Mechanism Of Apoptotic Cell Death Research Articles

Polystyrene nanoplastics as an ecotoxicological hazard: cellular and transcriptomic evidences on marine and freshwater in vitro teleost models

The contamination of marine and freshwater environments by nanoplastics is considered a global threat for aquatic biota. Taking into account the most recent concentration range estimates reported globally and recognizing a knowledge gap in polystyrene nanoplastics (PS-NPs) ecotoxicology, the present work investigated the harmful effects of 20 nm and 80 nm PS-NPs, at increasing biological complexity, on the rainbow trout Oncorhynchus mykiss RTG-2 and gilthead seabream Sparus aurata SAF-1 cell lines. Twenty nm PS-NPs exerted a greater cytotoxicity than 80 nm ones and SAF-1 were approximately 4-fold more vulnerable to PS-NPs than RTG-2. The engagement of PS-NPs with plasma membranes was accompanied by discernible uptake patterns and morphological alterations along with a nuclear translocation already within a 30-min exposure. Cells were structurally damaged only by the 20 nm PS-NPs in a time-dependent manner as indicated by distinctive features of the execution phase of the apoptotic cell death mechanism such as cell shrinkage, plasma membrane blebbing, translocation of phosphatidylserine to the outer leaflet of the cell membrane and DNA fragmentation. At last, functional analyses unveiled marked transcriptional impairment at both sublethal and lethal doses of 20 nm PS-NPs, with the latter impacting the “Steroid biosynthesis”, “TGF-beta signaling pathway”, “ECM-receptor interaction”, “Focal adhesion”, “Regulation of actin cytoskeleton” and “Protein processing in endoplasmic reticulum” pathways. Overall, a distinct ecotoxicological hazard of PS-NPs at environmentally relevant concentrations was thoroughly characterized on two piscine cell lines. The effects were demonstrated to depend on size, exposure time and model, emphasizing the need for a comparative evaluation of endpoints between freshwater and marine ecosystems.

Nanocarrier - Mediated Salinomycin Delivery Induces Apoptosis and Alters EMT Phenomenon in Prostate Adenocarcinoma.

Salinomycin (Sal) has been recently discovered as a novel chemotherapeutic agent against various cancers including prostate cancer which is one of the most commonly diagnosed cancers affecting male populations worldwide. Herein we designed salinomycin nanocarrier (Sal-NPs) to extend its systemic circulation and to increase its anticancer potential. Prepared nanoform showed high encapsulation and sustained release profile for salinomycin. The present study elucidated the cytotoxicity and mechanism of apoptotic cell death of Sal-NPs against prostate cancer both in vitro and in vivo. At all measured concentrations, Sal-NPs showed more significant cytotoxicity to DU145 and PC3 cells than Sal alone. This effect was mediated by apoptosis, as confirmed by ROS generation, loss of MMP and cell cycle arrest at the G1 phase in both cells. Sal-NPs efficiently inhibited migration of PC3 and DU145 cells via effectively downregulating the epithelial mesenchymal transition. Also, the results confirmed that Sal-NPs can effectively inhibit the induction of Prostate adenocarcinoma in male Wistar rats. Sal-NPs treatment exhibited a decrease in tumour sizes, a reduction in prostate weight, and an increase in body weight, which suggests that Sal-NPs is more effective than salinomycin alone. Our results suggest that the molecular mechanism underlying the Sal-NPs anticancer effect may lead to the development of a potential therapeutic strategy for treating prostate adenocarcinoma.

Mitochondrial Permeability Transition, Cell Death and Neurodegeneration.

Neurodegenerative diseases are chronic conditions occurring when neurons die in specific brain regions that lead to loss of movement or cognitive functions. Despite the progress in understanding the mechanisms of this pathology, currently no cure exists to treat these types of diseases: for some of them the only help is alleviating the associated symptoms. Mitochondrial dysfunction has been shown to be involved in the pathogenesis of most the neurodegenerative disorders. The fast and transient permeability of mitochondria (the mitochondrial permeability transition, mPT) has been shown to be an initial step in the mechanism of apoptotic and necrotic cell death, which acts as a regulator of tissue regeneration for postmitotic neurons as it leads to the irreparable loss of cells and cell function. In this study, we review the role of the mitochondrial permeability transition in neuronal death in major neurodegenerative diseases, covering the inductors of mPTP opening in neurons, including the major ones-free radicals and calcium-and we discuss perspectives and difficulties in the development of a neuroprotective strategy based on the inhibition of mPTP in neurodegenerative disorders.

Diaryl-substituted benzimidazole derivatives as antiproliferative agents: Design, synthesis, in vitro activity, and SAR analysis

In this study, a series of 1,2-disubstituted benzimidazoles, designed to mimic nucleotides and interact favorably with amino acids in protein active sites, were synthesized and characterized. These compounds were characterized using IR, NMR spectral data, and elemental analyses, leveraging the recognition of substituted benzimidazoles as pharmacophores in the literature due to their documented anticancer activities. The compounds involved chloro and substituted phenyl rings, known for their anticancer activities, as key pharmacophores. Cytotoxicity assessments using the MTT assay were performed against MCF7, MDA-MB-231, and normal mouse fibroblasts (L929) for all synthesized compounds (11–19). Among them, compound 13 exhibited the most significant activity, inducing cell cycle arrest at the G0/G1 phase in MCF7 cells, suggesting its potential as an antitumor agent through apoptosis. To understand the underlying mechanisms of apoptotic cell death induced by compound 13, we investigated the involvement of apoptotic proteins such as Bax, Bcl-2, and p53. Our findings highlight Compound 13 as a promising scaffold for further optimization in the development of effective anticancer drugs.

Evaluation of the toxicological effects of Neltuma alpataco (Prosopis alpataco) pod alkaloid extract

The pods of Neltuma spp. have shown potential as a source of protein and energy in livestock. However, prolonged consumption of some of these species can lead to neurological symptoms in ruminants. This study aimed to determine the alkaloid content, as well as the in vitro and in vivo effects of an alkaloid-enriched extract (AEE) from N. alpataco pods. High performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) identified juliprosine and juliprosopine as primary alkaloids, with juliprosine being most abundant. AEE from N. alpataco demonstrated dose-dependent cytotoxicity on glioma cells after 48 h, with a 50% cytotoxic concentration (CC50) of 24.69 μg/mL. However, the release of LDH was observed only at the highest tested concentration, indicating cellular damage. Further examination through phase-contrast microscopy and dual acridine orange/ethidium bromide fluorescence staining revealed morphological changes consistent with an apoptotic mechanism of cell death, ultimately leading to secondary necrosis. Finally, the LD50 after intraperitoneal injection in mice was determined to be 12.98 mg/kg. Taken together, these findings demonstrated for the first time the in vivo and in vitro toxicity of the AEE from N. alpataco pods.

Molecular mechanism of anticancer effect of heat shock protein 90 inhibitor BIIB021 in human bladder cancer cell line

Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity has been a promising pharmacological strategy for blocking of bladder cancer pathogenesis. BIIB021 is a next-generation HSP90 inhibitor which interrupts ATP hydrolysis process of HSP90 and inhibits the stabilization and correct folding of client proteins. In current study, we aimed to investigate the molecular mechanism of the anticancer activity of BIIB021 in human bladder cancer T24 cells. Our results revealed that nanomolar concentration of BIIB021 decreased viability of T24 cell. BIIB021 downregulated HSP90 expression in T24 cells and inhibited the refolding activity of luciferase in the presence of T24 cell lysate. PCR array data indicated a significant alteration in transcript levels of cancer-related genes involved in metastases, apoptotic cell death, cell cycle, cellular senescence, DNA damage and repair mechanisms, epithelial-to-mesenchymal transition, hypoxia, telomeres and telomerase, and cancer metabolism pathways in T24 cells. All findings hypothesize that BIIB021 could exhibit as effective HSP90 inhibitor in the future for treatment of bladder cancer patients.

Exploring the cytotoxicity of dinuclear Ru(II)p-cymene complexes appended N,N'-bis(4-substituted benzoyl)hydrazines: insights into the mechanism of apoptotic cell death.

Cancer is a perilous life-threatening disease, and attempts are constantly being made to create multinuclear transition metal complexes that could lead to the development of potential anticancer medications and administration procedures. Hence, this work aims to design, synthesize, characterize, and assess the anticancer efficacy of ruthenium p-cymene complexes incorporating N,N'-bis(4-substituted benzoyl)hydrazine ligands. The formation of the new complexes (Ru2H1-Ru2H3) has been thoroughly established by elemental analysis, and FT-IR, UV-vis, NMR, and HR-MS spectral techniques. The solid-state molecular structures of the complexes Ru2H1 and Ru2H3 have been determined using the SC-XRD study, which confirms the N, O, and Cl-legged piano stool pseudo-octahedral geometry of each ruthenium(II) ion. The stability of these complexes in the solution state and their lipophilicity profile have been determined. Furthermore, the title complexes were tested for their in vitro anticancer activity against cancerous H460 (lung cancer cells), SkBr3 (breast cancer cells), HepG2 (liver cancer cells), and HeLa (cervical cancer cells) along with non-cancerous (HEK-293) cells. The IC50 results revealed that complex Ru2H3 exhibits potent activity against the proliferation of all four cancer cells and outscored the effect of the standard metallodrug cisplatin. This may be attributed to the presence of a couple of lipophilic electron-donating methoxy groups in the ligand scaffold and also the ruthenium(II) p-cymene motifs. Advantageously, all the complexes (Ru2H1-Ru2H3) displayed cytotoxic specificity only towards cancerous cells by leaving the off-target non-cancerous cells undamaged. Acridine orange/ethidium bromide (AO/EB) staining, Hoechst 33342, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) staining assays were used to investigate the apoptotic pathway and ROS levels in mitochondria. The results of western blot analysis confirmed that the complexes triggered apoptosis through an intrinsic mitochondrial pathway by upregulating Bax and downregulating Bcl-2 proteins. Finally, the extent of apoptosis triggered by the complex Ru2H3 was quantified with the aid of flow cytometry using the Annexin V-FITC/propidium iodide (PI) double-staining technique.

Molecular Mechanism of Apoptotic Cell Death in Cyanidin-3-glucoside-induced Cytotoxic Potential on Human Liver Carcinoma (HepG2) Cell Line

Molecular Mechanism of Apoptotic Cell Death in Cyanidin-3-glucoside-induced Cytotoxic Potential on Human Liver Carcinoma (HepG2) Cell Line

In Vitro Evaluation of Vancomycin-Induced Toxicity in Human Primary Knee Chondrocytes.

Septic arthritis as a complication of orthopaedic joint surgery can have catastrophic outcomes for patients. To minimise infection risk associated with elective orthopaedics, topical vancomycin during surgery has become increasingly common. Evidence suggests that high concentrations of vancomycin, following direct application of the drug to the joint, are toxic towards various local cell types in the joint, including chondrocytes. However, the mechanism of this vancomycin tissue toxicity is yet to be determined. The aim of this study was to evaluate the toxicity of vancomycin on chondrocytes and the mechanisms of cell death involved. Human primary knee chondrocytes were exposed to vancomycin (1.25-10mg/mL) for 24h and their viability assessed using the resazurin reduction assay in vitro. Specific cell death mechanisms and their contributors, including reactive oxygen species (ROS) production and apoptosis, were measured. This study showed that high concentrations of vancomycin (5 and 10mg/mL) were toxic towards human primary knee chondrocyte cells, while lower concentrations (1.25 and 2.5mg/mL) were not. Cell death studies found that this occurred through an apoptotic pathway. This study provides additional support that vancomycin in high doses is toxic towards chondrocytes and preliminary evidence that this toxicity occurs via apoptotic cell death mechanisms.

Standardized Polyalthia longifolia leaf extract induces the apoptotic HeLa cells death via microRNA regulation: identification, validation, and therapeutic potential.

Polyalthia longifolia var. angustifolia Thw. (Annonaceae), is a famous traditional medicinal plant in Asia. Ample data specifies that the medicinal plant P. longifolia has anticancer activity; however, the detailed mechanisms of action still need to be well studied. Recent studies have revealed the cytotoxicity potential of P. longifolia leaf against HeLa cells. Therefore, the current study was conducted to examine the regulation of miRNAs in HeLa cancer cells treated with the standardized P. longifolia methanolic leaf extract (PLME). The regulation of miRNAs in HeLa cancer cells treated with the standardized PLME extract was studied through Illumina, Hi-Seq. 2000 platform of Next-Generation Sequencing (NGS) and various in silico bioinformatics tools. The PLME treatment regulated a subset of miRNAs in HeLa cells. Interestingly, the PLME treatment against HeLa cancer cells identified 10 upregulated and 43 downregulated (p < 0.05) miRNAs associated with apoptosis induction. Gene ontology (GO) term analysis indicated that PLME induces cell death in HeLa cells by inducing the pro-apoptotic genes. Moreover, the downregulated oncomiRs modulated by PLME treatment in HeLa cells were identified, targeting apoptosis-related genes through gene ontology and pathway analysis. The LC-ESI-MS/MS analysis identified the presence of Vidarabine and Anandamide compounds that were previously reported to exhibit anticancer activity. The findings of this study obviously linked the cell cytotoxicity effect of PLME treatment against the HeLa cells with regulating various miRNAs expression related to apoptosis induction in the HeLa cells. PLME treatment induced apoptotic HeLa cell death mechanism by regulating multiple miRNAs. The identified miRNAs regulated by PLME may provide further insight into the mechanisms that play a critical role in cervical cancer, as well as novel ideas regarding gene therapeutic strategies.

Some Wild Mushrooms with High Antioxidant Capacity Exhibit Potent Anticancer Activity on Cancer Cells using the Apoptotic and Antimigration Cell Death Mechanisms.

This study aims to provide in vitro experimental evidence that wild mushrooms have the potential to be used as a pharmaceutical that could be effective against various types of cancer. Throughout human history, besides food, traditional medicine and natural poisons obtained from mushrooms have been used for the treatment of many diseases. Clearly, edible and medicinal mushroom preparations have beneficial health effects without the known severe adverse side effects. This study was designed to reveal the cell growth inhibitory potential of five different edible mushrooms and the biological activity of Lactarius zonarius was shown here for the first time. The mushrooms fruiting bodies were dried and powdered then extracted with hexane, ethyl acetate, and methanol. The mushroom extracts were screened for possible antioxidant activities by the free radical scavenging activity (DPPH) method. Antiproliferative activity and cytotoxicity of the extracts were investigated in vitro on A549 (human lung carcinoma), HeLa (human cervix carcinoma), HT29 (human colon carcinoma), Hep3B (human hepatoma), MCF7 (human breast cancer), FL (human amnion cells), and Beas2B (normal human cells) cells lines by using MTT cell proliferation assay, lactate dehydrogenase (LDH) assay, DNA degradation, TUNEL, and cell migration assay. Using proliferation, cytotoxicity, DNA degradation, TUNEL, and migration assay, we displayed that hexane, ethyl acetate, and methanol extracts of the Lactarius zonarius, Laetiporus sulphureus, Pholiota adiposa, Polyporus squamosus, and Ramaria flava were effective on the cells even so at low doses (< 45.0 - 99.6 µg/mL) by acting in a way that represses migration, as a negative inducer of apoptosis. It was also demonstrated that mushroom extracts with high antioxidant effect have within the acceptable cytotoxic activity of 20%-30% on the cell membrane at concentrations higher than 60 µg/mL. Overall, all of the mushroom extracts with high antioxidant effects had strong antiproliferative activity and low toxicity for cells. These findings, at least, highlight that these mushroom extracts can be used for the treatment of cancer disease, especially as a supportive therapy against colon, liver, and lung cancer.

In Vitro and In Silico Analysis of the Anticancer Effects of Eurycomanone and Eurycomalactone from Eurycoma longifolia.

Eurycomanone and eurycomalactone are known quassinoids present in the roots and stems of Eurycoma longifolia. These compounds had been reported to have cytotoxic effects, however, their mechanism of action in a few cancer cell lines have yet to be elucidated. This study was aimed at investigating the anticancer effects and mechanisms of action of eurycomanone and eurycomalactone in cervical (HeLa), colorectal (HT29) and ovarian (A2780) cancer cell lines via Sulforhodamine B assay. Their mechanism of cell death was evaluated based on Hoechst 33342 assay and in silico molecular docking toward DHFR and TNF-α as putative protein targets. Eurycomanone and eurycomalactone exhibited in vitro anticancer effects manifesting IC50 values of 4.58 ± 0.090 µM and 1.60 ± 0.12 µM (HeLa), 1.22 ± 0.11 µM and 2.21 ± 0.049 µM (HT-29), and 1.37 ± 0.13 µM and 2.46 ± 0.081 µM (A2780), respectively. They induced apoptotic cancer cell death in dose- and time-dependent manners. Both eurycomanone and eurycomalactone were also predicted to have good inhibitory potential as demonstrated by the docking into TNF-α with binding affinity of -8.83 and -7.51 kcal/mol, respectively, as well as into DHFR with binding affinity results of -8.05 and -8.87 kcal/mol, respectively. These results support the evidence of eurycomanone and eurycomalactone as anticancer agents via apoptotic cell death mechanism that could be associated with TNF-α and DHFR inhibition as among possible protein targets.

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.

Abstract 3950: Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors

Abstract Background: Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for ~80% of all lung cancers. Up to 69% of NSCLC are mutated for the tumor suppressor gene TP53, and of these, 84% are missense mutations, where wild-type (WT) TP53 tumor suppressor function is lost and in most cases, emergent cancer-promoting oncogenic activities are acquired. Due to the poor draggability of mutated p53 protein, the focus of therapeutics targeting NSCLC tumors with oncogenic missense mutant p53 has been on vulnerabilities that are not present in normal cells expressing WT p53. One such vulnerability involves oncogenic mutant p53-dependent attenuation of the oxidative stress response. The ability of cancer cells to survive oxidative stress depends on the regulated synthesis and activity of cellular antioxidants, including glutathione (GSH). Proteasome inhibitors (PIs) are known to induce cellular stress at multiple levels, including proteotoxic, autophagic, and oxidative stress, but the clinical utility of PIs has been limited by lack of efficacy in patients with solid tumors, including unselected lung cancer patients. Methods and Results: We now show bortezomib (BTZ) and other PIs are preferentially cytotoxic in oncogenic mutant p53-expressing lung cancer cells in culture. Furthermore, BTZ-treated oncogenic mutant p53-expressing cells display elevated levels of reactive oxygen species and BTZ’s cytotoxic effects were rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ cytotoxicity. Upon BTZ treatment of oncogenic mutant p53-expressing NSCLC cells, but not in WT p53 expressing NSCLC cells, we also observed the transcriptional induction of pro-apoptotic BH3-only BCL-2 family protein NOXA, consistent with an apoptotic mechanism of BTZ-induced cell death. Furthermore, in combination with BTZ, the use of BH3-mimetics such as venetoclax (ABT-199) or navitoclax (ABT-263) to inhibit the function of pro-survival proteins BCL-2/BCL-XL synergistically enhanced BTZ cytotoxicity, but only in oncogenic mutant p53-expressing NSCLC cells. In vivo mouse xenograft studies demonstrated that combination treatments of BTZ + venetoclax or BTZ + carboplatin (a standard of care chemotherapy for NSCLC) significantly attenuated oncogenic mutant p53-expressing tumor growth. Conclusions: Our data suggest that the preferential cytotoxicity of BTZ, and potentially other PIs, in oncogenic mutant p53-expressing NSCLC cells depends on the induction of toxic oxidative stress leading to NOXA-dependent apoptosis. Thus, combining PIs with drugs that limit antioxidant responses and/or sensitize cells to NOXA-induced apoptosis (e.g., BH3-mimetics) should synergistically and selectively kill NSCLC cells expressing oncogenic mutant p53 and open novel therapeutic avenues for the treatment of NSCLC. Citation Format: Kranthi Kumar Chougoni, Victoria L. Neely, Boxiao Ding, Eziafa Oduah, Khanh Nguyen, Bin Hu, Jennifer Koblinski, Bradford Windle, Hisashi Harada, Steven Grossman. Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3950.

Abstract 6317: Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells

Abstract Cancer treatment has improved in the past decades due to advances in targeted therapies and surgical techniques. Immunotherapy, for example, has greatly improved outcomes for people with several types of cancer that typically have a poor prognosis. However, the response rate depends on the type of cancer being treated, with an average response rate between 20 to 50%. Thus, novel approaches to overcome such therapeutic challenges are warranted. Emerging therapies include fusion proteins and immunoconjugates that deliver a cytotoxic payload to the tumor cell with more specificity and potentially less avenues for resistance than exerted by the antibody or toxin alone. Carcinoembryonic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in a high percentage of solid tumors including colon and rectum, lung, prostate, ovary, thyroid, or liver. Thus, oncologic therapies against CEA may promote anti-tumor activity while sparing off-target toxicity to unaffected normal tissue. Our newly developed construct, designated GrB-Fc-huMFE, is a completely human fusion protein that contains the single-chain variable fragment huMFE antibody recognizing a juxta-membrane epitope of CEA remaining after proteolytic cleavage of CEA from the cell surface. The construct rapidly internalized into target cells resulting in granzyme B (GrB)-mediated mitochondrial depolarization and irreversible activation of both apoptotic and necrotic cascade mechanisms of cell death. In vitro cytotoxicity of GrB-Fc-huMFE against an initial subset of CEA+ tumor cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar IC50 cytotoxicity against CEA-negative cell lines. Significantly, GrB-Fc-huMFE cytotoxicity in vitro was unaffected by exogenously-added CEA extracellular domain at doses up to 1000 μg/ml, suggesting that the construct is not competitively absorbed by soluble CEA proteolytically released by tumors and present in the circulation of patients. An in vivo efficacy study against established CEA+ (A-549) tumors in nude mice resulted in significant growth inhibition compared to vehicle controls (1200 mm3 vs &amp;lt;40 mm3). Complete tumor regression was observed in 7/10 tumors in mice treated (IP) with doses of 100 mg/kg of GrB-Fc-huMFE in a Q3Dx5 dose administration schedule. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Pharmacokinetic, stability and in vitro tests against a broader panel of tumor cell lines are ongoing and will be reported. Altogether, GrB-Fc-huMFE appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical and eventual clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Walter N. Hittelman, Michael G. Rosenblum. Targeting cell-surface CEA with a novel human fusion construct delivers the serine protease granzyme B to solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6317.

Ruthenium(II)-Dithiocarbazates as Anticancer Agents: Synthesis, Solution Behavior, and Mitochondria-Targeted Apoptotic Cell Death.

The reaction of the Ru(PPh3 )3 Cl2 with HL1-3 -OH (-OH stands for the oxime hydroxyl group; HL1 -OH=diacetylmonoxime-S-benzyldithiocarbazonate; HL2 -OH=diacetylmonoxime-S-(4-methyl)benzyldithiocarbazonate; and HL3 -OH=diacetylmonoxime-S-(4-chloro)benzyl-dithiocarbazonate) gives three new ruthenium complexes [RuII (L1-3 -H)(PPh3 )2 Cl] (1-3) (-H stands for imine hydrogen) coordinated with dithiocarbazate imine as the final products. All ruthenium(II) complexes (1-3) have been characterized by elemental (CHNS) analyses, IR, UV-vis, NMR (1 H, 13 C, and 31 P) spectroscopy, HR-ESI-MS spectrometry and also, the structure of 1-2 was further confirmed by single crystal X-ray crystallography. The solution/aqueous stability, hydrophobicity, DNA interactions, and cell viability studies of 1-3 against HeLa, HT-29, and NIH-3T3 cell lines were performed. Cell viability results suggested 3 being the most cytotoxic of the series with IC50 6.9±0.2 μM against HeLa cells. Further, an apoptotic mechanism of cell death was confirmed by cell cycle analysis and Annexin V-FITC/PI double staining techniques. In this regard, the live cell confocal microscopy results revealed that compounds primarily target the mitochondria against HeLa, and HT-29 cell lines. Moreover, these ruthenium complexes elevate the ROS level by inducing mitochondria targeting apoptotic cell death.

Modulation of Mitochondrial Function in Elderly Brain: Involvement of Autophagy and Apoptosis

Mitochondria are crucial cellular organelles that regulate energy production, oxidative balance, and calcium homeostasis, among other things. Aging is a natural, multifactorial, and multi-organic process, in which both pathological and physiological changes occur gradually over time. Autophagy capacity and mitochondrial processes such as mitophagy, biogenesis, and dynamics are associated with aging. These processes are important for maintaining the structural integrity of mitochondria and thus cell life since mitochondrial dysfunction leads to an impairment in energy metabolism and an increased production of reactive oxygen species, both of which may stimulate mechanisms of cellular senescence and apoptotic cell death. Furthermore, in both model organisms and humans, a decline in mitochondrial activity can contribute to age-related disease phenotypes. As people age, mitochondrial damage and malfunction may induce apoptosis, thus explaining the rise in cell death. Many aging processes are believed to be driven by oxidative stress. Taken together, the evidence strongly suggests a relation between mitochondrial function and a wide spectrum of aging processes. Although there is no general consensus among the studies reviewed, it appears that aging reduces mitochondrial biogenesis and dynamics, as well as the mitophagy capacity of the organism involved. This review describes the involvement of autophagy and apoptosis in the modulation of mitochondrial function in the brain.

Apoptosis induction in leukemic cells by L-asparaginase preparation from Bacillus indicus: bench-scale production, purification and therapeutic application.

With the emergence of multiple side effects on the usage of commercial L-asparaginase formulations, keen interest is provoked to investigate new sources of L-asparaginases that possess antileukemic properties with minimal side effects. The present study reports the cost-effective bench-scale production, homogeneity purification and apoptosis induction potential of a new L-asparaginase preparation from Bacillus indicus against human leukemia cells. The enzyme is highly specific toward the natural substrate L-asparagine. The study initiated with the enzyme production using cost-effective substrates in which a 3.28-fold enhancement of enzyme activity was achieved in comparison with an unoptimized medium using the central composite experimental design approach. The scale-up of the process in a 3.7-L batch bioreactor resulted in 16.42 ± 0.17IU/mL of L-asparaginase activity in 24h. The crude extracellular enzyme was purified to homogeneity using anion exchange chromatography followed by gel filtration chromatography. A single band of approximately 35kDa molecular weight was obtained on SDS-PAGE, while native PAGE analysis confirmed it to be a tetramer of four identical subunits. The circular dichroism spectroscopic study revealed the α + β mixed type of secondary structure with 38.7% α-helices and 27.4% β pleated sheets. The antitumor toxicity exhibited on the MOLT-4 leukemia cells by the new L-asparaginase was revealed using the MTT assay and acridine orange/propidium iodide dual staining for live/dead cells. The flow cytometry analysis established the potential of the purified L-asparaginase to induce the apoptotic cell death mechanism in MOLT-4 leukemia cells. Conclusively, the L-asparaginase of Bacillus indicus is a highly promising candidate that can be introduced as a new enzyme therapeutic against various leukemia disorders.

Unveiling Antioxidant and Antiproliferative Effects of Prosopis juliflora Leaves against Human Prostate Cancer LNCaP Cells.

Herbal medications or formulations are regularly recommended by clinicians as a potential therapeutic method for a variety of human ailments, including cancer. Although Prosopis juliflora extracts have shown promise in anticancer activity, the effects on prostate cancer and the accompanying molecular mechanisms of action are still unexplored. This research aims at the antioxidant, antiproliferative, and apoptosis-inducing properties of Prosopis juliflora methanolic leaves extract in human prostate cancer LNCaP cells. The antioxidant ability of the extract was assessed using the DPPH (2, 2-diphenyl-2-picrylhydrazyl) and two additional reducing power tests. Antitumor activity was determined using MTT cell viability tests and LDH cytotoxicity assays. The probable mechanism of apoptotic cell death was further investigated utilizing a caspase-3 activation assay and qRT-PCR mRNA expression investigations of apoptotic-related genes. The results revealed that the methanol extract of Prosopis juliflora leaves contains alkaloids, flavonoids, tannins, glycosides, and phenols, all of which have substantial antioxidant activity. In vitro anticancer tests demonstrated that extract therapy resulted in a dose-dependent reduction in cell viability of LNCaP prostate cancer cells, but normal HaCaT cells showed no cytotoxic effects. Furthermore, plant extract therapy increased caspase-3 activation and mRNA expression of apoptotic-related genes, suggesting that this could be a mechanism for cancer cell growth suppression. The significance of Prosopis juliflora as a source of new antioxidant compounds against prostate cancer was emphasized in the current study. However, more study is needed to demonstrate the efficacy of Prosopis juliflora leaves extract in the treatment of prostate cancer.

Reinventing the Penumbra - the Emerging Clockwork of a Multi-modal Mechanistic Paradigm.

The concept of the ischemic penumbra was originally defined as the area around a necrotic stroke core and seen as the tissue at imminent risk of further damage. Today, the penumbra is generally considered as time-sensitive hypoperfused brain tissue with decreased oxygen and glucose availability, salvageable tissue as treated by intervention, and the potential target for neuroprotection in focal stroke. The original concept entailed electrical failure and potassium release but one short of neuronal cell death and was based on experimental stroke models, later confirmed in clinical imaging studies. However, even though the basic mechanisms have translated well, conferring brain protection, and improving neurological outcome after stroke based on the pathophysiological mechanisms in the penumbra has yet to be achieved.Recent findings shape the modern understanding of the penumbra revealing a plethora of molecular and cellular pathophysiological mechanisms. We now propose a new model of the penumbra, one which we hope will lay the foundation for future translational success. We focus on the availability of glucose, the brain's central source of energy, and bioenergetic failure as core pathophysiological concepts. We discuss the relation of mitochondrial function in different cell types to bioenergetics and apoptotic cell death mechanisms, autophagy, and neuroinflammation, to glucose metabolism in what is a dynamic ischemic penumbra.