Levels Of Bcl-2 Family Proteins Research Articles

ETFDH mutation involves excessive apoptosis and neurite outgrowth defect via Bcl2 pathway

The most common mutation in southern Chinese individuals with late-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD; a fatty acid metabolism disorder) is c.250G > A (p.Ala84Thr) in the electron transfer flavoprotein dehydrogenase gene (ETFDH). Various phenotypes, including episodic weakness or rhabdomyolysis, exercise intolerance, and peripheral neuropathy, have been reported in both muscular and neuronal contexts. Our cellular models of MADD exhibit neurite growth defects and excessive apoptosis. Given that axonal degeneration and neuronal apoptosis may be regulated by B-cell lymphoma (BCL)-2 family proteins and mitochondrial outer membrane permeabilization through the activation of proapoptotic molecules, we measured the expression levels of proapoptotic BCL-2 family proteins (e.g., BCL-2-associated X protein and p53-upregulated modulator of apoptosis), cytochrome c, caspase-3, and caspase-9 in NSC-34 cells carrying the most common ETFDH mutation. The levels of these proteins were higher in the mutant cells than in the wide-type cells. Subsequent treatment of the mutant cells with coenzyme Q10 downregulated activated protein expression and mitigated neurite growth defects. These results suggest that the activation of the BCL-2/mitochondrial outer membrane permeabilization/apoptosis pathway promotes apoptosis in cellular models of MADD and that coenzyme Q10 can reverse this effect. Our findings aid the development of novel therapeutic strategies for reducing axonal degeneration and neuronal apoptosis in MADD.

Role of layilin in regulating mitochondria-mediated apoptosis: a study on B cell lymphoma (BCL)-2 family proteins

BackgroundMalignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells.ResultsWe compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions.ConclusionThis study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis.

5-Aminolevulinic acid-mediated photodynamic therapy in combination with kinase inhibitor lapatinib enhances glioblastoma cell death.

5-Aminolevulinic acid (ALA) is an intraoperative imaging agent approved for protoporphyrin IX (PpIX) fluorescence-guided resection of glioblastoma (GBM). It is currently under clinical evaluation for photodynamic therapy (PDT) after the completion of GBM surgery. We previously showed that lapatinib, a clinical kinase inhibitor of epidermal growth factor receptor 1 & 2 (EGFR and HER2), enhanced PpIX fluorescence in a panel of GBM cell lines by blocking ABCG2 (ATP-binding cassette super-family G member 2)-mediated PpIX efflux, which suggests its potential for improving ALA for GBM surgery and PDT. Here we show that lapatinib enhanced PDT-induced cytotoxicity by promoting GBM cell death with the induction of apoptosis followed by necrosis. While the induction of tumor cell apoptosis was massive and rapid in the H4 cell line with no detectable Bcl-2 and a low level of Bcl-xL, it was delayed and much less in extent in A172, U-87 and U-118 cell lines with higher levels of pro-survival Bcl-2 family proteins. Lapatinib treatment alone neither reduced GBM cell viability nor had any significant effect on EGFR downstream signaling. Its enhancement of ALA-PDT was largely due to the increase of intracellular PpIX particularly in the mitochondria, resulting in the activation of mitochondria-mediated apoptosis in H4 cells. Our present study demonstrates that lapatinib inhibits ABCG2-mediated PpIX efflux and sensitizes GBM cells to ALA-PDT by inducing tumor cell death.

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

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

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Differential expression pattern of Bcl-2 family members in B and T cells in systemic lupus erythematosus and rheumatoid arthritis

ObjectiveThis study aimed to evaluate the expression level of anti-apoptotic Bcl-2 family proteins in B and T cells in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in relation to disease activity and the effect of various Bcl-2 family inhibitors (BH3 mimetics) as potential treatment.MethodsWe included 14 SLE patients, 12 RA patients, and 13 healthy controls to study anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 expression and cell survival in different B and T cell subsets using stimulation assays and intracellular flow cytometry. Effect of various BH3 mimetics was assessed by cell viability analyses.ResultsIn SLE, significant differences in Bcl-2 family members were confined to the B cell compartment with decreased induction of Bcl-XL (p ≤ 0.05) and Mcl-1 (p ≤ 0.001) upon CpG stimulation. In RA, we did not observe any differences in expression levels of Bcl-2 family proteins. Expression patterns did not correlate with disease activity apart from decreased induction of Mcl-1 in B cells in active SLE. After in vitro stimulation with CpG, plasmablasts were more viable after treatment with three different BH3 mimetics compared to naïve or memory B cells in control and patient cells. After activation, Mcl-1 inhibition was most effective in reducing plasmablast and T cell viability, however, less in patients than controls.ConclusionOur study provides evidence for the increased differential expression pattern of Bcl-2 family members in B and T cell subsets of patients with SLE compared to controls. Tested BH3 mimetics showed higher efficacy in controls compared to both autoimmune diseases, though nonsignificant due to low patient numbers.

LP-118, a Selective Bcl-2 Inhibitor with Tuned Bcl-Xl Activity, Causes Myeloid Differentiation and Cell Death in Acute Myeloid Leukemia (AML)

Introduction Targeted therapy against bcl-2 has proven efficacious in AML, notably with bcl-2 inhibitor venetoclax. Bcl-2 is one of several proteins in the bcl-2 protein family of anti-apoptotic proteins. Primary and adaptive resistance to venetoclax-induced apoptosis is common and can be due to increased levels of other bcl-2 family proteins besides bcl-2, notably bcl-xL and mcl-1. LP-118, a next generation oral, highly potent, selective bcl-2 inhibitor with tuned bcl-xL activity, has demonstrated preliminary efficacy in venetoclax relapsed/refractory (R/R) acute lymphocytic leukemia cell lines. Methods Cell-based viability assays (MTS) were performed in primary AML cells and AML cell lines. Colony forming unit (CFU) assays were performed on primary AML cells and CD34+ stem cells from healthy donor umbilical cord blood. An in vivo study was conducted in a MOLM-13 AML mouse model (Charles River Laboratories; 8-week-old male NGC mice were injected with AML cells day 1, started treatment day 4). Immunoblot studies were performed to measure on-target activity of LP-118 and bcl-2 family proteins. Flow cytometry and cytospins were performed on primary AML cells and AML cell lines to test for maturation. Results A 24-hour (hr) continuous exposure of LP-118 against AML cell lines demonstrated growth inhibition in all except OCI-AML3 and U937 (also resistant to venetoclax), with an IC50 in the nM range of 2.2 in MOLM-13 (venetoclax IC50 9), 3.5 in MV4-11, 0.75 in HL-60 (venetoclax IC50 2), 86.4 in K562, 7.8 in KG-1 (venetoclax IC50 30), and 4.3 in Kasumi-1 (venetoclax IC50 20). A 72-hr continuous exposure of LP-118 against primary AML cells demonstrated growth inhibition with an IC50 of 12.9uM. Venetoclax R/R primary AML cells were sensitive to LP-118 between 5 and 100uM after 72-hr continuous exposure. Primary AML cells also exhibited a 63% decrease in CFUs at 10uM dose from vehicle, while CD34+ stem cells maintained colony forming capability by CFU assay. Induction of apoptosis was observed with caspase-3 and PARP cleavage on immunoblot in AML cell lines and primary AML cells. Relative baseline levels of bcl-2 family proteins by immunoblot did not correlate with AML cell line sensitivity or resistance to LP-118. Following 4- and 24-hr exposure to LP-118 in MOLM-13, MV4-11, and Kasumi-1 cell lines, BCL-2, BCL-xL, MCL-1, and BCL-W protein levels were not increased relative to vehicle by immunoblot, but BCL2A1 levels increased 3-fold. Similar results were seen following a 24-hour exposure to LP-118 in primary AML cells. Myeloid differentiation was demonstrated in primary AML cells (n=3) and AML cell lines MOLM-13 (n=2) and Kasumi-1 (n=6) by flow cytometric detection of CD11b expression after 48- and 72-hr continuous exposure to LP-118, as well as, morphologically on cytospin images. CD11b was also detected after 48-hr continuous venetoclax exposure on Kasumi-1 cells. Limiting-cell RNA-sequencing is ongoing to identify differentially expressed genes following LP-118-induced myeloid differentiation. In a MOLM-13 mouse model, single-agent LP-118 (50mg/kg PO daily) had a statistically significant median overall survival (mOS) of 24 days compared to control (20 days), but venetoclax (100mg/kg daily for 3 days then weekly) did not (mOS 21 days). There was no significant difference in OS between LP-118+azacitidine (5mg/kg IP days 1-5 of 21-day cycle) and venetoclax+azacitidine. After 60 days, the median OS of LP-118+gilteritinib (15.24 mg/kg PO daily) was not reached and showed increased OS compared to azactidine+gilteritinib and venetoclax+azacitidine. By splenic histopathology, blasts are notably reduced in mice treated with azacitidine+gilteritnib, but spleens are effaced by neoplastic blasts in all other treatment groups. Conclusions LP-118 shows effective cell killing in multiple AML cell lines and primary samples with various molecular and cytogenetic profiles, including a patient sample with a PTPN11 mutation, against which venetoclax is known to be ineffective. Compared to venetoclax, LP-118 is effective at lower IC50 values in AML cell lines and is active in venetoclax R/R primary AML samples. LP-118 also proves efficacious in vivo, exhibiting improved OS and decreased disease burden. Finally, LP-118 induces myeloid differentiation, to our knowledge a novel mechanism of action of bcl-2/bcl-xL inhibition in AML. These data support the recently initiated AML clinical trial (NCT04771572).

Nintedanib induces senolytic effect via STAT3 inhibition

Selective removal of senescent cells, or senolytic therapy, has been proposed to be a potent strategy for overcoming age-related diseases and even for reversing aging. We found that nintedanib, a tyrosine kinase inhibitor, selectively induced the death of primary human dermal fibroblasts undergoing RS. Similar to ABT263, a well-known senolytic agent, nintedanib triggered intrinsic apoptosis in senescent cells. Additionally, at the concentration producing the senolytic effect, nintedanib arrested the cell cycle of nonsenescent cells in the G1 phase without inducing cytotoxicity. Interestingly, the mechanism by which nintedanib activated caspase-9 in the intrinsic apoptotic pathway differed from that of ABT263 apoptosis induction; specifically, nintedanib did not decrease the levels of Bcl-2 family proteins in senescent cells. Moreover, nintedanib suppressed the activation of the JAK2/STAT3 pathway, which caused the drug-induced death of senescent cells. STAT3 knockdown in senescent cells induced caspase activation. Moreover, nintedanib reduced the number of senescence-associated β-galactosidase-positive senescent cells in parallel with a reduction in STAT3 phosphorylation and ameliorated collagen deposition in a mouse model of bleomycin-induced lung fibrosis. Consistently, nintedanib exhibited a senolytic effect through bleomycin-induced senescence of human pulmonary fibroblasts. Overall, we found that nintedanib can be used as a new senolytic agent and that inhibiting STAT3 may be an approach for inducing the selective death of senescent cells. Our findings pave the way for expanding the senolytic toolkit for use in various aging statuses and age-related diseases.

EA.hy926 Cells and HUVECs Share Similar Senescence Phenotypes but Respond Differently to the Senolytic Drug ABT-263.

Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-β-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.

Synthesis and evaluation of iridium(III) complexes on antineoplastic activity against human gastric carcinoma SGC-7901 cells.

The study was intended to determine the antineoplastic effects of two new iridium(III) complexes [Ir(ppy)2(PTTP)](PF6) (1) (ppy = 2-phenylpyridine) and [Ir(piq)2(PTTP)](PF6) (2) (piq = 1-phenylisoquinoline, PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene). In MTT assay, the ligand PTTP displayed ineffective inhibition on cell growth in SGC-7901, BEL-7402, HepG2 as well as NIH3T3 cell lines, while complexes 1 and 2 showed high cytotoxic activity on SGC-7901 cells with an IC50 value of 0.5 ± 0.1µM and 4.4 ± 0.6µM, respectively. Cellular uptake, cell cloning experiments, wound healing assay and cell cycle arrest indicated that the two complexes can inhibit the cell proliferation in SGC-7901 and induce cell cycle arrest at G0/G1 phase. Additionally, reactive oxygen species (ROS) and mitochondrial membrane potential suggested that the two complexes induced cell apoptosis through disrupting mitochondrial functions. Further, western blot analysis illustrated that the two complexes caused apoptosis via regulating expression levels of Bcl-2 family proteins. Moreover, complex 1 could suppress tumor growth in vivo with an inhibitory rate of 49.41%. Altogether, these results demonstrated that complexes 1 and 2 exert a potent anticancer effect against SGC-7901 cells via mitochondrial apoptotic pathway and have a potential to be developed as antineoplastic drug candidates for human gastric cancer.

Retraction Note: Dioscin induces prostate cancer cell apoptosis through activation of estrogen receptor-\u03b2

Recent researches have shown that estrogen receptor-β (ERβ) activator may be a potent anticancer agent for prostate cancer (PCa), and our previous study also indicated that dioscin can upregulate the expression of ERβ in MC3T3-E1 cell. In the present work, the activity and mechanism of dioscin, a natural product, against PCa were investigated. The results showed that dioscin markedly inhibited cell viability, colony formation, motility and induced apoptosis in PC3 cells. Moreover, dioscin disrupted the formation of PC3 cell-derived mammospheres and reduced aldehyde dehydrogenase (ALDH) level and the CD133+/CD44+ cells, indicating that dioscin had a potent inhibitory activity on prostate cancer stem cells (PCSCs). In vivo results also showed that dioscin significantly suppressed the tumor growth of PC3 cell xenografts in nude mice. Furthermore, mechanism investigation showed that dioscin markedly upregulated ERβ expression level, subsequently increased prolyl hydroxylase 2 level, decreased the levels of hypoxia-inducible factor-1α, vascular endothelial growth factor A and BMI-1, and thus induced cell apoptosis by regulating the expression levels of caspase-3 and Bcl-2 family proteins. In addition, transfection experiment of ERβ-siRNA further indicated that diosicn showed excellent activity against PCa in vitro and in vivo by increasing ERβ expression level. The co-immunoprecipitation (Co-IP) results further suggested that dioscin promoted the interaction of c-ABL and ERβ, but did not change c-ABL expression. Moreover, the molecular docking assay showed that dioscin processed powerful affinity toward to ERβ mainly through the strong hydrogen bonding and hydrophobic effects, and the actions of dioscin on ERβ activation and tumor cells inhibition were significantly weakened in the mutational (Phe-336, Phe-468) PC3 cells. Collectively, these findings proved that dioscin exerted efficient anti-PCa activity via activation of ERβ, which should be developed as an efficient candidate in clinical for treating this cancer in the future.

Leucine Reconstitutes Phagocytosis-Induced Cell Death in E. coli-Infected Neonatal Monocytes-Effects on Energy Metabolism and mTOR Signaling.

MΦ differentiate from circulating monocytes (Mo). The reduced ability of neonatal Mo to undergo apoptosis after E. coli infection (phagocytosis-induced cell death (PICD)) could contribute to sustained inflammatory processes. The objective of our study was to investigate whether immune metabolism in Mo can be modified to gain access to pro-apoptotic signaling. To this end, we supplemented Mo from neonates and from adults with the branched amino acid leucine. In neonatal Mo, we observed increased energy production via oxidative phosphorylation (Oxphos) after E. coli infection via Seahorse assay. Leucine did not change phagocytic properties. In neonatal Mo, we detected temporal activation of the AKT and mTOR pathways, accompanied with subsequent activation of downstream targets S6 Kinase (S6K) and S6. FACS analyses showed that once mTOR activation was terminated, the level of anti-apoptotic BCL-2 family proteins (BCL-2; BCL-XL) decreased. Release of cytochrome C and cleavage of caspase-3 indicated involvement of the intrinsic apoptotic pathway. Concomitantly, the PICD of neonatal Mo was initiated, as detected by hypodiploid DNA. This process was sensitive to rapamycin and metformin, suggesting a functional link between AKT, mTOR and the control of intrinsic apoptotic signaling. These features were unique to neonatal Mo and could not be observed in adult Mo. Supplementation with leucine therefore could be beneficial to reduce sustained inflammation in septic neonates.

Effects of Amyloid Precursor Protein Overexpression on NF-κB, Rho-GTPase and Pro-Apoptosis Bcl-2 Pathways in Neuronal Cells.

Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of amyloid-beta peptides, play a pivotal role in AD pathophysiology. This study focuses on the evaluation of the effects of amyloid precursor protein (APP) overexpression on NF-κB, Rho-GTPase and Bcl-2 mediated pro-apoptotic pathways in neuronal cells. A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells. In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group. APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.

Diclofenac Sodium Triggers p53-Dependent Apoptosis in Human Corneal Epithelial Cells via ROS-Mediated Crosstalk.

Diclofenac sodium (DFS), a nonsteroidal anti-inflammatory drug, is frequently used in ophthalmology, but it causes negative effects on corneas. The mechanisms underlying the toxicities to corneas remains unclear. The present study was designed to assess the cytotoxicity of DFS to human corneal epithelial (HCEP) cells in vitro and further investigate its related mechanisms. The HCEP cells were treated with DFS at different concentrations ranging from 0.003 125% to 0.1%. DFS showed a dose- and time-dependent cytotoxicity to HCEP cells including abnormal morphology and declined viability. The 0.05% DFS-treated HCEP cells presented cell cycle arrest at S phase, reactive oxygen species (ROS) overproduction, and positive staining of phosphorylated H2AX, suggesting that DFS caused ROS-mediated DNA damage. The upregulation of p53 expression, formation of apoptotic body, phosphatidylserine externalization, and DNA ladder demonstrated that the p53-dependent apoptosis pathway was involved in the cytotoxicity of DFS. Furthermore, DFS activated caspase-8, caspase-9, and caspase-3 altered the expression levels of Bcl-2 family proteins including tBid, Bax, and Bcl-2, as well as increased poly(ADP-ribose) polymerase (PARP) cleavage. DFS also induced ΔΨm disruption, resulting in the release of cytochrome c and apoptosis-inducing factor into the cytoplasm. Additionally, the DFS-induced apoptosis was alleviated by p53 inhibitor. Taken together, DFS triggered p53-dependent apoptosis in HCEP cells via ROS-mediated crosstalk between the extrinsic and intrinsic pathways.

Effect of Calomelanone, a Dihydrochalcone Analogue, on Human Cancer Apoptosis/Regulated Cell Death in an In Vitro Model.

Calomelanone, 2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, possesses anticancer activities. This study was conducted to investigate the cytotoxic effect of calomelanone, a dihydrochalcone analogue, on human cancer cells and its associated mechanisms. The cytotoxic effect of calomelanone was measured by MTT assay. Annexin V-FITC/propidium iodide and DiOC6 staining that employed flow cytometry were used to determine the mode of cell death and reduction of mitochondrial transmembrane potential (MTP), respectively. Caspase activities were measured using specific substrates and colorimetric analysis. The expression levels of Bcl-2 family proteins were determined by immunoblotting. Reactive oxygen species were also measured using 2′,7′-dihydrodichlorofluorescein diacetate and dihydroethidium (fluorescence dyes). Calomelanone was found to be toxic towards various human cancer cells, including acute promyelocytic HL-60 and monocytic leukemic U937 cells, in a dose-dependent manner at 24 h and human hepatocellular HepG2 cells at 48 h. However, the proliferation of HepG2 cells increased at 24 h. Calomelanone was found to induce apoptosis in HL-60 and U937 at 24 h and HepG2 apoptosis at 48 h via the intrinsic pathway by inducing MTP disruption. This compound also induced caspase-3, caspase-8, and caspase-9 activities. Calomelanone upregulated proapoptotic Bax and Bak and downregulated antiapoptotic Bcl-xL proteins in HepG2 cells. Moreover, signaling was also associated with oxidative stress in HepG2 cells. Calomelanone induced autophagy at 24 h of treatment, which was evidenced by staining with monodansylcadaverine (MDC) to represent autophagic flux. This was associated with a decrease of Akt (survival pathway) and an upregulation of Atg5 (the marker of autophagy). Thus, calomelanone induced apoptosis/regulated cell death in HL-60, U937, and HepG2 cells. However, it also induced autophagy in HepG2 depending on duration, dose, and type of cells. Thus, calomelanone could be used as a potential anticancer agent for cancer treatment. Nevertheless, acute and chronic toxicity should be further investigated in animals before conducting investigations in human patients.

A Cerium Vanadate Nanozyme with Specific Superoxide Dismutase Activity Regulates Mitochondrial Function and ATP Synthesis in Neuronal Cells.

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress.

Erianthridin Induces Non-small Cell Lung Cancer Cell Apoptosis through the Suppression of Extracellular Signal-regulated Kinase Activity.

Due to the high mortality of lung cancer, natural derivative compounds have been promoted as versatile sources for anticancer drug discovery. Erianthridin, a phenanthrene compound isolated from Dendrobium formosum, exhibits intriguing apoptosis-inducing effects in non-small cell lung cancer cells. Apoptotic nuclei staining assays showed that apoptotic cells with DNA fragmentation and apoptotic bodies were apparent, and an increase in annexin V-FITC-positive cells were found in cells treated with erianthridin. The apoptosis protein markers for cleaved caspase-3 and cleaved poly-ADP-ribose polymerase were significantly upregulated in response to erianthridin. A mechanistic investigation revealed that erianthridin was able to attenuate extracellular signal-regulated kinase activity and thereby mediate apoptosis through the modulation of Bcl-2 family protein levels. U0126, an extracellular signal-regulated kinase inhibitor, augmented the apoptosis-inducing effect of erianthridin; in contrast, overexpression of exogenous extracellular signal-regulated kinase substantially abrogated erianthridin activity. Furthermore, an in vitro 3D tumorigenesis assay showed that erianthridin was able to potentially suppress lung cancer cell proliferation. This study is the first to report a promising cytotoxic effect of erianthridin, which provides preclinical evidence for further research and development of this compound.

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI &amp;lt; 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI &amp;gt; 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Pharmacological Induction of NOXA Sensitizes High-Risk B Cell Acute Lymphoblastic Leukemia Cells to Venetoclax

Background: Venetoclax (VEN), a specific BCL2 inhibitor, exerts anti-leukemic effects in various high-risk (HR) B-ALL subtypes, such as ALL with mixed lineage leukemia (MLL) gene rearrangements (MLLr ALL) (PMID: 26711339), Philadelphia chromosome-positive (Ph+) (PMID 30546081) or hypodiploid B-ALL (PMID 30862722). Nevertheless, despite high rationale for targeting BCL2 in these subtypes of B-ALL, VEN monotherapy is not effective enough to completely eliminate leukemic cells. For this reason identification of other drugs that could sensitize leukemic cells to VEN may become beneficial treatment strategy in HR ALL. Previously, we showed that the enzymes of the thioredoxin system are upregulated in primary B-ALL cells and that auranofin (AUR), a thioredoxin reductase inhibitor, effectively kills leukemic cells in vitro and in vivo. Importantly, elements of the thioredoxin system are not only balancing redox homeostasis within the cells, but may also interact with other pathways, including anti-apoptotic signaling. Considering above, we hypothesized that AUR may potentiate VEN efficacy in HR B-ALL. Methods: To evaluate cytostatic/cytotoxic effects of VEN+AUR combination by MTT assay and propidium iodide (PI)-staining we used HR B-ALL cell lines, including SEM (MLLr ALL), BV-173 (Ph+ ALL) and NALM-16 (hypodiploid ALL). Patient derived xenograft cells (PDX) were generated through long-term propagation of primary B-ALL samples in immune-deficient NSG mice. Ex vivo drug testing in co-culture system was performed using primary bone marrow-derived mesenchymal stem cells (BM-MSC) and murine stromal OP9 cell line. NOXA genomic knockout (KO) in SEM cells was established by CRISPR/Cas9 system. Chromatin accessibility within PMAIP1 gene (encodes for NOXA) was detected using ATAC-seq. Results: We observed that AUR sensitizes HR B-ALL cell lines to VEN, as determined by MTT and PI-staining. Further, we mimicked the bone marrow support of stromal cells towards B-ALL and evaluated its impact on the response to VEN+AUR. For this reason we employed an ex vivo co-culture system of B-ALL PDX cells with primary BM-MSC or an OP9 cell line. In all tested PDX samples representing diagnostic/relapsed MLLr ALL (n=8), Ph+ ALL (n=2) and Ph-like ALL (n=2) we observed synergistic effect of this combination (Fig. 1A). Next, we determined the efficacy of VEN+AUR combination in vivo using a PDX model of MLLr B-ALL. We observed that administration of VEN+AUR diminished the progression of leukemia during a 3 week-long treatment more effectively than any single drug alone, which reflected in longer survival of NSG mice (Fig. 1B). Subsequently, we aimed to uncover the mechanism responsible for the synergistic action of VEN+AUR. In cells treated with both drugs we observed enhanced caspase activation and changes in the levels of BCL2 family proteins involved in apoptotic signaling. In particular, we found that AUR strongly upregulates a pro-apoptotic NOXA protein, both in HR B-ALL cell lines and in MLLr ALL PDX samples (Fig. 1C). To evaluate whether NOXA induction is functionally relevant for the cell death mediated by VEN+AUR, we generated SEM cells with a NOXA genomic KO. Lack of NOXA significantly abolished VEN-single agent as well as VEN+AUR combination cytotoxicity, demonstrating its dependence on NOXA expression (Fig. 1D). We then showed that NOXA is regulated at the transcriptional level, as co-treatment with AUR and the transcription inhibitor, actinomycin D, abolished AUR-mediated NOXA induction at mRNA and protein levels in SEM cells. Additionally, to test whether AUR-treatment itself provokes changes in chromatin accessibility within the NOXA encoding gene (PMAIP1) we performed ATAC-seq. We observed a clear increase in accessibility at PMAIP1 in response to AUR, which correlated with transcriptional induction of NOXA. Moreover, ChIP-qPCR revealed that increased ATAC peaks within PMAIP1 were associated with an increase in H3 lysine 27 acetylation (H3K27ac) - an epigenetic mark associated with open chromatin conformation. Conclusions: Our results demonstrate that FDA-approved drug, AUR, is a promising candidate to be used in combination with VEN for the therapy of HR B-ALL subtypes. Importantly, NOXA induction by AUR plays a central role in the VEN+AUR synergistic cytotoxicity. More studies elucidating the mechanism of NOXA upregulation by AUR are underway. Disclosures Milne: OxStem Oncology (OSO), a subsidiary company of OxStem Ltd.: Other: Founding shareholder .

Isolinderalactone Induces Cell Death via Mitochondrial Superoxide- and STAT3-Mediated Pathways in Human Ovarian Cancer Cells.

The mortality rate of ovarian cancer (OC) worldwide increases with age. OC is an often fatal cancer with a curative rate of only 20–30%, as symptoms often appear after disease progression. Studies have reported that isolinderalactone (ILL), a furanosesquiterpene derivative extracted from the dried root of Lindera aggregata, can inhibit several cancer cell lines’ growth. However, the molecular mechanisms underlying ILL activities in human OC cells remain unexplored. This study investigated the antitumor activities of ILL in human OC cells by inducing mitochondrial superoxide (mtSO) and JAK-signal transducer and activator of transcription 3 (STAT3)-dependent cell death. ILL caused cell death in SKOV-3 and OVCAR-3 cells and increased the cell proportion in the subG1 phase. Additionally, ILL significantly induced mtSO production and reduced ROS production. Moreover, ILL downregulated mitochondrial membrane potential and the expression levels of anti-apoptotic Bcl-2 family proteins and superoxide dismutase (SOD)2. Results showed that ILL decreased phosphorylation of serine 727 and tyrosine 705 of STAT3 and expression of survivin, a STAT3-regulated gene. Furthermore, ILL-induced cell death was reversed by pretreatment of Mito-TEMPO, a mitochondria-specific antioxidant. These results suggest that ILL induces cell death by upregulation of mtSO, downregulation of mitochondrial SOD2, and inactivation of the STAT3-mediated pathway.