Bcl-xL mRNA Expression Research Articles

GdX inhibits the occurrence and progression of breast cancer by negatively modulating the activity of STAT3

ABSTRACT Aim To elucidate the biological functionality and regulatory mechanisms of GdX in breast cancer (BC). Methods The examination of GdX expression in human BC tissues and cell lines was conducted through immunohistochemical (IHC) and Western blot. Cell proliferation capacity was assessed via the CCK-8 and colony formation assay, while cell migration was determined through the wound healing assay. The expression levels of BCL-XL, Cyclin D1, and C-myc gene were quantified using RT-qPCR and Western blot. In vivo tumor growth was evaluated in nude mice xenografted with MDA-MB-231 cells overexpressing GdX, and a mouse model with GdX-deficient BC was established to observe the impact of GdX on BC formation and metastasis. Dual-luciferase reporter assay and immunofluorescence were employed to confirm the interaction between GdX and STAT3. Western blot was employed to validate the influence of GdX overexpression on the phosphorylation process of STAT3. Results GdX exhibited low expression in the cancer tissues of BC patients and cell lines. MDA-MB-231 and MCF-7 cells overexpressing GdX displayed a notable reduction in proliferation and diminished migratory capabilities, accompanied by downregulated mRNA and protein expression of BCL-XL, Cyclin D1, and C-myc. In the xenograft mouse model, heightened GdX expression correlated with a decelerated in vivo tumor growth. Furthermore, in mice deteleing GdX, both the quantity and weight of tumors increased, along with evident pulmonary metastasis. Mechanistically, STAT3 emerged as a downstream target gene of GdX. Conclusions GdX exerts its inhibitory effects on the initiation and progression of BC by negatively modulating the phosphorylation of STAT3.

Curcumin Combined with Thalidomide Inhibits Proliferation of KG-1 Cells and Its Related Mechanisms

To investigate the effects of curcumin combined with thalidomide on the proliferation and apoptosis of acute myeloid leukemia KG-1 cells, and its correlation with B-cell lymphoma-xL (Bcl-xL), signal transducer and activator of transcription 3 (STAT3). MTT assay was used to detect the proliferation of KG-1 cells and screen the optimal combined concentration of curcumin and thalidomide. The effects of curcumin, thalidomide and their combination on the proliferation and apoptosis of KG-1 cells were analyzed by MTT method and flow cytometry, respectively. The mRNA expression levels of STAT3 and Bcl-xL in single-drug group, two-drug combination group and control group (untreated cells) were detected by real-time quantitative PCR. Both curcumin and thalidomide inhibited the proliferation of KG-1 cells in a concentration-dependent manner in the range of 20-100 μmol/L (r =0.657, r =0.681). The IC50 value of curcumin and thalidomide at 48 h was (42.07±0.50) μmol/L and (57.01±2.39) μmol/L, respectively. The cell proliferation inhibition rate of curcumin (40 μmol/L) + thalidomide (60 μmol/L) was (86.67±1.53)%, which was significantly higher than (51.67±1.15)% of curcumin (40 μmol/L) and (55.33±1.53)% of thalidomide (60 μmol/L) (both P < 0.05). Treated with curcumin and thalidomide alone or in combination, the apoptosis rate of KT-1 cells was (18.67±2.08)%, (21.33±2.52)%, and (46.67±1.53)%, respectively, which was significantly higher than (0.72±0.03)% of control group (all P < 0.05). The cell apoptosis rate of two-drug combination group was significantly higher than that of single-drug group (both P < 0.05). Compared with the control group, the mRNA expressions of STAT3 and Bcl-xL in single-drug group, two-drug combination group were significantly decreased (both P < 0.05). Compared with single-drug group, the mRNA expressions of STAT3 and Bcl-xL in two-drug combination group were also significantly decreased (both P < 0.05). Curcumin combined with thalidomide can synergistically down-regulate the expression of STAT3 and Bcl-xL, inhibit the proliferation of KG-1 cells, and induce apoptosis.

Exploration of mechanism of total triterpenoids from fruits of Chaenomeles speciosa against senescent GES-1 cells induced by D-galactose based on Gln/GLS1/α-KG metabolic axis and mitochondrial apoptosis signaling pathway

Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of β-galactosidase(SA-β-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-β-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P&lt;0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P&lt;0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P&lt;0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P&lt;0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P&lt;0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.

Merremia emarginata Extract Potentiates the Inhibition of Human Colon Cancer Cells (HT-29) via the Modulation of Caspase-3/Bcl-2-Mediated Pathways.

Background This study investigates Merremia emarginata's curative effectiveness against colon cancer cells.M. emarginata, often known as Elika jemudu, is a Convolvulaceae family plant. The inhibitory ability of anticancer herbal extracts against cancer cell growth and mediators is tested. Aim This study aims to evaluate the potent anticancer activity of M. emarginata against coloncancer cellline (HT-29). Materials and methods M. emarginataleaves were gathered and processed using solvent extraction. Anticancer activity on colon cancer cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) test and cysteine aspartic acid protease-3 (caspase 3), B-cell lymphoma 2 (Bcl-2), and B-cell lymphoma-extra large(Bcl-xL)mRNA expressions. The data was reported as the mean ± SD of three separate experiments done in triplicate. The statistical analysis was carried out using one-way analysis of variance (ANOVA), with a p-value less than 0.05 indicating statistical significance. Results The cell viability test showed a gradual decrease in cell growth and proliferation as the concentration increased. The ethanolic extract of M. emarginata was found to be cytotoxic against colon caller cell lines. The extract was able to induce apoptosis of canceras revealed by Bcl-2, Bcl-xL, and caspase-3 (p<0.05and p<0.001) signaling pathways. Conclusion M. emarginata extracts showed good anticancer activity against colon cancer cell lines. Further work is required to establish and identify the chemical constituent responsible for its anticancer activity.

Significance of the Apoptotic Regulators BAX, BCL2, BCL- XL and MCL1 in Newly Diagnosed AML

Introduction: In acute myeloid leukemia (AML), cytogenetics and molecular mutations remain important prognostic tools for tailoring treatment after induction therapy. Abnormalities in multiple components of apoptotic pathways have been identified as potential drivers of AML. Identifying other biomarkers may help us further refine prognosis and identify other therapeutic strategies. B-cell lymphoma 2 (Bcl2), Bcl-xL and myeloid cell leukemia-1 (Mcl1) are commonly expressed prosurvival proteins in hematologic malignancies, whereas Bax acts as a promoter of apoptosis. We retrospectively evaluated the significance of Bax, Bcl2, Bcl-xL and Mcl1 expression in newly diagnosed AML patients using the Beat AML program 1. Methods: We analyzed RNA-expression data from The Beat AML study (672 tumor specimens collected from 562 patients) 1. Data was extracted from the public database. Different expression levels of the above genes were compared using t-tests. Results were considered significant with p-value &amp;lt; .05. Kaplan-Meier curves were used to estimate survival. Results: For the correlative survival analyses, Bax, Bcl2, Bcl-xL and Mcl1 mRNA expression and clinical information were both obtained from the BeatAML cohort 1. Patients were categorized into high expressers (≥ median) and low expressers (&amp;lt;median). In total, 461 patients had available gene expression data. The mean age of diagnosis was 57 years, and 56.6% of patients were male. The most common specific diagnosis at inclusion was AML with mutated NPM1 (22%), followed by AML with myelodysplasia related changes (21%) and AML, NOS (11%). Complete response to induction was seen in more than half (56%) of the patients for which response was recorded (n=369). Others developed refractory AML (32%). No data was available for 12%. Standard chemotherapy (induction therapy with idarubicin and cytarabine (7+3) and consolidation therapy with high dose cytarabine) was used in 98.4% of these patients. Over half of the analyzed samples were peripheral blood (52%), and the remaining samples were either bone marrow aspirate (46%) or from leukapheresis (2%). Most of the specimens were collected at initial acute leukemia diagnosis (62.2%), others were collected with residual disease or post chemotherapy (25.1%). The mean survival for this cohort was 13.7 months. Above median expression of Mcl1 was associated with longer survival (33 vs. 20 months; p = 0.0262). Above median expression of Bax was also associated with longer survival (32 vs. 18.2 months; p = 0.0114). In the Bax analysis, gene expression was further subdivided into 4 quartiles which showed correlation of expression levels with length of survival (Figure 1). The prognostic significance of the ratio Bax/ (Mcl1+Bcl2+Bcl-xL) was also analyzed given the mechanism of action of these various regulators. A higher ratio was associated with longer survival (40.7 vs. 18.7 months; p = 0.0095) (Figure 2) Conclusions Bcl-2, Bcl-xL and Mcl-1 are commonly expressed antiapoptotic proteins, whereas Bax is a promoter of apoptosis. The results with Bax are consistent with previous literature. Our results with Mcl1 expression differed from what we may have expected. This may be due to the sample size or methodology. However, it may also be due to the fact that a ratio of the actors is needed to help predict prognosis (as we analyzed in Figure 2). The overall survival between Bcl-2 and Bcl-xL was compared independently, however, they did not appear to have a significant difference in survival between high and low expression groups. We are currently working to see if this ratio can be validated in a prospective cohort of relapsed/ refractory AML patients treated on clinical trial and this data will be presented at the meeting. The Bcl-2 inhibitor venetoclax has demonstrated promising results in treating AML. However, venetoclax appears to have limited activity as a single agent. Our study suggests that potentially a combination of agents targeting factors such as Bcl-2, Bcl-xL and Mcl-1 may be associated with improved survival in patients with AML. Further validation of these biomarkers is warranted, and Mcl-1 inhibitors are currently in clinical trials.

Tangeretin's Anti-apoptotic Signaling Mechanisms in Oral Cancer Cells: In Vitro Anti-cancer Activity.

Introduction Citrus fruit peels contain Tangeretin, a natural chemical flavonoid that reinforces plant cell walls and serves as a defense mechanism. Apoptosis, growth inhibition, anti-oxidant, anti-diabetic, and anti-cancer activities are only a few of its many qualities. Tangeretin's principal function is to shield healthy cells or tissues from the harmful effects of chemotherapy. The purpose of this study was to investigate the apoptotic activity of Tangeretin's impact on KB (oral cancer cells) cell lines. Materials and method This study employed Tangeritin, in investigating its effects on oral cancer cells. Oral cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM), with 10% fetal bovine serum at 37°C in a 5% CO2 environment. Cell viability was assessed by seeding oral cancer cells in 96-well plates, exposing them to varying Tangeritin concentrations (50 µM, 100 µM, and 200 µM) with growth inhibition of KB cell viability in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayand morphological changes in cells were observed under an inverted light microscope at 10x magnification. The results were reported as mean±standard error mean (SEM) using one-way analysis of variancethrough IBM SPSS Statistics for Windows, Version 23 (Released 2015; IBM Corp., Armonk, New York, United States). Result MTT assay showed a significant reduction in KB cell viability when treated with Tangeretin. With asignificant decrease in mRNA levels of the anti-apoptotic genes Bcl-2 and Bcl-xL. At 50 µM, 100 µM, and 200 µM, the levels of Bcl-2 were 0.85 ± 0.09, 0.62 ± 0.05, and 0.67 ± 0.05, respectively. Similarly, the mRNA expression of Bcl-xL was 0.82 ± 0.07 for 50 µM, 0.7 ± 0.06 for 100 µM, and 0.77 ± 0.06for 200 µM. The mRNA expression levels of Bax were 1.1 ± 0.09 for 50 µM, 1.4 ± 0.12for 100 µM, and 1.3 ± 0.11 for 200 µM, respectively. Conclusion Tangeretin showed a promising apoptotic activity in KB cells suggesting its utility as an anti-cancer compound. It prevented the growth and proliferation of cancer cells by acting on pro-apoptotic and anti-apoptotic genes. However, this conclusion is mostly based on the in vitro study. Therefore in vivo animal studies were needed to confirm the findings.

Abstract 5986: The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study

Abstract INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV&amp;lt;20 mm3) in 60% population in comparison to either of the cohorts post 30 days of treatment (control: 2000 mm3, P- 1200 mm3; N- 1500 mm3, p&amp;lt;0.05). Compared to control sequential P+N treatment in vivo i) reduced anti-apoptotic BcLxL mRNA expression to 2.5 fold ii) Cleaved PARP increased 10 folds and iii) BcLxL levels decreased 2 fold. CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [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 5986.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. To identify overexpressed oncogenes as potential drug targets for MCC. NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Pediatric Preclinical Testing Consortium Evaluation of the MCL1 Inhibitor, AMG 176, Against Xenograft Models of Acute Lymphoblastic Leukemia

Introduction: Children diagnosed with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, however the outcome for high-risk subtypes remains poor. Evasion of apoptosis is a hallmark of cancer, with dysregulation of the BCL-2 family of proteins often reported (Delbridge et al, Nat Rev Cancer 16:99-109, 2016). The BCL-2 family consists of the anti-apoptotic MCL1, BCL-2 and BCLXL as well as pro-apoptotic proteins. AMG 176, a first-in-class MCL1 inhibitor, previously demonstrated potent apoptosis-inducing activity in the nanomolar range against cell lines and patient explants of human hematological malignancies (Caenepeel et al, Cancer Discov 8:1582-97, 2018). Furthermore, discontinuous oral administration of AMG 176 inhibited the growth of human acute myeloid leukemia and multiple myeloma tumor xenografts in vivo at well-tolerated doses. AMG 176 has entered clinical evaluation for patients with hematological malignancies. Therefore, it was of interest for the Pediatric Preclinical Testing Consortium (PPTC) to evaluate the in vivo efficacy of AMG 176 against its patient-derived xenograft (PDX) models of pediatric ALL. Methods:MCL1, BCL-2 and BCLXL mRNA expression was quantified by RNA-seq (http://pedcbioportal.kidsfirstdrc.org). For in vitro cytotoxicity assays, cells were treated with AMG 176 (10 μM - 1 pM) or DMSO control for 24 h and cell viability was determined by AlamarBlue assay and expressed as IC50values. AMG 176 was evaluated in vivo against a panel of 38 ALL PDXs in a single mouse trial format. Each PDX was inoculated via tail vein injection into one NSG mouse (1-5 x 106 cells per mouse). Engraftment and drug responses were determined by enumerating the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB) at weekly intervals. Treatment with AMG 176 (provided by Amgen, Inc.) began once the %huCD45+ was ≥1% in each mouse and consisted of 60 mg/kg via oral gavage 2 days on, 5 days off for 2 weeks. The experimental endpoint was pre-defined as the %huCD45+ reaching 25% in the PB. Drug efficacy was assessed by event-free survival (EFS) and stringent objective response measures (Houghton et al, Pediatr Blood Cancer 49:928-40, 2007). For the pharmacokinetic (PK) study naïve NSG mice were treated with either vehicle or AMG 176 (60 mg/kg) and groups of 3 mice harvested up to 24 h post-treatment. Plasma samples were collected for analysis of AMG 176 using acetonitrile protein precipitation and LC-MS/MS with gradient elution on a Phenomenex Kinetex XB-C18 (3.0 x 50 mm, 2.6 µm) analytical column. Results:MCL1 mRNA expression ranged from 21-282 FPKM across a panel of 90 ALL PDXs representing 6 broad ALL subtypes (B-cell precursor, BCP-ALL; Ph+-ALL; Ph-like ALL; mixed-lineage leukemia-rearranged, MLLr-ALL; T-cell ALL, T-ALL; and early T-cell precursor ALL, ETP-ALL). Thirty eight of the 90 ALL PDXs were selected for in vivo evaluation of AMG 176 activity based on their profiles of MCL1, BCL-2 and BCLXL mRNA expression. AMG 176 was well tolerated in the efficacy study, with only 3 mice experiencing >10% weight loss. Mouse EFS values ranged from 3.5 to 47.8 days from treatment initiation, however no PDX achieved an objective response (remission) with all but one PDX exhibiting progressive disease. The Ph-like ALL PDX, ALL-102 (MCL1 expression 71.8 FPKM) scored a stable disease and had the highest EFS value (47.8 days). In an effort to explain the poor in vivo activity of AMG 176 several follow-up studies were performed. First, the in vitro IC50 values elicited by the AMG-176 stock against 5 leukemia cell lines (MOLM-13, 1.6 µM; NB-4, 2.0 µM; Kasumi-1, 1.6 µM; THP-1, 1.2 µM; and U-937,1.3 µM) were comparable to published data. Second, C18 LC-MS analysis revealed major and minor peaks consistent with theoretical peaks with negligible degradation. Third, PK study results demonstrated that the AMG 176 plasma concentration peaked at 8.5 µM, one hour after administration, with an AUC of 87.7 µM*hr and t1/2 of 4.1 h. This AUC was markedly lower than that in athymic nude mice treated at the same dose (1,120 µM*hr). Conclusions: AMG 176 exhibited modest in vivo activity against a large range of pediatric ALL PDXs, with the best response defined as stable disease in a single PDX. PK data suggest that AMG 176 systemic exposures in NSG mice may not be sufficient to elicit meaningful and prolonged regressions in pediatric ALL PDXs. (Supported by NCI Grants CA199000 and CA199222)

Potential Therapeutic Effect of TLR4-Primed Mesenchymal Stem Cells in Lessening Kidney Damages in Rat Model of Diabetic Nephropathy

Background and Aims: Substantial damage to the kidney tissue and diabetic nephropathy (DN) can be caused by chronic hyperglycemic conditions and exposure to a high level of blood glucose. In the current study, we explored the capability of adipose-derived mesenchymal stem cells (ADSCs) and Toll-like receptor-4-primed mesenchymal stem cells (TLR4-primed MSCs) on kidney regeneration, resolution of inflammation, and alleviation of diabetic nephropathy in DN in the rats.&#x0D; Materials and Methods: STZ-induced diabetic rat models were divided into 5 subgroups, including 1) DN group, 2) DN group received insulin, 3) DN group received human foreskin fibroblast (DN-HFF), 4) DN group received single pulse of 1×106 cells of MSCs and 5) DN group received single pulse of TLR4-primed MScs. Thereafter, biochemical and histological analysis was performed on DN-model groups. Biochemical analysis exhibited a blood urea nitrogen level recovery in both MSCs and TLR4-primed MSCs-treated groups. Administration of MSCs also up-regulated mRNA expression of Bcl-xL, while the expression of Bax was significantly down-regulated.&#x0D; Results: Histological and molecular results showed that TLR4-primed MSCs have an anti-inflammatory and anti-apoptotic effect on inflamed kidneys and effectively reduced DN indicators in the TLR4-primed MSCs group compared to the unprimed MSCs group.&#x0D; Conclusion: Priming with LPS improves the therapeutic effects of MSCs in the rat model of DN, consequently lessening the symptoms of DN rats. This study proposes that primed MSCs can be served as a potential therapeutic approach in diabetes mellitus and DN management.

Elevated Expression of miRNA-181a-5p in Placenta Tissue of Preeclampsia

This study intends to assess miR-181a-5p expression profile in preeclampsia (PE). PE tissues from 30 patients and 30 normal pregnancy patients were obtained to measure miR-181a-5p level by RTPCR. Cell was transfected with miR-181a-5p mimics followed by analysis of cell migration by woundhealing assay, cell proliferation by CCK-8 assay as well as IGF2BP2 expression by RT-PCR and Bcl-2, Bax, caspase3, and Bcl-xl level by western blot. miR-181a-5p was significantly upregulated in PE patients. Mimics transfection significantly decreased cell migration and proliferation. Interestingly, miR-181a-5p bound to IGF2BP2 3′UTR. Further, miR-181a-5p upregulation decreased IGF2BP2 expression dose-dependently, upregulated the mRNA expression of Bax, caspase3 and Bcl-xl and downregulated Bcl-2. IGF2BP2 upregulated Bcl-2 and downregulated Bax and caspase3, which were reversed by miR-181a-5p. miRNA-181a-5p overexpression restrains placental trophoblast cells by down-regulation of IGF2BP2, thereby participating in PE pathogenesis.

Effect of Extract of Acanthopanax Senticosus Fruit on Breast Cancer Cells

Objectives: Acanthopanax senticosus is a tree used in traditional medicine for various diseases. In this study, we investigated the anti-cancer effects of a water extract of Acanthopanax senticocus fruit (ASF) on 2 human breast cancer cell lines (MCF-7 and MDA-MB-231).Methods: The MTT assay was used to assess cell proliferation. The expression of apoptosis-related genes was assessed by quantitative real-time PCR.Results: ASF treatment caused a dose-dependent inhibition of cell growth in both estrogen-independent MDA-MB-231 and estrogen-dependent MCF-7 breast cancer cells. ASF decreased mRNA expression of the apoptotic suppressor gene Bcl-xL, and increased mRNA expression of proapoptotic genes. ASF increased the mRNA expression of p21 and RIP-1 in both cell types. ASF decreased the mRNA expression of survivin in the MCF-7 cell line.Conclusions: ASF exhibits anti-cancer activity involving apoptotic cell death.

Leukemia/lymphoma-related factor (LRF) or osteoclast zinc finger protein (OCZF) overexpression promotes osteoclast survival by increasing Bcl-xl mRNA: A novel regulatory mechanism mediated by the RNA binding protein SAM68

AbstractRANKL induces NFATc1, a key transcriptional factor to induce osteoclast-specific genes such as cathepsin K, whereas transcriptional control of osteoclast survival is not fully understood. Leukemia/lymphoma-related factor (LRF) in mouse and osteoclast zinc finger protein (OCZF) in rat are zinc finger and BTB domain-containing protein (zBTB) family of transcriptional regulators, and are critical regulators of hematopoiesis. We have previously shown that differentiation and survival were enhanced in osteoclasts from OCZF-Transgenic (Tg) mice. In the present study, we show a possible mechanism of osteoclast survival regulated by LRF/OCZF and the role of OCZF overexpression in pathological bone loss. In the in vitro cultures, LRF was highly colocalized with NFATc1 in cells of early stage in osteoclastogenesis, but only LRF expression persisted after differentiation into mature osteoclasts. LRF expression was further enhanced in resorbing osteoclasts formed on dentin slices. Osteoclast survival inhibitor such as alendronate, a bisphosphonate reduced LRF expression. Micro CT evaluation revealed that femurs of OCZF-Tg mice showed significantly lower bone volume compared to that of WT mice. Furthermore, OCZF overexpression markedly promoted bone loss in ovariectomy-induced osteolytic mouse model. The expression of anti-apoptotic Bcl-xl mRNA, which is formed by alternative splicing, was enhanced in the cultures in which osteoclasts are formed from OCZF-Tg mice. In contrast, the expression of pro-apoptotic Bcl-xs mRNA was lost in the culture derived from OCZF-Tg mice. We found that the expression levels of RNA binding splicing regulator, Src substrate associated in mitosis of 68 kDa (Sam68) protein were markedly decreased in OCZF-Tg mice-derived osteoclasts. In addition, shRNA-mediated knockdown of Sam68 expression increased the expression of Bcl-xl mRNA, suggesting that SAM68 regulates the expression of Bcl-xl. These results indicate that OCZF overexpression reduces protein levels of Sam68, thereby promotes osteoclast survival, and suggest that LRF/OCZF is a promising target for regulating pathological bone loss.

DNA Fragmentation and mRNA Expression of Bcl-2, Bcl-xL, p53, p21 and HSP70 Genes in Nondysplastic and Dysplastic Oral Lichen Planus.

Background:Oral lichen planus (OLP) is a chronic inflammatory disease. Apoptosis of the basal keratinocytes is a causative factor for OLP pathogenesis but the detailed mechanism of apoptosis among nondysplastic and dysplastic OLP lesions is yet unraveled.Aims:This study aims to evaluate the involvement of cellular DNA fragmentation and alteration in the expression of Bcl-2 and B-cell lymphoma extra-large (Bcl-xL), p53, p21 and heat shock protein 70 (HSP70) in nondysplastic and dysplastic OLP lesions.Materials and Methods:Untreated, fifteen OLP patients each with nondysplastic and dysplastic lesions were enrolled for this study. Their DNA fragmentation was analyzed by the agarose gel electrophoresis method. The mRNA expression of Bcl-2, Bcl-xL, p53, p21 and HSP70 were measured using semi-quantitative reverse transcription-polymerase chain reaction.Results:Elevated DNA fragmentations were found in dysplastic lesions compared to nondysplastic type. Significantly higher expression of Bcl-2, Bcl-xL, p53 and p21 were found in both types of OLP lesion compared to the control. Expression of Bcl-2 and Bcl-xL were significantly elevated in nondysplastic lesions, whereas significantly overexpression of p53 and p21 were found in dysplastic lesions. Anti-stress protein HSP70 was overtly expressed in dysplastic lesions compared to other groups.Conclusion:Reduced expression of Bcl-2 and Bcl-xL, with elevated DNA fragmentation, may be associated with increased apoptosis in dysplastic lesions which aid in the resolution of the chronic inflammatory process. Higher expression of p53 and p21 in dysplastic lesions reflect its malignant potentiality. Overexpression of HSP70 in dysplastic lesions is a useful marker for higher cellular stress.

Ginsenoside Rg5 Sensitizes Paclitaxel-Resistant Human Cervical-Adeno-Carcinoma Cells to Paclitaxel-And Enhances the Anticancer Effect of Paclitaxel.

In cervical cancer chemotherapy, paclitaxel (PTX) chemoresistance has become a major difficulty, and it also affects the survival rate of numerous tumor patients. Thus, for the reversal of chemoresistance, it is imperative to develop combinatory drugs with petite or almost no side effects to sensitize cells to paclitaxel. Ginsenoside Rg5 (GRg5) may act as a chemosensitizer by reversing multidrug resistance. The present study aimed to determine the potential of GRg5 as a chemosensitizer in PTX-resistant human cervical adeno-carcinoma cell lines (HeLa cells). MTT assay was carried out to assess whether GRg5 can potentiate the cytotoxic effect of PTX in PTX- resistant HeLa cells; using flow cytometry-based annexin V-FITC assay, cellular apoptosis was analyzed; the rate of expression of the cell cycle, apoptosis and major cell-survival-signaling-related genes and its proteins were examined using RT-PCR and Western blotting technique. We found increased mRNA expression of Bak, Bax, Bid, and PUMA genes, whereas the mRNA expression of Bcl2, Bcl-XL, c-IAP-1, and MCL-1 were low; GRg5 combination triggered the efficacy of paclitaxel, which led to increased expression of Bax with an enhanced caspase-9/-3 activation, and apoptosis. Moreover, the study supports GRg5 as an inhibitor of two key signaling proteins, Akt and NF-κB, by which GRg5 augments the susceptibility of cervical cancer cells to PTX chemotherapy. GRg5 drastically potentiated the antiproliferative and pro-apoptotic activity of paclitaxel in PTX-resistant human cervical cancer cells in a synergistic mode. Moreover, in the clinical context, combining paclitaxel with GRg5 may prove to be a new approach for enhancing the efficacy of the paclitaxel.

LGG-18. Inhibition of Bcl-xL targets the senescent compartment of pilocytic astrocytoma

Abstract INTRODUCTION: Pilocytic astrocytoma (PA) is a mitogen-activated protein kinase (MAPK)-driven disease. Treatment of sub-totally resected PA remains challenging and relapses occur even after MAPK-targeted therapies such as MEK inhibition. Oncogenic activation of the MAPK-pathway drives the majority of cells into oncogene-induced senescence (OIS). OIS might represent a complementary vulnerability exploitable by senolytic agents. Here we investigated the senolytic properties of BH3-mimetics in PA. METHODS: Four patient-derived PA cell lines, DKFZ-BT66, -BT308, -BT317 (KIAA1549:BRAF-fusion) and DKFZ-BT314 (BRAF V600E-mutation) were treated with different BH3-mimetics, chemotherapeutics and MEK-inhibitors in proliferation (expression of SV40 large T (TAg)) and OIS (repression of TAg) states. Inhibition of metabolic activity (CellTiterGlo® 2.0) and reduction of viability (trypan blue) was assessed after 72 hours. Target expression was determined using gene expression data and Western blot. On-target activity was verified by immunoprecipitation. Dependence on Bcl-xL was investigated by shRNA-mediated knockdown and BH3-profiling. Gene expression data of primary PA and 751 cancer cell lines (GDSC dataset) was analyzed. RESULTS: BH3-mimetics inhibiting Bcl-xL (Bcl-xLi; Navitoclax, A-1131852, A-1155463, AZD4320) showed activity in 3/4 models (DKFZ-BT66, -BT314 and -BT317) in the OIS state (IC50s &amp;lt;300nM). Other BH3-mimetics, chemotherapeutics and MEK-inhibitors had no effect on all models in OIS. Bcl-xL mRNA expression levels were similar, on-target activity and dependence on Bcl-xL protein was comparable in all models. Bcl-xLi-resistance of DKFZ-BT308 was linked to upregulation of genes of the HALLMARK_XENOBIOTICS_METABOLISM gene set involved in drug metabolism. Correlation of these genes with IC50s of navitoclax was validated in an independent dataset (GDSC). CONCLUSION: Treatment with Bcl-xLi is a promising strategy targeting the senescent part of PA. IC50s of the clinically available drug navitoclax were &amp;gt;20-fold below achievable plasma concentration indicating translatability. Genes from the gene set HALLMARK_XENOBIOTICS_METABOLISM could represent a predictive biomarker.

Anti-Proliferative Effect of Hydroethanolic Leaf Extract of Citrullus colocynthis (L) on Retinoblastoma Cell Line

Introduction: Citrullus colocynthis has many pharmacological, biological, and therapeutic properties making it a very important medicinal plant. The cucurbitacin produced/derived from the plant play an important role in anticancer activities. The retinoblastoma Y-79 cell lines are studied in attachment culture. A proper study of these cells can pave the way for control of its proliferative property. Cell viability was assayed using a modified colorimetric technique that is based on the ability of the live cells to convert MTT, a tetrazolium compound into purple formazan crystals. There was an effect on the proliferation and gene expressions of the retinoblastoma Y-79 cell line. Bioactive extracts of these plants are being majorly used as a potential chemopreventive element as an alternative approach for cancer progression management.&#x0D; Materials and Methods: Human retinoblastoma cell line (Y-79)cell line was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cell viability was assayed using a modified colorimetric technique that is based on the ability of live cells to convert MTT.&#x0D; Results: The experimental study showed antiproliferative effects against the retinoblastoma Y-79 cells. The experimental study showed the effect on cell viability, Bcl2 mRNA expression and BclxL mRNA expression in Y-79 cells.&#x0D; Conclusion: The hydroethanolic leaf extract obtained from the Citrullus colocynthis exhibited an antiproliferative effect on the retinoblastoma Y-79 cell line by affecting the expression of the Bcl2 mRNA and BclxL mRNA and hindering the cell viability.

Cytotoxic Effect of Hydroethanolic Extract of Citrullus colocynth (L) Fruit against Human Lung Cancer Cell Line

Citrullus colocynthis is a traditional medicinal plant that belongs to the cucurbitaceae family. The extract of the fruit is rich in therapeutic phytochemicals. Lung cancer is a disease with high mortality and morbidity rates. Recently the research has been focused on herbal medicine as a treatment approach for cancer because of its no or less side effects. The aim of the study is to know about the anticancer effect of hydroethanolic extract of Citrullus colocynthis fruit against human lung cancer cell line through apoptosis pathway. Cell viability test was done by MTT assay. The mRNA expression of Bcl2 and Bcl-xL was done using the real-time PCR. The obtained data was analysed statistically by one way analysis of variance and Duncan multiple range test with graph prism version 5 to analyse the significance. The significance was considered at p&lt;0.05 level in Duncan’s test. Citrullus colocynthis fruit extract increased the inhibition of growth of lung cancer cells. The activity of Bcl2 and Bcl xL was significantly down regulated at 400μg. The study concludes that Citrullus colocynthis fruit extract has anticancer activity on A549 human lung cancer cell line through apoptosis pathway.

Bcl-xL expression and regulation in the progression, recurrence, and cisplatin resistance of oral cancer

BackgroundBcl-xL is an anti-apoptotic molecule, but its role in the progression and recurrent/ drug-resistant oral squamous cell carcinoma (OSCC) is poorly understood. Materials and methodsA total of one hundred twenty-five human OSCC tissue specimens including twenty-nine adjacent normals (AN), sixty-nine primary tumors (PT), twenty-seven recurrent chemoradiation resistance (RCRT) samples, and oral tongue SCC derived cisplatin-resistant (CisR SCC-4/-9) cells were used, for this study. Protein/mRNA expression levels of Bcl-xL and its regulation by ERK1/2, Stat-3, p53, NFκB, AP-1 (components: c-Jun, c-Fos, and Fra-2) molecules, and cell viability were measured by immunohistochemistry, Western blot, RT-PCR, and MTT analysis. Further, the individual and synergistic effects of Fra-2 (siRNA) and nimbolide were tested in CisR SCC-4/-9 cells. ResultsProgressive increase of Bcl-xL expression and its transcriptional-deregulation was observed with OSCC progression and resistance. Among all the possible upstream regulators of Bcl-xL, such as ERK1/2, Stat-3, p53, AP-1, and NFκB, the TF AP-1 (r = 0.644, p = 0.0001) showed maximum association with Bcl-xL mRNA expression. Though differential expression of AP-1 components were detected in OSCC specimens, with more striking positive-correction of c-Jun (r = 0.381, p = 0.049), c-Fos (r = 0.139, p = 0.488, ns) and Fra-2 (r = 0.664, p = 0.0001) with Bcl-xL expression observed stronger in RCRT tumor subgroup. Further, knockdown of Fra-2 and the application of plant-based phytochemical nimbolide decreased Bcl-xL expression and induced apoptosis in CisR SCC-4/-9 cells. ConclusionCollectively, we have demonstrated the role of Bcl-xL and AP-1 (Fra-2), causing OSCC progression and cisplatin resistance. Targeting Bcl-xL upstream pathway along with the application of nimbolide might be beneficial in eliminating drug-resistant OSCC.

Reduced cytotoxicity in doxorubicin-exposed HepG2 cells pretreated with menthol due to upregulation of P-glycoprotein.

The aim of the present study was to examine changes in the expression and activity of P-glycoprotein (P-gp) in human hepatocellular carcinoma HepG2 cells after exposure to menthol, and their relationship to the cytotoxicity of and apoptotic responses to doxorubicin (DOX), a substrate of P-gp, in the cells. The expression of P-gp in HepG2 cells was significantly increased by menthol treatment. Intracellular accumulation of DOX in HepG2 cells was significantly lower in the menthol-treated group than in the control group, but this phenomenon was abolished in the presence of verapamil. Decreased cell viability by DOX was significantly attenuated by 24-h menthol treatment prior to DOX exposure, which coincided with the changes in mRNA expression of Bcl-xl and caspase-3. These results demonstrate that menthol causes hepatocellular carcinoma cells to acquire resistance to DOX by increasing its efflux through the upregulation of P-gp.