Antitumor activity of pazopanib (P) and trametinib (T) in preclinical models of osteosarcoma (OS).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e22509-e22509
Author(s):  
Giulia Chiabotto ◽  
Maria Laura Centomo ◽  
Alessandra Merlini ◽  
Lorenzo D'Ambrosio ◽  
Dario Sangiolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antitumor Activity ◽  
Western Blot ◽  
Cell Lines ◽  
Fold Change ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Log2 Fold Change ◽  
In Vivo Antitumor Activity

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

scholarly journals Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246197
Author(s):  
Jorge Marquez ◽  
Jianping Dong ◽  
Chun Dong ◽  
Changsheng Tian ◽  
Ginette Serrero
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Negative Regulator ◽  
Target Validation ◽  
Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

scholarly journals Sapphyrins induce apoptosis in hematopoietic tumor–derived cell lines and show in vivo antitumor activity

2005 ◽  
Vol 4 (6) ◽  
pp. 968-976 ◽  
Author(s):  
Louie Naumovski ◽  
Jason Ramos ◽  
Mint Sirisawad ◽  
Jun Chen ◽  
Patti Thiemann ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cell Lines ◽  
In Vivo Antitumor Activity

„IN VITRO“ AND „IN VIVO“ ANTITUMOR ACTIVITY OF PLATINUM(II) COMPLEXES WITH MALONIC ACID ON BREAST AND COLORECTAL CARCINOMA CELL LINES

2021 ◽  
Author(s):  
Andjela Franich ◽  
◽  
Milica Dimitrijević Stojanović ◽  
Snežana Rajković ◽  
Marina Jovanović ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Colorectal Carcinoma ◽  
Cell Lines ◽  
Tumor Model ◽  
Carcinoma Cells ◽  
Spectroscopic Techniques ◽  
Murine Cell Line ◽  
In Vivo Antitumor Activity

Four Pt(II) complexes of the general formula [Pt(L)(5,6-epoxy-1,10-phen)], where L is anion of malonic (mal, Pt1), 2-methylmalonic (Me-mal, Pt2), 2,2-dimethylmalonic (Me2-mal, Pt3) or 1,1- cyclobutanedicarboxylic (CBDCA, Pt4) acid while 5,6-epoxy-1,10-phen is bidentately coordinated 5,6-epoxy-5,6-dihydro-1,10-phenanthroline were synthesized and characterized by elemental microanalysis, IR, UV-Vis and NMR (1H and 13C) spectroscopic techniques. In vitro anticancer activity of novel platinum(II) complexes have been investigated on human and murine cancer cell lines, as well as normal murine cell line by MTT assay. The obtained results indicate that studied platinum(II) complexes exhibited strong cytotoxic activity against murine breast carcinoma cells (4T1), human (HCT116) and murine (CT26) colorectal carcinoma cells. Complex Pt3 display stronger selectivity toward carcinoma cells in comparison to other tested platinum(II) complexes exhibiting beneficial antitumor activity mainly via the induction of apoptosis, as well as inhibition of cell proliferation and migration. Further study showed that Pt3 complex also carry significant in vivo antitumor activity in orthotopical 4T1 tumor model without detected liver, kidney, lung, and heart toxicity. All results imply that these novel platinum(II) complexes have a good anti-tumor effect on breast and colorectal cancer in vivo and in vitro and the affinity to become possible candidates for treatment in anticancer therapy.

scholarly journals Targeted Brain Tumor Therapy by Inhibiting the MDM2 Oncogene: In Vitro and In Vivo Antitumor Activity and Mechanism of Action

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1592
Author(s):  
Surendra R. Punganuru ◽  
Viswanath Arutla ◽  
Wei Zhao ◽  
Mehrdad Rajaei ◽  
Hemantkumar Deokar ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Tumor Therapy ◽  
Single Agent ◽  
M Cell ◽  
In Vivo Antitumor Activity ◽  
The Brain

There is a desperate need for novel and efficacious chemotherapeutic strategies for human brain cancers. There are abundant molecular alterations along the p53 and MDM2 pathways in human glioma, which play critical roles in drug resistance. The present study was designed to evaluate the in vitro and in vivo antitumor activity of a novel brain-penetrating small molecule MDM2 degrader, termed SP-141. In a panel of nine human glioblastoma and medulloblastoma cell lines, SP-141, as a single agent, potently killed the brain tumor-derived cell lines with IC50 values ranging from 35.8 to 688.8 nM. Treatment with SP-141 resulted in diminished MDM2 and increased p53 and p21cip1 levels, G2/M cell cycle arrest, and marked apoptosis. In intracranial xenograft models of U87MG glioblastoma (wt p53) and DAOY medulloblastoma (mutant p53) expressing luciferase, treatment with SP-141 caused a significant 4- to 9-fold decrease in tumor growth in the absence of discernible toxicity. Further, combination treatment with a low dose of SP-141 (IC20) and temozolomide, a standard anti-glioma drug, led to synergistic cell killing (1.3- to 31-fold) in glioma cell lines, suggesting a novel means for overcoming temozolomide resistance. Considering that SP-141 can be taken up by the brain without the need for any special delivery, our results suggest that SP-141 should be further explored for the treatment of tumors of the central nervous system, regardless of the p53 status of the tumor.

scholarly journals In Vitro and in Vivo Antitumor Activity of Scutebarbatine A on Human Lung Carcinoma A549 Cell Lines

Molecules ◽  
2014 ◽  
Vol 19 (7) ◽  
pp. 8740-8751 ◽  
Author(s):  
Xiao-Kun Yang ◽  
Ming-Yuan Xu ◽  
Gui-Sen Xu ◽  
Yu-Lan Zhang ◽  
Zhao-Xia Xu
Keyword(s):  
Antitumor Activity ◽  
Cell Lines ◽  
Lung Carcinoma ◽  
A549 Cell ◽  
Human Lung ◽  
In Vivo Antitumor Activity ◽  
Human Lung Carcinoma

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.

Mer Receptor Tyrosine Kinase Is Over-Expressed In and Contributes to Oncogenesis In Acute Myeloid Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1390-1390
Author(s):  
Alisa B. Lee-Sherick ◽  
Kristen M. Eisenman ◽  
Susan Sather ◽  
Deborah DeRyckere ◽  
Jennifer Schlegel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Cell Survival ◽  
Cell Lines ◽  
Wild Type ◽  
Knock Down ◽  
Acute Myelogenous

Abstract Abstract 1390 The abnormal activation of tyrosine kinases in pediatric leukemias has been associated with a poor prognosis, and provides a potential focus for targeted therapy. Pediatric acute myelogenous leukemia (AML) is known to be particularly difficult to treat successfully. The development of therapy for AML targeted against a specific cancer-promoting signaling pathway would potentially allow for a more efficacious clinical response with less therapy-associated toxicity. The Mer Tyrosine Kinase (TK), a transmembrane receptor in the TAM family, is known to regulate intracellular pathways promoting cell survival and proliferation in a number of malignancies, but has not previously been explored in AML. We assessed the prevalence of Mer TK expression in AML. Western blot and flow cytometric analysis demonstrated aberrant expression of Mer TK in 80% (13 of 15) of AML cell lines. Similarly, greater than 85% (24 of 28) of samples from newly diagnosed pediatric AML patients expressed Mer TK on leukemic blasts. In addition, 5 of 6 pediatric patients with relapsed or refractory AML had increased or equivalent Mer expression by flow cytometry relative to diagnostic samples. To assess whether Mer plays a role in proliferation in AML, we investigated downstream signaling pathways in the Nomo-1 and Kasumi-1 AML cell lines. Phosphoarray and western blot analysis demonstrated increased phospho-Erk 1/2, phospho-Akt, phospho-mTOR and phospho-MSK1 following treatment with Gas6, the Mer ligand. These data demonstrate activation of pathways which are known to aid in malignant cell survival. To assess the effect of Mer TK inhibition on myeloblast phenotype, we used two different shRNA constructs to decrease expression of Mer by >50% in the Nomo-1 and Kasumi-1 cell lines. The ability of these cell lines to evade apoptosis was determined by flow cytometry following staining with propidium iodide and Yo-Pro-1-iodide. Compared to wild-type Nomo-1 and Kasumi-1, the cell lines expressing decreased levels of Mer demonstrated two to four times more apoptosis in response to serum starvation (p<0.5). Additionally, myeloblast proliferative capacity was assessed using methylcellulose colony forming assays. Compared to wild-type, the AML cell lines expressing reduced levels of Mer demonstrated a 40–70% decrease in total colony forming units (p<0.5). To explore how knockdown of Mer affects myeloblast survival in vivo, we used a mouse xenograft model. Sub-lethally irradiated NSG mice were injected intravenously with wild-type Nomo-1 or Mer knock-down Nomo-1 lines and tumor-free survival was determined. Kaplan-Meier curves were generated and demonstrated a statistically significant difference in survival between mice injected with wild-type Nomo-1 cells and those injected with a Nomo-1 Mer knock-down cell line (20 versus 43 days, p<0.1). These data demonstrate a role for Mer in acute myelogenous leukemogenesis in vivo and suggest that inhibition of Mer TK may have a clinically significant effect in patients as a targeted therapy in the treatment of human AML. Disclosures: No relevant conflicts of interest to declare.

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