Abstract
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 in vitro. 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 atg5 MEF cell line, knockout of atg5, 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”.
Highlights
Head and neck squamous cell carcinomas (HNSCCs) occur frequently with more than 500,000 new cases diagnosed worldwide each year [1]
Thereafter, we examined autophagy induction under the amino acid-depleted (AAD) culture condition in the presence or absence of macrolides by tracing LC3B-II expression (Fig 1B)
This may be because AZM is more potent than CAM in blocking autophagy flux as we previously described in pancreatic cell lines; other molecular mechanisms may be involved [13]
Summary
Head and neck squamous cell carcinomas (HNSCCs) occur frequently with more than 500,000 new cases diagnosed worldwide each year [1]. HNSCC cells with increased activation of EGFR-related signals require a larger metabolic demand. To meet the amino acid demand, cancer cells increase the expression level of amino acid transporters such as LAT1, as well as the activation of autophagy for degradation of their cytoplasmic proteins and organelles to recycle the intracellular amino acids [6, 7]. Autophagy is a self-eating process wherein cells capsulate their own cytoplasmic proteins and organelles in double-membrane vesicles to form autophagosomes. They are transported to the lysosome following membrane fusion to each other (autolysosomes). Autophagy is indispensable for cell survival and is enhanced under a nutrient-starved condition, in solid tumors because of their excessive metabolic requirements and hypovascular condition [10, 11]
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