Melatonin enhances arsenic trioxide-induced cytotoxicity by modulating autophagy in an acute promyelocytic leukemia cell line

Abstract

Background: Arsenic trioxide (ATO)-containing therapeutic strategies are widely used in the treatment of acute promyelocytic leukemia (APL). Growing evidence has shown that melatonin enhances the radio- or chemo-sensitivity of numerous cancer cells. However, whether melatonin is capable of enhancing the cytotoxic effects of ATO in APL cells remains unknown.

Methods: The present study conducted a 24 h melatonin exposure followed by additional 12, 24 or 48 h ATO exposure in the APL cell line NB4 with or without autophagy-related protein 7 (ATG7) silencing by RNA interference. Cell cytotoxicity was evaluated by Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays. Cell apoptosis was assessed by Annexin-V/propidium iodide assay and western blotting against cleaved caspase 3, Bax and Bcl-2. Autophagy was evaluated by western blotting against LC3.

Results: Pre-treatment with a non-cytotoxic dose of melatonin significantly enhanced ATO-mediated reduced cell viability and increased LDH release. Furthermore, melatonin pre-treatment also enhanced ATO-mediated increase in early and late apoptosis, as well as the expression of Bax and cleaved caspase 3, while further decreasing ATO-mediated reduced expression of Bcl-2. Concomitantly, melatonin pre-treatment increased LC3II expression and enhanced the ATO-mediated elevation in LC3II expression. However, autophagy inhibition by ATG7 silencing blocked the enhancing effects of melatonin on ATO-induced apoptosis and cytotoxicity. These findings indicated that melatonin pre-treatment enhances ATO-induced cytotoxicity by modulating ATG7-mediated autophagy.

Conclusions: Melatonin could represent a valuable adjuvant to ATO in APL treatment, particularly in patients with ATO-resistant APL.

Keywords: Acute promyelocytic leukemia (APL); arsenic trioxide (ATO); autophagy; cytotoxicity; melatonin.

Figure 1

Effects of melatonin and ATO treatment, alone or in combination, on cellular cytotoxicity and LDH release in NB4 cells. Cell viability was evaluated in cells treated with (A) 0, 0.1, 0.5, 1 and 2 µM ATO or (B) 0, 0.5, 1, 2 and 4 mM melatonin for 24 or 48 h. (C) Cell viability and (D) LDH levels were determined in cells pre-treated with 1 mM melatonin followed by 2 µM ATO treatment for additional 6, 12, 24 and 48 h. Values are expressed as the mean ± standard error of the mean from ≥3 independent experiments. *P<0.05, **P<0.01 compared with the control groups; ^#P<0.05 compared with the ATO alone-treated group. ATO, arsenic trioxide; LDH, lactate dehydrogenase.

Figure 2

Effects of melatonin and ATO treatment, alone or in combination, on the expression levels of apoptosis-related proteins in NB4 cells. Cells were pre-treated with 1 mM melatonin with or without 2 µM ATO treatment for additional 48 h. (A) Representative images of Annexin V/propidium iodide staining. (B) Annexin V positive cells, indicative of early and late apoptosis, were quantified. (C) Representative images and quantitative analysis of caspase 3 and cleaved caspase 3 expression, as detected by western blotting. (D) Representative images and quantitative analysis of the expression levels of Bax and Bcl-2. The ratio of cleaved caspase 3/total caspase 3 was calculated. GAPDH was used as the internal control. Value are expressed as the mean ± standard error of the mean (n=3), *P<0.05 and **P<0.01 compared with the control or indicated groups. ATO, arsenic trioxide.

Figure 3

Effects of melatonin and ATO treatment, alone or in combination, on autophagy and apoptosis in NB4 cells with or without ATG7 silencing. Cells were treated as described in Figure 2. Autophagy inhibition was performed by ATG7 RNA interference. Representative images (A) and quantitative analysis (B) showing the expression levels of LC3 upon treatments with melatonin, ATO or their combinations, (C,D) with or without ATG7 silencing, and (E,F) the expression levels of caspase 3 and cleaved caspase 3 in cells treated with melatonin and/or ATO with or without ATG7 silencing. The ratio of cleaved caspase 3/total caspase 3 was calculated. GAPDH was used as the internal control. Each value is expressed as the mean ± standard error of the mean (n=3). *P<0.05 and **P<0.01 compared with the control or indicated groups. ns, no statistically significant difference; ATO, arsenic trioxide; ATG7, autophagy-related protein 7; LC3, light chain 3.

Figure S1

The efficiency of ATG 7 silencing. (A) Representative images, (B) quantitative analysis showing the efficiency of ATG 7 silencing. Each value was expressed as the mean ± standard error of the mean (n=3). **P<0.01 compared with the control siRNA (sc-Control) groups.

Figure 4

Effects of ATG7 silencing on the cytotoxicity of ATO treatment with or without melatonin pre-treatment. (A) Cell viability and (B) LDH levels were determined in cells treated with 2 µM ATO with or without ATG7 silencing. (C) Cell viability and (D) LDH levels were determined in cells pre-treated with 1 mM melatonin followed by 2 µM ATO for additional 48 h with or without ATG7 silencing. Each value was expressed as the mean ± standard error of the mean (n=3). **P<0.01 compared with the control groups; ^#P<0.05 compared with the indicated groups. ns, no statistically significant difference; ATO, arsenic trioxide; ATG7, autophagy-related protein 7; LDH, lactate dehydrogenase.