -8, and -9 activity. The obtained results of caspase activity were confirmed by cell imaging. Moreover, using the flow cytometer, the effects of the test compounds on mitochondrial potential change were assessed. The test results showed that novel pyrazole-platinum(II) complexes exhibited stronger anti-proliferative activity against two breast cancer cell lines than reference cisplatin. Compounds PtPz1, PtPz2, and PtPz3 with methyl substituents at the pyrazole ring showed stronger activity than pyrazole or ethylpyrazole containing complexes. Studies have shown that inhibition of cell survival occurs by arresting the G1 cell cycle and inducing apoptosis. Our analysis associated with the response of MCF-7 and MDA-MB-231 cells to treatment with PtPz1–PtPz6 showed that it leads the cells through the external and intrinsic (mitochondrial) apoptotic pathway via indirect DNA damage.

Dibromoacetic acid induced Cl.Ly1 + 2/−9 T-cell apoptosis and activation of MAPKs signaling cascades

Dibromoacetic acid (DBA), a haloacetic acid by-product of disinfection of drinking water, can cause many adverse effects in test animals, including immunotoxicity. However, the underlying molecular mechanism for the immunomodulatory effects remains unclear. The present study was undertaken to help in defining some potential mechanisms for this type of toxicity. Here, Cl.Ly1+2/−9 T-cells were exposed to varying levels of DBA and then several parameters, including cell survival, apoptosis, changes in mitochondrial potentials, and effects on select kinases (i.e., p38, ERK1/2, JNK1/2) were examined. The data showed that DBA significantly decreased Cl.Ly1+2/−9 cell viability in a dose-related manner. DBA also induced apoptosis, a decrease in mitochondrial trans-membrane potential, and up-regulated the protein expression of cleaved caspase-3. Moreover, DBA increased the phosphorylation of all three mitogen-activated protein kinases (MAPKs) evaluated. Pre-treatment with specific p38, ERK1/2, and JNK1/2 inhibitors (SB203580, U0126, SP600125, respectively) attenuated the inducible phosphorylation events. DBA also induced up-regulation of mRNA levels of the MAPKs downstream transcription factors ATF-2 and Elk-1. When taken together, the results suggest that DBA could induce murine Cl.Ly1+2/−9 T-cells apoptosis through mitochondria-dependent way, and activate the MAPKs pathways and downstream transcription factors ATF-2 and Elk-1.