Glucocorticoid Therapy Targets Proliferation, Differentiation and Bcl-2 Dependent Survival Signaling in ALL Blasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1841-1841
Author(s):  
Peter Rhein ◽  
Stefanie Scheid ◽  
Richard Ratei ◽  
Christian Hagemeier ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Induction Therapy ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Apoptotic Pathway ◽  
Therapy Targets ◽  
Cell Cycling ◽  
Induced Apoptosis

Abstract In the multicentric ALL-BFM (Berlin-Frankfurt-Munster) study, all patients are uniformly treated during the first week of induction therapy which uses glucocorticoids (GC) as the principal therapeutic agent. The GC response assessed at day 8 of therapy provides one of the basic parameters for further risk stratification. In spite of the clinical significance, molecular mechanisms of GC action in vivo are largely unknown. Our recent genome-wide analysis of gene expression in blasts persisting during induction therapy identified a common set of genes differentially expressed in blasts at day 8 (d8) and at diagnosis (d0) (n=457, false discovery rate <0.05). Expression changes indicated therapy-induced inhibition of cell cycling, expression shift towards normal mature B cells and downregulation of the apoptosis regulator Bcl-2. In the current study, we validated the key differences between d8 and d0 blasts at protein and cellular levels. DNA distribution and percentage of cycling blasts was determined by flow cytometry in a series of matched d8 and d0 samples (13 pts) and demonstrated the decreased proliferative activity of d8 cells (4.3-fold, p=0.014). Protein expression, investigated by flow cytometry in a total of 84 pts, demonstrated statistically significant expression decrease of the progenitor cell antigenes CD10, CD34 and TdT and expression increase of the B-cell antigene CD20 and the inflammatory response molecules CD11b and IFNGR1 (p<0.05). We were also able to confirm the lower expression values of the Bcl-2 protein in d8 blasts (p<0.05, n=57). Investigation of GC-sensitive B-lineage leukemia cell lines demonstrated that BCL-2 downregulation by GC was a pre-requisite of GC-induced apoptosis. Moreover, we found a considerable cross-correlation between viability, cell cycling and Bcl-2 expression levels. Upregulation of the Bcl-2 expression via IL-7 receptor signaling prevented GC-induced apoptosis in these cell lines. Collectively, GC therapy interferes with differentiation and proliferation programs in leukemic blasts which are closely related to the Bcl-2 specific apoptotic pathway.

Mechanisms of Apoptosis Induction by BH3 Inhibitor ABT-737 in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 244-244
Author(s):  
Michael Andreeff ◽  
Rooha Contractor ◽  
Peter P. Ruvolo ◽  
Xingming Deng ◽  
Ismael Samudio ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phosphorylation Site ◽  
Leukemia Cell ◽  
Mek Inhibitor ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
In Cells

Abstract Bcl2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus, strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). In this report, we investigate mechanisms of action of the novel small molecule Bcl2 inhibitor ABT-737 in AML. ABT-737 effectively killed AML patient blast cells and colony-forming cell lines at nanomolar concentrations with no effect on normal hematopoietic cells. Notably, CD34+38−123+ AML stem cells are highly sensitive to the compound. ABT-737-induced apoptosis is initiated by disruption of Bcl2:Bax dimers and activation of the intrinsic apoptotic pathway. ABT-737 works synergistically with chemotherapeutic agents such as ara-C and doxorubicin. To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50&gt;500 nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25 nM). Consistent with a mechanism whereby increased Bcl2 phosphorylation impedes ABT-737 suppression of Bcl2 dimerization with Bax, ABT-737 potently blocked Bcl2:Bax association in cells expressing exogenous WT Bcl2 and AAA mutant Bcl2 but not in cells expressing exogenous phosphomimetic EEE mutant Bcl2. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.08±0.003. OCI-AML3 cells exhibit the highest expression of Mcl-1 among the acute leukemia cell lines tested. We propose that loss of Mcl-1 expression as a result of suppression of ERK may also be involved in the ability of PD98059 to enhance ABT-737-induced apoptosis. siRNA to Mcl-1 strikingly sensitized OCI-AML3 cells to ABT-induced apoptosis (14% apoptosis in parental cells at 2.5μM ABT-737, 64% apoptosis in siRNA-transfected cells at 10-fold lower concentration of 0.25μM). We have further demonstrated that ABT-737 reduced leukemia burden and significantly (p=0.0018) prolonged survival of mice in an in vivo mouse model. These findings suggest that: 1) ABT-737 reduces apoptosis through disruption of Bcl2:Bax heterodimers; 2) its activity is limited by Bcl2 phosphorylation and Mcl-1 overexpression; 3) combination with MEK inhibition results in inhibition of Bcl2 phosphorylation, downregulation of Mcl-1 and dramatic enhancement of ABT-737-induced apoptosis in AML.

scholarly journals Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines

2019 ◽  
Vol 20 (22) ◽  
pp. 5567
Author(s):  
Jan Torben Schille ◽  
Ingo Nolte ◽  
Eva-Maria Packeiser ◽  
Laura Wiesner ◽  
Jens Ingo Hein ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
In Vivo Studies ◽  
Mediated Effects ◽  
Multinucleated Cells ◽  
Prostate Carcinoma Cell ◽  
Castrate Resistant ◽  
Induced Apoptosis

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.

TGF-β Neutralizing Antibody 1D11 Inhibits LIF-JAK-Stat3 Signaling and Enhances Cytarabine Induced Apoptosis in AML Cells in Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 926-926
Author(s):  
Yoko Tabe ◽  
Yuexi Shi ◽  
Zhihong Zeng ◽  
Linhua Jin ◽  
Yixin Zhou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Neutralizing Antibody ◽  
Leukemia Cells ◽  
U937 Cells ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 926 We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al.,Br J Haematol.2008). Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of leukemic BM microenvironment that promote leukemia cell survival and chemoresistance. Our preliminary data indicate that TGF-β neutralizing antibody 1D11 (Genzyme) prevents accumulation of AML cells in a quiescent G0 state under co-culture condition with BM-derived mesenchymal stromal cells (MSC) (Jin et al., ASH abstract 2010). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG1 fraction, 21% O2: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O2: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G0/G1 phase (21% O2; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O2; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p = 0.03). The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, ×107 photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003). We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells. In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials. Disclosures: Konopleva: Genzyme: Research Funding.

scholarly journals Small Molecule Kinase Inhibitor Sunitinib Specifically Targets Juvenile Myelomonocytic Leukemia Cells with SHP2(PTPN11) Mutation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3353-3353
Author(s):  
Chunxiao He ◽  
Yuming Zhao ◽  
Junbin Huang ◽  
Yao Guo ◽  
Hongman Xue ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Drug Screening ◽  
Leukemia Cell ◽  
Juvenile Myelomonocytic Leukemia ◽  
Oncogenic Signaling ◽  
Leukemia Cell Lines

Abstract Juvenile myelomonocytic leukemia (JMML) is a highly fatal malignant disease in early childhood. It is still unknown of the specific pathogenesis, and there is shortage of effective targeted therapeutic approaches. Gain of function SHP2 mutation encoded by PTPN11 gene is found in approximately 35% of JMML patients, which maybe contributed to its pathogenesis. JMML patients with SHP2 mutation have lower survival rate and higher recurrence rate. All of the above make development of new therapies imperative. Currently, there is no stable cell line that can accurately reflect the characteristics of JMML abnormal cells for research on JMML. In this study, we established two leukemia cell lines that depend on mutated SHP2 for survival, and discovered promising drugs that targeted mutated-SHP2-dependent oncogenic signaling pathway through drug screening method. HCD-57 cells are murine erythroleukemia cells that solely depend on exogenic erythropoietin (EPO) for survival. We constructed SHP2-D61Y and SHP2-E76K transformed HCD-57 cell lines through retroviral vectors, the survival of which dependent on mutated SHP2 mediated signaling pathway. Based on these cells, we established a drug screening platform and screened small molecule compound library containing 2862 FDA-approved drugs and 1707 kinase inhibitors. We performed cell viability, flow cytometry, Wright-Giemsa staining, and western blot to evaluate cells after drug treatment. To further assess therapeutic potential, we established in-vivo transplantation model that SHP2-D61Y transformed HCD-57 cells were implanted into immunodeficient NCG mice, and verified the effectiveness of the in-vitro screened drugs. We found that the survival and proliferation of HCD-57 cells transduced by SHP2-D61Y and SHP2-E76K no longer required EPO, but completely relied on the abnormal activation of signaling pathway mediated by mutated SHP2. Western blot results showed that the phosphorylation status of ERK1/2 and AKT of HCD-57 cells expressing SHP2 mutation were abnormally increased, consistent with SHP2-mutated JMML. Thus, we have obtained the leukemia cell lines that can represent the characteristics of activated signaling pathway in JMML with SHP2 mutation. Through drug screening, we observed that drug sunitinib (Sutent ®) selectively inhibits SHP2-mutated HCD-57 cell lines. CCK-8-based cell viability assay demonstrated a dose-dependent inhibition of SHP2-D61Y and SHP2-E76K transformed HCD-57 cell and no effects on the parental HCD-57 cells. Live cell counting with trypan blue revealed that the proliferation of SHP2-mutated HCD-57 cells was totally halted after one day upon treatment with 250 nM sunitinib, whereas the HCD-57 cells were unaffected. Wright-Giemsa staining demonstrated that SHP2-mutated HCD-57 cells showed no normal morphology change and no mitotic activity under sunitinib treatment, otherwise parental HCD-57 cells showed normal mitotic activity. Sunitinib induced apoptosis and cell cycle arrest at G1 phase in SHP2-mutated HCD-57 cells by flow cytometry, but had little effect on the parental HCD-57 cells. Sunitinib effectively downregulates the phosphorylation of ERK and AKT in SHP2-mutated cells, revealing the mechanism of sunitinib targeting SHP2-mutated cells. In addition, after transplantation of SHP2-D61Y transformed HCD-57 cells for 3 weeks, the spleen of NCG mice increased from an average of 45 mg to more than 300 mg; flow cytometry analysis showed that the implanted cells accounted for over 75% of the total nucleated cells in the bone marrow and spleen. Compared with the vehicle control, the number of monocytes in these mice was reduced to the normal range by treatment with sunitinib, and the spleen weights were reduced by about 50%. Histochemical staining showed disappearance of the myeloid infiltration in the spleen, liver and bone marrow. The above results all indicate that sunitinib has strong in-vivo anti-leukemia activity. Furthermore, western blot analysis showed that the administration of sunitinib significantly inhibited the phosphorylation expression level of AKT and ERK, indicating the effectivity of sunitinib in vivo. In conclusion, our data demonstrated that HCD-57 cell line is an effective tool for studying oncogenic signaling pathway and screening drugs that targeted JMML with SHP2 mutation. Sunitinib can be an effective drug for the targeted treatment of JMML in the future. Disclosures No relevant conflicts of interest to declare.

Perifosine, an Oral Bioactive Novel Akt Inhibitor, Induces In Vitro and In Vivo Antitumor Activity in Waldenstrom Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2488-2488 ◽  
Author(s):  
Xavier Leleu ◽  
Xiaoying Jia ◽  
Anne-Sophie Moreau ◽  
Evdoxia Hatjiharisi ◽  
Hai Ngo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antitumor Activity ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Akt Pathway ◽  
Low Grade ◽  
Akt Inhibitor ◽  
Induced Apoptosis

Abstract Background: Waldenstrom’s Macroglobulinemia (WM) is a low-grade lymphoplasmacytic lymphoma with limited options of therapy. The PI3k/Akt pathway is a critical regulator of cell survival. Our previous studies using proteomic analysis have demonstrated upregulation of members of the PI3k/Akt pathway in WM. We examined whether the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) induces cytotoxicity in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Primary CD19+ malignant cells were obtained from patients after informed consent. Inhibition of proliferation was measured using the MTT assay; DNA synthesis was measured using the thymidine uptake assay and apoptosis using Apo2.7 flow cytometry. Bone marrow stromal cells (BMSC) confer growth and resistance to conventional treatments. We therefore, tested the effect of perifosine on WM cells co-cultured with BMSC. Immunoblotting for signaling pathways was performed at different time (30 minutes to 16 hrs) and doses of therapy. In vivo activity of perifosine was assessed using a SCID-irradiated model with subcutaneous tumors in which perifosine was administered by oral gavage daily (35 mg/kg/day). A two-sided t-test was used to determine statistical differences. Results: Perifosine inhibited phosphorylation of Akt in a dose- and time- dependent fashion, as well as downstream GSK3a/b and ribosomal phospho-S6. Perifosine inhibited Akt activity as confirmed by Akt kinase assay. Perifosine induced significant cytotoxicity and inhibition of DNA synthesis with an IC50 of 5-20uM in all cell lines tested. Similar effects were observed in primary CD19+ patient WM cells. Perifosine induced apoptosis in WM cells as demonstrated by flow cytometry. The mechanism of apoptosis induced by perifosine was through activation of SAPK/JNK pathway, followed by caspase-8, -9 and PARP cleavage. The JNK inhibitor SP600125 abrogated perifosine-induced apoptosis. The growth inhibitory effects of perifosine were significant even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, perifosine did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells or in hematopietic stem cells in a methylcellulose colony forming cell assay, indicating lack of toxicity on normal cells. Interestingly, MAPK members such as MEK/ERK were activated by perifosine. The MEK inhibitor U0126 significantly enhanced perifosine-induced cytotoxicity in WM cells, indicating that this combination may be synergistic in vivo. Finally, perifosine induced significant reduction in WM tumor growth in vivo, as compared to control cohort treated with vehicle only at week 6 (p=0.05). Conclusion: Perifosine has significant antitumor activity in WM both in vitro and in vivo. These results provide the framework for clinical evaluation of perifosine in WM. Supported in part by the Leukemia and Lymphoma Society, the Lymphoma Research Foundation and an American Society of Hematology Scholar Award. * XL and XJ are co-first authors.

CXCR4 Promotes the Tumorogenicity of Multiple Myeloma, Including Increased Motility, Clonogenicity, up-Regulation of VLA-4, Protection From Chemotherapy and Aggressive Tumor Development In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1801-1801
Author(s):  
Katia Beider ◽  
Amnon Peled ◽  
Lola Weiss ◽  
Merav Leiba ◽  
Avichai Shimoni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Tumor Development ◽  
Soft Agar ◽  
Induced Apoptosis

Abstract Abstract 1801 Background: Multiple myeloma (MM) is by large incurable neoplasm of plasma cells, characterized by accumulation in the bone marrow (BM), in close contact to cellular and extracellular matrix (ECM) components. Chemokine receptor CXCR4 is expressed by the majority of patients' MM cells. It promotes myeloma cell migration and homing to the BM compartment, supports the tumor cells survival and protects the myeloma cells from chemotherapy-induced apoptosis. Further investigation is required to define the specific molecular mechanisms regulated by the CXCR4/CXCL12 axis in MM. However, surface CXCR4 is commonly down-regulated in the MM cell lines. In order to overcome this limitation, the aim of the current study was to produce a reliable model for studying the functional role of high CXCR4 in MM by generating MM cell lines with stable expression of surface CXCR4. Results: To over-express CXCR4, we transduced CXCL12-expressing MM cell lines ARH77 and RPMI8226 with lentiviral vector and generated cell lines with high and stable levels of surface CXCR4. Enhanced CXCR4 expression significantly increased the in vitro survival and growth of the 2 MM cell lines in serum-deprivation conditions (p<0.01). Furthermore, elevated expression of surface CXCR4 prominently increased MM cells motility and promoted CXCL12-dependent transwell migration of the transduced MM cell lines. Highly CXCR4-expressing RPMI8226 and ARH77 cells demonstrated 40% migration in response to CXCL12 (50 ng/ml), versus only 0–5% migration of MM cells with low expression of surface CXCR4 (p<0.01). Furthermore, adhesion of MM cells to either ECM proteins or BMSCs localize the malignant PCs within the BM microenvironment, promote growth and survival of MM cells and play a critical role in myeloma bone disease and tumor invasion. In accordance, we observed induced adhesion of the transfected RPMI8226-CXCR4 cells to ECM components fibronectin and laminin and to BM fibroblasts. Moreover, we found that enhanced CXCR4 not only functionally activates, but rather significantly elevates the surface levels of VLA-4 integrin on the RPMI8226 cells. In addition, we found that CXCR4-expressing MM cells were less sensitive to melphalan- and bortezomib-induced apoptosis, when they were co-cultured with BM fibroblasts. Testing the molecular signaling pathways regulated by CXCR4, we found that elevated CXCR4 increased the basic level of pERK1/2 and pAKT in the MM cells, and promoted their prolonged activation in response to CXCL12 stimulation. Finally, the ability to produce colonies in the soft agar semi-solid culture reflects the tumorigenic capacity of cancer cells and cancer stem cells. Differentiated MM cells thus rarely produce colonies in soft agar. Here, we demonstrate that up regulation of CXCR4 promoted ARH77 and RPMI8226 colony formation, significantly increasing colonies number and size. Lastly, we determined the role of CXCR4 in MM tumor development in vivo. CXCR4-expressing ARH77 and RPMI8226 cells were subcutaneously injected into NOD/SCID mice. CXCR4-expressing cells, but not parental cell lines, produced detectable tumors already 10 days after the injection. Rapid tumor growth was further observed in both CXCR4-expressing cell lines. These findings indicate that CXCR4 provided aggressive phenotype and supported MM growth in vivo. Conclusions: Taken together, our findings clearly demonstrate the important pathophysiologic role of CXCR4 in MM development and progression. Furthermore, for the first time, we provide the evidence for CXCR4 oncogenic potential in MM, showing that CXCR4 promotes the clonogenic growth of MM cells. Our model may further serve to elucidate CXCR4-regulated molecular events potentially involved in the pathogenesis of MM, and strongly support targeting CXCR4 as therapeutic tool in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lp16-PSP, A Member of YjgF/YER057c/UK114 Protein Family Induces Apoptosis and p21WAF1/CIP1 Mediated G1 Cell Cycle Arrest in Human Acute Promyelocytic Leukemia (APL) HL-60 Cells

2017 ◽  
Author(s):  
Thomson Patrick Joseph ◽  
Warren Chanda ◽  
Abdullah Faqeer Mohammad ◽  
Sadia Kanwal ◽  
Samana Batool ◽  
...  
Keyword(s):  
Cell Lines ◽  
Lentinula Edodes ◽  
Colorimetric Assay ◽  
Leukemia Cell ◽  
G1 Arrest ◽  
Dependent Manner ◽  
In Vivo Models ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Lp16-PSP from Lentinula edodes strain C91-3 has been reported previously in our laboratory to have selective cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to characterize the Lp16-PSP-induced cell death using HL-60 as model cancer. The results of phase contrast microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the activation of caspase-8, -9 and -3 cascade highlighting the involvement of Fas/FasL-related pathway. Whereas, western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic pathway might be involved in Lp16-PSP-induced apoptosis either. Low doses of Lp16-PSP resulted in the anchorage-independent growth inhibition, induction of G1 phase arrest accompanied by the increased expression of p21WAF1/CIP1 along with the decreased expression of cyclin D, E, and cdk6. Our findings suggest that induction of apoptosis and p21WAF1/CIP1 mediated G1 arrest might be one of the mechanisms of the action of Lp16-PSP, however, further investigations on multiple leukemia cell lines and in vivo models are of ultimate need.

scholarly journals Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Simona Laurino ◽  
Pellegrino Mazzone ◽  
Vitalba Ruggieri ◽  
Pietro Zoppoli ◽  
Giovanni Calice ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Inverse Correlation ◽  
Gastric Cancer Cells ◽  
Aberrant Expression ◽  
Cisplatin Sensitivity ◽  
Induced Apoptosis

Gastric cancer (GC) is characterized by poor efficacy and modest clinical impact of current therapies, in which apoptosis evasion is relevant. Intracellular calcium homeostasis dysregulation is associated with apoptosis escaping, and aberrant expression of calcium regulator genes could promote GC drug resistance. Since we previously found a prognostic value for TRPV2 calcium channel expression in GC, we aimed to characterize the role of TRPV2 in cisplatin resistance. Using the TCGA-STAD dataset, we performed a differential gene expression analysis between GC samples in upper and lower tertiles of TRPV2 expression, and then through a gene set analysis, we highlighted the enriched ontology and canonical pathways. We used qRT-PCR to assess TRPV2 expression in three GC cell lines and flow cytometry to evaluate cisplatin-induced cell death rates. Calcium green-1-AM assay was used to estimate differences in intracellular Ca2+ concentrations after inhibition of TRPV2. We engineered AGS cell line to overexpress TRPV2 and used confocal microscopy to quantify its overexpression and localization and flow cytometry to evaluate their sensitivity to cisplatin. Consistent with our hypothesis, among enriched gene sets, we found a significant number of those involved in the regulation of apoptosis. Subsequently, we found an inverse correlation between TRPV2 expression and sensitivity to cisplatin in GC cell lines. Moreover, we demonstrated that inhibition of TRPV2 activity by tranilast blocks the efflux of Ca2+ ions and, in combination with cisplatin, induced a significant increase of apoptotic cells (p = 0.004). We also demonstrated that TRPV2 exogenous expression confers a drug-resistant phenotype, and that tranilast is able to revert this phenotype, restoring cisplatin sensitivity. Our findings consistently suggested that TRPV2 could be a potential target for overcoming cisplatin resistance by promoting apoptosis. Notably, our data are a prerequisite for the potential reposition of tranilast to the treatment of GC patients and anticipate the in vivo evaluation.

scholarly journals Apoptin Causes Apoptosis in HepG-2 Cells by Inducing Stress Injury in the Endoplasmic Reticulum

2021 ◽  
Author(s):  
Yiquan Li ◽  
Bing Bai ◽  
Chao Shang ◽  
Jianan Cong ◽  
Jinbo Fang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Endoplasmic Reticulum ◽  
Fluorescence Staining ◽  
Apoptotic Pathway ◽  
Mitochondrial Apoptotic Pathway ◽  
Expression Levels ◽  
Mitochondrial Injury ◽  
Induced Apoptosis

Abstract Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. In our previous study, we demonstrated that Apoptin induced significant changes in the expression levels of endoplasmic reticulum stress (ERS) related proteins and caused a strong and lasting ERS response. The aim of this study was to explore the effects of ERS injury induced by Apoptin on the endoplasmic reticulum (ER) and the apoptotic pathway in mitochondria. ERS injury induced the intracellular levels of calcium (Ca2+) were determined by electron microscopy, flow cytometry and fluorescence staining. Mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. The relationship between Ca2+ level and mitochondrial injury on Apoptin-treating cells was analyzed using Ca2+ chelator, flow cytometry and fluorescence staining. Western blotting was used to investigate the levels of key proteins in the ER and the apoptotic pathway in mitochondria. We also investigated the in vivo effects of ERS injury on the ER and the mitochondrial apoptotic pathway via the immunohistochemical analysis of tumor tissues from HepG-2 cells acquired from nude mice undergoing xenografts. In vitro and in vivo experiments showed that Apoptin caused ERS injury and an imbalance in Ca2+, damaged the structure of the mitochondria, and increased the expression levels of Caspase-12, CHOP, AIF, HtrA2, Smac/Diablo, and Cyto-C. In summary, Apoptin induced apoptosis in HepG-2 cells via ERS and the mitochondrial apoptotic pathway. This study showed that Apoptin induced apoptosis in HepG-2 cells via ERS injury.

scholarly journals miRNA-193a-3p Regulates the AKT2 Pathway to Inhibit the Growth and Promote the Apoptosis of Glioma Cells by Targeting ALKBH5

2021 ◽  
Vol 11 ◽  
Author(s):  
Yong Cui ◽  
Qi Wang ◽  
Jing Lin ◽  
Lei Zhang ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Antitumor Effects ◽  
Induced Apoptosis ◽  
U87 Cells

Emerging evidence indicates that microRNA (miR)-193a-3p is involved in the tumor progression of various cancers. However, the biological functions and precise molecular mechanisms of miR-193a-3p in gliomas have not been well documented. Accordingly, this study focused on the tumor suppressor role and molecular mechanisms of miR-193a-3p in glioma cells. miR-193a-3p expression was determined by qRT-PCR in glioma tissues and cell lines. U251 and U87 glioma cells were transfected with a miR-193a-3p mimic. The effects of miR-193a-3p on cell growth and apoptosis were investigated using MTT, colony-forming, and flow cytometry assays. Overexpression of miR-193a-3p in U87 cells also significantly suppressed tumorigenicity and induced apoptosis in the xenograft mouse model. Luciferase assays were conducted to determine if ALKBH5 is a direct target of miR-193a-3p in glioma cells. Immunoprecipitation was used to explore the interaction between ALKBH5 and RAC-serine/threonine-protein kinase 2 (AKT2) in glioma cells. miR-193a-3p was downregulated in glioma tissues and cell lines. miR-193a-3p treatment suppressed proliferation and promoted apoptosis in both U251 and U87 cells. Bioinformatics analysis and luciferase reporter assay identified a novel miR-193a-3p target, ALKBH5. Notably, the antitumor effect of miR-193a-3p transfection in glioma cells may be due to the miR-193a-3p–induced inhibition of AKT2 expression caused by the suppression of ALKBH5 expression. Furthermore, immunoprecipitation indicated that ALKBH5 physically interacted with AKT2 through an RNA-independent mechanism in glioma cells. miR-193a-3p directly targets ALKBH5 to inhibit the growth and promote the apoptosis of glioma cells by suppressing the AKT2 pathway both in vitro and in vivo, and the physical interaction between ALKBH5 and AKT2 is essential for suppressing cell apoptosis by upregulating miR-193a-3p in glioma cells. Our study revealed that the antitumor effects of miR-193a-3p on glioma cells is due to ALKBH5 mediation of the AKT2-induced intrinsic apoptosis signaling pathway.

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