Stimulated Cell Death Research Articles

Nimotuzumab and irinotecan synergistically induce ROS-mediated apoptosis by endoplasmic reticulum stress and mitochondrial-mediated pathway in cervical cancer.

Irinotecan (CPT-11), a chemotherapeutic agent used to treat several types of cancer, induces cytotoxic effects on healthy cells. The epidermal growth factor receptor (EGFR) plays a crucial role in various forms of cancer. Nimotuzumab (NmAb), a monoclonal antibody that targets the EGFR, is utilized in some countries to treat malignancies that have an overexpression of EGFR. Yet, there is a lack of literature on the potential anticancer properties of the CPT-11 and NmAb combination on in vitro human cervical cancer cells. This study investigates the apoptosis mode of the CPT-11 and NmAb combination on cervical HeLa cancer cells. The Annexin V/propidium iodide staining examination demonstrated that the combination of CPT-11 and NmAb resulted in a decrease in the number of viable cells and more potent induction of cell apoptosis than the effects of CPT-11 or NmAb alone in HeLa cells. Furthermore, the combined treatment resulted in elevated levels of reactive oxygen species (ROS) and Ca2+ compared to the treatment with CPT-11 or NmAb alone. Cells that were pretreated with N-acetyl-l-cysteine, a substance that scavenges ROS, and then treated with CPT-11, NmAb, or a combination of CPT-11 and NmAb exhibited higher numbers of viable cells compared to those treated with CPT-11 or NmAb alone. The combination of CPT-11 and NmAb resulted in significantly higher caspase-3, -8, and -9 activity levels than CPT-11 or NmAb alone, as measured by flow cytometer assay. The combination of CPT-11 and NmAb in HeLa cells resulted in elevated endoplasmic reticulum stress-, mitochondria-, and caspase-mediated proteins compared to treatment with CPT-11 or NmAb alone. According to these observations, NmAb enhances the effectiveness of CPT-11 in fighting cancer by stimulating cell death in the HeLa cells. Therefore, NmAb has the potential to improve the efficacy of CPT-11 as a future cervical cancer treatment in humans.

ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 μM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells

Ultrasound-mediated cell membrane permeabilization – sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake – a critical regulator of cell death and proliferation – offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene's mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP's clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.

Curcumin Induces Autophagy-mediated Ferroptosis by Targeting the PI3K/AKT/mTOR Signaling Pathway in Gastric Cancer.

As a very common malignancy of the digestive system, the incidence and mortality rates of gastric cancer (GC) are increasing year by year. The critical role of ferroptosis in cancer development has been well-documented. The polyphenol compound curcumin shows prominent anti-tumor effects in multiple cancer types, including GC. However, whether curcumin participates in GC tumorigenesis by regulating ferroptosis remains unknown. Gastric cancer cells AGS and HGC-27 were treated with curcumin (0, 10, and 20 μM). Cell viability and death were evaluated through CCK-8 and LDH release assays. LC3B expression in cells was estimated through immunofluorescence staining. Intracellular ferrous iron (Fe2+), GSH, MDA, and lipid ROS levels were assessed by corresponding assay kits. The cellular levels of autophagy markers (ATG5, ATG7, Beclin 1, and LC3B), ferroptosis markers (ACSL4, SLC7A11, and GPX4), and phosphorylated (p)-PI3K, p-AKT, and p-mTOR were determined through western blotting. Curcumin attenuated cell viability but stimulated cell death in GC cells. Curcumin enhanced autophagy in GC cells, as demonstrated by the increased levels of ATG5, ATG7, Beclin 1, and LC3B. Besides, curcumin upregulated iron, MDA, GSH, and ACSL4 levels while downregulated lipid ROS, SLC7A11, and GPX4 levels, suggesting its stimulation on ferroptosis in GC cells. Curcumin decreased p-PI3K, p-AKT, and p-mTOR levels in cells. Importantly, the ferroptosis inhibitor ferrostatin-1 overturned the impacts of curcumin on GC cell viability, death, and ferroptosis. Curcumin suppresses GC development by inducing autophagy-mediated ferroptosis by inactivating the PI3K/AKT/mTOR signaling.

Abstract 7576: Exemestane and its primary metabolite 17-hydroexemestane inhibit synergically the tumor growth of ER/AR positive breast cancer tumors

Abstract Exemestane, an aromatase inactivator of the third generation, plays a crucial role in breast cancer therapy by targeting the P450 aromatase enzyme and, thus, decreasing estrogen synthesis. Exemestane (Aromasin™) is currently the only steroidal aromatase inactivator widely used in clinical routine treatment of ER+ breast cancer in all phases of the disease in a global perspective. However, the complex mechanisms underlying its therapeutic effects, besides being an aromatase inhibitor, remain incompletely understood. In this study, we employed a combination of human samples and in vitro data to unveil a compelling insight: Exemestane (EXE) and its primary metabolite, 17β-hydroxyexemestane (HEXE), exhibit potent inhibitory effects on tumor growth when present together in patient serum. Our biochemical analysis establishes a critical threshold—20% HEXE metabolite of the total EXE in patient serum—to trigger a tumor growth inhibition exceeding 90%, as evidenced by Ki67 staining. Mechanistically, our data reveals that both HEXE and EXE bind to the Androgen Receptor (AR), triggering a synergistic activation that induces a transcriptional program leading to cell death while diminishing the intracellular signaling activated by the oncogene Ras. Notably, patients with tumors characterized by a minimum of 20% AR+ epithelial cancer cells stand to benefit the most from exemestane and HEXE. Intriguingly, the binding of AR to chromatin in tumors gives rise to a molecular signature capable of distinguishing responsive from non-responsive patients to both EXE and HEXE. Collectively, our findings elucidate a dual therapeutic role for Exemestane in selected patients: it not only restrains estrogen-driven proliferation by estrogen suppression but also stimulates cell death by establishing a specific interactome with the AR at the genomic level. These insights suggest that both EXE and HEXE are necessary to achieve the best therapeutic effects in ER+/AR+ breast cancer tumors. Moreover, our findings suggest that AR-expression and targeting should be investigated further to potentially add novel strategies to our existing algorithms in ER+/AR+ MBC. Citation Format: Gemma Santacana-Font, Darek Kedra, Maria del Carmen García-Macías, Laurens Cornelus-Reitsma, Marianne Lyngra, Torill Sauer, Vessela Kristensen, Antoni Hurtado, Jürgen Geisler. Exemestane and its primary metabolite 17-hydroexemestane inhibit synergically the tumor growth of ER/AR positive breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7576.

Association of Serum Vitamin D with Risk of Breast Carcinoma: An Observational Case-control Study from Western Maharashtra, India

Introduction: Breast carcinoma is one of the most prevalent types of carcinoma and the leading cause of death among all carcinomas. Recently, vitamin D deficiency has been reported as a risk factor for breast carcinoma. Vitamin D, as an anticarcinoma agent, prevents cellular differentiation, stimulates cell death, reduces angiogenesis, tumour progression, and metastasis. Aim: To investigate the relationship between vitamin D deficiency and the risk of breast carcinoma. Materials and Methods: An observational study was conducted at the Armed Forces Medical College, Pune, Maharashtra, India, between November 2018 and October 2020. A total of 57 cases diagnosed with breast carcinoma and 57 healthy controls were analysed. Physical and reproductive health parameters were compared, along with vitamin D status using student’s t-test and Mann-Whitney U test. Logistic regression was used to assess the risk of breast carcinoma. Results: Out of 114 women, 57 were cases and 57 were controls with a mean age of 52 vs 48 years. The mean value of serum vitamin D levels was significantly lower (19.45 vs 27.91 ng/mL, p<0.001) than controls. The percentage of serum Vitamin D deficiency was significantly higher in cases (28 (49.1%) vs 12 (21.1%), p<0.001) compared to controls. Vitamin D concentration <20 ng/mL was significantly associated with a higher risk of breast carcinoma (OR 10.8, 95% CI 3.1-37.6). Multiparity ≥3 was associated with a decreased risk of breast carcinoma (OR 2.250, 95% CI 0.599-8.447) compared to parity ≤2 (OR 3.241, 95% CI 0.916-11.466). In luminal A and triplenegative subtypes, severe vitamin D deficiency (<20 ng/mL) was observed (p=0.045) compared to other subtypes. Conclusion: The present study findings showed that women diagnosed with breast carcinoma had low vitamin D levels, which were linked to an increased risk and prognosis of breast carcinoma. Furthermore, multiparity lowers the risk of breast carcinoma.

Octanoic Acid and Decanoic Acid Inhibit Tunicamycin‐Induced ER Stress in Rat Aortic Smooth Muscle Cells

ER stress is a crucial factor in the progression of vascular cell diseases. Notably, octanoic acid (OA; C8:0) and decanoic acid (DA; C10:0), prominent components of medium‐chain fatty acids (MCFAs), may provide potential health benefits. However, their effects on vascular smooth muscle cells (VSMCs) remain unknown. Given the link between ER stress and vascular cell pathological conditions, the primary goal of this research is to investigate the protective effects of OA and DA against ER stress induction in rat aortic smooth muscle cells (RASMCs). To achieve this objective, RASMCs were pretreated with OA and DA at concentrations of 250 and 500 μM for 24 h. Subsequently, the cells were exposed to 1 μg/mL of tunicamycin, an ER stress inducer, for an additional 24 h. Apoptosis was assessed using DAPI staining, while DCFH2‐DA probe was used to measure ROS levels. Furthermore, the gene expression of ER stress markers, such as CHOP, GRP78, ATF4, and eIF2α, as well as contractile markers like αSMA and MYH11, was assessed using real‐time reverse transcription polymerase chain reaction. Moreover, the αSMA protein level was measured using immunocytochemistry techniques. The study revealed that OA and DA significantly mitigated cell death caused by tunicamycin, decreased ROS production, and inhibited the gene expression of ER stress markers (CHOP, GRP78, and eIF2α). Notably, OA and DA also inhibited the expression of contractile genes (α-SMA and MYH11) and reduced the number of α‐SMA‐positive cells in tunicamycin‐treated RASMCs. These findings indicate that OA and DA offer protection against ER stress–stimulated cell death and ROS generation in VSMCs, thereby supporting their potential therapeutic applications for safeguarding these cells.

M2 Muscarinic Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.

Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.

Effects of progesterone and selective ligands of membrane progesterone receptors in HepG2 cells of human hepatocellular carcinoma

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors of the adiponectin and progestin receptor (mPRs) family. The effect of progesterone on cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of a high level of mRNA and mPRβ protein in HepG2 tumor cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. May be mPRβ has an important function in HepG2 cells. The main goal of the work was to study the function of this protein and the mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. The work studied the effects of progesterone, LS-01 and LS-02 on the proliferation and death of HepG2 cells, as well as the activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 hours of incubation with cells suppressed their viability and stimulated the appearance of phosphatidylserine on the outer surface of membranes, which was detected by binding to V-FITC annexin, but they did not affect DNA fragmentation of cell nuclei. Progesterone significantly reduced the expression of proliferation marker genes and stimulated the expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRβ. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

Effects of Progesterone and Selective Ligands of Membrane Progesterone Receptors in HepG2 Cells of Human Hepatocellular Carcinoma.

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors (mPRs) of adiponectin and progestin receptor families. The effect of progesterone on the cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of high level of mPRβ mRNA and protein in the HepG2 cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. It could imply that mPRβ has an important function in the HepG2 cells. The main goal of the work was to study functions of this protein and mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. Effects of progesterone, LS-01, and LS-02 on proliferation and death of HepG2 cells were studied in this work, as well as activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 h of incubation with the cells suppressed their viability and stimulated appearance of phosphatidylserine on the outer surface of the membranes, which was detected by binding of annexin V, but they did not affect DNA fragmentation of the cell nuclei. Progesterone significantly reduced expression of the proliferation marker genes and stimulated expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on the p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRβ. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

P10.22.B COMBINATION OF ARTS MIMETICS WITH AUTOPHAGY INHIBITORS AS A NOVEL THERAPEUTIC STRATEGY TO OVERCOME APOPTOSIS RESISTANCE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant brain tumour in adults. The current standard of care for GBM patients consists of maximal surgical resection followed by chemotherapy and radiation. Despite such an aggressive treatment approach, the average 5-year survival rate for GBM patients is approximately 5%. One of the main reasons for poor therapy response in the GBM is resistance to programmed cell death, otherwise known as apoptosis. One of the mechanisms behind apoptosis resistance relies on the upregulation of inhibitor of apoptosis proteins (IAPs) which are known to hinder pro-apoptotic proteins and allow cell survival under abnormal conditions such as severe DNA damage. However, there are natural IAP antagonists such as ARTS which are capable of stimulating cell death by promoting the degradation of IAPs. To harness the pro-apoptotic function of IAP antagonists, small peptide mimetics of ARTS have been developed. This project investigates the efficacy of utilizing ARTS mimetics as a novel strategy to overcome apoptosis resistance in the GBM treatment. MATERIAL AND METHODS This study utilizes patient-derived cells with exogenous gain-of-function (GOF) overexpression to introduce full-length ARTS protein as well as pharmacological small peptide mimetics of ARTS to evaluate the response of GBM cells following the promotion of ARTS activity. We also employ a patient-derived orthotopic xenograft mouse model to test novel combinatorial strategies in vivo, where we used a nanoparticle drug delivery system to ensure treatment delivery across the blood-brain barrier (BBB). RESULTS We found that patient-derived GBM cells were highly resistant to ARTS-induced apoptosis. Further molecular investigation unveiled a novel role of ARTS in the upregulation of autophagy as a resistance mechanism. A combination of ARTS GOF or mimetic peptides with autophagy inhibitors mitigated resistance to ARTS-mediated apoptosis and drastically decreased GBM cell proliferation. This combination was further tested in the patient-derived xenograft mouse model, which demonstrated the successful delivery of ARTS mimetic peptides and autophagy inhibitors through the BBB via nanoparticle lipid carriers. Animals treated with a combination of the autophagy inhibitor Spautin-1 and ARTS mimetic peptides demonstrated significantly prolonged survival, confirming the therapeutic potential of such combinatorial approach for GBM treatment. CONCLUSION Altogether, these findings uncover a previously unknown role of ARTS in autophagy regulation and provide evidence that the combination of IAP-antagonist mimetics with autophagy inhibitors can be an effective strategy against highly aggressive GBM brain tumours. This work was supported by the Health Sciences Centre Foundation (HSCF).

Suppressive Effect of Chemically Induced Hypoxia on Glioblastoma Cell Proliferation.

Glioblastoma is a tumor characterized by pronounced hypoxia. Hypoxia produces diverse effects on tumor cells, and the results of experimental studies available so far are contradictory. In vitro hypoxia can be modeled in two ways: by reducing the level of atmospheric oxygen (physically induced hypoxia) or by using hypoxia-inducing chemicals such as cobalt chloride (II) (CoCl2) (chemically induced hypoxia). In the present work, we analyzed the effect of CoCl2 on the viability, proliferation, and apoptosis of cells of three glioblastoma cell lines: 1321N1, T98g, and U373 MG. It was shown that CoCl2 induced a dose-dependent decrease in cell viability and proliferation, and at high concentrations (200 and 400 μM) stimulated cell death. CoCl2 had no effect on the cytotoxic activity of doxorubicin in two cell lines T98g and U373 MG, and enhanced the effect of the chemotherapeutic agent on the 1321N1 cell line, though no synergistic cytotoxic effect of the two agents was observed.

Molecular basis for nuclear accumulation and targeting of the inhibitor of apoptosis BIRC2.

The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2BIR2 and BIRC2BIR3, to the histone H3 tail and report the structure of the BIRC2BIR3-H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4+ T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2.

Wogonin, as a potent anticancer compound: From chemistry to cellular interactions.

Chinese native medicine Scutellaria baicalensis Georgi, also referred to as Chinese skullcap or Huang-Qin, is frequently used to treat cancer, viral infections, and seizures. This plant's abundance of flavones (wogonoside) and their related aglycones (wogonin) is responsible for many of its pharmacologic effects. A significant ingredient in S. baicalensis that has been the subject of the most research is wogonin. Numerous preclinical investigations revealed that wogonin suppresses tumor growth by cell cycle arrest, stimulating cell death and preventing metastasis. This review focuses on a complete overview of published reports that suggest chemopreventive action of wogonin and the mechanistic insights behind these neoplastic activities. It also emphasizes the synergistic improvements made by wogonin in chemoprevention. The factual data in this mini-review stimulate additional research on chemistry and toxicological profile of wogonin to confirm its safety issues. This review will encourage researchers to generalize the merits of wogonin to be used as potential compound for cancer treatment.

Targeting a novel apoptotic pathway in human disease.

Apoptotic pathways have always been regarded as a key-player in preserving tissue and organ homeostasis. Excessive activation or resistance to activation of cell death signaling may indeed be responsible for several mechanisms of disease, including malignancy and chronic degenerative diseases. Therefore, targeting apoptotic factors gained more and more attention in the scientific community and novel strategies emerged aimed at selectively blocking or stimulating cell death signaling. This is also the case for the TMEM219 death receptor, which is activated by a circulating ligand, the Insulin-like growth factor binding protein 3 (IGFBP3) and induces a caspase-8-dependent apoptosis of the target cells. Interestingly, stimulation of the IGFBP3/TMEM219 axis exerts an anti-proliferative effect, while blockade of the TMEM219 deleterious signal protects TMEM219-expressing cells of the endocrine pancreas, lung, and intestine from damage and death. Here, we summarize the most updated reports on the role of the IGFBP3/TMEM219 apoptotic axis in disease conditions, including intestinal disorders and diabetes, and we describe the advancements in designing and testing novel TMEM219-based targeting approaches in emerging potential clinical applications.

EDNA-stimulated cell dispersion from Caulobacter crescentus biofilms upon oxygen limitation is dependent on a toxin-antitoxin system.

In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin-antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment.

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro.

Persistence is a bet-hedging strategy in bacterial populations that increases antibiotic tolerance and leads to the establishment of latent infections. In this study, we demonstrated that a synthetic non-toxic taxane-based reversal agent (tRA), developed as an inhibitor of ABC transporter systems in mammalian cancer cells, enhanced antibiotic killing of persister populations from different pathogens, including Burkholderia, Pseudomonas, Francisella, and Yersinia. Acting as an inhibitor of bacterial efflux at 100 nM, tRA99020 enhanced antibiotic efficiency and suppressed the production of natural products of Burkholderia species polyketide synthase (PKS) function. We demonstrate that the metabolites produced by PKS in response to stress by different antibiotics act as inhibitors of mammalian histone deacetylase activity and stimulate cell death. Applying a single-molecule fluorescence in situ hybridization (smFISH) assay, we analyzed on a single-cell level the activation profiles of the persistence regulating pks gene in Burkholderia thailandensis treated with tRA99020 and antibiotics. We posit that a multi-pronged approach encompassing antibiotic therapies and inhibition of efflux systems and fatty acid catabolism will be required for efficient eradication of persistent bacterial populations.

Atractylenolide I inhibited the development of malignant colorectal cancer cells and enhanced oxaliplatin sensitivity through the PDK1-FoxO1 axis.

Colorectal cancer (CRC) is a type of ordinary malignancy of the gastrointestinal tract. Atractylenolide I (AT-I) has been shown to inhibit the process of CRC. However, the specific mechanism by which AT-I inhibits CRC is not yet well understood. Cell Counting Kit-8 and colony formation assays were conducted to examine cell proliferation. The cell apoptosis was detected by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL). Cell invasion and migration were evaluated by wound-healing and Transwell assay. The angiogenesis capabilities of the cells were examined by tube formation experiments. Western blot was conducted to examine the apoptosis and angiogenesis-associated proteins, pyruvate dehydrogenase kinase 1 (PDK1), and Forkhead box protein O1 (FoxO1) expression. We found that AT-I inhibited the proliferative, migratory and invasive abilities of Human colorectal cancer cell line HCT116 cells but stimulated cell death by promoting cell apoptosis via the PDK1/FoxO1 axis. In addition, the upregulation of PDK1 decreased the inhibitory effect of AT-I on HCT116 angiogenesis, and AT-I increased oxaliplatin sensitivity via the PDK1/FoxO1 axis. Collectively, AT-I inhibited the malignant development of CRC cells and increased oxaliplatin sensitivity by decreasing PDK1 and inhibiting FoxO1 phosphorylation. Thus, AT-I has protective potential and could be a promising agent for CRC treatment.

Lithium Enhances Autophagy and Cell Death in Skin Melanoma: An Ultrastructural and Immunohistochemical Study.

Lithium is an inhibitor of glycogen synthase kinase 3 beta, which is traditionally used in the treatment of bipolar disorders and has antitumor effects. The aim of the current study was to determine if lithium salt causes autophagy and apoptosis in skin melanoma cells to enhance cell death. Light microscopy, transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to study the mechanism of action of lithium carbonate in B16 melanoma cells in vivo. Proliferating cell nuclear antigen immunofluorescence assay revealed that the proliferation of B16 melanoma cells was suppressed by lithium treatment for 7 days. Electron microscopy demonstrated a significant increase in the number of autophagic vacuoles in lithium-treated cells relative to control. In addition, levels of autophagy markers LC3 beta and LAMP1 found in lithium-treated tumor xenografts were higher than levels of these markers in the control tumors. Lithium induced caspase-3 expression and apoptotic cell death in tumor cells. Thus, lithium carbonate is the compound that inhibits cell proliferation and stimulates cell death in melanoma cells through induction of autophagy and apoptosis. Stimulation of autophagy by lithium could contribute to the development of autophagic cell death in tumor cells.

Anticancer activities of Brachydin C in human prostate tumor cells (DU145) grown in 2D and 3D models: Stimulation of cell death and downregulation of metalloproteinases in spheroids.

Brachydin C (BrC) has demonstrated in vitro cytotoxic and antiproliferative effects in prostate cancer cells. In the present study, we compare the anticancer effects of BrC in DU145 cells grown in common bidimensional cultures (2D) and multicellular tumor spheroids (MCTS), often denominated 3D in vitro models, that can better mimic the microenvironment of tissues. BrC IC50 values obtained in the resazurin assay after 24 h of treatment were 47.31 μM (2D) and 229.8μM (3D) and these cytotoxic effects were time-dependent only in 3D. BrC (5.0-60 μM) interfered with the growth of MCTS and reduced cell viability after 11 days of treatment, a result that is not attributable to oxidative stress evaluated using the CM-H2 DCFDA probe. BrC (6.0μM) impaired horizontal (wound-healing) and vertical cell migration and invasion (transwell assay) in 2D and BrC (5.0-60 μM) in 3D (ECM Gel®). BrC modulated the expression of genes BIRC5, TNF-α, CASP3, NKX3.1, MMP9, MMP11, CDH1, and ITGAM and downregulated proteins CASP7, BAX, and TNF-α in Western blotting analysis. In conclusion, BrC stimulated cell death and decreased epithelial-mesenchymal transition. Furthermore, DU145 MCTS displayed higher resistance to BrC-induced cell death than 2D cultures, a difference that should be considered in future approaches in prostatic cancer studies.