Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines

Abstract

Abstract. Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance. IntroductionSoft tissue sarcomas are tumors arising from mesenchymal cell precursors that are committed towards the morpho genesis of soft tissues such as fat, muscle and deep skin tissues. Rhabdomyosarcoma (RMS) is considered a myogenic tumor and is classified as the most frequent sarcoma affecting children and adolescents (1). The current classification defines five different histotypes, with embryonal (eRMS) and alveolar (aRMS) subsets being the most frequently observed in children <5 years and in adolescents, respectively (2,3). The eRMS variant is the most treatable and most common subtype representing ~80% of RMS, while aRMS is more aggressive and characterized by a poorer prognosis. The genetic alterations characterizing eRMS commonly involve the loss of heterozygosis on chromosome region 11p15.5 (4), gain of chromosomes (5,6) and mutations on genes involved with growth factor signaling pathways (7)(8)(9)(10)(11)(12)(13)(14)(15). This leads to uncontrolled cell growth and the interruption of proper myogenic differentiation. Conversely, the aRMS subset is commonly characterized by the expression of Pax3-forkhead box O1 (FOXO1), a fused transcription factor derived from the chromosomal translocation t(2;13)(q35;q14), which juxtaposes the DNA-binding domain of Pax3 to the potent transactivation domain of FOXO1 (16). In the absence of the original Pax3 transactivation domain, the chimeric protein drives in a constitutive manner the transcription of numerous genes involved in muscle embryogenesis, such as c-MET and FGFR4, essentially maintaining the muscle precursors in a long-lasting proliferative state and thereby facilitating tumor initiation, aggressiveness and me...

Melatonin action in tumor skeletal muscle cells: an ultrastructural study

“…In this work we have first confirmed previous findings showing the ability of Mel administered at pharmacological concentrations (Rodriguez et al, 2013) in triggering cell death of RMS cells (Codenotti et al, 2015); moreover, since the dissolution solvent represents an important variable potentially influencing the Mel action, typically the absolute EtOH (Salucci et al, 2014a;Salucci et al, 2014b) or DMSO (Martín-Renedo et al, 2008; Jardim-Perassi et al, 2014), here we have established that both these solvents did not influence the efficacy of neurohormone activity as death inducer in RH30 cell line.…”

“…Cells were also routinely tested for mycoplasma contamination using the LookOut Mycoplasma PCR Detection Kit (MP0035 Lookout, Sigma), according to the manufacturer's instructions. RH30 cells were cultured in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1% antibiotics (Penicillin and Streptomycin) and maintained at 37 • C in humidified air with 5% CO 2 (Codenotti et al, 2015).…”

“…After washing with 0.1 M phosphate buffer, adherent and suspended cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h. The suspended cell population was depositated on polylysine-coated coverslips and the samples were post-fixed with 1% osmium tetroxide (OsO 4 ) in 0.1 M phosphate buffer solution (PBS) for 1 h. After alcohol dehydration, the samples were critical point dried, gold sputtered and observed using a Philips 515 scanning electron microscope (Codenotti et al, 2015).…”

“…Sample were incubated with specific primary antibodies, Rabbit anti-Bax, polyclonal (Rabbit, sc-526, Santa Cruz Biotecnology, Dallas, USA) or Rabbit anti-Bcl-2, polyclonal (Rabbit, sc-492, Santa Cruz Biotecnology, Dallas, USA) followed by peroxidase-conjugated secondary antibodies (anti-mouse IgG from Santa Cruz Biotechnology, Dallas, USA; antirabbit IgG from Thermo Scientific, Erembodegem, Belgium). The immunocomplexes were visualized using the enhanced chemiluminescence reagent (GeneSpin, Milan, Italy) and immune-reactive bands were quantified using densitometry analyses (Software Gel Pro Analyzer, version 4) (Codenotti et al, 2015).…”

“…Over the past years, Mel, beside its strong and documented antioxidant effects (Salucci et al, 2014b;Ganie et al, 2015), has been shown to act as an anticancer drug in different tumors (Sánchez-Hidalgo et al, 2012;Batista et al, 2014;Codenotti et al, 2015), for example via induction of the mitochondrial apoptosis (Bejarano et al, 2009) or attenuation of telomerase activity both in vivo and in vitro (Leon-Blanco et al, 2003).…”