Macrolide antibiotics enhance the antitumor effect of lansoprazole resulting in lysosomal membrane permeabilization‑associated cell death

Abstract

The proton pump inhibitor lansoprazole (LPZ) inhibits the growth of several cancer cell lines, including A549 and CAL 27. We previously reported that macrolide antibiotics such as azithromycin (AZM) and clarithromycin (CAM) potently inhibit autophagic flux and that combining AZM or CAM with the epidermal growth factor receptor inhibitors enhanced their antitumor effect against various cancer cells. In the present study, we conducted the combination treatment with LPZ and macrolide antibiotics against A549 and CAL 27 cells and evaluated cytotoxicity and morphological changes using cell proliferation and viability assays, flow cytometric analysis, immunoblotting, and morphological assessment. Combination therapy with LPZ and AZM greatly enhanced LPZ-induced cell death, whereas treatment with AZM alone exhibited negligible cytotoxicity. The observed cytotoxic effect was not mediated through apoptosis or necroptosis. Transmission electron microscopy of A549 cells treated with the LPZ + AZM combination revealed morphological changes associated with necrosis and accumulated autolysosomes with undigested contents. Furthermore, the A549 cell line with ATG5 knockout exhibited complete inhibition of autophagosome formation, which did not affect LPZ + AZM treatment-induced cytotoxicity, thus excluding the involvement of autophagy-dependent cell death in LPZ + AZM treatment-induced cell death. A549 cells treated with LPZ + AZM combination therapy retained the endosomal Alexa-dextran for extended duration as compared to untreated control cells, thus indicating impairment of lysosomal digestion. Notably, lysosomal galectin-3 puncta expression induced due to lysosomal membrane permeabilization was increased in cells treated with LPZ + AZM combination as compared to the treatment by either agent alone. Collectively, the present results revealed AZM-induced autolysosome accumulation, potentiated LPZ-mediated necrosis, and lysosomal membrane permeabilization, thus suggesting the potential clinical application of LPZ + AZM combination therapy for cancer treatment.

Azithromycin enhances the cytotoxicity of DNA‐damaging drugs via lysosomal membrane permeabilization in lung cancer cells

“…Recently, we also showed that AZM enhanced the anti–cancer effect of lansoprazole (LPZ), a proton pump inhibitor, by upregulating LMP, although AZM or LPZ alone did not induce LMP in A549 cells. 35 Taken together, these results suggest that AZM induces lysosomal membrane fragility in cancer cells. Furthermore, it has been reported that malignant transformation alters lysosomal structure and functions and makes cancer cells more sensitive to lysosome‐targeting reagents, 36 , 37 suggesting that AZM could be a good candidate for cancer therapy.…”

“…Further studies are required to clarify the molecular mechanisms that connect LMP and apoptosis. Recently, we also showed that AZM enhanced the anti–cancer effect of lansoprazole (LPZ), a proton pump inhibitor, by upregulating LMP, although AZM or LPZ alone did not induce LMP in A549 cells 35 . Taken together, these results suggest that AZM induces lysosomal membrane fragility in cancer cells.…”

Induction of synergistic non‑apoptotic cell death by simultaneously targeting proteasomes with bortezomib and histone deacetylase 6 with ricolinostat in head and neck tumor cells

“…2.0 (Intron Biotechnology, Inc.) according to the manufacturer's instructions. As a positive control for necroptosis induction, HT-29 cells (ATCC), a colorectal adenocarcinoma cell line, were authenticated using STR profiling, pre-treated with Z-VAD-fmk (20 µM) in DMEM for 30 min, followed by additional treatment with CHX (10 µg/ml) and human TNF-α (20 ng/ml) for 8 h, as described previously (17).…”

“…Cells were seeded onto a 60-mm dish (1x10 6 cells/dish) and pre-cultured for 24 h. After treatment with BTZ and/or RCS for 48 h, adherent cells were harvested by trypsinization. The cells were spread on glass slides by centrifugation (1,000 x g for 5 min at room temperature, then stained with May-Grünwald-Giemsa (Muto Pure Chemicals Co., Ltd.), as previously described (17). Samples were examined by digital light microscopy using a BZ-X810 microscope (Keyence Corporation).…”

Nonantimicrobial Actions of Macrolides: Overview and Perspectives for Future Development

“…The interactions of macrolides with lipids are outlined in section B. Interactions with Lipids. AZM-induced lysosomal dysfunction was shown to result in lysosomal biogenesis and accumulation in several cancer cell lines, and although alone it did not cause cell death, it potentiated the anticancer action of a proton-pump inhibitor causing cell necrosis (Takeda et al, 2020). AZM lysosomal accumulation has also been shown in fibroblasts isolated from patients with IPF, which has been suggested to be related to AZMinduced apoptosis (Krempaska et al, 2020).…”