Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

<div><p>Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents <i>in vitro</i>. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP^-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP^+/+ MEF cell lines. Using a tet-off <i>atg5</i> MEF cell line, knockout of <i>atg5</i>, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”.</p></div

Effects of autophagy inhibition on macrolide-induced cytotoxicity in tet-off <i>atg5</i> MEF cell line.

<p>(A) m5-7 cells were pre-treated with/without doxycycline (Dox, 10 ng/mL) for 7 days for complete autophagy inhibition. Subsequently, the cells were further treated with/without macrolides for 24 hrs under the complete culture or AAD culture conditions. The cellular proteins were separated by 15% SDS-PAGE, and immunoblotted with LC3B Ab. Immunoblotting with anti-GAPDH mAb was used as an internal control. The positive control was the cell lysate derived from PANC-1 cells treated with AZM (50 μM) for 24 hrs and used in a previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164529#pone.0164529.ref013" target="_blank">13</a>]. (B) After Dox treatment, m5-7 cells were cultured in the complete culture medium or AAD culture medium containing 10% FBS for 24 hrs. Viable cell numbers were assessed and expressed as percentage to the viable cells cultured in the complete culture medium at each indicated culture period. Data are presented as means ± SEM. *p < 0.05 (Dox (-) vs Dox (+)). (C) Cell growth inhibition of m5-7 cells cultured under the AAD culture condition with or without macrolides for 24 hrs. Data are presented as means ± SEM. *p < 0.05 (vs control). ‘n.s.’ indicates ‘not significant’.</p

CHOP induction by macrolides under AAD culture condition in accordance with intracellular amino acid starvation in CAL 27 cells.

<p>(A) After treatment of CAL 27 cells with/without macrolides in the AAD culture medium containing 10% FBS for various lengths of time, the cells were lysed and cellular proteins were separated by 11.25% SDS-PAGE and immunoblotting with anti-phosphor-p70S6K (Thr389) and anti-p70S6K Abs. The positive control was the cell lysate derived from Detroit 562 cells having constitutively activated PI3K mutation. Immunoblotting with anti-GAPDH mAb was used as an internal control. (B) Addition of amino acids into the culture medium canceled CHOP induction: After 4 hrs of depletion of amino acids in the culture medium in the presence of AZM/CAM (50 μM), amino acids were added at final concentrations of 1% MEM non-essential amino acids and 2% MEM essential amino acids, and subsequently cultured for the indicated time periods. Thereafter, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotting with anti-CHOP mAb. The positive control was the CAL 27 cell lysates cultured in the complete culture medium with thapsigargin (300 nM) for 6 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. (C) CAL 27 cells were cultured with thapsigargin (300 nM) for 6 hrs with/without the addition of amino acids into the cell culture medium as described above, and immunoblotted with anti-CHOP mAb. Immunoblotting with β-actin mAb was used as an internal control.</p

Proposed scheme for cell death induction by macrolide antibiotics under amino acid-depleted culture condition.

<p>Under the amino acid-depleted culture condition, autophagy is induced as an adaptive response. AZM/CAM block autophagy flux leading to the shortage of the intracellular amino acid pool and failure of cellular amino acid homeostasis. This results in the induction of an ER-stress-related pro-apoptotic transcription factor, CHOP.</p

Apoptosis induction after treatment with macrolides under AAD culture condition.

<p>(A) CAL 27 cells in the AAD culture medium containing 10% FBS with or without macrolides were treated with various concentrations of either Z-VAD-fmk (upper panel) or necrostatin-1 (lower panel). Data are presented as means ± SEM. *p < 0.05 (vs without Z-VAD-fmk/necrostatin-1). (B) Immunoblotting with PARP Ab from the cell lysates of CAL 27 cells cultured in the complete culture medium or AAD culture medium with or without macrolides for 12 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from HL-60 cells treated with vitamin K2 for 48 hrs was used as a positive control for apoptosis induction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164529#pone.0164529.ref017" target="_blank">17</a>]. (C) Flow cytometry with annexin V/PI double staining after 24-hr treatment of CAL 27 cells with AZM/CAM (50 μM) in the complete culture medium or AAD culture medium containing 10% FBS. Numbers indicate the percentage of the cells in each area.</p