Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation.

Abstract

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.

• Authors: Caitlin A Schonewolf, Monal Mehta, Devora Schiff, Hao Wu, Bruce G Haffty, Vassiliki Karantza, Salma K Jabbour
• Journal: World journal of gastrointestinal oncology (World J Gastrointest Oncol) Vol. 6 Issue 3 Pg. 74-82 (Mar 15 2014) ISSN: 1948-5204 [Print] China
• PMID: 24653797 (Publication Type: Journal Article)