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The synthetic retinoid-polyamine conjugate RASP induces S/G2 cell cycle arrest and DNA damage response on HaCat cells
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The synthetic retinoid-polyamine conjugate RASP induces S/G2 cell cycle arrest and DNA damage response on HaCat cells

Katerina Grafanaki,Christos Kontos, Aigli Korfiati, Athanasios Shaukat, Dionissios Papaioannou,Andreas Scorilas, Constantinos Stathopoulos and Denis Drainas

Department of Biochemistry, School of Medicine, University of Patras, Greece
Department of Biochemistry and Molecular Biology, University of Athens, Greece
Department of Medical Physics, School of Medicine, University of Patras, Greece
Department of Chemistry, University of Patras, Greece

Introduction

The novel synthetic N1,N12-Βis(all-trans-retinoyl)spermine retinoid-spermine conjugate (RASP) was tested for its apoptotic effect on HaCaT cells based on its previously reported anti-cancer properties and its minimum side-effects [1, 2, 3]. 

Materials & Methods

HaCaT cells were grown in the presence of 1μΜ (ΙC50)
Apoptosis was monitored at 24 and 48h by confocal microscopy and DNA damage assays
FACS analysis was used to monitor the effect of RASP on apoptosis and cell cycle regulation  
Transcriptomics analysis was performed using DNA microarrays for mRNA expression and NGS for detection of miRNAs, followed by qRT-PCR verification and bioinformatics analysis using the InSybio suite (www.insybio.com)

Results

The RASP-rhodamine conjugate is localized in the cytoplasm and induces apoptosis.
Approximately 10% of the cells undergo apoptosis after 24h in the presence of 1μΜ  RASP.
Confocal microscopy analysis indicated enhanced mitochondrial breakdown in the presence of RASP. Accordingly, RASP induced extensive DNA fragmentation as indicated from representative assays.
Subsequent FACS analysis suggests that RASP exhibits cell cycle arrest predominantly in the S/G2 phase probably due to extensive DNA damage.
Transcriptomics and bioinformatics analyses showed modulation of expression of essential genes that regulate cell cycle progression.
Cyclins A, D and E were found upregulated, with the exception of cyclin B which was found downregulated.
p53 and p21 were moderately modulated, while p27 was significantly upregulated.
The cell cycle regulators BTG2 and c-myc were found downregulated while expression of BTG3 and TOB1 genes were found upregulated.
The expression of several miRNAs targeting the above-mentioned genes was found to be correlated.
Studies have shown that miR-21, miR-23a cluster and miR-27a cooperatively regulate the expression of tumor suppressor gene BTG2.
MiR-182, miR-130a, miR-92a are upregulated in melanoma whereas RASP downregulates them.

Conclusions

RASP represents a new generation retinoid analog with established anti-cancer activity.
RASP induces apoptosis in moderate concentrations and shortly after administration.
It effectively induces apoptosis probably because of its DNA damage ability.
As a response, cell cycle arrest occurs in S/G2 phase due to extensive DNA damage accumulation and thus, cells do enter in mitosis.
RASP effect at the molecular level resembles anti-cancer drugs that exert their apoptotic effect via extensive cytotoxicity due to DNA damage. 
Its effect on HaCaT cells points towards its potential use as effective therapeutic compound against melanoma and skin cancer. 
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