Anti-apoptotic Molecules Research Articles

P0182 The systemic administration of the anti-MCL1, S63845, reduces inflammation and fibrosis in a murine model of DSS-Chronic Colitis

Abstract Background A recent RNA sequencing analyses performed in ileal resections from complicated Crohn´s disease patients revealed the up-regulation of the anti-apoptotic molecule, MCL1 in fibrotic lesions. Immunohistochemical studies localized MCL1 expression in several cell types, including fibroblasts and leukocytes1. We analyse here the role played by MCL1 on inflammation and fibrosis in a murine model of chronic colitis. Methods C57Bl/6 mice (20–25 g) were administered intraperitoneally with Vehicle or S63845 (25 mg/kg), five days a week during the two DSS cycles (a cycle consists in 7 days drinking water with 2% DSS, followed by ten days drinking water). Body weight and Disease Activity Index (DAI) were monitored daily, and mice were sacrificed 34 days after the beginning of the experiment. A sample from the colon was paraffin embedded for both histological analysis (epithelial damage was quantified by the Obermeier score in H&E-stained slides and collagen deposition was quantified by the QuPath software in Syrius Red- stained slides) and immunohistochemistry (CD68, CD3, MPO, TUNEL and Cleaved-caspase 3). Other sample was used for RT-PCR studies. Unless otherwise mentioned, data are expressed as mean±SEM. Results DSS-Vh treated mice exhibited a reduction in body weight (16.9±1.0) and increase in DAI score (2.5±0.6) which reached a statistical significance (P<0.001&, P<0.01&, respectively) at day 11, compared with body weight and DAI at day 0 (19.4±0.4 and 0, respectively). In S63845-DSS treated mice, no significant changes in body weight and DAI were detected all along the experimental period when compared with the respective day 0. The administration of S63845, compared with Vh in DSS-treated mice induced: a) a reduction in epithelial damage (1.3±0.8 vs 1.7±0.4) and a significant diminution in the thickness of the submucosa (19.6±1.7 vs 42.4±3.2, respectively, P =0.0008*) and collagen deposition in the same area (median an IQ range: 54.2 (55.9-68.5) vs 61.9 (55.9-68.5) respectively, P = 0.051#). b) a lower immunostaining for both MPO and CD3; c) a similar immunostaining for CD68, TUNEL and Cleaved-caspase 3 which was very scarce for the last two and d) a lower mRNA expression of inflammatory cytokines (il1β, il6 and chi3l1), chemokines (cxcl1 and cxcl5) and profibrotic markers (col1a1, col3a1, col4a1, and tgfβ1). *t-test; #Mann-Whitnney; &Repeated measures ANOVA Conclusion The systemic administration of the MCL1 inhibitor, S63845 prevented the infiltration of both neutrophils and CD3 lymphocytes and failed to induce apoptosis in the affected intestine while it reduced fibrosis in a murine model of DSS-chronic colitis

Integrative genomic analyses combined with molecular dynamics simulations reveal the impact of deleterious mutations of Bcl-2 gene on the apoptotic machinery and implications in carcinogenesis.

Unlike other diseases, cancer is not just a genome disease but should broadly be viewed as a disease of the cellular machinery. Therefore, integrative multifaceted approaches are crucial to understanding the complex nature of cancer biology. Bcl-2 (B-cell lymphoma 2), encoded by the human BCL-2 gene, is an anti-apoptotic molecule that plays a key role in apoptosis and genetic variation of Bcl-2 proteins and is vital in disrupting the apoptotic machinery. Single nucleotide polymorphisms (SNPs) are considered viable diagnostic and therapeutic biomarkers for various cancers. Therefore, this study explores the association between SNPs in Bcl-2 and the structural, functional, protein-protein interactions (PPIs), drug binding and dynamic characteristics. Comprehensive cross-validated bioinformatics tools and molecular dynamics (MD) simulations. Multiple sequence, genetic, structural and disease phenotype analyses were applied in this study. Analysis revealed that out of 130 mutations, approximately 8.5% of these mutations were classified as pathogenic. Furthermore, two particular variants, namely, Bcl-2G101V and Bcl-2F104L, were found to be the most deleterious across all analyses. Following 500ns, MD simulations showed that these mutations caused a significant distortion in the protein conformational, protein-protein interactions (PPIs), and drug binding landscape compared to Bcl-2WT. Despite being a predictive study, the findings presented in this report would offer a perspective insight for further experimental investigation, rational drug design, and cancer gene therapy.

A new perspective on apoptosis: Its impact on meat and organoleptic quality in different animals.

Apoptosis serves as the initial phase in the conversion of muscle to meat, driving key biochemical and morphological changes in the postmortem muscle. To effectively improve and control meat quality across different animal species, it is important to gather more information on the mechanisms by which apoptotic potential, mediated through the interaction of apoptosis-related molecules, influences meat quality variations. The apoptotic potential, determined by the balance between apoptotic and anti-apoptotic molecules, such as Ca2+, cytochrome c, caspases, and heat shock proteins, varies among different species. A moderate to rapid apoptotic rate can improve textural properties in species with a higher proportion of type I fibers, such as cattle. In contrast, in species with a predominance of type IIB fibers, such as pork and poultry, rapid apoptosis can lead to undesirable quality traits. Therefore, understanding these species-specific apoptotic responses is critical for improving and maintaining meat quality across various species.

The Suppression of Signal Transducer and Activator of Transcription-3 in A549human Lung Carcinoma Cells Induced by Marine Sponge Callyspongia aerizusa.

Lung cancer is recognized as a highly lethal disease, demanding swift and accurate solutions. Previous analysis showed the cytotoxic impact of Callyspongia aerizusa (C. aerizusa) extract containing ergost-22-en-3-one and ergost-7-en3-ol against A549 lung cancer cells, with an IC50 value of 9.38μg/mL. However, the extract did not have cytotoxicity towards Het-1A esophagus epithelial cells. Several reviews also validated the upregulation of pro-apoptotic molecules and the inhibition of anti-apoptotic molecules linked to the caspase-dependent signaling pathway. The objective of this research was to extend the understanding of the effects of C. aerizusa extract on A549 lung carcinoma, examining its influence on various signaling pathways, malignancy, migration, and invasion. PCR was used to measure mRNA expression, targeting PTEN, Akt, mTOR, STAT-3, IL-6, VEGF, and HIF1α. Additionally, Western Blot analysis was adopted to assess PTEN, p-Akt, Akt, p-mTOR, and p-STAT-3 protein expressions. Wound healing and invasion assays were performed to measure the migration and invasion capabilities of A549 cells post-treatment with C. aerizusa extract. The mRNA expression analysis showed an increase in Akt and m-TOR but a decrease in PTEN and STAT-3 after 24hours of treatment with C. aerizusa extract. At the protein level, there was a downregulation of p-Akt, Akt, p-mTOR, and p-STAT-3, while PTEN increased during 24-hour treatment. Wound healing and invasion assay results showed a weakened ability of A549 cells after a 24-hour treatment with C. aerizusa extract. Moreover, IL-6 and HIF-1α mRNA expression levels decreased during 24hours, while VEGF mRNA had a slight decrease compared to untreated cells. In conclusion, the ergosteroids present in marine sponge C. aerizusa extract signified a remarkable reduction in malignancy, migration, and invasion capabilities in A549 lung carcinoma cells. These results suggested their promising candidacy for future anti-angiogenesis in anticancer therapy.

Chiral Amino Acids Mediate Mitochondria-Dependent Apoptosis of Human Proximal Tubular Epithelial Cells Under Oxidative Stress.

Amino acids are the basic structural units of life, and their intake levels affect disease and health. In the case of renal disease, alterations in amino acid metabolism can be used not only as a clinical indicator of renal disease but also as a therapeutic strategy. However, the biological roles and molecular mechanisms of natural chiral amino acids in human proximal tubular epithelial cells (HK-2) remain unclear. In this study, cell viability assays revealed that chiral acidic amino acids (Glu and Asp) and aromatic amino acids (Trp and Phe) inhibited cell growth. The molecular mechanisms indicated that cell growth was closely related to ROS levels. Specifically, chiral Glu, Asp, Trp, and Phe induced oxidative stress and mitochondria-dependent apoptosis in HK-2 cells. This was manifested by elevated levels of intracellular ROS, 8-OHdG, and MDA, increased activities of antioxidant enzymes CAT, SOD, and GPx, decreased mitochondrial membrane potential, increased cytoplasmic Ca2+ concentration, and cell acidification. The expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Cyt-C, and Bax were increased, and the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, L-Glu, D-Asp, L-Trp, and D-Phe exhibited a more pronounced inhibition of cell growth and elicited more substantial alterations in gene expression compared to the other configurations.

Disruption of survivin protein expression by treatment with YM155 accelerates the resolution of neutrophilic inflammation.

Prolonged survival of neutrophils is essential for determining the progression and severity of inflammatory and immune-mediated disorders, including gouty arthritis. Survivin, an anti-apoptotic molecule, has been described as a regulator of cell survival. This study aims to examine the effects of YM155 treatment, a survivin selective suppressant, in maintaining neutrophil survival in vitro and in vivo experimental settings of neutrophilic inflammation. BALB/c mice were injected with monosodium urate (MSU) crystals and treated with YM155 (intra-articularly) at the peak of inflammatory response. Leukocyte recruitment, apoptosis neutrophil and efferocytosis were determined by knee joint wash cell morphology counting and flow cytometry. Resolution interval (Ri) was quantified by neutrophil infiltration, monitoring the amplitude and duration of the inflammation. Cytokine production was measured by ELISA. Mechanical hypernociception was assessed using an electronic von Frey aesthesiometer. Efferocytosis was evaluated in zymosan-induced neutrophilic peritonitis. Survivin and cleaved caspase-3 expression was determined in human neutrophils by flow cytometry. Survivin was expressed in neutrophils during MSU-induced gout, and the treatment with YM155 reduced survivin expression and shortened Ri from ∼8 h observed in vehicle-treated mice to ∼5.5 h, effect accompanied by increased neutrophil apoptosis and efferocytosis, both crucial for the inflammation resolution. Reduced IL-1β and CXCL1 levels were also observed in periarticular tissue. YM155 reduced histopathological score and hypernociceptive response. In human neutrophils, lipopolysaccharide (LPS) increased survivin expression, whereas survivin inhibition with YM155 induced neutrophil apoptosis, with activation of caspase-3. Survivin may be a promising therapeutic target to control neutrophilic inflammation.

Abstract B036: WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but resistance limits its clinical effectiveness. Therefore, understanding immune resistance mechanisms and identifying predictive markers are essential. Our transcriptomic analysis of PD-1 blockade-treated patients and non-responsive tumor models identifies WEE1 as a key resistance factor, promoting cancer stem cell (CSC)-like traits and immune resistance phenotypes including low T cell infiltrated into tumor and anti-apoptotic properties to cytotoxic T cells (CTLs). WEE1 drives AKT hyperactivation, enhancing expression of resistance factors such as CYCLIN A which contributes to CSC-like properties, and the anti-apoptotic molecule MCL-1, while also reducing T cell infiltration via CXCL10 downregulation. This signaling axis is conserved across various cancers. Notably, WEE1 inhibition sensitizes tumors to ICB by disrupting these refractory properties and reinvigorating antitumor immunity. These findings underscore the novel role of WEE1 in driving immune resistance and CSC-like characteristics, supporting the use of WEE1 inhibitors as a strategy to overcome resistance to anti-PD-1 therapy. Citation Format: Hyo-Jung Lee, Suyeon Kim, Eunho Cho, Tae Woo Kim. WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B036.

Photoprotective Effect of Ultrasonic-Assisted Ethanol Extract from Sargassum horneri on UVB-Exposed HaCaT Keratinocytes.

The present study investigated the photoprotective effect of the ultrasonic-assisted ethanol extract (USHE) from Sargassum horneri, a brown seaweed containing fucosterol (6.22 ± 0.06 mg/g), sulfoquinovosyl glycerolipids (C23H43O11S, C25H45O11S, C25H47O11S, C27H49O11S), and polyphenols, against oxidative damage in ultraviolet B (UVB)-exposed HaCaT keratinocytes. USHE indicated antioxidant activity in ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. After screening experiments, 15.6, 31.3, and 62.5 µg/mL concentrations of USHE and ascorbic acid as positive control were selected to be used throughout the investigation. USHE increased cell viability by markedly reducing the production of intracellular reactive oxygen species (ROS) in UVB-exposed HaCaT keratinocytes. Additionally, USHE reduced the apoptosis and sub-G1 cell population and increased the mitochondrial membrane potential. Moreover, USHE modulated the protein expression levels of anti-apoptotic molecules (Bcl-xL, Bcl-2, and PARP) and pro-apoptotic molecules (Bax, cleaved caspase-3, p53, cleaved PARP, and cytochrome C). This modulation accorded with the upregulation of cytosolic heme oxygenase (HO)-1, NAD(P)H quinone oxidoreductase 1 (NQO 1), and nuclear factor erythroid-2-related factor 2 (Nrf2), collectively known as components of the antioxidant system. These findings suggest that USHE has a photoprotective effect on UVB-exposed HaCaT keratinocytes and can be utilized to develop cosmeceuticals for UVB protection.

Synthesis of nanohybrid consisting of taurine derived carbon dots and nanoceria for anticancer applications

Here, we first synthesized carbon dots (CDs) from taurine and then a nanohybrid with ceria (CeO2) nanoparticles using thermal decomposition method for checking their antineoplastic efficacy against human triple negative mammary carcinoma cells, MDA-MB-231. The in vitro study demonstrated significant dose-dependent antineoplastic activity of the nanohybrids within a range of concentration from 10 to 50 μg/mL after 48 h of treatment. The cellular morphology analysis clearly depicted substantial amount of cell death which seems to stem from increased intracellular reactive oxygen species (ROS) production. However, the maximum anticancer activity of the nanohybrid as compared to bare CDs and CeO2 is supposed to be due to the combined anticancer effect of both CDs and CeO2 (a well-established antitumor agent). Further we have performed molecular docking study to reveal the anticancer mechanism of CDs which exhibited high binding capacity towards several proapoptotic and antiapoptotic protein molecules. The binding affinity values were found to be – 8.7 kcal/mol, – 7.9 kcal/mol, – 9.6 kcal/mol, – 9.5 kcal/mol, – 12 kcal/mol and – 11.1 kcal/mol for BAD, BCl-2, p53, Caspase-8, Caspase-9 and Caspase-3 respectively. Taken together, our synthesized CDs-CeO2 nanohybrid could be thought as a potential anticarcinogenic option in the field of breast cancer therapeutics.

FHOD3 Promotes the Progression of Lung Cancer by Regulating the Caspase-3-Mediated Signaling Pathway.

Lung cancer causes hundreds of thousands of deaths each year worldwide. FHOD3 was reported to accelerate the progression of brain cancer. However, its role in lung cancer is not clear. This study aimed to investigate the role of FHOD3 in lung cancer. The clinical significance of FHOD3 in lung cancer was analyzed based on the data from the TCGA database. The expression level of FHOD3 was detected by qPCR technology. Cell proliferation was detected by CCK-8 assay, and cell invasion was detected by transwell assay. The activity of caspase-3 was determined by the ELISA method, cell apoptosis was iden-tified by TUNEL assay, and protein expression was measured by western blotting technology. Based on the TCGA data, FHOD3 was overexpressed in tumor tissues compared to the normal tissues. Patients with higher FHOD3 expression exhibited a worse survival rate. The expression levels of FHOD3 in lung cancer cell lines were much higher than that in normal cells. When FHOD3 was knocked down, the ability of cell proliferation and invasion was sig-nificantly inhibited. Cell apoptosis rate was increased reversely. The activity of caspase-3 was increased significantly. In addition, the expression level of cleaved caspase-3 was increased. The expression levels of Bax, caspase-8, and ICAD were also increased significantly. However, the expression of antiapoptotic molecule Bcl-2 was decreased reversely. This suggests that the caspase-3-mediated apoptosis signaling pathway was activated by FHOD3 knockdown. FHOD3 was overexpressed and negatively associated with survival rate in lung cancer patients. FHOD3 regulates cell proliferation, invasion, and apoptosis through the caspase-3-mediated signaling pathway. This study indicates that FHOD3 is an important gene contributing to the progression of lung cancer and might be a new drug target for the therapy of lung cancer.

Protective effect of fructooligosaccharide against oxidative stress and apoptosis induced by Aeromonas hydrophila in Megalobrama amblycephala

This research aimed to investigate the effects of dietary fructooligosaccharides (FOS) on attenuating the Aeromonas hydrophila (A. hydrophila)-induced oxidative stress and apoptosis in blunt snout bream Megalobrama amblycephala. Fish were divided into three groups as follows: C1 (Control), T1 (A. hydrophila), and T2 (A. hydrophila + 4 g/kg FOS). The results showed that the activities of antioxidant enzymes increased, the liver morphology had disorderly arrangement, and extensive cell necrosis occurred because of A. hydrophila-infection. While the dietary FOS improved the above-mentioned liver damage. Additionaly, FOS elevated mRNA levels of pro-apoptotic molecules, including caspase-8 and 9, and down-regulated mRNA levels of the anti-apoptotic molecule Bcl-2, which is triggered by A. hydrophila-infection. The transcriptome analysis showed that the oxidative stress-related DEGs pathways were activated in intestine of blunt snout bream by A. hydrophila-infection. The FOS-added group led to the enrichment of more pathways to health. Further WGCNA co-expression network analysis showed that the screened single genes were clustered into 49 modules. The two modules with the highest association to the five traits (10 hub genes) were chosen to build the network by combining the physiological and biochemical characteristic. In summary, this research offers a foundation for the exploring of A. hydrophila-restoration genes in dietary FOS, and also lays a theoretical foundation for aquaculture in the future.

Study of TRAIL and SAHA Co-Treatment on Leukemia K562 Cell Line.

TRAIL (Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand) is an attractive agent being considered a potential cancer treatment. It attaches to its death receptors, leading many cancer cells to apoptosis. However, some malignancies indicate substantial resistance to TRAIL, challenging anticancer scientists. Herein, combination therapy with TRAIL plus SAHA (Suberoyl Anilide Hydroxamic Acid) was conducted to evaluate the capability of SAHA to overcome TRAIL resistance in the leukemia K562 cell line. First, the IC50 for SAHA was calculated (2 µM) at 12, 24, 48, and 72 h of treatment using MTT assay. Second, the K562 cells were treated with concentrations of 50 and 100 nM of TRAIL and 2 μM of SAHA separately and together for 24, 48, and 72 h and the survival of these cells was evaluated by Flowcytometry following the annexin-V and PI staining. To demonstrate the non-toxicity of the combined treatment for normal cells, the HEK-293 cell line was treated with the TRAIL 100 nM and SAHA 2 μM combined and separated at the same periods. In the end, by performing real-time PCR, the amount of candidate genes' expression implicated in TRAIL resistance, and the levels of BCR-ABL expression was measured. The drug dosages were not toxic to normal cells. SAHA plus TRAIL strongly triggered apoptosis in K562 cells after 24, 48, and 72 h of exposure. Furthermore, it was shown that DR4, DR5, and CHOP expressions were enhanced, and PI3K, Akt, ERK, STAT3, c-FLIPL, NF-κB, and BCR-ABL expressions were decreased by SAHA in K562 cells. Our study indicated that SAHA combined with TRAIL can increase the sensitivity of K562 leukemic cells to TRAIL by suppressing intracellular anti-apoptotic molecules and augmenting the expressions of DR4/DR5 and CHOP.

The Effects of Severe Symptoms of SARS-CoV-2 Infections on the Anti/Proapoptotic Molecules: A 6-Month Cohort Study.

The plausible effects of SARS-CoV-2 infection on the expression of anti/proapoptotic molecules have been suspected. This cohort study examined the expression of p53, Bcl-2, Bid, Bak, and Bax molecules, the genes associated with induction or inhibition of apoptosis, in the SARS-CoV-2-infected patients with severe and mild symptoms in an Iranian population. In this 6-month cohort study, the expression of p53, Bcl-2, Bid, Bak, and Bax molecules was evaluated at onset of diagnosis, 24 h after symptom onset, and 6 months later in the nasopharyngeal cells of SARS-CoV-2-infected hospitalized patients and outpatients in comparison with healthy controls using the real-time PCR technique. At the onset of the study, the relative expression of p53, Bcl-2, Bid, Bak, and Bax significantly increased in the SARS-CoV-2-infected hospitalized patients and decreased after 6 months. The healthy controls showed potential positive correlations among the molecules, but the patients did not show these correlations. Since SARS-CoV-2 needs host cell survival, it appears that the virus induces the expression of Bcl-2 as an antiapoptotic molecule, and the host cells upregulate the proapoptotic molecules to neutralize the effects. Dysregulation of correlation expression of the molecules among the patients proved that SARS-CoV-2 affects the expression of the molecules involved in apoptosis. SARS-CoV-2 could be considered an important factor that regulates the expression of several molecules participating in cancer pathogenesis.

Flow cytometric expression of Bcl-2, Mcl-1, and their ratios correlates with primary and secondary cytogenetic changes and their combinations in multiple myeloma.

Response to BH3 mimetics in multiple myeloma (MM) correlates with CCND1-rearrangement or expression of anti-apoptotic molecules, particularly Bcl-2 and Mcl-1. Our study investigates the relationship between cytogenetic abnormalities (CGAs) and intracellular Bcl-2 and Mcl-1 expression in myeloma plasma cells(MPCs) using flow cytometry (FCM). We measured median fluorescence intensity (MFI) of Bcl-2 and Mcl-1 in 163 bone marrow samples (143 MM, 20 controls) across various cell types. Both Bcl-2MFI and Mcl-1MFI were significantly higher in MPCs compared to other cells, with Bcl-2 MFI exceeding Mcl-1 MFI in MPCs. Bcl-2 expression peaked in CCND1-rearranged cases, while Mcl-1 expression was highest in cases with 1q21 gain/amplification. Notably, 65-74% of cases with other CGAs exhibited moderate to strong Bcl-2 or Mcl-1 expression, indicating potential utility of BH3 mimetics in this group, while 25% showed dim to absent expression of one or both markers, suggesting potential futility in these patients. Our study highlights FCM's potential for rapid Bcl-2 and Mcl-1 quantification, surpassing traditional methods. We propose that direct measurement of Bcl-2 and Mcl-1 expression in PCs by FCM, combined with cytogenetic characterization, could improve therapeutic decision-making regarding the use of BH3 mimetics in MM, potentially enhancing outcomes and overcoming resistance.

Vitamin K3 derivative inhibits androgen receptor signaling in targeting aggressive prostate cancer cells.

Prostate cancer (PCa) is the second critical cause of cancer-related deaths, with African Americans dying at higher rates in the U.S. The main reasons for the higher mortality rate are ethnic differences and lack of understanding of prostate cancer biology and affordable treatments, as well as the financial burden of African American men to obtain the most effective and safe treatments. The effect of micronutrients, including Vitamin K, on various cancer cell lines has been widely studied, but the potential anticancer effect of VK3-OCH3, an analog of vitamin K3 (Menadione), on African American prostate cancer has not been evaluated. In this study, we compared the anticancer effect of VK3-OCH3 on targeting African American derived PCa cell lines namely RC77-T and MDA-PCa-2b. Our results show that VK3-OCH3 significantly inhibits the proliferation of both RC77-T and MDA-PCa-2b African American PCa cells and promotes apoptosis, and the underlying mechanism of cell death appears to be similar in both the cell lines. Notably, VK3-OCH3 inhibits colony-forming ability and induces apoptosis by blocking the cell cycle at G0 in African American PCa cells. VK3-OCH3 also acts as an anti-metastatic agent by inhibiting the migration ability of the metastatic properties of African American PCa cells. The cell death of African American PCa cells mediated by VK3-OCH3 is associated with the production of free radicals, such as intracellular and mitochondrial reactive oxygen species (ROS). Interestingly, antioxidants such as N-Acetylcysteine (NAC) and Glutathione (GSH) effectively negated the oxidative stress induced by VK3-OCH3 on PCa cell lines derived from African American patients. Of note, VK3-OCH3 reduces androgen receptor and prostate-specific antigen expression in these PCa cells. Furthermore, molecular dynamic studies reiterated that VK3-OCH3 strongly binds to the androgen receptor, suggesting that the androgen receptor is the potential molecular target of VK3-OCH3. In addition, Western blot analysis showed that VK3-OCH3 reduces the expression of androgen receptor, TRX2, and anti-apoptotic signaling molecules such as Bcl-2 and TCTP in the MDA-PCa-2b metastatic PCa cellular model. In conclusion, our results suggested that VK3-OCH3 is a promising anticancer agent that could potentially reduce the mortality rates of African American PCa patients, warranting further preclinical and translational studies.

The caspase-inhibitor Emricasan efficiently counteracts cisplatin- and neomycin-induced cytotoxicity in cochlear cells

Cisplatin is a chemotherapeutic agent widely used to treat solid tumors. However, it can also be highly ototoxic, resulting in high-frequency hearing loss. Cisplatin causes degeneration of hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear, which are essential components of the hearing process and cannot be regenerated in mammals. As the affected cells primarily die by apoptosis, we tested several anti-apoptotic small molecules to protect these cells from drug-induced toxicity. We found that the general caspase inhibitor Emricasan could significantly counteract the toxic effects of cisplatin in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, phoenix auditory cells, and primary SGNs. Importantly, the anti-cytotoxic effect in neuronal cells was even more pronounced than the effect of sodium thiosulfate (STS), which is currently the only approved prevention option for cisplatin-induced ototoxicity. Finally, we tested the protective effect of Emricasan treatment in the context of another ototoxic drug, i.e., the aminoglycoside antibiotic neomycin, and again found a significant increase in cell viability when the cultures were co-treated with Emricasan. These results suggest a promising strategy to prevent ototoxicity in patients by temporarily blocking the apoptotic pathway when applying cisplatin or aminoglycoside antibiotics.Key messagesAnti-apoptotic small molecules can reduce cisplatin-induced toxicity.Emricasan can effectively exert its anti-apoptotic effect on cochlear cells.Strong protection from cisplatin- and neomycin-induced cytotoxicity with Emricasan.Sodium thiosulfate and Emricasan provide similar protective effects to cisplatin-treated cells.Emricasan is more potent than sodium thiosulfate in reducing neomycin-induced cytotoxicity.

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Protective effect of clusterin against interleukin-1β-induced apoptosis and inflammation in human knee osteoarthritis chondrocytes.

Chondrocyte apoptosis is recognized as one of the pathological features involved in cartilage degeneration driving the onset and progression of knee osteoarthritis (OA). This study aimed to determine the molecular mechanism underlying the effect of clusterin (CLU), anti-apoptotic molecule, in human knee OA chondrocytes. Primary knee OA chondrocytes were isolated from the cartilage of knee OA patients and divided into five groups: (1) the cells treated with interleukin (IL)-1β, (2) CLU alone, (3) a combination of IL-1β and CLU, (4) LY294002 (PI3K inhibitor) along with IL-1β and CLU, and (5) the untreated cells. Production of apoptotic, inflammatory, anabolic, and catabolic mediators in knee OA chondrocytes was determined after treatment for 24 h. Our in vitro study uncovered that CLU significantly suppressed the production of inflammatory mediators [nitric oxide (NO), IL6, and tumor necrosis factor (TNF)-α] and apoptotic molecule (caspase-3, CASP3). CLU significantly upregulated messenger ribonucleic acid (mRNA) expressions of anabolic factors [SRY-box transcription factor-9 (SOX9) and aggrecan (ACAN)], but significantly downregulated mRNA expressions of IL6, nuclear factor kappa-B (NF-κB), CASP3, and matrix metalloproteinase-13 (MMP13). Anti-apoptotic and anti-inflammatory effects of CLU were mediated through activating PI3K/Akt signaling pathway. The findings suggest that CLU might have beneficial effects on knee OA chondrocytes by exerting anti-apoptotic and anti-inflammatory functions via PI3K/Akt pathway, making CLU a promising target for potential therapeutic interventions in knee OA.

Synergistic effect of chimeric antigen receptor modified with Bcl-2 on enhanced solid tumour targeting.

Engineered T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic effects on haematological malignancies. However, CART cells are less effective on solid tumours mainly due to their weak persistence, which might be caused by activation-induced cell death (AICD). To overcome this limitation, CART cell with the antigen, Epidermal growth factor receptor variant III (EGFRvIII), targeting was modified to carry the anti-apoptotic molecule B cell lymphoma 2 (Bcl-2), and the final construct was named as EGFRvIII·CART-Bcl2 cells. Compared with the EGFRvIII·CART cells, EGFRvIII·CART-Bcl2 cells revealed higher capacities of proliferation, anti-apoptosis and tumour cell killing in vitro. Moreover, EGFRvIII·CART-Bcl2 cells had a longer persistence rate and exerted better anti-tumour effects than EGFRvIII·CART cells in cervical carcinoma xenograft model. Taken together, our findings suggest that incorporating anti-apoptotic molecules into CART cells may enhance its therapeutic effects against solid tumours.

Single cell RNA sequencing of human eosinophils from nasal polyps reveals eosinophil heterogeneity in chronic rhinosinusitis tissue

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the US, yet the actual roles of eosinophils in CRSwNP are largely unclear. ObjectiveTo reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue by single cell RNA-Sequencing (scRNA-Seq) analysis of NP tissue. MethodsSinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched prior to processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by the microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by gene ontology (GO) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry. ResultsAfter filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses) and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were down-regulated, and 354 genes were up-regulated in NP eosinophils, compared to all PB eosinophils (>1.5-fold, Padj<0.05). Up-regulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling and anti-apoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue. ConclusionsElevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part via controlling inflammation and hyperproliferation of other cells.