Decrease in tumorigenic activity of murine hepatoma cells after treatment with antioxidants and melatonin

We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed.     

Antioxidant action on the level of reactive oxygen species in normal and transformed fibroblasts

The level of reactive oxygen species (ROS) in normal (3T3) and transformed (3T3-SV40) murine fibroblasts treated with antioxidants for 15 min was studied using a carboxy-H₂DCFDA fluorescent probe. It was shown that N-acetylcysteine (NAC) decreased the ROS level in both cell types. Antioxidant alpha-lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) caused a pro-oxidant effect. ALA and DHLA in the concentration range from 0.1–1.25 mM increased the ROS level in a dose-dependent manner in both cell types. The ability of ALA and DHLA to activate hydrogen peroxide production is discussed.     

Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice

Melatonin has many protective effects against ischemic stroke, but the underlying neuroprotective mechanisms are not fully understood. Our aim was to explore the relationship between melatonin's neuroprotective effects and activation of the MT2 melatonin receptor in a murine ischemic-stroke model. Male ICR mice were subjected to a transient middle cerebral ischemic/reperfusional injury, and melatonin (5 and 10 mg/kg, ip) was administrated once daily starting 2 h after ischemia. More than 80% of the mice died within 5 days after stroke without treatment. Melatonin treatment significantly improved the survival rates and neural functioning with modestly prolonged life span of the stroke mice by preserving blood-brain barrier (BBB) integrity via a reduction in the enormous amount of stroke-induced free radical production and significant gp91(phox) cell infiltration. These protective effects of melatonin were reversed by pretreatment with MT2 melatonin receptor antagonists (4-phenyl-2-propionamidotetralin (4P-PDOT) and luzindole). Moreover, treatment with melatonin after stroke dramatically enhanced endogenous neurogenesis (doublecortin positive) and cell proliferation (ki67 positive) in the peri-infarct regions. Most ki67-positive cells were nestin-positive and NG2-positive neural stem/progenitor cells that coexpressed two neurodevelopmental proteins (adam11 and adamts20) and the MT2 melatonin receptor. RT-PCR revealed that the gene expression levels of doublecortin, ki67, adamts20, and adam11 are markedly reduced by stroke, but are restored by melatonin treatment; furthermore, pretreatment with 4P-PDOT and luzindole antagonized melatonin's restorative effect. Our results support the hypothesis that melatonin is able to protect mice against stroke by activating MT2 melatonin receptors, which reduces oxidative/inflammatory stress. This results in the preservation of BBB integrity and enhances endogenous neurogenesis by upregulating neurodevelopmental gene/protein expression.     

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.     

Comparison of potential protective effects of melatonin, indole-3-propionic acid, and propylthiouracil against lipid peroxidation caused by potassium bromate in the thyroid gland

Potassium bromate (KBrO3) is a prooxidant and carcinogen, inducing thyroid tumors. Melatonin and indole-3-propionic acid (IPA) are effective antioxidants. Some antioxidative effects of propylthiouracil (PTU)--a thyrostatic drug--have been found. The aim of the study was to compare protective effects of melatonin, IPA, and PTU against lipid peroxidation in the thyroids, collected from rats treated with KBrO3, and in homogenates of porcine thyroids, incubated in the presence of KBrO3. Wistar rats were administered KBrO3 (110 mg/kg b.w., i.p., on the 10th day of the experiment) and/or melatonin, or IPA (0.0645 mmol/kg b.w., i.p., twice daily, for 10 days), or PTU (0.025% solution in drinking water, for 10 days). Homogenates of porcine thyroids were incubated for 30 min in the presence of KBrO3 (5 mM) plus one of the antioxidants: melatonin (0.01, 0.1, 0.5, 1.0, 5.0, 7.5 mM), or IPA (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM), or PTU (0.01, 0.1, 0.5, 1.0, 5.0, 7.5, 10.0 mM). The level of lipid peroxidation products (MDA + 4-HDA) was measured spectrophotometrically in thyroid homogenates. In vivo pretreatment with either melatonin or with IPA or with PTU decreased lipid peroxidation caused by KBrO3--injections in rat thyroid gland. Under in vitro conditions, PTU (5.0, 7.5, and 10.0 mM), but neither melatonin nor IPA, reduced KBrO3-related lipid peroxidation in the homogenates of porcine thyroids. In conclusion, melatonin and IPA may be of great value as protective agents under conditions of exposure to KBrO3.     

Activity of matrix metalloproteinases in normal and transformed mouse fibroblasts exposed to antioxidants

The effects of two antioxidants on the activity of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts were examined. We compared the action of N-acetylcysteine (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, whereas, in transformed 3T3-SV40 cells, the MMP-9 activity was higher. NAC treatment for 2–6 h completely suppressed MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect did not depend on NAC concentration within the range of 1–10 mM. ALA (1.2 mM) did not affect the cells very dramatically; it decreased the MMP-2 activity in both types of cells. MMP-9 activity in the presence of ALA was decreased in 3T3 cells and slightly increased in 3T3-SV40 cells. The activity of the membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, the altered activity of MMP in the presence of antioxidant may influence the intracellular signaling and cell functions.     

Efficacy of some antioxidants supplementation in reducing oxidative stress post sodium tungstate exposure in male wistar rats

This study aimed to evaluate the protective efficacy of some antioxidants against sodium tungstate induced oxidative stress in male wistar rats. Animals were sub-chronically exposed to sodium tungstate (100 ppm in drinking water) for three months except for control group. In the same time, many rats were supplemented orally with different antioxidants (alpha-lipoic acid (ALA), n-acetylcysteine (NAC), quercetin or naringenin (0.30 mM)) for five consecutive days a week for the same mentioned period before. Exposure to sodium tungstate significantly (P < 0.05) inhibit blood δ-aminolevulinic acid dehydratase (ALAD) activity, liver and blood reduced glutathione (GSH) levels and an increase in oxidized glutathione (GSSG) and thiobarbituric acid reactive species (TBARS) levels in tissues. ALA acid and NAC supplementation post sodium tungstate exposure increased GSH and also, was beneficial in the recovery of altered superoxide dismutase and catalase activity, besides, significantly reducing blood and tissue reactive oxygen species and TBARS levels. The results suggest a more pronounced efficacy of ALA acid and NAC supplementation than quercetin or naringenin supplementation post sodium tungstate exposure in preventing induced oxidative stress in rats.     

Effect of alpha-lipoic acid on 3T3 and 3T3-SV40 fibroblasts: Comparison with N-acetylcysteine

In this study, we have investigated the intracellular level of reduced glutathione, cell-cycle phase distribution, and microfilament and microtubule structures in normal (3T3) and transformed (3T3-SV40) fibroblasts exposed to alpha-lipoic acid (ALA) in concentrations of 0.7–5 mM. It was found that ALA treatment increased the glutathione content in transformed cells, but did not affect its level in normal cells; moreover, it also induced the cell-cycle arrest of 3T3 cells (but not 3T3-SV40 cells) and disrupted actin microfilaments in cells of both lines. The ALA effect was compared to that of N-acetylcysteine (NAC), another antioxidant we examined previously. The findings allow us to assert that each of these antioxidants impacts on distinct target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. However, intermediate steps of ALA and NAC action may be common (altered intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).     

Blockade of Endogenous Reactive Oxygen Species by N-Acetyl-L-Cysteine Suppresses the Invasive Activity of Rat Hepatoma Cells by Modulating the Expression of Hepatocyte Growth Factor Gene

We have already reported that exogenously added reactive oxygen species (ROS) could potentiate the invasive activity of rat hepatoma cell line of AH109A by activating autocrine loop of hepatocyte growth factor (HGF)-c-Met pathway. In this report, we examined the involvement of endogenous ROS in the invasive activity of hepatoma cells by using a cell-permeable antioxidant, N-acetyl-L-cysteine (NAC). NAC could certainly scavenge intracellular ROS when directly added to the media at the concentration of 1 or 5 mM and could significantly suppress hepatoma cell invasion, although it showed a little effect on hepatoma cell proliferation at these concentrations. NAC also decreased the content of HGF mRNA and the secretion of HGF at these concentrations, leading to suppression of their invasion. In the present study, blockade of endogenous ROS by NAC proved to efficiently suppress the invasive activity of hepatoma cells by down-regulating HGF gene expression, suggesting the importance of endogenous ROS in cellular signaling of tumor cell invasion.     

N-acetylcysteine reduces susceptibility of transformed and embryonic cells to lytic activity of natural killer cells

Changes in the sensitivity of several transformed and embryonic cells to the lytic activity of natural killers (NK) due to N-acetylcysteine (NAC) has been studied. We found that epidermoid carcinoma A431 cells and murine hepatoma MH22a cells, as well as the transformed mouse fibroblasts 3T3-SV40 treated with 10 mM NAC that we investigated previously normalized their phenotype to the various extent. Like normal cells these cells exposed to NAC are not recognized and destroyed by NK. Murine embryonic fibroblasts (MEFs) similar to transformed cells were destroyed by NK. MEFs treated with 10 mM NAC lost their susceptibility to NK, as did transformed cells. The loss of cell sensitivity to NK cytolytic activity was accompanied by the reorganization of the actin cytoskeleton and generation of well-pronounced stress-fibers.     

Melatonin and protection from whole-body irradiation: survival studies in mice

The radioprotective ability of melatonin was investigated in mice exposed to an acute whole-body gamma radiation dose of 815 cGy (estimated LD50/30 dose). The animals were observed for mortality over a period of 30 days following irradiation. The results indicated 100% survival for unirradiated and untreated control mice, and for mice treated with melatonin or solvent alone. Forty-five percent of mice exposed to 815 cGy radiation alone, and 50% of mice pretreated with solvent and irradiated with 815 cGy were alive at the end of 30 days. Irradiated mice which were pretreated with 125 mg/kg melatonin exhibited a slight increase in their survival (60%) (p=0.3421). In contrast, 85% of irradiated mice which were pretreated with 250 mg/kg melatonin were alive at the end of 30 days (p=0.0080). These results indicate that melatonin (at a dose as high as 250 mg/kg) is non-toxic, and that high doses of melatonin are effective in protecting mice from lethal effects of acute whole-body irradiation.     

The hormone melatonin stimulates renoprotective effects of "early outgrowth" endothelial progenitor cells in acute ischemic kidney injury

Endothelial progenitor cells (EPCs) protect the kidney from acute ischemic injury. The aim of this study was to analyze whether pretreatment of murine "early outgrowth" EPCs (eEPCs) with the hormone melatonin increases the cells' renoprotective effects in the setting of murine acute ischemic renal failure. Male (8-12 wk old) C57Bl/6N mice were subjected to unilateral ischemia-reperfusion injury postuninephrectomy (40 min). Postischemic animals were injected with either 0.5×10(6) untreated syngeneic murine eEPCs or with cells, pretreated with melatonin for 1 h. Injections were performed shortly after reperfusion of the kidney. While animals injected with untreated cells developed acute renal failure, eEPC pretreatment with melatonin dramatically improved renoprotective actions of the cells. These effects were completely reversed after cell pretreatment with melatonin and the MT-1/-2 antagonist luzindole. In vitro analysis revealed that melatonin reduced the amount of tumor growth factor-β-induced eEPC apoptosis/necrosis. Secretion of vascular endothelial growth factor by the cells was markedly stimulated by the hormone. In addition, migratory activity of eEPCs was enhanced by melatonin and supernatant from melatonin-treated eEPCs stimulated migration of cultured mature endothelial cells. In summary, melatonin was identified as a new agonist of eEPCs in acute ischemic kidney injury.     

5-氨基γ-酮戊酸及其己酯衍生物对几种细胞株的光动力作用

5 氨基γ 酮戊 (ALA)及其己酯 (He ALA)具有内源生成光敏剂的特点 ,在肿瘤光动力探测及治疗中显示出了优势。ALA及He ALA对神经母细胞瘤、肝癌细胞及成纤维细胞的光动力作用被研究比较。由特征荧光光谱证实 ,经ALA或He ALA培养后 ,三种细胞内均可生成原卟啉 (PpIX)产物。激光扫描荧光显微镜显示 ,在经ALA或He ALA培养后的神经母细胞瘤中 ,PpIX均以弥散方式分布在细胞质中。PpIX在三种细胞中的积聚动力学过程不同 ,随着ALA或He ALA培育时间的增长 ,PpIX在肝癌细胞及成纤维原细胞中的积累增加 ,而在神经母细胞瘤中PpIX在 8h后已达到饱和。此外 ,在同样的培育条件下 ,神经母细胞瘤中PpIX的生成浓度明显高于肝癌细胞及成纤维细胞。经ALA培养及光照射后 ,可使近 90 %的神经母细胞瘤失活 ;而在同样条件下却只能杀伤 5 0 %左右的肝癌细胞及成纤维细胞。揭示了神经母细胞瘤对ALA光动力作用有极高的敏感性 ,并适于光动力治疗。与ALA相比 ,He ALA可在三种细胞内造成与ALA相近的杀伤率 ,但所用的药物浓度却比ALA低 10倍 ,显示He ALA具有极高的光动力灭活效率。因此在内源光动力治疗中 ,He ALA是一种极具开发前景的新药物。     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin prevents δ-aminolevulinic acid-induced oxidative DNA damage in the presence of Fe2+

-aminolevulinic acid (ALA), a heme precursor which accumulates during lead poisoning and acute intermittent porphyria, is reported to cause liver cancer. The carcinogenic mechanisms of ALA may relate to its ability to generate free radicals through metal-catalyzed oxidation which cause oxidative DNA damage. The aim of this study was to compare the efficacy of melatonin, trolox (vitamin E) and mannitol in altering DNA damage induced by ALA. Herein, we found, in the presence of Fe²⁺, that ALA-induced formation of 8-hydroxydeoxyguanosine in calf thymus DNA was dose and time-dependent. Melatonin, mannitol and trolox, all of which are free radical scavengers, inhibited the formation of 8-hydroxydeoxyguanosine in a concentration-dependent manner. The concentration of each (melatonin, mannitol and trolox) required to reduce DNA damage by 50%, i.e., the IC₅₀, was 0.52, 0.84 and 0.90 mM, respectively.     

Melatonin and activity rhythm responses to light pulses in mice with the Clock mutation

Melatonin and wheel-running rhythmicity and the effects of acute and chronic light pulses on these rhythms were studied in Clock(Delta19) mutant mice selectively bred to synthesize melatonin. Homozygous melatonin-proficient Clock(Delta19) mutant mice (Clock(Delta19/Delta19)-MEL) produced melatonin rhythmically, with peak production 2 h later than the wild-type controls (i.e., just before lights on). By contrast, the time of onset of wheel-running activity occurred within a 20-min period around lights off, irrespective of the genotype. Melatonin production in the mutants spontaneously decreased within 1 h of the expected time of lights on. On placement of the mice in continuous darkness, the melatonin rhythm persisted, and the peak occurred 2 h later in each cycle over the first two cycles, consistent with the endogenous period of the mutant. This contrasted with the onset of wheel-running activity, which did not shift for several days in constant darkness. A light pulse around the time of expected lights on followed by constant darkness reduced the expected 2-h delay of the melatonin peak of the mutants to approximately 1 h and advanced the time of the melatonin peak in the wild-type mice. When the Clock(Delta19/Delta19)-MEL mice were maintained in a skeleton photoperiod of daily 15-min light pulses, a higher proportion entrained to the schedule (57%) than melatonin-deficient mutants (9%). These results provide compelling evidence that mice with the Clock(Delta19) mutation express essentially normal rhythmicity, albeit with an underlying endogenous period of 26-27 h, and they can be entrained by brief exposure to light. They also raise important questions about the role of Clock in rhythmicity and the usefulness of monitoring behavioral rhythms compared with hormonal rhythms.     

Subchronic methionine load induces oxidative stress and provokes biochemical and histological changes in the rat liver tissue

Melatonin has antitumor activity via several mechanisms including its anti-proliferative and pro-apoptotic effects. Moreover, it has been proven that melatonin in combination with chemotherapeutic agents enhances chemotherapy-triggered apoptosis in several types of cancer. Therefore, this study was intended to evaluate whether melatonin is able to strengthen the anti-cancer potential of different chemotherapeutic drugs in human colorectal adenocarcinoma HT–29 cells. We found that treatment with 20 µM cisplatin (CIS) or 1 mM 5-fluorouracil (5-FU) for 72 h induced a decrease in HT-29 cell viability. Furthermore, 1 mM melatonin significantly (P < 0.05) increased the cytotoxic effects of 5-FU. Likewise, simultaneous stimulation with 1 mM melatonin and 1 mM 5-FU significantly (P < 0.05) enhanced the ratio of cells with an overproduction of intracellular reactive oxygen species and substantially augmented the population of apoptotic cells compared to the treatment with 5-FU alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in CIS-treated HT-29 cells, as suggested by a slight increment in the fraction of early apoptotic cells that was observed only after 48 h. Consistently, co-stimulation of HT-29 cells with 20 µM CIS or 1 mM 5-FU in the presence of 1 mM melatonin further increased caspase-3 activation. Apart from this, the cytostatic activity displayed by CIS due to S phase arrest was not affected by concomitant stimulation with melatonin. Overall, our results indicate that melatonin increases the sensitivity of HT-29 cells to 5-FU treatment and, consequently, the indolamine could be potentially applied to colorectal adenocarcinoma treatment as a potent chemosensitizing agent.     

Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin → melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

New actions of melatonin on tumor metabolism and growth

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.