Assessment Of Bcl-2 Research Articles

Silver nanoparticles induced hepatoxicity via the apoptotic/antiapoptotic pathway with activation of TGFβ-1 and α-SMA triggered liver fibrosis in Sprague Dawley rats

Despite the extraordinary use of silver nanoparticles (AgNPs) in medicinal purposes and the food industry, there is rising worry about potential hazards to human health and the environment. The existing study aims to assess the hepatotoxic effects of different dosages of AgNPs by evaluating hematobiochemical parameters, oxidative stress, liver morphological alterations, immunohistochemical staining, and gene expression to clarify the mechanism of AgNPs’ hepatic toxic potential. Forty male Sprague Dawley rats were randomly assigned into control and three AgNPs intraperitoneally treated groups 0.25, 0.5, and 1 mg/kg b.w. daily for 15 and 30 days. AgNP exposure reduced body weight, caused haematological abnormalities, and enhanced hepatic oxidative and nitrosative stress with depletion of the hepatic GSH level. Serum hepatic injury biomarkers with pathological hepatic lesions where cholangiopathy emerges as the main hepatic alteration in a dosage- and duration-dependent manner were also elevated. Furthermore, immunohistochemical labelling of apoptotic markers demonstrated that Bcl-2 was significantly downregulated while caspase-3 was significantly upregulated. In conclusion, the hepatotoxic impact of AgNPs may be regulated by two mechanisms, implying the apoptotic/antiapoptotic pathway via raising BAX and inhibiting Bcl-2 expression levels in a dose-dependent manner. The TGF-β1 and α-SMA pathway which triggered fibrosis with incorporation of iNOS which consequently activates the inflammatory process were also elevated. To our knowledge, there has been no prior report on the experimental administration of AgNPs in three different dosages for short and long durations in rats with the assessment of Bcl-2, BAX, iNOS, TGF-β1, and α-SMA gene expressions.Graphical abstract

Abstract 4656: Assessment of BCL-2, BCL-xL and MCL-1 in bone marrow biopsy samples using VENTANA BenchMark XT automated slide stainer

Abstract Immunophenotyping is essential for diagnosis and classification of most hematopoietic malignancies, and often followed by ancillary studies to understand the predictive and prognostic value of biomarkers. Immunohistochemistry (IHC) provides in-situ coupling of antigen expression with morphology of precursor B- from precursor T-lymphoblastic leukemia/lymphoma. The number of different IHC antibodies that can be applied to bone marrow (BM) biopsies is increasing concurrently with development of flow cytometry and molecular diagnostic methods. IHC detects scattered tumor cells in Hodgkin’s Lymphoma, Multiple Myeloma (MM) and Acute Myelogenous Leukemia (AML), where key biomarkers may not be detectable by current flow cytometry methods. The goal of this study was to detect BCL-2, Bcl-xL and MCL-1 that are able to predict pre-clinical sensitivity to the drug Venetoclax. We examined the expression of these biomarkers in MM bone samples decalcified using commercially available reagents to determine whether decalcification methods impact BCL-2 family IHC Staining. Results: MM bone samples (n=59) decalcified in Decal STAT® for 20 min, stained for BCL-2 (124) and Bcl-xL (54H6). MM bone samples (n=7) decalcified in Decal STAT® for 60 min stained for BCL-2 and Bcl-xL. Finally, eleven MM bone samples decalcified in Richard Allan Decal Solution® stained for BCL-2 and Bcl-xL. MCL-1 stained poorly with commercially available decalcification solutions. For MCL-1 to be used, additional staining optimization is required. Conclusion: BCL-2 and BCL-xL proteins can be detected in MM bone samples decalcified with commercially available solutions. With these preliminary results, future studies will pursue the potential for sensitivity to "Venetoclax (ABT-199/GDC-0199)". Citation Format: Ashley Streator, Eneida C. Villanueva, Burton F. Holmes, Liping Zhang. Assessment of BCL-2, BCL-xL and MCL-1 in bone marrow biopsy samples using VENTANA BenchMark XT automated slide stainer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2017-4656

Bcl-2/IgH Expression of Diffuse Large B-cell Lymphoma Cells in Minimal Bone Marrow Infiltration

We sought to investigate the role of the Bcl-2 translocation at the chromosomal and protein levels in minimal bone marrow (BM) infiltration by diffuse large B-cell lymphomas (DLBCL). The presence of the Bcl-2/IgH fusion gene was detected in BM samples and paraffin-embedded lymph node (LN) samples from 103 patients with DLBCLs using FISH. Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using immunocytochemistry (ICC) and immunohistochemistry (IHC), respectively. Bcl-2/IgH translocation in paraffin-embedded LN tissue sections was observed in 43 (41.7%) patients by FISH. Of the 43 patients, the Bcl-2/IgH rearrangement in the bone marrow specimens occurred in 34 patients. The Bcl-2/IgH recombination rate in stage III cancers was not significantly different compared to the rate observed in stage I/II cancers (p = 0.101), respectively showing no statistical differences between stage IV and I/II (p = 0.179). In 64.7% (22/34) of the cases with t(14;18), Bcl-2 was detected based on ICC analysis. Positive Bcl-2 ICC staining and the t(14;18) translocation were positively correlated (p < 0.001). We then applied our FISH method to slides with at least one abnormal cell and were subjected to FISH analysis after staining. During the follow-up, no infiltration by cytomorphology for 16 DLBCL patients whose bone marrow presented Bcl-2/IgH gene rearrangement at diagnosis, and two cases were positive by morphology compared to FISH-positive results 6 months later; 9 out of 16 patients (56.3%) presented with positive Bcl-2/IgH results earlier than the morphology evaluation after 12 months. Utilizing both FISH and cytologic morphology, the assessment of Bcl-2/IgH translocation status could contribute to the better detection of minimal bone marrow infiltration and relapse receiving treatment by DLBCL cells.

P2-12-08: Bcl-2 as a Prognostic Marker in Breast Cancer Patients Receiving Endocrine Therapy.

Abstract Background: In breast cancer patients with estrogen receptor (ER) positive tumors, endocrine treatment reduces recurrence. However, 30% of patients eventually relapse in spite of therapy. Additional markers to identify this group of therapy resistant, prognostically unfavourable patients are therefore needed. The expression of Bcl-2 has been shown to be a prognostic factor in breast cancer patients regardless of ER-status, but assessment of Bcl-2 by immunohistochemistry (IHC) needs to be standardized since different scoring systems and different cut-points have been used. In a pilot study comprising 258 ER positive tumors from tamoxifen-treated patients, a simple scoring system enabled us to find suitable cut-points, selecting patient-groups with poor prognosis. The aim of the current study was to validate these results in a similar population from a large randomised study. Material and methods: Bcl-2 was assessed in 1082 (78%) of the 1396 postmenopausal Danish patients who in the BIG 1–98 trial were randomised to 5 years of tamoxifen (N=325), letrozole (N=335) or a sequence of these agents (N=422). Tissue microarrays were made from formalin fixed paraffin embedded primary tumors. An IHC score for expression of cytoplasmic Bcl-2 (clone 124, Dako) was performed according to both the number of positive cells (0%=0, 1–10%=1, 11–50%=2, 51–100%=3) and the intensity of the staining (weak, moderate, strong) as in the pilot study. The associations between Bcl-2 status and clinicopathological variables were assessed. The prognostic value of Bcl-2 according to cut-points determined in the pilot study was analysed by Cox proportional hazards models. The primary endpoint was disease free survival (DFS). Results: Patients were divided into three subgroups according to predefined cut-points: Bcl-2 low (score 0 or 1 (n= 24)), Bcl-2 intermediate (score 2 or score 3/weak or moderate (n= 216)) and Bcl-2 high (score 3/strong (n= 842)). Bcl-2 expression was significantly associated with ER (P=0.0003) and progesterone receptor (PgR) (P&amp;lt;0.0001), and negatively associated with Ki-67 (P=0.0021) and tumor grade (P=0.005). In univariate analysis patients in the Bcl-2 low group had a bad prognosis compared to the Bcl-2 high group (HR=2.3; 95% CI: 1.2 to 4.1). In the multivariable analysis including ER, PgR, Ki-67, positive lymph nodes, age, tumor size and histological type and grading, association between Bcl-2 and DFS was not statistically significant (P=0.35). Discussion: We found Bcl-2 to be a prognostic factor in the univariate analysis (P=0.024) but not to be an independent predictor of poor survival of ER positive patients treated with tamoxifen and/or letrozole. In this population a low or absent expression of Bcl-2 was uncommon (2.2 %), as expected since Bcl-2 is an ER regulated protein. The results of the pilot study could not be confirmed in this validation study. However, the strong positive relation to other prognostic factors like tumor grade and high proliferative index indicates that Bcl-2 could contribute to factors characterising a group of tumors with a bad prognosis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-08.

Bcl-2/IgH expression in minimal bone marrow infiltration by follicular lymphoma cells

The purpose of this study was to investigate the roles of bcl-2 chromosomal translocation and Bcl-2 protein expression in follicular lymphoma (FL) minimal bone marrow (BM) infiltration. We identified the same bcl-2/IgH fusion gene in paraffin-embedded lymph node (LN) samples and BM samples using immunohistochemistry (IHC), immunocytochemistry (ICC), cytologic morphology and fluorescence in situ hybridization (FISH). The presence of the Bcl-2/IgH fusion gene in the BM samples and paraffin-embedded LN samples from 56 patients with follicular lymphomas was detected using FISH. The Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using ICC and IHC, respectively. Approximately 78.6% (44/56) of the paraffin‑embedded LN tissue sections that underwent FISH analysis had a bcl-2/IgH translocation. The primary lesion was also positive for the bcl-2/IgH fusion gene, as were the BM minimal infiltrates. The bcl-2/IgH rearrangement occurred in 88.6% (39/44) of the BM specimens. The bcl-2/IgH recombination rate in stage III/IV cancers was significantly different to that observed in stage I/II cancers (p=0.041). In 59% (23/39) of the cases with t(14;18), Bcl-2 was found to be present as assessed by ICC. Positive Bcl-2 ICC staining and the t(14;18) translocation (as detected using FISH) were positively correlated (p=0.028). We then applied the FISH method to slides that had previously been morphologically evaluated using Wright-Giemsa staining; any slides with at least one abnormal cell were subjected to FISH analysis following staining. The assessment of bcl-2/IgH translocation status may contribute to the better detection of minimal BM infiltration by FL cells. Utilizing FISH and cytologic morphology techniques allows for earlier and more accessible BM examination.

Lycium barbarum Polysaccharides Induce Apoptosis in Human Prostate Cancer Cells and Inhibits Prostate Cancer Growth in a Xenograft Mouse Model of Human Prostate Cancer

Lycium barbarum polysaccharides (LBPs) are important functional constituents in red-colored fruits of L. barbarum (Guo Qi Zi, a well-known traditional Chinese medicinal plant commonly known as Goji berry or wolfberry). The influence of LBP on human prostate cancer cells was systematically investigated in vitro and in vivo. The in vitro effects of LBP on two cell lines (PC-3 and DU-145) were examined by using trypan blue exclusion staining, single-cell gel electrophoresis, flow cytometry, terminal dUTP nick-end labeling assay, and immunohistochemical assay (assessment of Bcl-2 and Bax expression). The in vivo effect of LBP on PC-3 cells was assessed in the nude mouse xenograft tumor model. The in vitro results showed that LBP can dose- and time-dependently inhibit the growth of both PC-3 and DU-145 cells. LBP caused the breakage of DNA strands of PC-3 and DU-145 cells; the tail frequency and tail length were significantly higher than that of control cells. LBP also markedly induced PC-3 and DU-145 cell apoptosis, with the highest apoptosis rates at 41.5% and 35.5%, respectively. The ratio of Bcl-2/Bax protein expression following LBP treatments decreased significantly with a dose-effect relationship, which suggested that LBP can regulate the expression of Bcl-2 and Bax to induce apoptosis of PC-3 and DU-145 cells. The in vivo experimental results indicate that LBP might significantly inhibit PC-3 tumor growth in nude mice. Both the tumor volume and weight of the LBP treatment group were significantly lower than those of the control group.

“No Turning Bax” in the Combined Battle against Prostate Cancer:

In this issue of Clinical Cancer Research , Khor et al. ([1][1]) investigate Bcl-2 and Bak as potential predictors of clinical outcome in prostate cancer patients enrolled in the Radiation Oncology Group Protocol 92-02. Prostate cancer is the most commonly diagnosed cancer in men in the United

471 POSTER Functional assessment of Bcl-2 disordered loop through plasmon surface resonance technology

471 POSTER Functional assessment of Bcl-2 disordered loop through plasmon surface resonance technology

A phase I study of antisense Bcl-2 oligonucleotide (G3139) in combination with carboplatin (C) and paclitaxel (T) in patients with advanced solid tumors

13006 Background: G3139 is an 18-mer oligonucleotide that targets the mRNA of Bcl-2, a protein that inhibits apoptosis conferring treatment resistance, and is synergistic with taxanes in studies using Bcl-2 over-expressing xenografts. Here we conduct a study combining G3139 with C + T in order to define the recommended phase II dose, as well as evaluate the direct biologic effect of G3139 on tumor. Methods: Patients with advanced solid tumors were treated with a dose escalation of G3139 (3–7 mg/kg/day) by continuous infusion days 1–7, with C (AUC 5) and T (150 mg/m2) given on day 4, repeated in 3 week cycles. At the planned expanded cohort (G3139 7 mg/kg/day dose), 12 patients underwent paired tumor biopsies (baseline and day 4 pre-chemotherapy) for assessment of Bcl-2/Bax expression by RT-PCR and IHC, as well as intratumoral G3139 levels using ELISA. Likewise, peripheral blood mononuclear cells (PBLs) were assessed using flow cytometry and RT-PCR for changes in Bcl-2/Bax expression. Results: 34 patients have been treated: 11 melanoma, 7 bladder, 3 prostate, 2 esophageal, 3 non-small cell lung, and 1 head/neck, kidney, breast, pancreas, liver, gastrointestinal stromal, biliary, and unknown primary cancers. G3139/C/T appeared well tolerated at the doses administered with the main toxicities observed being attributed to the C + T chemotherapy (myelosuppression and thrombocytopenia). The maximal tolerated dose is yet to be achieved. Assessment of paired tumor biopsies on 12 patients show decreases in Bcl-2 transcription in laser capture microdissected tumor using RT-PCR, with concordance observed using IHC. Likewise, decrease in Bcl-2 gene expression in PBLs was seen. 2 confirmed PRs were observed, including a bladder patient with baseline Bcl-2 tumor expression 10-fold greater than any other patient assessed. Conclusions: G3139/C/T is well tolerated, with main toxicities attributable to the chemotherapy alone. Bcl-2 is suppressed in tumor and PBLs supporting that a biologically active dose of G3139 is being achieved. Currently, the C + T is being escalated to define a MTD. Complete clinical data, G3139 pharmacokinetics, and Bcl-2/Bax data will be presented in detail. No significant financial relationships to disclose.

Inhibition of Either Phosphatidylinositol 3-kinase/Akt or the Mitogen/Extracellular-regulated Kinase, MEK/ERK, Signaling Pathways Suppress Growth of Breast Cancer Cell Lines, but MEK/ERK Signaling is Critical for Cell Survival

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

Assessment of Bcl-2 Expression as Modulator of Fas Mediated Apoptosis in Acute Leukemia

Apoptosis is the primary mechanism through which most chemotherapeutic agents induce tumor cell death. The balance in the expression of pro (Fas/CD95) and anti-apoptotic protein (Bcl-2) may control the response of leukemic cells to chemotherapy and subsequently affect the patient's prognosis. The aim of this study was to determine the levels of Bcl-2 and Fas expression on blast cells from patients with acute leukemia and to correlate the degree of expression to the clinical and laboratory prognostic factors and the patient's outcome. Forty newly diagnosed patients with acute leukemia (16 ALL, 24 AML) were included in the study. Ten normal subjects of matched age and sex were studied as a reference control group. The degree of Bcl-2 and Fas expression on acute leukemia blast cells were assessed before the start of therapy and on mononuclear cells after 1 year of follow up, using flow cytometry. The degree of Bcl-2 and Fas expression were significantly higher in AML (P<0.01,<0.05, respectively) and ALL (P<0.01, <0.05, respectively) as compared to controls. The expression of Fas and Bcl-2 was related to FAB type with the highest Bcl-2 and lowest Fas expression in M5 and T-ALL (P<0.01, for all). In ALL, patients responding to induction chemotherapy revealed lower Bcl-2 and higher Fas expression when compared to non-responders (P<0.05). In contrast, in AML the difference between responders and non-responders to induction chemotherapy regarding Bcl-2 and Fas expressions was not statistically significant (P>0.05). Bcl-2 and Fas expression were significantly elevated in the relapsed acute leukemia group (in both AML and ALL) when compared to those in remission (P<0.01, <0.05, respectively). Bcl-2 and Fas expression at diagnosis was not significantly different when those surviving were compared to the group who had died, either in the ALL or AML groups (P>0.05). Bcl-2 expression was significantly correlated to bone marrow blast cell counts (R=0.6, P<0.01), blast cell distribution ratio (R=0.4, P<0.05) and lymphadenopathy (R=0.33, P<0.05). Whereas Fas expression was significantly correlated to bone marrow blast cell counts (R=0.52, P<0.01). In conclusion, assessment of Bcl-2 and Fas expression at diagnosis in acute leukemia (1) could predict responsiveness to induction chemotherapy in ALL but not in AML group but (2) could not predict patients out come both in ALL and AML groups.

Role of Mitochondrial Membrane Potential in Concanavalin A-Induced Apoptosis in Human Fibroblasts

Lectins induce apoptosis in a wide variety of cell types but the mechanisms of apoptotic induction are unknown. We examined the role of mitochondrial membrane potential (Ψm) in concanavalin A-induced apoptosis in human diploid fibroblasts. Cells were treated with Con A for 0.5, 1, 3, 5, and 24 h. Con A induced a time-dependent increase of the proportion of TUNEL+ve cells over 24 h. Ψmwas examined by staining cells with the mitochondria-specific fluorescent cationic dye JC-1. Comparison of JC-1 fluorescence within mitochondria by flow cytometry showed that after 3 h, Con A reduced Ψmin a subpopulation of apoptotic cells with smaller cell volume and with apoptotic nuclear morphology. In contrast, Ψmwas unchanged in a separate population of viable cells with normal volume and normal nuclear morphology. Cyclosporin A protected cells against reduction of Ψmand also against nuclear condensation and morphological apoptosis. Measurement of intracellular calcium ion concentration ([Ca2+]i) by ratio fluorimetry of fura 2-loaded cells showed that Con A did not affect [Ca2+]iin viable cells but induced a progressive depletion of [Ca2+]iwith generation of calcium oscillations in apoptotic cells. Assessment of Bcl-2 in Con A-treated cells demonstrated an initially strong increase in Bcl-2 protein and mRNA but the appearance of degraded Bcl-2 protein at 3 and 5 h after treatment, indicating an inadequate protective response to the Con A stimulation. Collectively, these data indicate that lectin-induced apoptosis in fibroblasts is associated with breakdown of Ψm, loss of [Ca2+]ihomeostasis, and induced Bcl-2 expression.