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

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”.

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis

“…In addition, both the LC3B-I and LC3B-II bands became faint under AAS culture conditions, although AAS should induce more autophagy. We previously observed the same phenomenon when the cells had markedly upregulated autophagy along with leading LC3B-II degradation ( 18 ). As the expression level of LC3B-II is determined by the balance of synthesis and breakdown, the degradation of LC3B-II appeared to occur more rapidly than the induction of LC3B-II under AAS culture conditions.…”

“…It is now well known that the depletion of amino acids in the cell culture medium promotes the induction of autophagy for the adaptation to the shortage of the intracellular amino acid pool ( 18 – 21 ). It has also been reported that EGFR-TKIs, such as GEF induce cytoprotective autophagy in various EGFR-expressing cell lines ( 9 – 13 ).…”

“…We have also reported that macrolide antibiotics, such as azithromycin (AZM) and clarithromycin (CAM) exert an inhibitory effect on autophagy flux in myeloma and squamous cell carcinoma cell lines ( 17 , 18 ). Of note, AZM and CAM exert cytotoxic effects under amino acid-depleted culture conditions in these cell lines along with the upregulation of the pro-apoptotic transcription factor, CHOP/GADD153, although they exhibit no cytotoxicity in complete culture medium ( 18 ). Mammalian target of rapamycin (mTOR), is a master regulator that combines amino acid availability to cell growth and autophagy ( 19 – 21 ).…”

Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K

“…Numerous studies on different cancer preclinical models [23][24][25][26][27][28][29][30][31] have addressed the role of Cla as an autophagy inhibitor, without clarifying the underlying molecular mechanism. We here demonstrate that the Cla-dependent modulation of autophagy in human CRC cells is biphasic: Cla initially stimulates autophagosome initiation and expansion, which is related to a decrease in both Akt and ERK1/2 phosphorylation.…”

“…A number of in vitro [15][16][17][18][19] and in vivo [19][20][21][22] preclinical studies have shown that inhibiting autophagy enhances CRC cell death and could be used to restore chemosensitivity. In this context, macrolide antibiotics, such as clarithromycin (Cla), erythromycin (Er), and azithromycin, have been proven to exert antitumor activity in several preclinical models of cancer, including CRC, by modulating the autophagic flux [23][24][25][26][27][28][29][30][31] . Macrolide antibiotics were effective either alone or in combination with conventional treatments [24][25][26][27]29,31 .…”