Hypoxia significantly reduces aminolaevulinic acid-induced protoporphyrin IX synthesis in EMT6 cells

Abstract

SummaryWe have studied the effects of hypoxia on aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) synthesis in EMT6 monolayer cultures characterized by different cell densities and proliferation rates. Specifically, after ALA incubation under hypoxic or normoxic conditions, we detected spectrofluorometrically the PpIX content of the following populations: (a) low-density exponentially growing cells; (b) high-density fed-plateau cells; and (c) high-density unfed-plateau cells. These populations were selected either for the purpose of comparison with other in vitro studies (low-density exponentially growing cells) or as representatives of tumour regions adjacent to (highdensity fed-plateau cells) and further away from (high-density unfed-plateau cells) capillaries. The amount of PpIX per cell produced by each one of these populations was higher after normoxic ALA incubation. The magnitude of the effect of hypoxia on PpIX synthesis was dependent on cell density and proliferation rate. A 42-fold decrease in PpIX fluorescence was observed for the high-density unfed-plateau cells. PpIX production by the low-density exponential cells was affected the least by ALA incubation under hypoxic conditions (1.4-fold decrease), whereas the effect on the high-density fed-plateau population was intermediate (20-fold decrease).

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…However, cells treated under these conditions were often compared to cells cultured under air, incorrectly inferring that this oxygenation was representative of "healthy" tissues, and studies have often used short-term decreases in O 2 concentration [4,5], rather than longer periods associated with disease. Additionally, air has been used to represent post-reperfusion oxygenation when modelling reperfusion events in vitro [55,56].…”

“…Non-melanoma skin cancers are the most common form of cancers diagnosed annually in the western world and, despite low mortality, the associated high morbidity [15] provides a significant incentive to identify improved, cosmetically acceptable treatments. Previous studies have considered the effects of hypoxia on the outcome of photodynamic therapies [5,16], although these were limited in their scope and did not attempt to recapitulate the O 2 https://doi.org/10.1016/j.freeradbiomed.2018.08.025 Received 22 July 2018; Accepted 20 August 2018 environment in which superficial epidermoid carcinomas reside. The use of a physiologically accurate in vitro model would therefore support the successful translation of experimental findings into in vivo investigations.…”

“…There is extensive literature on the effects of O 2 concentration on the efficacy of drug and radiation treatments in cancer cell killing, both in vivo and in vitro [4][5][6][7][8][9]. However, most studies have characterized these effects in vitro in cells that have been cultured, long-term, in a hyperoxic environment (i.e.…”

“…air) with subsequent short-term exposure to different O 2 concentrations during various treatments (drugs, photodynamic irradiation, hypoxia-reperfusion, etc.) [4,5,7,[9][10][11][12]. The consequences of long-term exposure to hyperoxic or physioxic environments on the subsequent cell killing effects of cancer treatments (e.g.…”

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

“…The oxygen tension and pH of the cellular environment were shown to affect the production of PpIX in exponentially growing bladder cancer cells (Wyld et al, 1998). Georgakoudi et al (1999) reported the effect of the combination of hypoxia, cell density and proliferation rate on PpIX production in EMT6 cells in monolayer cell culture. They demonstrated that PpIX is synthesized less effectively under hypoxia than in normoxic conditions, with the magnitude of the effect of hypoxia being strongly dependent on cell density and proliferation status.…”