Role of Melatonin in Cancer Treatment

Mechanism of Action of MLT

MLT and its metabolites interact with intracellular proteins (calmodulin), (RZR) (ROR) family nuclear-membrane receptors, and receptors located in the cell membrane (8). Different forms of high-affinity (MT1) and low-affinity melatonin receptors (MT2, MT3) have been identified (9). MT1 and MT2 were initially designated as Mel1a and Mel1b, and later classified as MT1 and MT2 by the International Union of Basic and Clinical Pharmacology (IUPHAR) (10). MT1 and MT2 are included in the G-protein-coupled receptor (GPCR) group (a family of guanidine triphosphate-binding proteins) and share many of their amino acid sequences (11). Both MT1 and MT2 are involved in adenyl cyclase and cyclic AMP (cAMP) inhibition. A decrease in cAMP production reduces the uptake of linoleic acid. Linoleic acid, oxidized to 13-Hydroxy octadecadienoic acid (13-HODE) by 15-lipoxygenase, serves as an energy source for tumor signaling molecules and tumor growth. Inhibition of lineoleic acid uptake by MLT is regarded as a mechanism of its antiproliferative effects (9).

A third member of the MLT receptor family is the X-linked orphan G protein-coupled receptor (GPR50) (12), which shares 45% homology with MT1 and MT2 receptors. Its ligand and physiological function remain unclear, although an involvement in key hypothalamic functions, including regulation of the hypothalamo pituitary axes has been suggested (13). Moreover, orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by doing so, they regulate the function of the latter (14). In this respect, deletion of the large c-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins with MT1 (15).

An MT3 receptor/binding site has also been investigated. Mass spectrometry and enzymatic data have confirmed that MT3 is quinone reductase 2 (QR2), a known detoxifying enzyme. The induction of this enzyme expression is associated with a decreased susceptibility to cancer initiation and progression (16, 17).

It has been shown that MLT has oncostatic properties on a wide variety of tumors, including prostate cancer, sarcoma, colorectal cancer, hepatocarcinoma, melanoma, neural tumors, laryngeal carcinoma, skin carcinoma, and ovarian, breast and cervical cancer (18). The oncostatic effects of MLT have been particularly studied in hormone-dependent tumors. Extensive evidence on the oncostatic activity of MLT is based on in vitro studies carried out in cell lines derived from human tumors and murine tumoral models. A general conclusion of these studies is that MLT inhibits cell proliferation and induces apoptosis in most tumor cell lines. Mechanisms of cancer inhibition by MLT include antioxidant effects; regulation of estrogen receptor expression and transactivation; modulation of the enzymes involved in the local synthesis of estrogens; modulation of the cell cycle, differentiation and apoptosis; inhibition of telomerase activity; antiangiogenesis; prevention of circadian disruption; activation of the immune system; and epigenetic factors. There is abundant information regarding the involved mechanisms provoking the effects of MLT on many tumor types, as well as reviews summarizing the results of research in a number of different malignant neoplasms (18-23).

The anticarcinogenic actions of MLT relate in part to its antioxidative and free radical-scavenging activity. The antiestrogenic properties of MLT depend on its ability to reduce the expression of estrogenic receptor alpha (ERα) and to inhibit the binding of the estradiol (E2-ER) complex to the estrogen response element (ERE) on DNA (18, 23). These effects are exerted through MLT binding to specific (MT1) membrane receptors (discussed more extensively in the next section). On the other hand, the inactivation of calmodulin by MLT, is another means by which this hormone may interact with the estrogen-signalling pathway (24). MLT is both a selective estrogen receptor and an enzyme modulator. Its preferential inhibition of ERα explains the oncostatic properties of MLT on estrogen-dependent tumors (18). Other mechanisms of action, including pro-apoptotic effects of MLT on tumor cells and inhibition of telomerase activity are only partially known (25-27).

MLT exerts direct antiangiogenic effects through inhibition of vascular endothelial growth factor (VEGF), as well as indirect effects by (a) inhibiting other tumor growth factors, such as epidermal growth factor (EGF), endothelin-1 (ET-1) and insulin-like growth factor 1 (IGF-1), which are strong mitogens and stimulate cancer angiogenesis (28), and (b) by neutralizing reactive oxygen species (ROS). Research on the antiangiogenic properties of MLT is of significant importance, with possible future clinical applications. MLT is also synthesized by lymphoid organs (bone marrow, thymus, lymphocytes) and is considered to be an immunoenhancer. The administration of MLT stimulates the production of natural killer cells, monocytes, leukocytes, interleukins (IL-2, IL-6, IL-12), interferon-gamma and tumor necrosis factor-alpha (TNFα) through its binding to specific membrane and nuclear receptors present in these cells (29). Finally, novel roles for MLT in epigenetic modulation of gene transcription have been suggested (18).

MLT and Cancer

MLT exerts oncostatic activity through several biological mechanisms including antiproliferative actions, stimulation of anticancer immunity, modulation of oncogen expression, and anti-inflammatory, antioxidant and antiangiogenic effects. In addition, MLT inhibits human cancer cell growth in culture and recent clinical studies seem to confirm its anticancer properties in vivo (30).

Preclinical studies on MLT and cancer. The oncostatic and cytotoxic effects of MLT have been evaluated in different cell culture lines and experimental animal models, the results of which suggest that MLT possesses antiproliferative activity towards a variety of cancer cells. However, a marked variability among the findings of these studies has been possibly observed, due to methodological differences, including study parameters, culture conditions, and different MLT concentrations, doses, and duration of treatment (9).

In relation to breast cancer, most studies have been performed on chemically induced adenocarcinoma in rats and the cell tumor line MCF-7 (31). It has been shown that oncostatic actions of melatonin on hormone-dependent mammary tumors are mainly based on its antiestrogenic actions (32). MLT at a physiological concentration (1 nM) and in the presence of serum or estradiol: (a) reversibly inhibits cell proliferation; (b) increases the expression of p53 and p21/WAF1 proteins and modulates the length of the cell cycle; and (c) reduces the metastatic capacity of these cells and counteracts the stimulatory effect of estradiol on cell invasiveness; this effect is mediated, at least in part, by a melatonin-induced increase in the expression of the cell surface adhesion proteins E-cadherin and beta(1)-integrin.

The direct oncostatic effects of melatonin depend on its interaction with the tumor cell estrogen-responsive pathway. In this sense, it has been demonstrated that melatonin down-regulates the expression of ERα and inhibits the binding of the E2-ER complex to the ERE in DNA (33). Recently, it has been demonstrated that MLT inhibits estrogen production in breast adipose fibroblasts, by inhibiting transcription of the cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) gene that encodes the key enzyme aromatase (34). Other studies have shown that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells (35).

The inhibitory effect of MLT on colon oncogenesis has been demonstrated in different murine colon carcinoma-derived cell lines and rat models (36, 37). The decreased expression of MT1 in human colorectal cancer points to a role of MLT in this disease (38), although the oncostatic action of MLT on intestinal cancer also appears to be dependent on both MT2 and RZR/ROR receptors (39). Moreover, MLT at pharmacological concentrations has been shown to influence the expression of angiogenic and angiostatic chemokine genes in different hepatocellular carcinoma cell lines (40). Moreover, MLT suppresses the hepatocellular tumor-promoting activity of oxfenzadole in male rats through a reduction in ROS production by the activation of CYPs (41).

Studies performed in vivo and in vitro have shown that exogenous MLT exerts oncostatic effects on melanoma cells, especially through its ability to stimulate IL-2 production (23, 42), and protects cells from ultraviolet A (UVA) and B (UVB) actions (43). The radioprotective action of MLT is related to its antioxidant properties (44).

Several other investigations have assessed the signaling pathways and the expression of MLT receptors in cells lines of endometrial cancer (45), glioma (46), renal cancer (47), leukemia and hematological cancer (48, 49), prostate cancer (50), gastric adenocarcimoma (51), oral cavity tumors (52), Ewing sarcoma and bone tumors (53, 54) among others. The results of these studies have provided the basis for the potential utility of melatonin in cancer management.

Clinical studies on MLT and cancer management. Long-term disruption of circadian rhythm and reduced MLT secretion has been related to increased cancer risk (55-58). A meta-analysis of 13 observational studies reported a significant increase in the incidence of breast cancer among female airline cabin crew and female night workers, with a standardized incidence ratio of 1.44 [95% confidence interval (CI) 1.26-1.65] and 1.51 (95% CI=1.36-1.68), respectively (58). In recognition of this relationship, an expert Working Group convened by the International Agency for Cancer Research has concluded that shiftwork that involves circadian disruption is probably carcinogenic to humans (59).

Several clinical studies have been conducted in order to determine the role of MLT in the treatment of different cancer types, in conjunction with different oncological strategies on the basis of the potent antioxidant, immune-modulating, hormone-modulating, and antiproliferative actions of MLT. In order to present data supported by the best available evidence, only results from controlled randomized studies, systematic reviews and meta-analyses are reviewed here.

A recent meta-analysis of eight randomized controlled clinical trials involving 761 patients with solid tumors in which MLT was used in daily doses of 20 mg orally, as adjunct treatment concurrent with chemotherapy or radiotherapy for cancer, showed a statistically significant improvement in the complete and partial remission rate [risk ratio (RR)=1.95, 95% CI=1.49-2.54, p<0.00001] and 1-year survival rate (RR=1.90, 95% CI=1.28-2.83, p=0.001), as well as a significant decrease of radiochemotherapy-related adverse effects, such as thrombocytopenia, neurotoxicity, and fatigue (60).

These results confirm previous findings of a systematic review and meta-analysis reported by Seely et al. (61). This study, using data collected from 21 clinical trials, evaluated the effects of MLT in conjunction with chemotherapy, radiotherapy, supportive care, and palliative care regarding 1-year survival, complete and partial responses, stable disease, and chemotherapy-associated toxicities in 3697 patients with solid tumors (breast, colorectal, lung, renal cell, hepatocellular carcinoma, glioblastomas, and others types of cancers), most of them with advanced or metastatic disease. In the majority of trials, MLT was given at a dose of 20 mg. The pooled RR for 1-year mortality was 0.63 (95% CI=0.53-0.74, p<0.001). Complete response (RR=2.33, 95% CI=1.29-4.20), partial response (RR=1.90, 95% CI=1.43-2.51), and stable disease (RR=1.51, 95% CI=1.08-2.12) were all significantly improved by the addition of MLT. Moreover, MLT was found to significantly reduce occurrences of alopecia, anemia, asthenia, and thrombocytopenia.

In addition, in a systematic review of randomized controlled trials, followed by a meta-analysis, carried out by Mills et al. (62) in 2005, which included 10 trials published between 1992 and 2003, with 643 patients with solid tumor cancers, MLT reduced the risk of death at one year (RR=0.66, 95% CI=0.59-0.73). It is noteworthy that in these studies, effects were consistent with MLT dose and type of cancer.

Several randomized trials have been conducted in patients with breast cancer, colorectal cancer, and lung cancer. It has been shown that MLT may stimulate ER expression and enhance the effects of tamoxifen. In a randomized study of tamoxifen vs. tamoxifen plus MLT (20 mg/day orally in the evening) in 40 post-menopausal patients with ER-negative metastatic breast cancer, no complete response was documented (63). However, the partial response rate was significantly higher in patients treated with the combination of tamoxifen and MLT than in those who received tamoxifen alone (36.5% vs. 9.5%, p<0.05) as was 1-year survival (63.2% vs. 23.8%, p<0.01). No MLT-related toxicity was observed; on the contrary, most patients receiving MLT experienced a relief of anxiety and of depression. This preliminary study suggests that the association of MLT may also make tamoxifen effective for patients with an ER-negative metastatic breast cancer.

In patients with advanced colorectal cancer who progressed in response to 5-fluorouracil (5-FU) plus folates, the administration of IL-2 plus MLT was evaluated in 50 patients the survival time (64). Patients were randomized to receive supportive care alone or low-dose subcutaneous IL-2 (3 million IU/day for 6 days/week for 4 weeks) plus MLT (40 mg/day orally). No spontaneous tumor regression occurred in patients receiving supportive care alone. A partial response was achieved in 3/25 patients treated with immunotherapy. The percentage of survival at one year, was significantly higher for patients treated with immunotherapy than for those treated with supportive care alone (36% vs. 12%, p<0.05). This study suggests that low-dose subcutaneous IL-2 plus MLT may be effective as a second-line therapy to induce tumor regression and to increase survival at one year in patients with metastatic colorectal cancer patients, progressing under 5-FU and folates. In another study of 30 patients with metastatic colorectal cancer progressing after at least one previous chemotherapeutic line containing 5-FU, weekly treatment with low-dose irinotecan plus MTL (20 mg/day), as compared with irinotecan alone, was associated with a higher percentage of patients with disease control (partial remission plus stable disease, 85.7% vs. 43.7%, p<0.05) (65). This preliminary study shows that the efficacy of irinotecan in pre-treated metastatic colorectal cancer may be enhanced by a concomitant daily administration of MLT.

A number of randomized trials have been conducted in patients with non-small cell lung cancer (NSCLC). In NSCLC patients resistant to first-line chemotherapy containing cisplatin, the administration of 10 mg/day of MLT (n=31) vs. supportive care alone (n=32) was associated with significantly higher percentages of disease stabilization and 1-year survival. Patients treated with MLT also showed a significant improvement in performance status (66). However, in patients with advanced lung cancer, treatment with MLT (40 mg/day) vs. placebo for 21 consecutive days, starting two days before chemotherapy with carboplatin and etoposide, did not protect against the myelotoxic effects of these cytotoxic drugs (67). In another randomized study of 60 patients with locally advanced or metastatic NSCLC, immunotherapy with low-dose IL-2 plus MLT (40 mg/day starting seven days before IL-2) vs. chemotherapy with cisplatin and etoposide, was associated with a significantly higher mean progression-free period and a higher percentage of survival at one year compared to chemotherapy (68). Toxicity was also substantially lower in immunotherapy-treated patients than in those treated with chemotherapy.

In a study of 70 patients in poor clinical condition with advanced NSCLC who were randomized to received first-line chemotherapy with cisplatin and etoposide or chemotherapy plus MLT (20 mg/day), higher tumor responses (32.3% vs. 17.1%) and percentage of 1-year survival (44.1% vs. 19.4%, p<0.05) were obtained for patients treated with MLT plus chemotherapy than for those treated with chemotherapy alone (69). In 100 consecutive patients who were randomized to receive chemotherapy alone (cisplatin and etoposide) or chemotherapy and MLT, both the overall tumor regression rate and the 5-year survival results, were significantly higher in patients concomitantly treated with MLT (70). In particular, no patient treated with chemotherapy alone was alive after two years, whereas 5-year survival was achieved in three out of 49 (6%) patients treated with chemotherapy and MLT. These data suggest a promising biochemotherapeutic strategy in the treatment of solid neoplasms.