Upregulation Of Bcl-2 Research Articles

Azadirachta indica A. Juss Fruit Mesocarp and Epicarp Extracts Induce Antimicrobial and Antiproliferative Effects against Prostate (PC-3), Breast (MCF-7), and Colorectal Adenocarcinoma (Caco-2) Cancer Cell Lines through Upregulation of Proapoptotic Genes.

Effective alternative strategies and methodological approaches are critically necessary for cancer prevention and therapy. In this study, we investigated the antitumor potential of neem fruit mesocarp and epicarp extracts. The chemical composition of the derived extracts was characterized using GC–MS. Data were collected on the antimicrobial activity of the extracts in addition to the cytotoxicity effect evaluated against PC-3, MCF-7, and Caco-2 cancer cell lines, compared with the normal Vero cells. Cell-cycle arrest, apoptosis, and expression of apoptosis-related genes were assessed on PC-3 cells. Both extracts had significant antiproliferative effects on all tested cell lines in a dose-dependent manner, with the mesocarp extract being more potent. Both extracts also showed high antibacterial and antifungal activities. These results were related to the chemical constituents of the extracts identified by the GC–MS analysis. The extract of neem fruit mesocarp caused cell-cycle arrest at G2/M phase of PC-3 cells. The cytotoxicity of neem mesocarp extract is strongly correlated with the induction of apoptosis, where it caused downregulation of the antiapoptotic BCL2 gene but upregulation of the proapoptotic P53 and BAX genes. This study showed that neem fruit extract is potential anticancer material in the future.

Nose to brain delivery of melatonin lipidic nanocapsules as a promising post-ischemic neuroprotective therapeutic modality

Ischemic stroke accounts for about 87% of all strokes, causing long-term disability in adults, and is the second leading cause of death worldwide. In search of new therapeutic modalities, the use of neuroprotective agents loaded in nanocarriers to be delivered by noninvasive means (i.e. via intranasal route) became a popular approach. In the current study, melatonin (MEL) was loaded in lipidic nanocapsules (LNCs) prepared using the phase inversion method, and characterized in terms of size, polydispersity, zeta potential, in vitro drug release, viscosity, storage stability, and ex vivo permeation across sheep nasal mucosa. Moreover, MEL-LNCs were tested for efficacy in cerebral ischemia/reperfusion (I/R/) injury model through histopathological assessment, and analysis of oxidative stress markers, pro-inflammatory cytokines, and apoptotic markers. Results showed that LNCs exhibited particle size ranging from 18.26 to 109.8 nm, negative zeta potential, good storage stability, spherical morphology, and a burst release followed by a sustained release pattern. LNCs exhibited 10.35 folds higher permeation of MEL than the drug solution across sheep nasal mucosa. Post-ischemic intranasal administration of MEL-LNCs revealed lowering of oxidative stress manifested by a decrease in malondialdehyde levels, and elevation of glutathione and superoxide dismutase levels, lowering of the inflammatory markers tumor necrosis factor-α, NO, myeloperoxidase, and significant inhibition of Caspase-3 activity as an apoptotic marker. Western blot analysis delineated a recovery of protein expression Nrf-2 and HO-1 with downregulation in the parent inflammatory markers nuclear factor kappa B p65, inducible nitric oxide synthase, Bax, and Cytochrome C expressions, and upregulation of B-cell lymphoma-2 Bcl-2, hence promoting neuronal survival. This was supported by histological evidence, revealing significant restoration of hippocampal neurons. In light of the above, it can be concluded that MEL-LNCs could be a promising delivery system for nose to brain delivery for treatment of cerebral ischemia.

TMBIM6 promotes diabetic tubular epithelial cell survival and albumin endocytosis by inhibiting the endoplasmic reticulum stress sensor, IRE1α

Reduced albumin reabsorption in proximal tubular epithelial cells (PTECs), resulting from decreased megalin plasma membrane (PM) localization due to prolonged endoplasmic reticulum (ER) stress, potentially contributes to albuminuria in early diabetic kidney disease (DKD). To examine this possibility, we investigated the cytoprotective effect of TMBIM6 in promoting diabetic PTEC survival and albumin endocytosis by attenuating ER stress with an IRE1α inhibitor, KIRA6. Renal TMBIM6 distribution and expression were determined by immunohistochemistry, western blotting, and qPCR, whereas tubular injury was evaluated in db/db mice. High-glucose (HG)-treated HK-2 cells were either treated with KIRA6 or transduced with a lentiviral vector for TMBIM6 overexpression. ER stress was measured by western blotting and ER-Tracker Red staining, whereas apoptosis was determined by performing TUNEL assays. Megalin expression was measured by immunofluorescence, and albumin endocytosis was evaluated after incubating cells with FITC-labeled albumin. Tubular injury and TMBIM6 downregulation occurred in db/db mouse renal cortical tissues. Both KIRA6 treatment and TMBIM6 overexpression inhibited ER stress by decreasing the levels of phosphorylated IRE1α, XBP1s, GRP78, and CHOP, and stabilizing ER expansion in HG-treated HK-2 cells. TUNEL assays performed with KIRA6-treated or TMBIM6-overexpressing cells showed a significant decrease in apoptosis, consistent with the significant downregulation of BAX and upregulation of BCL-2, as measured by immunoblotting. Both KIRA6 and TMBIM6 overexpression promoted megalin PM localization and restored albumin endocytosis in HG-treated HK-2 cells. TMBIM6 promoted diabetic PTEC survival and albumin endocytosis by negatively regulating the IRE1α branch of ER stress.

Downregulation of hsa-miR-548d-3p and overexpression of HOXA9 in diffuse large B-cell lymphoma patients and the risk of R-CHOP chemotherapy resistance and disease progression.

Routine categorization of DLBCL patients into GCB and non-GCB groups by Hans' criteria could not accurately predict chemotherapy resistance and disease progression in patients treated with standard R-CHOP therapy. There is a need to identify better biomarker predictors to enhance assisted selection of chemotherapy regimens for DLBCL patients. To identify dysregulated miRNAs and mRNAs that are predictive of resistance to R-CHOP chemotherapy or disease progression in patients with DLBCL. miRNA and mRNA profiling were performed on archival FFPE samples of the DLBCL patients. miRabel and miRNet bioinformatic tools were applied to determine experimental validated miRNA-mRNA target interaction. The significance of the genomic predictive values was assessed using adjusted odds ratios (AOR) and 95% confidence intervals (CI). 19/36 were R-CHOP therapy-resistant whilst 17/36 were R-CHOP therapy-sensitive. Ten dysregulated miRNAs and 12 dysregulated mRNAs were identified in therapy-resistant DLBCL patients. These dysregulated miRNAs and mRNA cause therapy resistance and disease progression in DLBCL patients, most likely via upregulation of the anti-apoptotic protein bcl2, activation of the JAK/STAT signalling pathway and dysregulation of p53 pathway. Downregulation of hsa-miR-548d-3p and overexpression of HOXA9 mRNA were significantly associated with therapy resistance and disease progression in DLBCL patients [hsa-miR-548d-3p AOR: 0.258, 95%CI: 0.097-0.684, p= 0.006]. DLBCL patients with downregulation of hsa-miR-548d-3p and overexpression of HOXA9 mRNA are more likely to experience R-CHOP therapy resistance and disease progression.

Repositioning Linagliptin for the Mitigation of Cadmium-Induced Testicular Dysfunction in Rats: Targeting HMGB1/TLR4/NLRP3 Axis and Autophagy.

Cadmium, a ubiquitous environmental toxicant, disrupts testicular function and fertility. The dipeptidyl peptidase-4 inhibitor linagliptin has shown pronounced anti-inflammatory and anti-apoptotic features; however, its effects against cadmium-evoked testicular impairment have not been examined. Herein, the present study investigated targeting inflammation, apoptosis, and autophagy by linagliptin for potential modulation of cadmium-induced testicular dysfunction in rats. After 60 days of cadmium chloride administration (5 mg/kg/day, by gavage), testes, epididymis, and blood were collected for analysis. The present findings revealed that linagliptin improved the histopathological lesions, including spermatogenesis impairment and germ cell loss. Moreover, it improved sperm count/motility and serum testosterone. The favorable effects of linagliptin were mediated by curbing testicular inflammation seen by dampening of HMGB1/TLR4 pathway and associated lowering of nuclear NF-κBp65. In tandem, linagliptin suppressed the activation of NLRP3 inflammasome/caspase 1 axis with consequent lowering of the pro-inflammatory IL-1β and IL-18. Jointly, linagliptin attenuated testicular apoptotic responses seen by Bax downregulation, Bcl-2 upregulation, and suppressed caspase 3 activity. With respect to autophagy, linagliptin enhanced the testicular autophagy flux seen by lowered accumulation of p62 SQSTM1 alongside upregulation of Beclin 1. The observed autophagy stimulation was associated with elevated AMPK (Ser487) phosphorylation and lowered mTOR (Ser2448) phosphorylation, indicating AMPK/mTOR pathway activation. In conclusion, inhibition of testicular HMGB1/TLR4/NLRP3 pro-inflammatory axis and apoptosis alongside stimulation of autophagy were implicated in the favorable actions of linagliptin against cadmium-triggered testicular impairment.

MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus.

Rearrangements that drive ectopic MEF2C expression have recurrently been found in patients with human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL). Here, we show high levels of MEF2C expression in patients with ETP-ALL. Using both in vivo and in vitro models of ETP-ALL, we demonstrate that elevated MEF2C expression blocks NOTCH-induced T cell differentiation while promoting a B-lineage program. MEF2C activates a B cell transcriptional program in addition to RUNX1, GATA3, and LMO2; upregulates the IL-7R; and boosts cell survival by upregulation of BCL2. MEF2C and the Notch pathway, therefore, demarcate opposite regulators of B- or T-lineage choices, respectively. Enforced MEF2C expression in mouse or human progenitor cells effectively blocks early T cell differentiation and promotes the development of biphenotypic lymphoid tumors that coexpress CD3 and CD19, resembling human mixed phenotype acute leukemia. Salt-inducible kinase (SIK) inhibitors impair MEF2C activity and alleviate the T cell developmental block. Importantly, this sensitizes cells to prednisolone treatment. Therefore, SIK-inhibiting compounds such as dasatinib are potentially valuable additions to standard chemotherapy for human ETP-ALL.

Rutin Attenuates Acrylamide Induced Neuropathic Pain via Inhibition of Proinflammatory Cytokines, Up-Regulation of Bcl-2 and Down-Regulation of Bax

Rutin is a flavonoid of the flavonol type found in many typical plants, such as buckwheat, passion flower, apple and tea. Acrylamide (ACR) is a known industrial toxic chemical that produces neurotoxicity characterized by progressive neuronal degeneration. Rats were randomly divided into Control, ACR, Pregabalin and Rutin treated groups. Male wistar rats were treated with ACR (50 mg/kg/ i.p.) for 4 weeks which produce typical symptoms of neuropathy in rats. Pregabalin (10 mg/kg) and Rutin (50 & 100 mg/kg) were administered orally for 4 weeks after one hour of ACR administration. ACR enhanced the production of reactive oxygen species (ROS). Treatment with Rutin significantly improved neurological score. Rutinsignificantly (p<0.001) attenuated acrylamide induced oxidative stress markers. The expression of Bcl-2 was up-regulated and TNF-α, IL-6 and Bax were down-regulated by rutin treatment. From our results, it can be concluded that rutinshowed an ameliorative effect against ACR induced neurotoxicity in rats through its antioxidant, anti-inflammatory and antiapoptotic actions.

The effect of quercetin in the maturation media on cumulus-granulosa cells and the developmental competence of bovine oocytes

The present study was designed to investigate the effects of Quercetin on the developmental competence of bovine oocytes and cumulus-granulosa cells (CGs). Two groups of immature cumulus-oocyte complexes (COCs) were subjected to IVM with or without Quercetin. The viability, nuclear status, early and late apoptosis of oocytes and CGs were evaluated using gene expression analysis and staining methods. Embryonic development was assessed morphologically by recording Post-IVF survival, cleavage, and blastocyst rates. The proportion of oocytes reaching the MII stage was greater and the number of early-apoptotic oocytes was lower in the group matured with Quercetin compared to the Control (p < 0.05). Relative upregulation of OCT-4, IGF2R and Bcl-2, and downregulation of CHOP was seen in treated oocytes. Also, downregulation of Bax and upregulation of Glut-4 was detected in treated CGs. The treated oocytes experienced higher post-IVF survival and cleavage rates compared to the untreated group (p < 0.05); more cleaved embryos reached ≥16-cell stage and blastocyst at days 4th and 7th respectively. In addition, total blastocyst rate was significantly improved. It is concluded that supplementing maturation media with Quercetin can enhance the quality of bovine oocytes and endow them with protective potential against early apoptotic damage possibly through CHOP regulation of BCL2 gene family, triggering expression of a gene in CGs to maintain the intactness of oocyte against apoptotic signals and providing oocytes with more energy substrates. It also boosts the subsequent development of oocyte to blastocyst and improves the efficacy of bovine embryo production in vitro.

Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

Over-expression of survivin could prevent the oxidative stress and toxicity of rotenone in SH-SY5Y cells.

Objective(s):It is important to find novel therapeutic molecular targets for curing Parkinson’s disease (PD). Accordingly, this study aimed to evaluate the effect of over-expression of the survivin gene, a gene frequently reported as neuroprotective, on the in vitro model of PD. Materials and Methods: Survivin was over-expressed in SH-SY5Y cells. Next, the cells were treated with rotenone (500 nM) for 24 hr. Then, viability and the total antioxidant capacity were assessed. The expression levels of 15 important genes of key cellular processes (oxidative stress, apoptosis, cell cycle, and autophagy) were assessed. The studied genes included survivin, superoxide dismutase, catalase, BAX, bcl2, caspase 3, caspase 8, caspase 9, p53, SMAC, β-catenin, mTOR, AMPK, ATG7, RPS18. The apoptosis level and the frequency of cell cycle stages were assessed by flow cytometry. For analyzing the data, the ANOVA test followed by Tukey’s test was used to evaluate the significant differences between the experimental groups. P<0.05 was considered significant. Results:Survivin could significantly decrease the rotenone-induced apoptosis in SH-SY5Y cells. The rotenone treatment led to down-regulation of catalase and up-regulation of bax, bcl2, caspase 3, caspase 8, P53, β-catenin, and ATG7. Survivin could significantly neutralize the effect of rotenone in most the genes. It could also increase the total antioxidant capacity of SH-SY5Y cells. Conclusion:Survivin could prevent the toxic effect of rotenone on SH-SY5Y cells during the development of in vitro PD model via regulating the genes of key cellular processes, including anti-oxidation, apoptosis, cell cycle, and autophagy.

A Chinese Medicine Compound Alleviates Cisplatin-Induced Acute Kidney Injury via Its Antiapoptosis and Anti-Inflammation Effects in Mice.

Cisplatin, also known as cis-diamine dichloroplatinum (CDDP), is a widely used chemotherapeutic drug. However, its application is limited by the occurrence of serious nephrotoxicity. Currently, no effective therapy is available for combating CDDP-induced acute kidney injury (AKI). In the present study, we investigated the efficacy of Jianpi Yishen Tang (JPYST), a traditional Chinese medicine (TCM) compound commonly used to treat chronic kidney disease, against CDDP-induced AKI. In the CDDP + JPYST group, male mice were pretreated with JPYST (18.35 g/kg/day) for 5 consecutive days before receiving a single dose of CDDP (20 mg/kg), all mice were sacrificed 72 h after the CDDP injection. Results showed that JPYST suppressed CDDP-induced kidney dysfunction and tubular damage scores in the mice. Mechanistically, JPYST treatment attenuated CDDP-induced renal tubular cell apoptosis in AKI mice, as manifested by a marked decreased in TUNEL-positive cell counts, downregulation of the pro-apoptotic proteins Bax, Bad and caspase 3, and upregulation of the antiapoptotic protein Bcl-2 in kidney tissues. Meanwhile, JPYST decreased the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum and renal tissues of mice following CDDP administration. These factors are involved in suppressing the activation of phospho-NF-κB p65 in tubular epithelial cells. Taken together, these findings demonstrated that JPYST exerts renoprotective effects against CDDP-induced AKI through antiapoptosis and anti-inflammation effects, and these are associated with downregulation of NF-κB activation. Therefore, JPYST has potential for development of treatment therapies against CDDP-induced AKI.

STAT5 does not drive steroid resistance in T-cell acute lymphoblastic leukemia despite the activation of BCL2 and BCLXL following glucocorticoid treatment.

Physiological and pathogenic interleukin-7-receptor (IL7R)-induced signaling provokes glucocorticoid resistance in a subset of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Activation of downstream STAT5 has been suggested to cause steroid resistance through upregulation of anti-apoptotic BCL2, one of its downstream target genes. Here we demonstrate that isolated STAT5 signaling in various T-ALL cell models is insufficient to raise cellular steroid resistance despite upregulation of BCL2 and BCL-XL. Upregulation of anti-apoptotic BCL2 and BCLXL in STAT5-activated T-ALL cells requires steroid-induced activation of NR3C1. For the BCLXL locus, this is facilitated by a concerted action of NR3C1 and activated STAT5 molecules at two STAT5 regulatory sites, whereas for the BCL2 locus this is facilitated by binding of NR3C1 at a STAT5 binding motif. In contrast, STAT5 occupancy at glucocorticoid response elements does not affect the expression of NR3C1 target genes. Strong upregulation of BIM, a NR3C1 pro-apoptotic target gene, upon prednisolone treatment can counterbalance NR3C1/STAT5-induced BCL2 and BCL-XL expression downstream of IL7- induced or pathogenic IL7R signaling. This explains why isolated STAT5 activation does not directly impair the steroid response. Our study suggests that STAT5 activation only contributes to steroid resistance in combination with cellular defects or alternative signaling routes that disable the pro-apoptotic and steroid-induced BIM response.

Study of the dynamics of in vitro infection with bovine gammaherpesvirus type 4 and apoptosis markers in susceptible cells

Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.

Abstract 3253: Probing the effects of retinoblastoma binding protein 6 (RBBP6) knockdown on the sensitivity of cisplatin in cervical cancer cells

Abstract RBBP6 is considered a potential cancer biomarker due to its association with cell proliferation and the fact that it is overexpressed at cervical cancer sites where there is marked apoptosis and elevated p53. More information is emerging regarding the role of RBBP6 in cancer treatment, specifically its potential to sensitize cancer cells to radiation and certain chemotherapeutic agents via BCL-2 gene regulation. Cisplatin is an FDA-approved chemotherapeutic agent that still presents with acquired resistance in certain cervical cancer cases through p53 repression and BCL-2 upregulation, which is why there is ongoing research in trying to understand the molecular mechanisms involved in the response of cervical cancer cells to this drug. The present study therefore aims to investigate the possible relationship between cisplatin and RBBP6 expression in cervical cancer cells. RBBP6 was silenced in Hela, CaSki and Vero cells using the RNAi technology, followed by measurement of wild-type p53 and BCL-2 at mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were therefore analyzed for apoptosis induction and real time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to cisplatin-only group and real time growth monitoring revealed a reduced growth rate in RBBP6-knockdown cells treated with cisplatin. Although wild-type p53 remained unchanged in the co-treatment group of cancer cells, BCL-2 was completely repressed, suggesting that RBBP6 is necessary for the responsiveness of cervical cancer cells to via BCL-2 downregulation. Findings from this study suggest that RBBP6 expression promotes sensitivity of HeLa cells to cisplatin through BCL-2 downregulation. Knockdown of RBBP6 limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of RBBP6 among individual patients. Citation Format: Pontsho Moela, Harshini Mehta, Melvin Ambele. Probing the effects of retinoblastoma binding protein 6 (RBBP6) knockdown on the sensitivity of cisplatin in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3253.

Abstract 1797: Bcl-2 up-regulation mediates taxane resistance downstream of APC loss

Abstract Patients with triple negative breast cancer (TNBC) are treated with traditional chemotherapy, such as paclitaxel (PTX). Despite the efficacy of taxanes, many tumors will recur due to drug resistance. Therefore, the need to understand the mechanism behind drug resistance is critical to improve patient outcome and survival. Our lab was the first to show that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor caused resistance to PTX in the MDA-MB-157 human TNBC cell line. In the absence of APC, apoptosis induction was decreased, as measured through cleaved caspase 3 and annexin/PI staining. To understand the molecular mechanisms behind APC-mediated PTX response, we analyzed the BCL-2 family of proteins and found a robust increase of the pro-survival family member, Bcl-2. The BH3 mimetic, ABT-199 (Venetoclax), which specifically targets Bcl-2, has been used as a single or combination therapy in multiple hematologic malignancies. In addition, ABT-199 has shown promise in multiple subtypes of breast cancer. Therefore, we used ABT-199 in combination with PTX to address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance. Combination treatment in three CRISPR-mediated APC knockout TNBC cell lines (MDA-MB-157, MDA-MB-231, and SUM159) caused changes in cell proliferation and apoptosis. Combined, these data suggest restored sensitivity to PTX using ABT-199. Our studies are the first to show that Bcl-2 inhibition can restore PTX-sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC. Citation Format: Sara Maloney, Sarah Zabala, Grace Richmond, Jenifer R. Prosperi. Bcl-2 up-regulation mediates taxane resistance downstream of APC loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1797.

BCL-2 isoform β promotes angiogenesis by TRiC-mediated upregulation of VEGF-A in lymphoma.

Bcl-2 (B-cell lymphoma 2), the first identified anti-apoptosis factor, encodes two transcripts, the long isoform α and the short isoform β. The current understanding of the Bcl-2 function mainly focuses on Bcl-2α, while little is known about the function of Bcl-2β, which lacks the transmembrane domain and contains 10 unique amino acids at the C-terminus instead. Here, we analyzed the expressions of BCL-2 two isoforms in diffused large B-cell lymphoma (DLBCL) and found a significant positive correlation between them. Then, with the CRISPR/Cas9-based transcriptional activator (CRISPRa), we generated mouse B-cell lymphomas with Bcl-2 upregulation from the endogenous locus, in which both Bcl-2α and Bcl-2β levels were increased. Bcl-2β itself promoted angiogenesis both in vitro and in vivo through increased vascular endothelial growth factor A (VEGF-A). Inhibiting VEGF receptors with Axitinib reduced angiogenesis induced by Bcl-2β overexpression. Co-immunoprecipitation and mass spectrometry analysis revealed that Bcl-2β interacted with the T-complex protein ring complex (TRiC). Disruption of TRiC significantly impaired the angiogenesis-promoting activity of Bcl-2β, indicated by reduced VEGF-A protein level and HUVEC tube formation. Thus, our study suggests that Bcl-2 isoform β plays a role in promoting tumor angiogenesis through the Bcl-2β-TRiC-VEGF-A axis.

The extract of Polygala fallax Hemsl. slows the progression of diabetic nephropathy by targeting TLR4 anti-inflammation and MMP-2/9-mediated anti-fibrosis in vitro

BackgroundPolygala fallax Hemsl. is a plant that is commonly used as a folk medicine by Guangxi ethnic minorities, and it is also widely used in the clinical treatment of chronic diseases in China. The extract of P. fallax (EPF) contains key biologically active components from the roots and stems. However, the role of P. fallax or EPF in diabetic nephropathy (DN) is unclear. PurposeThis study aimed to investigate the effects and mechanisms of EPF on high glucose (HG)-induced human glomerular mesangial cell (HMC) injury, inflammation, fibrosis, and apoptosis in vitro. MethodsFor the in vitro study, MTT and ELISA assays were performed with HG-treated HMCs, as well as MMP, Hoechst, flow cytometry, qRT-PCR, and western blot analyses. The expression of the TLR4/NF-κB pathway, along with its downstream inflammatory, apoptosis, and fibrosis factors, was measured. The expression of the TLR4/NF-κB pathway and its downstream inflammatory factors were also measured after the addition of TLR4 inhibitors. ResultsOur results suggest that EPF can reverse the hyperproliferation and apoptosis of HMCs induced by HG. In addition, the extract inhibited the increase in inflammatory factors IL-6, TNF-α, IL-1β, MCP-1, and IL-18 in cells treated with HG. The mRNA and protein expression of TLR4, MyD88, NF-κB, Col IV, FN, MMP-9, and MMP-2 were downregulated by EPF. In addition, EPF significantly reduced the loss of MMP and the upregulation of Bcl-2/Bax mRNA and protein levels after HG treatment. ConclusionThese results demonstrated that EPF protects against diabetes-induced renal injury in vitro. EPF protected against HG-induced HMCs proliferation, apoptosis, fibrosis, and inflammation likely via inhibition of TLR4-dependent NF-κB signaling. This herbal extract may also be a novel treatment for DN.

Thalidomide Mitigates Apoptosis via Endoplasmic Reticulum Stress in Diabetic Nephropathy.

Previous studies have shown that endoplasmic reticulum (ER) stress is related to the apoptosis in the development of diabetic nephropathy (DN) and thalidomide (Thd) has renalprotective effects by suppressing inflammation and proliferation of MCs in DN. However, the effect of Thd on the apoptosis of MCs in DN remains largely unclear. The present research is designed to explore the effect of Thd on apoptosis in DN and the related mechanisms. The study is designed to examine the effect and mechanism of Thd on apoptosis in type 2 diabetic mice and high glucose (HG)-induced MCs. We first evaluated the ER stress markers and apoptosis-related proteins with the treatment of Thd in type 2 diabetic mice and MCs in vitro under HG conditions. MTT assay was used to assess cell viability. Additionally, we evaluated the effect of Thd treatment upon MC apoptosis through flow cytometry. Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate genes and protein expression related to ER stress and apoptosis. The levels of blood urea BUN, CREA, Urine albumin, and UACR in diabetic mice were observed to be significantly reduced after 8 weeks of intervention with Thd. And also, there were upregulated glucose-regulated protein 78 (GRP78), Caspase-12, and downregulated B-cell lymphoma 2 (Bcl-2) in glomeruli of DN mice. In vitro, compared with the HG group, MC apoptosis reduced dramatically with Thd treatment along with upregulation of Bcl-2 and downregulation of Bax. At the same time, ER stress markers GRP78, C/EBP homologous protein (CHOP), and Caspase-12 were also mitigated following the Thd treatment. The present study indicates that Thd might reduce the ER stress in DN via downregulating GRP78, CHOP, and Caspase12 expressions, ultimately mitigating MCs apoptosis.

Simvastatin Alleviates Vascular Cognitive Impairment Caused by Lacunar Cerebral Infarction Through Protein Kinase B/Nuclear Factor Erythroid 2–Related Factor 2 (AKT/Nrf2) Signaling Pathway

Lacunar cerebral infarction (LACI) is one of the main causes of vascular cognitive impairment (VCI). Herein, this study explored the potential effect of Simvastatin (Sim) on VCI secondary to LACI and Akt/Nrf2 signaling transduction and apoptosis. We established a rat model of VCI and the animals were administered with Sim (40 mg/kg and 80 mg/kg) every day for 28 days. After that, the cognition and memory abilities of rats were assessed together with analysis of morphological changes of hippocampal neurons by immunohistochemistry staining and level of anti-apoptotic related proteins and Akt and Nrf2 signaling proteins by western blot. Compared with normal saline (control group), Sim administration significantly improved the capacity spatial learning and relieved the memory impairment with an improvement in morphological defects. Importantly, Sim treatment restored the p-Akt, t-Nrf2, n-Nrf2 and HO-1 expression along with up-regulation of Bcl-2 and down-regulation of Bax. In conclusion, Sim improves cognitive and morphological disorders induced by LACI possibly through regulating Akt/Nrf2 signaling pathway. These evidence might promote the development of Sim-based treatment for VCI and LACI.

SOCS5 contributes to temozolomide resistance in glioblastoma by regulating Bcl-2-mediated autophagy

ABSTRACT Temozolomide (TMZ) is the primary chemotherapeutic drug for treating glioblastoma (GBM); however, the final clinical outcome is considerably limited by the poor response and resistance to TMZ. Although autophagy is thought to be associated with chemotherapy resistance and cancer cell survival, the precise molecular mechanisms underlying this process remain elusive. The suppressor of cytokine signaling (SOCS) family is widely distributed in vivo and exerts a range of effects on tumors; however, the expression pattern and role of SOCS in GBM remains unknown. In this study, we determined that high SOCS5 expression level was associated with poor prognosis and TMZ resistance in GBM. TMZ induced an increase in SOCS5 expression level and upregulated autophagy during the acquisition of drug resistance. The observed increase in the extent of autophagy was mediated by SOCS5. Mechanistically, SOCS5 enhances the transcription of Bcl-2. Knockdown of SOCS5 inhibited TMZ chemoresistance in GBM cells through the inhibition of Bcl-2 recruited autophagy; upregulation of Bcl-2 blocked this effect. In summary, our findings revealed the involvement and underlying mechanism of SOCS5 in TMZ resistance. SOCS5 plays a critical role in GBM chemoresistance and may serve as a novel prognostic marker and therapeutic target for chemotherapeutically treating drug-resistant GBM.