Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1α and hypoxia-mediated mitophagy

Abstract

The antiangiogenic effects of sustained sorafenib treatment in hepatocellular carcinoma (HCC) lead to the occurrence of hypoxia-mediated drug resistance resulting in low therapy efficiency and negative outcomes. Combined treatments with coadjuvant compounds to target the hypoxia-inducible factor-1α (HIF-1α) represent a promising therapeutic approach through which sorafenib efficiency may be improved. Melatonin is a well-documented oncostatic agent against different cancer types. Here, we evaluated whether melatonin could enhance sorafenib cytotoxicity and overcome the hypoxia-mediated resistance mechanisms in HCC. The pharmacological melatonin concentration (2 mM) potentiated the oncostatic effects of sorafenib (5 μM) on Hep3B cells even under hypoxia. Melatonin downregulated the HIF-1α protein synthesis through the inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase beta-1 (p70S6K)/ribosomal protein S6 (RP-S6) pathway, although the indole enhanced Akt phosphorylation by the mTORC1/C2 negative feedback. Furthermore, melatonin and sorafenib coadministration reduced the HIF-1α-mitophagy targets expression, impaired autophagosome formation and subsequent mitochondria and lysosomes colocalization. Together, our results indicate that melatonin improves the Hep3B sensitivity to sorafenib, preventing HIF-1α synthesis to block the cytoprotective mitophagy induced by the hypoxic microenvironment, an important element of the multifactorial mechanisms responsible for the chemotherapy failure.

Keywords: hepatocarcinoma; hypoxia-inducible factor-1α; hypoxia-mediated mitophagy; melatonin; sorafenib.

Figure 1. Dose-dependent Hep3B cells viability response to sorafenib and melatonin

Cells were incubated under normoxia (Nx) or hypoxia (Hx) for 48 h in the absence or presence of sorafenib (2.5, 5 or 10 μM) and/or melatonin (1 or 2 mM). Viability was analyzed by MTT. Data are expressed as a percentage of mean values ± SD of experiments performed in triplicate. *p<0.05 vs normoxic or hypoxic non-treated cells.

Figure 2. Effect of melatonin and sorafenib on HIF-1α expression and role of HIF-1α in the hypoxia-mediated resistance

Hep3B cells incubated under normoxia (Nx) or hypoxia (Hx) were treated with sorafenib (5 μM) and/or melatonin (2 mM). (A) HIF-1α protein levels were measured by Western blot 24 h post-treatment. (B) Representative immunoblots of HIF-1α and HIF-2α 48 h post-treatment (upper panel). Viability was analyzed by MTT assays 48 h after the different treatments (lower panel). Data are expressed as a percentage of mean values ± SD of experiments performed in triplicate. *p<0.05 vs control siRNA hypoxic cells. (C) Representative immunoblots from the melatonin effects on HIF-1α dynamics. Hep3B cells were preincubated under hypoxia for 3 h (Lane 2), and melatonin was subsequently added for 1.5 and 3 h (Lanes 3 and 4). After melatonin removal, cells were incubated again under hypoxia conditions for additional 1.5 and 3 h (Lanes 5 and 6) to restore the initial hypoxic microenvironment. Lane 1 shows normoxic HIF-1α basal levels.

Figure 3. Effect of melatonin on HIF-1α transcription, protein synthesis and degradation

Hep3B were incubated under normoxia (Nx) or hypoxia (Hx) in the absence or presence of melatonin (2 mM). (A) HIF-1α mRNA levels were measured by qRT-PCR at 0, 1.5, 3 and 6 h after treatment. Data are expressed as mean values of arbitrary units (a.u.) ± SD of three independent experiments. *p<0.05 vs normoxia at the same time. (B) ROS production was measured using DCF quantification under normoxia and hypoxia plus melatonin and/or NAC (5 mM) treatment for 0, 3 and 6 h. Data are expressed as a percentage of mean values ± SD of experiments performed in triplicate. *p<0.05 vs normoxia at the same time, #p<0.05 significant differences between melatonin-treated vs untreated cells under hypoxia. (C) Effect of melatonin on HIF-1α synthesis was assayed by Western blot. Cells were incubated under normoxia or hypoxia for 0, 1.5, 3 and 6 h in the absence or presence of MG132 (10 μM), DMOG (1 mM) or melatonin. (D) Effect of melatonin on HIF-1α degradation analyzed by Western blot. Cells were incubated under normoxia or hypoxia for 6 h with or without MG132 (10 μM), CHX (100 μM) or melatonin.

Figure 4. Impact of melatonin on PI3K/Akt/mTOR, HIF-1α synthesis pathway assayed by Western blot

(A) Cells were incubated under hypoxia (Hx) for 0, 1, 3 and 6 h with or without melatonin (2 mM). Lane 1 of each panel shows normoxic basal protein levels. (B) and (C) Effect of rapamycin (20 nM) and LY294002 (50 μM) alone or in combination with melatonin on HIF-1α and PI3K/Akt/mTOR-related proteins measured by Western blot.

Figure 5. Effect of melatonin and sorafenib on hypoxia-induced mitophagy and role of mitophagy in sorafenib hypoxia-mediated resistance

Hep3B cells were incubated under normoxia (Nx) and hypoxia (Hx) in the absence or presence of sorafenib (5 μM) and/or melatonin (2 mM). (A) BNIP3 and NIX levels were analyzed by Western blot 24 h post-treatment. Lane 1 shows basal protein levels under normoxia. (B) Cell viability from non-silenced and silenced cells was analyzed by MTT assay 48 h post-treatment. Data are expressed as a percentage of mean values ± SD of experiments performed in triplicate. *p<0.05 vs control siRNA hypoxic cells, #p<0.05 significant differences between BNIP3 and control siRNAs cells under the different treatments. (C) p62 and LC3-II were analyzed by Western blot 6, 12 and 24 h after treatment. Lane 1 of each panel shows its normoxic basal levels. Immunoblots were quantified using ImageJ software. Data are expressed as mean values of arbitrary units (a.u.) ± SD of three independent experiments. †p<0.05 vs normoxia, *p<0.05 vs hypoxia, #p<0.05 vs significant differences between cotreated vs sorafenib-treated cells under hypoxia at the same time. (D) Immunoblots of p62 and LC3-II from Hep3B cells incubated with the indicated treatments for 12 h and with or without Baf-A1 (5 μM) for the last 3 h. (E) Confocal images show mitochondria and lysosomes localization in Hep3B up to 24 h, using Tom20 (red) and LAMP2 (green) antibodies respectively. DAPI (blue) denotes cell nucleus. Magnification is 63X and scale bar represents 15 μm.

Figure 6. Model of melatonin inhibition of hypoxia-induced mitophagy in HCC

Melatonin inhibits HIF-1α synthesis blocking subsequent hypoxia-mediated mitophagy induced by sustained sorafenib treatment. Melatonin downregulates mTORC1 and its effectors, p70S6K and RP-S6, for HIF-1α protein synthesis inhibition. Thus, HIF-1α inhibition by melatonin diminishes the transcription of the mitophagy initiators BNIP3 and NIX, preventing the prosurvival mitophagy.