scholarly journals The effects of trans-chalcone and chalcone hydrate on the growth of Babesia andTheileria

2018 ◽  
Author(s):  
Gaber El-Saber Batiha ◽  
Amany Magdy Beshbishy ◽  
Dickson Stuart Tayebwa ◽  
Oluyomi Stephen Adeyemi ◽  
Hazem Shaheen ◽  
...  
Keyword(s):  
Adverse Effects ◽  
Cell Lines ◽  
Combined Treatment ◽  
Transmitted Disease ◽  
Companion Animals ◽  
Diminazene Aceturate ◽  
Imidocarb Dipropionate ◽  
Nih 3T3

AbstractBackgroundChemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated.Methodology/Principal findingsThe fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on five of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti–infected mouse model. The cytotoxicity of compounds was tested on Madin– Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC–diminazene aceturate and TC–clofazimine were more potent against B. microti infection in mice than their monotherapies.Conclusions/SignificanceIn conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.Author summaryProtozoa of the genus Babesia are the second most common blood-borne parasites of mammals after the trypanosomes. Babesia and Theileria are the etiological agents of piroplasmosis, a tick-transmitted disease causing substantial losses of livestock and companion animals worldwide and has recently gained attention as one of the emerging zoonosis in humans. Diminazene aceturate and imidocarb dipropionate are still the first choices for the treatment of animals. However, these drugs cause many adverse effects. Furthermore, they are not approved for human medicine. Therefore, the development of alternative treatment remedies against babesiosis is urgently required. In the present study we evaluated the effects chalcone hydrate (CH) and trans-chalcone (TC), against the growth of four species of Babesia and T. equi. Furthermore, we studied the chemotherapeutic potential of TC on B. microti in mice. The effects of the combined treatment of TC with DA, CF and AQ revealed that TC was found to diminish the adverse effects of these drugs

scholarly journals Radiosensitisation of Hepatocellular Carcinoma Cells by Vandetanib

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1878 ◽  
Author(s):  
Sami Znati ◽  
Rebecca Carter ◽  
Marcos Vasquez ◽  
Adam Westhorpe ◽  
Hassan Shahbakhti ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Combination Therapy ◽  
Cell Lines ◽  
Radiation Treatment ◽  
Combined Treatment ◽  
New Combination ◽  
Migration And Invasion ◽  
Syngeneic Mouse Model

Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models. Methods: Vandetanib and radiation treatment were combined in HCC cell lines grown in vitro and in vivo. In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC. Results: Vandetanib IC 50 s were measured in 20 cell lines and the drug was found to significantly enhance radiation cell kill and to inhibit both cell migration and invasion in vitro. In vivo, combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment. Conclusion: In 2D and 3D studies in vitro and in a syngeneic model in vivo, the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.

Looking for synergy or additivity between 188Re and sorafenib on hepatoma cell lines.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 247-247
Author(s):  
Marc Pracht ◽  
Nicolas Lepareur ◽  
Julien Edeline ◽  
Laurence Lenoir ◽  
Valerie Ardisson ◽  
...  
Keyword(s):  
Cell Line ◽  
Hepg2 Cell ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
External Beam Radiotherapy ◽  
Combined Treatment ◽  
Hepatoma Cell Lines ◽  
Heparg Cell Line

247 Background: In case of non resectable HCC, radioembolization and sorafenib (S) are therapeutic options respectively for intermediate and advanced stages. In some other cancers, there is an increase of efficacy when external beam radiotherapy is done concomitantly with systemic chemotherapy or targeted therapies. So we wondered if there could be a synergistic or an additive activity when S is combined with a radionuclide. Methods: Hepatoma cell lines N1S1 (murine HCC), HepG2 (human hepatoblastoma) and HepaRG (human HCC) were treated with increasing concentrations of rhenium-188 (188Re) or S. On each cell line, we have studied the cellular toxicities of S and 188Re using Tetrazolium dye test, extra-cellular medium LDH level and morphologic analysis. This was done for different dosage of S and 188Re. We measured the lethal concentration killing 25% of cells (LC25) with the results of the Tetrazolium dye test. Secondly, we looked for synergy or additivity on cellular toxicity of these two compounds according to cell lines by combined treatment. Synergy or additivity was estimated with the combination index (CI) method (synergy if CI lower than 1, additivity if CI = 1, antagonism if CI upper to 1) based on the Tetrazolium dye test’s results. Results: Monotherapy dose-dependent toxicities were observed for all three cell lines with 188Re and for the N1S1 and HepG2 cell lines only with S. Combined treatment with 188Re and S showed synergy on HepaRG and N1S1 cell lines and additivity on the HepG2 cell line. Conclusions: The additive, and even synergistic, interest of a combined treatment with 188Re and S is demonstrated in vitro (for the first time to our knowledge) on hepatoma cell lines. This results, in particular for the HepaRG cell line (human HCC), could be explained by the down-regulation of the hepatic drug transporters which are responsible for the Sorafenib efflux in case of simultaneous DNA damages due to a radionuclide exposition. This promising approach now needs to be confirmed in vivo. [Table: see text]

Role of the Histone Deacetylase Inhibitor Givinostat (ITF2357) in Treatment of CRLF2 Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2534-2534 ◽  
Author(s):  
Angela Maria Savino ◽  
Jolanda Sarno ◽  
Luca Trentin ◽  
Margherita Vieri ◽  
Grazia Fazio ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Combined Treatment ◽  
Cytokine Receptor ◽  
Human Leukemia ◽  
Xenograft Models ◽  
Stat Pathway

Abstract B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) represents 35% of all cancers in pediatric age group. The cure rate for this disease approaches 90% with current treatment regimens, however only a third of patients with relapse are cured. Therefore, there is an urgent need to focus on subgroups of patients with hallmarks of bad prognosis that could benefit from novel therapeutic approaches. Alterations of Cytokine Receptor-like Factor 2 (CRLF2), a negative prognostic factor in pediatric BCP-ALL, have been identified in up to 10% of patients. However these patients represent half of the high risk Ph-like ALL and of Down Syndrome-associated BCP-ALL. Rearrangements of CRLF2 result in the overexpression of this component of the heterodimeric cytokine receptor for thymic stromal lymphopoietin (TSLP) and is associated with activating mutations of the JAK-STAT pathway. Together these cause hyperactivation of JAK/STAT and PI3K/mTOR signaling. Inhibition of CRLF2/JAK2 signaling has the potential to become a therapeutic targeted intervention for this subgroup of poor prognostic patients. Previous studies have shown that the HDAC inhibitor Givinostat/ITF2357 has potent anti-tumor activity against hematological malignancies, particularly JAK2V617F mutated myeloproliferative neoplasms (MPN) such as polycythemia vera, for which it has already a clinic application and established safety profile. We therefore studied the in vitro and in vivo efficacy of Givinostat in cases with CRLF2 rearrangements. Here we demonstrated that Givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged MHH-CALL4 and MUTZ5 cell lines positive for exon 16 JAK2 mutations. Of note, the observed IC50 values for MHH-CALL4 were lower than those for the SET2 cell line positive control bearing JAK2V617F mutation, both for proliferation (IC50: 0.08±0.05µM vs. 0.14±0.03µM) and apoptosis (IC50: 0.17±0.03µM vs. 0.22±0.04µM). We next investigated the effect of Givinostat on blasts from CRLF2 rearranged BCP-ALL patient samples. For this purpose we developed xenograft models of human CRLF2 rearranged ALL to expand cells from patients and to recapitulate human leukemia in recipient mice. ALL blasts isolated from xenografts were co-cultured on OP9 stroma to perform ex vivo assays. Consistent with our findings in cell lines, Givinostat (0.2µM) reduced the % of live cells (Annexin V/Sytox negative) in all xenografts treated with the drug. In particular, after 72 hours, Givinostat was able to kill up to >90% of blast cells in all xenografts in contrast with the vehicle-treated samples which showed 25-60% of blasts still alive after treatment. The induction of cell death in Givinostat treated primografts was confirmed on primary samples from diagnosis using CyTOF which allowed us to observe that CD10+/CRLF2+ blasts were preferentially killed by the drug whereas CD45 high expressing cells (normal residue) remained unaffected by the treatment. Moreover, at low doses (0.2 µM), Givinostat downregulated genes of the JAK/STAT pathway (STAT5A, JAK2, IL7Rα, CRLF2, BCL2L1 and cMYC) and inhibited the basal and ligand induced signaling, reducing the phoshporylation of STAT5 in all tested primografts (mean fold decrease of pSTAT5: 2.4+0.6). Most importantly, to understand if the transcriptional downregulation of CRLF2 resulted in a functional effect, the downmodulation of CRLF2 protein was observed by flow cytometry (mean fold decrease 3.55+1.38). In vivo, Givinostat significantly reduced engraftment of human blasts in xenograft models of CRLF2 positive BCP-ALL (ranging from 1.9 to 34 fold decrease in bone marrow). Furthermore, Givinostat augmented the effect of chemotherapy in inhibiting proliferation and inducing apoptosis in CRLF2 rearranged cell lines and in primografts, in vitro. After 72 hours, the combined treatment reached 4.6-8.8 fold lower % of remaining viable blasts than chemotherapy alone (6.3-35.3% viable cells in chemotherapy-treated samples vs 1.4-4.3% of combination), 2.5-8.5 fold lower than Givinostat alone (4.3-36.4% vs 1.4-4.3%) and 2.4-13 fold lower than Methyl-prednisolone (5.2-39.1 vs 1-16.3%). In conclusion, Givinostat may represent a novel and effective tool, in combination with current chemotherapy, to treat this difficult to handle subset of ALL and these data strongly argue for the translation of Givinostat in combination with conventional therapy into human trials. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

scholarly journals The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2947-2947
Author(s):  
Xu-Wen Guan ◽  
Wang Hua-Qing ◽  
Li Jia ◽  
Feng-Ting Liu
Keyword(s):  
Cell Death ◽  
Tumor Growth ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Combined Treatment ◽  
B Cell Lymphoma ◽  
Surface Expression ◽  
Cd20 Expression

Abstract Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP. We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo. Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment. Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups. Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. Disclosures No relevant conflicts of interest to declare.

DDRE-03. THERAPEUTIC TARGETING OF THE ERK AND CDK PATHWAYS IN PRECLINICAL MODELS OF BRAIN METASTASES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi74-vi75
Author(s):  
Mohini Singh ◽  
Naema Nayyar ◽  
Ashish Dahal ◽  
Priscilla Brastianos
Keyword(s):  
Cell Cycle ◽  
Brain Metastases ◽  
Cell Lines ◽  
Combined Treatment ◽  
Durable Response ◽  
Cdkn2a Mutation ◽  
Response To Chemotherapy ◽  
Cell Arrest

Abstract Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system. BM develop in 40-50% of advanced lung adenocarcinoma (LUAD), but the lack of durable response to chemotherapy, immunotherapy, or targeted therapy results in death within a year of BM diagnosis. Several advances have been made in identifying genetic drivers of primary cancers. The cell cycle, RAS and ERK pathways have all been implicated in as critical oncogenic regulators, with aberrations linked to driving the progression and metastasis of LUAD. Abemaciclib is a targeted CDK4/6 inhibitor, and LY3214996 is selective ERK1/2 inhibitor, and have shown efficacy in preclinical tumor models as well as in clinical trials. Furthermore, both therapeutics can interfere with the cell cycle, abemaciclib through targeting CDK4/6 and LY3214996 through cyclinD1. Here we present data assessing abemaciclib and LY3214996, as single and combined agents, in cell lines across different KRAS and CDKN2A mutational backgrounds. Seven days post-intracranial inoculation of NSCLC and NSCLC-BM line, mice received either abemaciclib, LY3214996, or a combination P.O. daily for 21 days, and were monitored pre- and post-treatment for tumor growth with bioluminescent imaging. In vitro we demonstrated a dose-dependent reduction in cell growth with each treatment, as well as cell arrest in G1 phase. In vivo, whereas cell lines with a combined KRAS mutation and CDKN2A mutation/deletion had no significant reduction in BM growth, cell lines with a CDKN2A del or BRAF mutation had significant BM reduction, with single agents and combined treatment. Further research is necessary to elucidate under what genetic contexts abemaciclib, LY3214996 or the combination are most effective. Nonetheless, this work highlights that abemaciclib and LY3214996 should be further explored for CDKN2A or BRAF mutant BM.

Treatment with arsenic trioxide (ATO) and MEK1 inhibitor activates the p73-p53AIP1 apoptotic pathway in leukemia cells

Blood ◽  
2004 ◽  
Vol 104 (2) ◽  
pp. 519-525 ◽  
Author(s):  
Paolo Lunghi ◽  
Antonio Costanzo ◽  
Massimo Levrero ◽  
Antonio Bonati
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Apoptotic Cell ◽  
Combined Treatment ◽  
Dominant Negative ◽  
Apoptotic Pathway

Abstract Arsenic trioxide (ATO) induces differentiation and apoptosis of malignant cells in vitro and in vivo and has been used in the treatment of a variety of hematologic malignancies. We found that in NB4 acute promyelocytic and in K562 erythroleukemia cell lines treatment with the MEK1 inhibitors PD98059 and PD184352 greatly enhances apoptotic cell death induced by ATO alone. Combined treatment results in the induction of the p53AIP1 (p53-regulated apoptosis-inducing protein 1) gene in both cell lines. Because NB4 and K562 cell lines carry an inactive p53, we investigated the possible role of p73, a p53 paralogue that has been shown to regulate several p53 target genes including p21, Bax, and p53AIP1. We found that MEK1 inhibitors reduce the levels of dominant-negative (ΔN) p73 proteins and promote the accumulation of endogenous p73α through its transcriptional activation and its tyrosine phosphorylation, resulting in p21 up-regulation and significant inhibition of cell growth. ATO reduces ΔNp73 levels and promotes a p300-mediated acetylation of endogenous p73, thus favoring cell cycle arrest and apoptosis. Finally, the combined treatment with MEK1 inhibitors and ATO enhances the affinity of phosphoacetylated p73 for the p53AIP1 promoter in vivo, as determined by chromatin immunoprecipitation experiments, leading to p53AIP1 up-regulation and increased apoptosis. (Blood. 2004; 104:519-525)

Arsenic Trioxide (ATO) Interacts Synergistically with MEK Inhibitors to Induce Apoptosis in STI571-Resistant Bcr-Abl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2177-2177
Author(s):  
Paolo Lunghi ◽  
Laura Mazzera ◽  
Attilio Corradi ◽  
Anna Maria Cantoni ◽  
Stefano Jottini ◽  
...  
Keyword(s):  
Cell Lines ◽  
Combined Treatment ◽  
Myelogenous Leukemia ◽  
Histopathological Analysis ◽  
Mitochondrial Depolarization ◽  
Mek Inhibitors ◽  
Imatinib Resistant ◽  
Mutant Forms

Abstract Abstract 2177 Poster Board II-154 Resistance toward Imatinib and other Bcr-Abl tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. Previous preclinical studies reported that the MEK inhibitors PD184352 or PD0325901 (Pfizer), strikingly enhances ATO-mediated apoptosis in Acute Myelogenous Leukemia and in Multiple myeloma. The aim of this study was to investigate whether the combined treatment with PD184352 (PD) and ATO has cytotoxic effects on murine Ba/F3 cells expressing wild-type (wt) or various imatinib-resistant mutant forms of Bcr-Abl, including T315I. We first analyzed the pharmacologic interactions between PD and ATO using a fixed-ratio experimental design in Bcr-Abl Ba/F3p210wt, Ba/F3p210T315I and Ba/F3p210Y253F cell lines and found that the combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in all cell lines tested (Chou-Talalay method): the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in PD plus ATO treated cells were 0.72± 0.19, 0.61± 0.04 and 0.69± 0.09 in BCR-ABL Ba/F3p210wt, Ba/F3p210T315I, and Ba/F3p210Y253F respectively. Synergistic interaction between ATO and PD0325901, a derivative of PD184352, was demonstrated in all tested cell lines. In order to investigate the molecular effectors involved in PD/ATO-induced apoptosis we first evaluated its effects on Bcr-Abl protein expression and CrkL phosphorylation, a well-known downstream target of Bcr-Abl. Immunoblotting analyses demonstrated that treatment for 24 to 48 hours of Ba/F3p210wt and Ba/F3p210T315I cells with PD (1μM) or ATO (1μM) alone or in combination had no effects on levels of total Bcr-Abl or phospho—CrkL thereby indicating that the combination PD/ATO does not act via Bcr-Abl oncogenic signaling. Therefore, we studied whether p53 and the p53-related gene p73 are molecular targets of the combined treatment in Ba/F3p210wt and Ba/F3p210T315I cell lines. We found that monotreatment with neither PD nor ATO 1 μM (or their combination) was able to induce p53 accumulation, whereas the combination PD/ATO promoted the accumulation of the proapoptotic and antiproliferative TA-p73α protein and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔN-p73α in both cell lines. Consistent with these results, we found that PD greatly enhanced the ATO-induced Puma expression, mitochondrial depolarization, caspase-3 activation, and apoptosis in Ba/F3p210wt and Ba/F3p210T315I cells and functional knock-out of p73 gene expression by small interfering (si)RNAs significantly reduced (P< .05 Dunnett test) the PD/ATO induced mitochondrial depolarization. To determine whether the PD plus ATO efficacy observed in vitro for BaF3 cells expressing mutant forms of Bcr-Abl was recapitulated in vivo, we studied PD/ATO combination in a mouse model of Imatinib-resistant, Bcr-Abl—dependent disease. Severe combined immunodeficient mice were injected intravenously with Ba/F3 cells expressing Bcr-Abl-T315I isoform. Mice with Bcr-Abl-T315I—induced leukemia were treated with the MEK inhibitor PD0325901 (10 mg/kg; orally) plus ATO (3.75 mg/kg; intraperitoneally) or Imatinib (50 mg/kg, twice daily; intraperitoneally) or vehicle for three weeks. Untreated or Imatinib-treated mice harboring the T315I isoform developed aggressive disease, with massive liver and splenic infiltration, typically resulting in death in 32 days. However, mice harboring the T315I isoform showed significantly prolonged survival when treated with PD/ATO (43 days, P=.001, Kaplan-Meier method and compared using the log-rank test). Moreover, histopathological analysis of 20 days Imatinib-treated mice revealed infiltration of the liver and spleen. In contrast, histopathological analysis of organs from PD/ATO-treated mice demonstrated normal tissue architecture. Consistent with these results immumoblottig analysis of the lysates from livers and spleens revealed a marked expression of Bcr-Abl protein in mice treated with Imatinib or vehicle. Our preclinical in vitro and in vivo studies suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl-positive leukemias, including those harboring the T315I mutation. Disclosures: No relevant conflicts of interest to declare.

Expression of cyclin D3 confers resistance to erlotinib

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 18054-18054
Author(s):  
W. J. Petty ◽  
W. R. Voelzke ◽  
V. A. Memoli ◽  
K. H. Dragnev ◽  
J. J. Urbanic ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Cell Lines ◽  
A549 Cells ◽  
Cyclin D3 ◽  
Sensitive Cell ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

18054 Background: Transcriptional repression of cyclin D1 occurs during responses to erlotinib (E) both in vitro and in vivo. Cyclin D3 has overlapping function with cyclin D1 but has distinct transcriptional regulation. Methods: The expression of cyclin D3 was compared in E sensitive cell lines (H358, H441) and an E resistant cell line (A549). Cyclins D1, D2, and D3 were independently overexpressed in E sensitive NIH 3T3 cells by plasmid transfection. Biopsy tissues from a proof-of-principal clinical trial of E were assessed for cyclin D3 expression. Results: A549 cells were resistant to E and expressed high levels of cyclin D3 RNA and protein compared to E sensitive cell lines. Overexpression of cyclin D1 and cyclin D3 conferred partial resistance to E in NIH 3T3 cells while cyclin D2 had no significant effect. Comparison of cyclin D3 immunostaining in tumor biopsies from patients before and after treatment with E revealed an increase in the percentage of cyclin D3 expressing cells following treatment with E. Conclusions: Cyclin D3 confers resistance to E in vitro and in vivo. Drugs such as retinoids and rexinoids that target cyclin D3 expression may prove useful for enhancing sensitivity to E. No significant financial relationships to disclose.

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