Expression Of BCL2L1 Research Articles

Cardioprotective effects of cinnamoyl imidazole on apoptosis and oxidative stress in hypoxia/reoxygenation-induced H9C2 cell lines

BackgroundThis study explored the effects of cinnamoyl imidazole on alleviating oxidative stress and apoptosis in hypoxia/reoxygenation (H/R)-induced H9C2 cells, using computational analysis with in-vitro validation. MethodsComputational techniques, including SwissADME and Swiss Target Prediction, were employed to predict the ADME properties and to identify targets of cinnamoyl imidazole. Differential gene expression (DEG) analysis was conducted on myocardial infarction (MI) datasets obtained from the Gene Expression Omnibus. Gene enrichment and molecular simulation studies were done to focus on apoptotic pathways. The computational findings were validated through In vitro experiments on H9C2 cardiomyocytes subjected to 8 h of hypoxia followed by 24 h of reoxygenation. Antioxidant enzyme levels (catalase, GST, GSH-Px, and SOD), mitochondrial membrane potential (ΔΨm), caspase-3 activity, and the expression of CASP3, MAPK8, JAK2, and BCL2L1 were assessed. ResultsCinnamoyl imidazole has demonstrated favourable pharmacokinetic properties, characterized by high gastrointestinal absorption and low toxicity with negative toxicity for organ endpoints. Molecular docking studies revealed the strong binding affinities for CASP3, MAPK8, and JAK2. In vitro results showed a significant increase in cell viability (94.7 % at 10 μM, p < 0.001) and antioxidant enzyme activity, along with a 64.3 % reduction in caspase-3 activity at 1000 μM (p < 0.01). Cinnamoyl imidazole treatment preserved mitochondrial membrane potential, downregulated pro-apoptotic genes CASP3 and MAPK8, and upregulated the anti-apoptotic gene BCL2L1. ConclusionCinnamoyl imidazole effectively mitigates oxidative stress and apoptosis in H/R-induced H9C2 cells, enhancing cell viability and antioxidant defenses while maintaining mitochondrial integrity.

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall invitro maturation efficiency and early embryonic stages.

Lipid peroxidation products induce carbonyl stress, mitochondrial dysfunction, and cellular senescence in human and murine cells.

Lipid enals are electrophilic products of lipid peroxidation that induce genotoxic and proteotoxic stress by covalent modification of DNA and proteins, respectively. As lipid enals accumulate to substantial amounts in visceral adipose during obesity and aging, we hypothesized that biogenic lipid enals may represent an endogenously generated, and therefore physiologically relevant, senescence inducers. To that end, we identified that 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal (4-HHE) or 4-oxo-2-nonenal (4-ONE) initiate the cellular senescence program of IMR90 fibroblasts and murine adipose stem cells. In such cells, lipid enals induced accumulation of γH2AX foci, increased p53 signaling, enhanced expression of p21Cip1, and upregulated the expression and secretion of numerous cytokines, chemokines, and regulatory factors independently from NF-κB activation. Concomitantly, lipid enal treatment resulted in covalent modification of mitochondrial proteins, reduced mitochondrial spare respiratory capacity, altered nucleotide pools, and increased the phosphorylation of AMP kinase. Lipid-induced senescent cells upregulated BCL2L1 (Bcl-xL) and BCL2L2 (Bcl-w). and were resistant to apoptosis while pharmacologic inhibition of BAX/BAK macropores attenuated lipid-induced senescence. Insitu, the 4-HNE scavenger L-carnosine ameliorated the development of the cellular senescence, while in visceral fat of obese C57BL/6J mice, L-carnosine reduced the abundance of 4-HNE-modified proteins and blunted the expression of senescence biomarkers CDKN1A (p21Cip1), PLAUR, BCL2L1, and BCL2L2. Taken together, the results suggest that lipid enals are endogenous regulators of cellular senescence and that biogenic lipid-induced senescence (BLIS) may represent a mechanistic link between oxidative stress and age-dependent pathologies.

Integrative single-cell RNA-seq and spatial transcriptomics analyses reveal diverse apoptosis-related gene expression profiles in EGFR-mutated lung cancer

In EGFR-mutated lung cancer, the duration of response to tyrosine kinase inhibitors (TKIs) is limited by the development of acquired drug resistance. Despite the crucial role played by apoptosis-related genes in tumor cell survival, how their expression changes as resistance to EGFR-TKIs emerges remains unclear. Here, we conduct a comprehensive analysis of apoptosis-related genes, including BCL-2 and IAP family members, using single-cell RNA sequence (scRNA-seq) and spatial transcriptomics (ST). scRNA-seq of EGFR-mutated lung cancer cell lines captures changes in apoptosis-related gene expression following EGFR-TKI treatment, most notably BCL2L1 upregulation. scRNA-seq of EGFR-mutated lung cancer patient samples also reveals high BCL2L1 expression, specifically in tumor cells, while MCL1 expression is lower in tumors compared to non-tumor cells. ST analysis of specimens from transgenic mice with EGFR-driven lung cancer indicates spatial heterogeneity of tumors and corroborates scRNA-seq findings. Genetic ablation and pharmacological inhibition of BCL2L1/BCL-XL overcome or delay EGFR-TKI resistance. Overall, our findings indicate that BCL2L1/BCL-XL expression is important for tumor cell survival as EGFR-TKI resistance emerges.

Synergy of retinoic acid and BH3 mimetics in MYC(N)-driven embryonal nervous system tumours

BackgroundCertain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches.MethodsWe performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing.ResultsGroup 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo.ConclusionsRA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.

TRIM33 plays a critical role in regulating dendritic cell differentiation and homeostasis by modulating Irf8 and Bcl2l11 transcription

The development of distinct dendritic cell (DC) subsets, namely, plasmacytoid DCs (pDCs) and conventional DC subsets (cDC1s and cDC2s), is controlled by specific transcription factors. IRF8 is essential for the fate specification of cDC1s. However, how the expression of Irf8 is regulated is not fully understood. In this study, we identified TRIM33 as a critical regulator of DC differentiation and maintenance. TRIM33 deletion in Trim33fl/fl Cre-ERT2 mice significantly impaired DC differentiation from hematopoietic progenitors at different developmental stages. TRIM33 deficiency downregulated the expression of multiple genes associated with DC differentiation in these progenitors. TRIM33 promoted the transcription of Irf8 to facilitate the differentiation of cDC1s by maintaining adequate CDK9 and Ser2 phosphorylated RNA polymerase II (S2 Pol II) levels at Irf8 gene sites. Moreover, TRIM33 prevented the apoptosis of DCs and progenitors by directly suppressing the PU.1-mediated transcription of Bcl2l11, thereby maintaining DC homeostasis. Taken together, our findings identified TRIM33 as a novel and crucial regulator of DC differentiation and maintenance through the modulation of Irf8 and Bcl2l11 expression. The finding that TRIM33 functions as a critical regulator of both DC differentiation and survival provides potential benefits for devising DC-based immune interventions and therapies.

Abstract 355: BCLxL inhibition enhances copanlisib response in colorectal cancer

Abstract Background: Anti-apoptotic signaling, including through BCLxL, is a potential mechanism of resistance to PI3K inhibition in PIK3CA mutant cancers. We have previously demonstrated enhanced activity of PI3K inhibition in combination with BCLxL inhibition in PIK3CA mutated colorectal cancer (CRC) models. Here we examine the potential impact of BCLxL expression in PIK3CA mutant CRCs and the mechanism of treatment response with the combination of copanlisib and navitoclax. Methods: PIK3CA mutant CRCs were identified within TCGA colorectal adenocarcinoma data set and two cohorts were generated based on BCL2L1 (gene that encodes protein BCLxL) expression. Differentially expressed genes and gene set enrichment analysis were performed. CRC cell lines SW48 and SW48PK (Horizon Discovery, SW48 with a PIK3CAH1047R/+ mutation) were treated with copanlisib (copan) (1-100 nM) and navitoclax (250 nM). Immunoblotting was performed quantified using ImageJ to evaluate PI3K and apoptotic signaling. Flow cytometry using annexin V and propidium iodide (PI) was performed as a measure of apoptosis. Immunohistochemistry (IHC) using Ki67 was performed as a measure of cell proliferation on SW48PK xenografts and quantified as percent positive nuclei at 40x field of view (FOV). Results: Two datasets comprised of PIK3CA mutant CRC patients with either high or low BCL2L1 expression containing 39 and 47 patients, respectively, were used in the TGCA analysis. There was no difference in age, gender, or staging between these two datasets. An increase in DNA repair (false discovery rate q-value= 0.034) and interferon alpha signaling pathways (q= 0.022) was seen in PIK3CA mutant and high BCL2L1 expression compared to PIK3CA mutant and low BCL2L1 expression patients. PIK3CA mutant patients with low expression of BCL2L1 had a greater disease-free and overall survival (p= 0.037 and p= 0.042, respectively). Combination of copan and navitoclax treatment of the SW48 and SW48PK cell lines resulted in a reduction in activated ribosomal protein S6 (pRPS6) as compared with control or navitoclax alone at both 6 and 24. Cleaved PARP increased in SW48PK cells treated with 100 nM copan plus navitoclax versus control at both 6 and 24 hour timepoints (p= 0.03, p= 0.049). SW48 and SW48PK cells that were treated with copan and navitoclax for 24 hours had an increase in annexin v, a marker for apoptosis, compared to cells treated with control or single agents. In SW48PK xenografts, a significant reduction in Ki67 percent positive nuclei was seen in combination compared to control (median percent positive nuclei: 20.5, control: 33.2, p&amp;lt;0.001). Conclusions: High BCLxL expression is a sign of poor prognosis for PIK3CA mutant CRC. Inhibiting BCLxL can enhance the sensitivity of PI3K inhibition in PIK3CA mutant cancers and deserves further investigation clinically. Citation Format: Alexa E. Schmitz, Rebecca A. DeStefanis, Alyssa K. DeZeeuw, Autumn M. Olson, Anna L. Lippert, Kennedy J. Maduscha, Katherine A. Johnson, Sean Kraus, Cheri A. Pasch, Dustin A. Deming. BCLxL inhibition enhances copanlisib response in colorectal cancer [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 355.

Abstract 5826: Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated lung cancer

Abstract Despite an initial marked response to targeted therapy with tyrosine kinase inhibitors (TKIs) in EGFR-mutated lung cancer, the duration of response is limited due to the inevitable development of acquired resistance. Anti- and pro-apoptotic genes play an important role in the survival of tumor cells. Currently, variations in these gene expression levels are not well characterized due to the complex interplay between the inducement of expression changes by EGFR-TKIs and the presence of inter- and intra-tumor heterogeneity. Here, a comprehensive analysis of apoptosis-related genes, including BCL-2 family members and the IAP family members, was conducted via single-cell RNA sequence (scRNA-seq) and spatial transcriptomics. scRNA-seq captured changes in apoptosis-related gene expression after EGFR-TKI treatment using different EGFR-mutated lung cancer cell lines. Notably, BCL2L1 expression increased, whereas MCL1 expression decreased after EGFR-TKI treatment. Other anti- and pro-apoptotic genes did not display consistent trends in the cell lines used. Additionally, scRNA-seq was carried out on samples collected from patients with EGFR-mutated lung cancer after the development of resistance to EGFR-TKIs in order to validate the results obtained from cell line data and to compare gene expression levels between tumor and non-tumor cells. BCL2L1 exhibited high expression specifically in tumor cells; in contrast, MCL1 expression was lower in tumor cells compared to non-tumor cells. Moreover, BAD and XIAP were highly expressed in a tumor-specific manner. To further corroborate the scRNA-seq findings and delineate the spatial heterogeneity in tumors, spatial transcriptomics was performed using EGFR-driven lung cancer transgenic mouse specimens in three conditions: prior to treatment, during therapeutic response after short-term treatment, and following acquired resistance to EGFR-TKIs. Spatial transcriptomics data demonstrated that an increased BCL2L1 expression was observed in tumor tissues and that MCL1 expression was significantly higher in tissue adjacent to tumors than in tumors. Finally, we demonstrated that genetic ablation of BCL2L1 and pharmacological inhibition of BCL2L1 overcame or delayed resistance to EGFR-TKIs in vitro and in vivo. Overall, BCL2L1 is particularly important for tumor cell survival during the emergence of drug tolerance and the development of acquired resistance to EGFR-TKIs. Citation Format: Motohiro Izumi, Masanori Fujii, Vivian Ho, Ikei S. Kobayashi, Susumu S. Kobayashi. Comprehensive analysis with single-cell RNA sequence and spatial transcriptomics unravels distinctive expression profile of apoptotic-related genes in EGFR-mutated lung cancer [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 5826.

Abstract 2787: Heme oxygenase 1 pulls the brake on stemness-induced properties by prostate cancer-bone crosstalk

Abstract Prostate cancer (PCa) is the second most diagnosed cancer in men globally, and it is positioned as the fifth leading cause of cancer-related death in males. PCa aggressiveness and mortality is intricately linked to the metastatic disease, which is propelled by PCa stem cells (PCSCs). We have previously showcased the strong antitumoral role heme oxygenase-1 (HO-1) exerts in PCa. However, little is known about the association of HO-1 with the stemness capacity of PCa cells. In this work, we first performed clonogenic assays in PCa cells (PC3 and C4-2B) to assess colony formation, which evidenced a reduction on the stem-like properties of tumor cells treated with hemin (FDA approved drug and specific HO-1 inducer and activator). Next, we employed an indirect co-culture system of PCa cells (PC3) grown with bone progenitors (MC3T3) to emulate the dynamic PCa-bone crosstalk. PC3 cells were pre-treated or not with hemin. We performed comprehensive RNA-seq analyses and assessed the transcriptome of PC3 cells focusing on genes associated with stemness (CD44, CD133 and other pluripotency markers), metastasis and a stem-like signature previously identified by our group (ADAM15, BCL2L1, LTBR, MBNL2, SPINT1). PC3 cells that were co-cultured with MC3T3 displayed upregulated PCSC and pluripotency markers, as well as BCL2L1 and LTBR, when compared with PC3 cells cultured alone. Intriguingly, pre-treatment of PC3 cells with hemin prior to the co-culture impaired the upregulation of these genes, including the PCSC marker CD44, underscoring the protective effect of HO-1 induction against the pro-stemness effect triggered by bone progenitors secreted factors. Furthermore, we extended our analysis using PCa patients’ survival and transcriptomic publicly available data (TCGA-PRAD (n=565), SU2C-PRAD (n=444), FHCRC-PRAD (n=176)), which revealed that ADAM15, BCL2L1, LTBR and SPINT1 expression was higher in bone metastases vs. primary PCa (p&amp;lt;0.001). Moreover, POU5F1, ALDH1L2, ADAM15, BCL2L1, LTBR and SPINT1 presented higher expression in bone metastases vs. other sites of metastasis (p&amp;lt;0.01). We also performed multiple survival analyses including progression-free, metastasis-free, biochemical relapse-free and overall survival (TCGA-PRAD, GSE116918, GSE70770) which revealed that CD44 expression is associated with increased risk of metastasis (p&amp;lt;0.05), while patients with high expression of SOX2, ALDH1B1 and ALDH1L2 showed shorter times to biochemical relapse (p&amp;lt;0.05) Altogether, HO-1 expression modulates stemness and metastasis-related genes in PCa cells, steering tumor cells towards a more differentiated state and counteracting the pro-stemness effect inherent to the communication with bone progenitors. These findings support the antitumoral role of HO-1 in PCa and underscores CD44 as an HO-1-modulated compelling candidate for further investigations. Citation Format: Agustina Ayelen Sabater, Ines Achinelli, Pablo Sanchis, Nicolas Anselmino, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Javier Cotignola, Elba Vazquez, Geraldine Gueron, Ayelen Toro. Heme oxygenase 1 pulls the brake on stemness-induced properties by prostate cancer-bone crosstalk [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 2787.

Abstract 706: Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy

Abstract Pediatric soft-tissue sarcomas are a group of solid tumors that account for about 7% of childhood cancer cases. Rhabdomyosarcoma (RMS), the most common subtype, further stratifies into PAX3 or PAX7 and FOXO1 fusion-negative (FN) or fusion-positive (FP) RMS. FP RMS is the higher risk subtype and is more aggressive, metastatic, and less responsive to treatment. Front-line treatments for RMS FP patients have had limited progress in the recent years and existing treatments such as radiotherapy lacks tumor cell selectivity, resulting in toxic side effects. To address this, we conducted a high-throughput drug screen to identify radiosensitizers to enhance efficacy of RMS radiotherapy. Using three patient-derived FP RMS tumoroids, we screened a comprehensive drug library with and without radiation, comprising 528 drugs at varying stages of pre-clinical and clinical trials. A validation screen of the top 29 drugs was conducted on a larger cohort of samples including eight patient-derived FP RMS tumoroids and three non-cancer models. The screening revealed that drugs targeting the anti-apoptotic protein B-cell lymphoma-extra-large (BCL-xL) exhibited both potent radiosensitization and cytotoxicity selectively for FP RMS. Notably, the sensitivity to BCL-xL inhibitors correlated with elevated BCL2L1 expression and PAX3 fusion status. Additionally, drugs targeting polo-like kinase 1 (PLK1) and other cell cycle-related genes exhibited robust radioprotective effects across both RMS and non-cancer models. Our findings propose the therapeutic potential of BCL-xL inhibition, either as a standalone treatment or in combination with radiotherapy. In parallel, we show the protective potential of PLK1 inhibition to mitigate radiation toxicity. In the long term, we aspire to integrate the identified radiosensitizing and radioprotecting compounds with existing treatment regiments for RMS, maximizing precision and efficacy in RMS radiotherapy. Citation Format: Sammy Se Whee Park, Nona Struyf, Tom Erkers, Sören Lehmann, Michael T. Meister, Jarno Drost, Frank C. Holstege, Nikolas Herold, Päivi Östling, Jakob Stenman, Olli Kallioniemi, Jan-Inge Henter, Brinton Seashore-Ludlow, Kasper Karlsson. Advancing radiotherapy strategies for high-risk rhabdomyosarcoma: Unveiling BCL-xL inhibition as a potent sensitizer to radiotherapy [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 706.

Noscapine and Apoptosis in Breast and Other Cancers.

Breast cancer is the second leading contributor to the age-standardized mortality rate, for both sexes and all ages worldwide. In Europe and the United States, it is the second leading cause of mortality, with an incidence rate of about 2.6 million cases per year. Noscapine, a well-known alkaloid used as a cough suppressant, demonstrated anti-tumor effects by triggering apoptosis in various cancer cell lines and has the potential to become another ally against breast, ovarian, colon, and gastric cancer, among other types of malignancy. Apoptosis plays a crucial role in the treatment of cancer. Noscapine affected BAX, CASP8, CASP9, NFKBIA, and RELA gene and protein expression in the MCF-7 and MDA-MB-231 cell lines. Gene expression was higher in tumor than in normal tissue, including the BAX expression levels in lung, ovary, endometrium, colon, stomach, and glioblastoma patients; BCL2L1 expression in endometrium, colon, and stomach patients; CASP8 gene expression levels in lung, endometrium, colon, stomach, and glioblastoma patients; RELA in colon, stomach, and glioblastoma patients; and NFKBIA in glioblastoma patients. It can be concluded that noscapine affected genes and proteins related to apoptosis in cancer cell lines and several types of cancer patients.

Combinatorial BCL-2/ BCL2L1 Expression Predicts Clinical Response to Ruxolitinib in Myelofibrosis

Introduction: JAK inhibitors (JAKi) marked a revolutionary breakthrough in the treatment of patients (pts) with myelofibrosis (MF). The BCL-2 family of proteins have been implicated in both disease development and mechanisms of response/resistance to JAKi. This is further reinforced by preliminary evidence of therapeutic efficacy of combining JAK2 and BCL-xL inhibition. Aims&amp;Methods: In the current study, we investigated the expression of three major BCL-2 family members in MF pts treated with ruxolitinib (RUX) at baseline and during treatment. Gene expression of BCL-2, BCL2L1 (encoding BCL-xL) and MCL-1 was analyzed by qPCR normalized to endogenous 18S gene using cDNA obtained from peripheral blood (PB) granulocytes; the relative gene expression (expressed as fold-change, FC) was calculated with reference to healthy controls using the Ct (2−ΔΔCt) method. Results: The study included 19 pts (10 males; median age 61 years) with MF (10 primary, 9 secondary) who received treatment with RUX. Sixteen pts were JAK2-mutated, 2 CALR-mutated, and 1 MPL-mutated. Treatment response was fully evaluable across the entire treatment period. After a median treatment time of 67 (range, 6-121) months, 7 (37%) pts achieved a spleen response (defined as a &amp;gt;50% reduction in palpable splenic length lasting &amp;gt;6 months) and were considered as responders, as compared to 12 (63%) non-responders. We did not find any significant correlation of baseline BCL-2/ BCL2L1/ MCL-1 expression with clinical and molecular characteristics, the only exception being the association between EZH2 mutation and higher BCL2L1 expression. First, we ascertained whether BCL2 family protein expression levels at baseline predicted response to RUX. We found that BCL2 and BCL2L1 were significantly more expressed in responders compared to non-responders, with median FC values of 0.14 vs 0.04 ( P=.0130) and 2.46 vs 0.51 ( P=.0096), respectively (Fig. 1A). A similar, not statistically significant, trend was observed for MCL-1 expression. Accordingly, in univariate regression logistic analysis, both BCL2 and BCL2L1 FCs correlated significantly with the probability of response to RUX. Then, we incorporated BCL-2 and BCL2L1 gene expression at baseline into a combinatorial score (CS) resulting from the product of the respective FCs (FC BCL-2 * FC BCL-xL). The CS outperformed the individual BCL2 family proteins expression in predicting RUX response, as confirmed in regression logistic analysis. ROC analysis identified 0.06 as the optimal cut-off value of the CS. In univariate analysis, pts with a baseline CS &amp;gt;0.06 had a significantly higher probability of responding to RUX (OR 27.5, 95% CI 2.1-236.7; P=.0037), with a superior cumulative incidence of RUX response at 24 weeks (50% vs 9%, respectively; P=.0045) (Fig. 1B). Finally, we prospectively assessed the changes in gene expression during treatment with RUX. Six out of seven responder pts had an available PB sample at the time of best response, while all non-responders were evaluated at some point during RUX therapy. Among responders, 5 lost response after a median time of 34 months, and 3/5 had an available sample for analyses at the time of response loss. Although we did not find any significant difference comparing gene expression at baseline and on treatment, we observed a trend for BCL-2 upregulation in responders at the time of best response. Furthermore, non-responders showed a down-regulation of MCL-1 expression during treatment ( P=.05). By comparing gene expression of responder pts at the time of best vs loss of response, we discovered a down-regulation for all the three genes, with a statistical significance that was reached only for BCL-2 ( P=.0471). Conclusion: Despite study limitations (mainly the small cohort), this preliminary data suggests that the expression levels of BCL2 family proteins may represent a predictor of response to RUX. Specifically, we developed a simple, combinatorial score includingbaseline BCL-2 and BCL2L1 expression that accurately discriminated pts who responded to RUX. Moreover, we showed that BCL2 family protein expression changes during RUX treatment, with potential correlations with both response achievement and loss. Further studies are needed in order to confirm and expand our data.

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Introduction Regulated cell death (RCD) is a process commonly dysregulated in patients (pts) with acute myeloid leukemia (AML), resulting in the survival of genetically unstable cells. Apoptosis, as the best-known form of RCD, interacts with neddylation, a process that contributes to tumorigenesis and apoptosis resistance. Aims This study was conducted to determine the expression of the selected genes involved in the neddylation pathway and to assess their clinical importance along with selected interacting proapoptotic genes. In addition, we correlated the expression levels of these genes with known prognostic factors in AML and determined their impact on the survival of AML pts. Methods The expression of selected proapoptotic genes BIK, BAX, BAK, BCL2L11, BBC3 , PMAIP1 CASP3, CASP7, and neddylation-related genes: CUL1, CUL2 , CUL4A, CUL5, CUL7, CUL9, NEDD8 was determined in duplicates in bone marrow samples of newly diagnosed AML pts. Real-Time PCR was carried out using the TaqMan chemistry and the QuantStudio7 thermal cycler (Applied Biosystems-Thermo Fisher Scientific). Gene expression was normalized using reference genes selected by the Normirazor tool. The normalization was performed by ∆Ct = Ct (reference) - Ct (mRNA of interest). This approach results in higher values for higher mRNA expression. Differential expression analysis was performed using a Welch t-test. Kaplan-Meier method with the log-rank test was used to assess overall survival (OS). Results In a prospective study, we included 40 newly diagnosed AML pts (mean age 62; range 26-87) with a median (m) follow-up of 6.7 months (95% CI: 4.5-9.1). High-risk AML was present in 52.5%, intermediate-risk in 32.5%, and low-risk in 15% of pts, according to European LeukemiaNet (ELN) 2022 risk stratification. NPM1 mutation was present in 23%, whereas FLT3 ITD or TKD mutation in 25% of pts. Intensive treatment was introduced in 43% of pts, while 25% were treated with azacitidine+venetoclax. Non-intensive treatment with azacitidine in monotherapy was administered to the remaining pts. BAK expression was detected in 70%, BIK in 98%, while expression of the other genes was found in all pts. In the high-risk group, we detected lower expression of both proapoptotic BAX (fold change - FC=0.7, p=0.01) and BIK gene (FC=0.4, p=0.01), as well as lower expression of CUL9 (FC=0.7, p=0.03). Accordingly, in pts with NPM1 mutation we revealed upregulation of BIK (FC=6.1, p=0.001), and CUL9 (FC=1.6, p=0.01). Whereas pts with FLT3 mutation had lower expression of proapoptotic BBC3 (FC=0.5, p=0.01), and neddylation-related CUL1 (FC=0.6, p=0.01). In cases of initial bone marrow blasts infiltration above median value (&amp;gt;49%) BCL2L11 CUL5 , NEDD8 and PMAIP1were downregulated ( p=0.001-0.02), while CUL7 ( p=0.00) and CASP3 ( p=0.04) were upregulated. Patients with initial white blood cell count &amp;gt;20 G/l had lower expression of BCL2L11 ( p=0.005), and CUL4A ( p=0.02), while higher expression of CASP7 ( p=0.04). The mOS in the study cohort was not reached. OS was longer in pts with higher (&amp;gt; -2.061, cutoff was determined with CutOff Finder) BAX expression (log-rank: p=0.03) [Figure 1]. The mOS was not reached in this group, whereas the mOS in pts with lower BAX expression was 1.3 months (95% CI: 0.6-1.3). In univariate Cox regression analysis, higher BAX expression (HR 0.31, 95%CI: 0.11-0.84, p=0.022), higher albumin level (HR 0.13, 95%CI: 0.04-0.41, p=0.0004), and intensive treatment (HR 0.18, 95%CI: 0.04-0.69, p=0.012) were factors influencing the outcome. In the multivariate model for OS, higher expression of BAX (HR 0.03, 95%CI: 0.003-0.33, p=0.004) retained its significant protective effect in the context of established prognostic factors [Table 1]. Conclusions To our knowledge, we were the first to evaluate such a complex spectrum of neddylation and apoptosis-related genes in AML. We revealed dysregulation of these genes in AML. Our data indicate that upregulation of BAX at the diagnosis is associated with longer OS. Moreover, we reported significantly lower BAX expression in ELN high-risk group. Thus, indicating BAX may be a potential prognostic factor in AML. Our preliminary results did not prove significant differences in the effect of neddylation gene expression levels on the prognosis of AML patients. As studies are emerging on the potential of neddylation-targeted therapies, we are conducting further research to verify the effect of neddylation on AML.

Metformin reduces the cellular DNA repair capacity and enhances the effect of curcumin on the induction of apoptosis in AGS gastric cancer cells

BackgroundCancer results from the accumulation of mutations in critical genes, such as DNA repair genes. But these genes are a double-edged sword, because the basis of current cancer treatment is DNA damage from chemotherapy and radiation, and the repair system can slow the healing process by repairing the induced damage. Therefore, any substance that can reduce the DNA repair capacity of cancer cells can make the cells more sensitive to treatment. Metformin and curcumin, as low-complication compounds, can play this role well.MethodsIn the present work, changes in the expression of CASP3, BAX, and BCL2L1 apoptotic genes, and nine genes involved in DNA repair pathways (XRCC1, XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, XRCC7, BRCA1 and BRAC2) were measured comparatively by real-time PCR in AGS gastric cancer cell line under single and co-treatments with metformin and curcumin.ResultsOur findings showed that co-treatment of metformin and curcumin induced decreasing the expression of anti-apoptotic BCL2L1 and increasing expression of proapoptotic CASP3 and BAX. Metformin decreased the expression levels of seven genes, while curcumin did not alter the expression levels. The co-treatment of metformin and curcumin showed that although the XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, XRCC7, BRCA1, and BRCA2 were down-regulated, there was no difference between metformin and co-treatment for mRNA levels.ConclusionOur results suggest that metformin increases the sensitivity of cancer cells to anticancer drugs by suppressing several DNA repair pathways and that curcumin may induce apoptosis.

GABA regulates the proliferation and apoptosis of head and neck squamous cell carcinoma cells by promoting the expression of CCND2 and BCL2L1

Gamma-aminobutyric acid (GABA) is a multifunctional molecule that is widely present in the nervous system and nonneuronal tissues. It plays pivotal roles in neurotransmission, regulation of secretion, cell differentiation, proliferation, and tumorigenesis. However, the exact mechanisms of GABA in head and neck squamous cell carcinomas (HNSCCs) are unknown. We took advantage of RNA sequencing in this work and uncovered the potential gene expression profiles of the GABA-treated HNSCC cell line HN4–2. We found that the expression of CCND2 and BCL2L1 was significantly upregulated. Furthermore, GABA treatment inhibited the cell apoptosis induced by cisplatin and regulated the cell cycle after treatment with cisplatin in HN4–2 cells. Moreover, we also found that GABA could upregulate the expression of CCND2 and BCL2L1 after treatment with cisplatin. Our results not only reveal the potential pro-tumorigenic effect of GABA on HNSCCs but also provide a novel therapeutic target for HNSCC treatment.

The pharmacological mechanism of β-elemene in the treatment of esophageal cancer revealed by network pharmacology and experimental verification

The study aimed to investigate the mechanism of action of β-elemene (ELE) in the treatment of esophageal cancer (EC). In this study, public databases were used to predict related targets in ELE and EC. The network analysis was performed to identify key targets of ELE in EC treatment. Further, bioinformatics and DAVID databases were used for GO and KEGG enrichment analysis, respectively. Ultimately, molecular docking and in vitro cell experiments were conducted to validate the results of network pharmacology enrichment. As a result, 34 candidate targets for ELE in the treatment of EC were obtained, and five key targets (STAT3, EGFR, CTNNB1, BCL2L1 and CASP9) were identified. GO functional annotation yielded 2200 GO entries (p < 0.05). KEGG signaling pathway enrichment analysis screened 100 pathways (p < 0.05). Molecular docking results showed that ELE had similar affinity with five key targets. In vitro experiments showed that the expressions of STAT3, EGFR and BCL2L1 were significantly decreased, and the expression of CASP9 in the ELE intervention group was significantly increased compared with that in the control group. All in all, ELE may play a key role in the treatment of EC by regulating the expression of STAT3, EGFR, BCL2L1 and CASP9.

Inhibitory effect of miR342 on the progression of triple-negative breast cancer cells in vitro and in the mice model.

Breast cancer is the most common cancer in women worldwide, and the triple-negative subtype is the most invasive, with limited therapeutic options. Since miRNAs are involved in many cellular processes, they harbor great value for cancer treatment. Therefore, in this study, we have investigated the anti-proliferative and anti-invasive roles of miR342 in 4T1 triple-negative cells in vitro and also studied the effect of this miRNA on tumor progression and the expression of its target genes in vivo. 4T1 cells were transduced with conditioned media of miR342-transfected Hek-LentiX cells. MTT and clonogenic assays were used to assess the viability and colony-forming ability of 4T1 cells. Apoptosis and invasion rates were respectively evaluated by annexin/7-AAD and wound healing assays. At last, in vivo tumor progression was evaluated using H&E staining, real-time PCR, and immunohistochemistry. The viability of transduced-4T1 cells reduced significantly 48 hours after cell seeding and colony forming ability of these cells reduced to 50% of the control group. Also, miR342 imposed apoptotic and anti-invasive influence on these cells in vitro. A 30-day follow-up of the breast tumor in the mice model certified significant growth suppression along with reduced mitotic index and tumor grade in the treatment group. Moreover, decreased expression of Bcl2l1, Mcl1, and ID4, as miR342 target genes, was observed, accompanied by reduced expression of VEGF and Bcl2/Bax ratio at the protein level. To conclude, our data support the idea that miR342 might be a potential therapeutic target for the treatment of triple-negative breast cancer (TNBC).

Amsacrine downregulates BCL2L1 expression and triggers apoptosis in human chronic myeloid leukemia cells through the SIDT2/NOX4/ERK/HuR pathway

Accumulating evidence indicates that the anticancer activity of acridine derivatives is mediated through the regulation of anti-apoptotic and pro-apoptotic BCL2 protein expression. Therefore, we investigated whether the cytotoxicity of amsacrine with an acridine structural scaffold in human chronic myeloid leukemia (CML) K562 cells was mediated by BCL2 family proteins. Amsacrine induced apoptosis, mitochondrial depolarization, and BCL2L1 (also known as BCL-XL) downregulation in K562 cells. BCL2L1 overexpression inhibited amsacrine-induced cell death and mitochondrial depolarization. Amsacrine treatment triggered SIDT2-mediated miR-25 downregulation, leading to increased NOX4-mediated ROS production. ROS-mediated inactivation of ERK triggered miR-22 expression, leading to increased HuR mRNA decay. As HuR is involved in stabilizing BCL2L1 mRNA, downregulation of BCL2L1 was noted in K562 cells after amsacrine treatment. In contrast, amsacrine-induced BCL2L1 downregulation was alleviated by restoring ERK phosphorylation and HuR expression. Altogether, the results of this study suggest that amsacrine triggers apoptosis in K562 cells by inhibiting BCL2L1 expression through the SIDT2/NOX4/ERK-mediated downregulation of HuR. Furthermore, a similar pathway also explains the cytotoxicity of amsacrine in CML MEG-01 and KU812 cells.

Cell starvation regulates ceramide-induced autophagy in mouse preimplantation embryo development

Ceramide induces autophagy upon starvation via downregulation of nutrient transporters. To elucidate the mechanism by which starvation regulates autophagy in mouse embryos, the present study investigated nutrient transporter expression and the effect of C2-ceramide on in vitro embryo development, apoptosis, and autophagy. The transcript levels of the glucose transporters Glut1 and Glut3 were high at the 1- and 2-cell stages, and gradually decreased at the morula and blastocyst (BL) stages. Similarly, expression of the amino acid transporters L-type amino transporter-1 (LAT-1) and 4F2 heavy chain (4F2hc) gradually decreased from the zygote to the BL stage. Upon ceramide treatment, expression of Glut1, Glut3, LAT-1, and 4F2hc was significantly reduced at the BL stage, while expression of the autophagy-related genes Atg5, LC3, and Gabarap and synthesis of LC3 were significantly induced. Ceramide-treated embryos exhibited significantly reduced developmental rates and total cell numbers per blastocyst, and increased levels of apoptosis and expression of Bcl2l1 and Casp3 at the BL stage. Ceramide treatment significantly decreased the average mitochondrial DNA copy number and mitochondrial area at the BL stage. In addition, ceramide treatment significantly decreased mTOR expression. These results suggest that ceramide-induced autophagy promotes apoptosis by following downregulation of nutrient transporters during mouse embryogenesis.

Changes in the expression of cancer- and metastasis-related genes and proteins after metformin treatment under different metabolic conditions in endometrial cancer cells

Research questionHyperinsulinemia and elevated estrogen levels are known risk factors for endometrial cancer (EC) development and are associated with obesity, type 2 diabetes mellitus (T2DM), insulin resistance, among others. Metformin, an insulin-sensitizing drug, displays anti-tumor effects in cancer patients, including EC, but the mechanism of action is still not completely understood. In the present study, the effects of metformin on gene and protein expression were investigated in pre- and postmenopausal EC in vitro models in order to identify candidates that are potentially involved in the drug's anti-cancer mechanism. DesignAfter treating the cells with metformin (0.1 and 1.0 mmol/L), changes in the expression of >160 cancer- and metastasis-related gene transcripts were evaluated with RNA arrays. A total of 19 genes and 7 proteins were selected for a follow-up expression analysis, including further treatment conditions, in order to evaluate the influence of hyperinsulinemia and hyperglycemia on metformin-induced effects. ResultsChanges in the expression of BCL2L11, CDH1, CDKN1A, COL1A1, PTEN, MMP9 and TIMP2 were analyzed on gene and protein level. The consequences resulting from the detected expression changes as well as the influence of varying environmental influences are discussed in detail. With the presented data, we contribute to a better understanding of the direct anti-cancer activity of metformin as well as its underlying mechanism of action in EC cells. ConclusionsAlthough further research will be necessary to confirm the data, the influence of different environmental settings on metformin-induced effects could be highlighted with the presented data. Additionally, gene and protein regulation were not similar in the pre- and postmenopausal in vitro models.