Acidic extracellular pH neutralizes the autophagy-inhibiting activity of chloroquine

Abstract

Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.

Significantly enhanced tumor cellular and lysosomal hydroxychloroquine delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin

“…However, its clinical usefulness is severely limited because highly effective doses are required [13][14][15] to compensate for its nonselective distribution in vivo and its inability to cross tumor cell membranes effectively in the acidic tumor microenvironment. 22 Here we tried to avert all 3 problems by encapsulating HCQ in PEGylated liposomes coated with TR peptide. We show using in vitro and in vivo approaches that HCQ/Lip-TR specifically targets B16F10 tumors and efficiently accumulates HCQ within tumor cells and lysosomes.…”

“…3D), the TR peptide appears to help overcome the pH barrier that normally prevents efficient internalization of HCQ under acidic conditions. Indeed, acidic extracellular pH inhibits chloroquine endocytosis, 22 and its structure is similar to that of HCQ. Taken together, our results suggest that the TR peptide supports efficient delivery of HCQ/ Lip-TR preferentially to tumors expressing high levels of the ITGAV-ITGB3/integrin a v b 3 receptor in an acidic microenvironment.…”

“…21 Recently, Pellegrini et al found that the acidic tumor microenvironment could seriously inhibit the ability of chloroquine derivatives such as HCQ to cross cell membranes. 22 Their observations suggest that even though chloroquine could be well distributed to tumors, the cellular uptake is still limited to well-perfused regions but cannot be achieved in acidic regions, predicting possible limitations in efficacy of chloroquine and its derivatives in autophagy inhibition. Thus, methods are urgently needed to deliver HCQ selectively to tumors, overcome the barrier of the acidic tumor microenvironment and improve HCQ concentration within lysosomes.…”

Therapeutic antitumor efficacy of tumor-derived autophagosome (DRibble) vaccine on head and neck cancer

“…Chloroquine can affect lysosomal activity and induce accumulation of LC3-II only at the physiological pH 7.4, but not at acidic pH conditions. 36 Therefore, we speculated that culture medium was at acidic pH when cells had been cultured for more than 36 hours. Ammonium chloride did not inhibit lysosomal activity under acidic extracellular pH.…”

The acidic microenvironment as a possible niche of dormant tumor cells

“…Acute and chronic exposure to low pH promotes high level of autophagic activity, and this sustains cell survival and aggressiveness, while the pharmacological inhibition of autophagy in such acidic tumor cells reduces their ability to survive [55]. Furthermore, it was demonstrated that acidosis blocks the inhibitory activity of chloroquine on autophagy, thus chloroquine does not result effective in tumor treatment [56]. Up-regulation of autophagy-mediated degradation and recycling of cellular substrates by acidic cancer cells can support metabolic pathways considering that an acidic microenvironment is generally characterized by low nutrients and oxygen.…”