Involvement Of Melatonin In Autophagy-mediated Mouse Hepatoma H22Cell Survival

Melatonin (MLT) is a kind of indole secreted by human pineal glands. It has manyimportant physiological and pathological functions, including regulating sleep and circadianrhythms, preventing mutagenesis, oxidation, providing cardiovascular protection, modulatingimmune, controlling reproduction, regulating mental disorders and delay aging etc. Actingthrough its receptors MT1and MT2, MLT could exert effects of anti-proliferation on manykinds caners, containing beast cancer, lung caner, endometrial, prostate cancers, melanoma,hepatoma, and colon. Autophagy contributes to eliminate overexpressed, toxic, and long-termproteins or organelles, protecting essential cellular components. In cancer cells, autophagicresponses have the same signaling pathway with normal physiological status, whileautophagic pathway is more complicated because of some impairments of the sharedPI3K/Akt/mTOR signaling pathway and various interactions with other pathways. Autophagyis related to cancer through several signaling pathways including PI3K/Akt/mTOR, LKB1/AMPK/mTOR, p53, BCL-2, endoplasmic reticulum stress, and Calcium channel. MLT canenhance cellular autophagic process, while its effects don't act on the autophagic process, buton its inducible factors. Inducing autophagy by MLE may be through activation of mTOR orBeclin-1.Objective To investigate roles of MLT on inducing autophagy in cancers.Methods The model of H22tumor in BALB/c mice was established. Briefly, micewere subcutaneously injected0.1mL of cell suspensions containing1.0×106cells/ml in thedorsal area. After inoculation with H22cells on day0, mice were randomly assigned intothree groups (n=10for each group). Tumor-bearing mice were given an intraperitonealinjection with MLT (10,20mg/kg per day for14days) or sterile normal saline (0.2mL perday for14days). Tumor size was determined by caliper measurement of the largest andperpendicular diameters every3days. Tumor volumes were calculated according to theformula: V=a×b2×0.52, where a is the largest superficial diameter and b is the smallest superficial diameter. On day15, mice were sacrificed; tumors were dissected and weighed. Todetect the Beclin1and LC3expression, tumors tissues excised were fixed in10%formalin orfrozen in80°C. The Beclin-1, LC3expression and Akt phosphorylation was detected byWestern-Blot and Immunofluorescence. The apoptosis of H22cells was detected by flowcytometry. The formation of autophagic vacuoles was further assessed and confirmed bytransmission electron microscopic test. To inhibit autophagy, H22cells were pretreated withBeclin-1RNAi(LV-control, or LV-shbeclin-1) prior to administration of MLT(100μM) orco-treated with MLT(100μM) and3-MA(10mmol/L).Results First, tumor volume was more suppressed in MLT-treated groups than salinecontrol. It was clear that MLT-treated mice showed significant inhibition to the tumor growth.Second, we demonstrated that MLT could induce autophagy in H22-bearing mice,promote expression of Beclin-1and LC-3, and inhibit Akt/mTOR-mediated signaling pathway.Only a few LC3-positive puncta were observed in tumor tissue from saline-treatedH22-bearing mice. However, in the tumor tissue from MTL-treated H22-bearing mice, LC3punctuated staining was increased. In contrast to saline-treated H22-bearing mice,MLT-treated animals showed increased autophagic morphology, such as autophagic vacuolesin the cytoplasm. These results indicate that MLT induced autophagic formation.Finally, we studied effects of blocking autophagic pathways on inducing autophagy byMLT. To identify whether the role of MLT-induced autophagy in hepatoma cells is aprotective or death promoting mechanism, we evaluated the consequences of the disruption ofautophagy by treatment with Beclin-1RNAi or3-MA (an inhibitor of autophagy) on theanti-tumor effects of MLT. Our results show that the level of Beclin-1was significantlydecreased by Beclin-1RNAi. Compared with LV-control, Beclin-1RNAi decreasedMLT-inhibited cell viability and enhanced MLT-induced apoptosis. Further studies showedthat co-treatment with MLT (100μM) and3-MA(10mmol/L) significantly decreased cellviability, compared with treatment with MLT alone. Co-treatment with MLT and3-MA alsosignificantly increased the apoptotic population, especially late apoptotic cells, compared withthecells treated with MLT alone.Conclusion The model of H22tumor BALB/c mice was established and anti-tumorefficacy of MLT was directly observed in tumors excised from H22-bearing mice. MLT couldinduce autophagy in H22-bearing mice, promote expression of Beclin-1and LC-3, and inhibit Akt/mTOR-mediated signaling pathway. Induction of autophagy was associated withinhibition of the PI3K/Akt/mTOR signaling pathway. Furthermore, the disruption of theautophagic process enhances the biological effects of melatonin in H22cells in vitro.