Bcl-xL mRNA Expression Research Articles

Effect of Metformin on Proliferation, Differentiation and Apoptosis of THP-1 Cells

To investigate the effect of metformin on proliferation, differentiation and apoptosis of THP-1 cells and explore its possible mechanism. THP-1 cells were cultured with different concentrations of metformin for 24 h and 48 h. The cell proliferation was evaluated by CCK-8, the cell apoptosis was analyzed by Annexin V/7-AAD double labeling, the expression of CD14 and CD11b (surface differentiation antigens on THP-1 cells) was evaluated by flow cytometry, the BCL-XL, BAX, BIM and caspase-3 mRNA expressions of THP-1 cells were detected by real time quantitative PCR. Metformin could significantly inhibit the growth of THP-1 cells in a time- and dose- dependent manner. After treated with 20 mmol/L metformin for 24 h, the expressions of CD14 and CD11b in THP-1 cells didn't change much (P>0.05), the early apoptosis rates in exprimental and control groups were (2.02±0.85)% and (4.46±1.33)% respectively, the late apoptosis rates in experimental and control groups were (1.43±0.83)% and (3.31±0.59)% respectively. In process of inducing effect of 20 mmol/L metformin on THP-1 cells, the expressions of BCL-XL and BIM did not significantly changed, while the expressions of BAX and caspase-3 significantly increased (P<0.01). Metformin can effectively inhibit proliferation and induce apoptosis of THP-1 cells. However, it has no significant effect on differentiation of THP-1 cells, its mechanism inducing apoptosis maybe related with up-regulating BAX and caspase-3.

Fucosylated chondroitin sulfate from sea cucumber inhibited islets of langerhans apoptosis via inactivation of the mitochondrial pathway in insulin resistant mice

The effects of fucosylated chondroitin sulphate from sea cucumber (CHS) on inhibition of pancreatic islet apoptosis in high-fat high-sucrose diet (HFSD)-induced insulin resistant mice were studied for 19 weeks. CHS treatment significantly (p<0.05) decreased fasting blood glucose, insulin, and tumor necrosis factor (TNF)-α levels, and increased the serum adiponectin level, improved insulin resistance, and repaired HFSD-injured islets of langerhans, compared with controls. CHS supplementation significantly (p<0.05) inhibited pancreas cell apoptosis via reduction of cytochrome c, caspase 9, and caspase 3 mRNA expressions, cytochrome c release into the cytoplasm, and caspase 9 and cleaved-caspase 3 protein expressions, compared with controls. These effects were associated with upregulation of Bcl-2 and Bcl-xL mRNA and protein expressions, and downregulation of Bax and t-Bid mRNA and protein expressions. Combination treatments with CHS and rosiglitazone synergistically protected pancreatic islets. CHS improves insulin resistance and protects islets of langerhans from apoptosis via inactivation of the intrinsic mitochondrial pathway.

황칠나무 줄기 추출물의 MCF-7과 MDA-MB-231 유방암 세포주에 대한 세포증식억제 효과

Objectives : Dendropanax morbifera is known as a tree that has been used in traditional medicine for various diseases. However, its biological activities in cancer have not yet been clearly elucidated. In this study, we investigated the anti-cancer effects of water extract of Dendropanax morbifera (DP) on 2 human breast cancer cell lines (estrogen dependent MCF-7 and estrogen independent MDA-MB-231). Methods : The MTT assay and flow cytometry were used to assess cell proliferation, along with cell cycle analysis. Nitric oxide production was detected by Griess assay. The expression of apoptosis related gene was assessed by quantitative real-time PCR. Results : Our data revealed that DP inhibits the cell growth in a dose dependent manner (0, 50, 100, 250, and 500 &#x3BC;g/ml) of both estrogen independent MDA-MB-231 and estrogen dependent MCF-7 breast cancer cells. Also, LPS induced nitric oxide production was significantly reduced by DP. Cell cycle analysis showed an increased G1 phase in the MCF-7 cell and G2/M phase in the MDA-MB-231 cell. DP decreased mRNA expression of apoptotic suppressor gene Bcl-xL, and increased mRNA expression of pro-apoptotic genes. DP increased mRNA expression of p21, and Rip1 in both cell. And DP decreased mRNA expression of survivin in the MCF-7 cell. Conclusions : Taken together, these results indicate that DP extract are source of anti-cancer potential and could be developed botanical drug.

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases.

Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4’,6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and IκB-α expression levels were the highest, and Bcl-2, Bcl-xL, XIAP, cIAP-1, cIAP-2, survival, and NF-κB expression levels were the lowest. These results proved that UDCA could induce apoptosis of HSC-3 cancer cells through caspase activation, and the higher concentration of UDCA had stronger effects in vitro. UDCA might be a good nutrient for oral cancer prevention.

Oxidative stress-induced apoptotic insults to rat osteoblasts are attenuated by nitric oxide pretreatment via GATA-5-involved regulation of Bcl-X L gene expression and protein translocation.

Nitric oxide (NO) has biphasic effects on regulating osteoblast survival and death. This study was aimed to evaluate the effects of NO pretreatment on hydrogen peroxide (HP)-induced insults of rat osteoblasts and the possible mechanisms. Exposure of osteoblasts prepared from rat calvarias to HP significantly increased intracellular reactive oxygen species levels, decreased alkaline phosphatase activity and cell survival, and ultimately induced cell apoptosis. However, NO pretreatment lowered HP-induced oxidative stress and apoptotic insults. In parallel, HP increased Bax levels and its translocation from the cytoplasm to mitochondria. NO pretreatment caused significant attenuations in HP-induced modulations in Bax synthesis and translocation. In contrast, pretreatment with NO enhanced levels and translocation of antiapoptotic Bcl-XL protein in rat osteoblasts. RNA analyses further revealed that HP inhibited Bcl-XL mRNA expression without affecting Bax mRNA levels. In comparison, NO induced Bcl-XL mRNA production and alleviated HP-caused inhibition of this mRNA expression. As to the mechanism, HP suppressed RNA and protein levels of transcription factor GATA-5 in rat osteoblasts. Pretreatment with NO induced GATA-5 mRNA and protein expressions and simultaneously attenuated HP-induced inhibition of this gene's expression. Consequently, GATA-5 knockdown using RNA interference inhibited Bcl-XL mRNA expression and concurrently lowered NO's protection against HP-induced apoptotic insults. Therefore, this study showed that NO can protect rat osteoblasts from HP-induced apoptotic insults. The protective mechanisms are mediated by GATA-5-mediated transcriptional induction of Bcl-X L gene, and translocational modulation of Bcl-XL and Bax proteins.

Continuous infusion of angiotensin II modulates hypertrophic differentiation and apoptosis of chondrocytes in cartilage formation in a fracture model mouse.

Although components of the renin-angiotensin system (RAS) are reported to be expressed in cultured chondrocytes and cartilage, little is known about the precise function of Angiotensin II (Ang II) in chondrocytes. In this study, we employed a rib fracture model mouse to investigate the effect of Ang II on chondrocytes. Ang II type 1 receptor (AT1R) was expressed in chondrocytes in the growth plate of mouse tibia. Continuous infusion of Ang II to rib-fractured mice resulted in a significant increase in the volume of cartilage, suggesting Ang II-induced hypertrophic differentiation of chondrocytes. It was also confirmed by a significant increase in the mRNA expression of Sox9 and runt-related transcription factor 2 (Runx2), which are genes related to chondrocyte differentiation, and type X collagen, matrix metalloproteinase (MMP)-13 and Indian hedgehog (Ihh), which are hypertrophic chondrocyte-specific molecular markers. Chondrocyte hypertrophy with upregulation of these genes was attenuated by administration of olmesartan, an AT1R blocker, but not by hydralazine. Moreover, Ang II infusion significantly suppressed apoptosis of chondrocytes, accompanied by significant induction of mRNA expression of bcl-2 and bcl-xL. Olmesartan, but not hydralazine, significantly attenuated the reduction of apoptotic cells and the increase in anti-apoptotic genes induced by Ang II infusion. Overall, the present study demonstrated that Ang II promoted hypertrophic differentiation of chondrocytes and reduced apoptosis of hypertrophic chondrocytes independently of high blood pressure. The present data indicate the role of Ang II in cartilage, and might provide a new concept for treatment of cartilage diseases.

Non-canonical NF-κB Plays a Pivotal Role in Non-Hodgkin's Lymphoma.

This study tried to dissect BAFF/BAFF-R-mediated non-canonical NF-κB signaling in the drug resistance of B-cell non-Hodgkin's lymphoma. FQ-PCR was employ to determine the mRNA expression of BAFF, BAFF-R, Bcl-xL, and RIPK2 in the circulating blood of B-cell non-Hodgkin's lymphoma patients. Further correlation studies were performed with the gene expression in the circulating blood and tumor tissue. MTT assay as used to determine BAFF's role on lymphoma cell proliferation. Western blot was employed to determine protein expression after BAFF stimulation. The mRNA expression of BAFF, BAFF-R, Bcl-xL, and RIPK2 in the circulating blood of the resistant group was higher than that of the non-resistant group, which was statistically significant. The mRNA expression of the target genes was positively correlated. The mRNA expression was positively correlated with disease progression, which was statistically significant. The Bcl-xL mRNA expression in the resistant group was relatively higher than that in the non-resistant group, which was also statistically significant. However, the mRNA expression of other genes only showed increased tendency compared with non-resistant group. There was no significant change between target genes in different tumor tissues. With increased BAFF concentration and prolonged exposure, the proliferation of the tumor cells increased significantly, which was statistically significant. Western blot showed the expression of BAFF, BAFF-R, Bcl-xL, and RIPK2 all increased with increased BAFF concentration, which was also statistically significant. In B-cell, non-Hodgkin's lymphoma, BAFF may activate non-canonical NF-κB signaling to regulate drug resistance.

Hypoxic Preconditioning Protects Cord Blood-Mesenchymal Stromal Cells from Ischemic Damage in Vitro

Objectives: We have previously shown that cord blood mesenchymal stromal cell (CBMSC)-secreted factors act anti-apoptotic and pro-mitotic on endothelial cells and cardiomyocytes to ischemia-like conditions. However CB-MSCs themselves suffer ischemic damage in the infarcted heart, hampering their potential clinical efficacy. To increase CB-MSC tolerance to ischemia, we developed a specific hypoxic preconditioning (HP) protocol and investigated the molecular mechanisms involved. Methods: Human CBMSCs were subjected to different durations (12, 24 or 36 hour) of hypoxic preconditioning (1% O2 in glucose/serum-containing medium) prior to a final phase of simulated ischemia (24 hour, 1% O2 in glucose/serum-free medium). Cell loss, metabolic activity (WST-8 conversion), apoptosis (nuclear fragmentation and shrinking) and proliferation (BrdU incorporation) were measured. The effects of HP on Akt-, ERK1/2 and STAT3-phosphorylation and Bcl-2, Bcl-XL and Bag-1 mRNA expression were determined by Western blot and quantitative real time PCR. Results: Ischemic damage of CBMSCs was lowest after 24 hour HP. Here, the percentage of remaining cells was higher (74 ± 7% versus non-HP 39 ± 11%, p < 0.05) and metabolic activity was enhanced (35 ± 2% versus non-HP 18 ± 6%, p < 0.05). Nuclear fragmentation index was lower (11.9 ± 0.2 versus non-HP 13.8 ± 0.3, p < 0.05), nuclear shrinking was less pronounced (mean nuclear area 179 ± 5 µm2 versus non-HP 155 ± 5 µm2, p < 0.05), and the proliferation index was enhanced (49 ± 1 versus non-HP 16 ± 3, p < 0.05). While phosphorylation of Akt and STAT3 was increased in preconditioned cells, ERK1/2 phosphorylation and Bcl-2 mRNA expression were downregulated in response to HP, and mRNA expression of Bcl-XL and Bag-1 was not altered. Conclusions: Hypoxic preconditioning protects CBMSCs from the deleterious impact of combined oxygen/glucose/serum-deprivation via STAT3-dependent signaling. This might by a useful strategy to increase their tolerance to ischemia and improve their therapeutic efficacy in clinical applications.

Effects of thonningianin A in natural foods on apoptosis and cell cycle arrest of HepG-2 human hepatocellular carcinoma cells.

The anti-cancer activities of Thonningianin A on the HepG-2 human hepatocellular carcinoma cell line were evaluated by MTT assay, flow cytometry, quantitative real-time PCR and western blotting. Results showed that Thonningianin A effectively inhibited the proliferation of HepG-2 cells by inducing apoptosis, as evidenced by increase in the sub-G1 cell population, DNA fragmentation, and increase in the content of reactive oxygen species. Activation of caspase-9 and the subsequent activation of caspase-3 indicated that Thonningianin A-induced apoptosis is caspase-dependent. Thonningianin A also disrupted the mitochondrial membrane potential (Δψm) and down-regulated the Bcl-xL mRNA expression in HepG-2 cells. Thonningianin A induced cell cycle arrest by changing the cyclin D1 and CDK4 mRNA expression levels. Moreover, western blotting showed that Thonningianin A significantly down-regulated the NF-kappa-B cell survival pathway, along with up-regulation of the expression level of phosphorylated P38 and down-regulation of the expression level of phosphorylated ERK. The anti-cancer activity of Thonningianin A was confirmed by the characteristic patterns of DNA fragmentation and cell cycle arrest, suggesting that Th A is an effective antitumor ingredient in natural plant foods, and is worthy of further study.

Cooperative Targeting of Bcl-2 Family Proteins By ABT-199 (GDC-0199) and Tyrosine Kinase Inhibitors to Eradicate Blast Crisis CML and CML Stem/Progenitor Cells

Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells.Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy.To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation.To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing.Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. DisclosuresCarter:AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

Obinutuzumab (GA101) Significantly Inhibits Cell Proliferation and Induces Programmed Cell Death in Primary Mediastinal B-Cell Lymphoma (PMBL): Obinutuzumab May be a Future Targeted Agent for the Treatment of PMBL

BACKGROUND: Primary Mediastinal large B-cell lymphoma (PMBL) is a rare form of Non Hodgkin Lymphoma (NHL) representing 2% of mature B-cell non-Hodgkin lymphoma in patients less than 18 years of age (Lones/Cairo et al, JCO 2000; Burkhardt et al, BJH 2005). PMBL has histological features somewhere between Diffuse Large B-Cell Lymphoma (DLBCL) and classical HL (cHL) (Abramson et al, Blood 2005). Gene expression studies suggested that the molecular signature of PMBL had a striking resemblance to the expression profile of cHL (Rosenwald et al, JEM 2003). We have recently reported that a significant decrease in EFS among children and adolescent PMBL patients compared with other stage III non-PMBL pediatric DLBCL patients following FAB/LMB 96 therapy, suggesting that children and adolescent with PMBL required alternative treatment strategies (Gerrard/Cairo et al, Blood 2013). PMBL has been demonstrated to have an over-activated NF-kB pathway by gene expression profiling (Rosenwald et al, JEM 2003). Since over 95% of PMBL express CD20, targeting the CD20 receptor with a CD20 antibody is of high clinical interest. Obinutuzumab (GA101) is novel glycoengineered anti-CD20 targeted monoclonal antibody recognizing a unique CD20 type II epitope and it has been demonstrated to have greater efficacy in reducing tumor size, inducing remission and improving survival in other B-NHL xenograft models (Mössner et al, Blood 2010). Obinutuzumab has been recently approved by FDA for first line treatment of chronic lymphocytic leukemia (CLL) in combination with chlorambucil.OBJECTIVES: We hypothesize that obinutuzumab may be a future potential targeted agent for the treatment of PMBL, and therefore, we investigated whether obinutuzumab treatment results in significant changes in signaling pathways, genes expression, programmed cell death and cell proliferation in PMBL.METHODS: Karpas-1106P cells (DSMZ) were treated with obinutuzumab (generously provided by Dr. Klein, Roche) at every 24 hours (1-100ug/ml). qRT-PCR, western blot, MTS, Caspase 3/7 assay (Promega) and FACS analysis were performed. The BeadChip array (Illumina, HumanHT-12) was used for gene expression profiling.RESULTS: There was a significant decrease of cell proliferation in obinutuzumab-treated Karpas cells with 10ug/ml (0.69 ± 0.025, p<0.005) vs control (1.00 ± 0.000) at 48 hours. Concomitantly, there was a significant increase in programmed cell death in 10ug/ml obinutuzumab treated Karpas (37.80 ± 10.096, p<0.05) vs control (1.19 ± 0.762) at 48 hours. We also observed a significant decrease of CD20 expression (0.74± 0.010, p<0.05) with 10ug/ml obinutuzumab treatment at 48 hours. A total of 133 differentially expressed genes were identified by gene expression profiling (>1.5-fold, 0.57%) and 77.5% of genes including apoptosis related genes (CASP2 and PAK2) and MAPK signaling pathways (RASA1 and JUN) and EGR1 were upregulated and 22.5% of genes including ID3, GRAP and RAB6B were downregulated in obinutuzumab treated Karpas vs control (Fig 1). There were significant decreases of p-STAT6 (0.72± 0.011, p=0.01), p-Akt (0.69± 0.011, p<0.05), p-ikBα (0.70± 0.017, p<0.05) and p-Erk (0.56± 0.019, p<0.05) with 10ug/ml obinutuzumab treatment at 48 hours (Fig 2). Additionally, There were significant down-regulation of mRNA expression of Bcl-xL (0.91±0.011, p<0.04) and Bax (0.66±0.022, p<0.02) vs control.CONCLUSIONS: We observed that obinutuzumab significantly inhibited cell proliferation and induced programmed cell death and downregulated downstream of PI3k/Akt and NF-kB signaling pathways. Gene expression analysis indicated obinutuzumab induced changes in the expression of genes in Karpas that were involved in apoptosis and signaling pathways including CASP2, EGR1 and ID3. Future studies 1) will investigate the efficacy of combination therapies to enhance programmed cell death, and 2) will assess the proteomic signature induced by obinutuzumab in obinutuzumab sensitive and resistant PMBL, and furthermore, 3) will focus on the in vivo effects of obinutuzumab in a NOD/SCID PMBL xenograft mouse model. Obinutuzumab may be a future potential targeted agent for the adjuvant treatment of PMBL lymphoma. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Pim Kinase Inhibitor AZD1208 Enhances Apoptosis Induction By Clinically Active FLT3 Inhibitors in FLT3-ITD Acute Myeloid Leukemia Cells in Vitro and in Vivo through Synergistic Downregulation of Mcl-1 and of Bcl-xL

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses.The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo.Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L.To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p<0.0001), S (p<0.0001) or C (p<0.001), and of MV4-11 (p<0.0001) and MOLM14 (p<0.05) cells treated with AZD1208 combined with Q, in relation to each drug alone. Apoptosis was additionally confirmed by loss of mitochondrial membrane potential. Synergistic induction of apoptosis was not seen in Ba/F3-WT or 32D-WT cells, with wild-type FLT3, indicating a FLT3-ITD-specific effect. Synergistic (p<0.01) induction of apoptosis was seen in three FLT3-ITD AML patient samples treated in vitro with AZD1208 combined with Q. In an in vivo model, synergistic decrease in tumor volume was seen with combined AZD1208 and Q therapy in mice with subcutaneously implanted MV4-11 cells, with FLT3-ITD, but not with KG1a cells, with wild-type FLT3.Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment.In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. DisclosuresNo relevant conflicts of interest to declare.

Mesenchymal stem cells promote liver regeneration and prolong survival in small-for-size liver grafts: involvement of C-Jun N-terminal kinase, cyclin D1, and NF-κB.

BackgroundThe therapeutic potential of mesenchymal stem cells (MSCs) has been highlighted recently for treatment of acute or chronic liver injury, by possibly differentiating into hepatocyte-like cells, reducing inflammation, and enhancing tissue repair. Despite recent progress, exact mechanisms of action are not clearly elucidated. In this study, we attempted to explore whether and how MSCs protected hepatocytes and stimulated allograft regeneration in small-for-size liver transplantation (SFSLT).MethodsSFSLT model was established with a 30% partial liver transplantation (30PLT) in rats. The differentiation potential and characteristics of bone marrow derived MSCs were explored in vitro. MSCs were infused transvenously immediately after graft implantation in therapy group. Expressions of apoptosis-, inflammatory-, anti-inflammatory-, and growth factor-related genes were measured by RT-PCR, activities of transcription factors AP-1 and NF-κB were analyzed by EMSA, and proliferative responses of the hepatic graft were evaluated by immunohistochemistry and western blot.ResultsMSCs were successfully induced into hepatocyte-like cells, osteoblasts and adipocytes in vitro. MSCs therapy could not only alleviate ischemia reperfusion injury and acute inflammation to promote liver regeneration, but also profoundly improve one week survival rate. It markedly up-regulated the mRNA expressions of HGF, Bcl-2, Bcl-XL, IL-6, IL-10, IP-10, and CXCR2, however, down-regulated TNF-α. Increased activities of AP-1 and NF-κB, as well as elevated expressions of p-c-Jun, cyclin D1, and proliferating cell nuclear antigen (PCNA), were also found in MSCs therapy group.ConclusionThese data suggest that MSCs therapy promotes hepatocyte proliferation and prolongs survival in SFSLT by reducing ischemia reperfusion injury and acute inflammation, and sustaining early increased expressions of c-Jun N-terminal Kinase, Cyclin D1, and NF-κB.

Apoptosis inducing effects of Kuding tea polyphenols in human buccal squamous cell carcinoma cell line BcaCD885.

Tea polyphenols are functional substances present in tea. Kuding tea as a traditional drink also contains these compounds. After 25, 50 and 100 μg/mL of Kuding tea polyphenol treatment for 48 h, cell proliferation of human buccal squamous cell carcinoma cell line BcaCD885 was inhibited, and the 100 μg/mL of Kuding tea polyphenol showed the highest inhibitory rate at 72.3%. Compared to the lower concentration, the 100 μg/mL of Kuding tea polyphenols significantly (p < 0.05) induced apoptosis as determined by flow cytometry analysis, the content of sub-G1 cancer cells was 32.7%. By RT-PCR and western blot assays, Kuding tea polyphenol significantly induced apoptosis in BcaCD885 cancer cells (p < 0.05) by upregulating caspase-3, caspase-8, caspase-9, Fas/FasL, Bax, p53, p21, E2F1, p73 and downregulating Bcl-2, Bcl-xL, HIAP-1, and HIAP-2 mRNA and protein expressions. Kuding tea polyphenols thus present apoptosis inducing effects in vitro.

Activation of p38 MAPK-regulated Bcl-xL signaling increases survival against zoledronic acid-induced apoptosis in osteoclast precursors

The nitrogen-containing bisphosphonate zoledronic acid (ZA) induces apoptosis in osteoclasts and inhibits osteoclast-mediated bone resorption. It is widely used to treat osteoporosis. However, some patients are less responsive to ZA treatment, and the mechanisms of resistance are still unclear. Here, we identified that murine osteoclast precursors may develop resistance to ZA-induced apoptosis. These resistant cells survived the apoptotic effect of ZA following an increase in anti-apoptotic Bcl-xL. Pharmacologically inhibiting Bcl-xL facilitated ZA-induced apoptosis. Treatment with ZA activated p38 MAPK, increasing Bcl-xL expression and cell survival. Nuclear import of β-catenin regulated by p38 MAPK determined Bcl-xL mRNA expression and cell survival in response to ZA. ZA also inactivated glycogen synthase kinase (GSK)-3β, a negative upstream regulator of β-catenin, in a p38 MAPK-mediated manner. Synergistic pharmacological inhibition of p38 MAPK with ZA attenuated receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and facilitated ZA-induced apoptosis. These results demonstrate that elevated Bcl-xL expression mediated by p38 MAPK-regulated GSK-3β/β-catenin signaling is required for cell survival of ZA-induced apoptosis in both osteoclast precursors and osteoclasts. Finally, we demonstrated that inhibiting p38 MAPK-mediated pathway enhanced ZA effect on increasing the bone mineral density of ovariectomized mice. This result suggests that targeting these pathways may represent a potential therapeutic strategy.

P0063 Inhibition of STAT3 signalling and tumour cell growth in melanoma by atractylenolide II treatment

Background Advanced melanoma accounts for most deaths from skin cancer. STAT3 (signal transducer and activator of transcription 3) has been reported to be constitutively activated in melanoma and contributes to tumour cell proliferation, survival, angiogenesis and metastasis, and immune evasion. Methods This study assessed inhibition of STAT3 signalling and tumour cell growth in melanoma by treatment with atractylenolide II (AT-II), a sesquiterpene isolated from the Chinese medicinal herb Atractylodis macrocephalae rhizoma (Baizhu in Chinese). Findings In this study we showed that AT-II dose-dependently and time-dependently inhibited cell proliferation and migration and induced apoptosis in both murine B16 and human A375 melanoma cells. AT-II significantly suppressed subcutaneous B16 xenograft tumour growth in mice and also potently and dose-dependently inhibited STAT3 and Src phosphorylation and activation in melanoma cells in vivo and in vitro. Results of cellular experiments also showed that AT-II lowered protein and mRNA expression of STAT3 target genes VEGF, MCL-1, Bcl-XL, MMP-2, MMP-9, and IL-10, which are involved in angiogenesis, cell apoptosis, invasion, and immune evasion, respectively. Additionally, STAT3 overexpression diminished AT-II-mediated apoptotic effects and inhibiting effects on expressions of STAT3 target genes in A375 cells. Interpretation Our results suggest that AT-II inhibits STAT3 signalling and tumour cell growth in melanoma in vivo and in vitro.

Fucosylated chondroitin sulphate from sea cucumber inhibits high-fat-sucrose diet-induced apoptosis in mouse pancreatic islets via down-regulating mitochondrial signaling pathway

Hyperglycaemia can induce pancreatic islets apoptosis. We previously found that fucosylated chondroitin sulphate from Cucumaria frondosa (Cf-CHS) exhibited anti-hyperglycaemic effects; however, its effects on pancreatic islets are lacking. This study investigated the effects of Cf-CHS on inhibition pancreatic islets apoptosis in high-fat high-sucrose diet (HFSD)-induced insulin resistant mice for 19weeks. Results showed that Cf-CHS significantly repaired HFSD-injured pancreatic islets, decreased blood glucose, insulin, TNF-α levels, and increased adiponectin level. Cf-CHS significantly reduced Bid, Bax, cytochrome c, caspase 9, and caspase 3 mRNA expressions, and increased Bcl-2 and Bcl-xL mRNA expressions. Cf-CHS also caused significant down-regulation of t-Bid, Bax, cytochrome c in cytoplasm, caspase 9, and cleaved-caspase 3 proteins, and up-regulation of Bcl-2 and Bcl-xL proteins. Furthermore, Cf-CHS enhanced the effects of rosiglitazone (RSG). These indicate that Cf-CHS inhibits pancreatic islets apoptosis via inhibition mitochondrial pathway. These findings may provide a dietary intervention hyperglycaemia-induced pancreatic islets apoptosis.

Effects of radiation on apoptosis rate and expression of apoptosis-related genes in well-differentiated nasopharyngeal carcinoma cells

Objective To investigate the effects of radiation on the apoptosis rate and expression of 7 apoptosis-related genes in well-differentiated human nasopharyngeal carcinoma cell line CNE-1.Methods CNE-1 cells were cultured in vitro.The apoptosis rates of CNE-1 cells under 0-,2-,4-,6-,and 8-Gy radiation were measured by flow cytometry.The mRNA expression levels of Bcl-2,Bcl-xl,Bcl-w,Bax,Bak,Bad,and Bid were measured by RT-PCR.The Pearson test was used for analyzing the correlation of mRNA expression with apoptosis rate and radiation dose and the correlation between apoptosis rate and survival fraction.Results The early apoptosis rate of CNE-1 cells increased gradually as the radiation dose ranged from 0 to 6 Gy,but decreased when the radiation dose was 8 Gy; the late apoptosis rate of CNE-1 cells increased as the radiation dose ranged from 0 to 8 Gy.The mRNA expression of Bax was upregulated as CNE-1 cells were irradiated,and it was positively correlated with the early/late apoptosis rate and radiation dose (P =0.000 for all comparisons).The mRNA expression of Bcl-xl was downregulated,and it was negatively correlated with the early/late apoptosis rate (P =0.005 and 0.039) ; but it showed no correlation with radiation dose (P =0.369).The mRNA expression of Bcl-2 was upregulated and reached the peak level when the radiation dose was 4 Gy,and then it fell as the radiation dose increased.The mRNA expression of Bcl-w,Bcl-2,Bad,and Bid were not correlated with the early/late apoptosis rate (P =0.058 -0.894).There was no correlation between the apoptosis rate and survival fraction in CNE-1 cells (P =0.064).Conclusions Bax and Bcl-xl have some correlation with apoptosis rate in CNE-1 cells,but no correlation between the apoptosis rate and survival fraction was observed. Key words: Cell line, nasopharyngeal carcinoma; Apoptosis; Bcl gene expression

Sequence-Specific Talens-Induced DLEU1 Gene Disruption Significantly Suppresses Apoptosis and Increases Phosphorylation Of AKT and Ikba In Pediatric Burkitt Lymphoma (PBL): DLEU1 May Act As a Tumor Suppressor Gene In PBL

BackgroundPediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJHaem, 2012). We previously identified, in a subset analysis, that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leukemia, 2009; Nelson/Cairo/Sanger et al, BJHaem, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene on 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 has been implicated in regulating programmed cell death in patients by inducing TUBB2C and UBR1 expression. Sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro and in vivo experiments with high efficiency as a new experimental and therapeutic tool (Sander et al, Nat Bio, 2011). We have previously reported TALENs mediated transient downregulation of DLEU1 in Raji cells (Figure 1), and demonstrated a decrease in the spontaneous apoptotic rate and an increase in cell proliferation in transiently DLEU1 down-regulated Raji cells (Lee/Cairo et al, ASH, 2012). ObjectivesWe hypothesize that DLEU1 may act as a tumor suppressor gene and therefore investigated whether the silencing of DLEU1 results in changes in proliferation, programmed cell death and associated signaling pathways induced by Rituximab and Cyclophosphamide alone and in combination in TALEN-mediated DLEU1 knockout Raji BL cells. MethodsA stable, single DLEU1 knockout clone (DLEU1 KO) was isolated from Raji cells transfected with DLEU1 TALENs (Lee/Cairo et al, ASH, 2012). Genomic DNA and total RNA were extracted using a DNA extraction kit (Promega) and Trizol (Invitrogen) for verification of endogenous DLEU1 gene disruption by TALENs. qRT-PCR was performed by CFX96 (Bio-rad) and the protein level was measured by western blotting. DLEU1 KO cells were plated (1x104 cells) into 48 or 96 well plates and then treated with Rituximab (0, 1, 10 and 100ug/ml) and/or Cyclophosphamide (0, 1, 5 and 10mM) and cell proliferation and apoptosis were measured by MTS (Promega) and Caspase 3/7 (Promega) assay. Statistical significance was determined by a one tailed paired Student t-test. ResultsDLEU1 KO (mono-allelic deletion) in Raji (BL) exhibited significantly reduced DLEU1 mRNA expression compared to mock control cells (83% reduction, p<0.006). There was significantly reduced expression of c-myc, TUBB2C and UBR1 mRNA (85%, p<0.001; 60%, p<0.04; 25%, p<0.006), respectively in DLEU1 KO compared to mock control cells. DLEU1-KO showed significant reduction of Caspase 3/7 activities (25% at 24hrs, p<0.007; 33% at 48hrs, p<0.03) and significant increase in cell proliferation (20% at 24hrs, p<0.04; 25% at 48hrs, p<0.03). DLEU1-KO showed a significant increase in expression of anti-apoptotic genes, Bcl-2, Bcl-xL and Mcl-1 mRNA (14%, p<0.03; 30%, p<0.04; 17%, p<0.04), respectively and a significantly a decrease in mRNA expression of pro-apoptotic genes, Bax and Bad (18%, p<0.04; 14%, p<0.04), respectively. There was a significant increase in phosphorylation of IkBa (2.6 fold, p<0.007) and Akt (1.5 fold, p<0.05) in DLEU1 KO. There was a significant reduction in Caspase-dependent apoptosis with RTX (15% reduction, p<0.05), CTX alone (24% reduction, p<0.03), and in combination (35% reduction, p<0.03) in DLEU1-KO compared to mock control. A significant increase in cell proliferation was observed with RTX (10% increase, p<0.04), CTX alone (9.4% increase, p<0.05), and combination (7.7% increase, p<0.02) in DLEU1-KO. RTX treated DLEU1-KO showed a significant increase in expression in Bcl-2 and Bcl-xL mRNA (1.3 fold, p<0.05 and 2.0 fold, p<0.02), respectively and a significant decrease in mRNA expression of Bax (1.2 fold, p<0.03). ConclusionsWe demonstrate that 1) TALENs mediated DLEU1 gene knockout resulted in downregulation of DLEU1 mRNA and consequently, inhibition of BL apoptosis and increase in cell proliferation, through several mechanism(s), and 2) the silencing of DLEU1 resulted in decrease in apoptosis in RTX, CTX alone and in combination treatment. We hypothesize that deletion of DLEU1 in childhood BL may result in chemotherapy resistance secondarily to a loss of a tumor suppressor gene. Disclosures:No relevant conflicts of interest to declare.▪

Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures:No relevant conflicts of interest to declare.