The role of miRNA-29b1 on the hypoxia-induced apoptosis in mammalian cardiomyocytes.

Cardiomyocyte apoptosis is a complex biological process involving the interaction of many factors and signaling pathways. In hypoxic environment, cardiomyocytes may trigger apoptosis due to insufficient energy supply, increased production of oxygen free radicals, and disturbance of intracellular calcium ion balance. The present research aimed to investigate the role of microRNA-29b1 (miR-29b1) in hypoxia-treated cardiomyocytes and its potential mechanism involved. We established an in vitro ischemia model using AC16 and H9C2 cardiomyocytes through hypoxia treatment (1% O2, 48 h). Cell apoptosis was evaluated by flow cytometry using Annexin V FITC-PI staining assay. Moreover, we used Western blot and immunofluorescence analysis to determine the expression of Bcl-2, Bax caspase-3 and Cx43 proteins. We found that miR-29b1 protected AC16 and H9C2 cells from hypoxia-induced injury as evidence that miR-29b1 attenuated the effects of hypoxia treatment on AC16 and H9C2 cell apoptosis after hypoxia treatment. In conclusion, our findings suggest that miR-29b1 may have potential cardiovascular protective effects during ischemia-related myocardial injury.

Targeted inhibition of DHODH is synergistic with BCL2 blockade in HGBCL with concurrent MYC and BCL2 rearrangement.

High-grade B-cell lymphoma (HGBCL), the subtype of non-Hodgkin lymphoma, to be relapsed or refractory in patients after initial therapy or salvage chemotherapy. Dual dysregulation of MYC and BCL2 is one of the important pathogenic mechanisms. Thus, combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine. It has been shown to be a potential therapeutic target for multiple diseases. In this study, the DHODH inhibitor brequinar exhibited growth inhibition, cell cycle blockade, and apoptosis promotion in HGBCL cell lines with MYC and BCL2 rearrangements. The combination of brequinar and BCL2 inhibitors venetoclax had a synergistic inhibitory effect on the survival of DHL cells through different pathways. Venetoclax could upregulate MCL-1 and MYC expression, which has been reported as a resistance mechanism of BCL2 inhibitors. Brequinar downregulated MCL-1 and MYC, which could potentially overcome drug resistance to venetoclax in HGBCL cells. Furthermore, brequinar could downregulate a broad range of genes, including ribosome biosynthesis genes, which might contribute to its anti-tumor effects. In vivo studies demonstrated synergetic tumor growth inhibition in xenograft models with brequinar and venetoclax combination treatment. These results provide preliminary evidence for the rational combination of DHODH and BCL2 blockade in HGBCL with abnormal MYC and BCL2.

Mastering Death: The Roles of Viral Bcl-2 in dsDNA Viruses.

Proteins of the Bcl-2 family regulate cellular fate via multiple mechanisms including apoptosis, autophagy, senescence, metabolism, inflammation, redox homeostasis, and calcium flux. There are several regulated cell death (RCD) pathways, including apoptosis and autophagy, that use distinct molecular mechanisms to elicit the death response. However, the same proteins/genes may be deployed in multiple biochemical pathways. In apoptosis, Bcl-2 proteins control the integrity of the mitochondrial outer membrane (MOM) by regulating the formation of pores in the MOM and apoptotic cell death. A number of prosurvival genes populate the genomes of viruses including those of the pro-survival Bcl-2 family. Viral Bcl-2 proteins are sequence and structural homologs of their cellular counterparts and interact with cellular proteins in apoptotic and autophagic pathways, potentially allowing them to modulate these pathways and determine cellular fate.

Disarib, a Specific BCL2 Inhibitor, Induces Apoptosis in Triple-Negative Breast Cancer Cells and Impedes Tumour Progression in Xenografts by Altering Mitochondria-Associated Processes.

Targeted cancer therapy aims to disrupt the functions of proteins that regulate cancer progression, mainly by using small molecule inhibitors (SMIs). SMIs exert their effect by modulating signalling pathways, organelle integrity, chromatin components, and several biosynthetic processes essential for cell division and survival. Antiapoptotic protein BCL2 is highly upregulated in many cancers compared with normal cells, making it an ideal target for cancer therapy. Around 75% of primary breast cancers overexpress BCL2, providing an opportunity to explore BCL2 inhibitors as a therapeutic option. Disarib is an SMI that has been developed as a selective BCL2 inhibitor. Disarib works by disrupting BCL2-BAK interaction and activating intrinsic apoptotic pathways in leukemic cells while sparing normal cells. We investigated the effects of Disarib, a BCL2 specific inhibitor, on breast cancer cells and xenografts. Cytotoxicity and fluorometric assays revealed that Disarib induced cell death by increasing reactive oxygen species and activating intrinsic apoptotic pathways in Triple-Negative Breast Cancer cells (MDA-MB-231 and MDA-MB-468). Disarib also affected the colony-forming properties of these cells. MDA-MB-231- and MDA-MB-468-derived xenografts showed a significant reduction in tumours upon Disarib treatment. Through the transcriptomics approach, we also explored the influence of BCL2 inhibitors on energy metabolism, mitochondrial dynamics, and epithelial-to-mesenchymal transition (EMT). Mitochondrial dynamics and glucose metabolism mainly regulate energy metabolism. The change in energetics regulates tumour growth through epithelial-mesenchymal transition, and angiogenesis. RNA sequencing (RNAseq) analysis revealed that BCL2 inhibitors ABT-199 and Disarib maintain Oxphos levels in MDA-MB-231. However, key glycolytic genes were significantly downregulated. Mitochondrial fission genes were seen to be downregulated both in RNAseq data and semi quantitative real time polymerase chain reaction (qRTPCR) in Disarib-treated TNBC cells and xenografts. Lastly, Disarib inhibited wound healing and epithelial-to-mesenchymal transition. This study showed that Disarib disrupts mitochondrial function, activates the intrinsic apoptotic pathway in breast cancer, and inhibits epithelial-to-mesenchymal transition both in vitro and in vivo. These findings highlight Disarib's potential as a multifaceted therapeutic strategy for patients with Triple-Negative Breast Cancer.

Bcl-2 Up-Regulation Mediates Taxane Resistance Downstream of APC Loss.

Triple-negative breast cancer (TNBC) patients are treated with traditional chemotherapy, such as the taxane class of drugs. One such drug, paclitaxel (PTX), can be effective in treating TNBC; however, many tumors will develop drug resistance, which can lead to recurrence. In order to improve patient outcomes and survival, there lies a critical need to understand the mechanism behind drug resistance. Our lab made the novel observation that decreased expression of the Adenomatous Polyposis Coli (APC) tumor suppressor using shRNA caused PTX resistance in the human TNBC cell line MDA-MB-157. In cells lacking APC, induction of apoptosis by PTX was decreased, which was measured through cleaved caspase 3 and annexin/PI staining. The current study demonstrates that CRISPR-mediated APC knockout in two other TNBC lines, MDA-MB-231 and SUM159, leads to PTX resistance. In addition, the cellular consequences and molecular mechanisms behind APC-mediated PTX response have been investigated through analysis of the BCL-2 family of proteins. We found a significant increase in the tumor-initiating cell population and increased expression of the pro-survival family member Bcl-2, which is widely known for its oncogenic behavior. ABT-199 (Venetoclax), is a BH3 mimetic that specifically targets Bcl-2. ABT-199 has been used as a single or combination therapy in multiple hematologic malignancies and has shown promise in multiple subtypes of breast cancer. To address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance, we combined treatment of PTX and ABT-199. This combination treatment of CRISPR-mediated APC knockout MDA-MB-231 cells resulted in alterations in apoptosis, suggesting that Bcl-2 inhibition restores PTX sensitivity in APC knockout breast cancer cells. Our studies are the first to show that Bcl-2 functional inhibition restores PTX sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC.

Myeloid Targeted Human MLL-ENL and MLL-AF9 Induces cdk9 and bcl2 Expression in Zebrafish Embryos.

Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consistent with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery.

Novel Lipid Nanocomplex Co-Carrying Bcl2 siRNA and Quantum Dots for EGF Receptor-Targeted Anti-Cancer Theranosis.

Many different types of nanoparticles have been suggested for tumor-targeted theranosis. However, most systems were prepared through a series of complicated processes and could not even overcome the blood-immune barriers. For the accurate diagnosis and effective treatment of cancers, herein we suggested the lipid micellar structure capturing quantum dot (QD) for cancer theranosis. The QD/lipid micelles (QDMs) were prepared using a simple self-assembly procedure and then conjugated with anti-epidermal growth factor receptor (EGFR) antibodies for tumor targeting. As a therapeutic agent, Bcl2 siRNA-cholesterol conjugates were loaded on the surface of QDMs. The EGFR-directed QDMs containing Bcl2 siRNA, so-called immuno-QDM/siBcl2 (iQDM/siBcl2), exhibited the more effective delivery of QDs and siBcl2 to target human colorectal cancer cells in cultures as well as in mouse xenografts. The effective in vivo targeting of iQDM/siBcl2 resulted in a more enhanced therapeutic efficacy of siBcl2 to the target cancer in mice. Based on the results, anti-EGFR QDM capturing therapeutic siRNA could be suggested as an alternative modality for tumor-targeted theranosis.

Curcumin as a Potential Therapeutic Agent for Mitigating Carbon Monoxide Poisoning: Evidence from an Experimental Rat Study.

BACKGROUND Carbon monoxide (CO) is a poisonous gas and causes tissue damage through oxidative stress. We aimed to investigate the protective value of curcumin in CO poisoning. MATERIAL AND METHODS Twenty-four female Spraque Dawley rats were divided into 4 subgroups: controls (n=6), curcumin group (n=6), CO group (n=6), and curcumin+CO group (n=6). The experimental group was exposed to 3 L/min of CO gas at 3000 ppm. Curcumin was administered intraperitoneally at a dosage of 50 mg/kg. Hippocampal tissues were removed and separated for biochemical and immunohistochemical analysis. Tissue malondialdehyde (MDA) levels, nitric oxide (NO) levels, and superoxide dismutase (SOD) and catalase (CAT) activities were assayed spectrophotometrically, and serum asymmetric dimethylarginine (ADMA) were measured using the ELISA technique. Tissue Bcl-2 levels were detected by the immunohistochemistry method. RESULTS Tissue CAT and SOD activities and NO levels were significantly lower, and MDA and serum ADMA levels were higher in the CO group than in the control group (P<0.001). The curcumin+CO group had higher CAT activities (P=0.007) and lower MDA than the CO group (P<0.001) and higher ADMA levels than the control group (P=0.023). However, there was no significant difference observed for tissue SOD activity or NO levels between these 2 groups. In the curcumin+CO group, the Bcl-2 level was higher than that in the CO group (P=0.017). CONCLUSIONS The positive effect of curcumin on CAT activities, together with suppression of MDA levels, has shown that curcumin may have a protective effect against CO poisoning.

Low BCL-xL expression in triple-negative breast cancer cells favors chemotherapy efficacy, and this effect is limited by cancer-associated fibroblasts.

Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.

Piperine-coated zinc oxide nanoparticles target biofilms and induce oral cancer apoptosis via BCl-2/BAX/P53 pathway.

BACKGROUND: Dental pathogens play a crucial role in oral health issues, including tooth decay, gum disease, and oral infections, and recent research suggests a link between these pathogens and oral cancer initiation and progression. Innovative therapeutic approaches are needed due to antibiotic resistance concerns and treatment limitations. METHODS: We synthesized and analyzed piperine-coated zinc oxide nanoparticles (ZnO-PIP NPs) using UV spectroscopy, SEM, XRD, FTIR, and EDAX. Antioxidant and antimicrobial effectiveness were evaluated through DPPH, ABTS, and MIC assays, while the anticancer properties were assessed on KB oral squamous carcinoma cells. RESULTS: ZnO-PIP NPs exhibited significant antioxidant activity and a MIC of 50 µg/mL against dental pathogens, indicating strong antimicrobial properties. Interaction analysis revealed high binding affinity with dental pathogens. ZnO-PIP NPs showed dose-dependent anticancer activity on KB cells, upregulating apoptotic genes BCL2, BAX, and P53. CONCLUSIONS: This approach offers a multifaceted solution to combatting both oral infections and cancer, showcasing their potential for significant advancement in oral healthcare. It is essential to acknowledge potential limitations and challenges associated with the use of ZnO NPs in clinical applications. These may include concerns regarding nanoparticle toxicity, biocompatibility, and long-term safety. Further research and rigorous testing are warranted to address these issues and ensure the safe and effective translation of ZnO-PIP NPs into clinical practice.

Comparative analysis of Bcl-2 family protein overexpression in CAR T cells alone and in combination with BH3 mimetics.

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Gene expression and anticancer evaluation of Kigelia africana (Lam.) Benth. Extracts using MDA-MB-231 and MCF-7 cell lines.

In recent years, a cancer research trend has shifted towards identifying novel therapeutic compounds from natural assets for the management of cancer. In this study, we aimed to assess the cytotoxic activity of Kigelia Africana (KA) extracts on breast cancer (MDA-MB-231 and MCF-7) and noncancerous kidney cells (HEK-293T) to develop an efficient anticancer medication. We used gas chromatography mass spectrometry (GC-MS to analyze the constituents of EKA and HKA extracts meanwhile the crystal violet and the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assays were used to examine the possible cytotoxic effects of plant extracts on our cancer cell lines along with non-cancerous control. The quantitative real-time PCR (RT-PCR) was run on cell samples to evaluate the differential expression of cell proliferative markers of cancer (BCL-2 and TP53). These phytochemicals have been reported to have binding affinity for some other growth factors and receptors as well which was evaluated by the in-silico molecular docking against Bcl2, EGFR, HER2, and TP53. Our Morphological observation showed a significant difference in the cell morphology and proliferation potential which was decreased under the effect of plant extracts treatment as compared to the control samples. The ethanol extract exhibited a marked antiproliferative activity towards MDA-MB-231 and MCF-7 cell lines with IC50 = 20 and 32 µg/mL, respectively. Quantitative RT-PCR gene expression investigation revealed that the IC50 concentration of ethanolic extract regulated the levels of mRNA expression of apoptotic genes. With the target and active binding site amino acids discovered in the molecular docking investigation, TP53/Propanoic acid, 3-(2, 3, 6-trimethyl-1, 4-dioxaspiro [4.4] non-7-yl)-, methyl ester (-7.1 kcal/mol) is the best-docked ligand. The use of this plant in folk remedies justifies its high in vitro anti-cancer capabilities. This work highlights the role of phytochemicals in the inhibition of cancer proliferation. Based on all these findings, it can be concluded that EKA extract has promising anti-proliferative effect on cancerous cells but more study is required in future to further narrow down the active ingredients of total crude extract with specific targets in cancer cells.

[Diagnosis and differential diagnosis of large B-cell lymphoma with IRF4 rearrangement].

Objective: To analyze the clinicopathological features and differential diagnosis of large B-cell lymphoma with IRF4 rearrangement, aiming enhance its recognition and prevent misdiagnosis. Methods: The clinicopathological features, immunophenotype, and fluorescence in situ hybridization (FISH) results of six cases diagnosed with IRF4 rearrangement-positive B-cell lymphoma at the Affiliated Hospital of Xuzhou Medical University from 2015 to 2023 were retrospectively analyzed. Additionally, a comprehensive review of the literature was conducted. Results: Six patients with IRF4 rearrangement-positive large B-cell lymphoma were included. Patients 1 to 5 included three males and two females with a median age of 19 years ranging from 11 to 34 years. Four patients presented with head and neck lesions, while the other one had a breast nodule; all were in clinical Ann Arbor stages I to Ⅱ. Morphologically, entirely diffuse pattern was present in two cases, purely follicular pattern in one case, and diffuse and follicular patterns in other two cases. The tumor cells, predominantly centroblasts mixed with some irregular centrocytes, were of medium to large size, with a starry sky appearance observed in two cases. Immunophenotyping revealed all cases were positive for bcl-6 and MUM1, with a Ki-67 index ranging from 70% to 90%, and CD10 was positive in two cases. IRF4 rearrangement was confirmed in all cases by FISH analysis, with dual IRF4/bcl-6 rearrangements identified in two cases, leading to a diagnosis of LBCL-IRF4. Case 6, a 39-year-old female with a tonsillar mass and classified as clinical Ann Arbor stage Ⅳ, displayed predominantly diffuse large B-cell lymphoma (DLBCL) morphology with 20% high-grade follicular lymphoma characteristics. Immunohistochemistry showed negative CD10 and positive bcl-6/MUM1, with a Ki-67 index of approximately 80%. Triple rearrangements of IRF4/bcl-2/bcl-6 were identified by FISH, leading to a diagnosis of DLBCL with 20% follicular lymphoma (FL). All six patients achieved complete remission after treatment, with no progression or relapse during a follow-up period of 31-100 months. Conclusions: Large B-cell lymphoma with IRF4 rearrangement is a rare entity with pathological features that overlap with those of FL and DLBCL. While IRF4 rearrangement is necessary for diagnosing LBCL-IRF4, it is not specific and requires differentiation from other aggressive B-cell lymphomas with IRF4 rearrangement.

Toll-like receptor signaling in multiple myeloma cells promotes the expression of pro-survival genes B-cell lymphoma 2 and MYC and modulates the expression of B-cell maturation antigen.

Infections are common in plasma cell cancer multiple myeloma (MM) due to disease-related immune deficiencies and cancer treatment. Myeloma cells express Toll-like receptors (TLRs), and TLR activation has been shown to induce proliferative and pro-survival signals in cancer cells. MM is a complex and heterogeneous disease, and expression levels of TLRs as well as downstream signaling components are likely to differ between patients. Here, we show that in a large cohort of patients, TLR1, TLR4, TLR6, TLR9, and TLR10 are the most highly expressed in primary CD138+ cells. Using an MM cell line expressing TLR4 and TLR9 as a model, we demonstrate that TLR4 and TLR9 activation promoted the expression of well-established pro-survival and oncogenes in MM such as MYC, IRF4, NFKB, and BCL2. TLR4 and TLR9 activation inhibited the efficacy of proteasome inhibitors bortezomib and carfilzomib, drugs used in the treatment of MM. Inhibiting the autophagosome-lysosome protein degradation pathway by hydroxychloroquine (HCQ) diminished the protective effect of TLR activation on proteasome inhibitor-induced cytotoxicity. We also found that TLR signaling downregulated the expression of TNFRSF17, the gene encoding for B-cell maturation antigen (BCMA). MYC, BCL2, and BCL2L1 were upregulated in approximately 50% of primary cells, while the response to TLR signaling in terms of TNFRSF17 expression was dichotomous, as an equal fraction of patients showed upregulation and downregulation of the gene. While proteasome inhibitors are part of first-line MM treatment, several of the new anti-MM immune therapeutic drugs target BCMA. Thus, TLR activation may render MM cells less responsive to commonly used anti-myeloma drugs.

Evaluation of infliximab-induced genotoxicity and possible action on BCL-2 and P53 genes.

Although the last pandemic created an urgency for development of vaccines, there was a continuous and concerted effort to search for therapeutic medications among existing drugs with different indications. One of the medications of interest that underwent this change was infliximab (IFM). This drug is used as an anti-inflammatory, predominantly in patients with Crohn 's disease, colitis ulcerative, and rheumatoid arthritis. In addition to these patients, individuals infected with Coronavirus Disease (COVID-19) were administered this chimeric monoclonal antibody (IMF) to act as an immunomodulator for patients in the absence of comprehensive research. Consequently, the present study aimed to examine the genotoxic effects attributed to IFM treatment employing different assays in vivo using mouse Mus musculus. Therefore, IFM was found to induce genotoxic effects as evidenced by the comet assay but did not demonstrate genotoxic potential utilizing mouse bone marrow MN test. The results of evaluating the expression of the P53 and BCL-2 genes using RT-qPCR showed stimulation of expression of these genes at 24 hr followed by a decline at 48 hr. Although the comet assay provided positive results, it is noteworthy that based upon negative findings in the micronucleus test, the data did not demonstrate significant changes in the genetic material that might affect the therapeutic use of IFM. The stimulation of expression of P53 and BCL-2 genes at 24 hr followed by a decline at 48 hr suggest a transient, if any, effect on genetic material. However, there is still a need for more research to more comprehensively understand the genotoxic profile of this medication.

Cytotoxicity of alkaloids isolated from Peganum harmala seeds on HCT116 human colon cancer cells.

BACKGROUND: The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. METHODS AND RESULTS: P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed with flow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3ß) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3ß and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48 h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3ß and Bcl-2 and upregulation of Bax and p53 were observed. CONCLUSION: The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.

The regulation of the apoptotic pore-An immunological tightrope walk.

Apoptotic pore formation in mitochondria is the pivotal point for cell death during mitochondrial apoptosis. It is regulated by BCL-2 family proteins in response to various cellular stress triggers and mediates mitochondrial outer membrane permeabilization (MOMP). This allows the release of mitochondrial contents into the cytosol, which triggers rapid cell death and clearance through the activation of caspases. However, under conditions of low caspase activity, the mitochondrial contents released into the cytosol through apoptotic pores serve as inflammatory signals and activate various inflammatory responses. In this chapter, we discuss how the formation of the apoptotic pore is regulated by BCL-2 proteins as well as other cellular or mitochondrial proteins and membrane lipids. Moreover, we highlight the importance of sublethal MOMP in the regulation of mitochondrial-activated inflammation and discuss its physiological consequences in the context of pathogen infection and disease and how it can potentially be exploited therapeutically, for example to improve cancer treatment.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status.

ABSTRACT: Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.

Machine learning-assisted SERS approach enables the biochemical discrimination in Bcl-2 and Mcl-1 expressing yeast cells treated with ketoconazole and fluconazole antifungals.

Antifungal medications are important due to their potential application in cancer treatment either on their own or with traditional treatments. The mechanisms that prevent the effects of these medications and restrict their usage in cancer treatment are not completely understood. The evaluation and discrimination of the possible protective effects of the anti-apoptotic members of the Bcl-2 family of proteins, critical regulators of mitochondrial apoptosis, against antifungal drug-induced cell death has still scientific uncertainties that must be considered. Novel, simple, and reliable strategies are highly demanded to identify the biochemical signature of this phenomenon. However, the complex nature of cells poses challenges for the analysis of cellular biochemical changes or classification. In this study, for the first time, we investigated the probable protective activities of Bcl-2 and Mcl-1 proteins against cell damage induced by ketoconazole (KET) and fluconazole (FLU) antifungal drugs in a yeast model through surface-enhanced Raman spectroscopy (SERS) approach. The proposed SERS platform created robust Raman spectra with a high signal-to-noise ratio. The analysis of SERS spectral data via advanced unsupervised and supervised machine learning methods enabled unquestionable differentiation (100 %) in samples and biomolecular identification. Various SERS bands related to lipids and proteins observed in the analyses suggest that the expression of these anti-apoptotic proteins reduces oxidative biomolecule damage induced by the antifungals. Also, cell viability assay, Annexin V-FITC/PI double staining, and total oxidant and antioxidant status analyses were performed to support Raman measurements. We strongly believe that the proposed approach paves the way for the evaluation of various biochemical structures/changes in various cells.

Molecular Engineering of Functional SiRNA Agents.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (<5 EU/µg RNA). Furthermore, we demonstrate that three representative BioRNA/siRNAs against GFP, BCL2, and PD-L1 are biologically active and can specifically and efficiently silence their respective targets with the potential to effectively produce downstream antiproliferation effects by PD-L1-siRNA. With these promising results, we aim to advance the field of synthetic biology by offering a novel platform to bioengineer functional siRNA agents for research and drug development.