Low Bcl-2 Expression Research Articles

Unveiling the therapeutic potential of anthocyanin/cisplatin-loaded chitosan nanoparticles against breast and liver cancers

BackgroundLiver and breast cancers are among the leading causes of cancer-related deaths worldwide, prompting researchers to seek natural anticancer agents and reduce chemotherapy side effects. Red beetroot (Beta vulgaris Linnaeus), rich in polyphenols and powerful antioxidants, has shown potential in cancer prevention. This study aimed to evaluate the impact of red beetroot-derived anthocyanin (Ant), Ant-loaded chitosan nanoparticles (Ant NPs), cisplatin (Cis), Cis-loaded chitosan (Cis NPs), and Cis + Ant-loaded chitosan NPs on human hepatoma HepG2 and breast adenocarcinoma MCF7 cell lines.MethodsNPs preparation was evaluated by zeta potential, FTIR, and SEM. The cytotoxic, apoptotic, antioxidant, anti-inflammatory, anti-metastatic, and anti-angiogenic effects were assessed by MTT assay, qPCR, AO/EB staining, and flow cytometry.ResultsTreatment with Ant, Ant NPs, Cis, Cis NPs, and Cis + Ant NPs caused cytotoxicity in HepG2 and MCF7 with best effect in Cis-treated cells. The anticancer effects were attributed to mitochondrial-dependent apoptosis (with high Bax and low Bcl2 expression), chromatin disintegration, and cell cycle arrest in G2/M and S phases. All treatments inhibited migration by downregulating the migration-related gene MMP9 and upregulating the anti-migratory gene TIMP1 and decreased the angiogenesis-related gene VEGF and the inflammatory gene TNFα with best results in Cis NPs-treated cells. Interestingly, Ant, Ant NPs, and Cis + Ant NPs increased the antioxidant status (high GSH and upregulated expression of Nrf2 and OH-1) and decreased drug resistance-related MAPK1 and MDR1 genes compared to Cis and Cis NPs-treated cells.ConclusionsAnthocyanin and cisplatin-loaded chitosan nanoparticles effectively combat breast and liver cancers by inducing cancer cell apoptosis, enhancing antioxidant defenses, and reducing inflammation. They also inhibit tumor spread and blood vessel formation through downregulation of MMP9 and VEGF, highlighting their therapeutic potential.

Патогенетические аспекты рецидивирующей эндометриальной гиперплазии без атипии у женщин репродуктивного возраста

Aim: to determine the most informative immunohistochemical markers of recurrent endometrial hyperplasia without atypia in women of childbearing age. Methods. Prospective analysis of 89 patients of childbearing age was performed at the Department of Obstetrics and Gynecology with Perinatology, Medical Institute of the Peoples' Friendship University of Russia. During the study the patients were divided into two cohorts based on the EH course: the index group (n=45) was represented by patients with the morphologically verified recurrent endometrial hyperplasia without atypia (one or more episodes within a year), the control group (n=44) was represented by patients with the newly diagnosed endometrial hyperplasia without atypia. The average age of patients in the studied cohort was 44±3.0 years (comparison group – 42±2.0 years). Results. Immunohistochemical markers of recurrence were identified during the study: high Ki-67 expression (p<0.05), low Bcl-2 expression (p<0.05) that enhanced proliferative and reduce antiproliferative processes in the endometrium. Immunohistochemical signs of chronic endometritis (СD138+) were found in 40% of patients suffering from recurrent endometrial hyperplasia (in 18.2% of controls). It should be also noted that there was local hyperestrogenism associated with high expression of estrogen in the glandular epithelium and stroma. Conclusion. Thus, the Ki-67 marker expression and low Bcl-2 expression contribute to recurrence of endometrial hyperplasia without atypia. Furthermore, the combination of endometrial hyperplasia and morphologically verified chronic endometritis provides a further predictor of the endometrial proliferative process recurrence. Immunohistochemistry makes it possible to predict not only the risk of recurrent endometrial hyperplasia, but also the disease course. However, there is currently no universal marker allowing one to predict the chance of hyperplasia diagnosis and recurrence. Design: a comparative prospective study.

Proteogenomics Coupled with Ex Vivo Profiling for Therapeutic Targeting in AML

AML is the most common acute leukaemia in adults and incidence is on the rise, partly due to population ageing. Despite the emergence of new targeted therapies for AML patients, survival rates remain dismal (~27% at 5 years). Current challenges include the rapid development of resistance and the inability to predict which therapy is optimal for each patient, especially for those without ‘actionable’ mutations. These challenges are compounded by the lack of complete testing platforms for capturing critical patient-specific molecular profiles beyond somatic mutations, such as changes in the proteome and phosphoproteome. Due to the heterogeneous nature of AML, individualised screening and personalised treatment plans are likely required to improve treatment and survival outcomes for AML patients. By combining genetic profiling, phosphoproteomic and proteomic analysis, we have developed a pipeline for profiling AML patient samples. Using this pipeline, we have identified novel therapies for different leukaemia subtypes ( Leukemia, 2021). In this study, we have integrated genomic, proteomic, and phosphoproteomic profiling together with ex vivo drug sensitivity profiling for a functional read-out of drug sensitivity or resistance, to identify optimal therapeutic regimes for individual AML patients. Ex vivo drug sensitivity profiling has been performed on 71 patients. Genomic profiling was performed using the SOPHiA Genetics Myeloid Panel. The mutation frequency identified was similar to previous reports ( TCGA, N Engl J Med 2013). The drug screen panel included AML standard of care treatments (e.g., cytarabine, midostaurin) as well as investigational agents. Cell survival was measured at 24 hours using an ATPlite assay or Annexin V staining. Results identified both known and novel associations with mutation subgroups. Consistent with previous reports ( Jawhar, Blood 2017), IDH2 mutations were associated with reduced sensitivity to midostaurin (p=0.0039). Samples with NPM1 mutation were significantly more sensitive to Aurora kinase/CDK inhibitor JNJ7706621 compared to NPM1 wildtype (p=0.0007). This association also held true in the BeatAML cohort (Vizome, p<0.0001). NPM1 has documented roles in cell cycle regulation, and these results suggest that mutant NPM1 may sensitise to cell cycle targeted therapies. Multi-omic analyses of 23 intermediate/high risk patients, 9 of which had no actionable mutations, identified further drug vulnerabilities, with >2 therapies identified for each patient. Validation of this approach is exemplified by individual case studies where our profiling platform correlates with clinical outcome. In the first case, the patient presented with a KRAS mutation, predictive of resistance to midostaurin. Proteomic profiling identified low expression of BCL2, predictive of resistance to venetoclax. Phosphoproteomic profiling however identified activation of DNA repair and CDK proteins, suggesting sensitivity to DNA repair and CDK inhibitors. Ex vivo drug sensitivity profiling of the patient blasts identified resistance to midostaurin and venetoclax, but sensitivity to PARP and CDK inhibitors. Concurrently, the patient received a combination of standard chemotherapy and targeted therapy (cytarabine, midostaurin, venetoclax) and was primary refractory to treatment. In a second case, proteogenomics and ex vivo screening predicted sensitivity to midostaurin and venetoclax. The patient received cytarabine, midostaurin, and venetoclax therapy and achieved remission. In our third case study, ex vivo screening predicted sensitivity to standard of care agents (cytarabine, midostaurin, venetoclax). While the patient achieved remission with standard of care chemotherapy, they subsequently relapsed. At relapse, ex vivo screening identified reduced sensitivity to cytarabine but a high level of venetoclax sensitivity. The patient was placed on FLAG with venetoclax and was bridged to transplant. Therefore, our approach can predict treatment sensitivities and resistance to standard of care therapies, and may also identify alternative clinically available therapies. The results of these and further case studies will be presented at the meeting.

Expression of p53, p63, p16, Ki67, Cyclin D, Bcl-2, and CD31 Markers in Actinic Keratosis, In Situ Squamous Cell Carcinoma and Normal Sun-Exposed Skin of Elderly Patients

Background: Age and cumulative exposure to ultraviolet (UV) light are primary contributors to skin cancer development. Regulatory proteins within the cell cycle are essential for the homeostasis of squamous epithelium. Methods: This study assessed the expression of immunohistochemical markers p53, p63, p16, Ki67, Cyclin D, Bcl-2, and CD31 in keratinocyte intraepithelial neoplasia (actinic keratosis and squamous cell carcinoma in situ) compared to normal skin. The objective was to distinguish disease-specific changes from those attributable to ageing and sun exposure in elderly skin. Results. Analysis included 22 actinic keratoses (AK), 7 in situ squamous cell carcinomas (SCC), and 8 normal skin biopsies. The mean age was 78.1 years for the AK/SCC group and 73.8 years for controls, with no significant age difference noted between the groups. The AK/SCC group exhibited a higher occurrence of amorphous masses, higher intensity of p53, lower Bcl-2 expression in the epidermis, higher Bcl-2 expression in the dermis, and higher CD31 expression in the dermis, all of which were statistically significant (p < 0.05). Conclusions: The study identifies distinct differences in the presence of amorphous masses and the expression levels of p53, Bcl-2, and CD31 between sun-exposed skin and in situ cutaneous squamous cell carcinomas, including actinic keratoses.

Correlation of Flow Cytometric BCL2 and MCL1 Expression with Cytogenetic Characteristics and Outcome in Multiple Myeloma

Introduction: Clonal plasma cells mediate apoptosis resistance through increased expression of anti-apoptotic proteins which increase survival and mediate resistance to intrinsic mitochondrial pathway. The key anti-apoptotic members are BCL-2, MCL-1, BCL-XL, BCL-B and BCL-W. High BCL2 expression or t(11;14) is shown to be predictive of response to Venetoclax in Multiple Myeloma (MM). Venetoclax may only be effective in a subset of patients, namely those who have relatively high BCL2 and low MCL1. Resistance to Venetoclax can be mediated by mutation in BCL2 or gain in copy number of MCL1, which maps to 1q21. The relationship between cytogenetic abnormalities and expression of BCL2 and MCL1 is not clear except for an occasional study which has shown MM with CCDN1 translocation has high BCL2 and low MCL1 expression and MM with MMSET and MAF translocation has high MCL1 and low BCL2 expression. Also, previous studies have used either immunohistochemistry or real time quantitative PCR for studying BCL2 and MCL1 expression, both of which are met with challenges in interpretation. We aimed to correlate the intracellular expression of BCL2 and MCL1 by flow-cytometry (FCM) with cytogenetic characteristics and outcome. Methodology: FCM was performed in bone-marrow of 143 consecutive MM cases with ≥10% plasma cells (PCs) and 20 bone marrow samples with non-clonal PCs by lyse-stain-wash method followed by intracellular staining of BCL2 (Alexa Fluor 647 conjugated anti-human BCL2 antibody) and MCL-1 (PE-conjugated anti-human MCL1 antibody). The sequential gating strategy used in the analysis of intracellular BCL2 and MCL1 expression in PCs and T lymphocytes is shown in Figure 1. The percentage of PCs positive for BCL2 and MCL1 compared to the tube without these antibodies [Fluorescence Minus One (FMO) cutoff threshold], the mean fluorescence intensity (MFI) expression of BCL2 and MCL1 in PCs and T cells, and the ratio of expression of BCL2 and MCL1 on PCs and T cells (BCL2 PC/T, MCL1 PC/T), MFI BCL-2 PC/MFI MCL1 PC were studied and further correlated with 5-7 panel FISH cytogenetics by using Kruskal Wallis test or Mann-Whitney U test. The overall survival/OS and progression-free survival/PFS was calculated using Kaplan-Meir Survival Curve analysis. Result and discussion: During the study, FCM was performed in 143 MM patients (103 Newly Diagnosed MM and 40 Relapsed Refractory MM). The cytogenetic findings includes IGH:: FGFR3 (n=29), IGH:: CCND1 (n=8), IGH:: MAF (n=3), gain1q21 (n=76), 1q21-amplification (n=30), 1p32-deletion (n=17) and TP53-deletion (n=21). A median of 38,779 PCs was acquired by FCM. The BCL2 and MCL1 expression was seen in 4-100% (median-96.9%) and 0-100% (median-78.8%) of clonal PCs, respectively. The median BCL2 MFI and MCL1 MFI were 43,289 and 6,481 respectively. The median BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 PC in clonal PCs were 1.90, 4.35, and 07.1, respectively, whereas in non-clonal PCs they were 0.87, 3.02 and 3.21, respectively.BCL2 PC/T, MCL1 PC/T and BCL2 PC/MCL1 P were significantly higher in clonal PCs than non-clonal PCs, with a p-value of 0.002, 0.001, and 0.009, respectively. The median MCL1 MFI was significantly lower (3,825 vs. 6,559; p=0.020) and median BCL2 PC/MCL-1 PC (12 vs 6.7; p=0.012) was significantly higher in IGH:: CCND1 compared to others; while an opposite pattern was seen in gain1q21 (7,420 vs 5,525; p=0.032 and 5.5 vs 8.6; p=0.046). The dim expression (<25-percentile) of BCL2 and MCL1 was noted in 25% (n=36) MM cases. Survival analysis did not yield a significant difference in OS or PFS between patients with dim, moderate or strong expression of BCL2 or MCL1. Conclusions: Our study demonstrates an objective assessment of BCL2 and MCL1 expression by FCM. BCL2 PC/T,MCL1 PC/T and BCL2 PC /MCL1 PC were significantly higher in myeloma cells compared to non-clonal PCs. Low MCL1 expression or high BCL2/MCL1 ratio was associated with IGH:: CCND1; while strong MCL1 and low BCL2/MCL1 ratio was most frequent in 1q21 amplification. We identified dim-expression (< 25-percentile) of BCL2 or MCL1 in 25% of cases of MM, where the benefit of anti-BCL2/MCL1 targeted therapy needs to be studied. Routine testing of BCL2 and MCL1 expression by FCM may be beneficial before starting Venetoclax/similar targeted therapy.

Correlation of ultrasound features with Bcl-2 protein expression in basal cell carcinomas (BCCs).

The expression of the Bcl-2 protein is frequently observed in basal cell carcinomas (BCCs), making it a significant biological marker and potential therapeutic target. Skin ultrasonography offers a noninvasive means of obtaining anatomical information about cutaneous tumors. The purpose of this study was to investigate the correlation between ultrasound features and Bcl-2 expression in BCCs, to provide a reference for developing pharmacological treatment plans. According to the Bcl-2 protein expression, 74 BCCs confirmed by surgical pathology were divided into high Bcl-2 expression BCCs (HB-BCCs) and low Bcl-2 expression BCCs (LB-BCCs). Preoperative lesion ultrasound features were analyzed retrospectively based on Liang's criteria, which included the following features: shape, surface, keratinization, base, infiltration level, internal echogenicity, distribution of hyperechoic spots, posterior echogenic changes, internal Doppler signal, and lesion size (maximum diameter and infiltration depth). The differences of two groups were compared using a chi-square test or a paired t-test. Based on ultrasound features, cystic areas were more frequent in LB-BCCs (χ2 = 7.015, P = .008). Furthermore, LB-BCCs exhibited greater infiltration depth than HB-BCCs (4.86 ± 2.12 mm vs. 2.72 ± 1.40 mm, P = .000), had a higher propensity to infiltrate the subcutaneous tissue (χ2 = 12.422, P = .002), and displayed a more abundant internal Doppler signal within the lesions (χ2 = 24.696, P = .000). Conversely, maximum diameter of the lesions, shape, surface, keratinization, base, hyperechoic spots distribution, and posterior echogenic changes of the lesions did not differ significantly between the two groups. Ultrasound features are correlated with Bcl-2 protein expression level in BCCs. LB-BCCs show greater infiltration depth, subcutaneous infiltration, more cystic changes and more abundant internal Doppler signal than HB-BCCs, which may suggest a potential basis for drug selection in BCC chemotherapy.

Cannabis sativa a Potential Anticancer Treatment in Melanoma Cancer Cells

Introduction Melanoma cancer is the most aggressive skin cancer type with a poor prognosis. Chemotherapy has been used in the past in treating melanoma cancer, however, the method usually results in toxicity. Therefore, a call has been made to develop cancer treatments using medicinal plants to reduce drug toxicity. The aim of this study was to establish the role of RBBP6 in melanoma cancer development and progression. The hypothesis of this study was that RBBBP6 is highly expressed in melanoma cancer and serves as an early biomarker. Results obtained supported the hypothesis since high expression of RBBP6 in melanoma cancer was observed in untreated control melanoma cells. Methodology 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Cytosmart fluorescence, and quantitative Polymerase Chain Reaction (qPCR) were used. Results The IC50 values of Cannabis sativa EtOH and MeOH extracts were obtained at 51.31315 and 57.34135 µg/mL, respectively, for A375 cells. Normal cells were used to check if there are any serious cytotoxic effects, IC50 values for Cannabis sativa EtOH and MeOH extracts were obtained at 79.577850 and 59.5754 µg/mL, respectively. RMG-1 cells showed IC50 at 84.748 and 34.66475 µg/mL for Cannabis sativa EtOH and MeOH extracts, respectively. CYTOSMART LUX3 bright field microscope was used for morphological analysis of both A375 and RMG-1 cells and changes were observed after treating with Cannabis sativa EtOH and MeOH extracts. Apoptosis analysis to determine the mode of cell death was done using a CYTOSMART fluorescence microscope. Early apoptotic effects were observed in both Cannabis sativa EtOH and MeOH extracts on A375 cells. Early apoptotic effects were also observed after treating RMG-1 cells with EtOH extract. Caspase 3/7 activity was done on both A375 and RMG-1 cells, and caspase 3/7 activity were increased after treating with Cannabis sativa EtOH and MeOH extracts. Real Time Polymerase Chain Reaction (RT-PCR) showed that RBBP6 and BCl2 were down-regulated after treating with Cannabis sativa EtOH and MeOH extracts. Conclusion siRNA co-treatment with EtOH and MeOH extracts resulted in low expression of both RBBP6 and BCl2. P53 was upregulated after treatment with the extracts and co-treatment with the extracts and siRNA.

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation, apoptosis, and migration of human ectopic endometrial stromal cells: based on JAK2/STAT3 signaling pathway

Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.

Abstract 1000: Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer

Abstract Purpose: Young women’s breast cancers (YBCs), defined herein as breast cancer (BC) diagnosed at age ≤ 40 years, are etiologically and biologically distinct from postmenopausal BCs, demonstrating a higher proportion of estrogen receptor (ER) negative subtypes and a worse prognosis. Studies have not assessed whether risk factors such as recent parity and body mass index (BMI) are linked to protein expression profiles in YBCs. Accordingly, we examined protein expression assessed with GeoMx® Digital Spatial Profiling (DSP) in 640 YBCs prepared as tissue microarrays (TMAs) in the Young Women’s BC Study (YWBCS). Methods: The YWBCS is a multi-institutional cohort of 1,297 women with incident BC diagnosed at age <40 years identified through medical record review, with risk factor data collected via questionnaires. TMAs including 1,505 cores of 640 YBCs that were collected prior to therapy and were suitable for molecular analysis were evaluated for 72 markers with NanoString GeoMx DSP. Associations of log-transformed biomarker expression with BMI and time from last live birth to BC diagnosis were carried out using linear mixed modeling approaches, accounting for multiple tumor tissue cores per individual. Results: Mean age at BC was 35.5 years (range 17-40). Tumor subtypes based on clinical immunostaining were the following: luminal B 46%; luminal A 36%; triple negative 12% and HER2+ 6%. Of 72 biomarkers tested, 53 (74%) were evaluable after QC and normalization. GeoMx ER and PR protein levels were higher in luminal A and luminal B YBCs than in HER2+ and TNBC (p=2x10-94 and 2x10-52, respectively), supporting the accuracy of the GeoMx assay. Compared to nulliparas or those with most recent birth > 3 years before BC, those who gave birth ≤ 3 years prior to BC had tumors with higher expression of PR (8x10-6) and ER (p=0.006), and lower expression of BCL-2 (p=0.006). In contrast, women with last birth ≥ 6 years prior to BC had higher expression of CD8 (p=0.006) and PD-1 (p=0.001) than nulliparas and those with more recent births. Compared to women with BMI between 18.5 and 29.9, those with higher or lower BMIs had higher expression of CD34 (p=0.0003), CD4 (p=0.0009) and CD14 (p=0.001). BCL-2 expression was higher among women with BMI < 18.5 compared to those with higher values (p=0.005). Women with BMI of 30+ had higher β2-microglobulin levels than those with lower BMIs (p=0.003). Inverse dose-response associations (with higher BMI related to lower levels) were observed for expression of ER (p=0.01), pan cytokeratin (p=0.004) and four proteins in the p13K/AKT signaling pathway (p ≤ 0.01 for each). Conclusions: Preliminarily, we identify differences in hormonal and immune markers in YBCs in relation to timing of births and BMI, suggesting that these factors may contribute to etiologic heterogeneity. Multivariate modeling is ongoing to incorporate molecular subtype and other risk factors. Citation Format: Robert A. Vierkant, Laura M. Pacheco-Spann, Stacey J. Winham, Jennifer M. Kachergus, Ji Shi, Craig Snow, Fergus J. Couch, Janet E. Olson, Chen Wang, Derek C. Radisky, Amy C. Degnim, E. Aubrey Thompson, Laura C. Collins, Kathryn J. Ruddy, Ann H. Partridge, Mark E. Sherman. Associations of parity and body mass index with NanoString digital spatial protein profiling in early onset breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1000.

Abstract 6249: Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewing sarcoma

Abstract Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma. Citation Format: Guillem Pascual-Pasto, Claudia Resa-Pares, Angel M. Carcaboso. Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6249.

Abstract P3-05-51: Patients with low expression of BCL 2 experience more late recurrence than patients who do not, which neutralized the protective effect of low BCL2 expression in patients with estrogen receptor positive breast cancer

Abstract Background) BCL2 is a key factor for the regulation of cellular apoptosis and its overexpression inhibits apoptotic cell death and activates cellular proliferation, resulting tumor progression. Although BCL2 are expected to be associated with an adverse prognosis in breast cancer, previous studies have reported inconsistent results and some reported even favorable features. The aim of this study is to investigate what characteristics of BCL2 yield this discrepant result in patients with breast cancer. Materials and methods) Female patients with breast cancer who completed primary treatment against breast cancer between 2003 and 2018 at Wonju Severance Hospital, Korea, were included. Clinocopathological characteristics including BCL2 expression were collected. Patients were categorized into two groups, BCL2 expression in more or less than 10% of tumor cells. Kaplan-Meier curves were generated to compare recurrence-free interval (RFI) and overall survival (OS). Result) The final cohort included 616 patients with a mean age of 54.79±11.2 (25-86) years. Patients with estrogen receptor positive breast cancer were more frequent in patients with tumor in which BCL2 was expressed in less than 10% of tumor cells (70.7% vs. 57.7%). Patients with tumor in which BCL2 was expressed in less than 10% of tumor cells showed better survival than their counterpart. Subgroup analysis according to the estrogen receptor and HER2 overexpression status was done. In patients with tumor in which HER2 was not overexpressed, patients with tumors in which BCL2 expressed less than 10% of tumor cells showed better survival(p=0.007). Survival difference maintained in patients with triple negative disease(p=0.010). However, in patients with estrogen receptor positive breast cancer, survival of two groups of patients with high and low BCL2 expression become not significant over time as late recurrence occurred (p=0.227). In contrast, patients with HER2 positive disease breast cancer showed worse prognosis than their counterpart when their tumor showed BCL2 expression in less than 10% of tumor cells. Conclusion) BCL2 overexpression showed different contribution to the patients’ survival according to the subtypes. Patients with HER2 overexpression showed better survival when BCL2 was overexpressed and patients with HER2 negative disease does not. First, similar survival was resulted from late recurrence in patients with estrogen receptor positive disease. Second, apoptotic capacity of tumor cells may not be major survival factor if tumor cells are exactly targeted. Citation Format: Hyang Suk Choi, Hany Hany Noh, In-Jeong Cho In-Jeong Cho, Seung Taek Lim, Jong-In Lee, Airi Han. Patients with low expression of BCL 2 experience more late recurrence than patients who do not, which neutralized the protective effect of low BCL2 expression in patients with estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-51.

Expression of HSP27 and BAX/BCL-2 apoptosis factor in silicosis rat model of fibrosis

To study the expression of heat shock protein 27(HSP27), BAX and BCL-2 apoptosis in silicosis rat model, and to explore the correlation between HSP27 and BAX and BCL-2 apoptosis. Silicosis model was established by the oropharyngeal and endotracheal intubation. Forty SPF healthy adult Wistar male rats were randomly divided into 4 groups, with 10 rats in each group. Silicosis group for 6 weeks(feeding for 6 weeks), silicosis group for 8 weeks(feeding for 8 weeks): oropharyngeal and tracheal perfusion of 50 mg/mL SiO_2 suspension 1.0 mL/mouse; Model control group for 6 weeks and model control group for 8 weeks: 1.0 mL saline was infused into the oropharynx and trachea. Immunohistochemical staining was used to detect the expression of HSP27, BAX and BCL-2 in the right lower lung of silicosis model group at 6 and 8 weeks and model control group at 6 and 8 weeks. Western blot was used to detect the protein expression of HSP27, BAX and BCL-2 in the left lower lobe lung tissue of silicosis model group at 6 and 8 weeks and model control group at 6 and 8 weeks, respectively. Immunofluorescence staining was used to detect the colocalization of HSP27 with pro-apoptotic factor BAX and HSP27 with anti-apoptotic factor BCL-2. Compared with the model control group at 6 weeks and 8 weeks, the expression of HSP27 and pro-apoptotic factor BAX in fibrotic region increased, and the expression of anti-apoptotic factor BCL-2 decreased in silicosis model group at 6 weeks and 8 weeks(P<0.05). Immunofluorescence staining showed that there was colocalization of HSP27 and pro-apoptotic factor BAX in the fibrotic region. Correlation analysis showed that the correlation coefficient between HSP27 and pro-apoptotic factor BAX was r=0.94, indicating a positive correlation between them, while the correlation coefficient between HSP27 and anti-apoptotic factor BCL-2 was r=-0.81, indicating a negative correlation between them. High expression of HSP27 and pro-apoptotic factor BAX and low expression of anti-apoptotic factor BCL-2 exist in silicosis rats, and their expression is correlated.

Tanshinone IIA inhibits ischemia-reperfusion-induced inflammation, ferroptosis and apoptosis through activation of the PI3K/Akt/mTOR pathway.

Ischemia-reperfusion (I/R) is a common clinical process, and the lung is one of the most sensitive organs of I/R injury, which often leads to acute lung injury (ALI). Tanshinone IIA (Tan IIA) has anti-inflammatory, antioxidant, and anti-apoptotic activities. However, the effects of Tan IIA on lung I/R injury remain uncertain. Twenty-five C57BL/6 mice were randomly divided into five groups: control (Ctrl), I/R, I/R + Tan IIA, I/R + LY294002 and I/R + Tan IIA + LY294002 group. Tan IIA (30μg/kg) was injected intraperitoneally 1h before injury in the I/R + Tan IIA and I/R + Tan IIA + LY294002 groups. These data showed that Tan IIA significantly improved I/R-induced histological changes and scores of lung injury, decreased lung W/D ratio, MPO and MDA contents, reduced infiltration of inflammatory cells, and decreased the expression of IL-1β, IL-6 and TNF-α. Meanwhile, Tan IIA significantly increased the expression of Gpx4 and SLC7A11, and decreased the expression of Ptgs2 and MDA. Moreover, Tan IIA significantly reversed the low expression of Bcl2, and the high expression of Bax, Bim, Bad and cleave-caspase 3. Furthermore, Tan IIA caused a significant increase in the phosphorylation levels of PI3K, Akt and mTOR in the lungs. However, these beneficial effects of Tan IIA on I/R-induced lung inflammation, ferroptosis and apoptosis were offset by LY294002. Our data suggest that Tan IIA significantly ameliorates I/R-induced ALI, which is mediated through activation of PI3K/Akt/mTOR pathway.

Antibacterial, Antifungal, and Anticancer Effects of Camel Milk Exosomes: An In Vitro Study

Camel milk (CM) has potent antibacterial and antifungal effects and camel milk exosomes (CM-EXO) have been shown to inhibit the proliferation of a large variety of cancer cells including HepaRG, MCF7, Hl60, and PANC1. However, little is known regarding the effects of CM-EXO on bacteria, fungi, HepG2, CaCo2, and Vero cells. Therefore, this study aimed to evaluate the antibacterial, antifungal, and anticancer effects of CM-EXO. EXOs were isolated from CM by ultracentrifugation and characterized by transmission electron microscope and flow cytometry. Unlike CM, CM-EXO (6 mg/mL) had no bactericidal effects on Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, and Enterococcus feacalis) but they had bacteriostatic effects, especially against Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis), and fungistatic effects on Candida albicans. HepG2, CaCo2, and Vero cells were respectively treated with CM-EXOs at low (6.17, 3.60, 75.35 μg/mL), moderate (12.34, 7.20, 150.70 μg/mL), and high (24.68, 14.40, 301.40 μg/mL) doses and the results revealed that CM-EXOs triggered apoptosis in HepG2 and CaCo2 cells, but not in normal Vero cells, as revealed by high Bax expression and caspase 3 activities and lower expression of Bcl2. Interestingly, CM-EXOs also induced the elevation of intracellular reactive oxygen species and downregulated the expression of antioxidant-related genes (NrF2 and HO-1) in cancer cells but not in normal cells. CM-EXOs have antibacterial and antifungal effects as well as a selective anticancer effect against HepG2 and CaCo2 cells with a higher safety margin on normal cells.

Low Bcl-2 is a robust biomarker of sensitivity to nab-paclitaxel in Ewing sarcoma

Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) shows potent preclinical anticancer activity in pediatric solid tumors such as Ewing sarcoma, rhabdomyosarcoma and neuroblastoma, but responses in clinical trials have been modest. In this work, we aimed to discover a rational biomarker-based approach to select the right candidate patients for this treatment. We assessed the efficacy of nab-paclitaxel in 27 patient-derived xenografts (PDX), including 14 Ewing sarcomas, five rhabdomyosarcomas and several other pediatric solid tumors. Response rate (partial or complete response) was remarkable in rhabdomyosarcomas (four of five) and Ewing sarcomas (four of 14). We addressed several predictive factors of response to nab-paclitaxel such as the expression of the secreted protein acidic and rich in cysteine (SPARC), chromosomal stability of cancer cells and expression of antiapoptotic members of the B-cell lymphoma-2 (Bcl-2) family of proteins such as Bcl-2, Bcl-xL, Bcl-W and Mcl-1. Protein (immunoblotting) and gene expression of SPARC correlated positively, while immunoblotting and immunohistochemistry expression of Bcl-2 correlated negatively with the efficacy of nab-paclitaxel in Ewing sarcoma PDX. The negative correlation of Bcl-2 immunoblotting signal and activity was especially robust (r = 0.8352; P = 0.0007; Pearson correlation). Consequently, we evaluated pharmacological strategies to inhibit Bcl-2 during nab-paclitaxel treatment. We observed that the Bcl-2 inhibitor venetoclax improved the activity of nab-paclitaxel in highly resistant Bcl-2-expressing Ewing sarcoma PDX. Overall, our results suggest that low Bcl-2 expression could be used to select patients with Ewing sarcoma sensitive to nab-paclitaxel, and Bcl-2 inhibitors could improve the activity of this drug in Bcl-2-expressing Ewing sarcoma.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

Increased Perforin- and IL-21-Expressing NK Cells in Patients with Early-Stage Chronic Lymphocytic Leukemia

Objective: CD5+CD19+ cells have a low proliferation capacity and elevated expression of anti-apoptotic protein BCL-2, mostly in the G0/ G1 cell phase and accumulate in the pathogenesis of chronic lymphocytic leukemia (CLL). Natural killer (NK) cells have the ability to kill the intracellular pathogen-infected or cancer cells and secrete cytotoxic enzymes. This study evaluated the frequency, expression of cytotoxic enzymes, and intracellular cytokine levels of NK cells in CLL patients. Materials and Methods: In this study, peripheral blood mononuclear cells were isolated from peripheral blood samples of CLL patients (n=29) and healthy controls (n=16) by density gradient centrifugation method. The frequency of the total NK cells and the intracellular levels of perforin, granzyme, interferon (IFN)-γ, interleukin (IL)-21, IL-4 and IL-17 in NK cells were investigated. Results: Elevated total NK cell frequency were found in the CLL patients compared to the healthy controls, and negatively correlated with CD5+CD19+ malign B cell frequency. Increased perforin expression was observed in patients’ total NK cells. Additionally, increased levels of IL-17 and IL-21 in total NK cell of CLL patients were obtained compared to healthy subjects. Conclusion: The findings suggest that in the early stage of CLL, increased total NK cell frequency, and elevated perforin and IL-21 levels in NK cells might have a protective impact against the progression of the disease for CLL patients.

Novel therapeutic mechanism of action of metformin and its nanoformulation in Alzheimer's disease and role of AKT/ERK/GSK pathway

IntroductionBrain Insulin-resistance plays a critical role in pathogenesis of Alzheimer's disease (AD). Current study explored the therapeutic mechanism of metformin (insulin sensitizer) and its solid-lipid nanoformulation (SLN) in rat-model of AD. In our study, SLN was prepared using microemulsion method. AD was induced with ICV-Aβ whereas the control-group (sham) received ICV-NS. Treatment arms included, disease-control (no treatment), Metformin (50 mg/kg, 100 mg/kg and 150 mg/kg), SLN-metformin 50 mg/kg and memantine 1.8 mg/kg (positive-control). Animals were tested for cognitive performance (EPM, MWM) after 21 days of therapy and sacrificed. Aβ (1–42), hyperphosphorylated tau, pAKTser473, GSK-3β, p-ERK (ELISA), metformin level(HPLC), neuronal injury score(H&E), Bcl2 and Bax(IHC) was evaluated in isolated brain. In our study, metformin-SLN were of spherical shape (size<200 nm) with 94.08% entrapment efficiency. Metformin was detectable in brain. Compared to sham, the disease-control group showed significantly higher (p ≤ 0.05) memory impairment(MWM and EPM), hyperphosphorylated tau, Aβ(1–42), neuronal-injury, Bax and lower Bcl-2 expression. Treatment with metformin and nanoformulation significantly reverse these parameters. AKT-ERK-GSK3β-Hyperphosphorylated tau pathway was found to be involved in the protective efficacy of metformin. To conclude, both metformin and its SLN were found to be effective as therapeutic agents in AD which act through the AKT-ERK-GSK3β-Hyperphosphorylated tau pathway. We need population based studies to confirm the same.

MiR-328-5p Induces Human Intervertebral Disc Degeneration by Targeting WWP2.

Intervertebral disc degeneration (IDD) development is regulated by miRNA, including inflammatory reactions, cell apoptosis, and degradation of extracellular matrix. Nucleus pulposus cells apoptosis has a absolute influence in the development of IDD. This experiment explores the mechanism of miR-328-5p regulating IDD. Through the analysis of miRNA and mRNA microarray database, we screened the target genes miR-328-5p and WWP2. We verified the expression of miR-328-5p, WWP2, and related apoptotic genes in normal and degenerative nucleus pulposus tissues by qRT-PCR. The expressions of WWP2, Bcl-2, and Bax were detected by qRT-PCR and western blot after transfection to nucleus pulposus cell. The nucleus pulposus cell proliferation and apoptosis after transfection were confirmed by CCK8 and flow cytometry. Luciferase reporter assay and bioinformatics analyzed the targeting relationship between miR-328-5p and WWP2. Firstly, the qRT-PCR experiments confirmed the significant increase of miR-328-5p expression, while significant reduction of WWP2 in a degenerative tissues compared to the normal tissues. Surprisingly, miR-328-5p expression was positively, while that of WWP2 negatively correlated with the degeneration grade of IDD. And we also identified the high expression of Bax and Caspase3, while low expression of Bcl-2 in a degenerative tissues. After miR-328-5p mimic transfected into nucleus pulposus cell, qRT-PCR and western blot confirmed that WWP2 and Bcl-2 expressions were downregulated, while Bax and Caspase3 expressions were upregulated, and the same results were obtained by knocking down WWP2. CCK8 and flow cytometry confirmed that miR-328-5p inhibited the proliferation and induced apoptosis of nucleus pulposus cells. WWP2 is a target gene of miR-328-5p by bioinformatics and luciferase reporter assay. In summary, miR-328-5p targets WWP2 to regulate nucleus pulposus cells apoptosis and then participates in the development of IDD. Furthermore, this study may provide new references and ideas for IDD treatment.

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.