Metabolism and X-ray irradiation-induced responses of the human non-small cell lung cancer cell line H358 in hypoxia

Abstract

With a survival rate of around 15 %, lung cancer is the deadliest cancer of our today´s world. One of its main treatment pillars - radiation therapy - is severely limited by hypoxia, a state of low environmental oxygen concentration which is a phenomenon present in most solid tumors. In this present work, the effect of hypoxia (1 % and 0.1 % oxygen) compared to normoxic conditions (20 % oxygen) on growth and metabolism of the non-small cell lung cancer (NSCLC) cell line H358 was assessed. Furthermore, experiments to evaluate effects of the combination of cell growth in hypoxia and subsequent irradiation with X-rays on the induction and repair of DNA double strand breaks, the activation of the nuclear factor klight-chain-enhancer of activated B-cells (NF-κB) pathway and subsequent interleukin-6 (IL-6) and -8 (IL-8) production, cell cycle arrest and cell survival were conducted. H358 cells grown in hypoxia showed an approximately 4 h longer doubling time compared to normoxic cells as well as a lower glucose uptake and a higher lactate production per living cell. 2 Gy X-ray irradiation induced slightly less DNA double strand breaks in hypoxic cells compared to normoxic cells. However, these results were not observed to be significant. 8 Gy X-ray irradiation resulted in a stronger NF-κB pathway activation via the nuclear translocation of p65-containing dimers in hypoxia and resulted in a higher IL-6 production as well as a higher production of IL-8 per living H358 cell independent of the growth condition. The same X-ray dose induced a G2 cell cycle arrest again independent of the growth condition, however this arrest was observed to be timewise delayed and less prominent in hypoxic cells. Hypoxia itself was observed to additionally induce a G1 cell cycle arrest in H358 cells. Colony forming ability assays showed that hypoxia leads to higher radioresistance for lower X-ray doses (<4 Gy) compared to normoxic H358 cells when cells are plated immediately after irradiation and a trend reversal for cells given a 24 h recovery phase before plating, as this led to a higher radioresistance of hypoxic cells at X-ray doses >4 Gy. The results of this work help to better understand the behavior of NSCLC cells in hypoxia with and without X-ray irradiation and thereby provide a basis to unravel radioresistance mechanisms in NSCLC cells and possible connected pathways.