HiPerFect Transfection Reagent Research Articles

32P Immunoregulatory loop between let-7a and CCAT1 lncRNA coordinated by c-Myc underlies the PD-1/PD-L1 immunoresistance in triple negative breast cancer patients

PD-1/PD-L1 blockade therapeutic strategy has revolutionized the oncological treatment landscape. Patients with chemo-resistant immunogenic triple-negative breast cancer (TNBC) tumors were among the top listed applicable candidates for PD-1/PD-L1 inhibitors. Yet, TNBC patients developing innate and adaptive immune-resistance have started to appear in the clinics. Thus it became essential to identify novel predictive biomarkers to discriminate between resistant and responsive TNBC patients in a precision immune-oncological approach. Our research group is currently focusing on molecular engines controlling PD-L1 in TNBC patients and cell lines. CCAT1 long noncoding RNA and let-7a microRNA are among the recently identified regulators of PD-L1. The aim of this study was to investigate the synchronization between CCAT1 and let-7a in regulating PD-L1 in TNBC patients and cell lines. TNBC patients (n=17) were recruited into the study. MDA-MB-231 cells were cultured and transfected with let-7a mimics and CCAT1 siRNAs using HiPerFect Transfection Reagent. Total RNA was extracted using Biozol reagent, reverse transcribed and quantified using qRT-PCR. MTT and colony-forming assays were performed. TNBC patients showed a marked repression of let-7a, inversely correlated with elevated CCAT1, c-Myc and PD-L1 expression levels. Ectopic expression of let-7a and/or knockdown of CCAT1 resulted in a significant repression of PD-L1 in MDA-MB-231 cells and thus halt TNBC cellular viability and colony-forming ability. let-7a mimics resulted in a repression of c-Myc-regulated CCAT1 while CCAT1 siRNAs induced let-7a levels. Mechanistically, it was found that the impact of let-7a on CCAT1 is mediated by c-Myc. let-7a mimics resulted in repression of c-Myc and downstream CCAT1 lncRNA. This study highlights Let-7a/c-Myc/CCAT1 as a novel immunoregulatory loop for PD-L1 in TNBC patients and cell lines. Nonetheless, this study underlines the significance of let-7a and CCAT1 as novel predictive immunomodulatory biomarkers in PD-L1-overexpressing TNBC patients.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

The effect of U3 snoRNA knockdown on the chondrocyte proteome

Purpose: Osteoarthritis (OA) is the most common age related musculoskeletal disease leading to impairment in movement, with a total of 8.75 million people affected in UK only. During OA cartilage in joints start to deteriorate uncovering underlying bone, the friction of which causes pain. Accounting the studies up to date the mechanisms of cartilage degeneration are still not fully understood. Although, extracellular matrix composes 95% of the hyaline cartilage the only residing cells in the cartilage are chondrocytes. With regard to this, understanding biological processes regulating the homoeostasis of chondrocytes and defining the biomarkers of early OA attracts huge interest. Small nucleolar RNAs (snoRNAs) represent a class of regulatory RNAs, most of which involved in chemical modification of ribosomal RNAs and spliceosomal RNAs. One of the important snoRNA which associated with 18s rRNA maturation is U3, snoRNA binds to 18s pre-RNA in order to cleave 5’ ETS at A0 and A1 sites. Although, U3 possess conservative motives and their functions are similar among eukaryote species, the most of the work was carried out on Xenopus and Saccharomyces cerevisiae. In that context, it was interesting to investigate the role of snoRNAs in human cell lines. For that purpose we choose SW1353 cells, a human chondrosarcoma cell lineage which holds certain similarities with primary human chondrocytes. Methods: SW1353 cells were cultured in phenol red free Dulbecco's Modified Eagle medium supplemented with 10% Fetal bovine serum, 0.2% Amphotericin B, 1% Penicillin-Streptomycin and 1% L-Glutamin. Transfection of U3 antisense oligonucleotide and scrambled oligonucleotide (ASO) was carried out using HiPerfect transfection reagent. Total RNA from cells was extracted using TRIzol reagent and knockdown of U3 was confirmed by qPCR. Proteins of cell lysates and proteins secreted into media of U3 knockdown and scrambled cells were analysed on Q-exactive Orbitrap mass spectrometer. Proteins were identified using Mascot and quantified with ProgenesisQI™ software. Results: Amount of identified proteins derived from cell lysates were in the range of 951 and 473 proteins after adjustment to 1% FDR. Meanwhile, proteins from media showed far less proteins identified, with the highest 41 and the lowest 10 proteins in sample. The number of differently expressed (DE) proteins with at least 2-fold change and p< 0.05 was 124, of which 4 were downregulated in knockdown and 120 upregulated in cell lysate samples. However, media proteins showed difference in only few proteins (p<0.05), namely FLNB, EIF4B, SERPINE2, ALYREF and ECM1, all upregulated in knockdown samples. Among the top molecular and cellular functions in Ingenuity Pathway Analysis, DE proteins were accounted to cell death and survival, with 71 proteins mapped to the aforementioned cellular function. Conclusions: In current study we have showed the importance of U3 snoRNA in homeostasis of SW1353 cells, the knockdown of U3 leads to the upregulation of proteins involved in cell death pathway. Moreover, suppression of U3 expression increased activation in glycolysis pathway, we assume that is to compensate the reduction of mature and functional ribosomes.

MicroRNA-16 inhibits hypoxia-induced vascular endothelial growth factor expression in ARPE-19 cells

Purpose: To investigate the effect of microRNA-16 on hypoxia-induced VEGF expression of ARPE-19 cells.Methods: ARPE-19 cells were cultivated under normoxia and hypoxia state. At 0 h, 12 h, 24 h, and 48 h after cultivation, the supernate of the culture medium was separated to test the VEGF secretion by ELISA, and the cells were purified to measure the expression of VEGF mRNA and microRNA-16 by qRT-PCR; microRNA-16 mimic was then transfected into ARPE-19 cells by the Hiperfect transfection reagent, a liposome transfection system. Scramble group and the non-transfected group were set as the controls. VEGF secretion and the level of VEGF mRNA were measured in these three groups.Results: The VEGF secretion of the hypoxia ARPE cells was significantly higher than the initial state (p < 0.01). Compared with the normoxia cells, the VEGF secretion of the hypoxia cell was significantly increased (p < 0.01), and the division of VEGF secretion between these two groups increased by time. VEGF mRNA of the hypoxia ARPE cell was significantly higher than the initial state (p < 0.01). Compared with the normoxia cells, VEGF mRNA of the hypoxia cells was significantly increased (p < 0.01), and the division of VEGF mRNA between these two groups increased by time. After cultivating under hypoxia, the expression of microRNA-16 in ARPE-19 cells decreased significantly compared with the normal group, and the division of these two groups augmented by time. MicroRNA-16 was successfully transfected into ARPE-19 cells by the Hiperfect transfection system. Twenty four hours and 48 h after the transfection, the VEGF secretion of the miR-16 transfected cell was decreased significantly (p < 0.01) compared with the scramble and the non-transfected group under hypoxia, while VEGF mRNA level had no significant difference among these three groups.Conclusions: Hypoxia can increase the expression of VEGF mRNA and the secretion of VEGF protein of ARPE-19 cells. At the same time, microRNA-16 expression can be down-regulated by hypoxia. Transfection of microRNA-16 exogenously can down-regulate the VEGF protein secretion but cannot affect the expression of VEGF mRNA.

PCSK9 Promotes oxLDL-Induced PC12 Cell Apoptosis Through the Bcl-2/Bax-Caspase 9/3 Signaling Pathway.

Hyperlipidemia is a risk factor for neurodegenerative diseases. Proprotein convertase subtilisin / Kexin type 9 (PCSK9) degrades hepatic low-density lipoprotein receptor (LDLR) to regulate lipid metabolism. It is unclear if PCSK9 plays a role in neurodegenerative diseases. This study was designed to determine whether PCSK9 is crucial between hyperlipidemia and Alzheimer's disease. The interrelationship between PCSK9 and neuronal apoptosis was explored in PC12 cells in response to treatment with oxidized low-density lipoprotein (oxLDL). Cultured PC12 cells were serum-starved and incubated with different concentrations of oxLDL for 24 h. Intracytoplasmic lipid droplets were observed by oil red O staining. Morphological assessment of apoptotic cells was performed using Hoechst 33258 staining and flow cytometry analysis. The expression of mRNA and protein was detected by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analyses, respectively. Transfection of small interfering RNA (siRNA) into PC12 cells was conducted using HiperFect Transfection Reagent. Concentrations of Aβ40 and Aβ42 were detected by enzyme-linked immunosorbent assay (ELISA) kit. Intracellular lipid content, the number of apoptotic cells, and PCSK9 expression were increased in PC12 cells after oxLDL treatment. Transfection with PCSK9 siRNA reduced the oxLDL-induced apoptosis of PC12 cells. We further confirmed the involvement of Bcl-2/Bax-Caspase (9, 3) signaling pathway in the regulation of PC12 cells apoptosis.β-Secretase 1, another target gene of PCSK9, was downregulated in PC12 cells in response to oxLDL treatment. Aβ40 and Aβ42 contents were also decreased. PCSK9 promotes oxLDL-induced PC12 cell apoptosis through the Bcl-2/Bax-Caspase 9/3 signaling pathway.

Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma.

To investigate the effect of microRNA on insulin-like growth factor binding protein-3 (IGFBP-3) and hence on insulin-like growth factor-II (IGF-II) bioavailability in hepatocellular carcinoma (HCC). Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mirVana miRNA Isolation Kit. microRNA-17-5p (miR-17-5p) expression was mimicked and antagonized in HuH-7 cell lines using HiPerFect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cDNA followed by quantification of miR-17-5p and IGFBP-3 expression using TaqMan real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-II protein was measured in transfected HuH-7 cells using IGF-II ELISA kit. Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where miR-17-5p was extensively underexpressed in HCC tissues (P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients (P = 0.0041) compared to healthy donors. Forcing miR-17-5p expression in HuH-7 cell lines showed a significant downregulation of IGFBP-3 mRNA expression (P = 0.0267) and a significant increase in free IGF-II protein (P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of miR-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone (P = 0.0474). These data suggest that regulating IGF-II bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miRNAs.

TM4SF1 Promotes Gemcitabine Resistance of Pancreatic Cancer In Vitro and In Vivo.

BackgroundTM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR.MethodsThe expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines.ResultsThe mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging.ConclusionThese findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.

Combination of BCL11A siRNA with vincristine increases the apoptosis of SUDHL6 cells.

BackgroundB cell chronic lymphocytic leukemia/lymphoma 11 A (BCL11A) is associated with human B cell malignancy initiation. Our previous study has shown that downregulation of BCL11A mRNA by small interfering RNA (siRNA) is capable of inducing apoptosis in the SUDHL6 cell line. To further explore the effects of BCL11A siRNA on the enhanced cytotoxicity of a chemotherapeutic drug, we investigated the effects of BCL11A siRNA combined with vincristine (VCR) on SUDHL6 cell proliferation and apoptosis.MethodsChemically synthesized BCL11A siRNA was transfected into SUDHL6 cells using the HiPerFect Transfection Reagent in combination with VCR. Cell proliferation was measured by the CCK8 assay. The morphology of apoptotic cells was observed with Hoechst 33258 staining. The rate of cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide double staining using fluorescence-activated cell sorting (FACS) analysis.ResultsAfter BCL11A siRNA plus VCR treatment, cell proliferation was significantly decreased in comparison with VCR or BCL11A siRNA treatment alone and negative control siRNA plus VCR treatment (P <0.05). The apoptotic rate of BCL11A siRNA plus VCR treated cells was significantly increased compared with BCL11A siRNA and VCR treatment alone and negative control siRNA plus VCR treatment (P <0.05).ConclusionsThe combination of BCL11A siRNA and VCR increases apoptosis in SUDHL6 cells. Our study implies that BCL11A siRNA in combination with VCR may be a useful approach for improving effective treatment for B cell lymphoma.

Up Regulation Of Peroxiredoxin 2 Which Is Correlated With Disappearance Of Philadelphia Chromosome In CML Cells Might Have Therapeutic Applications During Imatinib Therapy

BackgroundBCR/ABL oncogenic tyrosine kinase is known to induce high levels of intracellular ROS which may further induce genomic instability with malignant transformation and even imatinib (IM) resistance. There are several lines of evidence that the production of reactive oxygen species (ROS) and the expression and activity of antioxidant enzymes make up the primary redox control of leukemia cells and cell survival. Therapies targeting the redox environment such as over-expression of antioxidants or antioxidant treatment, could inhibit tumor cell growth even resistant cells. In this study, we investigated the change of peroxiredoxin (Prx), which have a function of protection against oxidative stress, and survival of BCR-ABL positive cells during IM therapy. MethodsBCR-ABL1 positive cell lines were cultured using 20% IMDM medium. We evaluated cell growth by MTT assay, and also evaluated BCR/ABL expression by western blot analysis. We also evaluated changes of intracellular ROS level and antioxidant enzymes, by immunoblot assay. siRNAs against Prx2 (100-200nM) were transfected into K562 cells using HiPerFect transfection Reagent (Qiagen) according to manufacturers' protocol. During siRNA transfection, 1uM of imatinib was treated for 48hours. 72 hours after siRNA transfection, expression of BCR/ABL and Prx2 were detected by western blot assay and proliferation was determined by MTT assay method. Intracellular ROS was measured by flow cytometry after 10uM DCF-DA staining for 20mins. ResultsDuring IM treatment, we observed the significant decrease of cell viability and decrease of intracellular ROS level in BCR-ABL1 positive cell lines. These finding were well correlated with eradication of BCR/ABL oncogene by western blot results. The expression levels of Prx2 was restored with IM treatment and restoration of redox enzymes is correlated with reduction of BCR-ABL protein in Philadelphia positive cells. siRNAs transfected cells were not decreased cell survival even though eradication of BCR/ABL oncogene during IM treatment. ConclusionSignificant change of ROS and Prx2 levels according to IM treatment are closely correlated with bcr/abl kinase level in both BCR-ABL1 positive cell lines. This finding was correlated with restoration of the level of PRX2. Those results has been implicated the possibility of therapeutic application by modulation redox system. Strategy of over-expression of Prx2 might be able to control sensitivity of the BCR-ABL1 positive cells to drug-induced apoptosis. Understanding the molecular mechanisms of changes on ROS and redox enzymes may be potential tools to develop more potent new drugs in Ph+ CML or Ph+ ALL patients especially for IM resistant. Disclosures:No relevant conflicts of interest to declare.

Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells.

ObjectiveB-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells.MethodsChemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining.ResultsAfter the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl-2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl-2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05).ConclusionsBcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

BBF2H7-Mediated Sec23A Pathway Is Required for Endoplasmic Reticulum-to-Golgi Trafficking in Dermal Fibroblasts to Promote Collagen Synthesis

Collagen fibers, structural elements responsible for mechanical strength in skin, are synthesized constitutively in response to cytokines such as IGF-I. However, little is known about their intracellular trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus during synthesis. We demonstrate herein that the BBF2 human homolog on chromosome 7 (BBF2H7)-mediated Sec23A pathway is involved in regulation of intracellular procollagen trafficking. The mRNA and protein expression of BBF2H7, Sec23A, and type I and III collagen (COL1 and COL3) was induced by IGF-I stimulation. In addition, the cleaved form of BBF2H7 was detected in IGF-I-treated cultures, indicating that activation occurs concurrently with its expression. Knockdown with small interfering RNAs targeting BBF2H7 caused a significant reduction in the expression of COL1 and COL3, regardless of IGF-I treatment. Both mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways via IGF-I receptor activation were required for BBF2H7 induction. Using immunofluorescence microscopy, we showed that Golgi apparatus dysmorphology is due to coat protein complex II vehicle hypoplasia caused by the absence of BBF2H7 and Sec23A. The BBF2H7-mediated Sec23A pathway was required for ER-to-Golgi procollagen trafficking to promote collagen synthesis. This role of growth factors such as IGF-I, which to our knowledge is previously unreported, suggests antiaging strategies.

Imatinib and Bortezomib Induce the Expression and Distribution of Anaphase-Promoting Complex Cdh1 in Blast Crisis of Chronic Myelogenous Leukemia

Abstract 4428 Introduction:CML is well-known as the best understood human malignancy and a paradigm for cancer research from bench to bedside. However, treatment options for imatinib-resistant patients are still limited. Imatinib (IM) has already established as the standard first-line therapy for patients with chronic-phase CML. Bortezomib (BOR) not only prolonged life span for relapsed multiple myeloma(MM) patients, but also induced cell apoptosis in CML. However, the efficacy of TKIs and bortezomib on imatinib-resistant CML remains obscure.The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. Cdh1, the adaptor protein of APC/C, in conjunction with APC/C in G1-phase, is required for preventing unscheduled proliferation and protecting primary mammalian cells from genomic instability. Cdh1-deficient cells showed a large variety of chromosomal aberrations. On the contrary, Cdc20, the substrate of Cdh1, could contribute to genetic aberration. However, it's marginally addressed about the relationship between interaction of Cdh1-Cdc20 with IM resistance and genetic aberrations. Methods:We treated K562-WT and IM-resistant K562 cells with IM/nilotinib(AMN) and BOR, verified the effect of TKIs and BOR by FACS, and explored the change of the expression and the subcellular localization of Cdh1 by immunoblot and immunofluorescence microscopic analysis. Then, we enrolled ten patients with newly diagnosed CML-BC and dissected Cdh1-Skp2-p27 cascade in primary CML cells by immunoblot analysis. We explored the subcellular localization and interaction in space between Cdh1 and Skp2 by immunofluorescence in IM-sensitive or resistant primary cells and cell line. Moreover, the single siRNAs of Cdh1 and Skp2 were designed and were transiently transfected with HiPerFect Transfection Reagent. The expressions of Cdh1-Skp2-p27 pathway proteins were detected by immunoblot, the change of cell cycle and apoptosis were detected by flow cytometry. Result:We found that IM and BOR induced cell cycle quiescence and arrest, and changed the expression and nuclear relocation of Cdh1 in CML-BC cells. Moreover, AMN and BOR resulted in up-regulation of Cdh1 in IM-resistant cells. Our study revealed Cdh1 and Skp2 were co-localized more abundantly in the cytoplasm of IM-sensitive cells, but co-distributed to nuclears in IM-resistant cells. Furthermore, Cdh1 was lower level in IM-resistant CML-blast crisis (BC) than that of IM-sensitive patients. On the other side, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells. Conclusion:Expression and dynamic distribution of Cdh1, induced by IM and BOR, provide a novel interpretation of underlying mechanism to inhibit BCR-ABL downstream cascade via Cdh1-Skp2-p27 axis. Furthermore, we demonstrated BOR still exerted the regulation effect on expression and relocation of Cdh1 even in IM-resistant CML-BC cells, which provided evidences for synergy combination of TKI and proteasome inhibitor for overcoming IM-resistance. Besides, our results revealed Cdh1 tends to be related to genomic stability in CML-BC. Disclosures:No relevant conflicts of interest to declare.Footnotes:* Asterisk with author names denotes non-ASH members.

Imatinib and bortezomib induce the expression and distribution of anaphase-promoting complex adaptor protein Cdh1 in blast crisis of chronic myeloid leukemia

Anaphase promoting complex cofactor Cdh1 plays a critical role in tumor suppression and genomic stability in cancer. However, its role in chronic myeloid leukemia (CML) remains unclear. We treated both wild-type and imatinib-resistant K562 cells with imatinib or nilotinib and bortezomib, respectively. The siRNAs of Cdh1 and Skp2 were designed and transiently transfected with HiPerFect transfection reagent into CML cells. Expression of Cdh1-Skp2-p27 pathway proteins were detected by Western blotting. Cell cycle, cell apoptosis and cellular morphology were detected by flow cytometry and Wright staining. Our study revealed that Cdh1 was expressed at lower levels in imatinib-resistant CML blast crisis (BC) patients than imatinib-sensitive ones. Moreover, imatinib and bortezomib induced cell cycle quiescence or arrest, upregulation and nuclear relocation of Cdh1 in CML cells. Furthermore, nilotinib and bortezomib resulted in upregulation of Cdh1 in imatinib-resistant CML cells. Conversely, Cdh1 silencing resulted in stabilization of Skp2 and Cdc20, subsequently promoting G1-S transition and formation of multinucleated cells. Our study shows that TKIs and bortezomib can regulate the cell cycle and cell apoptosis via regulation of the expression and redistribution of Cdh1 in CML-BC, which sheds light on the orchestration of crosstalk between TKIs and bortezomib in imatinib-resistant CML-BC. Additionally, Cdh1 tends to play an important role in maintenance of genomic stability, the detailed mechanisms deserve further study.

Radiation-triggered Tumor Necrosis Factor (TNF) α-NFκB Cross-signaling Favors Survival Advantage in Human Neuroblastoma Cells

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.

Protein Interactions of Phosphatase and Tensin Homologue (PTEN) and Its Cancer-associated G20E Mutant Compared by Using Stable Isotope Labeling by Amino Acids in Cell Culture-based Parallel Affinity Purification

The tumor suppressor PTEN (phosphatase and tensin homologue) negatively regulates the PI3K pathway through its lipid phosphatase activity and is one of the most commonly lost tumor suppressors in human cancers. Though the tumor suppressive function involves the lipid phosphatase-dependent and -independent activities of PTEN, the mechanism leading to the phosphatase-independent function of PTEN is understood poorly. Some PTEN mutants have lipid phosphatase activity but fail to suppress cell growth. Here, we use a cancer-associated mutant, G20E, to gain insight into the phosphatase-independent function of PTEN by investigating protein-protein interactions using MS-based stable isotope labeling by amino acids in cell culture (SILAC). A strategy named parallel affinity purification (PAP) and SILAC has been developed to prioritize interactors and to compare the interactions between wild-type and G20E PTEN. Clustering of the prioritized interactors acquired by the PAP-SILAC approach shows three distinct clusters: 1) wild-type-specific interactors, 2) interactors unique to the G20E mutant, and 3) proteins common to wild-type and mutant. These interactors are involved mainly in cell migration and apoptosis pathways. We further demonstrate that the wild-type-specific interactor, NUDTL16L1, is required for the regulatory function of wild-type PTEN in cell migration. These findings contribute to a better understanding of the mechanisms of the phosphatase-dependent and -independent functions of PTEN.

Abstract 4071: Sestrins modulate the expression of the metabolic stress sensor AMPK in breast cancer cells

Abstract Introduction: Sestrins (SESN) are stress response mediators that accumulate in cells exposed to genotoxic stresses, such as ultraviolet and ionizing radiation (IR). The three mammalian SESN (1-3) were recently shown to modulate signalling events. SESN1/2 are suggested to interact with and increasing the activity of the metabolic stress sensor AMP-activated protein kinase (AMPK). SESN1/2 have been postulated to act as scaffolding proteins that can bind targets of the AMPK metabolic pathway including AMPKα and TSC2. However, the direct mechanism in which SESN1/2 increase AMPK activity is still being elucidated. Furthermore, the ability of SESNs to modulate the expression of AMPK subunits in untreated or IR treated cells has not been examined. Methods: Immunoprecipitation of the AMPK subunits (α1-2, β1-2, γ1-3) with subunit-specific antibodies was preformed to determine the function AMPK heterotrimer complexes in MCF7 breast cancer cells. Overexpression of full length SESN2 cDNA was achieved in MCF7 cells using tetracycline inducible promoter system. SESN1/2 siRNA was introduced into cells using HiPerFect transfection reagent and the Qiagen protocol. Protein and mRNA levels were measured with western blotting and RT-PRC, respectively. Cells were treated with 0 – 8 Gy IR, and cell survival was measured using clonogenic assays. Results: Through immunoprecipitation we identified that the most prominent AMPK functional complex in MCF7 cells is the α1β1γ1 heterotrimer. SESN2 overexpression increases both mRNA and protein expression levels of AMPKα1 and AMPKβ1, and was co-immunoprecipitated with the AMPKβ1 subunit. Similarly, increasing SENS2 expression increased the phosphorylation levels of AMPKα (Thr172) and AMPKβ1 (Ser108), as well as phosphorylation of the AMPK substrate ACC. Conversely, siRNA against SESN2 decreased both AMPKα1 and AMPKβ1 mRNA and protein levels, while SESN1 siRNA blocked basal and IR-induced phosphorylation of AMPKα (Thr172). In addition, SESN2 overexpression sensitized MCF7 cells to the cytotoxic effects of IR. Conclusions: To date our work suggests that upregulation of SESNs in breast cancer cells modulates AMPK by stabilizing the AMPK (α1β1γ1) heterotrimer and by enhancing the subunit expression of AMPK. We observed that SESN2 was shown to directly interact with the AMPKβ1 subunit and also enhance its Ser108 phosphorylation, an additional marker of AMPK activity. Furthermore, silencing SESN1 expression blocks basal IR-induced AMPKα (Thr172) phosphorylation, while SESN2 overexpression acts as a radiation sensitizer in MCF7 cells. Taken together, this data indicates that SESNs not only regulate AMPK activity, but also play a role in stabilizing this enzymes basal and stress-induced expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2011-4071

Effect of RNA interference-mediated down-regulation of JMJD2A on human breast cancer cell line MCF-7

Objective To study the effect of specific small interfering RNA (siRNA) silencing of Jumonji Domain Containing 2A (JMJD2A) on human breast cancer cell line MCF-7. Methods The chemically synthetic siRNA duplexes targeting JMJD2A was transfected into MCF-7 cells by HiPerFect Transfection Reagent. After siRNA interfering cells were harvested, the expression of JMJD2A was detected by real-time reverse transcription-polymerase chain reaction ( RT-PCR), and Western blotting. The activity of cell proliferation and cell cycle were examined by WST-8 and flow cytometry (FCM) respectively , and the abilities of cell invasion and metastasis were evaluated using Transv ell chambers. Results JMJD2A siRNA effectively inhibited the expression of JMJD2A mRNA and protein (P<0. 05). After siRNA interference, the cell proliferation was suppressed [ 1.45±0. 05 vs 1.73±0. 02(48h),P <0. 05] , and cell cycle was arrested at G0/G1 phase (53. 80±1. 80 vs 44. 84±1. 86,P<0. 05) with a significant decrease of cells in S phase (36. 55±1. 52 vs 47. 06±1. 26,P<0. 05). The ability of cell invasion and metastasis was depressed (P<0. 05). Conclusion siRNA interfering to JMJD2A effectively reverses some of the malignant biological characters of human breast cancer cell line MCF-7, implying that JMJD2A might be involved in the progression and metastasis of breast cancer cells. Key words: RNA interference; Breast carcinoma; Proliferation; Cell cycle; Metastasis

Role for the Proapoptotic Factor BIM in Mediating Imatinib-induced Apoptosis in a c-KIT-dependent Gastrointestinal Stromal Tumor Cell Line

The c-KIT receptor tyrosine kinase is constitutively activated and oncogenic in the majority of gastrointestinal stromal tumors. The identification of selective inhibitors of c-KIT, such as imatinib, has provided a novel therapeutic approach in the treatment of this chemotherapy refractory tumor. However, despite the clinical importance of these findings and the potential it provides as a model system for understanding targeted therapy, this approach has not yielded curative outcomes in most patients, and the biochemical pathways connecting c-KIT inhibition to cell death are not completely understood. Here, we show that inhibition of c-KIT with imatinib in gastrointestinal stromal tumors (GISTs) triggered the up-regulation of the proapoptotic protein BIM via both transcriptional and post-translational mechanisms. The inhibition of c-KIT by imatinib increased levels of the dephosphorylated and deubiquitinated form of BIM as well as triggered the accumulation of the transcription factor FOXO3a on the BIM promoter to activate transcription of BIM mRNA. Furthermore, using RNA interference directed against BIM, we demonstrated that BIM knockdown attenuated the effects of imatinib, suggesting that BIM functionally contributes to imatinib-induced apoptosis in GIST. The identification and characterization of the pathways that mediate imatinib-induced cell death in GIST provide for a better understanding of targeted therapy and may facilitate the development of new therapeutic approaches to further exploit these pathways.

A Role for HSP70 in Protecting against Indomethacin-induced Gastric Lesions

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E(2) for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin.

SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.