HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis

Abstract Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls. In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

Download Full-text

HOXB7 Is Critically Involved in Multiple Myeloma-Induced Angiogenic Switch.

Blood ◽

10.1182/blood.v114.22.125.125 ◽

2009 ◽

Vol 114 (22) ◽

pp. 125-125

Author(s):

Paola Storti ◽

Gaetano Donofrio ◽

Marina Bolzoni ◽

Simona Colla ◽

Irma Airoldi ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Protein Level ◽

Potential Role ◽

Tumor Mass ◽

Angiogenic Switch ◽

Angiogenic Inhibitor

Abstract Abstract 125 It is known that bone marrow (BM) angiogenesis is increased in multiple myeloma (MM) patients in relationship with disease progression and supports MM cell growth. MM-induced angiogenesis is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment. However, the molecular mechanisms at the basis of the angiogenic process are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is involved in tumor-associated angiogenic switch in solid tumors. Interestingly, we have recently shown that HOXB7 is one of the overexpressed genes in BM micorenvironment cells in MM patients as compared to healthy subjects. In this study we have investigated the expression of HOXB7 by MM cells and its potential role in MM-induced angiogenesis in vitro and in vivo. First, by microarray analysis we evaluated HOXB7 expression in MM cells finding that HOXB7 was overexpressed in 43% of 23 human myeloma cell lines (HMCLs) tested as compared to normal plasmacells. HOXB7 expression was further confirmed by real time PCR and western blot analysis. On the other hand a small fraction of patients (about 10% in a representative database of 133 MM patients) showed HOXB7 overexpression with a preferential distribution based on the characteristics of molecular MM subtypes being significantly overexpressed in those cases which did not show any of the primary IGH translocations, corresponding to the TC2 and TC3 groups in the translocations/cyclins (TC) classification. In order to investigate the potential role of HOXB7 in MM cells either we enforced HOXB7 expression by lentivirus vectors in JJN3 HMCL (JJN3-HOXB7) or we silenced its expression by appropriate siRNA in the KMS20 HMCL that constitutively over-expressed HOXB7. Interestingly we found that cell proliferation was significant higher in JJN3-HOXB7 as compared to JJN3 transduced with the empty vector (JJN3-pWPI) as well as HOXB7 downregulation in KMS20 significantly reduced their cell proliferation and viability. Following we evaluated the whole transcriptional and specifically the pro-angiogenic profile of JJN3-HOXB7 as compared to JJN3-pWPI using Genechips® HG-U133Plus 2.0 and an angiogenesis oligonucleotide arrays. We found that the pro-angiogenic cytokines as VEGFA, FGF2, MMP2 and PDGFA were significantly upregulated whereas we identified the specific downregulation of the angiogenic inhibitor TSP2 in JJN3-HOXB7. Data were then validated at protein level by ELISA assay in cell-conditioned media (CM). Consistently, we found that CM of JJN3-HOXB7 significantly stimulated vessel formation as compared to JJN3-pWPI using both in vitro angiogenic model (Angiokit) and chorioallantoic membrane assay. In line with these observations, we found that HOXB7 silencing by siRNA in KMS20 reduced the expression of pro-angiogenic molecules, including VEGF and FGF2, and growth factors. Finally the potential role of HOXB7 in MM-induced angiogenesis was tested in vivo in NOD/SCID mice. We found that mice injected with JJN3-HOXB7 cells developed a significantly bigger tumor mass than mice inoculated with the JJN3-pWPI (p=0.0039) with higher number of vessels stained by CD31 antigen. Moreover, the transcriptional and angiogenic profiles of the tumor mass indicated the upregulation of VEGFA, FGF2, MMP2, PDGFA and WNT5a as well as the downregulation of TSP2, at both mRNA and protein level in mice colonized by JJN3-HOXB7 as compared to those inoculated with JJN3-pWPI, confirming in vitro observations. In conclusion our data demonstrate the critical role of HOXB7 in the production of pro-angiogenic molecules by MM cells and in the regulation of MM-induced angiogenic switch suggesting that HOXB7 could be a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽

10.3390/cancers12092341 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2341

Author(s):

Normann Steiner ◽

Karin Jöhrer ◽

Selina Plewan ◽

Andrea Brunner-Véber ◽

Georg Göbel ◽

...

Keyword(s):

Multiple Myeloma ◽

Tyrosine Kinase ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Tyrosine Kinase Receptor ◽

Bone Marrow Biopsies ◽

Flt3 Inhibitors ◽

Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

Download Full-text

p29ING4 Regulates the Production of Pro-Angiogenic Molecules by Human Myeloma Cells in Normoxic and Hypoxic Conditions Being Involved in Myeloma-Induced Angiogenesis.

Blood ◽

10.1182/blood.v108.11.515.515 ◽

2006 ◽

Vol 108 (11) ◽

pp. 515-515

Author(s):

Sara Tagliaferri ◽

Francesca Morandi ◽

Paolo Lunghi ◽

Simona Colla ◽

Mirca Lazzaretti ◽

...

Keyword(s):

Tumor Suppressor ◽

Mrna Expression ◽

Real Time ◽

Real Time Pcr ◽

Interleukin 8 ◽

Mrna Levels ◽

Quantitative Real Time Pcr ◽

Angiogenic Switch ◽

Hypoxic Conditions

Abstract Multiple myeloma (MM) cells produce several angiogenic molecules as VEGF, Angiopoietin-1 (Ang-1), interleukin-8 (IL-8) and osteopontin (OPN), however the molecular mechanisms underlying the angiogenic switch are not completely elucidated. The candidate tumor suppressor gene inhibitor of growth family member 4 (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the suppression of angiogenic related molecules including interleukin-8 (IL-8) and the hypoxia inducible factor (HIF)-1 alpha. In this study we investigate the potential involvement of p29ING4 in the angiogenic switch in MM. First using quantitative real time PCR we compared p29ING4 with VEGF, Ang-1, IL-8 and OPN mRNA levels in eight human myeloma cell lines (HMCLs). A significantly negative correlation was observed between ING4 and IL-8 and a trend of correlation with OPN. Following we transfected HMCLs JJN3, OPM-2 and RPMI-8226 with specific siRNA to completely block the expression of p29ING4 checking the effect on the expression and production of the myeloma-related angiogenic molecules VEGF, Ang-1, IL-8 and OPN by quantitative real time PCR and ELISA assay. p29ING4 suppression in HMCLs did not affect VEGF and Ang-1 production but induced a strong up-regulation of IL-8 mRNA and IL-8 protein secretion. Similarly an induction of OPN mRNA expression as well as OPN secretion was induced by siRNA anti-p29ING4. Moreover conditioned media of HMCLs transfected with siRNA anti p29ING4 significantly increased vessel formation in an experimental in vitro model of angiogenesis (ANGIO kit) as compared to controls. Further we investigate the role of p29ING4 in the production of angiogenic molecule by MM cells in hypoxic condition compared to normoxic one as well as its potential relationship with HIF-1alpha system. Hypoxia induced HIF-1alpha expression at nuclear level and its activity in HMCLs and p29ING4 suppression by siRNA further induced HIF-1alpha transcriptional activity with an increase of its target gene Nip-3 in HMCLs. In turn the block of HIF1-alpha by specific siRNA up-regulated p29ING4 and suppressed IL-8 and OPN mRNA expression by HMCLs suggesting a relationship between p29ING4 and HIF-1alpha activity. These in vitro observations have been extended in vivo by the finding of a significant correlation between bone marrow (BM) plasma IL-8 levels and p29ING4 mRNA expression in purified MM cells obtained from 40 newly diagnosed MM patients (R=−0.58 Spearman’s 2-tailed test: p=0.04), consistently MM patients with higher BM IL-8 levels have a significantly lower p29ING4 mRNA levels. Similarly MM patients positive for OPN expression with high OPN BM levels had a significant lower p29ING4 levels (p=0.02). Finally we found that MM patients with high microvalscular density (MVD>30) have significant lower p29ING4 levels as compared to those with low MVD (<30) (p=0.04) and that MM patients with histological high grade had significant lower p29ING4 mRNA levels (Mann-Whitney 2-tailed: p=0.05). In conclusion, our data indicate that the tumor suppressor p29ING4 regulate the production of angiogenic molecules by MM cells both in normoxic and hypoxic conditions being involved in MM-induced angiogenesis and potentially in tumor progression.

Download Full-text

EGFL6 Modulates WNT Signaling to Drive Esophageal Cancer Metastasis and Proliferation

10.21203/rs.3.rs-30021/v1 ◽

2020 ◽

Author(s):

Yanhong Wang ◽

Jing Kang ◽

Jihua Tian ◽

Hongyan Jia ◽

Juanjuan Wang ◽

...

Keyword(s):

Esophageal Cancer ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Expression Level ◽

Marker Genes ◽

Invasion And Migration ◽

Ec Cells

Abstract Background Esophageal cancer (EC) is the sixth deadliest cancer in the world. There has been no breakthrough in the research on EC in the past few decades. Epidermal growth factor-like protein 6 (EGFL6), as a member of the epidermal growth factor superfamily, plays an important role in the occurrence and development of some tumors. However, the role of EGFL6 in the EC has never explored. Methods Immunohistochemical staining was used to evaluate the expression level of EGEC6 protein in human EC and its adjacent non-tumor tissues, and analyzed the correlation between the expression level of EGFL6 protein and clinical pathological indexes and survival rate. In vitro, by constructing EGFL6 silence and overexpressed EC cells,used CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry to explore the effects of EGFL6 on the proliferation, invasion, migration and apoptosis of EC. By using real-time PCR or western blot to detect the related marker genes of epithelial-mesenchymal transformation (EMT), tumor stem cells (TSCs) and Wnt/β-catenin. In vivo, established a nude mouse EC transplantation tumor model. Results The results showed that the expression level of EGFL6 in EC is significantly higher than that in adjacent non-tumor tissues, and is related to poor prognosis of patients. In vitro, CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry results show that EGFL6 overexpression can promotes proliferation, invasion and migration of EC cells and inhibits apoptosis. EGFL6 silencing inhibits proliferation, invasion and migration of EC cells and promotes apoptosis. Real-time PCR and Western-blot detection of EMT-related markers found that EGFL6 can induce EC cells EMT. Real-time PCR detection of esophageal cancer stem cell-related genes showed that EGFL6 may maintain the expression of esophageal cancer stem cell-like cell population. Western-blot detection of Wnt/β-catenin signaling marker genes showed that EGFL6 participated in the expression of Wnt/β-catenin signaling pathway. In vivo experiments found that knockout of EGFL6 could inhibit the formation of subcutaneous tumors in nude mice. Conclusion Taken together, our study identified a novel role and mechanism of EGFL6 in EC and provided epigenetic therapeutic strategies for the treatment of EC.

Download Full-text

An in Vitro Study on the Role of Angiogenesis in Iron Deficiency Induced Reactive Thrombocytosis

Blood ◽

10.1182/blood-2018-99-115378 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2450-2450

Author(s):

Pedro De Alarcon ◽

Manu Gnanamony ◽

Jessica Garcia

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Iron Deficiency ◽

Real Time ◽

Real Time Pcr ◽

Tube Formation ◽

Reactive Thrombocytosis ◽

Serum Free

Abstract Introduction: Iron deficiency (ID) is one of the recognized causes of reactive thrombocytosis in children. Factors that are commonly associated with megakaryopoiesis such as thrombopoietin (TPO), interleukin 6 (IL-6) and IL-11 are not altered in patients with iron deficiency and thrombocytosis suggesting the role of alternate mechanisms in controlling this process. We have previously shown using an ID rat model that ID increased the number of megakaryocytes in the bone marrow. We have also shown an increase in VEGFR (FLT1) and CXCR4 staining in bone marrow slides of ID rats. This data suggests that angiogenesis plays a vital role in the development of reactive thrombocytosis in response to ID. In this report, we have expanded our study to identify specific angiogenic signaling molecules associated with ID and used functional assays to validate it. Methods: For this study, we used the megakaryoblast cell line MEG-01 as an in vitro model of megakaryopoiesis. MEG-01 cells were adapted to grow in chemically defined serum free medium containing iron (iron replete media). For iron deficiency, serum free iron free media was mixed with iron replete media at a 1% v/v concentration (iron deplete media). For our experiments, MEG-01 cells were grown in both iron replete and depleted media for 7 days. Cell viability was measured using the trypan blue exclusion assay. Messenger-RNA expression of iron-related markers (TFR1, TFR2, FLT1, FLT3, FTL, FTH1, TF, HMOX1 and HMOX2) and angiogenic markers (VEGFA, VEGFB, VEGFC, PDGF, ANGPTL1, ANGPTL2, FGF2) was studied using real time PCR. We performed functional validation of angiogenesis with an in vitro tube formation assay using human umbilical vein endothelial (HUVEC) cells. For statistical analysis of the data we performed the t test using graph pad prism software and we considered p<0.05 as statistically significant. Results: In low iron conditions, MEG-01 cells showed a significant increase in FLT1 (4 fold) and FLT3 (3 fold) expression using real time PCR (p<0.001). Iron deficiency also induced a 2 fold increase in the mRNA expression of angiogenic molecules VEGFB, VEGFC, FGF2 and PDGFA (p<0.001). Using the tube formation assay, we also show that conditioned media collected from iron deficient MEG-01 cells induced increased vessel formation in endothelial cells. Conclusion: In this study, we were able to validate our earlier in vivo findings on iron deficiency induced reactive thrombocytosis. We show that cells adapt to low iron conditions by upregulating FLT1, FLT3 and FTL. We also show that several markers in the angiogenesis pathway like VEGFB, VEGFC, FGF2 and PDGFA are upregulated in response to iron deficiency. We were also able to show that an increase in these angiogenic molecules induced increased vessel formation in endothelial cells. This report, along with our previous findings, points to the importance of the angiogenic pathway in reactive thrombocytosis induced by iron deficiency. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Cardiac miRNAs were involved in the regulation of pathophysiological mechanisms underlying HF in pediatric patients after ventricular assist device implantation

European Heart Journal ◽

10.1093/eurheartj/ehab724.0941 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

R Ragusa ◽

A Di Molfetta ◽

S Del Turco ◽

G Basta ◽

M Rizzo ◽

...

Keyword(s):

Cardiac Muscle ◽

Real Time ◽

Mirna Expression ◽

Real Time Pcr ◽

In Silico ◽

In Silico Analysis ◽

Adiponectin Receptors ◽

Silico Analysis

Abstract Background VAD use in heart failure (HF) children have undergone rapid progress in the last three decades through pump technological innovation and improvement of perioperative care. Studies in HF adults showed that VAD put native heart at rest and lead to molecular changes in cardiac muscle, including at microRNA (miRNA) level. However, little is known on changes induced by VAD implant in cardiac miRNA expression and their putative targets in HF children. Purpose The aims of this study were to evaluate: 1) modification of miRNA expression in cardiac muscle from HF children after VAD support; 2) the putative targets of selected miRNAs by in silico analysis; 2) the role of the identify miRNAs on putative targets by in vitro study. Methods Cardiac biopsies were collected from HF children at the moment of VAD implant [n=8; 20 (7.5–64.5) months, 2 males; 19 (15.75–32.25) LVEF%] and at the time of heart transplant after VAD support [n=5; 32 (5–204) months; 4 males; 13.5 (10–18) LVEF%]. Cardiac miRNA expression was evaluated by NGS. The potential miRNA targets were identified by bioinformatics analyses and their cardiac expression by real-time PCR was evaluated. HL-1 cell line was used for testing the regulatory role of selected miRNA on predicted targets by miRNA mimic transfection study. Results At NGS, 465 miRNA were found on average in each sample and the cardiac expression levels of miR19a-3p, miR-1246 and miR-199b-5p decreased in HF children after VAD support compared to pre-implant (Fig. 1A-B). In silico analysis showed that more than 5000 potential gene targets regulated by miR-19a-3p, miR-1246 and miR-199b-5p. Among them, adiponectin receptors (AdipoR1, AdipoR2, T-CAD) were identified as common targets for 3 miRNAs. Real-time PCR data showed that levels of all adiponectin receptors increased significantly whilst the expression of 3 miRNAs decreased after VAD support (Fig. 1C). Moreover, AdipoR2 and T-CAD were inversely related to miRNA levels (Fig. 1D). In vitro studies confirmed the regulatory role of miR-1246 and miR-199b-5p on AdipoR2 (Fig. 1E-F), whilst only miR-199b-5p reduced the expression of T-CAD (Fig. 1G). Finally, AdipoR1 expression levels are not modified compared to control by miRNAs mimic transfection (data not shown). Conclusion In HF children the use of VAD could modify the expression of several miRNAs potentially involved in the regulation of several pathophysiological mechanisms underlying HF. Specifically, the reductions of miR-1246, mir-19a-3p, miR-199b-5p were associated with an increase of the adiponectin receptors AdipoR2 and T-CAD mRNA, suggesting the existence of a miRNAs related fine tuning of the adiponectin system at cardiac tissue level by VAD implant, able to favour the protective effect of adiponectin in HF cardiac muscle. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): FP7-ICT-2009 Project, Grant Agreement 24863 Figure 1

Download Full-text

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽

10.1182/blood.v106.11.5078.5078 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5078-5078

Author(s):

Timothy J. Molloy ◽

Baulch-Brown Cindy ◽

Yi-Mo Deng ◽

Andrew Spencer ◽

David F. Ma

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Real Time ◽

Real Time Pcr ◽

Mevalonate Pathway ◽

Annexin V ◽

Quantitative Real Time Pcr ◽

Myeloma Cells ◽

Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

Download Full-text

Biological evaluation of transdichloridoplatinum( II) complexes with 3- and 4-acetylpyridine in comparison to cisplatin

Radiology and Oncology ◽

10.2478/raon-2013-0050 ◽

2013 ◽

Vol 47 (4) ◽

pp. 346-357 ◽

Cited By ~ 4

Author(s):

Lana Filipovic ◽

Sandra Arandelovic ◽

Nevenka Gligorijevic ◽

Ana Krivokuca ◽

Radmila Jankovic ◽

...

Keyword(s):

Western Blot ◽

Mrna Expression ◽

Real Time ◽

Real Time Pcr ◽

Inductively Coupled Plasma ◽

Gelatin Zymography ◽

Biological Evaluation ◽

Emission Spectrometry ◽

Quantitative Real Time Pcr

Abstract Background. In our previous study we reported the synthesis and cytotoxicity of two trans-platinum(II) complexes: trans-[PtCl2(3-acetylpyridine)2] (1) and trans-[PtCl2(4-acetylpyridine)2] (2), revealing significant cytotoxic potential of 2. In order to evaluate the mechanism underlying biological activity of both trans-Pt(II) isomers, comparative studies versus cisplatin were performed in HeLa, MRC-5 and MS1 cells. Materials and methods. The cytotoxic activity of the investigated complexes was determined using SRB assay. The colagenolytic activity was determined using gelatin zymography, while the effect of platinum complexes on matrix metalloproteinases 2 and 9 mRNA expression was evaluated by quantitative real-time PCR. Apoptotic potential and cell cycle alterations were determined by FACS analyses. Western blot analysis was used to evaluate the effect on expression of DNA-repair enzyme ERCC1, and quantitative real-time PCR was used for the ERCC1 mRNA expression analysis. In vitro antiangiogenic potential was determined by tube formation assay. Platinum content in intracellular DNA and proteins was determined by inductively coupled plasma-optical emission spectrometry. Results. Compound 2 displayed an apparent cytoselective profile, and flow cytometry analysis in HeLa cells indicated that 2 exerted antiproliferative effect through apoptosis induction, while 1 induced both apoptosis and necrosis. Action of 1 and 2, as analyzed by quantitative real-time PCR and Western blot, was associated with down-regulation of ERCC1. Both trans-complexes inhibited MMP-9 mRNA expression in HeLa, while 2 significantly abrogated in vitro tubulogenesis in MS1 cells. Conclusions. The ability of 2 to induce multiple and selective in vitro cytotoxic effects encourages further investigations of trans-platinum(II) complexes with substituted pyridines.

Download Full-text

Osteocytes and Weightlessness

Current Osteoporosis Reports ◽

10.1007/s11914-021-00713-8 ◽

2021 ◽

Author(s):

Donata Iandolo ◽

Maura Strigini ◽

Alain Guignandon ◽

Laurence Vico

Keyword(s):

Shear Stress ◽

Fluid Flow ◽

Simulated Microgravity ◽

Potential Role ◽

Signalling Molecules ◽

Stress Changes ◽

Bone Biopsies ◽

Microgravity Conditions

Abstract Purpose of Review Osteocytes are considered to be the cells responsible for mastering the remodeling process that follows the exposure to unloading conditions. Given the invasiveness of bone biopsies in humans, both rodents and in vitro culture systems are largely adopted as models for studies in space missions or in simulated microgravity conditions models on Earth. Recent Findings After a brief recall of the main changes in bone mass and osteoclastic and osteoblastic activities in space-related models, this review focuses on the potential role of osteocytes in directing these changes. The role of the best-known signalling molecules is questioned, in particular in relation to osteocyte apoptosis. Summary The mechanotransduction actors identified in spatial conditions and the problems related to fluid flow and shear stress changes, probably enhanced by the alteration in fluid flow and lack of convection during spaceflight, are recalled and discussed.

Download Full-text

Expression and Role of APP Gene in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.4691.4691 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4691-4691

Author(s):

Fanyi Meng ◽

Wei Wang ◽

Zoufang Huang ◽

Ming Huang ◽

Lixiang Liu

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Western Blot ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Myeloid Leukemia ◽

App Gene ◽

Acute Myeloid

Abstract Abstract 4691 Introduction Amyloid precursor protein(APP) gene was increasingly expressed in solid tumors, promoted the proliferation of tumor cells and the overexpression of APP was a bad prognostic factor to oral squamous cell carcinoma. However, little has been known about the clinical significance and role of APP in acute myeloid leukemia(AML). Methods The expressions of APP mRNA in 85 AML patients and 20 nonmalignant hematological diseases that worked as control were measured by real-time PCR and the expressions of APP in AML cell lines were examined by real-time PCR and western blot. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL60 cell by lipofectamine2000, after RNAi 24h, 48h and 72h, cell growth of HL60 was measured by trypan blue dye exclusion method and MTT, differentiation was observed by Wright-Giemsa staining, cell cycle was examined by PI/RNase staining, apoptosis induction was analyzed by Annexin V/PI and Hoechst33342 staining; apoptosis-related proteins NF-κB, bcl-2 and Caspase-3 were detected by Western blot after RNAi 48h; sensitivity of HL60 to adrimycin was measured by MTT. Results The expression of APP mRNA among AML subtypes was significantly different(P=0.019), M2 with t(8;21) was the highest expression subtype and M5b was the lowest. APP expression had no significant effect on AML clinical characteristic excepting AML subtypes. kasumi-1 was the highest expression cell in AML cell lines and U937 was the lowest(P<0.05), and the expression of APP in HL60/ADM was significantly lower than HL60(P<0.05). The APP expressions in AML cell lines was in agreement with its expressions in primary AML subtypes. After RNAi 24h, 48h, and 72h, no significant differences in proliferation, differentiation, apoptosis, cell cycle and sensitivity of HL60 to adriamycin were detected between interfering group and control groups. Conclusions The APP mRNA expression in M2 with t(8;21) was high and M5b was low. Down-regulation of APP expression had no significant effect on biological behaviour of HL60 and APP was not tightly related to pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text