Nitric oxide and P-glycoprotein modulate the phagocytosis of colon cancer cells

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.