Published online Mar 15, 2014. doi: 10.4251/wjgo.v6.i3.74
Revised: January 11, 2014
Accepted: January 15, 2014
Published online: March 15, 2014
AIM: To investigate whether the inhibition of autophagy by chloroquine (CQ) sensitizes rectal tumors to radiation therapy (RT) or concurrent chemoradiation (chemoRT).
METHODS: In vitro, HCT-116 and HT-29 colorectal cancer (CRC) cell lines were treated as following: (1) PBS; (2) CQ; (3) 5-fluorouracil (5-FU); (4) RT; (5) CQ and RT; (6) 5-FU and RT; (7) CQ and 5-FU; and (8) 5-FU and CQ and RT. Each group was then exposed to various doses of radiation (0-8 Gy) depending on the experiment. Cell viability and proliferative capacity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Clonogenic survival curves were constructed and compared across treatment groups. Autophagy status was determined by assessing the LC3-II to LC3-I ratio on western blot analysis, autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFP-labeled LC3 on fluorescence microscopy. Cell cycle arrest and cell death were evaluated by FACS and Annexin V analysis. All experiments were performed in triplicate and statistical analysis was performed by the student’s t test to compare means between treatment groups.
RESULTS: RT (2-8 Gy) induced autophagy in HCT-116 and HT-29 CRC cell lines at 4 and 6 h post-radiation, respectively, as measured by increasing LC3-II to LC3-I ratio on western blot. Additionally, electron microscopy demonstrated autophagy induction in HT-29 cells 24 h following irradiation at a dose of 8 Gy. Drug treatment with 5-FU (25 μmol/L) induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction. CQ (10 μmol/L) alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation (8 Gy; P < 0.001 and 0.00001, respectively). Significant decrease in clonogenic survival was seen only in the HT-29 cell line, when CQ was combined with RT at doses of 2 and 8 Gy (P < 0.5 and P = 0.05, respectively). There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT.
CONCLUSION: Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro, suggesting that addition of CQ to chemoRT improves CRC treatment response.
Core tip: Autophagy is implicated as a mechanism of resistance to cancer treatment. We hypothesized that chloroquine, a lysosomotropic autophagy inhibitor, would sensitize colorectal cancer (CRC) cell lines to both radiation therapy (RT) alone and concurrent chemoradiation. Our results showed that chloroquine decreased clonogenic survival of CRC cells when given in combination with RT or concurrent 5-fluorouracil and RT. Radiosensitization by chloroquine represents a novel therapeutic approach to enhance treatment efficacy in rectal cancer.