Kelly Cell Research Articles

Isobolographic interactions of cannabidiol and AM 1172 with cisplatin in human neuroblastoma and glioblastoma cell lines: An in vitro study.

Glioblastoma is the most aggressive brain cancer in humans with very poor prognosis and high mortality rate. Despite advances in treatment, glioblastoma almost always recurs and new therapeutic methods are urgently needed. This study aimed at assessing the cytotoxic and antiproliferative effects of AM 1172 and cannabidiol (two cannabinoid receptor ligands) in vitro, when used alone and in combination with cisplatin (a standard cytotoxic drug), in various human neuroblastoma (CHP-134, KELLY), human glioblastoma (U-87MG and T98G) and rat glioblastoma (C6) cell lines. Our experiments showed that AM 1172 and cannabidiol inhibited cell proliferation with IC50 values in the range of 2.29-17.21μM (for AM 1172) and 11.61-20.35μM (for cannabidiol), respectively. The selectivity index for AM 1172 ranged from 0.61 to 4.60 and that for cannabidiol ranged from 1.45 to 2.55 in the studied glioblastoma and neuroblastoma cell lines. With isobolographic analysis, it was found that AM 1172 combined with cisplatin exerted a synergistic interaction in the CHP-134cell line (p<0.01). In contrast, AM 1172 when combined with cisplatin produced an antagonistic interaction in the C6 cell line (p<0.01). The remaining combinations of AM 1172 with cisplatin in the U-87MG, KELLY and T98G cell lines were additive. In case of cannabidiol, its combination with cisplatin produced an antagonistic interaction in the T98G cell line (p<0.0001), whereas the combinations of cannabidiol with cisplatin in the CHP-134, U-87MG, KELLY, and C6 cell lines were additive in nature. The synergistic and additive interactions for the combination of AM 1172 and cannabidiol with cisplatin seem to be a promising direction in glioblastoma therapy. Unfortunately, the combinations producing antagonistic interactions (AM 1172+cisplatin in C6, and cannabidiol+cisplatin in T98G cell lines) should be avoided due to the antagonistic antiproliferative effect of two-drug mixtures.

Phenotypic changes on central nervous system (CNS) tumor cell lines cultured in vitro 2D and 3D models and treated with cisplatin

Despite aggressive therapy, most patients with brain tumors present disease relapse due to the cellular and molecular nature of these tumors. One of the models that best explains the heterogeneity observed in CNS tumors is the presence of cancer stem cells (CSCs). In this paper, we evaluated platinum-based response in brain tumor U-87 MG, LN-18, and KELLY cell lines cultured in monolayer (2D) and neurosphere (CSC enrichment– 3D) models. We evaluated mRNA expression of heat shock proteins (HSPA1A, HSPB1, HSPA1AL, TRAP1, and HSPD1), and DNA repair gene ERCC1. Changes in cell cycle and glycosylation profile were assessed by flow cytometry. After treatment with cisplatin, we found that the mRNA expression of HSPs markedly increased in the U-87 MG and LN-18 neurosphere cells. In KELLY monolayer cells, cisplatin induced upregulation of all genes. In KELLY neurosphere cells, only the HSPA1A, HSPB1, TRAP1, and HSPD1 genes were upregulated. The proportion of cells in the G0/G1 phase was significantly higher in U-87 MG neurosphere cisplatin-treated cells. A trend towards a greater proportion of cells in the S phase of U-87 MG monolayer cisplatin-treated cells was also observed. On the other hand, a significant decrease in the number of cells in the S phase and an increase in G2/M was observed in LN-18 monolayer cisplatin-treated cells. Glycosylation analysis using lectins showed a higher surface binding for PNA in the U-87 MG treated monolayer and a lower binding for Concanavalin A in the treated neurospheres. The binding of Isolectin GS-IB4, GSII, and SBA in KELLY monolayer cisplatin-treated cells was lower whereas the proportion of cells labeled with Concanavalin A was higher. In the KELLY neurosphere cisplatin-treated cells, the binding of Concanavalin A was lower than nontreated cells. Thus, our findings strongly supported the idea that definitions of phenotypic characteristics may help to establish better therapeutic strategies for brain tumors.

Single-Cell Gene Network Analysis and Transcriptional Landscape of MYCN-Amplified Neuroblastoma Cell Lines.

Neuroblastoma (NBL) is a pediatric cancer responsible for more than 15% of cancer deaths in children, with 800 new cases each year in the United States alone. Genomic amplification of the MYC oncogene family member MYCN characterizes a subset of high-risk pediatric neuroblastomas. Several cellular models have been implemented to study this disease over the years. Two of these, SK-N-BE-2-C (BE2C) and Kelly, are amongst the most used worldwide as models of MYCN-Amplified human NBL. Here, we provide a transcriptome-wide quantitative measurement of gene expression and transcriptional network activity in BE2C and Kelly cell lines at an unprecedented single-cell resolution. We obtained 1105 Kelly and 962 BE2C unsynchronized cells, with an average number of mapped reads/cell of roughly 38,000. The single-cell data recapitulate gene expression signatures previously generated from bulk RNA-Seq. We highlight low variance for commonly used housekeeping genes between different cells (ACTB, B2M and GAPDH), while showing higher than expected variance for metallothionein transcripts in Kelly cells. The high number of samples, despite the relatively low read coverage of single cells, allowed for robust pathway enrichment analysis and master regulator analysis (MRA), both of which highlight the more mesenchymal nature of BE2C cells as compared to Kelly cells, and the upregulation of TWIST1 and DNAJC1 transcriptional networks. We further defined master regulators at the single cell level and showed that MYCN is not constantly active or expressed within Kelly and BE2C cells, independently of cell cycle phase. The dataset, alongside a detailed and commented programming protocol to analyze it, is fully shared and reusable.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines

BackgroundHigh-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated.MethodsCell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined.ResultsCell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 μM and 0.22 μM, compared to SY5Y cells and SKNAS cells (IC50 0.96 μM and 0.83 μM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model.ConclusionTW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.

Novel Structural Abnormalities Involving Chromosomes 1, 17 and 2 Identified by Fluorescence In Situ Hybridization (FISH) and/or Cytogenetic Karyotyping in Kelly and SH-SY5Y Human Neuroblastoma Cell Lines, Respectively

Abstract: MYCN amplification and 1p36 deletion are important poor prognostic factors in neuroblastoma. 1p36 deletion and unbalanced translocations involving chromosomes 1, 17 and 2p were often reported in neuroblastoma cell lines. We aimed to investigate novel chromosomal abnormalities that are likely to affect neuroblastoma progression in Kelly and SH-SY5Y cell lines. Therefore, we analyzed the metaphase chromosomes using fluorescence in situ hybridization (FISH) method with probes specific to the chromosome bands 2p24 and 1p36 in SH-SY5Y and Kelly, respectively. Moreover, the rearrangements of chromosomes 1 and 17 from Kelly were re-examined by cytogenetic karyotyping. FISH analysis shows duplication of chromosome 2p24 on the long arm of a partner chromosome resulted from an unbalanced translocation der(?9)t(2;?9)(p24;q?34) in SH-SY5Y, suggesting that duplication of 2p24 locus containing MYCN gene may contribute to triggering MYCN amplification in neuroblastoma. On the other hand, FISH and karyotype analyses reveal three copies of chromosome 1 that consist of an intact chromosome 1, an extra derivative chromosome 1 with terminal and interstitial deletions (:p32→q25::q41→qter), and another including only interstitial deletion (pter→q25::q41→qter), leading to monosomy of the long arm segment 1q25-q41 in Kelly. These results suggest that chromosome 1q25-q41 may contain one or more tumour suppressor genes important for neuroblastoma progression. Together, FISH analysis shows that an additional 1p36 locus in Kelly is translocated to the short arm of extra chromosome 17, where the p53 tumour suppressor gene is located. Consequently, these novel structural abnormalities involving chromosomes 1, 17 and 2 could be contributed to the tumourigenicity of neuroblastoma cells.

Abstract LB-245: Preclinical study of epigenetic drug-based differentiation therapy for neuroblastoma

Abstract Neuroblastoma (NBL) is the most common extracranial solid tumor in children. We previously reported that the CpG island methylator phenotype (CIMP) of NBL was strongly associated with poor prognosis, and also suggested that CIMP may be a possible target for DNA demethylation therapy. Differentiation therapy with 13-cis-retinoic acid has already been established as the standard for high-risk NBLs in the USA. In this study, we aimed to establish an “epigenetic drug-based differentiation therapy” using a combination of a DNA demethylating agent (5-aza-2’-deoxycytidine: 5-Aza-CdR) and a differentiation agent (tamibarotene: TBT), a new synthetic retinoid. Treatment with 5-Aza-CdR suppressed the growth of 12 NBL cell lines by increasing the number of cells in the S-phase. Genome-wide DNA methylation analysis revealed that 5-Aza-CdR treatment induced global DNA hypomethylation, and that genes related to cell death and neurological processes were enriched as hypomethylated genes, suggesting that DNA demethylation therapy might assist the differentiation agent in inducing neuron differentiation. TBT induced differentiation of five NBL cell lines along with induction of neural extension and upregulation of differentiation markers, such as HOXD4, NGFR, and NTRK1. Pretreatment with 5-Aza-CdR increased the expression levels of differentiation markers, indicating that 5-Aza-CdR enhanced TBT-induced differentiation in vitro. Finally, the tumor suppression effect of 5-Aza-CdR and TBT in vivo was investigated using a mouse xenograft model of KELLY and NB-1 cell lines. Although a synergistic effect of 5-Aza-CdR and TBT was not apparent, they could induce significant tumor regression without severe side-effects. From these data, we concluded that 5-Aza-CdR and TBT had antitumor activity in vitro and in vivo, and that epigenetic drug-based differentiation therapy is a promising therapeutic strategy for NBL. Citation Format: Naoko Hattori, Akiko Mori, Kana Kimura, Emi Kubo, Kiyoshi Asada, Hiroshi Kawamoto, Toshikazu Ushijima. Preclinical study of epigenetic drug-based differentiation therapy for neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-245.

Isouronium and N-hydroxyguanidinium derivatives as Cell growth inhibitors: A comparative study

Based on the results obtained from a computational study on the suitability of the isouronium and N-hydroxyguanidinium cations as hydrogen bond donors/acceptors, the DNA binding of a series of isouronium derivatives was assessed by DNA thermal denaturation experiments and compared to related N-hydroxyguanidines. Due to the poor DNA binding observed, the nature of the diaromatic linker was explored by preparing the corresponding amide-linked bis-isouronium derivative and measuring its DNA affinity. Next, the inhibitory effects of the isouronium derivatives on cell viability were evaluated in two different cancer cell lines providing IC50 values in the range of 36.9–57.4 μM (HL-60, leukemia), and 17.3–33.9 μM (Kelly, neuroblastoma). These values are comparable to those previously found for the N-hydroxyguanidine series. Compounds with the –S- linker (3, 6, and 10) proved to be considerably active in the HL-60 cells and even more active in the Kelly cell line. No correlation was found between DNA minor groove binding and cell growth inhibition; hence, activity may depend on different modes of action. Further studies into the apoptotic potential of these compounds indicated that, besides inhibiting cell viability and proliferation, derivatives 9 and 10, are significant apoptosis-inducers in both cell lines. Results obtained with HL-60 cells suggest that G2/M arrest and subsequent apoptosis induced by compound 10 are associated with microtubular depolymerisation, loss of mitochondrial membrane potential and activation of the caspase cascade. Moreover, the effects of compound 10 on cell viability and apoptosis in two non-cancereous cell lines (NIH3T3 and MCF-10A) indicate none or minimal toxicity.

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Aromatic Bis-N-hydroxyguanidinium Derivatives: Synthesis, Biophysical, and Biochemical Evaluations

In this paper we report the synthesis of a new family of hydroxyguanidinium aromatic derivatives (4a-g) as potential minor groove binders and cytotoxic agents. Their DNA affinity was evaluated by thermal denaturation experiments using salmon sperm DNA. The antiproliferative effects of derivatives 4a, 4d, and 4f were evaluated in human promyelocytic HL-60, breast carcinoma MCF-7, and neuroblastoma Kelly cell lines using the AlamarBlue viability assay, and IC(50) values were obtained. All three compounds were active in the HL-60 cell line. In particular, 4b exhibits antiproliferative effects in all three cell lines while 4d reduced HL-60 and Kelly viability. Both 4b and 4d produced considerable antiproliferative activity in the Kelly cell line. Derivative 4d was chosen for further cell cycle and apoptosis studies using flow cytometric analysis of cellular DNA content.

Treatment of neuroblastoma and rhabdomyosarcoma using RGD-modified liposomal formulations of patupilone (EPO906)

BackgroundPatupilone (EPO906) is a microtubule stabilizer with a potent antitumor effect. Integrin αVβ3-binding (RGD) liposomes were loaded with EPO906, and their antitumor efficacy was evaluated in two pediatric tumor models, ie, neuroblastoma and rhabdomyosarcoma.MethodsIntegrin αVβ3 gene expression, RGD-liposome cellular association, and the effect of EPO906 and liposomal formulations of EPO906 on cell viability were assessed in vitro in human umbilical vein endothelial cells (HUVEC), in the RH-30 rhabdomyosarcoma cell line, and in the Kelly neuroblastoma cell line. In vivo, mice bearing neuroblastoma or rhabdomyosarcoma tumors were treated with EPO906, EPO906-liposomes, or EPO906-RGD-liposomes. Tumor growth, cumulative survival, and toxicity were monitored.ResultsIntegrin αVβ3 was highly expressed in HUVEC and RH-30, but not in Kelly cells. Accordingly, RGD-liposomes were highly associated with HUVEC and RH-30 cells in vitro, but not with the Kelly cells. EPO906 and its liposomal formulations inhibited HUVEC, RH-30, and Kelly cell viability to the same extent. In vivo, EPO906 1.5 mg/kg and liposomal EPO906 potently inhibited tumor growth in both xenograft models without triggering major toxicity. At this dose, liposomal EPO906 did not enhance the antitumor effect of EPO906 in neuroblastoma, but tended to have an increased antitumor effect in rhabdomyosarcoma. Using a lower dose of EPO906-RGD-liposomes significantly enhanced cumulative survival in rhabdomyosarcoma compared with EPO906 alone.ConclusionEPO906 shows a strong antitumor effect in neuroblastoma and rhabdomyosarcoma, without triggering major side effects. Its liposomal encapsulation does not alter its activity, and enhances cumulative survival when EPO906-RGD-liposomes are used at low dose in rhabdomyosarcoma.

Abstract 1715: High ALK gene copy number as a predictor of response to crizotinib in non-small cell lung cancer cell lines

Abstract Introduction: The anaplastic lymphoma kinase (ALK) gene translocation is a rare event, described in approximately 4-6% of lung adenocarcinoma. ALK translocation is a robust predictive factor for ALK inhibitors sensitivity. However, increased ALK gene copy number seems to be a frequent event, described in 13-17% of Non-small cell lung cancer (NSCLC). The goal of this study was to explore the predictive value of ALK High copy number for 2 ALK inhibitors, crizotinib and TAE684 in NSCLC cell lines. Methods: 27 NSCLC cell lines were screened for ALK copy number by FISH after cytospin. ALK high copy number (HCN) was defined as the presence of ≤ 5 copies of ALK. FISH with CEP2 was performed to determine the ploïdy status in cell lines with HCN. As crizotinib is a dual c-MET and ALK inhibitor, FISH c-MET was performed in selected cell lines for in vitro studies. C-MET scoring was based on Cappuzzo criteria. In vitro sensitivity was evaluated through WST1 assays and clonogenic tests. Kelly, an ALK mutated neuroblastic cell line, was added as a positive control. Cells were seeded in triplicates in 96-well plates 18h before the treatment with TAE 684 or crizotinib with concentrations varying from 50 to 4000nM and 100 to 10000nM respectively. The half maximal inhibitory concentration of the two drugs (IC50) was estimated after 72h of treatment with 4 and 5 parameters logistic regression models. In clonogenic tests, crizotinib was tested with three schedules of concentrations: low dose (50nM), intermediate (200nM) and high (1000nM). Results: Among NSCLC cell lines, 6 (22%) displayed more than 5 copies of ALK, 19 (70%) presented a gain of 3 or 4 ALK copy number, only one cell line exhibited normal ALK copies and one harbored EML4-ALK translocation. FISH with CEP2 revealed a polysomy of chromosome 2 in cases with ALK HCN. C-MET amplification was observed in only three cell lines. Based on ALK FISH status, 9 NSCLC cell lines were selected to undergo in vitro assays, 6 exhibiting ALK HCN (H2030, H661, A427, H3255, BEN, H1299) and 3 exhibiting low ALK copy number (LCN) (H1975, H1651, H1650). Kelly and A549 cell lines were added as sensitive and resistant controls respectively. In ALK HCN cell lines, two (H661 and A427) were as sensitive as Kelly for crizotinib through clonogenic and WST1 assays. In addition, ALK HCN cell lines exhibited lower IC50 for anti-ALK drugs than cell lines with ALK LCN (median IC50 with crizotinib values: 1750nM [300-2800nM] in ALK HCN cell lines versus 4500nM [800-8000nM] in ALK LCN cell lines, p=0.35). The trend of crizotinib activity was not due to c-MET inhibition. Conclusion: The in vitro assays suggest that ALK HCN may be a predictive marker for sensitivity to crizotinib. The predictive value of ALK HCN would be investigated in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1715. doi:1538-7445.AM2012-1715

Abstract 5595: ALK mutant-driven cell lines develop crizotinib resistance by secondary mutations and ALK-independent means

Abstract The clinical success of crizotinib against mutant ALK-expressing cancers has generated great interest; however, the emergence of drug resistance is limiting its durability in some patients. In NSCLC, crizotinib resistance is linked to secondary mutations within EML4-ALK at amino acids L1196M, C1156Y, L1152R. A mutation at amino acid F1174L reduced the sensitivity to criozotinib in a patient with an inflammatory myofibroblastic tumor harboring a RANBP2-ALK translocation. The goal of our studies was to evaluate mechanisms of crizotinib resistance in a range of ALK-mutant expressing cell lines. Cells from ALCL (Karpas299), NSCLC (NCI-H3122 and NCI-H2228) or neuroblastoma (SKNSH and KELLY) developed resistance to crizotinib after being exposed to incremental increases in the compound over several weeks. NPM-ALK expressing Karpas299 cells independently acquired point mutations in ALK at amino acids N1178H and I1171N. As the concentration of drug was increased, cells that harbored the NPM-ALK N1178H mutation also expressed a mutation at G1269A. Each mutation enhanced the temperature dependent auto-phosphorylation activity of the kinase and diminished the inhibitory effect of crizotinib on downstream ALK signaling. Mutations in Karpas299 cells were also accompanied by up regulation of a drug efflux transporter, BCRP, whose heightened expression level was dependent on the presence of the compound. In contrast no additional mutations were discovered in ALK in crizotinib resistant NCI-H3122 and NCI-H2228 (EML4-ALK) or SKNSH and KELLY (ALK F1174L) cell lines. Instead both NCI-H3122 and NCI-H2228 resistant cell lines displayed increased levels of phospho-HER2 and HER3 and activation of AKT and ERK that may be due to autocrine secretion of a growth factor. NSCLC resistant lines also displayed an apparent change in phenotype with a mesenchymal-to-epithelial transition. SKNSH and KELLY are inherently less sensitive to crizotinib. We found that these cells may survive exposure to high levels of the compound by altering RET expression. Our observations represent potential mechanisms of resistance demonstrated in a small number of cell lines. It will be important to align these results with emerging data from drug-resistant patients to understand the multiple mechanisms by which resistance might develop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5595. doi:1538-7445.AM2012-5595

The Role of p14ARF Methylation in Neuroblastoma Minimal Residual Disease

Objectives: The aim of the present study is to investigate the clinical significance of epigenetic changes in neuroblastoma, we evaluated the relationship between therapeutic variables and the pattern of gene methylation in neuroblastoma cell lines. Methods: In this minimal residual disease model, cytotoxic effects of blocked p14ARF by a demethylating agent on KELLY and SHSY-5Y human neuroblastoma cell lines were assessed following addition of the drugs used in the Turkish Pediatric Oncology Group (TPOG) chemotherapy protocol. Drug induced effects on cell viability, cell damage and apoptotic cell death ratio were assessed with trypan blue dying. Cytotoxic effects differed among the used neuroblastoma cell lines. For the investigation of mechanisms of this effect, p14 ARF gen methylation and expression levels, and MYCN expression levels were targeted. Expressions of mRNA and protein were determined with real-time PCR and ELISA, respectively. Results: The p14 ARF gene expression levels in KELLY 5-aza-CdR, Vincristin, Vincristin+5aza-CdR, Dacarbazine, Ifosfamide+5-aza-CdR, Doxorubicine+5-aza-CdR, Etoposide+5aza-CdR groups were found to be significantly higher than those measured in corresponding groups of SHSY-5Y (p<0.05). The percentages of p14ARF gene promoter unmethylation were higher in MYCN (-) groups than MYCN (+) groups (p=0.034). Conclusion: In this pioneering study we suggest not only a new therapeutic approach for early or late relapses following the standard treatment protocols, but also demonstrate the effect of chemoterapeutic agents on p14 ARF gene. The present study suggests that clinically aggressive neuroblastoma cell lines have aberrant methylation of p14ARF genes and provides a

Antiproliferative and newly attributed apoptotic activities from an invasive marine alga: Caulerpa racemosa var. cylindracea

Caulerpa racemosa (Forsskål) is a green marine alga which spreads from tropical to warm-water regions. Due to having invasive capacity C. racemosa var. cylindracea is a well-known biological pollution in Mediterranean Sea. One of the most important secondary metabolites of C. racemosa is Caulerpenyne (CPN). In the present study, antiproliferative and apoptotic effects of C. racemosa var. cylindracea extract and purified CPN on two well-known neuroblastoma cell lines, SHSY5Y and Kelly, are investigated. The antiproliferative and, additionally, newly attributed apoptotic effects of both C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines have been shown in the present study. IC 50 values are 0.59 ± 0.06; 1.06 ± 0.23 g wet alga/methanol and 5.64 ± 0.09; 6.02 ± 0.09 μM CPN for C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines, respectively. Percentages of apoptotic cells of SHSY5Y and Kelly in 0, 0.1 and 1 μM CPN conditions were 1.00 ± 0.71, 3.00 ± 0.71 and 49.40 ± 3.78, 39.60 ± 6.19 and 78.00 ± 2.74, 69.40 ± 3.78, respectively. In conclusion, the present study shows the antiproliferative effect of C. racemosa var. cylindracea extract and newly attributed apoptotic effects of C. racemosa var. cylindracea this extract. Compared to other alkylating anticancer drugs, CPN and also C. racemosa var. cylindracea extract might be considered as an alternative native source of antitumor drugs. Inasmuch as both C. racemosa extract and CPN have shown both antiproliferative and apoptotic effects on SHSY5Y and Kelly cell lines, the CPN and CPN derivatives might be considered as multifunctional agents in cell metabolism.

Ubiquitous aberrant RASSF1A promoter methylation in childhood neoplasia.

The role of RASSF1A has been elucidated recently in regulating apoptosis and cell cycle progression by inhibiting cyclin D1 accumulation. Aberrant RASSF1A promoter methylation has been found frequently in multiple adult cancer types. Using methylation-specific PCR and reverse transcription-PCR, we investigated epigenetic deregulation of RASSF1A in primary tumors, adjacent nontumor tissues, secondary metastases, peripheral blood cells, and plasma samples from children with 18 different cancer types, in association with their clinicopathologic features. Regardless of the tumor size, ubiquitous RASSF1A promoter methylation was found in 67% (16 of 24) of pediatric tumors, including neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, pancreatoblastoma, adrenocortical carcinoma, Wilms' tumor, Burkitt's lymphoma, and T-cell lymphoma. A majority (75%) of pediatric cancer patients with tumoral RASSF1A methylation was male. Methylated RASSF1A alleles were also detected in 4 of 13 adjacent nontumor tissues, suggesting that this epigenetic change is potentially an early and critical event in childhood neoplasia. RASSF1A promoter methylation found in 92% (11 of 12) of cell lines largely derived from pediatric cancer patients was significantly associated with transcriptional silencing/repression. After demethylation treatment with 5-aza-2'-deoxycytidine, transcriptional reactivation was shown in KELLY, RD, and Namalwa cell lines as analyzed by reverse transcription-PCR. For the first time, RASSF1A methylation was detected in 54% (7 of 13), 40% (4 of 10), and 9% (1 of 11) of buffy coat samples collected before, during, and after treatment, correspondingly, from pediatric patients with neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, rhabdomyosarcoma, Burkitt's lymphoma, T-cell lymphoma, or acute lymphoblastic leukemia. Concordantly, RASSF1A methylation was found during treatment in plasma of the same patients, suggesting cell death and good response to chemotherapy. RASSF1A methylation in tumor or buffy coat did not correlate strongly with age, tumor size, recurrence/metastasis, or overall survival in this cohort of pediatric cancer patients. Of importance, epigenetic inactivation of RASSF1A may potentially be crucial in pediatric tumor initiation.

Micronuclei and apoptosis in glioma and neuroblastoma cell lines and role of other lesions in the reconstruction of cellular radiosensitivity.

It is now well established that micronuclei frequency does not always rank cell lines according to radiosensitivity. There is, however, a growing interest in reconstructing cellular radiosensitivity (measured by colony assay) from concurrent micronucleus and apoptosis data. Using a variety of radiosensitive and radioresistant cell lines, we have derived a missing parameter--Poe, the probability of cell death by other events such as small deletions, chromosome aberrations, late apoptosis and necrosis which are undetectable by micronucleus and apoptosis assays performed at a single time point. In the radioresistant glioma cell lines G120, G60, G28, G44 and G62 (SF2 > or =0.59), a characteristic threshold dose exists above which cell loss due to undetectable lesions occurs. In the radiosensitive SK-N-SH and KELLY cell lines (SF2 < or =0.43), the Poe parameter is positive at very low doses, reaches a maximum and declines at higher doses. In the radiation resistant G28 cells, Poe was found to be below zero for doses up to 6 Gy. In the G62, G44 and G120 cell lines, the threshold doses to induce Poe events were 0.87, 3.04 and 3.85 Gy, respectively. Cell death by undetectable lesions is a cell-specific and time-dependent variable. Micronucleus and apoptosis assays performed concurrently and at a specific time point miss cell death due to other events and this may be the reason why reconstruction of cellular radiosensitivity from micronucleus and apoptosis data fails.