Dataset Information

Gene expression profile of murine cutaneous melanoma after systemic treatment with tumor necrosis factor (TNF) associated or not with melphalan (MEL).

ABSTRACT: Cutaneous melanomas are malignant radio and chemoresistant neoplasias presenting high morbidity and elevated mortality rates. Isolated Limb Perfusion (ILP) with Melphalan (Mel) is used in the treatment of non-resectable locally advanced melanomas of extremity sparing the limb from amputation in 40-50% of the cases. Adding the Tumor Necrosis Factor alpha (Tnfa) improves total complete response to 70-90%. The cellular and molecular mechanisms underlying the changes promoted by Mel and Tnfa are not completely understood. In this study we evaluated the impact of systemic Mel and Tnfa administration on tumor growth, analyzed the morphological changes promoted by each treatment, and searched for early molecular targets of Mel and Tnfa, alone or in combination, in a murine melanoma model. Morphological changes were analyzed by histological analysis, while microarray gene expression followed by quantitative RT-PCR were used to search for early molecular targets. We found that Mel systemic administration accounted for the impairment of tumor-growth (p<0.001) and improvement of survival. Tnfa co-administration augmented necrosis (p<0.024) and decreased mitotic rates (p=0.001). We identified a set of 118 potential molecular markers that might be correlated with the observed biologic response to treatment with Melphalan and Tnfa and which could represent potential therapeutic targets in melanoma.

ORGANISM(S): Mus musculus

PROVIDER: GSE21559 | GEO | 2011/12/31

SECONDARY ACCESSION(S): PRJNA126233

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Cutaneous melanomas are malignant radio and chemoresistant neoplasias presenting high morbidity and elevated mortality rates. Isolated Limb Perfusion (ILP) with Melphalan (Mel) is used in the treatment of non-resectable locally advanced melanomas of extremity sparing the limb from amputation in 40-50% of the cases. Adding the Tumor Necrosis Factor alpha (Tnfa) improves total complete response to 70-90%. The cellular and molecular mechanisms underlying the changes promoted by Mel and Tnfa are not completely understood. In this study we evaluated the impact of systemic Mel and Tnfa administration on tumor growth, analyzed the morphological changes promoted by each treatment, and searched for early molecular targets of Mel and Tnfa, alone or in combination, in a murine melanoma model. Morphological changes were analyzed by histological analysis, while microarray gene expression followed by quantitative RT-PCR were used to search for early molecular targets. We found that Mel systemic administration accounted for the impairment of tumor-growth (p<0.001) and improvement of survival. Tnfa co-administration augmented necrosis (p<0.024) and decreased mitotic rates (p=0.001). We identified a set of 118 potential molecular markers that might be correlated with the observed biologic response to treatment with Melphalan and Tnfa and which could represent potential therapeutic targets in melanoma. We used amplified RNA (aRNA) from 18 tumor samples (Control – 5 samples; Mel – 4 samples; Tnfa – 4 samples; and Mel+Tnfa – 5 samples). For replica hybridization with dye swap, aRNA from the tumors and from a pool composed of equal amounts of RNA extracted from the K1735M2, M2R, B16F10, and B16F1 melanoma cells were labeled with Alexa555 or Alexa647. Labeled aRNA samples were hybridized against a glass platform containing 16,128 immobilized sense oligonucleotides (Fox Chase Cancer Center, USA) corresponding to murine genes. Slides were pre-hybridized at 42ºC for approximately 6 hours in a solution containing Denhardt’s 5X (Ficoll 400 0.05g/mL, poli-vynil-pyrrolidone 0.05g/mL, bovine serum albumin 0.05g/mL), SSC (sodium saline citrate) 5X, 0.2% SDS (Sigma), and 1% BSA. For hybridization, we used a mix of 3.5µg of each labeled sample aRNA and reference aRNA. The hybridization solution contained Denhardt’s 5X, formamide 25% (Sigma), SSC 5X, SDS 0.1%, salmon sperm DNA 0.1mg/mL (GE Healthcare), Poly-A 0.1mg/mL (GE Healthcare), and Cot-1 0.1mg/mL (GE Healthcare), to a final volume of 100µL. Hybridizations were carried out on a Gene Tac hybridization station (Genomic Solutions) at 42ºC for about 16h. The slides were removed from the hybridization cassettes directly to a recipient containing a washing solution (SSC 2X, SDS 0.1%) at 42ºC, put in a slide rack, transferred to another recipient containing fresh solution, and washed under constant agitation at 42ºC for 5 min. After that, they were washed twice for 5min in a second wash solution (SSC 0.1X, SDS 0.1%) at room temperature and rinsed five times for 1min at room temperature with a SSC 0.1X solution. The slides were dried upside down in a centrifuge at 1500rpm for 5min. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA), and the spot intensities processed with the help of the ScanArray Express program (Packard BioScience), with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA) with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647, and data were extracted with ScanArray Express software (Packard BioScience) using the histogram method. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The Locally Weighted Scatter-plot Smoothing method (LOWESS), adjusted for linear and non-linear systematic variations, both of the intensity dependent type, mainly for low intensity spots, was employed for data normalization.

Project description:Purpose: Evaluate the molecular changes associated with melphalan-induced cardiotoxicity and rescue by N-acetyl-L-cysteine (NAC) supplementation. Methods: RNA-Seq analysis was performed to analyze global transcriptome profiles of hiPSC-CMs treated with DMSO (Control group), 20 µM melphalan (Mel group), and 20 µM melphalan with 1 mM NAC (Mel+NAC group), respectively. The Illumina TruSeq technology was used to prepare RNA-Seq libraries, and next-generation sequencing was done via an Illumina HWI-ST1276. RNA sequence reads were aligned to the human reference genome (GRCh38) using STAR v2.5. HTSeq v0.6.1 was used to count the read numbers mapped of each gene, and then Fragments Per Kilobase Million (FPKM) was calculated to estimate gene abundance. Differential expression analysis was performed using the DESeq2 R package (2_1.6.3). The resulting P-values were adjusted using the Benjamini and Hochberg's approach. Results: As detected by RNA-Seq, more genes were down-regulated than up-regulated by the treatment of melphalan, whereas NAC supplementation resulted in more genes being up-regulated than down-regulated. Among the top 10 up-regulated genes by melphalan treatment, 4 were direct p53 effectors (CDKN1A, EDXR, TNFRSF10C, and GDF15). And among the top 10 down-regulated genes by melphalan treatment, 5 were correlated to cell adhesion (CDH13, CNTN1, SDK1, CTNND2, and PARD3B). In addition, the up- and down-regulation of genes involved in oxidative stress (e.g., DUOX2 and NOX4) following melphalan treatment (Mel vs. control) was attenuated with NAC supplementation (Mel+NAC vs. Control). Similarly, the up- and down-regulation of genes involved in cardiac muscle contraction (e.g., Ca2+ handling proteins CACNA1C, RYR2 and CASQ2 and cardiac contractile proteins TNNC1, ACTC1, TNNC1) and cardiac conduction (e.g., ATP2B2 and ABCC9) following melphalan treatment (Mel vs. Control) was attenuated with NAC supplementation (Mel+NAC vs. Control). Conclusion: NAC ameliorates melphalan-induced alteration of hiPSC-CM transcriptomic profiles.

Dataset Information

Gene expression profile of murine cutaneous melanoma after systemic treatment with tumor necrosis factor (TNF) associated or not with melphalan (MEL).

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets