IM-9 Cells Research Articles

Unlocking the potential of senescence-related gene signature as a diagnostic and prognostic biomarker in sepsis: insights from meta-analyses, single-cell RNA sequencing, and in vitro experiments.

Cellular senescence is closely associated with the pathogenesis of sepsis. However, the diagnostic and prognostic value of senescence-related genes remain unclear. In this study, 866 senescence-related genes were collected from CellAge. The training cohort, GSE65682, which included 42 control and 760 sepsis samples, was obtained from the Gene Expression Omnibus (GEO). Feature selection was performed using gene expression difference detection, LASSO analysis, random forest, and Cox regression. TGFBI and MAD1L1 were ultimately selected for inclusion in the multivariate Cox regression model. Clustering based on the expressions of TGFBI and MAD1L1 was significantly associated with sepsis characteristics and prognoses (all P < 0.05). The risk signature served as a reliable prognostic predictor across the GSE65682, GSE95233, and GSE4607 cohorts (pooled hazard ratio = 4.27; 95% confidence interval [CI] = 1.63-11.17). Furthermore, it also served as a robust classifier to distinguish sepsis samples from control cases across 14 cohorts (pooled odds ratio = 5.88; 95% CI = 3.54-9.77). Single-cell RNA sequencing analyses from five healthy controls and four sepsis subjects indicated that the risk signature could reflect the senescence statuses of monocytes and B cells; this finding was then experimentally validated in THP-1 and IM-9 cells in vitro (both P < 0.05). In all, a senescence-related gene signature was developed as a prognostic and diagnostic biomarker for sepsis, providing cut-in points to uncover underlying mechanisms and a promising clinical tool to support precision medicine.

Analysis of signaling cascades from myeloma cells treated with pristimerin.

Multiple myeloma (MM) is the 2nd most frequently diagnosed blood cancer after non-Hodgkin's lymphoma. The present study aimed to identify the differentially expressed genes (DEGs) between the control and pristimerin-treated MM cell lines. We examined the GSE14011 microarray dataset and screened DEGs with GEO2R statistical tool using the inbuilt limma package. We used a bioinformatics pipeline to identify the differential networks, signaling cascades, and the survival of the hub genes. We implemented two different enrichment analysis including ClueGO and Metacore™, to get accurate annotation for most significant DEGs. We screened the most significant 408 DEGs from the dataset based on p-values and logFC values. Using protein network analysis, we found the genes UBC, HSP90AB1, HSPH1, HSPA1B, HSPA1L, HSPA6, HSPD1, DNAJB1, HSPE1, DNAJC10, BAG3, and DNAJC7 had higher node degree distribution. In contrast, the functional annotation provided that the DEGs were predominantly enriched in B-cell receptor signaling, unfolded protein response, positive regulation of phagocytosis, HSP70, and HSP40-dependent folding, and ubiquitin-proteasomal proteolysis. Using network algorithms, and comparing enrichment analysis, we found the hub genes enriched were INHBE, UBC, HSPA1A, HSP90AB1, IKBKB, and BAG3. These DEGs were further validated with overall survival and gene expression analysis between the tumor and control groups. Finally, pristimerin effects were validated independently in a cell line model consisting of IM9 and U266 MM cells. Pristimerin induced in vitro cytotoxicity in MM cells in a dose-dependent manner. Pristimerin inhibited NF-κB, induced accumulation of ubiquitinated proteins and inhibited HSP60 in the validation of bioinformatics findings, while pristimerin-induced caspase-3 and PARP cleavage confirmed cell death. Taken together, we found that the identified DEGs were strongly associated with the apoptosis induced in MM cell lines due to pristimerin treatment, and combinatorial therapy derived from pristimerin could act as novel anti-myeloma multifunctional agents.

Radotinib inhibits multiple myeloma cell proliferation via suppression of STAT3 signaling.

Multiple myeloma (MM) is a hematological cancer causing from accumulated abnormal plasma cells. STAT3 overexpression in MM appears to be mediated by a variety of factors, and it may be associated with an adverse prognosis and play a role in microenvironment-dependent treatment resistance. Unfortunately, MM remains an incurable disease, as relapse is very common. Therefore, there is urgent need to develop new treatment options for MM. Radotinib is a novel anti-cancer drug, currently approved in South Korea for the treatment of chronic myeloid leukemia patients. It is an oral, multitargeted inhibitor of receptor tyrosine kinases, including BCR-ABL, c-KIT, PDGFR, and Src family kinases. However, little is known about the effects of radotinib on multiple myeloma cells. However, little is known about the effects of radotinib on multiple myeloma cells. But even tinip almost not known about the impact of multiple myeloma cells. Moreover, nothing is known about how it affects STAT3 and JAK2. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Herein, Moreover, nothing is known about how it. Moreover, not all is known about how the affects STAT3 and JAK2. We investigated the effect of radotinib on the STAT3 signaling pathway in MM cells, including several MM cell lines and mouse models. So we investigated the effect of radotinib on MM cells, including several MM cell lines and mouse models. Interestingly, radotinib induced apoptosis, and inhibited cell proliferation in MM cells including RPMI-8226, MM.1S, U266B1, and IM-9 cells. Moreover, radotinib treatment significantly increased the number Annexin V-positive cells and G0/G1-phase cells. In addition, radotinib treatment in various MM cells strongly suppressed the activity and expression of STAT3 and JAK2 proteins. We also observed that diverse proteins related to the STAT3 signaling pathway, including c-Myc, Bcl-xL, Mcl-1, cyclin D1 and cyclin D3, were powerfully inhibited by radotinib treatment in MM cells. Furthermore, radotinib significantly suppressed MM cell growth in a xenograft animal model using IM-9 cells. In conclusion, radotinib may play an important role as a candidate agent for MM treatment.

Withaferin A inhibits cell proliferation of U266B1 and IM-9 human myeloma cells by inducing intrinsic apoptosis.

Withaferin A, a withanolide obtained from Withania somnifera exhibits remarkable pharmacological properties. Withaferin A has been reported to exert cytotoxic effects against human multiple myeloma cells. Nevertheless, the in-depth understanding of the withaferin A induced antiproliferative effects against human myeloma cells is still unclear. The results showed that withaferin A inhibited the viability of six different myeloma cells with a lowest IC50 of 9 μM against the U266B1 and IM-9 cell lines. Withaferin A inhibited the viability and colony formation of the U266B1 and IM-9 cells in a dose and time-dependent manner. The DAPI and annexin V/PI staining assays revealed that withaferin A exerts anticancer effects against the human myeloma cells via induction of apoptosis. The induction of apoptosis in U266B1 and IM-9 cells was associated with upregulation of Bax and cytochrome c, downregulation of Bcl-2 and activation of PARP, caspase-3 and capase-9 cleavage. Additionally, withaferin A triggered the production of ROS in human myeloma cells indicative of ROS mediated apoptosis in human myeloma cells. The treatment of the U266B1 and IM-9 with ascorbic acid (antioxidant) could prevent the withaferin A mediated ROS production and the withaferin A induced antiproliferative effects. Collectively, the results show that withaferin A inhibits human myeloma cell proliferation via ROS mediated intrinsic apoptosis.

Dual Effect Exhibited by Insulin in Myeloma and Lymphoblastoid Cells

Many reports have documented the role of INS (insulin) as growth factors in a variety of cancers. Epidemiological studies revealed that INS therapy causes increased mortality in multiple myeloma (MM) patients with pre-existing or steroid-induced type 2 diabetes. However, there is limited experimental evidence of this association. In the present study, the dual effect of INS on the viability of myeloma RPMI8226 and lymphoblastoid IM9 cells was revealed. In serum-containing medium exogenous INS serves as a growth factor, whereas INS decreases the number of cells under serum-free medium. In the last case, the main mechanism of decreasing the cell population is apoptosis through up-regulation of Cas-3 and downregulation of Bcl-2 expression. INS has also been shown to be involved in the regulation of necrotic cell death.

The c-Abl inhibitor, radotinib induces apoptosis in multiple myeloma cells via mitochondrial-dependent pathway

Multiple myeloma (MM) is a hematological cancer resulting from accumulated abnormal plasma cells. Unfortunately, MM remains an incurable disease, as relapse is very common. Therefore, there is urgent need to develop new treatment options for MM. Radotinib is a novel anti-cancer drug, currently approved in South Korea for the treatment of chronic myeloid leukemia patients. Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and the platelet-derived growth factor receptor. Generally, the mechanism of inhibition of non-receptor tyrosine kinase c-Abl has played an essential role in the inhibition of cancer progression. However, little is known regarding the effects of the c-Abl inhibitor, radotinib on MM cells. In this study, we analyzed the effect of radotinib on multiple myeloma cells. Interestingly, radotinib caused apoptosis in MM cells including RPMI-8226, MM.1S, and IM-9 cells, even in the absence of c-kit expression in 2 of these lines. Radotinib treatment significantly increased the number Annexin V-positive cells and decreased the mitochondrial membrane potential in MM cells. Additionally, we observed that cytochrome C was localized in the cytosol of radotinib-treated MM cells. Moreover, radotinib decreased the expression of Bcl-2 and Bcl-xL, and increased the expression of Bax and Bak in MM cells. Furthermore, radotinib promoted caspase pathway activation by inducing the expression and activity of caspase-3, -7, and -9. Expression of cleaved PARP-1 was also increased by radotinib treatment in various MM cells. In addition, radotinib significantly suppressed MM cell growth in a xenograft animal model using RPMI-8226 cells, and killed ex vivo myeloma cells from patients. In conclusion, radotinib may play an important role as a candidate agent or chemosensitizer for the treatment of MM.

The Inhibiting Effect of Autophagy Inhibitor ROC-325 on Multiple Myeloma

To investigate the effects of autophagy inhibitor ROC-325 and its combination with bortezomib on the proliferation, apoptosis and autophagy of multiple myeloma cell lines. Multiple myeloma cells were treated with ROC-325 at different concentration. The cell proliferation was detected by CCK-8. Apoptosis was determined by Caspase-3/7 and Caspase-9 activity assays. Autophagy was detected by monodansylcadaverine staining. The apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62, Beclin-1, and LC3A/B) were analyzed by Western blot. The combined effect with bortezomib on bortezomib-resistant cell line was detected by CCK-8. ROC-325 inhibited the proliferation of RPMI 8226, RPMI 8226-BTZ100, U266 and IM9 cells in a dose-dependent manner (r=-0.8275, r=-0.9079, r=-0.9422, r=-0.9305), the 72 h IC50 values were 2.795, 4.020, 5.432 and 4.755 μmol/L, respectively. The activity assays of Caspase-3/7 and Caspase-9 showed that their relative activity was increased gradually in proportion to the drug concentration with the statistically significant difference (r=0.9648, r=0.9377, r=0.9318; r=0.9087, r=0.9431, r=0.8914). MDC staining results showed that the number of autophagic vacuoles increased with the rise of ROC-325 concentration (r=0.9565, r=0.9373, r=0.9233). ROC-325 could increase the expression of apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62 and LC3-Ⅱ/LC3-Ⅰ), but decrease the expression of Beclin-1 detected by Western blot. The CCK-8 assay showed that ROC-325 combined with bortezomib had synergistic effect on the inhibition of drug resistant cell line RPMI 8226-BTZ100. ROC-325 can inhibit the proliferation, induce the apoptosis of myeloma cells through the mitochondrial pathway, inhibit the autophagy of myeloma cells by affecting the fusion of autophagosomes and lysosomes, and overcome bortezomib resistance by the combination of ROC-325 with bortezomib.

A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin.

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.

SPAG6 promotes cell proliferation and inhibits apoptosis through the PTEN/PI3K/AKT pathway in Burkitt lymphoma.

The main purpose of the present study was to elucidate the role of sperm-associated antigen 6 (SPAG6) in the occurrence and development of Burkitt lymphoma (BL) and explore the underlying molecular mechanisms. A correlation was observed between the expression of SPAG6 and the prognosis of patients with lymphoma using The Cancer Genome Atlas (TCGA) database analysis. It was demonstrated that the levels of SPAG6 in BL cells were higher compared with that in IM-9 cells by reverse transcription-PCR and western blot assays. Moreover, silencing of SPAG6 significantly decreased proliferation and increased apoptosis of Daudi and Raji cells, whereas SPAG6 overexpression exerted the opposite effects on CA46 and NAMALWA cells. When investigating the possible mechanism, it was first observed that the level of phosphatase and tensin homolog (PTEN) protein was significantly increased, while that of phosphorylated (p-)AKT protein was markedly reduced in the SPAG6-knockdown group compared with the blank control group in Daudi and Raji cells by western blot analysis. It was further ascertained whether the phosphoinositide 3-kinase (PI3K)/PTEN/protein kinase B (AKT) pathway mediates the effects of SPAG6 on cell proliferation and apoptosis, and the results demonstrated that silencing of SPAG6 suppressed the viability of Daudi and Raji cells, whereas PTEN knockdown using siRNA or SF1670 (a specific PTEN inhibitor) reversed the inhibitory effect on cell proliferation and the promoting effect on cell apoptosis induced by SPAG6 depletion in vitro as well as in vivo. These data revealed that SPAG6 may promote the proliferation and inhibit the apoptosis of BL cells via the PTEN/PI3K/AKT pathway. The results of the present study suggest that SPAG6 may play a key role in the progression of BL and may be of value as a predictive prognostic biomarker in patients with BL.

Zinc oxide nanorod based immunosensing platform for the determination of human leukemic cells

Zinc oxide (ZnO) based nanostructures owing unique physical properties – high photoluminescence, biocompatibility and other characteristics, therefore, they attract attention as building blocks suitable for biosensor development. In this research as a target we have used human leukemic cell line IM9 (IM9). IM9 was derived from the patient with a multiple myeloma and expressed cluster of differentiation proteins СD19 on the surface of 85–95% here investigated cancer cells. As a control sample healthy human's peripheral blood mononuclear cells (PBMC) were used and the expression of CD19 protein was found only in 5–9% of these cells. Two types of antibodies labeled by fluorescein isothiocyanate (FITC) were used for the labeling of human leukemic cells: FITC-conjugated mouse antibodies against Human CD19 protein (anti-CD19-FITC*) and FITC-conjugated mouse antibodies against Human IgG1 protein (anti-IgG1-FITC*). In order to demonstrate the applicability of zinc oxide nanorods (ZnO-NRs) based platforms three types of ZnO-NRs-based structures were investigated: (i) ZnO-NRs modified by anti-CD19-FITC*; (ii) ZnO-NRs modified by IM9 cells, which were pre-incubated with anti-CD19-FITC*; (iii) ZnO-NRs modified by PBMC cells, which were pre-incubated with anti-CD19-FITC*. It was demonstrated that IM9 cells after specific interaction with anti-CD19-FITC* bind to ZnO-NRs (ZnO-NRs/IM9 +anti-CD19-FITC*) and photoluminescence based signal significantly increase in comparison with that observed in control samples, which contained PBMC cells incubated with anti-CD19-FITC* (ZnO-NRs/PBMC+anti-CD19-FITC*). The photoluminescence results are in good correlation with the data obtained by flow cytometry. This study illustrate that ZnO-NRs exhibit a photoluminescence signal suitable for the determination of anti-CD19-FITC* labeled IM9 cell line at concentrations – from 10 till 500 cells adsorbed per 1 mm2 of ZnO-NRs platform.

Defining the function of a novel TNF receptor superfamily member

Background: &#x0D; Insulin Growth Factor Like 1 Receptor (IGFLR1) is a putative TNF receptor superfamily member of unknown function. Transcripts for IGFLR1 are most abundant in T cells in mice and several leukocyte subsets in humans. A major limitation to understanding IGFLR1 function is the lack of biochemical data regarding IGFLR1 expression and processing in leukocytes. &#x0D; Experimental Design: &#x0D; A panel of human leukocyte and keratinocyte cell lines was screened for IGFLR1 transcript expression by quantitative PCR. Immunoblotting was then used to detect IGFLR1 protein and characterize which isoform(s) were expressed. Chimeric IGFLR1 construct that could be used to mimic IGFLR1 activation was also generated. This chimeric protein, hNGFR-DmrB2- hIGFLR, contains two DmrB domains that allow for multimerization in the presence of AP20187. &#x0D; Results: &#x0D; IGFLR1 transcript expression varied between hematopoietic cell lines with relatively low expression in MOLT4 cells (T cell line), intermediate expression in IM9 and ARH77 cells (B cell lines) and high expression in MKB cells (T cell line). Immunoblotting in these cell lines revealed a similar protein signal expression pattern. Immunoblotting also suggested that the most abundant isoform of IGFLR1 was likely isoform c, which lacks ligand-binding and transmembrane domains. With respect to the hNGFR-DmrB2-hIGFLR1 construct, the chimeric protein was detected in CHO cells by immunoblotting and biochemical analyses confirmed that addition of AP20187 induced multimerization of this protein. These findings confirm the functionality of hNGFR-DmrB2-hIGFLR1. &#x0D; Potential Impact: &#x0D; Biochemical studies of endogenously expressed IGFLR1 in these cell lines suggest differential mRNA splicing may play an important role in the production and function of IGFLR1 isoforms. Future studies using the hNGFR-DmrB2-hIGFLR1 construct in the aforementioned human cell lines can be performed to study IGFLR1 signaling.

Effects of HDAC Inhibitor Scriptaid on IM9 Cell Line and Its Mechanism

To study the effect of HDAC inhibitor Scriptaid on multiple myeloma IM9 cells and preliminarily clarify the mechanism of Scriptaid-induced cell apoptosis. The cell viability, cell cycle and cell apoptosis were measured by CCK8 assay and flow cytometry respectively, the relative target gene expression levels were detected by RT-PCR, the effect of Scriptaid on p21 promoter activity was detected by using luciferase reporter assay. Scriptaid inhibited IM9 cell viability in a dose-dependent manner. Scriptaid induced IM9 cell cycle arrest at G2/M phase in a dose-dependent manner. Scriptaid triggered IM9 cell apoptosis was obviously, the mRNA levels of apoptosis-related proteins Caspase 9, Caspase 3 and PARP1 were also activated. The apoptosis-associated factors BAD, PTEN and p21 increased following treatment with different dose of Scriptaid, meanwhile, p21 promoter activity was also activated significantly. HDAC inhibitor Scriptaid can promote IM9 cell apoptosis by transcriptional activation of p21 promoter in concentration-dependent manner.

The combination of ionizing radiation and proteasomal inhibition by bortezomib enhances the expression of NKG2D ligands in multiple myeloma cells.

Bortezomib, which is a potent proteasome inhibitor, has been used as a first-line drugs to treat multiple myeloma for a few decades, and radiotherapy has frequently been applied to manage acute bone lesions in the patients. Therefore, it was necessary to investigate what the benefits might be if the two therapies were applied simultaneously in the treatment of multiple myeloma. Since it was known that radiotherapy and proteasome inhibitors could increase the expression of NKG2D ligands through induction of protein synthesis and suppression of protein degradation of NKG2D ligands, respectively, we supposed that the combined treatment might further enhance the expression of NKG2D ligands. In this study, we analyzed the expression level of NKG2D ligands using multiplex PCR and flow cytometry after treatment of IM-9 and RPMI-8226 myeloma cells with bortezomib and ionizing radiation; we then assayed the susceptibility to NK-92 cells. Although the expression of only some kinds of NKG2D ligands were increased by treatment with bortezomib alone, five kinds of NKG2D ligands that we assayed were further induced at the surface protein level after combined treatment with ionizing radiation and bortezomib. Furthermore, combined treatment made myeloma cells more susceptible to NK-92 cells, compared with treatment with bortezomib alone. In conclusion, the combination therapy of ionizing radiation plus the proteasome inhibitor bortezomib is a promising therapeutical strategy for enhancing NK cell–mediated anticancer immune responses.

Nickel(II) bis(isatin thiosemicarbazone) complexes induced apoptosis through mitochondrial signaling pathway and G0/G1 cell cycle arrest in IM-9 cells

Three novel complexes (1, 3 and 4) ligating N-substituted isatin thiosemicarbazone derivatives have been synthesized and their structural and biological characteristics have been compared with those of the known analogs (2, 5–7 and 8). In addition, the structure of the representative ligands (L1, L3 and L4) and complex (4) was confirmed by single crystal X-ray diffraction method. All the complexes (1–8) were assessed for their cytotoxic property against a panel of four human cancer cells such as HepG-2 (liver), MOLM-14 (acute monocytic leukemia), U937 (histiocytic lymphoma). and IM-9 (myeloma). Complex 4 exhibited prominent cytotoxic property against MOLM-14, U937 and IM-9 cell lines. Moreover, the results were compared with the well-known anticancer drugs like doxorubicin, cisplatin and daunorubicin. Besides, complex 4 enhanced the apoptotic cell death in IM-9 cell line and induced cell cycle arrest at G1 phase. Western blot analysis revealed the down-regulation of Bcl-2 (b-cell lymphoma-2), up-regulation of Bax (bcl-2 associated X protein), release of cytochrome c and activation of caspases-3 in IM-9 cells by complex 4. Importantly, complex 4 was not toxic to the normal Vero cell line (IC50 > 300 μM). In addition, complex 4 showed the concentration dependent cleavage of supercoiled (SC) DNA to its nicked circular (NC) form.

Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth.

Yes-associated protein 1 (YAP1) interacts with numerous transcription factors, including TEA-domain family proteins (TEAD) and p73. YAP1 is negatively regulated by the tumor suppressor Hippo pathway. In human cancers, the deregulation of the Hippo pathway and YAP1 gene amplification lead to the activation of YAP1, which induces epithelial-mesenchymal transition (EMT) and drug resistance. YAP1 inhibitors are expected to be useful in cancer therapy. On the other hand, in certain cancers, YAP1 upregulates p73-dependent gene transcription and behaves as a tumor suppressor. Moreover, as YAP1 regulates self-renewal and differentiation of tissue stem cells and plays an important role in tissue homeostasis, YAP1 activators may contribute to the regenerative medicine. With this in our mind, we screened for YAP1 activators by using human retinal pigment epithelial ARPE-19 cells expressing the TEAD-responsive fluorescence reporter under the coexpression of YAP1. From an extensive chemical compound library (n = 18,606) 47 candidate YAP1 activators were identified. These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator was effective against the human multiple myeloma IM-9 cells and chronic myeloid leukemia K562 cells.Implications: YAP1 activation limits growth, induces apoptosis, and may be useful at suppressing hematological cancers. Mol Cancer Res; 16(2); 197-211. ©2017 AACR.

Abstract A136: KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib

Abstract Purpose: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for myeloma treatment. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance remain to be understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To overcome bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant and bortezomib-sensitive IM-9 cell lines in response to bortezomib, and expressions of KDM5A genes were significantly increased. Methods: The differential gene expression profiles of bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cell lines in response to bortezomib were performed using Affymetrix GeneChip. To confirm the results, real-time PCR and Western blot analysis were performed. Bortezomib-resistant IM-9 cells were treated with or without bortezomib and&amp;lt;/&amp;gt;or histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride (2-PCPA). Moreover, level of histone methylation was examined by Western blot analysis. Results: At concentrations that effectively inhibited proteasome activity (maximum dose with 100nM), bortezomib induced cell death in bortezomib-sensitive IM-9 cells, but not in bortezomib-resistant IM-9. In comparison of differential gene expression profiles between bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cells, we showed overexpression of KDM5A, which is associated with chromatin-mediated reversible drug-tolerant state, Moreover, 2-PCPA overcomes bortezomib resistance in a dose-dependent manner. The histone demethylation at H3K4, H3K9, and H3K27 was suppressed with 2-PCPA. Conclusion: Histone lysine-specific demethylase inhibitor, 2-PCPA, with bortezomib may be useful for overcoming bortezomib resistance in myeloma cells by suppression of KDM3A and KDM5A activities. Citation Format: Yasuhito Terui, Yuji Mishima, Kiyohiko Hatake. KDM5A activity inhibition by 2-PCPA: overcoming tolerance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A136.

The MIP-1α autocrine loop contributes to decreased sensitivity to anticancer drugs.

Several autocrine soluble factors, including macrophage inflammatory protein-1α (MIP-1α), tumor necrosis factor-α, and hepatocyte growth factor, promote cell survival and growth in multiple myeloma (MM) cells. We hypothesized that inhibition of the MIP-1α autocrine loop may enhance the cytotoxic effect of anticancer drugs in MM cell lines. In the present study, an MIP-1α neutralizing antibody suppressed cell proliferation and enhanced the cytotoxic effect of melphalan or bortezomib on MM cells. In addition, melphalan resistance cells (RPMI8226/L-PAM and HS-sultan/L-PAM cells) secreted MIP-1α and neutralizing antibody of MIP-1α partially overcame melphalan resistance. Moreover, combination treatment with MIP-1α neutralizing antibody and melphalan or bortezomib inhibited extracellular signal regulated kinase 1/2 (ERK1/2), Akt, and mammalian target of rapamycin (mTOR) activation, Bcl-2, Bcl-xL, and Survivin expression, and upregulated the expression of Bim and cleaved Poly (ADP-ribose) polymerase (PARP). Treatment of IM9 cells with MIP-1α siRNA suppressed the activation of ERK1/2, Akt, and mTOR, and enhanced the cytotoxic effect of melphalan and bortezomib. These results indicate that MIP-1α neutralizing antibodies or MIP-1α siRNA enhance the cytotoxic effect of melphalan and bortezomib by suppressing the chemokine receptor/ERK and chemokine receptor/Akt/mTOR pathways. The inhibition of MIP-1α may thus provide a new therapeutic approach to control tumor progression and bone destruction in patients with MM.

Identification of cellular responses to low-dose radiation by the profiling of phosphorylated proteins in human B-lymphoblast IM-9 cells

Purpose: The aim of this study was to explore the potential for radiation-specific signaling of various LDIR-induced effects in human B-lymphoblast IM-9 cells.Materials and methods: Human lymphoblast IM-9 cells were exposed to ionizing radiation at 0.1 and 2 Gy using a 137Cs γ-irradiator at a dose rate of 0.8 Gy/min. Cell viability and DNA fragmentation were determined using MTT assay and TUNEL assay at 24 h after irradiation. Profiling of protein phosphorylation by radiation was identified using a phospho-antibody array at 4 h after irradiation and Dataset of the profiling was analyzed by IPA.Results: Cell survival and apoptotic signaling were not affected by 0.1 Gy of radiation, whereas 2 Gy induced cellular damage. The analysis of low-dose ionizing radiation (LDIR) or high-dose ionizing radiation (HDIR)-specific responses by IPA generated different results. Various cell maintenance functions were only apparent following the analysis of increased protein phosphorylation by LDIR, whereas several cancer formation- and development-related functions were only detected following the analysis of increased protein phosphorylation by HDIR.Conclusions: The LDIR-induced protein phosphorylation patterns might be involved in various cell survival responses or cellular maintenance functions, which provide important insight into our understanding of the different effects of LDIR and HDIR.

Trapping and proliferation of target cells on C60 fullerene nano fibres.

The ratio of the surface area to the volume of materials increases in inverse proportion to their size and therefore the surface area of nanostructures and nanomaterials is extremely large compared to that of macroscopic materials of the same volume, thanks to which it is supposed that chemical and biochemical reactions may be greatly enhanced and target molecules and cells may be efficiently trapped on the surface of nanomaterials. It is well known that C60 molecules are stable both physically and chemically and the affinity of C60 molecules with biomolecules is rather high. Here, we synthesise fibres composed of C60 and sulphur and immobilise the surface of the fibres with the primary antibody; i.e., epithelial cell adhesion molecules (anti-EpCAM), to trap target cells. The primary antibody is evenly immobilised on the fibres confirmed by a fluorescent secondary antibody attached to the primary one and then TE2 esophageal and DLD-1 colon cancer cells are successfully trapped by the primary antibody immobilised on the fibres thanks to its high affinity with TE2 and DLD-1 cells, whereas few IM9 B lymphoblast cells are captured on the fibres since the affinity of the primary antibody with IM9 cells is extremely low. Furthermore, those cells trapped by the primary antibody immobilised on the fibres proliferate faster than native cells thanks to the primary antibody acting as a growth factor. The present result suggests that different types of cells can be trapped and grown on nano fibres by immobilising appropriate antibody molecules on the surface of the fibres. Even an extremely small number of cells in sample fluids may be analysed and characterised for the detection of diseases such as cancer in the early stage by trapping and proliferating target cells on the fibres.

Consecutive stimulation of HBsAg promotes the viability of the human B lymphoblastoid cell line IM-9 through regulating the SIRT1-NF-κB pathway.

Patients with chronic HBV infection have been reported to suffer a significantly increased risk of NHL, but the underlying mechanisms remain to be clearly explained. The aim of the present study was to clarify the relationship between chronic HBV infection and NHL development. Fluorescence-activated cell sorting, Annexin V/7-aminoactinomycin D staining and MTS assay were used to analyze the rate of apoptosis and cell viability. In addition, western blotting was used to detect protein expression. The effects of the activator of SIRT1, SRT1720, and the inhibitor of SIRT1, nicotinamide, were also analyzed. The expression levels cytokines and chemokines were determined by multiplex assay. Hepatitis B surface antigen (HBsAg) was demonstrated to increase the viability of the human peripheral B lymphoblastoid cell line, IM-9, in a dose- and time-dependent manner. HBsAg also decreased histone H3 acetylation and p21 expression at the molecular level. HBsAg upregulated the expression of anti-apoptotic B-cell lymphoma-extra-large and B-cell lymphoma 2 proteins, and inactivated the intrinsic apoptosis pathway by reducing BCL2 associated X, apoptosis regulator expression and increasing the expression of sirtuin 1 (SIRT1) and nuclear factor-κB (NF-κB). HBsAg also altered the levels of certain chemokines and cytokines, including interleukin (IL)-4, -10 and -12, C-X-C motif chemokine 10 and C-C motif chemokine ligand 5. Inhibition of SIRT1 suppressed the effects induced by HBsAg. The anti-apoptotic effect of HBsAg in IM-9 cell lines occurred via the promotion of cell viability, inhibition of apoptosis, regulation of chemokines and cytokines, acetylation of histone H3 and alteration of SIRT1 and NF-κB expression. In conclusion, chronic stimulation with HBsAg promoted the viability of the human B lymphoblastoid cell line, IM-9, through regulation of the SIRT1-NF-κB pathway. This may be an underlying mechanism of HBV-associated NHL.