Figure 5. Autophagy and ATP-Dependent Improvement of Anticancer Chemotherapy by Hydroxycitrate.

(A) Autophagy-dependent release of ATP in vitro. ATP concentration (mean ± SEM, n = 7, pooled from two experiments) was measured in the supernatants of autophagy-competent or Atg5^KD CT26 cells treated with 20 mM hydroxycitrate (HC) and/or 2 μM mitoxantrone (MTX).

(B) Autophagy-dependent release of ATP in vivo. Autophagy-competent or Atg5^KD CT26 colorectal cancers expressing a luciferase variant detecting extracellular ATP were treated with MTX and/or HC, and ATP release was monitored until 48 hr post chemotherapy. Representative images (left panel) and corresponding box plots of quantification (right panel) expressed as fold change of photon flux ratio.

(C) Requirement of autophagy and extracellular ATP for the anti cancer effects of the MTX/HC combination. Tumor growth curves (mean ± SEM) from C57BL/6 mice bearing autophagy-competent or autophagy-deficient (Atg5^KD) MCA205 tumors or MCA205 tumors or over expressing a CD39 transgene (CD39⁺) received MTX and/or HC.

(D) Inhibition of HC-induced autophagy by IGF-1. U2OS cells stably expressing the autophagic marker GFP-LC3 were treated with HC alone or in combination with 10 mM insulin growth factor 1 (IGF-1).

(E–G) (E) Autophagy was measured by assessing the abundance of GFP-LC3 puncta per cell (in the presence of BafA1) and quantified in (F, mean ± SEM, n = 3). WT immunocompetent C57BL/6 mice bearing MCA205-derived tumors were treated with HC alone or in combination with recombinant IGF-1. Autophagy was assessed by immunoblotting in the tumor (F) and quantified in (G, mean ± SEM, n = 3).

(H) Reversal of the therapeutic effect of HC by IGF-1. WT immunocompetent C57BL/6 mice were inoculated subcutaneously with murine fibrosarcoma MCA205 cells and tumors were treated withHCand/or MTX, aloneor combined with intraperitoneal injectionsof recombinant IGF-1 protein (averaged ±SEM tumor growth curves).

Data were analyzed by ANOVA for multiple comparisons (A and B), unpaired Student's t test (E and G), linear mixed-effect modeling (C and H), and linear modeling (H). Levels of significance: ***p < 0.001, **p < 0.01, *p < 0.05 (comparisons with Co/PBS unless indicated by a segment); ^###p < 0.001, ^#p < 0.05 (comparisons between chemotherapy and chemotherapy + CRM); ns, not significant. For the indication of all statistical comparisons of (C) and (H), see Tables S1 and S2.