Unlike glycolytic
enzymes that directly catabolize
glucose to
pyruvate, the family of 6-phosphofructo-2-
kinase/
fructose-2,6-bisphosphatases (PFKFBs) control the conversion of
fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic
enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human
cancer cells, and derivatives of a PFKFB3 inhibitor,
3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced
ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional
glucose starvation induced by inhibition of PFKFB3 in
tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-
tumor effects of PFKFB3 inhibitors.
RESULTS: We found that selective inhibition of PFKFB3 with either
siRNA transfection or 3PO in HCT-116
colon adenocarcinoma cells caused a marked decrease in
glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62
protein expression,
acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in
reactive oxygen species since N-acetyl-
cysteine blocked both the conversion of LC3-I to LC3-II and the increase in
acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including
chloroquine, increased 3PO-induced apoptosis as measured by double staining with
Annexin V and
propidium iodide in both HCT-116 cells and
Lewis lung carcinoma (LLC) cells.
Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the
tumors.
CONCLUSIONS: We conclude that PFKFB3 inhibitors suppress
glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of
cancer.