Actinomycin D Arrests Cell Cycle of Hepatocellular Carcinoma Cell Lines and Induces p53-Dependent Cell Death: A Study of the Molecular Mechanism Involved in the Protective Effect of IRS-4.

Luis G Guijarro,Sofia Zoullas,Miguel Angel Álvarez-Mon,Eva Pérez-Cuevas,Miguel A Ortega,Jorge Monserrat,Melchor Álvarez-Mon,Julia Bujan,Eduardo Arilla-Ferreiro,Fernando Noguerales-Fraguas,M Val Toledo-Lobo,Miguel A Sáez,Patricia Sanmartin-Salinas

doi:10.3390/ph14090845

Abstract

Actinomycin D (ActD) is an FDA-approved NCI oncology drug that specifically targets and downregulates stem cell transcription factors, which leads to a depletion of stem cells within the tumor bulk. Recently, our research group demonstrated the importance of IRS-4 in the development of liver cancer. In this study, we evaluated the protective effects of IRS-4 against ActD. For this study, three hepatocellular carcinoma cell lines (HepG2, Huh7, and Chang cells) were used to study the mechanism of actinomycin D. Most assays were carried out in the Hep G2 cell line, due to the high expression of stem cell biomarkers. We found that ActD caused HepG2 cell necroptosis characterized by DNA fragmentation, decreased mitochondrial membrane potential, cytochrome c depletion, and decreased the levels of reduced glutathione. However, we did not observe a clear increase in apoptosis markers such as annexin V presence, caspase 3 activation, or PARP fragmentation. ActD produced an activation of MAP kinases (ERK, p38, and JNK) and AKT. ActD-induced activation of AKT and MAP kinases produced an activation of the Rb-E2F cascade together with a blockage of cell cycle transitions, due to c-jun depletion. ActD led to the inhibition of pCdK1 and pH3 along with DNA fragmentation resulting in cell cycle arrest and the subsequent activation of p53-dependent cell death in the HepG2 cell line. Only JNK and AKT inhibitors were protective against the effects of ActD. N-Acetyl-L-cysteine also had a protective effect as it restored GSH levels. A likely mechanism for this is IRS-4 stimulating GCL-GSH and inhibiting the Brk-CHK1-p53 pathway. The assessment of the IRS-4 in cancer biopsies could be of interest to carry out a personalized treatment with ActD.

Highlights

In the last decade, hepatocellular carcinomas (HCC) and hepatoblastomas (HB) had global incidences of 400–930 and 1.2–2.5 cases per million per year [1,2], respectively.Notably, both cancers are highly resistant to chemotherapy [3,4]
We observed a dose-dependent effect of Actinomycin D (ActD) on the loss of the mitochondrial membrane potential of HepG2 cells incubated for 24 h in the presence of 5% fetal bovine serum (FBS) using a JC1 probe (Figure 1A)
We studied the mRNA levels of c-fos and c-jun in HepG2 IRS-4 knockdown cells in the absence or presence of ActD (1 μM) for different periods of time

Summary

Introduction

Hepatocellular carcinomas (HCC) and hepatoblastomas (HB) had global incidences of 400–930 and 1.2–2.5 cases per million per year [1,2], respectively. Both cancers are highly resistant to chemotherapy [3,4]. One of the driving forces of liver cancer relapse post-treatment is the persistence of liver cancer stem cells (LCSCs). LCSCs have been shown to be extremely tumorigenic and resistant to therapy [4]. Despite the large disparity in LCSC characterization, stem cell signatures are maintained in several HCC cell lines including HepG2, HuH7, and Chang cells [5]. HepG2 cells present an upregulation of stem cell transcriptional factors and as a consequence maintain the expression of CD24, CD133, CD44, and SOX 2 [6,7]

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