Azathioprine desensitizes liver cancer cells to insulin-like growth factor 1 and causes apoptosis when it is combined with bafilomycin A1

Abstract

Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma)>Hep3B (derived from a hepatocellular carcinoma)>HuH6 (derived from a hepatoblastoma)>>HuH7 (derived from a hepatocellular carcinoma)=Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been proven to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis. In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs.

Keywords: 3-MA; 3-methyladenine; AKT; Apoptosis; Autophagy; BAF; Cancer; Desensitization; E2F; E2F transcription factor; EC50; ERK; FOXO1; GST; GTPγS; IRS; LC3; MAPK; N-acetylcysteine; NAC; PARP; PI3K; Rb; Senescence; TSC-2; Thiopurine; bafilomycin A1; extracellular signal-regulated kinase; forkhead box O1; glutathione S transferase; guanosine 5′-O-[gamma-thio]triphosphate; half maximal effective concentration; insulin receptor substrate; mTOR; mammalian target of rapamycin; microtubule-associated protein light chain 3; mitogen-activated protein kinase; phosphoinositide-3-kinase; poly (ADP-ribose) polymerase; protein kinase B; retinoblastoma protein; tuberous sclerosis 2 protein.