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

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.

Is Insulin Receptor Substrate4 (IRS4) a Platform Involved in the Activation of Several Oncogenes?

“…In myeloid progenitor cells, IRS-4 mediated AKT signaling after insulin stimulation without promoting an anti-apoptotic effect [ 69 ]; however, in pancreatic β-cells (INS-1 cells), IRS4 introduction compensated for the apoptosis induced by a decrease in the IRS2 level [ 70 ]. Some studies suggested that IRS4 overexpression in tumor cells makes them more resistant to treatment with trastuzumab and lapatinib [ 23 ], as well as actinomycin D [ 71 ]. It has recently been shown that IRS4 exerts an effect on tumor cell migration, a very important aspect of tumor growth and metastasis formation.…”

Mitochondrial abnormality in ovarian granulosa cells of patients with polycystic ovary syndrome

“…PcoS is one of the most common hormonal disorders of women during the reproductive years. in patients with PcoS, the mitochondrial state of Gcs is altered, the spindle assembly disordered, and the quality of oocytes decreases (15)(16)(17). any problems of Gcs in growth differentiation, metabolism and apoptosis will directly or indirectly affect the quality of follicular (18)(19)(20).…”

Elimination of apoptotic cells by non-professional embryonic phagocytes can be stimulated or inhibited by external stimuli

“…A similar cleavage-inhibiting effect of actinomycin D was documented in previous studies on in vitro cultured mouse, rabbit and bovine oocytes 10,29,30 , developing preimplantation embryos 31 and blastocysts 15,32 . Actinomycin D at nanomolar concentrations also inhibited proliferation in various cell lines and tissues 33,34 .…”