RSL3 enhances ROS-mediated cell apoptosis of myelodysplastic syndrome cells through MYB/Bcl-2 signaling pathway.

Myelodysplastic syndromes (MDS) are clonal hematopoietic malignancies and seriously threaten people's health. Current therapies include bone marrow transplantation and several hypomethylating agents. However, many elderly patients cannot benefit from bone marrow transplantation and many patients develop drug resistance to hypomethylating agents, making it urgent to explore novel therapy. RSL3 can effectively induce ferroptosis in various tumors and combination of RSL3 and hypomethylating agents is promising to treat many tumors. However, its effect in MDS was unknown. In this study, we found that RSL3 inhibited MDS cell proliferation through inducing ROS-dependent apoptosis. RSL3 inhibited Bcl-2 expression and increased caspase 3 and PARP cleavage. RNA-seq analysis revealed that MYB may be a potential target of RSL3. Rescue experiments showed that overexpression of MYB can rescue MDS cell proliferation inhibition caused by RSL3. Cellular thermal shift assay showed that RSL3 binds to MYB to exert its function. Furthermore, RSL3 inhibited tumor growth and decreased MYB and Bcl-2 expression in vivo. More importantly, RSL3 decreased the viability of bone marrow mononuclear cells (BMMCs) isolated from MDS patients, and RSL3 had a synergistic effect with DAC in MDS cells. Our studies have uncovered RSL3 as a promising compound and MYB/Bcl-2 signaling pathway as a potential target for MDS treatment.

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.

[Melatonin Enhances the Effect of ABT-737 in Acute Monocytic Leukemia THP-1 Cells].

Melatonin (N-acetyl-5-methoxytryptamine, MEL) is a hormone synthesized by the pineal gland. Due to its oncostatic effect, it can be considered as an antitumor agent and used for combination therapy. ABT-737, a Bcl-2 inhibitor, promotes cell death after treatment with agents that induce pro-apoptotic signals. In the present study, the combined effect of MEL and ABT-737 on changes in proliferative and mitotic activity, mitochondrial membrane potential, intracellular production of reactive oxygen species (ROS), and cytosolic Ca^(2+) was studied. Moreover, changes in the expression of anti- and pro-apoptotic proteins (Bcl-2 and Bax), autophagy markers (LC3A/B (I, II)), endoplasmic reticulum stress markers (chaperones BIP and PDI, CHOP) were studied under these conditions. The effect of MEL together with ABT-737 led to an increase in the level of cytosolic Ca^(2+), intracellular production of ROS and a decrease in the membrane potential of mitochondria. The content of Bcl-2 increased, while the level of Bax decreased. Activation of CHOP stimulated autophagy and led to a decrease in the synthesis of chaperones BIP and PDI. It is assumed that melatonin can enhance the effect of other chemotherapeutic agents and can be used in the treatment of tumors.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

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.

Hepatoprotective effects of zingerone on sodium arsenite-induced hepatotoxicity in rats: Modulating the levels of caspase-3/Bax/Bcl-2, NLRP3/NF-κB/TNF-α and ATF6/IRE1/PERK/GRP78 signaling pathways.

OBJECTIVE: Long-term exposure to arsenic has been linked to several illnesses, including hypertension, diabetes, hepatic and renal diseases and cardiovascular malfunction. The aim of the current investigation was to determine whether zingerone (ZN) could shield rats against the hepatotoxicity that sodium arsenite (SA) causes. METHODS: The following five groups of thirty-five male Sprague Dawley rats were created: I) Control; received normal saline, II) ZN; received ZN, III) SA; received SA, IV) SA + ZN 25; received 10 mg/kg body weight SA + 25 mg/kg body weight ZN, and V) SA + ZN 50; received 10 mg/kg body weight SA + 50 mg/kg body weight ZN. The experiment lasted 14 days, and the rats were sacrificed on the 15th day. While oxidative stress parameters were studied by spectrophotometric method, apoptosis, inflammation and endoplasmic reticulum stress parameters were measured by RT-PCR method. RESULTS: The SA disrupted the histological architecture and integrity of the liver and enhanced oxidative damage by lowering antioxidant enzyme activity, such as those of glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) level and increasing malondialdehyde (MDA) level in the liver tissue. Additionally, SA increased the mRNA transcript levels of Bcl2 associated x (Bax), caspases (-3, -6, -9), apoptotic protease-activating factor 1 (Apaf-1), p53, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), c-Jun NH2-terminal kinase (JNK), mitogen-activated protein kinase 14 (MAPK14), MAPK15, receptor for advanced glycation endproducts (RAGE) and nod-like receptor family pyrin domain-containing 3 (NLRP3) in the liver tissue. Also produced endoplasmic reticulum stress by raising the mRNA transcript levels of activating transcription factor 6 (ATF-6), protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and glucose-regulated protein 78 (GRP-78). These factors together led to inflammation, apoptosis, and endoplasmic reticulum stress. On the other hand, liver tissue treated with ZN at doses of 25 and 50 mg/kg showed significant improvement in oxidative stress, inflammation, apoptosis and endoplasmic reticulum stress. CONCLUSIONS: Overall, the study's data suggest that administering ZN may be able to lessen the liver damage caused by SA toxicity.

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.

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.

CXCR4-Mediated Codelivery of FLT3 and BCL-2 Inhibitors for Enhanced Targeted Combination Therapy of FLT3-ITD Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is often associated with poor prognosis and survival. Small molecule inhibitors, though widening the treatment landscape, have limited monotherapy efficacy. The combination therapy, however, shows suboptimal clinical outcomes due to low bioavailability, overlapping systemic toxicity and drug resistance. Here, we report that CXCR4-mediated codelivery of the BCL-2 inhibitor venetoclax (VEN) and the FLT3 inhibitor sorafenib (SOR) via T22 peptide-tagged disulfide cross-linked polymeric micelles (TM) achieves synergistic treatment of FLT3-ITD AML. TM-VS with a VEN/SOR weight ratio of 1/4 and T22 peptide density of 20% exhibited an extraordinary inhibitory effect on CXCR4-overexpressing MV4-11 AML cells. TM-VS at a VEN/SOR dosage of 2.5/10 mg/kg remarkably reduced leukemia burden, prolonged mouse survival, and impeded bone loss in orthotopic MV4-11-bearing mice, outperforming the nontargeted M-VS and oral administration of free VEN/SOR. CXCR4-mediated codelivery of BCL-2 and FLT3 inhibitors has emerged as a prospective clinical treatment for FLT3-ITD AML.

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.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.