Preclinical evidence of ET-743 as a potential chemotherapy option for the treatment of biliary carcinoma.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 193-193
Author(s):  
Francesco Leone ◽  
Caterina Peraldo-Neia ◽  
Giuliana Cavalloni ◽  
Marco Soster ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Culture ◽  
Dna Damage ◽  
Clinical Trials ◽  
Cell Lines ◽  
Primary Cell Culture ◽  
Primary Cell ◽  
Term Survival ◽  
Anti Tumor Activity

193 Background: The standard chemotherapy for unresectablebiliary tract carcinoma (BTC) is based on gemcitabine and platinum compounds. However, these combinations have not been shown to be effective in improving long-term survival. Thus, there is a real need to find new strategies that would impact in a significant way on clinical outcome. Ecteinascidin-743 (ET-743), a compound isolated from the marine tunicate Ecteinascidia turbinata. ET-743, is approved for the treatment of ovarian cancer and soft tissue sarcoma. Phase II and III clinical trials are ongoing for the treatment of different solid tumors. No preclinical data are available about the efficacy of ET-743 in BTC. In a phase I study, one patient received ET-743 plus capecitabine and experienced a long lasting complete metabolic response. Here, we investigated the antitumor activity of ET-743 in preclinical BTC models. Methods: Four BTC cell lines TFK1, EGI-1, HuH28 and TGBC1 were used to evaluate the effect of ET-743 on proliferation, cell cycle, apoptosis and on the activation of DNA damage proteins. The effect on proliferation was also investigated on a primary cell culture of a gallbladder carcinoma (GBC) resistant to gemcitabine and oxaliplatin. On the same cells, the inhibition of VEGF secretion mediated by ET-743 was analyzed by ELISA. The anti-tumor activity of ET-743 was tested on EGI-1 xenografts in NOD/SCID mice. Results: In vitro, ET-743 is able to markedly reduce cell proliferation of BTC cell lines through cell cycle blockage on G0/G1 phase and to inhibit the growth of primary cell culture derived from GBC patient. Moreover, ET-743 promotes apoptosis by caspase 3 activation, activates proteins involved in DNA damage and reduces VEGF secretion. In the in vivo model, ET-743 is able to slow tumor growth in BTC xenograft. The mechanism of anti-tumor activity involves DNA damage, the induction of hypoxia transcription factor-1, and angiogenesis inhibition. ET-743 has no significant effect on apoptosis in vivo. Conclusions: These data suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials of ET-743 in BTC patients.

In Vivo-like model of glial tumors: Creation of primary cell lines.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e14515-e14515
Author(s):  
Sofia V. Timofeeva ◽  
Oleg I. Kit ◽  
Anastasia O. Sitkovskaya ◽  
Irina V. Mezhevova ◽  
Svetlana Yu. Filippova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Primary Cell Culture ◽  
Tumor Tissue ◽  
Primary Cell ◽  
Glial Tumor ◽  
Ki 67 ◽  
Glial Tumors

e14515 Background: The choice of cell source for 3D bioprinting of in vivo-like models of glial tumors is crucial and must take into account the ability to proliferation and stable metabolism. Oral administration of 5-aminolevulinic acid (5-ALA) in patients prior to surgery increases the fluorescent contrast between tumor and surrounding tissue, but the effect of contrast agents on cells in vitro is unknown. The aim of the study was obtaining viable glial tumor tissues using 5-ALA, as well as the development of a stable primary cell culture for 3D bioprinting. Methods: Tumor tissue was obtained from patients with glioblastoma during surgery under visual control using the Opmi Pentero Blue E400 microscope and 5-ALA. Material was disaggregated on a BD Machine using Medicons 50 μm (BD). Glioblastoma cells were cultured in DMEM/F12 medium with L-glutamine (Gibco) containing 10% fetal bovine serum (Biolot, Russia), 1% non-essential amino acids (NEAA, Sigma-Aldrich) and 0.5% penicillin-streptomycin (Biolot) at 37C. Glial cell lines were characterized immunohistochemically using antibodies to the glial fibrillary acidic protein (GFAP) and proliferation index (Ki-67). Microsatellite analysis was performed using three dinucleotide repeat markers D2S123, D17S250, D5S346 and five mononucleotide loci BAT25, BAT26, NR21, NR24 and NR27. Results: The positive expression of GFAP on the cell processes of the star-like shape was clearly visualized, indicating a morphological feature of glial tumors. The Ki-67 labeling index was 70%. Changes were observed at the D17S250 locus (148-148/148-152) for the glial tumor primary cells after the sixth passage. Microsatellite instability was not observed in the primary cell culture. Conclusions: The accumulation of porphyrins from 5-ALA in glial tumor cells does not prevent the in vitro creation of a cell culture from tumor tissue. Microsatellite analysis showed that the obtained glioblastoma cell lines remain stable for at least 10 passages. Material obtained during resection using 5-ALA is a reliable source of stable glial tumor cell lines.

scholarly journals The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

2021 ◽  
Vol 22 (12) ◽  
pp. 6565
Author(s):  
Jennifer H. Foster ◽  
Eveline Barbieri ◽  
Linna Zhang ◽  
Kathleen A. Scorsone ◽  
Myrthala Moreno-Smith ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Tumor Weight ◽  
Cycle Arrest ◽  
P53 Status ◽  
Neuroblastoma Cell Lines ◽  
Anti Tumor Activity

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

scholarly journals TMOD-01. CHARACTERIZATION OF PATIENT-DERIVED PRIMARY CELL LINES AND XENOGRAFTS FOR GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi262-vi262 ◽  
Author(s):  
Noriyuki Kijima ◽  
Daisuke Kanematsu ◽  
Tomoko Shofuda ◽  
Masahiro Nonaka ◽  
Ryoichi Iwata ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Surface ◽  
Cell Lines ◽  
Primary Cell Culture ◽  
Surface Marker ◽  
Primary Cell ◽  
Cell Surface Marker ◽  
High Expression ◽  
Glioblastoma Patient ◽  
Primary Cell Lines

Abstract Patient-derived primary cell culture and xenograft are essential tools for translational research for glioblastoma. However, characteristics of each patient derived cell line and xenograft is not extensively studied. In this study, we aim to analyze the characteristics of our glioblastoma patient-derived cell lines and xenografts based on cell surface markers and their differentiation patterns. We have established 20 glioblastoma primary cell culture lines by serum free medium containing EGF and bFGF and found that primary cell culture lines could be classified based on the expression of CD133 and CD44. Four cell lines had high expression of both CD133 and CD44. Eleven cell lines had high expression of only CD44, three cell lines had high expression of only CD133, two cell lines had low expression of both CD133 and CD44. In addition when we induce differentiation, these cell lines showed differentiation to both glial and neuronal differentiation, but differentiation patterns were different depending on each cell line. Four cell lines showed predominant neuronal differentiation and others showed predominant glial differentiation. We next investigated in vivo characteristics of glioblastoma patient derived xenografts from these established cell lines. We have injected these cell lines into NOD/Shi-scid IL2Rγ KO mouse and histopathologically analyzed characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. These results suggest that glioblastoma patient derived primary cell lines and xenografts have different characteristics of cell surface marker expressions and differentiation patterns, thus can classify these cell lines depending on cell surface marker expressions and differentiation patterns. Further analysis is needed to examine the biological importance of the differences in cell surface marker expressions and differentiation patterns.

Rapid melanization of bomirski amelanotic melanoma cells in cell culture

1983 ◽  
Vol 3 (2) ◽  
pp. 189-194 ◽  
Author(s):  
A. Słominski
Keyword(s):  
Cell Culture ◽  
Primary Cell Culture ◽  
Growth Conditions ◽  
Melanoma Cells ◽  
Amelanotic Melanoma ◽  
Primary Cell ◽  
In Vitro Growth

Transfer of Bomirski amelanotic melanoma ceils from in vivo to in vitro growth conditions results in occurrence of rapid melanization in their cytoplasm. The melanized ceils from primary cell culture initiate tumours in hamsters, which do not contain traces of melanin and resemble typical amelanotic melanoma.

scholarly journals Do alterations in gene expressions influence tumorigenesis in the transmissible venereal tumor in dogs?

2020 ◽  
Vol 50 (11) ◽  
Author(s):  
Haline Ballestero Fêo ◽  
Luis Mauricio Montoya Flórez ◽  
Ricardo Seiti Yamatogi ◽  
Anderson do Prado Duzanski ◽  
João Pessoa Araújo Junior ◽  
...  
Keyword(s):  
Cell Culture ◽  
Tumor Suppressor Genes ◽  
Primary Cell Culture ◽  
Gene Mutations ◽  
Tp53 Gene ◽  
Primary Cell ◽  
Loss Of Function ◽  
Gene Expressions

ABSTRACT: Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1496-1496 ◽  
Author(s):  
Ilaria Iacobucci ◽  
Andrea Ghelli Luserna Di Rorà ◽  
Maria Vittoria Verga Falzacappa ◽  
Enrico Derenzini ◽  
Anna Ferrari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cell Lines ◽  
Single Agent ◽  
Time Dependent ◽  
Leukemia Cells ◽  
In Vivo Models

Abstract Abstract 1496 Introduction: Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods: Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results: Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions: Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

The DNA Methyltransferase Inhibitor Decitabine Induces DNA Damage, Cell Cycle Arrest and Apoptosis in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1833-1833
Author(s):  
Ken Maes ◽  
Miguel Lemaire ◽  
Jordan Gauthier ◽  
Hendrik De Raeve ◽  
Eline Menu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Cycle Arrest ◽  
Phase Arrest ◽  
Damage Response ◽  
M Phase

Abstract Abstract 1833 Multiple myeloma (MM) is still an incurable plasma cell malignancy, thus highlighting the need for alternative treatment options. Currently, strategies for therapy are being developed targeting epigenetic modification using epigenetic modulating agents like histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi). 5-aza-2'-deoxycitidine or decitabine (DAC) is a DNMTi and is FDA approved for treatment of myelodysplastic syndrome and has beneficial clinical effects against leukemia. The anti-tumor effects are ascribed to two non-mutual exclusive modes of action. Relative low doses are thought to lead to passive CpG demethylation resulting in re-expression of genes silence by DNA methylation and apoptosis, while relative high doses are cytotoxic by inducing a DNA damage response together with cell cycle arrest and apoptosis. In multiple myeloma (MM), preclinical data regarding the effects of DAC is, however, limited. Therefore, we investigated the cytotoxic effects of DAC in MM both in vitro and in vivo. In addition, we evaluated the combination of DAC with the pan-HDAC inhibitor JNJ-26481585. First, we assessed the effects of DAC on cell cycle progression and apoptosis on a panel of MM cell lines. We used one murine (5T33MMvt) and 5 human (OPM-2, RPMI 8226, LP-1, KMS-11 and NCI-H929) MM cell lines. In general, DAC could affect cell cycle progression by inducing either a G0/G1-phase arrest or a G2/M-phase arrest. The 5T33MMvt and LP-1 cells were arrested in the G2/M-phase, while OPM-2 and NCI-H929 cells underwent a G0/G1-phase arrest. Subsequently, apoptosis occurred in all cell lines. Interestingly, the 5T33MMvt cells were relatively sensitive, as nM doses of DAC were sufficient to induce massive apoptosis in a relative short incubation time (2 days). The human cell lines were less sensitive since higher doses (μM range) and longer incubation time (3–5 days) were necessary to induce apoptosis, with the OPM-2 cells being the least sensitive. To determine the potential mechanisms more in detail, we focused on the 5T33MMvt and OPM-2 cells. In both cell lines, DAC-mediated apoptosis was associated with caspase activation and PARP cleavage, Bim upregulation and posttranslational changes in Mcl-1 expression. The G2/M-phase arrest in the 5T33MMvt cells was accompanied by phosphorylation of CDK-1 and an increase in cyclinB1 expression. In both cell lines, p27 protein expression was increased, what may contribute to the cell cycle arrest. Furthermore, in the 5T33MMvt cells, a DNA damage response was activated as evidenced by a clear induction of ATM and H2AX phosphorylation. This was not the case for the OPM-2 cells, in which we observed no ATM activation and only a modest H2AX phosphorylation upon DAC treatment. In addition, the tumor suppressor p53 was phosphorylated on ser15 upon DAC treatment in both cell lines, indicating a potential role of p53. However, a p53 inhibitor, pifithrin-α, could not abrogate DAC-induced apoptosis indicating that p53 transactivation is not essential in this process. Next, we used the syngeneic 5T33 murine MM model (5T33MM) to investigate the in vivo effects of DAC. 5T33MM mice were daily treated with 0.1, 0.2 and 0.5 mg/kg DAC. We observed a significant decrease in serum M-protein, bone marrow plasmacytosis and spleno- and hepatomegaly compared to vehicle treated mice. These effects led to a significant increase in survival probability of DAC treated mice (p≤0.001). Lastly, we evaluated the possibility of combining DAC with a pan-HDAC inhibitor JNJ-26481585 (JNJ-585). DAC and JNJ-585 synergistically induced cell death in RPMI-8226, OPM-2 and 5T33MMvt cells. We further demonstrated the combinatory effects of DAC and JNJ-585 in the 5T33MM murine model. Here, we observed enhanced effects of DAC and JNJ-585 on serum M-protein, BM tumor load and survival (p≤0.001) compared to either agent alone. In conclusion, DAC shows potent anti-MM effects both in vitro and in vivo. Mechanistically, we observed induction of a DNA damage response and/or cell cycle arrest. Apoptosis was caspase-mediated but independent of the transactivation of p53. DAC was also efficient in the murine 5T33MM model in which DAC treatment led to a survival benefit. In addition, DAC showed useful in a combination with the HDAC inhibitor JNJ-585. Disclosures: No relevant conflicts of interest to declare.

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