Altered Mitochondrial Membrane Potential Research Articles

Cytotoxicity of ungeremine towards multi-factorial drug resistant cancer cells and induction of apoptosis, ferroptosis, necroptosis and autophagy

BackgroundSuccessful cancer chemotherapy is hampered by resistance of cancer cells to established anticancer drugs. Numerous natural products reveal cytotoxicity towards tumor cells. PurposeThe present study was aimed to determine the cytotoxicity of a betaine-type alkaloid, ungeremine, towards 9 cancer cell lines including various sensitive and drug-resistant phenotypes. The mode of action of this compound was further investigated. MethodsThe cytotoxicity, ferroptotic and necroptotic cell death were determined by the resazurin reduction assay. Caspase activation was evaluated using the caspase-Glo assay. Flow cytometry was applied for the analysis of cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Apoptotic, necroptotic and autophagic markers were determined by Western blotting. CCRF-CEM leukemia cells were used for all mechanistic studies. ResultsUngeremine displayed cytotoxic activity towards the 9 cancer cell lines tested, including drug-sensitive and MDR phenotypes. The IC50values obtained varied from 3.67 µM (in MDA-MB-231-BCRP breast carcinoma cells) to 75.24 µM (against in CEM/ADR5000 leukemia cells) for ungeremine and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin (control drug). Ungeremine induced ferroptosis, necroptosis, autophagy as well as apoptosis mediated by caspase activation, MMP alteration and increase ROS production. ConclusionThe present investigation showed that ungeremine is a promising cytotoxic compoundthat could be further explored in the future to develop new anticancer drugs to fight sensitive and resistant phenotypes.

Anticancer effect of fucoidan on cell proliferation, cell cycle progression, genetic damage and apoptotic cell death in HepG2 cancer cells.

The centre of the attraction of this article is inevitably associated with fucoidan polymers in terms of brown seaweed such as Turbinaria conoides. Fucoidan is a sulphated polysaccharide constitutes fucose as a major principle sugar along with other monosugars such as glucuronic acid, xylose and galactose. The core value of fucoidan in terms of various cancer types were substantially exhibited through targeting the key apoptotic molecules and subsequently mitigate the toxicity that are essentially included in the chemotherapeutic agents and radiation. The pragmatic investigation about the anti-cancer effect of fucoidan in a hepatoblastoma-derived (HepG2) cell line was thoroughly analyzed by the typical techniques such as cell viability, colony formation, cell migration, cell cycle progression, genetic damage and apoptosis along with their nuclear morphology and mitochondrial membrane potential. Following the analyzes, the cell viability was precisely evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. And hence, cell cycle arrest and apoptosis was appropriately examined staining with propidium iodide (PI) and annexin V-fluorescein isothiocyante (FITC) by flowcytometer, respectively. Primarily, genetic damage by fucoidan in HepG2 cell line was evaluated by following Trevigen's comet assay kit. In addition, alteration of nuclear content and mitochondrial membrane potential were also detected with Hoechst and mitochondrial membrane potential dye (JC-1: 5,5'6,6'-tetrachloro-1,1'3,3'tetraethylbenzimi-dazolycarbocyanine iodide) by fluorescence microscopy, respectively. The results of the present study showed that cells constituted with fucoidan/quercetin standard at 50, 100 and 200 μg/ml exhibited cell viability about 71, 60 & 40/80, 65 & 45%, respectively. The above recorded effect of fucoidan was a concentration-dependant inhibition on the basis of decline in colony forming and cell migration potential of HepG2 cancer cells. Compared with untreated control, fucoidan consituted cells were significantly (p ≤ 0.05) accumulated proliferative cells in the G0/G1 phase of the cell cycle in a concentration dependent manner. Increasing concentration of fucoidan (50,100 and 200 μg/ml) was remarkably enhanced the DNA damage which reflected through tail moment value of 3.8, 7.1 & 12.8 folds with respect to the untreated control. Fucoidan induced total apoptotic cells were observed ∼20-40% at 50-200 μg/ml concentrations. The apoptotic cell formation effected by change in the nuclear content and mitochondrial membrane potential was confirmed in HepG2 cancer cells under fluorescence microscopy. It was eventually concluded that the fucoidan display promising anti-cancer activity against HepG2 cancer cells by promoting the inhibition of cell proliferation, migration and cell arrest on concentration dependent-manner that was well correlated with genetic damage and apoptosis.

Arctium lappa L. root extract induces cell death via mitochondrial-mediated caspase-dependent apoptosis in Jurkat human leukemic T cells

Arctium lappa L. is a perennial herb traditionally consumed to improve well-being. It has been widely reported for its antioxidant properties; however, very little is known for its exact mechanisms underlying the anticancer activity. This study aimed to investigate the mechanisms of anticancer action for different A. lappa root extracts. Arctium lappa root was extracted with ethanol, hexane and ethyl acetate, then examined for in vitro anticancer activity against cancerous HeLa, MCF-7, Jurkat cell lines and non-cancerous 3T3 cell lines. Induction of apoptosis was determined by cellular morphological changes, mitochondrial membrane potential (ΔΨm), caspase-3/7 activity and DNA fragmentation. The active compounds present in the most potent root extracts were identified by LC-ESI-MS. Among all the extracts, ethyl acetate root extract has the highest potency with IC50 of 102.2 ± 42.4 μg/ml, followed by ethanolic root extract in Jurkat T cells, at 24 h. None of the extracts were cytotoxic against 3T3 cells, suggesting that the extracts were selective against cancerous cells only. Both ethyl acetate and ethanolic root extracts exhibited significant morphological changes in Jurkat T cells, including the detachment from adjacent cells, appearance of apoptotic bodies and cells shrinkage. The extracts treated cells also displayed an increase in caspase-3/7 activity and alteration in mitochondrial membrane potential. Only ethyl acetate root extract at IC50 induced DNA fragmentation in Jurkat T cells. LC-ESI-MS analysis of the extract revealed the presence of 8 compounds, of which only 6 compounds with various biological activities reported. These findings suggest that the ethyl acetate extract of A. lappa had strong anticancer potential and induced intrinsic apoptosis via loss of ΔΨm and activation of caspase-3/7 This study can provide new insight to the discovery of new promising lead compound in chemopreventive and chemotherapeutic strategies.

Measurement of Nanoparticle-Induced Mitochondrial Membrane Potential Alterations.

Mitochondria hold a critical role in cell metabolism and homeostasis. Mitochondrial injury plays central part in deciding cell fate especially in programmed cell death pathways. Various nanomaterials lead to different cell death modalities by inducing mitochondrial injury. Mitochondrial injury is manifested as multiple biochemical events ranging from altered energy production, mitochondrial outer membrane permeability, release of pro-apoptotic BCl-2 family proteins, loss of mitochondrial inner membrane potential, mitochondrial swelling, and disruption of mitochondrial structure leading to eventual lysis of mitochondria. Mitochondrial membrane permeability (loss of mitochondrial membrane potential) is a critical event in deciding cell fate. This chapter presents an overview of nanomaterial-induced loss of mitochondrial membrane potential and discusses potential nano-specific artifacts in these assays. Finally, a detailed methodology to accurately quantify and validate the loss of mitochondrial membrane potential after nanomaterial exposures is presented.

Highlight on Endothelial Activation and Beyond.

Highlight on Endothelial Activation and Beyond.

Alterations in mitochondrial membrane potential in peripheral blood mononuclear cells in Parkinson's Disease: Potential for a novel biomarker.

Progressive apoptosis in the dopaminergic neurons of substantia nigra lead to Parkinson's disease. Since neurons require substantially higher supply of energy, their mitochondria have a pivotal status in neuronal survival. These organelles have a key role to play in apoptosis and any impairment thereof may lead to apoptosis mediated cell death. To evaluate and compare the mitochondrial membrane potential (Δψ) in Parkinson's disease patients and healthy controls. We evaluated the mitochondrial membrane potential (Δψ) in the peripheral blood mononuclear cells by Flow cytometry using a lipophillic cationic dye JC-1 in Parkinson's disease patients (N = 61) and healthy controls (N = 37). JC-1 fluorescence was measured and represented as percentage positivity i.e., Mean±SEM in FL-2 (representing non-apoptotic aggregates) and FL-1 (indicating apoptotic cell population having depolarized or damaged mitochondria) channels. The ratio of % FL-2 and % FL-1, which is an indicator of cellular mitochondrial membrane potential, was found to be significantly higher in healthy controls (Mean±SEM = 60.48±18.42) as compared to patients (Mean±SEM = 24.30±4.671) in both stimulated and unstimulated conditions. Mitochondrial membrane potential is altered and hence its evaluation in peripheral blood mononuclear cells may serve as an early marker of apoptosis in PD and, therefore, may pave way for early interventions. Since Δψ has a role in the maintenance of electrochemical gradient, the disruption of which may lead to neuronal apoptosis, Δψ is intricately nested within etiopathogenesis of PD and may prove to be useful in design of diagnostics, prognostics and therapeutics for PD.

Anticancer potential of Ferula hezarlalehzarica Y. Ajani fraction in Raji lymphoma cell line: induction of apoptosis, cell cycle arrest, and changes in mitochondrial membrane potential.

BackgroundCancer is a major cause of mortality. The present study evaluates the antitumor effects of Ferula hezarlalehzarica Y. Ajani fractions on various cancer cell lines, including the Raji Burkitt’s lymphoma cells.MethodsWe evaluated the cytotoxic activity of various fractions of F. hezarlalehzarica against tumor cell lines by the MTT assay. Annexin V-PE/7-AAD and cell cycle analysis were assessed by flow cytometry. Expressions of genes associated with cell death and proliferation (Bax, Bcl-2, Fas, and c-Myc) were determined using real-time PCR. Alteration in mitochondrial membrane potential (MMP) was examined by JC-1 dye staining.ResultsThe hexane fraction of F. hezarlalehzarica showed the highest degree of cytotoxicity against Raji cells (IC50 = 31.6 μg/ml). Flow cytometry analysis showed that 200 μg/ml of the fraction induced apoptosis in >96% of Raji cells after 24 h. In cell cycle analysis, at the same concentration, the percentage of apoptotic cells in the sub G1phase increased to 95.25 ± 1.76% at 48 h of treatment. The fraction induced cell cycle arrestat the G0/G1phase. Exposure to 100 μg/ml of the fraction after 48 h increased the percentage of G0/G1 cells (76.3 ± 6.08%) compared to the negative control (<50%). Treatment with75μg/ml of fraction reduced the expressions of Bcl-2 (0.23 ± 0.008-fold) and c-Myc (0.68 ± 0.07-fold) and increased Bax (1.75 ± 0.31-fold) and Fas (5.02 ± 0.74-fold; p < 0.01). We observed a decrease in MMP (≈0.4, p < 0.05) at ≥100 μg/ml and this effect remained almost unchanged until 48 h.ConclusionsThe F. hezarlalehzarica hexane fraction induced apoptosis in Raji cells by changing the expression of apoptosis-related genes, cell cycle distribution, and MMP. These data suggested a potential effectiveness of F. hezarlalehzarica for inducing cell death in lymphoma cells. Graphical abstractᅟ

The Mitochondrial Genes BAK1, FIS1 and SFN are Linked with Alterations in Mitochondrial Membrane Potential in Barrett's Esophagus.

Barrett’s esophagus and esophageal cancer lack prognostic markers that allow the tailoring of personalized medicine and biomarkers with potential to provide insight into treatment response. This study aims to characterize mitochondrial function across the metaplasia-dysplasia-adenocarcinoma disease sequence in Barrett’s esophagus and examines the functional effect of manipulating mitochondrial genes. Mitochondrial genes of interest were validated in in vitro cell lines across the metaplasia (QH), dysplasia (GO) and adenocarcinoma (OE33) sequence and in in vivo patient tissue samples. These genes were subsequently knocked down in QH and OE33 cells and the functional effect of siRNA-induced knockdown on reactive oxygen species production, mitochondrial mass, mitochondrial membrane potential and cellular metabolism was investigated. Three global mitochondrial genes (BAK1, FIS1 and SFN) were differentially altered across the in vivo Barrett’s disease sequence. We also demonstrate that knockdown of BAK1, FIS1 and SFN in vitro resulted in significant alterations in mitochondrial membrane potential; however, no differences in reactive oxygen species or mitochondrial mass were observed. Furthermore, knockdown of these genes in esophageal adenocarcinoma cells significantly altered cellular metabolism. In conclusion, we found that differential expression of BAK1, FIS1, and SFN were altered across the Barrett’s disease sequence and manipulation of these genes elicited significant effects on mitochondrial membrane potential.

262 - Investigating the anti-oxidant effects of estrogen in RAW cell macrophages

The female sex hormone, estrogen (E2) is proposed to have cardioprotective effects due to its anti-oxidant properties. Previous studies have shown that as women enter the post-menopausal period, cardiovascular incidences increase dramatically. This may be due to a decrease in E2 and its protective properties. E2 receptors can be found on the surface of the RAW macrophage cell as well as the nucleus and mitochondria, where E2 can modulate both nuclear and mitochondrial genes in response to inflammatory stimuli. We hypothesize that in the RAW murine macrophage cell line E2 increases cell viability, decreases reactive oxygen species (ROS) and increases mitochondrial function. PCR analysis confirmed ER-α presence in RAW cells. 48 hours after plating RAW cells, they were treated with low (100nM) and high (1mM) levels of E2 in serum and phenol-free DMEM. The cells were pretreated with E2 or vehicle for 15hrs, followed by treatment with the inflammation-inducing endotoxin, Lipopolysaccharide (LPS) for 6hrs. The Calcein AM Assay showed greater cell concentrations when cells were pretreated with E2. ROS production measurement via DCF Fluorescence of post-LPS treatment showed that E2 pretreatment of cells decreased ROS production. Changes in ROS production may be due alterations in mitochondrial membrane potential. Therefore, mitochondrial membrane potential was measured using TMRM Fluorescence and showed that E2 pretreatment increased mitochondrial membrane potential. A greater mitochondrial membrane potential may infer a more efficient mitochondria. Seahorse measurement of mitochondrial function confirmed that low and high levels of E2 pretreatment increases mitochondrial function in RAW cells. We hope to further explore cellular localization of ROS production and the effects of estrogen on mitochondrial function after LPS treatment. This data suggests that estrogen is anti-oxidant and may exert these effects by modulating mitochondrial function. Overall this data suggests that the anti-oxidant properties of estrogen may be beneficial to cardiovascular health.

Alpha pinene modulates UVA-induced oxidative stress, DNA damage and apoptosis in human skin epidermal keratinocytes

AimsThis study aims to evaluate the protective effect of alpha pinene (AP), an essential oil monoterpene, against ultraviolet-A (UVA; 320–400 nm) induced cellular damages in human skin epidermal keratinocytes (HaCaT cells). Materials and methodsIn this study, HaCaT cells were subjected to single UVA-irradiation (10 J/cm2) in the presence and absence of AP (30 μM) then different cellular end points were analyzed. The protective effect of AP against UVA-induced cytotoxicity was evaluated by MTT-based metabolic assay. Generation of reactive oxygen species (ROS), alteration of mitochondrial membrane potential (MMP), DNA single- and double strand breaks (SSBs and DSBs) and apoptotic morphological changes during different treatment conditions were measured by fluorescence microscopy and spectrofluorometry. Modulatory role of AP against UVA-mediated inflammatory markers expression, nucleotide excision repair (NER) proteins and apoptotic markers expression during AP and/or UVA treatment were studied by western blot. Key findingsPretreatment with AP prevented UVA-induced cytotoxicity, generation of ROS, lipid peroxidation and DNA stand breaks probably through its antioxidant property. AP also inhibited UVA-induced inflammatory mediators such as NF-κB, TNF-α and IL-6 expression in HaCaT cells. Further, AP modulates NER proteins via activation of p53 and p21 thereby subsequently prevent the formation of UVA-induced cyclobutane pyrimidine dimers (CPDs). We also noticed that AP inhibits apoptotic cell death by preventing UVA-induced loss of mitochondrial membrane potential through modulating Bax/Bcl-2 expression in HaCaT cells. SignificanceThe present findings suggest that AP prevent UVA-induced oxidative stress, inflammation, DNA damages and apoptosis in human skin cells.

Proanthocyanidins exert a neuroprotective effect via ROS/JNK signaling in MPTP‑induced Parkinson's disease models invitro and invivo.

The pathological alterations of Parkinson's disease (PD) predominantly manifest as a loss of dopaminergic neurons in the substantia nigra, which may be caused by oxidative stress damage. Proanthocyanidins (PCs) are a class of compounds found in various plants, which have significant antioxidant and free radical-scavenging activity. The present study investigated the protective effects and underlying mechanisms of PCs in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in vitro and in vivo. MTT assays were used to detect cell viability, and flow cytometry and TUNEL assays were used to detect cell apoptosis. Mitochondrial membrane potential (MMP) alterations were investigated using a JC-1 MMP Assay kit. The pole test was used to measure motor behavior in a mouse model of PD. Levels of reactive oxygen species (ROS) were measured using the fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. Immunohistochemistry and western blotting were performed to detect the expression levels of proteins associated with PD. In vitro, it was demonstrated that in MPTP-treated PC12 cells, PCs increased cell viability and reduced cell apoptosis in a dose-dependent manner. In vivo, it was revealed that PC treatment inhibited striatal dopamine depletion, which resulted in significant improvements in PD-like movement impairment. Reactive oxygen species (ROS) production and MPTP-induced apoptosis were also inhibited. Furthermore, the results demonstrated that the neuroprotective activity of PCs may be mediated via the inhibition of ROS generation, as well as modulation of c-Jun N-terminal kinase activation. Taken together, these data revealed that PCs may exert neuroprotective effects in in vivo and in vitro PD models, and may have potential in the prevention or treatment of PD.

Licochalcone D directly targets JAK2 to induced apoptosis in human oral squamous cell carcinoma.

Licochalcone (LC) families have been reported to have a wide range of biological function such as antioxidant, antibacterial, antiviral, and anticancer effects. Although various beneficial effects of LCD were revealed, its anticancer effect in human oral squamous cancer has not been identified. To examine the signaling pathway of LCD's anticancer effect, we determined whether LCD has physical interaction with Janus kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) signaling, which is critical in promoting cancer cell survival and proliferation. Our results demonstrated that LCD inhibited the kinase activity of JAK2, soft agar colony formation, and the proliferation of HN22 and HSC4 cells. LCD also induced mitochondrial apoptotic events such as altered mitochondrial membrane potential and reactive oxygen speciesproduction. LCD increased the expression of apoptosis-associated proteins in oral squamous cell carcinoma (OSCC) cells. Finally, the xenograft study showed that LCD significantly inhibited HN22 tumor growth. Immunohistochemical data supported that LCD suppressed p-JAK2 and p-STAT3 expression and induced cleaved-caspase-3 expression. These results indicate that the anticancer effect of LCD is due to the direct targeting of JAK2 kinase. Therefore, LCD can be used for therapeutic application against OSCC.

HIV-1 TAT-mediated microglial activation: role of mitochondrial dysfunction and defective mitophagy

ABSTRACTWhile the advent of combination antiretroviral therapy (cART) has dramatically increased the life expectancy of HIV-1 infected individuals, paradoxically, however, the prevalence of HIV-1-associated neurocognitive disorders is on the rise. Based on the premise that the cytotoxic HIV-1 protein, transactivator of transcription (TAT), a known activator of glial cells that is found to persist in the central nervous system (CNS) despite cART, we sought to explore the role of defective mitophagy in HIV-1 TAT-mediated microglial activation. Our results demonstrated that exposure of mouse primary microglia to HIV-1 TAT resulted in cellular activation involving altered mitochondrial membrane potential that was accompanied by accumulation of damaged mitochondria. Exposure of microglia to HIV-1 TAT resulted in increased expression of mitophagy signaling proteins, such as PINK1, PRKN, and DNM1L, with a concomitant increase in the formation of autophagosomes, as evidenced by increased expression of BECN1 and MAP1LC3B-II. Intriguingly, exposure of cells to HIV-1 TAT also resulted in increased expression of SQSTM1, signifying thereby a possible blockade of the mitophagy flux, leading, in turn, to the accumulation of mitophagosomes. Interestingly, HIV-1 TAT-mediated activation of microglia was associated with decreased rate of extracellular acidification and mitochondrial oxygen consumption and increased expression of proinflammatory cytokines, such as Tnf, Il1b, and Il6. HIV-1 TAT-mediated defective mitophagy leading to microglial activation was further validated in vivo in the brains of HIV-1 transgenic rats. In conclusion, HIV-1 TAT activates microglia by increasing mitochondrial damage via defective mitophagy.Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A1; BECN1: beclin 1, autophagy related; cART: combined antiretroviral therapy; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-diamidino-2-phenylindole‎; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAND: HIV-1-associated neurocognitive disorders; HIV-1 TAT: human immunodeficiency virus-1 transactivator of transcription; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; mt-CO1: mitochondrially encoded cytochrome c oxidase; mt-ND6: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 6; NFKB1: nuclear factor kappa B subunit 1; NLRP3: NLR family pyrin domain containing 3; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor

Myricetin exerts potent anticancer effects on human skin tumor cells

Purpose: To evaluate the anticancer activity of myricetin against skin cancer A431 cell lines.Methods: Cell viability was determined by MTT and colony formation assays. Apoptosis was determined by DAPI and annexin V/PI staining. Cell cycle, ROS and MMP analysis were performed by flow cytometry. Cell migration and invasion were assessed by Boyden Chamber assay, while protein expression was determined using western blotting.Results: Myricetin showed considerable anticancer activity against skin A431 cancer cell lines. However, lower cytotoxic effects were observed in normal FR2 cells. The anticancer activity of myricetin was due to ROS-prompted alterations in mitochondrial membrane potential and initiation of apoptotic cell death. The expressions of Bcl-2 and Bax were altered in response to myricetin treatment. Myricetin also induced cell cycle arrest and suppressed the migration and invasion of A431 cells.Conclusion: These results suggest that myricetin may be an important lead molecule for the development of a suitable treatment of skin cancer.Keywords: Skin carcinoma, ROS, Apoptosis, Myricetin, Cell migration

Abstract LB-080: Discovery of an unconventional form of cell-death in colorectal cancer

Abstract We recently discovered unique, structurally constrained 4-pyridinyl - containing compounds that induce a form of caspase-independent cell death in colorectal cancer (CRC) cells. This was characterized by the simultaneous induction of dysregulated macropinocytosis and macroautophagy, a type of cell death we have termed ‘methuophagy' (this neologism arises from the Greek ‘methuo', to drink to intoxication and ‘phagy', self-eating). Our new class of small molecular probes (BAPT analogues) do not induce cell death via activation of the well characterized classical apoptotic pathways. In contrast, our BAPT compounds cause methuophagy by inducing the formation and accumulation of fluid-filled, heterogeneous vacuoles and autophagosomes, resulting in metabolic failure, loss of membrane integrity, and detachment of cells from the substratum. Methuophagy produces extensive autophagy without cell shrinkage, chromatin condensation, alteration of mitochondrial membrane potential or nuclear membrane damage. The optimization of the BAPT structure led to the discovery of BAPT-27, which has nanomolar potency in inducing methuophagy in broad spectrum of CRC cell lines and may serve as a prototype for a new class of drugs. BAPT-27 produces methuophagy and surmounts MDR in CRC cells independent of Ras, p53 or other apoptotic protein (i.e. Bax, Bak) or expression of ATP-binding cassette (ABC) transporters. Furthermore, BAPT-27 is non-toxic in normal cells and zebrafish. Based on our findings, we postulate that the induction of methuophagy by BAPT-27 produces endolysosomal trafficking defects that prevent recycling of lysosomes and cause lysosome-to-nucleus signaling defect, a mechanism that disturb cellular clearance and energy metabolism. The selected compounds will allow us to understand the biology of methuophagy-induced cell death and will be particularly beneficial for treatment of patients with apoptotic-resistant and aggressive CRCs. Citation Format: Amit K. Tiwari, Haneen Amawi, Karthikeyan Chandrabose, Paul Erhardt, Piyush Trivedi. Discovery of an unconventional form of cell-death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-080.

Hypomethylation of mitochondrial D-loop and ND6 with increased mitochondrial DNA copy number in the arsenic-exposed population

Groundwater arsenic contamination has become a serious global concern due to its adverse effects on human health. Arsenic-induced reactive oxygen species trigger oxidative stress inside mitochondria, which initiate a cascade of events including altered mitochondrial (mt) membrane potential, uncoupling of electron transport chain, and mtDNA damage. A case-control study was conducted to examine the association between arsenic exposure and differences in mtDNA methylation and to assess the downstream consequences. We recruited 221 arsenic-exposed individuals, including 106 individuals with skin lesions (WSL) and 115 subjects without any skin lesions (WOSL) from the Murshidabad district, West Bengal, India. The unexposed group included 101 individuals from the arsenic unexposed area in East Midnapore. We analyzed the status of mtDNA methylation in D-loop region and ND6 gene by methylation-specific PCR. Gene expression was studied by quantitative real-time PCR. Significant hypomethylation in both D-loop and ND6 was observed with a consequent increase in their target gene expression and higher mtDNA copy number in arsenic-exposed populations compared to controls. Further mechanistic insights regarding mitochondrial epigenetic alteration in arsenic exposure will be of critical importance for the prevention of adverse health effects.

Octaarginine-modified gold nanoparticles enhance the radiosensitivity of human colorectal cancer cell line LS180 to megavoltage radiation.

BackgroundThis study investigated the effectiveness and underpinning mechanisms of radiosensitization using octaarginine (R8)-modified gold nanoparticle–poly(ethylene glycol) (GNP-PEG-R8) in colorectal cancer cell line LS180 to megavoltage radiotherapy in vitro.MethodIn-house synthesized GNP-PEG was characterized by transmission electron microscopy, dynamic light scattering, ultraviolet–visible spectrophotometry, and X-ray photoelectron spectroscopy. Inductively coupled plasma mass spectroscopy was used to quantify internalization. Direct cytotoxicity was established using the Cell Counting Kit-8, while radiosensitivity was determined using the gold standard in vitro clonogenic assay. Cell-cycle distribution, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were analyzed by flow cytometry, further exploring the key mechanisms driving GNP-PEG-R8 radiosensitization.ResultsThe core GNP diameter was 6.3±1.1 nm (mean±SD). Following functionalization, the hydrodynamic diameter increased to 19.7±2.8 nm and 27.8±1.8 nm for GNP-PEG and GNP-PEG-R8, with respective surface plasmon resonance peaks of 515 nm and 525 nm. Furthermore, incorporation of the R8 significantly increased nanoparticle internalization compared to GNP-PEG (p<0.001) over a 1 h treatment period. Functionalized GNPs confer little cytotoxicity below 400 nM. In clonogenic assays, radiation combined with GNP-PEG-R8 induced a significant reduction in colony formation compared with radiation alone, generating a sensitizer enhancement ratio of 1.59. Furthermore, GNP-PEG-R8 plus radiation predominantly induced cell-cycle arrest in the G2/M phase, increasing G2/M stalling by an additional 10% over GNP-PEG, markedly promoting apoptosis (p<0.001). Finally, ROS levels and alterations in MMP were investigated, indicating a highly significant (p<0.001) change in both parameters following the combined treatment of GNP-PEG-R8 and radiation over radiation alone.ConclusionR8-modified GNPs were efficiently internalized by LS180 cells, exhibiting minimal cytotoxicity. This yielded significant radiosensitization in response to megavoltage radiation. GNP-PEG-R8 may enhance radiosensitivity by arresting cell cycle and inducing apoptosis, with elevated ROS identified as the likely initiator.

Silver Nanoparticles: Synthetic Routes, In Vitro Toxicity and Theranostic Applications for Cancer Disease.

The large use of nanomaterials in many fields of application and commercial products highlights their potential toxicity on living organisms and the environment, despite their physico-chemical properties. Among these, silver nanoparticles (Ag NPs) are involved in biomedical applications such as antibacterial agents, drug delivery vectors and theranostics agents. In this review, we explain the common synthesis routes of Ag NPs using physical, chemical, and biological methods, following their toxicity mechanism in cells. In particular, we analyzed the physiological cellular pathway perturbations in terms of oxidative stress induction, mitochondrial membrane potential alteration, cell death, apoptosis, DNA damage and cytokines secretion after Ag NPs exposure. In addition, their potential anti-cancer activity and theranostic applications are discussed.

A naturally occuring triterpene saponin ardisiacrispin B displayed cytotoxic effects in multi-factorial drug resistant cancer cells via ferroptotic and apoptotic cell death

IntroductionMultidrug resistance of cancer cells constitutes a serious problem in chemotherapy and a challenging issue in the discovery of new cytotoxic drugs. Many saponins are known to display anti-cancer effects. In this study, the cytotoxicity and the modes of action of a naturally occuring oleanane-type tritepene saponin, ardisiacrispin B isolated from the fruit of Ardisia kivuensis Taton (Myrsinaceae) was evaluated on a panel of 9 cancer cell lines including various sensitive and drug-resistant phenotypes. MethodsResazurin reduction assay was used to evaluate cytotoxicity and ferroptotic cell death of samples; caspase-Glo assay was used to detect the activation of caspases in CCRF–CEM leukemia cells. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells by annexin V/PI staining, analysis of mitochondrial membrane potential (MMP) and measurement of reactive oxygen species (ROS). ResultsArdisiacrispin B displayed significant cytotoxic effects in the 9 tested cancer cell lines with IC50 values below 10 µM. The IC50 values ranges were 1.20 µM (towards leukemia CCRF–CEM cells) to 6.76 µM [against heptocarcinoma HepG2 cells] for ardisiacrispin B and 0.02 µM (against CCRF–CEM cells) to 122.96 µM (against resistant CEM/ADR5000 leukemia cells) for doxorubicin. Collateral sensitivity of resistant HCT116p53−/− colon adenocarcinoma cells to ardisiacripsin B was observed. Ardisiacrispin B induced apoptosis in CCRF–CEM cells via activation of inititator caspases 8 and 9 and effector caspase 3/7, alteration of MMP and increase in ROS production. Ferroptosis also contributed to the cytotoxicity of ardisiacrispin B. ConclusionsThe studied oleanane-type triterpene saponin is a good cytotoxic molecule that deserve more detailed exploration in the future, to develop novel cytotoxic drugs to combat both sensitive and drug-resistant cancers.

Pre-Treatment with Either L-Carnitine or Piracetam Increases Ultrasound-Mediated Gene Transfection by Reducing Sonoporation-Associated Apoptosis

Sonoporation, the use of ultrasound to alter the permeability of cell membranes, is a non-viral technique used to facilitate gene delivery, possibly by opening transient pores in the cell membrane. However, sonoporation may have negative bio-effects on cells, such as causing apoptosis, which limits its efficacy in gene delivery. In this study, we investigated whether pre-treatment with either L-carnitine or piracetam could protect cells from undergoing apoptosis after sonoporation and the possible mechanisms. We found that either L-carnitine or piracetam can promote gene transfection without reducing cell viability, possibly by reducing cavitation-induced reactive oxygen species generation, reversing alterations of mitochondrial membrane potential, preventing caspase-3/7 activity and facilitating mitochondrial ATP production. In conclusion, pre-treatment with either L-carnitine or piracetam could protect cells from sonoporation-associated apoptosis by preserving mitochondrial function.