Bcl-2 Research Articles

Altered post-fracture systemic bone loss in a mouse model of osteocyte dysfunction

Abstract Femur fracture leads to loss of bone at uninjured skeletal sites, which may increase risk of subsequent fracture. Osteocytes, the most abundant bone cells, can directly resorb bone matrix and regulate osteoclast and osteoblast activity, but their role in systemic bone loss after fracture remains poorly understood. In this study we used a transgenic (TG+) mouse model that overexpresses human B-cell lymphoma 2 (BCL-2) in osteoblasts and osteocytes. This causes enhanced osteoblast proliferation, followed by disruption in lacunar-canalicular connectivity and massive osteocyte death by 10 weeks of age. We hypothesized that reduced viable osteocyte density would decrease the magnitude of systemic bone loss after femur fracture, reduce perilacunar remodeling, and alter callus formation. Bone remodeling was assessed using serum biomarkers of bone formation and resorption at 5 days post-fracture. We used micro-computed tomography, high resolution x-ray microscopy, mechanical testing, and Raman spectroscopy to quantify the magnitude of systemic bone loss, as well as changes in osteocyte lacunar volume, bone strength, and bone composition 2 weeks post-fracture. Fracture was associated with a reduction in circulating markers of bone resorption in non-transgenic (TG-) animals. TG+ mice exhibited high bone mass in the limbs, greater cortical elastic modulus and reduced post-yield displacement. After fracture, TG+ mice lost less trabecular bone than TG- mice, but conversely TG+ mice exhibited trends towards a lower yield point and reduced femoral cortical thickness after fracture, though these were not statistically significant. Lacunar density was greater in TG+ mice, but fracture did not alter lacunar volume in TG+ or TG- mice. These findings suggest that osteocytes potentially play a significant role in the post-traumatic systemic response to fracture, though the effects differ between trabecular and cortical bone.

Early apoptosis detection in canine granulosa cells through the analysis of BCL-2 and BAX proteins during the follicular development associated with oocyte maturation

Early apoptosis detection in canine granulosa cells through the analysis of BCL-2 and BAX proteins during the follicular development associated with oocyte maturation

In vivo, In vitro, and In silico Studies of Umbelliferone and Irinotecan on MDA-MB-231 Breast Cancer Cell Line and Drosophila melanogaster Larvae.

Deaths from cancer are still very common all over the world and continue to be the focus of scientific research. Chemotherapy is one of the primary treatments used to prevent deaths from cancer. Side effects of chemotherapeutic drugs and resistance of cells to drugs are essential problems that limit the treatment process. Drug combination therapy is regarded as a significant application that inhibits the growth of tumors and is anticipated to provide a solution for the issues encountered. The combination therapy aims at a synergistic effect that will limit drug resistance and cytotoxic effects with appropriate drug combinations. In this context, we aim to investigate the in vitro, in vivo, and in silico effects of single and combined doses of umbelliferone and irinotecan, known for their anticarcinogenic and curative effects, on MDA-MB-231 breast cancer cell lines and the model organism Drosophila melanogaster. Irinotecan is currently used as an anticarcinogenic drug. Anticarcinogenic effects of umbelliferone have also been detected. The in vivo, in vitro, and in silico impacts of single and combined doses use of these two agents are not yet available in the literature. This study aims to determine the anticarcinogenic effects of single and combined use of umbelliferone and irinotecan at the molecular level. It also attempts to determine the binding energies of chemicals to cancerrelated proteins through docking and molecular dynamic studies. The cytotoxic effects of individual and combinational doses of umbelliferone and irinotecan on the MDAMB- 231 cell line and D. melanogaster were calculated by XTT and probit analyses. IC50 values for the cancer cells, LC50, and LC99 values for D. melanogaster were found. Gene expression analysis was performed to determine the effects of chemical agents on miR-7, miR-11, and miR-14, and their expression levels were found. The sequences of miRNAs not found in the literature were determined, and their molecular imaging was performed. In addition, the binding energies of irinotecan and umbelliferone to Bcl-2, Bad, and Akt1 proteins, which are known to have apoptotic effects, were found by the molecular docking method. Molecular dynamics studies of Bad proteins and chemicals were also performed. The drug potential of chemicals was determined by ADME/T analysis. The cytotoxic effect on cells was calculated, and the IC50 value of umbelliferone was calculated as 158 μM, the IC50 value of irinotecan was calculated as 48,3 μM and the IC50 value was calculated as 20 μM. In the probit analysis performed to calculate the cytotoxic effects of drugs on D. melanogaster, the LC50 value of umbelliferone was 2,5 μM, and the LC99 value was 13,4 μM. The LC50 value of irinotecan was found to be 0,1 μM, and the LC99 value was 0,28 μM. It was concluded that single and combined doses of chemicals in the invasion experiment significantly affected the spread of cells. As a result of expression analysis, a significant increase in HsamiR- 7 (Homo sapiens miRNA-7), Hsa-miR-14 (Homo sapiens miRNA-14), and Hsa-miR-11(Homo sapiens miRNA-11) expression was observed in cells treated with umbelliferone irinotecan compared to the control groups. In our study, it can be concluded that the cytotoxic effects of individual and combination doses of umbelliferone and irinotecan on MDA-MB-231 cells and D. melanogaster larvae are significant. In addition, the effects of umbelliferone and irinotecan on the expression level of miR-7, which is a common D. melanogaster and human miRNA, should be widely investigated. Expression analyses and docking studies of Hsa-miR-11 and Hsa-miR-14, which have been newly studied and are not in data repositories, are important for cancer research. In particular, the expression and binding energy of these miRNAs in new drug combinations and the expression level in different cancer cell lines are important for future studies. Another crucial point is that in vivo tests using different model species validate the usage of drugs at both single and mixed dosages. Other: As a result of this study, the in vivo, in vitro, and in silico effects of single and combined doses of umbelliferone and irinotecan were determined. In future studies, it would be useful to determine the binding energies of umbelliferone and irinotecan to other cancer-related proteins and to find their interactions with different miRNAs. Additionally, studies on different model organisms are also important.

Venetoclax Dose Escalation Rapidly Activates a BAFF/BCL-2 Survival Axis in Chronic Lymphocytic Leukemia.

Venetoclax, a first-in-class BH3 mimetic drug targeting BCL-2, has improved outcomes for patients with chronic lymphocytic leukemia (CLL). Early measurements of the depth of the venetoclax treatment response, assessed by minimal residual disease, are strong predictors of long-term clinical outcomes. Yet, there are limited data concerning the early changes induced by venetoclax treatment that might inform strategies to improve responses. To address this gap, we conducted longitudinal mass cytometric profiling of blood cells from patients with CLL during the first five weeks of venetoclax monotherapy. At baseline, we resolved CLL heterogeneity at the single-cell level to define multiple subpopulations in all patients distinguished by proliferative, metabolic and cell survival proteins. Venetoclax induced significant reduction in all CLL subpopulations coincident with rapid upregulation of pro-survival BCL-2, BCL-XL and MCL-1 proteins in surviving cells, which had reduced sensitivity to the drug. Mouse models recapitulated the venetoclax-induced elevation of survival proteins in B cells and CLL-like cells that persisted in vivo, with genetic models demonstrating that extensive apoptosis and access to the B cell cytokine, BAFF, were essential. Accordingly, analysis of patients with CLL that were treated with venetoclax or the anti-CD20 antibody obinutuzumab exhibited marked elevation of BAFF and increased pro-survival proteins in leukemic cells that persisted. Overall, these data highlight the rapid adaptation of CLL cells to targeted therapies via homeostatic factors and support co-targeting of cytokine signals to achieve deeper and more durable long-term responses.

Experimental study on the treatment of diabetic cardiomyopathy in rats by using ultrasound-targeted microbubble destruction combined with coenzyme Q10 loaded long-circulating nanoliposomes

Objective: To investigate the therapeutic effects and mechanisms of Ultrasound-targeted microbubble destruction (UTMD) technology combined with CoQ10 loaded PEGylated nanoliposomes (CoQ10-PEG-lips) on diabetic cardiomyopathy (DCM) in rats. Methods: CoQ10-PEG-lips were prepared using the thin-film dispersion method combined with ultrasonic hydration, followed by quality assessment. Sixty healthy and clean male SD rats were selected, and 50 were randomly chosen using a random number table to establish a type 1 diabetes mellitus (DM) model via a single intraperitoneal injection of streptozotocin. The remaining 10 rats were assigned as the normal control group. A total of 47 rats successfully developed the DM model, and 40 were selected using the random number table method. Based on different intervention methods, these rats were then randomly divided into DM model group, CoQ10 solution group, CoQ10-PEG-Lips group, and CoQ10-PEG-Lips+UTMD group (n=10 per group). Normal control group and DM model group rats were injected with 1 ml of normal saline through caudal vein. CoQ10 solution group and CoQ10-PEG-Lips group were injected with 1 ml of CoQ10 solution or CoQ10-PEG-Lips solution containing 10 mg/kg of CoQ10 through caudal vein, respectively. Rats in the CoQ10-PEG-Lips+UTMD group were injected with 1 ml of CoQ10-PEG-Lips solution containing 10 mg/kg of CoQ10+100 mg of freeze-dried ultrasound microbubble powder through caudal vein and were given UTMD treatment at the same time, with the intervention given twice a week. After 12 weeks of intervention, cardiac function indexes [left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), and left ventricular ejection fraction (LVEF)]of each group were measured by ultrasonic cardiac function detection in vivo. Simultaneously, ex-vivo histopathological examination was conducted to assess myocardial cell morphology, cross-sectional area, collagen volume fraction (CVF), and apoptosis index (AI) in each group. Additionally, molecular biology techniques were employed to measure oxidative stress-related indicators and the expression of apoptosis-related pathway proteins. Results: The prepared CoQ10-PEG-lips had a well-rounded morphology, good dispersibility, and a high encapsulation efficiency of 87.45%±3.23%. After 12 weeks of intervention, the myocardial cell morphology in the CoQ10-PEG-Lips+UTMD group was intact, with orderly arrangement, closely resembling that of the normal control group. There were no statistically significant differences between the two groups in terms of LVEDs [(1.53±0.07) mm vs (1.42±0.04) mm], LVEDd [(2.93±0.15) mm vs (2.81±0.05) mm], or LVEF (80.76%±3.42% vs 84.60%±2.10%) (all P>0.05). Similarly, there were no significant differences between the two groups in myocardial cell cross-sectional area, CVF, or AI (all P>0.05). The CoQ10-PEG-Lips+UTMD group showed statistically significant differences in the above-mentioned indicators compared to the DM group, CoQ10 solution group, and CoQ10-PEG-Lips group (all P<0.05). In the CoQ10-PEG-Lips+UTMD group, the levels of myocardial superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) were significantly higher compared to the DM model group, CoQ10 solution group, and CoQ10-PEG-Lips group (all P<0.05), while malondialdehyde levels and the expression of Bcl-2-associated X protein (Bax) and caspase-3 were lower (all P<0.05). Conclusions: Using PEG-lips to encapsulate the poorly soluble drug CoQ10 in combination with UTMD technology enables targeted delivery of the drug to the myocardium, which can help reduce myocardial cell damage, fibrosis, and apoptosis caused by diabetes mellitus (DM) by inhibiting oxidative stress damage and the Bcl-2/Bax/caspase-3 apoptosis signaling pathway, ultimately improving cardiac function in DCM rats.

Computational metal-flavonoids complexes presentation of greenly synthesized silver nanoparticles combined flavonoids from Lens culinaris L. as anticancer agents using BcL-2 and IspC proteins

Lens culinaris L., has been widely recognized for its medical applications. LC-ESI-TOF-MS identified 22 secondary metabolites including phenolics, flavonoids, and anthocyanidin glycosides among its total extract (LCTE). The study aimed to apply LCTE as a biogenic material for reducing and capping the silver nanoparticles (LC-AgNPs). The ynthesized LC-AgNPs were characterized using different techniques. The UV absorption was observed at λ max 379 nm. LC-AgNPs were spherical, with 19.22 nm average size. The face cubic centre nature was demonstrated by HR-TEM and XRD. The LC-AgNPs were then evaluated for their anticancer and antimicrobial potentials. LC-AgNPs showed an extremely potent cytotoxic activity against MCF-7, HCT-116 and HepG2 cell lines (IC50= 0.37, 0.35 and 0.1 µg/mL, respectively). LC-AgNPs induced significant apoptotic effects in the three examined cancer cell lines. LC-AgNPs resulted in sequestration of cells in G1 phase of the cell cycle in both MCF-7 and HCT-116 cells, meanwhile it trapped cells at the G2 phase in HepG2 cells. Moreover, the antimicrobial activity of LC-AgNPs was highly confirmed against Klebsiella pneumoniae and Acinetobacter baumannii. Molecular docking study designated Kaempferol-3-O-robinoside-7-O-rhamnoside and Quercetin-3-D-xyloside as the topmost LCTE active constituents that caused inhibition of both Bcl-2 and IspC cancer targets in combination with the produced silver nanoparticles.

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.

Effect of fermentation on the constituents in the branches and leaves of Taxus media and non-small cell lung cancer

IntroductionNon-small cell lung cancer (NSCLC) is a prominent lung cancer disease worldwide. Currently, commonly used methods, such as surgery and radiotherapy, have significant side effects. Traditional Chinese medicine (TCM) has become a research hotspot because of its safe and effective characteristics. The branches and leaves of Taxus media are abundant in antitumor active compounds, and there has been no research conducted as yet regarding its anti–lung cancer molecular mechanism.ObjectiveThe aim of this study is to investigate the antitumor activity of two samples before and after fermentation of T. media, and to research the molecular mechanism of its inhibitory effect on NSCLC.MethodsThe chemical composition of pre-fermentation T. media (TM) and post-fermentation T. media qu (TMQ) were investigated using UHPLC-Q-Qrbitrap HRMS and high-performance liquid chromatography (HPLC). The anti-lung cancer activities of TM and TMQ were compared using an A549-induced tumor mouse model. An enzyme-linked immunosorbent assay (ELISA), hematoxylin and eosin (H&amp;amp;E) staining, immunohistochemistry, and immunofluorescence were used to determine the of TMQ mechanism of action.ResultsThe results indicated that TM and TMQ contained 83 compounds, consisting primarily of flavonoids, organic acids, and taxanes. Both taxanes and flavonoids in TMQ were higher than that in TM. Both TM and TMQ effectively inhibited the tumor growth in non-small cell lung cancer (NSCLC), and the inhibition rate was greater in TMQ (57.24%) than in TM (49.62%). TMQ administration downregulated the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and the glutathione (GSH) level and upregulated interferon-γ (IFN-γ), reactive oxygen species (ROS), and malondialdehyde (MDA) levels in the serum of tumor mice. TMQ treatment also increased the protein expression of Bax, Caspase-3, and Beclin-1 in tumor tissues. In contrast, the bcl-2, PI3K, Ki67, ULK1, and mTOR protein levels were suppressed by TMQ. Protein assay analyses reemphasized the superior antitumor effect of TMQ over TM. These cumulative findings demonstrated that the mechanism of action of TMQ was closely related to the activation of transcriptional misregulation in the cancer pathway that inhibited the cholinergic synaptic, AMPK, and PI3K/Akt/mTOR signaling pathways.ConclusionThis study demonstrated that fermentation increased the active ingredient contents and antitumor effects of T. media. In addition, post-fermentation TMQ was superior to TM as a herbal medicine for NSCLC treatment.

HIV Protein Nef Induces Cardiomyopathy Through Induction of Bcl2 and p21

HIV-associated cardiovascular diseases remain a leading cause of death in people living with HIV/AIDS (PLWHA). Although antiretroviral drugs suppress the viral load, they fail to remove the virus entirely. HIV-1 Nef protein is known to play a role in viral virulence and HIV latency. Expression of Nef protein can be detected in different organs, including cardiac tissue. Despite the established role of Nef protein in HIV-1 replication, its impact on organ function inside the human body is not clear. To understand the effect of Nef at the organ level, we created a new Nef-transgenic (Nef-TG) mouse that expresses Nef protein in the heart. Our study found that Nef expression caused inhibition of cardiac function and pathological changes in the heart with increased fibrosis, leading to heart failure and early mortality. Further, we found that cellular autophagy is significantly inhibited in the cardiac tissue of Nef-TG mice. Mechanistically, we found that Nef protein causes the accumulation of Bcl2 and Beclin-1 proteins in the tissue, which may affect the cellular autophagy system. Additionally, we found Nef expression causes upregulation of the cellular senescence marker p21 and senescence-associated b-galactosidase expression. Our findings suggest that the Nef-mediated inhibition of autophagy and induction of senescence markers may promote aging in PLWHA. Our mouse model could help us to understand the effect of Nef protein on organ function during latent HIV infection.

Noradrenaline Protects Human Microglial Cells (HMC3) Against Apoptosis and DNA Damage Induced by LPS and Aβ1-42 Aggregates In Vitro

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, characterized by the accumulation of amyloid-beta (Aβ) plaques and neuroinflammation. This study investigates the protective effects of noradrenaline (NA) on human microglial cells exposed to lipopolysaccharides (LPS) and Aβ aggregates—major contributors to inflammation and cellular damage in AD. The reduced Aβ aggregation in the HMC3 human microglial cells co-treated with Aβ and NA was confirmed by thioflavin T (ThT) assay, fluorescent ThT staining, and immunocytochemistry (ICC). The significantly increased viability of HMC3 cells after 48 h of incubation with NA at 50 µM, 25 µM, and 10 µM, exposed to IC50 LPS and IC50 Aβ, was confirmed by XTT and LDH assays. Moreover, we found that NA treatment at 25 μM and 50 μM concentrations in HMC3 cells exposed to IC50 LPS or IC50 Aβ results in an increased proliferation of HMC3 cells, their return to normal morphology, decreased levels of DNA damage, reduced caspase-3 activity, decreased expression of pro-apoptotic DDIT3 and BAX, and increased expression of anti-apoptotic BCL-2 genes and proteins, leading to enhanced cell survival, when compared to that of the HMC3 cells treated only with IC50 LPS or IC50 Aβ. Furthermore, we showed that NA induces the degradation of both extracellular and intracellular Aβ deposits and downregulates hypoxia-inducible factor 1α (HIF-1α), which is linked to impaired Aβ clearance and AD progression. These findings indicate that NA holds promise as a therapeutic target to address microglial dysfunction and potentially slow the progression of AD. Its neuroprotective effects, particularly in reducing inflammation and regulating microglial activity, warrant further investigation into its broader role in mitigating neuroinflammation and preserving microglial function in AD.

Apoptosis, Mitochondrial Autophagy, Fission, and Fusion Maintain Mitochondrial Homeostasis in Mouse Liver Under Tail Suspension Conditions

Microgravity can induce alterations in liver morphology, structure, and function, with mitochondria playing an important role in these changes. Tail suspension (TS) is a well-established model for simulating the effects of microgravity on muscles and bones, but its impact on liver function remains unclear. In the current study, we explored the regulatory mechanisms of apoptosis, autophagy, fission, and fusion in maintaining liver mitochondrial homeostasis in mice subjected to TS for 2 or 4 weeks (TS2 and TS4). The results showed the following: (1) No significant differences were observed in nuclear ultrastructure or DNA fragmentation between the control and TS-treated groups. (2) No significant differences were detected in the mitochondrial area ratio among the three groups. (3) Cysteine aspartic acid-specific protease 3 (Caspase3) activity and the Bcl-2-associated X protein (bax)/B-cell lymphoma-2 (bcl2) ratio were not higher in the TS2 and TS4 groups compared to the control group. (4) dynamin-related protein 1 (DRP1) protein expression was increased, while mitochondrial fission factor (MFF) protein levels were decreased in the TS2 and TS4 groups compared to the control, suggesting stable mitochondrial fission. (5) No significant differences were observed in the optic atrophy 1 (OPA1), mitofusin 1 and 2 (MFN1 and MFN2) protein expression levels across the three groups. (6) Mitochondrial autophagy vesicles were present in the TS2 and TS4 groups, with a significant increase in Parkin phosphorylation corresponding to the duration of the TS treatment. (7) ATP synthase and citrate synthase activities were significantly elevated in the TS2 group compared to the control group but were significantly reduced in the TS4 group compared to the TS2 group. In summary, the coordinated regulation of apoptosis, mitochondrial fission and fusion, and particularly mitochondrial autophagy preserved mitochondrial morphology and contributed to the restoration of the activities of these two key mitochondrial enzymes, thereby maintaining liver mitochondrial homeostasis in mice under TS conditions.

Baicalein alleviates palmitic acid-induced endothelial cell dysfunction via inhibiting endoplasmic reticulum stress.

Endothelial cells play a critical role in maintaining vascular function and kinetic homeostasis, but excessive accumulation of palmitic acid (PA) may lead to endoplasmic reticulum stress and trigger endothelial cell dysfunction. Baicalin (BCL), a natural plant extract, has received widespread attention for its biological activities in anti-inflammation and anti-oxidative stress. However, the mechanism of BCL on PA-induced endothelial cell dysfunction is unclear. Therefore, the aim of this study was to investigate whether BCL could inhibit PA-induced endoplasmic reticulum stress and thus attenuate endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVECs) were divided into Control, PA, PA + BCL-10 μM, PA + BCL-20 μM, and PA + BCL-50 μM groups. The PA group was treated with PA (200 μM), while the PA + BCL groups were co-treated with different concentrations of BCL (10 μM, 20 μM, 50 μM) for 24 hours. Cell viability was detected by MTT. Cell migration ability was determined by Transwell assay, apoptosis level by flow cytometry, and tube formation ability by tube formation assay. Finally, the levels of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and angiogenesis-related proteins (VEGFA and FGF2) were detected by western blot, MMP-9, as well as the protein levels of endoplasmic reticulum stress biomarkers (GRP78, CHOP, PERK, and ATF4). The results at the cellular level showed that cell viability, migration ability and tube formation ability of PA-induced HUVECs were significantly reduced, while apoptosis level was significantly increased. However, administration of different concentrations of BCL significantly enhanced PA-induced cell viability, migration ability and tube formation ability of HUVECs while inhibiting apoptosis. The results of protein levels showed that the protein levels of Bax and cleaved caspase-3 were observably up-regulated in the cells of the PA group, while the protein level of Bcl-2 was significantly down-regulated; compared with the PA group, the protein levels of Bax and cleaved caspase-3 were much lower and the Bcl-2 protein level was much higher in the PA + BCL group. Additionally, the protein levels of VEGFA, FGF2 and MMP-9 were raised and those of GRP78, CHOP, PERK and ATF4 were lowered in the PA + BCL group of cells in a concentration-dependent manner. BCL significantly attenuates PA-induced endothelial cell dysfunction by inhibiting endoplasmic reticulum stress.

“Sustainable synthesis of Camellia sinensis-mediated silver nanoparticles (CsAgNP) and their anticancer mechanisms in breast cancer cells”

The present investigation focuses on synthesizing eco-friendly and cost-effective silver nanoparticles (CsAgNP) utilizing Camellia sinensis ethanolic extract (CsE) as a reducing agent and investigating the potential enhancement in its anticancer efficacy as compared to CsE. The CsAgNP formation was confirmed through the color change from pale green to dark brown and further validated using UV–visible spectroscopy in the 400-450 nm range. The optimal CsAgNP synthesis parameters include 1:4 ratio of CsE: 1 mM AgNO3, 60 min of duration and 50 °C reaction temperature. The morphology and the size of nanoparticles were estimated using AFM, SEM and TEM where the results showed a smooth topography with a size <100 nm. The CsAgNP crystalline form was confirmed through SAED pictures and silver's presence confirmed through EDX analysis. FTIR study ascertained the capping agents and distortion in functional groups compared to CsE. The anticancer potency of CsAgNP and crude extract (CsE) was assessed against the T-47D breast cancer cells by MTT assay. CsAgNP displayed strong activity towards T-47D cells (IC50 8 μg/ml) compared to CsE and relatively low activity towards the normal HEK-293 cells. Further, fluorescence microscopy and flow cytometry data revealed that the CsAgNP promotes apoptosis and also induces G2-M phase cell cycle arrest. Furthermore, CsAgNP treatment decreases p53 and Bcl-2 protein expression, while increasing Bax, Cytochrome c and Caspase-3 levels, indicating mitochondrial-mediated apoptotic pathway activation. Thus, our research aims to investigate the potential of using Camellia sinensis to synthesize CsAgNP, a potent drug delivery system, to enhance anticancer effectiveness and advance cancer therapy in the future.

Lower-intensity CPX-351 plus venetoclax induction for adults with newly diagnosed AML unfit for intensive chemotherapy

Preclinical data suggest a rationale for combining CPX-351, a dual-drug liposomal encapsulation of daunorubicin and cytarabine, with venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor. This phase 1b study evaluated lower-intensity CPX-351 combined with venetoclax in adults with acute myeloid leukemia (AML) considered unfit/ineligible for intensive chemotherapy. In a dose-exploration phase using a 3+3 design, patients received stepwise dosing of CPX-351 intravenously on days 1 and 3 plus venetoclax 400 mg orally on days 2-21 per cycle to determine the recommended phase 2 dose (RP2D) for this combination. During the expansion phase, additional patients received CPX-351 plus venetoclax at the identified RP2D. The primary endpoints were the RP2D and safety of CPX-351 combined with venetoclax. Secondary endpoints included preliminary efficacy and pharmacokinetics. Overall, 35 patients were enrolled in the study. A RP2D of CPX-351 30 units/m2 (daunorubicin 13.2 mg/m2 and cytarabine 30 mg/m2) plus venetoclax 400 mg was established. The safety profile of the combination was consistent with the known safety profiles of CPX-351 and venetoclax. Complete remission (CR)/CR with incomplete hematologic recovery (CRi) was achieved by 17/35 (49%) patients, all after cycle 1; of these, 14 were negative for measurable residual disease. CR was achieved by 1/8 (13%) patients with a mutation in TP53, and CR/CRi was achieved by 15/26 (58%) patients with wild-type TP53. This study highlights that lower-intensity therapy of CPX-351 plus venetoclax as induction therapy provides a well-tolerated treatment option in adults with AML deemed unfit for intensive chemotherapy. This trial was registered at www.clinicaltrials.gov as #NCT04038437.

Mechanisms of Action of Sea Cucumber Triterpene Glycosides Cucumarioside A0-1 and Djakonovioside A Against Human Triple-Negative Breast Cancer.

Breast cancer is the most prevalent form of cancer in women worldwide. Triple-negative breast cancer is the most unfavorable for patients, but it is also the most sensitive to chemotherapy. Triterpene glycosides from sea cucumbers possess a high therapeutic potential as anticancer agents. This study aimed to identify the pathways triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line) by the glycosides cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi. Using flow cytometry, fluorescence microscopy, immunoblotting, and ELISA, the effects of micromolar concentrations of the compounds on cell cycle arrest, induction of apoptosis, the level of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and expression of anti- and pro-apoptotic proteins were investigated. The glycosides caused cell cycle arrest, stimulated an increase in ROS production, and decreased Δψm in MDA-MB-231 cells. The depolarization of the mitochondrial membrane caused by cucumarioside A0-1 and djakonovioside A led to an increase in the levels of APAF-1 and cytochrome C. This, in turn, resulted in the activation of caspase-9 and caspase-3 and an increase in the level of their cleaved forms. Glycosides also affected the expression of Bax and Bcl-2 proteins, which are associated with mitochondria-mediated apoptosis in MDA-MB-231 cells. These results indicate that cucumarioside A0-1 and djakonovioside A activate the intrinsic apoptotic pathway in triple-negative breast cancer cells. Additionally, it was found that treatment with Cuc A0-1 resulted in in vivo inhibition of tumor growth and metastasis of murine solid Ehrlich adenocarcinoma.

A lipid signature of BAK-driven apoptotic pore formation.

Apoptotic cell death is regulated by the BCL-2 protein family, with clusters of BAK or BAX homodimers driving pore formation in the mitochondrial outer membrane via a poorly understood process. There is growing evidence that, in addition to BAK and BAX, lipids play an important role in pore formation. Towards a better understanding of the lipidic drivers of apoptotic pore formation in isolated mitochondria, two complementary approaches were taken. Firstly, the lipids released during BAK-mediated pore formation were measured with targeted lipidomics, revealing enrichment of long chain polyunsaturated lysophospholipids (LPLs) in the released fraction. In contrast, the BAK protein was not released suggesting that BAK and LPLs locate to distinct microdomains. Secondly, added cholesterol not only prevented pore formation but prevented the clustering of BAK homodimers. Our data lead us to a model in which BAK clustering triggers formation of a separate microdomain rich in LPLs that can progress to lipid shedding and the opening of a lipid-lined pore. Pore stabilisation and growth may be due to BAK dimers then moving to the pore edge. Our BAK-lipid microdomain model supports the heterogeneity of BAK assemblies, and the observed lipid-release signature gives new insight into the genesis of the apoptotic pore.

Genetic Profiling in Chronic Lymphocytic Leukemia: The Role of TP53 Mutations and IGHV Status in Treatment Decision-Making – A Case Report

Introduction. The TP53 gene encodes the p53 protein, crucial for DNA damage response, apoptosis, and cell cycle regulation. In chronic lymphocytic leukemia (CLL), TP53 loss due to 17p deletion or mutation leads to poor chemoimmunotherapy response and shorter survival. Patients with unmutated immunoglobulin heavy chain variable (IGHV) status exhibit a more aggressive disease course, poorer outcomes, and reduced response to standard chemoimmunotherapy. These genetic variations significantly affect disease progression, treatment choice, and response to therapy. Case Report. In November 2023, a 61-year-old man presented to our clinic with complaints of generalized weakness and rapid fatigue since October 2023. CLL was diagnosed in 2018 through pathohistological and immunohistochemical analysis of the bone marrow. The patient received no therapy until 2023 due to the absence of active disease signs. From March 28, 2023, to July 25, 2023, the patient received chemoimmunotherapy with the bendamustine and rituximab (BR) regimen. Five courses of therapy were administered; however, further treatment was discontinued due to side effects that occurred during the fifth course of bendamustine administration. A subsequent diagnostic evaluation aimed at assessing treatment efficacy, prognosis, and genetic profiling included a cytomorphological analysis, which revealed that lymphocytes comprised 80% of the cellular composition. Immunocytological analysis confirmed the presence of monoclonal atypical lymphocytes, accounting for 70% of the cell population. Next-generation sequencing identified a pathogenic TP53 mutation, and fragment analysis confirmed an unmutated IGHV status. The presence of the TP53 mutation and unmutated IGHV status categorizes the patient as being in the very high-risk group, for which the use of Bruton’s tyrosine kinase inhibitors (BTKIs) and/or B-cell lymphoma-2 (BCL-2) inhibitors is recommended. Conclusions. This case highlights the significance of comprehensive genetic profiling in CLL patients using techniques such as fluorescence in situ hybridization, polymerase chain reaction, and next-generation sequencing. Such profiling detects molecular genetic alterations, facilitating personalized and effective treatment.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

Transforming growth factor-beta and Epithelial-mesenchymal transition is considered an attractive pivotal Genetic-Biomarker for Hepatocellular carcinoma with hepatitis C virus-infection patients

Abstract Introduction/Objective Hepatocellular carcinoma (HCC) is the most common cause of cancer worldwide. HCC is induced by hepatitis C-virus (HCV) which represents a major health problem in Egypt. The transforming growth factor- beta (TGF-β) is a potent cytokine that has a multifunctional pro-fibrotic activity, for enhancing liver-inflammation, fibrosis, tumor-progression, and metastasis. TGF-β has a vital role in the regulation of tumorigenesis, by controlling epithelial-mesenchymal-transition (EMT), apoptosis, proliferation, and differentiation process. The dysregulation of TGF-β, Wnt/β-catenin signaling pathways, by HCV-core protein is implicated in the development of HCC. Methods/Case Report The study aims to determine the effect of the HCV-infection in HCC patients with a set of growth-factors, EMT, and anti-apoptotic protein expression levels by using western blotting and immunohistochemistry staining analysis. Results (if a Case Study enter NA) These results observed an insignificant increase in the expression of TGF-β protein for HCC/HCV-infected cells compared to the HCV-uninfected cells, and the expression of the β-catenin protein level is elevated in the nucleus. Also, it showed that Bcl-2 protein is a significant increase in HCV-infected when compared to HCV-free groups. Positive staining for p53 protein was detected in the HCC/HCV-infected, more than in HCC/HCV-uninfected cells. This data indicated that the core-protein of HCV can switch TGF-β from a tumor suppressor to a tumor promoter by suppressing apoptosis of hepatocytes and increasing EMT expression by activation of Wnt/β-catenin signaling pathway. Therefore, HCV-core protein enhances the nuclear accumulation of β-catenin expression protein to activate downstream target genes, which regulate cell-growth and cell-cycle progression. Conclusion It is speculated that HCV may exert an anti-apoptotic effect, and thus enhance tumorigenesis. Also, the accumulation of nuclear Wnt/β-catenin and p53 correlates with TGF-β expression, typically a late event in hepato- carcinogenesis. Taken together, the expression level of TGF-β, Wnt/β-catenin, Bcl-2, and p53 genotype in HCV is considered an indicator of shortened survival in patients, and may be an attractive clinically pivotal genetic biomarker to mediate liver pathogenesis.

Quercetin ameliorates lupus symptoms by promoting the apoptosis of senescent Tfh cells via the Bcl-2 pathway

Systemic lupus erythematosus (SLE) is an autoimmune disorder that commonly affects the skin, kidneys, joints, and various other systemic tissues, with its development intricately linked to the process of immunosenescence. Quercetin (QC), a phytochemical that occurs naturally, demonstrates many different biological capabilities, such as antibacterial, antioxidant, and anti-inflammatory activities. Our investigation found that QC effectively reduced kidney damage and relieved mesenteric lymph nodes (mLNs) swelling in MRL/lpr lupus mice. Moreover, QC has been found to decrease the number of senescent follicular helper T (Tfh) cells, a pivotal kind of T cells that contribute to the progression of SLE. In vitro, QC exhibited the capacity to modulate mRNA expression levels, with the downregulation of IL-6, IL21-AS1, IL-27, BCL6, and BCL2L12, and the upregulation of FOXP1 and BIM. This modulation resulted in the suppression of Tfh cells differentiation and the enhancement of apoptosis in senescent CD4+ T cells. In addition, the HuProtTM Human Proteome Microarray revealed that QC can directly bind to BCL-2 protein and therefore promote the apoptosis of senescent CD4+ T cell. As a result, our investigative elucidate the potent inhibitory action of QC on the ontogeny of Tfh cells, along with its capacity to abrogate the immunosenescent phenotype. This positions QC as a promising therapeutic strategy for treating SLE.