RésuméIn malignant tumors, tumor macrophages (TAM) are abundant. They are alternatively activated in M2 macrophages. They promote tumor growth and their role in drug resistance has also been described. The aim of this thesis was to study the impact of polarized M1 and M2 macrophages on the response of cancer cells HepG2 and A549 to etoposide-induced apoptosis under normoxia and hypoxia.
Polarized macrophages were obtained from THP-1 monocytes differentiated with PMA and polarized in M1 and M2 macrophages by incubation with IFN-γ and LPS (M1) or IL-4 and IL-13 (M2). Macrophages were co-cultured in indirect contact with A549 or HepG2 cancer cells and both cell populations were incubated in the presence of etoposide under normoxia or hypoxia. Results showed that M1 macrophages were cytotoxic and increased the apoptosis induced by etoposide under normoxia and hypoxia. Under hypoxia, their cytotoxic effect was slightly reduced. Conversely, M2 macrophages protected cancer cells from apoptosis induced by etoposide. In HepG2 cells, the protective effect of macrophages M2 was added to that exerted by hypoxia. In A549 cells, unprotected by hypoxia, M2 macrophages had the same effect on cancer cell apoptosis under normoxia and hypoxia.
To identify the mediator(s) responsible for the effect of M2 macrophages on apoptosis, conditioned media were generated. Normoxic M2 conditioned media reproduced to a lesser extent the effects observed in indirect co-culture. Interestingly, hypoxic M2 conditioned media were more protective than normoxic M2 conditioned media. Protein or RNA depletion from hypoxic M2 conditioned media did not affect their ability to protect HepG2 cells from etoposide-induced apoptosis. Similarly, lipids extracted from conditioned media had no effect. However, control medium acidification to reach the conditioned medium pH reproduced the protection.
Incubation of HepG2 or A549 cells with medium acidified with HCl at a pH of 6.9-6.8 decreased etoposide-induced apoptosis. The protective effect increased in parallel with decreasing pH. The protection induced by acidosis is associated with a cell cycle arrest in the G1 phase, which decreased the sensitivity of cells to etoposide. Reduced etoposide-induced DNA damage caused a decrease in the abundance of p53 and in the expression of some p53 target proteins like Bak, an important pro-apoptotic protein implicated in intrinsic apoptosis.
The possible effect of acidification in co-culture was investigated. Because M1 and M2 macrophage conditioned media had the same pH but different effects on etoposide-induced apoptosis, the protection provided by M2 involved secreted molecules.
M2 macrophages protect cancer cells from etoposide-induced apoptosis. Under hypoxia, protective factors secreted by M2 macrophages and acidic pH act together to decrease cancer cell death induced by etoposide. Further experiments are needed to identify the protective mediators released by M2 macrophages.
|la date de réponse||17 déc. 2014|
|Superviseur||Martine Raes (Promoteur), Carine MICHIELS (Promoteur), JEAN-PIERRE GILLET (Président), Jean-Michel DOGNE (Jury), Muriel Moser (Jury) & Agnès Noël (Jury)|