Expression Of Bcl-2 Family Proteins Research Articles

Cucurbitacin E induces caspase-dependent apoptosis and protective autophagy mediated by ROS in lung cancer cells

Cucurbitacin E (CuE) is a triterpenoid with potent anticancer activities while the underlying mechanisms remain elusive. In the present study, the anticancer effects of CuE on 95D lung cancer cells were investigated. CuE decreased cell viability, inhibited colony formation, and increased reactive oxygen species (ROS) in a concentration-dependent manner, which were reversed by N-acetyl-l-cysteine (NAC). CuE induced apoptosis as determined by JC-1 staining, expression of Bcl-2 family proteins, cleavage of caspases, and TUNEL staining. NAC and Ac-DEVD-CHO partially reversed CuE-induced cleavage of caspase-3, caspase-7, and PARP. Furthermore, CuE caused accumulation of autophagic vacuoles and concentration- and time-dependent expression of LC3II protein. Autophagy inhibitors chloroquine and bafilomycin A1 enhanced CuE-induced LC3II expression and cell death. CuE-triggered protein expression of p-AKT, p-mTOR, Beclin-1, and p-ULK1 was partially reversed by NAC pretreatment. In addition, CuE treatment damaged F-actin without affecting β-tubulin as confirmed by immunofluorescence. In conclusion, CuE induced ROS-dependent apoptosis through Bcl-2 family and caspases in 95D lung cancer cells. Furthermore, CuE induced protective autophagy mediated by ROS through AKT/mTOR pathway. This study provides novel roles of ROS in the anticancer effect of CuE.

Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways.

Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption.

Synthesis, characterization, in vitro cytotoxicity and anticancer effects of ruthenium(II) complexes on BEL-7402 cells

Four new ruthenium(II) polypyridyl complexes [Ru(dmb)2(DQTT)](ClO4)2 (1) (DQTT=12-(1,4-dihydroquinoxalin-6-yl)-4,5,9,14-tetraazabenzo[b]triphenylene, dmb=4,4′-dimethyl-2,2′-bipyridine), [Ru(bpy)2(DQTT)](ClO4)2 (2) (bpy=2,2′-bipyridine), [Ru(phen)2(DQTT)](ClO4)2 (3) (phen=1,10-phenanthroline) and [Ru(dmp)2(DQTT)](ClO4)2 (4) (dmp=2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. The cytotoxic activity in vitro of the complexes was evaluated against human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines by MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method. The IC50 values of complexes 1–4 against BEL-7402 cells are 31.8±1.0, 35.8±1.6, 29.0±0.8 and 25.0±0.9μM, respectively. The morphological apoptosis was investigated with AO/EB (acridine orange/ethidium bromide) and Hoechst 33258 staining methods. The DNA damage was assayed by comet assay. The inhibition of cell migration was evaluated by the wound healing assay. The levels of ROS (reactive oxygen species) and the changes of mitochondrial membrane potential were studied under fluorescent microscope. The percentages in the cells of apoptotic and necrotic cells and the cell cycle arrest were determined by flow cytometry. The expression of Bcl-2 family proteins was investigated by western blot analysis. The results show that the complexes induce BEL-7402 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulation of the expression of Bcl-2 family proteins.

Synthesis, cytotoxicity in vitro, apoptosis, cell cycle arrest and comet assay of asymmetry ruthenium(II) complexes

A new ligand Adbdp and its four ruthenium(II) polypyridyl complexes [Ru(N-N)2(Adbdp)](ClO4)2 [N-N=dmb: 4,4′-dimethyl-2,2′-bipyridine 1, bpy: 2,2′-bipyridine 2, phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4, Adbdp=acenaphtheno[2,3-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2′,3′-c]phenazine] were synthesized and characterized by elemental analysis, ESI-MS and 1H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cells was studied by MTT method. The IC50 values are 24.2±1.9, 11.5±3.6, 3.4±0.3 and 9.7±0.5μM for complexes 1, 2, 3 and 4 toward A549 cells, respectively. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI-stained cell nuclei. The reactive oxygen species and mitochondrial membrane potential were performed under fluorescent microscope. The cell cycle distribution was studied by flow cytometry and the protein expression of Bcl-2 family proteins was analyzed by western blot. The results show that the complexes induce A549 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Differential Expression of Bcl-2 Family Proteins Determines the Sensitivity of Human Follicular Lymphoma Cells to Dexamethasone-mediated and Anti-BCR-mediated Apoptosis.

Bcl-2 family comprises proapoptotic and antiapoptotic proteins. The balance between these proteins is critical for the survival of the cells. Overexpression of the antiapoptotic protein, Bcl-2, is the hallmark of follicular lymphoma (FL). High expression of Bcl-2 provides survival advantage and may facilitate chemotherapeutic resistance in FL. In the present study, we examined expression profile of Bcl-2 family proteins such as Bcl-2, Bcl-xL, and Bim in human FL cell lines, HF1A3 and HF28. We assessed the correlation between the expression levels of these proteins and cells' sensitivity to dexamethasone (Dex)-mediated and B-cell receptor (BCR)-mediated apoptosis. Here, we show that Dex and anti-BCR-induced synergistic apoptosis which correlated with significant downregulation of Bcl-xL, inhibition of ERK1/2 phosphorylation and accumulation of nonphosphorylated Bim. However, HF28 cells were less sensitive than HF1A3 cells to Dex-induced and anti-BCR-induced apoptosis due to high Bcl-2 protein level. It is interesting to note that, a Bcl-2-specific inhibitor, ABT-199, sensitized HF28 cells to Dex-induced or anti-BCR-induced apoptosis. In addition, overexpression of Bcl-xL prevented Dex-mediated, anti-BCR-mediated, and ABT-199-mediated apoptosis, indicating that mitochondria were involved. In conclusion, these data show that the expression levels of Bcl-2 family proteins may serve to predict tumor response to BH3 mimetics and the sensitivity of FL cells to Dex-induced and anti-BCR-induced apoptosis. Moreover, our results show that BCR-targeted apoptosis might have therapeutic benefit against FL and B-cell lymphomas.

Overexpressed EDIL3 predicts poor prognosis and promotes anchorage-independent tumor growth in human pancreatic cancer.

Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein associated with vascular morphogenesis and remodeling, is commonly upregulated in multiple types of human cancers and correlates with tumor progression. However, its expression pattern and underlying cellular functions in pancreatic ductal adenocarcinoma (PDAC) remain largely unexplored. In current study, we observed that expression of EDIL3 was significantly up-regulated in PDAC compared with normal controls in both cell lines and clinical specimens. In addition, elevated EDIL3 expression was positively correlated with patients’ TNM stage and T classification. Kaplan-Meier analysis indicated that high EDIL3 expression was significantly associated with shorter overall survival times in PDAC patients. Multivariate Cox regression analysis confirmed EDIL3 expression, age, lymph node metastasis and histological differentiation as independent prognostic factors in PDAC. Knockdown of EDIL3 showed no significant influence on cell viability, migration, invasion and starvation-induced apoptosis, but compromised anoikis resistance and anchorage independent tumor growth of PDAC cells. Meanwhile, treatment with recombinant EDIL3 protein markedly promoted anoikis resistance and anchorage independent tumor growth. Mechanistically, we demonstrated that altered protein expression of Bcl-2 family might contribute to the oncogenic activities of EDIL3. In conclusion, this study provides evidences that EDIL3 is a potential predictor and plays an important role in anchorage independent tumor growth of PDAC and EDIL3-related pathways might represent a novel therapeutic strategy for treatment of pancreatic cancer.

Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells.

Dodecyl gallate (DG) is a gallic acid ester that has been shown to inhibit tumor growth. The aim of this study was to investigate the mechanism by which DG induces antiproliferative and apoptotic effects in MG-63 human osteosarcoma cells. Dose- and time-dependent cytotoxic effects of DG were determined using an MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) of DG in MG-63 cells was 31.15 µM at 24 h, 10.66 µM at 48 h, and 9.06 µM at 72 h. Flow cytometric analysis demonstrated that exposure to 20 and 40 µM DG resulted in an increase in the sub-G1 phase population and in S-phase cell cycle arrest. Furthermore, western blot analysis of apoptosis-related protein expression revealed an increase in the activation of caspases 8 and 3, cleavage of poly (ADPribose) polymerase (PARP), and disruption of mitochondrial membrane permeability was measured by flow cytometry. An increase in the Bax/Bcl-2 ratio and a decrease in the expression of inhibitor of apoptosis protein (IAP) family members, namely X-linked inhibitor of apoptosis protein and survivin, were also observed following DG treatment. These data provide insight into the molecular mechanisms governing the ability of DG to induce apoptosis in human osteosarcoma cells in vitro.

Results of a Phase I-II Study of Fenretinide and Rituximab for Patients with B-Cell Lymphomas

Background:Despite improved treatment options, indolent non-Hodgkin lymphoma (NHL) and Mantle Cell Lymphoma (MCL) remain incurable for most patients. Fenretinide (4-hydroxy(phenyl)retinamide; 4-HPR), an orally bioavailable synthetic retinoid, has been shown to induce apoptotic cell death in a variety of tumor types, presumably via intratumoral induction of oxygen free radicals. Our group has shown single-agent fenretinide anti-B-NHL activity as well as synergy with anti-CD20 antibody therapy when tested in vitro (Shan et al Clin Cancer Res. 2001) and in human xenograft models (Gopal et al, Blood 2004). These preclinical data support the first trial to evaluate this strategy of fenretinide and rituximab in patients with B-cell malignancies. Here we report results from a phase I-II study evaluating the safety and efficacy of fenretinide with rituximab for indolent NHL and MCL.Methods:This was an open-label, phase I/II study conducted at the Fred Hutchinson Cancer Research Center (FHCRC) and Seattle Cancer Care Alliance, Seattle, WA (ClinicalTrials.gov identifier: NCT00288067). The study was approved by the institutional review board at FHCRC and written informed consent was obtained from all patients. Major eligibility criteria included: CD20 positive lymphoid malignancy, radiographically measurable disease, ECOG PS ²2, and adequate organ function. The phase I portion evaluated the safety of dosing single agent fenretinide at 900 mg/m2 PO BID for days 1-5 of a 7 day cycle and allowed de-escalation for excess toxicity. The phase II portion added 375 mg/m2 IV rituximab weekly on weeks 5-9 then every 3 months. All patients could remain on therapy until disease progression or unacceptable toxicity. Response assessment occurred approximately every 3 months. Correlative studies included fenretinide pharmacokinetics, serum retinol concentrations, and evaluation of tumor expression of BCL-2 family proteins. Response was scored by standard criteria (Cheson 1999).Results:Thirty-two patients enrolled in the study: 7 patients in phase I, and 25 patients in phase II. The median age was 64 years (range, 40 - 78), 81% were male, and the median number of prior therapies was 1 (range, 0 - 10) with 22 (69%) having received prior rituximab and 8 (25%) with rituximab-refractory disease. Histologies included 13 CLL/SLL, 10 follicular (FL), 7 mantle cell (MCL), 1 lymphoplasmacytic, and 1 marginal zone. No dose limiting toxicities were observed in the phase I portion, and 900 mg/m2 was the dose level delivered in combination with rituximab for the phase II component. The most common treatment-related adverse events (AE) of any grade were reversible night blindness (n=18, 56%), other eye disorders (n=17, 53%), rash (n=12, 38%), and photosensitivity (n=8, 25%). The most common treatment related AEs of grade 3 or higher included: maculopapular rash, night blindness, and decreased lymphocyte count. Three patients (9%) discontinued treatment early due to toxicity (rash-2, GI toxicity-1). One patient with MCL in the phase I portion experienced stable disease (SD) lasting 35 months. In the phase II portion of the study, 5 (20%) patientÕs disease responded (Figure), with 2 (8%) achieving complete remission (both SLL/CLL), and 3 (12%) achieving partial remission (2 FL, 1 MCL). In addition 16 (64%) patients had SD. The median progression-free survival was 9 months (range, 6 - 31 months), and the median overall survival was not reached (range, 33 months to not reached). Median time to progression of responders was 14.5 months. The one-month median peak and trough concentrations of fenretinide were 11.45 µM and 2.5 µM, respectively. Correlative studies assessing Bcl-2, BAX, and apoptosis in the 13 patients with circulating tumor cells were not able to associate these data with response, or survival.Discussion:In this phase I/II study, the combination of fenretinide and rituximab was well tolerated with predictable, reversible toxicities with up to 4.5 years of continuous therapy. Though the ORR was modest (20%), the majority had of patients had disease control (ORR + SD=84%) which lasted for > 6 months. Further study of this novel combination should focus on identifying the subset of B-NHL that is most likely to respond or the rational addition of other agents to augment these anti-tumor effects. [Display omitted] DisclosuresPagel:Actinium Pharmacetuicals, Inc.: Equity Ownership. Gopal:Merck: Research Funding; Emergent/Abbott: Research Funding; Cephalon/Teva: Research Funding; BioMarin: Research Funding; Sanofi-Aventis: Honoraria; Millenium: Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Piramal: Research Funding; Biogen Idec, BMS: Research Funding.

Concomitantly Targeting BCL-2 with Venetoclax (ABT-199/GDC-0199) and MAPK Signaling with Cobimetinib (GDC-0973) in Acute Myeloid Leukemia Models

Pro-survival molecules including BCL-2 play critical roles in leukemia transformation and chemoresistance. ABT-199/GDC-0199 (venetoclax) is an orally available BH3-mimetic that binds with high affinity to BCL-2, but lacks affinity for BCL-XL and MCL-1. We have recently demonstrated anti-leukemia potency of venetoclax in acute myeloid leukemia (AML) models (Pan et al. Cancer Discovery 2014). However, venetoclax poorly inhibits MCL-1, causing resistance in leukemia cells that rely on MCL-1 for survival. The RAF/MEK/ERK (MAPK) cascade is a major effector pathway in AML that is activated by upstream mutant proteins such as FLT3, KIT and RAS. Additionally, the MAPK pathway regulates BCL-2 family proteins by stabilizing anti-apoptotic MCL-1 and inactivating pro-apoptotic BIM. In this study, we evaluated the anti-tumor effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib)..We initially examined activity of these agents in a panel of myeloid leukemia cell lines with diverse genetic alterations (Fig. 1A). The IC50 values of cobimetinib ranged from < 0.01 µM to > 1 µM after 72 hours of drug treatment but did not correlate with the basal level of p-ERK1/2. In 7 out of 11 cell lines, combination of the agents elicited synergistic growth inhibition. Notably synergism of venetoclax with cobimetinib was observed in venetoclax-resistant cell lines (MOLM14, OCI-AML3, NB4 and THP1). Ongoing analysis of pharmacodynamic markers include transcriptome assessment by RNA sequencing, functional proteomics by reverse phase protein array (RPPA) and quantification of BCL-2:BIM and MCL-1:BIM complexes using the electrochemiluminescent ELISA assay (Meso Scale Discovery, MSD-ELISA). The preliminary MSD data revealed that BCL-2:BIM complex was disrupted in most cell lines and accumulated following cobimetinib treatment, which may be due to the disruption of MCL-1:BIM complex by inhibition of MEK (Fig. 1B).In a long-term culture of primary AML blasts in serum-free stem cell growth medium supplemented with cytokines and StemRegenin 1 (SR1) to main the immature state of leukemia cells, the combination of venetoclax and cobimetinib induced distinct apoptotic cell death, with AML #1 sensitive to venetoclax but resistant to cobimetinib. Alternatively, AML #2 and #3 samples were resistant to venetoclax but sensitive to cobimetinib and the combination of both drugs (Fig. 1C). We next investigated signaling patterns and BCL-2 family protein expression in AML stem/progenitor cells using a 34-antibody panel and time-of-flight mass cytometry (CyTOF). In AML#1, BCL-2 was expressed in leukemia blasts, with enrichment in a progenitor AML population phenotypically defined as CD45dim CD34+ CD38+ CD123+ CD33+ (Fig. 1D). The high expression level of BCL-2 and low expression of MCL-1 and BCL-XL may account for sensitivity to venetoclax in AML#1. Both basal and G-CSF- or SCF-stimulated p-ERK was efficiently down-regulated by cobimetinib; however, G-CSF-evoked p-STAT3/5 and SCF-induced p-AKT were only slightly reduced (Fig. 1E). Notably we observed increased phosphorylation of STAT5 pathway upon treatment with cobimetinib, suggesting that active MAPK signals inhibit phosphorylation of the JAK-STAT pathway, as previously reported (Krasilnikov et al. Oncogene, 2003 and Lee at al. Cancer Cell, 2014).To test the efficacy of both compounds in vivo, we injected NSG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the cobimetinib single agent and venetoclax plus cobimetinib co-treated mice (Fig. 1F). The efficacy study is ongoing and median survival for cobimetinib and venetoclax co-treated mice has yet to be determined (Fig. 1G).In summary, our data demonstrates that combinatorial blockade of MAPK and BCL-2 pathways is synergistic in the majority of AML cell lines tested and can overcome intrinsic resistance to venetoclax. Ongoing studies will evaluate efficacy of this combination therapy in primary human AML xenografts and elucidate mechanisms of synergy. [Display omitted] DisclosuresLeverson:AbbVie: Employment, Equity Ownership. Dail:Genentech: Employment, Equity Ownership. Phillips:AbbVie: Employment, Other: Shareholder, Patents & Royalties. Chen:Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Jabbour:Pfizer: Consultancy, Research Funding. Sampath:Genentech: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Evaluation of Effects of Metformin in Primary Ovarian Cancer Cells.

Ovarian cancer is the third most common cause of cancer in Indian women. Despite an initial 70-80% response rate, most patients relapse within 1-2 years and develop chemoresistance. Hence, identification or repositioning of drugs to resensitise ovarian cancer cells to existing chemotherapy is needed. Traditionally immortalized cell lines have been used in research, but these may contain genetic aberrations and chromosomal abnormalities serving as poor indicators of normal cell phenotype and progression of early-stage disease. The use of primary cells, maintained for only short periods of time in vitro, may serve as the best representative for studying in vivo conditions of the tissues from which they are derived. In this study we have attempted to evaluate the effect of metformin (an antidiabetic drug) in primary ovarian cancer cells because of its promising effect in other solid tumours. Primary cultures of epithelial ovarian cancer cells established from ascitic fluid of untreated ovarian cancer patients were used. The cells were treated with metformin at doses standardized by MTT assay and its ability to induce apoptosis was studied. The cells were analysed for apoptosis and apoptosis related proteins by flow cytometry and western blotting respectively. Metformin induced apoptosis in ovarian cancer cells, provoking cell cycle arrest in the G0/G1 and S phase. It induced apoptosis in ovarian cancer cells by, down-regulating Bcl-2 and up-regulating Bax expression. Metformin was able to induce apoptosis in primary ovarian cancer cells by modulating the expression of Bcl-2 family proteins. These data are relevant to ongoing translational research efforts exploring the chemotherapeutic potential of metformin.

Cathelicidins positively regulate pancreatic β-cell functions.

Cathelicidins are pleiotropic antimicrobial peptides largely described for innate antimicrobial defenses and, more recently, immunomodulation. They are shown to modulate a variety of immune or nonimmune host cell responses. However, how cathelicidins are expressed by β cells and modulate β-cell functions under steady-state or proinflammatory conditions are unknown. We find that cathelicidin-related antimicrobial peptide (CRAMP) is constitutively expressed by rat insulinoma β-cell clone INS-1 832/13. CRAMP expression is inducible by butyrate or phenylbutyric acid and its secretion triggered upon inflammatory challenges by IL-1β or LPS. CRAMP promotes β-cell survival in vitro via the epidermal growth factor receptor (EGFR) and by modulating expression of antiapoptotic Bcl-2 family proteins: p-Bad, Bcl-2, and Bcl-xL. Also via EGFR, CRAMP stimulates glucose-stimulated insulin secretion ex vivo by rat islets. A similar effect is observed in diabetes-prone nonobese diabetic (NOD) mice. Additional investigation under inflammatory conditions reveals that CRAMP modulates inflammatory responses and β-cell apoptosis, as measured by prostaglandin E2 production, cyclooxygenases (COXs), and caspase activation. Finally, CRAMP-deficient cnlp(-/-) mice exhibit defective insulin secretion, and administration of CRAMP to prediabetic NOD mice improves blood glucose clearance upon glucose challenge. Our finding suggests that cathelicidins positively regulate β-cell functions and may be potentially used for intervening β-cell dysfunction-associated diseases.

Desialylation is associated with apoptosis and phagocytosis of platelets in patients with prolonged isolated thrombocytopenia after allo-HSCT.

BackgroundProlonged isolated thrombocytopenia (PT) is a frequent complication in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), and it is associated with an adverse prognosis. In this study, we hypothesized that desialylation on platelet surfaces was associated with PT after allo-HSCT. The mechanisms participating in this process may include NEU1 translocation, platelet apoptosis, and phagocytosis by macrophages.MethodsPT was defined as a peripheral platelet count less than 100 × 109/L without sustained anemia or leukopenia for more than 3 months after allo-HSCT. 34 patients were identified consecutively from a cohort of 255 patients who underwent allo-HSCT for hematologic malignancies between May and October 2014 at Peking University Institute of Hematology. Desialylation, enzyme expression, and phagocytosis were detected using flow cytometry, immunofluorescence, RT-PCR, Western blot, and so on.ResultsPlatelets from the PT patients had significantly fewer sialic acids (P = .001) and increased β-galactose exposure indicative of desialylation on the surface (P = .042), and serum from the PT patients showed a higher sialic acid concentration (8.400 ± 0.2209 μmol/L, P < .001). The sialidase NEU1 was over-expressed from mRNA to protein levels, and its catalytic activity was increased in platelets from the PT patients. Desialylation of GPIbα in the PT patients was correlated with changes in 14-3-3ζ distribution, which, relative to Bad activation, modulated the expression of Bcl-2 family proteins, depolarized the inner membrane of the mitochondria, and initiated the intrinsic mitochondria-dependent pathway of apoptosis. Macrophages derived from the THP-1 cell line preferred to phagocytize desialylated platelets from the PT patients in vitro. We also revealed that oseltamivir (400 μmol/L) could inhibit 50 % of the sialidase activity on platelets and could protect 20 % of platelets from phagocytosis in vitro.ConclusionsDesialylation of platelets was associated with platelet apoptosis and phagocytosis, whereas oseltamivir could reduce platelet destruction in the periphery, indicating a potential novel treatment for PT after allo-HSCT.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0216-3) contains supplementary material, which is available to authorized users.

Molecular mechanisms underlying the antitumor activity of (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide in human colorectal cancer cells in vitro and in vivo.

Upregulation of class I histone deacetylases (HDAC) correlates with poor prognosis in colorectal cancer (CRC) patients. Previous study revealed that (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide (Compound 11) is a potent and selective class I HDAC inhibitor, exhibited significant anti-proliferative activity in various human cancer cell lines. In current study, we demonstrated that compound 11 exhibited significant anti-proliferative and cytotoxic activity in CRC cells. Notably, compound 11 was less potent than SAHA in inhibiting HDAC6 as evident from the lower expression of acetyl-α-tubulin, suggesting higher selectivity for class I HDACs. Mechanistically, compound 11 induced cell-cycle arrest at the G2/M phase, activated both intrinsic- and extrinsic-apoptotic pathways, altered the expression of Bcl-2 family proteins and exerted a potent inhibitory effect on survival signals (p-Akt, p-ERK) in CRC cells. Moreover, we provide evidence that compound 11 suppressed motility, decreased mesenchymal markers (N-cadherin and vimentin) and increased epithelial marker (E-cadherin) through down-regulation of Akt. The anti-tumor activity and underlying molecular mechanisms of compound 11 were further confirmed using the HCT116 xenograft model in vivo. Our findings provide evidence of the significant anti-tumor activity of compound 11 in a preclinical model, supporting its potential as a novel therapeutic agent for CRC.

B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy.

The synthetic curcumin analog B5 is a potent inhibitor of thioredoxin reductase (TrxR) that has potential anticancer effects. The molecular mechanism underlying B5 as an anticancer agent is not yet fully understood. In this study, we report that B5 induces apoptosis in two human cervical cancer cell lines, CaSki and SiHa, as evidenced by the downregulation of XIAP, activation of caspases and cleavage of PARP. The involvement of the mitochondrial pathway in B5-induced apoptosis was suggested by the dissipation of mitochondrial membrane potential and increased expression of pro-apoptotic Bcl-2 family proteins. In B5-treated cells, TrxR activity was markedly inhibited with concomitant accumulation of oxidized thioredoxin, increased formation of reactive oxygen species (ROS), and activation of ASK1 and its downstream regulatory target p38/JNK. B5-induced apoptosis was significantly inhibited in the presence of N-acetyl-l-cysteine. Microscopic examination of B5-treated cells revealed increased presence of cytoplasmic vacuoles. The ability of B5 to activate autophagy in cells was subsequently confirmed by cell staining with acridine orange, accumulation of LC3-II, and measurement of autophagic flux. Unlike B5-induced apoptosis, autophagy induced by B5 is not ROS-mediated but a role for the AKT and AMPK signaling pathways is implied. In SiHa cells but not CaSki cells, B5-induced apoptosis was promoted by autophagy. These data suggest that the anticarcinogenic effects of B5 is mediated by complex interplay between cellular mechanisms governing redox homeostasis, apoptosis and autophagy.

Cytotoxic activity, DNA damage, cellular uptake, apoptosis and western blot analysis of ruthenium(II) polypyridyl complex against human lung decarcinoma A549 cell

A new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2Ru1 was synthesized and characterized. The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC50 values of 1.6±0.4, 9.0±0.8, 1.5±0.2 and 1.5±0.3μM, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can down-regulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad. The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins.

Abstract 3501: EGFR tyrosine kinase inhibitors combined with cytotoxic drugs for treatment of NSCLC with EGFR gene mutations: Efficacy and mechanisms

Abstract Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have improved overall survival in non-small-cell lung cancer (NSCLC) patients whose tumors have EGFR activating mutations. After treatment with EGFR-TKIs, NSCLC tumors with EGFR activating mutations become resistant to EGFR-TKIs, and development of novel treatment strategies is needed to improve survival in such patients.Next-generation EGFR-TKIs have been developed, and a clinical trial showed clinical activity of 3rd generation EGFR-TKIs in patients with acquired resistance, especially secondary mutation T790M to EGFR-TKIs. Moreover, clinical trials demonstrated survival benefit of combination with cytotoxic agents and EGFR-TKIs in patients with EGFR-mutated NSCLC. In contrast, the IMPRESS study showed that continuation of gefitinib in addition to cisplatin and pemetrexed didn't show survival benefit in patients with acquired resistance to gefitinib.Several basic studies reported that efficacy of targeted therapy combined with chemotherapy is schedule dependent. We, therefore, conducted an in vitro study to develop a novel therapeutic strategy for NSCLC with EGFR mutations. Methods: In vitro effects of docetaxel and an EGFR-TKI, gefitinib (AstraZeneca) or AZD9291 (Selleckchem), were examined in five lung cancer cell lines (PC9, PC9GR, HCC827, HCC827GR, and H1975) using a 3D culture model (PrimeSurface 96, SUMITOMO BAKELITE CO.). The NSCLC cells were treated with the combinationeither concurrently or sequentially (EGFR-TKIs after docetaxel or docetaxel after EGFR-TKIs). Combination Index (CI) values were calculated at various levels of cytotoxicity (the Chou-Talalay method). We evaluated changes in MAPK pathway activation and in expressions of Bcl2 family proteins and cleaved PARP (Cell Signaling) by western blot analysis. Results: Docetaxel followed by EGFR-TKIs weremore effective at CI, compared with EGFR-TKIs followed by docetaxel in two of the five cell lines with EGFR activating mutations, PC9GR (EGFR19del+T790M) and HCC827GR (EGFR19del+METamp) cells. The HCC827GR cells were likely to be more sensitive than PC9GR to the combination with docetaxel and gefitinib. The combo led to inactivation of the MAPK pathway and alteration of the Bcl2 family proteins. d) Conclusion: In conclusion, our results suggest that the sequential combination of docetaxel followed by EGFR-TKIs was effective against NSCLC with acquired resistance to EGFR-TKIs. Citation Format: Ryohei Yoshida, Synsuke Okumura, Takaaki Sasaki, Yoshinobu Ohsaki. EGFR tyrosine kinase inhibitors combined with cytotoxic drugs for treatment of NSCLC with EGFR gene mutations: Efficacy and mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3501. doi:10.1158/1538-7445.AM2015-3501

TKTL1 promotes cell proliferation and metastasis in esophageal squamous cell carcinoma.

Transketolase-like-1 (TKTL1), which is a rate-limiting enzyme in the non-oxidative part of the pentose-phosphate pathway, has been demonstrated to promote carcinogenesis through enhanced aerobic glycolysis. Dysregulation of TKTL1 expression also leads to poor prognosis in patients with urothelial and colorectal cancer. However, the expression pattern and underlying cellular functions in human esophageal squamous cell carcinoma (ESCC) remain largely unexplored. In this study, we measured TKTL1 expression in ESCC cell lines and paraffin-embedded ESCC tumor tissues. Our results revealed that TKTL1 expression was upregulated in all of the four ESCC cell lines and in 61.25% (98/160) of ESCC specimens detected, while only 27.5% (11/40) in normal epithelium. Silencing of TKTL1 expression decreased cell proliferation through inhibiting the expression of MKI67 and cyclins including Ccna2, Ccnb1, Ccnd1 and Ccne1. Meanwhile, down-regulation of TKTL1 also associated with increased apoptotic ratio and altered protein expression of Bcl-2 family in ESCC cells. Furthermore, knockdown of TKTL1 significantly reduced the invasive potential of ESCC cells through up-regulation of anti-metastasis genes (MTSS1, TIMP2 and CTSK) and down-regulation of pr-metastasis genes (MMP2, MMP9, MMP10 and MMP13). Taken together, our results indicate that TKTL1 is associated with a more aggressive behavior in ESCC cells and suppresses its expression or enzyme activity might represents a potential target for developing novel therapies in human ESCCs.

Abstract 1770: A role of nicotinamide phosphoribosyltransferase in growth of KRAS mutant non-small cell lung cancer

Abstract Background: KRAS mutations are frequently found in non-small cell lung cancer (NSCLC). The KRAS mutations could be predictive of resistance to targeted therapy like epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) or a prognostic factor in NSCLC. No successful targeted therapy currently exists to treat patients with KRAS mutant NSCLC, and novel therapeutic strategies are needed to prolong survival in patients with KRAS mutant NSCLC. Nicotinamide phosphoribosyltransferase (nampt) is a rate-limiting enzyme in NAD+ salvage, and we previously reported that nampt is a potent therapeutic target in NSCLC. Based on the analysis result of GEO database, we found that expression of nampt is associated with RAS-MAPK signaling pathway in NSCLC, and we therefore, conducted an in vitro study to evaluate a role of nampt in growth of KRAS mutant NSCLC. Methods: We used a panel of KRAS mutant NSCLC cell lines (A427, A549, H157, H23, H1792, H2009, H358, H441, Calu6, Calu1). The cells were treated with a nampt inhibitor, FK866, and acid phosphatase (APH) assay (Thermo SCIENTIFIC) was used to evaluate cell viability of the KRAS mutant cells. Changes in expression of MAPK signaling pathway and BCL2 family proteins (antibodies from Cell Signaling and Santa Cruz) were evaluated by western blot. Apoptosis of the cells treated with FK866 was detected using an Annexin V FITC apoptosis detection kit (BD Biosciences). FACS analysis was performed using a BD FACSAria II cytometer (BD Bioscience). Autophagic proteolysis was studied by Cyto-ID autophagy detection kit (Cosmo Bio), and the expression of the fluorescent signal was visualized by a FV1000 confocal microscope (OLYMPUS JAPAN). Results: We found that the sensitivity to FK866 was different among the KRAS mutant cell lines, and for instance, A427 cells were sensitive while H157 cells were resistant. FK866 suppressed growth of KRAS mutant NSCLC and decreased expression of p-ERK expression in the cell line sensitive to FK866. We will report alteration of apoptosis and autophagy in the KRAS mutant NSCLC treated with FK866. Conclusion: Nampt could be a potent therapeutic target in KRAS mutant NSCLC. Citation Format: Shunsuke Okumura, Takaaki Sasaki, Yoshinobu Ohsaki. A role of nicotinamide phosphoribosyltransferase in growth of KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2015-1770

Effects of daidzein in regards to cytotoxicity in vitro, apoptosis, reactive oxygen species level, cell cycle arrest and the expression of caspase and Bcl-2 family proteins.

In the present study, the in vitro cytotoxicity of daidzein was evaluated in human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. BEL-7402 cells were sensitive to daidzein treatment, with an IC50 value of 59.7±8.1 µM. Daidzein showed no cytotoxic activity toward A549, HeLa, HepG-2 and MG-63 cells. Daidzein increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Morphological and comet assays showed that daidzein effectively induced apoptosis in BEL-7402 cells. Additionally, daidzein caused cell cycle arrest at the G2/M phase in the BEL-7402 cell line. Daidzein downregulated the expression of Bcl-2, Bcl-x and Baid proteins and upregulated the levels of Bim protein in the BEL-7402 cells. The results demonstrated that daidzein induced BEL-7402 cell apoptosis through an ROS-mediated mitochondrial dysfunction pathway.

Celastrol induces apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway.

Celastrol is an active compound extracted from the root bark of Triptergium wilfordii Hook F., also known as 'Thunder of God Vine'. It is a well-known Chinese medicinal herb that was found to inhibit tumor cell growth and promote apoptosis in several tumor cell lines. However, research into its effects on osteosarcoma cell apoptosis is still extremely limited. The present study was undertaken to determine the effect of celastrol on viability and apoptosis of osteosarcoma cells and furthermore, to illuminate the molecular mechanism of celastrol-induced osteosarcoma cell apoptosis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to evaluate the viability of the cells following treatment with celastrol. The effect of celastrol on the apoptotic rate of the cells was evaluated by flow cytometry using Annexin V-PE/7-AAD staining assay. Fluorescence microscopy was used to detect the morphological changes in the human osteosarcoma U-2OS cell lines. The expression of Bcl-2 family proteins, caspase-3, caspase-8, caspase-9, cytochrome c and PARP was measured by western blotting. We found that celastrol significantly inhibited the growth of osteosarcoma cells in a dose-dependent manner, particularly U-2OS cells. Furthermore, we observed that celastrol upregulated the expression of the pro-apoptotic proteins Bax and cytochrome c and altered the ratio of Bax/Bcl-2, and triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and -9 activation and PARP cleavage. To conclude, the results indicate that celastrol inhibits the proliferation of human osteosarcoma cancer cells by inducing apoptosis via the mitochondrial-dependent pathway.