scholarly journals Anti-tumor effects of rivoceranib against canine melanoma and mammary gland tumour in vitro and in vivo mouse xenograft models

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Qiang Li ◽  
You-Seok Kim ◽  
Ju-Hyun An ◽  
Jin-Ah Kwon ◽  
Sang-Hyun Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammary Gland ◽  
Cell Line ◽  
Mouse Xenograft ◽  
Xenograft Models ◽  
And Migration ◽  
Canine Melanoma ◽  
Gland Tumour

Abstract Background Rivoceranib, a novel tyrosine kinase inhibitor, exhibits anti-tumour effects by selectively blocking vascular endothelial growth factor receptor-2 (VEGFR2) in cancer cells. Recently, the therapeutic effects of rivoceranib on solid tumours have been elucidated in human patients. However, the anti-tumour effects of rivoceranib against canine cancer remain unclear. Here, we investigated the anti-tumour effects of rivoceranib using in vitro and in vivo mouse xenograft models. Methods We performed cell proliferation, cell cycle, and migration assays to determine the effects of rivoceranib on canine solid tumour cell lines in vitro. Furthermore, apoptosis and angiogenesis in tumour tissues were examined using a TUNEL assay and immunohistochemistry methods with an anti-cluster of differentiation-31 antibody, respectively. Additionally, the expression levels of cyclin-D1 and VEGFR2 activity were determined using western blot analysis. Results Rivoceranib treatment showed anti-proliferative effects and mediated cell cycle arrest in the canine melanoma cell line (LMeC) and the mammary gland tumour (MGT) cell line (CHMp). In animal experiments, rivoceranib decreased the average volume of LMeC cells compared to that following control treatment, and similar results were observed in CHMp cells. Histologically, rivoceranib induced apoptosis and exerted an anti-angiogenic effect in tumour tissues. It also downregulated the expression of cyclin-D1 and inhibited VEGFR2 activity. Conclusion Our results show that rivoceranib inhibits proliferation and migration of tumour cells. These findings support the potential application of rivoceranib as a novel chemotherapeutic strategy for canine melanoma and MGTs.

scholarly journals Positive feedback between lncRNA FLVCR1-AS1 and KLF10 may inhibit pancreatic cancer progression via the PTEN/AKT pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jiewei Lin ◽  
Shuyu Zhai ◽  
Siyi Zou ◽  
Zhiwei Xu ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Positive Feedback ◽  
Molecular Mechanisms ◽  
Tumor Tissues ◽  
And Migration

Abstract Background FLVCR1-AS1 is a key regulator of cancer progression. However, the biological functions and underlying molecular mechanisms of pancreatic cancer (PC) remain unknown. Methods FLVCR1-AS1 expression levels in 77 PC tissues and matched non-tumor tissues were analyzed by qRT-PCR. Moreover, the role of FLVCR1-AS1 in PC cell proliferation, cell cycle, and migration was verified via functional in vitro and in vivo experiments. Further, the potential competitive endogenous RNA (ceRNA) network between FLVCR1-AS1 and KLF10, as well as FLVCR1-AS1 transcription levels, were investigated. Results FLVCR1-AS1 expression was low in both PC tissues and PC cell lines, and FLVCR1-AS1 downregulation was associated with a worse prognosis in patients with PC. Functional experiments demonstrated that FLVCR1-AS1 overexpression significantly suppressed PC cell proliferation, cell cycle, and migration both in vitro and in vivo. Mechanistic investigations revealed that FLVCR1-AS1 acts as a ceRNA to sequester miR-513c-5p or miR-514b-5p from the sponging KLF10 mRNA, thereby relieving their suppressive effects on KLF10 expression. Additionally, FLVCR1-AS1 was shown to be a direct transcriptional target of KLF10. Conclusions Our research suggests that FLVCR1-AS1 plays a tumor-suppressive role in PC by inhibiting proliferation, cell cycle, and migration through a positive feedback loop with KLF10, thereby providing a novel therapeutic strategy for PC treatment.

scholarly journals Suppression of long noncoding RNA MALAT1 inhibits the development of uveal melanoma via microRNA-608-mediated inhibition of HOXC4

2020 ◽  
Vol 318 (5) ◽  
pp. C903-C912 ◽  
Author(s):  
Shuai Wu ◽  
Han Chen ◽  
Ling Zuo ◽  
Hai Jiang ◽  
Hongtao Yan
Keyword(s):  
Cell Cycle ◽  
Uveal Melanoma ◽  
Noncoding Rna ◽  
Cell Cycle Progression ◽  
Melanoma Cells ◽  
Cell Cycle Distribution ◽  
Invasion And Migration ◽  
And Migration

This study explored the effects of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on the development of uveal melanoma. Moreover, the role of the MALAT1/microRNA-608 (miR-608)/homeobox C4 (HOXC4) axis was assessed by evaluating the proliferation, invasion, and migration, as well as the cell cycle distribution of uveal melanoma in vitro after knocking down MALAT1 or HOXC4 and/or overexpression of miR-608 in uveal melanoma cells (MUM-2B and C918). Moreover, the effects of the MALAT1/miR-608/HOXC4 axis in uveal melanoma in vivo were further evaluated by injecting the C918 cells into the NOD/SCID mice. HOXC4 was found to be a gene upregulated in uveal melanoma, while knockdown of its expression resulted in suppression of uveal melanoma cell migration, proliferation, and invasion, as well as cell cycle progression. In addition, the upregulation of miR-608 reduced the expression of HOXC4 in the uveal melanoma cells, which was rescued by overexpression of MALAT1. Hence, MALAT1 could upregulate the HOXC4 by binding to miR-608. The suppressed progression of uveal melanoma in vitro by miR-608 was rescued by overexpression of MALAT1. Additionally, in vivo assays demonstrated that downregulation of MALAT1 could suppress tumor growth through downregulation of HOXC4 expression via increasing miR-608 in uveal melanoma. In summary, MALAT1 downregulation functions to restrain the development of uveal melanoma via miR-608-mediated inhibition of HOXC4.

scholarly journals Raddeanin A Induces Apoptosis and Cycle Arrest in Human HCT116 Cells through PI3K/AKT Pathway Regulation In Vitro and In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Chunqin Meng ◽  
Yuhao Teng ◽  
Xiaodong Jiang
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Cycle Arrest ◽  
Colorectal Cell ◽  
Pathway Regulation ◽  
Hct116 Cells

This study aimed to investigate the in vitro and in vivo effects of Raddeanin A on apoptosis and the cell cycle in the human colorectal cell line, HCT116, and to explore the possible underlying mechanisms of action. We found the growth inhibition rate gradually increased as the drug concentration increased via the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, which indicated that Raddeanin A significantly inhibited the growth of HCT116 cells. Flow cytometry (FCM) showed that Raddeanin A concentration-dependently induced apoptosis in HCT116 cells. In addition, the percentage of cells in the G0/G1phase was noticeably increased, which indicated that Raddeanin A blocked cell cycle progression in HCT116 cells and caused arrest in the G0/G1phase. Moreover, the expression of proteins involved in the PI3K/AKT signaling pathway (e.g., p-PI3K and p-AKT) was decreased. The results showed that in vivo revealed that Raddeanin A significantly inhibited tumor growth in an HCT116-xenografted mouse model; apoptotic cells were also detected in the tumor tissue. The expression of the tissue proteins cyclinD1, cyclinE, p-PI3K, and p-AKT was decreased. The above results show that the Raddeanin A exerted a strong antitumor effect in the human colorectal cell line HCT116 both in vitro and in vivo. This effect may be caused by the induction of apoptosis and cycle arrest achieved through PI3K/AKT signaling pathway regulation.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

The Novel Kinesin Spindle Protein Inhibitor SB-743921 Exhibits Marked Activity In In Vivo and In Vitro In Models of Aggressive Diffuse Large B-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 118-118
Author(s):  
Danielle C Bongero ◽  
Luca Paoluzzi ◽  
Enrica Marchi ◽  
Neisa Roberto ◽  
Rafael Escandon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Line ◽  
Germinal Center ◽  
Mitotic Spindle ◽  
Tumor Volume ◽  
P Value ◽  
Kinesin Spindle Protein

Abstract Abstract 118 A mitotic spindle target that has emerged as unique and potentially restricted to the mitotic spindle is Eg5, also known as the kinesin spindle protein (KSP). SB-743921 induces mitotic spindle dysfunction and cell cycle arrest by inhibiting Eg5. Preliminary Phase 1 studies of SB-743921 have demonstrated that this compound is not associated with any neuropathy like other anti-mitotic agents. These studies have also demonstrated a potential signal in patients with relapsed and refractory lymphoma. We investigated the efficacy of SB-743921 in aggressive B-cell lymphomas to evaluate effectiveness and tolerability in germinal center (GCB) and post germinal center (ABC) diffuse large B-cell lymphomas (DLBCL). For cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition with Biotek Synergy HT. The IC50s were calculated using the Calcusyn software (Biosoft). Cell Cycle was assessed by staining with Vybrant DyeCycle Green (Invitrogen) followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. In vivo experiments were performed with five to 7-week-old severe combined immunodeficiency (SCID) beige mice (Taconic Laboratories, Germantown, NY) injected with 1 × 107 Ly1-DLBCL cells on the flank via a subcutaneous (SQ) route. When tumor volumes approached 80 mm3, mice were separated into cohorts of ten mice each. Tumors were assessed using the two largest perpendicular axes (l, length; w, width) as measured with standard calipers. Tumor volume was calculated using the formula 4/3 r3, where r=(l + w) / 4. Tumor-bearing mice were assessed for weight loss and tumor volume at least twice weekly. The IC50 values for SB-743921 across a panel of different DLBCL lines are listed in table 1. Cell cycle analysis showed that compared to the untreated group, after treatment with 100nM of SB 743921 the percentage of GCB cells in G2/M phase increased from 17.6% to 40.3% (+129%) in Ly7, 23.9% to 40.7 % (+70%) in Sudhl6 and from 17.55% to 32.4% (+85%) in Ly1. In comparison, the percent increase of cells in G2/M for the ABC lines was statistically less (p-value 0.001). For example, Ly10 increased from 15% to 27.6% (+45%), Riva from 29.3% to 36.95% (+26%) and Sudhl2 from 22.6% to 27.6% (+22%). Immunoblot analysis of DLBCL cells treated with SB-743921 probed for Eg5, CyclinB1, and phosphorylated BubR1 revealed that although all cells demonstrated a measurable increase in Eg5, the total Eg5 present varied from cell line to cell line. The In vivo xenograft experiment was conducted with the GCB Ly1 cell line and consisted of 4 cohorts; one control and 3 treatments with doses of 2.5 mg/kg, 5 mg/kg and 10 mg/kg. SB-743921 was administered by the intraperitoneal route on days 1, 5, and 9 on a 23 day cycle for 2 cycles. The graph below displays the inhibition of tumor growth in the cohorts after treatment with SB-74321. All 3 cohorts had a p-value of <0.001 relative to the control. In conclusion, SB-743921 is promising as a single agent for treatment of DLBCL. Future studies exploring the specific cell cycle features of different cell lines with respect to their check-point control will afford new opportunities to better understand the mechanisms of increased resistance in ABC compared to GCB. The data suggests SB 743921 overall is effective in the treatment of DLBCL both in vitro and in vivo. Further studies exploring potential synergistic interactions with conventional chemotherapeutic agents as well as establishing the most effective treatment schedules for the agent may provide a new approach to treating these diseases. Disclosures: Escandon: Cytokinetics: Employment. Wood:Cytokinetics: Employment. O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1653-1653
Author(s):  
Silvia Locatelli ◽  
Arianna Giacomini ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Michele Magni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Drug Combination ◽  
Akt Inhibitor ◽  
Sorafenib Treatment ◽  
Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

ANKHD1 Silencing Reduces In Vitro Clonogenicity and In Vivo Tumorogenicity Of Multiple Myeloma Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3168-3168
Author(s):  
Anamika Dhyani ◽  
João Agostinho Machado-Neto ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Tumor Size ◽  
Control Group ◽  
Mice Model ◽  
And Migration

Abstract Introduction ANKHD1 is a multiple ankyrin repeats containing protein, highly expressed in cancers, such as acute leukemia. Earlier studies showed that ANKHD1 is highly expressed and plays important role in proliferation and cell cycle progression of multiple myeloma (MM) cells. It was also observed that ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irresepective of TP53 mutational status of MM cell lines. Objective The present study aimed to study the effect ofANKHD1 silencing on MM growth both in vitro (clonogenicity, migration) and in vivo (xenograft tumor mice model). The purpose was to investigate the feasibility of ANKHD1 gene therapy for MM. Methods In the present study, ANKHD1 expression was silenced using short hairpin RNA (shRNA)-lentiviral delivery vector in MM cell lines (U266 and MM1S). For control MM cells were tranduced by lentiviral shRNA against LacZ. Downregulation of ANKHD1 expression was confirmed by qPCR and Western blot. Colony formation capacity and migration of control and ANKHD1 silenced MM cells was determined by methylcellulose and transwell migration assays, respectively. For in vivo MM growth, NOD-SCID mice were divided in two groups injected with control and ANKHD1 silenced cells, separately. Mice were observed daily for tumor growth. Once the tumor size reached 1 mm3, mice in both groups were sacrificed and tumor was excised to measure tumor volume and weight. Results Corroborating the results obtained in our earlier studies, in the present study also inhibition of ANKHD1 expression suppressed growth of MM cells in vitro. MM cell lines tranduced with ANKHD1 shRNA showed significantly low number of colonies ten days after plating in methylcellulose medium as compared to control (p<0.05). Similarly, in transwell migration assay, cell lines transduced with ANKHD1 showed significantly less migration as in response to 10% FBS at lower chamber as compared to control group (p<0.05) in both the cell lines analyzed. Further in xenograft MM mice model, the growth of tumor was visibly suppressed in mice injected with ANKHD1 silenced cells compared to control group. There was significant difference in tumor size (volume) between these 2 groups (P< 0.006). The tumor weight of the inhibition group was 0.71 ±0.2 g, significantly lighter than those of the control group (1.211 ± 0.5 g, P =0.02) Conclusion Our data indicates ANKHD1 downregulation significantly inhibits colony-forming ability and migration of both glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further, gene silencing of ANKHD1 also resulted in reduced in vivo tumor growth in NOD/SCID mice. Collectively, the result obtained indicates that ANKHD1 may be a target for gene therapy in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HB9 Represses Hematopoietic Stem Cell Proliferation and Induces Senescence

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1539-1539
Author(s):  
Deborah Ingenhag ◽  
Franziska Auer ◽  
Arndt Borkhardt ◽  
Julia Hauer
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Line ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Oncogenic Potential ◽  
Stem Cell Proliferation ◽  
Senescent Phenotype

Abstract Introduction: HB9 is a transcription factor encoded by homeobox gene B9 (HLXB9). It is physiologically expressed during early embryonic development as well as in pancreatic beta- and motor neuronal cell development. Ectopic HB9 expression is found in infant acute myeloid leukemia with translocation t(7;12), accounting for up to one third of infant AML cases with a poor 3-year EFS of 0% irrespective of the treatment approach. We previously showed that HB9 regulates cell-cell interaction/adhesion (Wildenhain et al. Leukemia, 2010) in hematopoietic cells and influences the prostaglandin signalling pathway (Wildenhain & Ingenhag et al. JBC, 2012). In this study we focussed on the oncogenic potential of HB9 in hematopoiesis. Methods: To investigate the oncogenic influence of HB9 expression on hematopoiesis, we developed an in vivo murine transplantation model. HB9-transduced lineage negative (Lin-) murine HSCs were transplanted into lethally irradiated wild-type mice and we monitored hematopoietic reconstitution and leukemia emergence by serial retroorbital bleedings for up to one year. Final analysis included comprehensive flow cytometric analysis of all hematopoietic compartments, with respect to dissemination of blast cells and cellular distribution. In vitro studies included proliferation as well as cell cycle analysis. Senescent phenotype was characterized by senescence-associated beta-galactosidase staining and cellular morphology. Knockdown of p53 was obtained via transfection of siRNA. Results: Transplantation of HB9- or mock-transduced murine Lin- cells into lethally irradiated wild-type recipient mice (n=10) showed >80% donor chimerism and HB9-transduced Lin- cells gave rise to all hematopoietic lineages (B-lineage: CD19+, T-lineage: CD3+, NK-lineage: Nk1.1+, granulocytic lineage: Gr-1+, Monocytic lineage: CD11b+) in the peripheral blood, indicating no lineage-related preference of HB9-expressing HSCs. Reconstitution of peripheral blood cell compartments in HB9 transplanted mice, however, was significantly decreased in all three lineages (CD3+: 9.5-fold, CD19+: 34.7-fold , Gr+: 1.8-fold) compared to the control group with respect to copy number, mRNA and protein expression. We did not observe an accumulation of hematopoietic stem (LT-HSC, ST-HSC, MPP) and precursor cells subsets (CLP, MEP, CMP, GMP) in the bone marrow of mice transplanted with HB9-positive Lin- cells. Finally, mice transplanted with HB9-transduced Lin- cells did not develop leukemia after 12 months follow-up. The decreased reconstitution capacity of HB9 expressing HSCs led us to the assumption that HB9 represses cellular proliferation in vivo. Thus we performed proliferation studies in vitro. Ectopic expression of HB9 in the murine NIH3T3 cell line revealed a complete inhibition of cell proliferation compared to mock control (n=3). The same effect was observed in human HT1080 cell line. Cell cycle analysis revealed a significant decrease of the S-phase (2-fold, p<0.05), stalling the cells in G1 and G2 phase of the cell cycle. In both cell line models HB9-transduced cells developed a senescent phenotype being multinuclear, flattened and enlarged. Staining for senescence-associated β-galactosidase activity was positive in HB9-transduced cells in contrast to complete absence in mock-transduced cells. Immunoblot analysis revealed that the HB9 dependent cell cycle arrest was mediated via p53-induced upregulation of p21. Knockdown experiments using p53-targeting siRNAs confirmed that the p53-signalling is responsible for the growth arrest because p53-knockdown was able to reverse the effect. Conclusion:In our study HB9 represses hematopoietic stem cell proliferation in vivo and induces a senescent phenotype in vitro. Senescence is an evasion mechanism in response to aberrant oncogene expression and induction of senescence is the first evidence for an oncogenic potential of HB9. Future studies elucidating the signal pattern of HB9-induced senescence will shed new light on the pathomechanism and potential therapeutic targets in the treatment of translocation t(7;12) positive AML. Disclosures No relevant conflicts of interest to declare.

EBAG9 is a tumor‐promoting and prognostic factor for bladder cancer.

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Bladder Cancer ◽  
Immunohistochemical Study ◽  
Tumor Formation ◽  
Loss Of Function ◽  
Xenograft Models ◽  
Prostate Cancers ◽  
And Migration

Upregulation of EBAG9 expression has been observed in severalmalignant tumors such as advanced breast and prostate cancers,indicating that EBAG9 may contribute to tumor proliferation. Inthe present study, we assess the role of EBAG9 in bladder cancer.We generated human bladder cancer EJ cells stably expressingFLAG-tagged EBAG9 (EJ-EBAG9) or empty vector (EJ-vector),and investigated whether EBAG9 overexpression modulates cellgrowth and migration in vitro as well as the in vivo tumor formationof EJ transfectants in xenograft models of BALB/c nude mice.EBAG9 overexpression promoted EJ cell migration, while theeffect of EBAG9 to cultured cell growth was rather minimal.Tumorigenic experiments in nude mice showed that the size of EJEBAG9-derived tumors was significantly larger than EJ-vectorderivedtumors. Loss-of-function study for EBAG9 using smallinterfering RNA (siRNA) in xenografts with parental EJ cellsshowed that the intra-tumoral injection of EBAG9 siRNA markedlyreduced the EJ tumor formation compared with controlsiRNA. Furthermore, immunohistochemical study for EBAG9expression was performed in 60 pathological bladder cancer specimens.Intense and diffuse cytoplasmic immunostaining wasobserved in 45% of the bladder cancer cases. Positive EBAG9immunoreactivity was closely correlated with poor prognosis ofthe patients (p 5 0.0001) and it was an independent prognosticpredictor for disease-specific survival in multivariate analysis(p 5 0.003). Our results indicate that EBAG9 would be a crucialregulator of tumor progression and a potential prognostic markerfor bladder cancer.

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