Cinobufagin, used for the treatment of cancer pain, promoted beta-END mRNA and protein expressions, but not DYN A, in the microglia of the spinal cord in a rat bone cancer model; this effect was mediated by the alpha7-nicotinic acetylcholine receptor and induced mechanical anti allodynia ^[1][130]. Moreover, cinobufagin relieved cancer pain by upregulating the expressions of both mu-opioid receptors and beta-END in the hind paw tumor and tissues placed close to the tumor in an experimental animal model of paw cancer pain ^[2][131]. Compared with the sham group, the concentration of beta-END decreased in the rostral ventromedial medulla and spinal cord in an experimental model of cancer-induced bone pain; electroacupuncture and wrist-ankle acupuncture did not affect beta-END concentrations in these regions ^[3][132]. Beta-END increased body weight gain, NK (natural killer) cell cytotoxicity, T cell proliferation, and the relative quantities of T cell subtypes but did not affect T cell release in an experimental rat model of bone cancer pain ^[4][133].

Pro-enkephalin and MET have been observed in human brain tumors (e.g., ganglioglioma, glioma, meningioma) and associated cyst fluids ^[5][134]. Moreover, an inverse correlation between MET and brain tumor malignancy degree has been reported: higher MET level, lower tumor degree ^[5][134]. MET promotes apoptosis in rat C6 glioma cells, increases the activity of caspases 3, 8, and 9 and the expressions of Bax, FasL, and Fas, decreases Bcl-2 expression, and increases Ca⁺⁺ influx into the cytoplasm and NFAT1 accumulation into the cell nucleus ^[6][135]. No effect on tumor cell viability and FasL upregulation was observed when NFAT1 was knocked down ^[6][135]. The results suggest that NFAT1 regulates downstream genes (e.g., FasL) and promotes apoptosis in tumor cells. MET-binding sites decrease with increasing malignancy of gliomas, and a shift from mu-opioid receptors in low-grade gliomas to delta-opioid receptors in high-grade gliomas has been reported ^[7][136]. These results suggest an inactivation of the MET/opioid receptor system in glioma, which blocks the inhibitory action exerted by MET on average astrocyte growth, promoting tumor progression. Moreover, pro-enkephalin expression was favored by norepinephrine and downregulated by endothelin-1 in C6 rat glioma cells ^[8][9][137,138], and MET favored the growth of human U-373 MG astrocytoma cells ^[10][139].

Beta-END-binding sites were reported in the human glioblastoma SF126 cell line ^[11][140], and beta-END was observed in human brain tumor cyst fluids ^[12][141].

Glutamate augmented the water content in C6 glioma cells, whereas DYN A_1-13, via kappa receptors, decreased it ^[13][142].

A polymorphism in the mu-opioid receptor gene has been associated with a reduced response to opioids in breast cancer; patients showing this polymorphism had better survival ^[14][143]. MET, LEU, and beta-END expressions have been reported in cells and stroma of human breast cancer samples ^[15][144]. The activation of hypoxia-inducible factor 1alpha by delta-opioid receptors promoted cyclooxygenase 2 expression, through phosphatidylinositol 3 kinase (PI3k)/protein kinase B (Akt) stimulation, in breast cancer cells (MCF-7, T47D) causing a paracrine activation of the vascular endothelial cells by prostaglandin E₂ receptors ^[16][18].

MET, but not LEU stimulated the migration of MDA-MB-468 human breast carcinoma cells ^[17][145], and END did not affect the migratory capacity of these cells ^[17][145]. A study has reported that the low-fasting plasma level of pro-enkephalin is correlated with an increased risk of breast cancer development in postmenopausal/middle-aged women ^[18][146]. The opioid growth factor (MET)/opioid growth factor receptor (receptor zeta) system blocked the proliferation of triple-negative breast cancer cell lines (BT-20, MDA-MD-231), and this effect was mediated by p21 cyclin-dependent inhibitory kinase pathways ^[19][147].

High plasma beta-END concentrations have been observed in healthy women, which were even higher in healthy postmenopausal women; however, lower levels were found in women with breast cancer, and no difference was observed between premenopausal and postmenopausal women who have breast cancer ^[20][148]. Moreover, chemotherapy only improved beta-END levels in postmenopausal women but without reaching the levels observed in healthy women ^[20][148]. Beta-END activates the survival/mitogenic signaling pathways (Akt, signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK)) in human MDA-MB-231 breast cancer cells. Increasing plasma beta-END levels have been correlated with increasing tumor burden in a mouse model of breast cancer ^[21][149]. This observation means that the peptide is involved in cancer progression, and, significantly, the high levels of plasma beta-END did not decrease pain in mice with breast tumors; quite the opposite, the pain increased in these animals ^[21][149]. Patients with breast carcinoma treated with a galactose-specific lectin standardized mistletoe extract showed an increase in the level of plasma beta-END and an enhancement of T lymphocytes and NK cells ^[22][150]. A correlation between plasma beta-END level and the activity of the last two cells was also reported ^[22][150] and between an increased plasma beta-END level and an improved quality of life in patients with breast cancer ^[23][151].

In female rats in which a mammary tumor was developed, the level of beta-END was higher in the midbrain, striatum, and pituitary; the level of MET was lower in the striatum, and that of DYN decreased in the hypothalamus and pituitary when compared with the levels observed in control animals ^[24][152]. Fetal alcohol exposure reduces beta-END levels, causing a hyper-stress response and inhibiting the immune action against tumors ^[25][153]. Fetal alcohol exposure and control rats treated with N-nitroso-N-methylurea to promote mammary cancer growth were studied, and neurons expressing beta-END were transplanted into the hypothalamus after tumor development to augment beta-END production ^[25][153]. This strategy blocked tumor development in fetal alcohol-exposed and control animals and reversed the effects mediated by fetal alcohol exposure regarding the susceptibility to breast cancer. In this sense, it has been suggested that beta-END regulates the stress response and promotes innate immunity preventing breast cancer development ^[26][154].

Moreover, it seems that the cancer-preventive effect mediated by beta-END was due to an inhibition of the sympathetic neuronal action, which increased the activities of NK cells and macrophages and the production of anti-inflammatory cytokines ^[27][155]. Thus, beta-END counteracts breast cancer development by favoring immune-mediated anticancer defenses ^[28][29][156,157]. In addition, beta-END alters the tumor microenvironment by inhibiting the production of catecholamines and inflammatory cytokines, which alter cell-matrix attachment, DNA repair, epithelial-mesenchymal transition, and angiogenesis ^[27][155].

The presence of DYN A_1-17 and DYN A_1-8 has been reported in Walker 256 tumors, a carcinosarcoma originated from the mammary gland of rats, but no opioid binding site was observed ^[30][158].

MET blocked cervical carcinoma progression in vivo; decreased myeloid-derived suppressor cell-infiltrated both tumor and circulation; induced apoptosis, and increased the expressions of caspase 3 and 8, Fas, and the signaling pathway mediator Bax ^[31][159]. This result suggests that MET is a promising antitumor research line in cervical cancer.

Electroacupuncture increased plasma beta-END levels in cervical cancer patients ^[32][160].

A low-dose of naltrexone (an opioid antagonist) blocked colorectal cancer progression in vivo and in vitro ^[33][161]. This treatment augmented the expressions of macrophage markers (CD68, F4/80), M1 macrophage phenotypic markers (CD80), and the level of cytokines (tumor necrosis factor-alpha) ^[33][161]. Moreover, a low-dose of naltrexone upregulated the expressions of the opioid growth factor receptor and apoptotic factors (PARP, caspases 3 and 9, Bax) and downregulated the expressions of Ki67 and Bcl-2, causing apoptosis in tumor cells ^[33][161].

The presence of enkephalin has been reported in rectal carcinoids ^[34][162]. MET enhanced in vivo colon carcinogenesis induced by azoxymethane ^[35][163]. However, MET exerted an antitumor action in vivo against colorectal tumors (MC38 cell line) by acting on the tumor microenvironment and the immune system ^[36][164]. MET augmented both immunogenicity and recognition of tumor cells; downregulated Kras (oncogene), Bcl2, and Bclxl (anti-apoptotic proteins); blocked the synthesis of inflammatory cytokines; reduced immune checkpoints (2b4, Flgl1, Lag3, Pd-11, Pd-1) in tumor cells, and increased CD4⁺T, CD8⁺T, and macrophages (M1) infiltration ^[36][164]. The antitumor effect exerted by MET was also mediated by effector T cells; the peptide upregulated the opioid growth factor receptor, and the specific inhibitor of this receptor, naltrexone, blocked the antitumor action induced by MET ^[36][164]. The data support that MET is a promising therapeutic agent against colorectal cancer by improving immunotherapy efficacy. Moreover, CD10 (a marker for liver metastasis in colorectal cancer) increased colorectal cancer cell metastasis by abrogating the antitumor action mediated by MET since the peptide blocked the growth, invasion, and survival of tumor cells after thiorphan (inhibitor of the enzyme that degrades enkephalins)-induced CD10 inactivation ^[37][165]. MET, via delta opioid receptors, decreased the phosphorylation of ERK/epidermal growth factor receptors and increased p38-dependent apoptosis ^[37][165]. LEU decreased the invasive capacity of murine colon 26-L5 adenocarcinoma cells ^[38][166].

Beta-END has been reported in adenocarcinomas derived from the colon mucosa; its expression was higher in adenocarcinomas than in the mucosal layer of normal colons ^[39][167], and the peptide has also been observed in rectal carcinoids ^[34][162]. The effects of ultraviolet A eye irradiation on colon carcinoma induced by dextran sodium sulfate and azoxymethane have been studied in an experimental mouse model ^[40][168]. Mu-opioid receptors, MET, and beta-END expressions increased in treated animals, and these expressions increased more when these animals received ultraviolet A eye irradiation ^[40][168]. Colon carcinoma symptoms were decreased after this irradiation, but these beneficial effects were reduced when beta-END inhibitors were administered and disappeared with naltrexone ^[40][168]. This fact suggests that the beneficial effects observed in colon carcinoma after the ultraviolet A eye irradiation were mediated by beta-END and MET. Hypothalamic neurons containing beta-END inhibited the development of preneoplastic/neoplastic lesions in an experimental colon cancer model induced by 1, 2-dimethylhydrazine ^[41][169]. Animals with hypothalamic beta-END neuronal transplants (70%) failed to develop tumors, and animals with transplanted beta-END neurons showed a lesser adenoma development in the colon and tissue lesions (e.g., aberrant crypt foci) and decreased expressions of Ki-67, tumor necrosis factor-alpha, and NF-κB nuclear translocation in colonic tissues ^[41][169].

Moreover, decreased levels of transcription factors linked to epithelial-mesenchymal transition (e.g., Twist, Snail, N-cadherin) and increased levels of E-cadherin were observed in the colon tissue of transplanted animals ^[41][169]. The data suggest that beta-END neuron transplants inhibited colon cancer progression by reducing the epithelial-mesenchymal transition and the inflammatory mechanisms. However, the administration of beta-END into the nucleus of the raphe magnus induced analgesia. It facilitated the metastasis, which was inhibited with naloxone, and when this nucleus was electrically stimulated and analgesia induced, the metastasis was considerably attenuated ^[42][170].

MET, DYN A_1-8, beta-END, and LEU (10⁻⁴ M; 10⁻⁶ M) did not affect the migration, chemotaxis, or invasion of colorectal tumor cells (HCT-116, HT-29) ^[43][171]. Moreover, MET, DYN A_1-8, and beta-END (10⁻⁶ M) did not alter the viability of the HT-29 tumor cell line ^[44][172].

MET blocked the cell growth of cutaneous squamous cell carcinomas by inducing the G0/G1 cell cycle arrest and by promoting apoptotic mechanisms through the caspase 3/Bax/Bcl-2 signaling pathway ^[45][173]. MET, through the opioid growth factor receptor, blocked the proliferation of A431 cells by inducing apoptosis, promoting autophagy in cutaneous squamous cell carcinoma cells, and activating dendritic cells ^[46][174]. MET also decreased immunosuppression by reducing the number of myeloid-derived suppressor cells, controlling the polarization of tumor-associated macrophages from M2 to M1, and inhibiting the JAK2/STAT3 tumor-promotion/immunosuppression signaling pathway, which is involved in macrophage polarization and myeloid-derived suppressor cell expansion ^[45][173]. Thus, MET exerts an antitumor effect against cutaneous squamous cell carcinoma.

MET blocked the growth of human gastric cancer HGC27 and SGC7901 cell lines ^[47][175]. The peptide arrested the cell cycle in the G0/G1 phase, reducing Ki67, cyclin D1, and c-myc mRNA and promoting apoptosis by upregulating the Bax expression through downregulating Bcl-2/surviving expressions and by activating PARP and caspase 3 ^[47][175]. MET also upregulated the expression of the opioid growth factor receptor. Another study has demonstrated that MET-induced apoptosis in human gastric tumor cells (HGC27, SGC7901) by inhibiting the PI3k/Akt/mammalian target of rapamycin (mTOR) signal pathway, reduced the number of M2-type macrophages and increased the M1-type ^[48][176]. Tumor cell apoptosis was blocked when the opioid receptor expression was knocked down ^[48][176]. The data suggest that MET is a promising antitumor agent against gastric cancer.

Plasma beta-END levels decreased in gastric cancer patients after transcutaneous electrical acupoint stimulation ^[49][177], and beta-END was observed in adenocarcinomas derived from the antral mucosa ^[50][178].

The activation of the mu-opioid receptor promoted head and neck squamous cell carcinoma growth in vitro and in vivo experiments, increasing the proliferation and migration of tumor cells (FaDu, MDA6868Tu) ^[51][179]. Thus, the mu-opioid receptor is a promising antitumor target to treat head and neck squamous cell carcinomas.

MET expression has been reported in human head and neck squamous cell carcinomas ^[52][180]. Reduced DNA synthesis and tumor weight/volume have been reported in the head and neck squamous cell carcinomas after treatment with MET, imiquimod, and naltrexone (low dose) ^[53][181]. The inhibitory action exerted by the MET/opioid growth factor receptor system is mediated through the p16 pathway; MET increases the expression of the cyclin-dependent kinase inhibitor p16 protein ^[54][182]. Downregulation of the opioid growth factor receptor favored the progression of head and neck squamous cell carcinoma ^[55][183], and LEU has been detected in head and neck paragangliomas ^[56][184].

Beta-END increased the production of the leukocyte migration inhibitory factor, reaching almost normal levels, in patients with squamous carcinoma of the head and neck; this means that the peptide regulates the immune system ^[57][185].

MET, DYN A_1-8, beta-END, and LEU (10⁻⁴ M; 10⁻⁶ M) did not affect the migration, chemotaxis, or invasion of squamous carcinoma cells (CAL-27, SCC-1) ^[58][186]. Moreover, MET, DYN A_1-8, and beta-END (10⁻⁶ M) did not affect either the differentiation of SCC-1 and CAL-27 tumor cells ^[59][187] or the viability of the latter cells ^[44][172].

MET has been observed in neuroendocrine tumors (paragangliomas) of the larynx ^[60][188].

Beta-END has been reported in tumor cells in an oat cell carcinoma of the larynx ^[61][189].

The presence of pro-enkephalin A has been reported in leukocytes from patients with chronic lymphoblastic leukemia ^[62][190], and MET increased CD10 expression and inhibited the metabolic activity of the leukemic NALM-1 cell line ^[63][191]. MET promoted apoptosis in K562 human erythroid leukemia cells ^[64][192], and the peptide favored pre-B acute lymphoblastoid cell migration (LAZ 221, NALM 6) and increased the CD9 surface expression (a leukemia cell marker) in the latter cells ^[65][193]. This migration, induced by MET, was considerably decreased when pre-B acute lymphoblastoid cells were incubated with antibodies against CD9 ^[65][193].

Beta-END has been reported in the cerebrospinal fluid of children with acute lymphoblastic leukemia; the highest level was observed at the end of the intensification chemotherapy, whereas treatment with glucocorticoids decreased beta-END levels ^[66][194]. However, plasma beta-END levels decreased in patients with solid tumors after treatment with the chemotherapeutic drug CDDP ^[67][195]. Plasma beta-END levels were higher in patients with acute leukemia than in healthy individuals, and stress factors (e.g., high temperature, anemia, hypoxic conditions, pain, cancer) increased the synthesis of beta-END, particularly in the white blood cells of patients with acute leukemia during chemotherapy treatment ^[68][196]. Beta-END promoted the growth of T-lymphoblastoid cells; however, this was not observed in promyelocyte and B-lymphoblastoid cells ^[69][197]. Finally, naloxone-resistant receptors for beta-END are downregulated after activation of the protein kinase C in the U937 cell line (isolated from a histiocytic lymphoma) ^[70][198].

The proliferation of hepatocellular carcinoma cells (Hep 3B, Hep G2, SK-HEP-1) was inhibited after treatment with MET due to the blockade of the DNA synthesis and not to necrotic/apoptotic mechanisms ^[71][199]. Moreover, silencing the opioid growth factor receptor promoted the proliferation of these cells ^[71][199]. MET concentration was higher in metastasis-positive human livers than in normal ones ^[37][165]. This finding is important since it suggests that the increase in MET could be an endogenous antitumor mechanism to counteract tumor progression. Two patients with hepatoblastoma were cured after surgical resection and treatment with naltrexone (low dose) and MET ^[72][200]. It seems that this treatment is a promising antitumor therapeutic strategy.

In an experimental animal model of liver cancer, neurons expressing beta-END transplanted into the hypothalamus prevented hepatocellular carcinoma formation and hepatocellular injuries ^[73][201]. This strategy inhibited carcinogen-induced liver histopathologies (e.g., fibrosis, collagen deposition, inflammatory infiltration) and augmented the concentration of NK cell cytotoxic agents in the liver ^[73][201].

Pro-enkephalin and MET have been reported in human lung cancer cells ^[74][202], and serum LEU level was higher in patients with bronchial carcinoma than in control individuals ^[75][203]. Morphine, via the opioid growth factor receptor, suppressed lung cancer cell proliferation (H1975); this suppression occurred in the cell cycle S phase ^[76][204]. By controlling the Wnt/beta-catenin pathway, MET exerted an antitumor effect against lung cancer in vitro and in vivo experiments, leading to cell cycle arrest at the G0/G1 phase ^[77][205]. Moreover, the antitumor action of the peptide was abolished in the knockdown of growth factor receptor, and MET augmented the infiltration of dendritic cells, CD4⁺ T and CD8⁺ T cells, macrophages (M1) and NK cells, and reduced the number of macrophages (M2) and myeloid inhibitory cells ^[77][205]. MET also upregulated the expression of interleukin-15, interleukin-21, interferon-gamma and downregulated interleukin-10, and tumor necrosis-beta 1 expression in the tumor microenvironment ^[77][205]. MET increased the expression of the opioid growth factor receptor and, by activating the caspase 3/Bax/Bcl-1 signaling pathway, promoted apoptosis in lung cancer cells ^[78][206]. These effects disappeared when the previous receptor was blocked. MET also increased the immunogenicity of lung cancer cells, NK cell activity, and the expression of NK cell-related cytokines (e.g., interferon-gamma, granzyme B) ^[78][206]. Previous findings suggest that MET is a promising antitumor agent against lung cancer. However, another study has demonstrated that methylnaltrexone (an opioid antagonist) counteracted Lewis lung carcinoma growth (cancer cells express mu-receptors) and decreased lung metastasis and that morphine (a mu-receptor agonist) favored tumor growth ^[79][207]. It is important to note that nicotine partially or reversed opioid-induced growth suppression in 9/14 lung cancer cell lines studied ^[80][208]. Moreover, MET blocked pulmonary metastasis and enhanced the activity of NK cells ^[81][209].

Beta-END has been reported in the bronchoalveolar lavage fluid of patients with lung cancer ^[82][210] and in the plasma of patients with this disease ^[83][211]. Beta-END has been reported in lung small-cell carcinomas and carcinoid tumors ^[84][212]. Human small-cell lung carcinoma cell lines (NCI-N417, NCI-H345, NCI-H69) express naloxone-insensitive endorphin binding sites that were insensitive to naloxone and other mu-, delta- or kappa-opioid receptor ligands ^[85][213]. The U1,690 cell line (small-cell lung carcinoma) expresses beta-END, and the peptide promotes the proliferation of this cell line through non-opioid binding sites; moreover, beta-END binding did not affect the synthesis of cAMP ^[86][214]. Beta-END also acts as a chemoattractant for small-cell lung carcinoma cells favoring migration and metastasis ^[87][215].

Pre-proDYN mRNA has been reported in small-cell lung carcinomas ^[88][216], and serum DYN A/B and MET levels increased in a non-small-cell lung cancer cell xenograft stress reduction mouse model ^[89][217]. Moreover, via Gαi, DYN B blocked cAMP formation in non-small-cell lung cancer cells ^[89][217]. Most lung tumor cells co-expressed pro-DYN and DYN, prohormone convertase 1, prohormone convertase 2, or carboxypeptidase E. In contrast, a few lung cancer cells only expressed one of the last markers ^[90][218]. DYN was observed in cancer cells infiltrating human lung tissues and nerve fibers in the bronchial submucosa ^[90][218]. Lung cancer cells express mu, kappa, and delta opioid receptors and contain several combinations of opioid peptides (DYN, ENK, beta-END). After opioid administration, cAMP concentration was decreased in these cells ^[80][208]. In addition, agonists of the previous three receptors (1-100 nM) blocked the growth of tumor cells in vitro, whereas nicotine (100-200 nM) totally or partially counteracted the growth blockade mediated by opioids ^[80][208].

Pro-opiomelanocortin gene delivery blocked the growth of alpha-melanocyte-stimulating hormone/melanocortin 1 receptor (MC1R)-deficient Lewis lung carcinoma cells as well as the growth of these cells in mice; apoptotic mechanisms mediated these effects through an MC1R-independent pathway ^[91][219]. The authors also demonstrated that, in this case, via an MC1R-dependent pathway, this delivery blocked tumor progression and metastasis of B16-F10 melanoma cells ^[91][219]. Pro-opiomelanocortin gene delivery attenuated the beta-catenin signaling pathway by decreasing protein concentrations of beta-catenin and its downstream proto-oncogenes (e.g., c-myc, cyclin D1) and blocked tumor vasculature ^[91][219].

Compared to normal skin, the expression of MET and LEU was decreased in melanocytic tumors ^[92][220]. MET and LEU have been detected in six of seven secondary neuroendocrine carcinomas of the skin, whereas both peptides were not found in skin primary neuroendocrine carcinomas (Merkel cell carcinoma) ^[93][221]. MET exerted an antitumor effect in mice xenografted with melanoma B16-BL6 cells which was inhibited with naloxone; the antitumor action was due to the immune system’s modulation and a cytotoxic effect on melanoma cells ^[94][222]. In the same experimental model, MET promoted cell cycle arrest. It increased the plasma levels of interferon-γ, tumor necrosis factor-alpha, and interleukin-2 ^[95][223]. MET promotes cell cycle arrest in the G0/G1 phase, decreases the number of cells in the S and G2/M phases, and increases the expression of the opioid growth factor receptor in B16 melanoma cells ^[95][223]. Tumor growth and tumor cell dissemination were counteracted with MET, and the peptide blocked A375 melanoma cell proliferation through apoptotic mechanisms ^[95][96][223,224]. MET also promoted cell cycle arrest in the G0/G1 phase, decreased the cell number in S and G2/M phases, and favored apoptosis in human A375 melanoma cells ^[96][224]. Imiquimod also upregulates the expression of the opioid growth factor receptor, increasing MET’s antitumor effect ^[97][225]. This synthetic immune response modifier has been successfully applied in melanoma treatment ^{[98][99][100][101][102]}[226,227,228,229,230]. MET did not affect adenylate cyclase activity in AB16 melanoma cells ^[103][231]. Moreover, MET decreased tumor weight and volume in vivo and increased the ratio of CD4⁺ to CD8⁺ T cells ^[95][223].

Beta-END was observed in 30 of 42 melanoma samples ^[104][232]. Beta-END was found in six of seven secondary neuroendocrine carcinomas of the skin but was absent in primary neuroendocrine carcinomas (Merkel cell carcinoma) ^[93][221]. B16 melanoma cells synthesize and release beta-END ^[105][233]. Tumor growth was studied in mu-opioid receptor-deficient and wild-type mice administered with B16 melanoma cells producing beta-END ^[105][233]. B16 cells decreased tumor growth and increased the infiltration of immune cells into the tumor in mu-opioid receptor-deficient animals. Opioids in the B16 cell supernatant reduced the proliferation of normal leukocytes but not those from mu-opioid receptor-deficient animals ^[105][233]. A correlation was observed between beta-END levels and tumor progression in melanoma tissues ^[105][233]. In this sense, beta-END immunoreactivity was lower in benign melanocytic naevi than in metastatic and advanced melanomas ^[104][232]. Moreover, a low-dose ultraviolet exposure promoted beta-END synthesis in epidermal keratinocytes and increased the plasma level of the peptide ^[106][234]. Thus, mu-opioid peptides modulate the immune response and control the development of tumors. Moreover, beta-END did not affect adenylate cyclase activity in AB16 melanoma cells ^[103][231].

U50,488, a kappa-opioid receptor agonist, favored Fas-induced apoptosis without Fas receptor expression increase and decreased cell proliferation in human multiple myeloma LP-1 cells expressing mu- and kappa-opioid receptors ^[107][235]. However, this study demonstrated that the antiproliferative effect mediated by U50,488 was independent of the kappa receptor. This effect was due to a G0/G1 phase blockade, and cell cycle inhibitors (e.g., p53, p27Kip1, p21Cip1) were not upregulated ^[107][235]. DYN or morphine did not regulate apoptosis or cell proliferation in LP-1 cells ^[107][235].

MET has been located in mouse neuroblastoma Neuro2a cells, and the peptide was released from these cells with a high K⁺ stimulation ^[108][236]. MET arrested the growth of human SK-N-SH neuroblastoma cells ^[109][237].

Neuroblastoma Kelly, NMB, and IMR-32 cell lines express Beta-END-binding sites ^[110][238]. Tumors of transplanted neuroblastoma S20Y cells in mice and treated with complete or intermittent opioid receptor blockade with naltrexone showed an upregulation of beta-END and MET levels and MET-binding sites ^[111][239]. In this study, MET decreased the tumor mitotic index, which was counteracted with naltrexone. Thus, a complete blockade with naltrexone increased tumor cell proliferation, whereas an intermittent blockade inhibited cancer cell proliferation ^[111][239].

Pre-pro-DYN mRNA and pre-pro-ENK have been reported in neuroblastoma SK-N-MC and SCLC H69 cell lines ^[88][216] and opioid binding sites in the neuroblastoma S20Y cell line ^[112][240]. A cysteine protease-degrading DYN A_1-13 and DYN A_1-17 has been reported in the membrane of mouse neuroblastoma N18 cells ^[113][241]. DYN exerted a cytotoxic action, promoted apoptosis, and downregulated the expression of the anti-apoptotic protein Bcl-2 in SH-SY5Y neuroblastoma cells; previous effects were inhibited with the anesthetic isoflurane ^[114][242]. MET, DYN A_1-8, and beta-END did not affect the differentiation of SK-N-SH neuroblastoma cells at the concentration administered (10⁻⁶ M) ^[59][187]. DYN A, at high concentration, binds to neuropeptide Y receptors (Y1 and Y2) in SK-N-MC and SMS-MSN neuroblastoma cell lines, and it has been suggested that DYN A could exert an antagonistic effect on these cells ^[115][243]. Moreover, this binding was not mediated either by changes in receptor-G protein interaction or by receptor phosphorylation.

Enkephalin has been reported in tumor cells in ovarian carcinoids ^[116][244] and MET and the opioid growth factor receptor in human ovarian cancer cells ^[117][245]. MET, in a dose-dependent manner, exerted an inhibitory proliferative effect on ovarian tumor cells (HEY, CAOV-3, SW626), whereas the neutralization of MET promoted cell proliferation and the silencing of the opioid growth factor receptor favored tumor cell replication; MET, via the opioid growth factor receptor, delayed cells moving by upregulating the cyclin-dependent inhibitory kinase pathways ^[117][118][16,245]. A low dose of naltrexone inhibited ovarian tumor progression and, in combination with cisplatin, exerted an enhanced inhibitory effect ^[119][246].

Beta-END has been observed in ovarian sex cord-stromal tumors ^[120][247] and ovarian carcinoids ^[116][244]. A positive correlation has been reported between survival time/disease-free time and plasma beta-END level in patients with ovarian cancer ^[121][248]. Lower beta-END concentrations were observed in patients with recurrence than those without recurrence ^[121][248]. Moreover, beta-END and MET blocked the proliferation of human ovarian KF cancer cells; this effect was counteracted with naloxone, and it seems that both peptides blocked protein/RNA synthesis but not DNA synthesis ^[122][249].

A lipid conjugation of MET increased the tumor-suppression activity of the peptide against human pancreatic adenocarcinomas ^[123][250], and the MET/opioid growth factor receptor system increased the cyclin-dependent kinase inhibitor p21 protein expression to attenuate the progression of human pancreatic cancer ^[124][251]. Moreover, the administration of MET ameliorated clinical symptoms and survival in patients with advanced pancreatic cancer ^[125][252], and a high plasma level of MET has been observed in patients with pancreatic cancer ^[126][253].

DYN A1-8, MET, beta-END, and LEU (10⁻⁴ M; 10⁻⁶ M) did not affect the migration, chemotaxis, or invasion of pancreatic tumor cells (MIA PaCa-2, PANC-1, BxPC-3) ^[58][186] and the first three peptides mentioned did not alter the viability of the MIA PaCa-2 tumor cell line at the concentration administered (10⁻⁶ M) ^[44][172].

Mouse insulinoma beta TC3 cells show a high expression of pro-DYN mRNA and its derived peptides (DYN A_1-8, DYN B_1-13, alpha-neo-END) ^[127][254]. These cells also expressed prohormone convertase 1 and 2 mRNAs but not convertase 5 mRNA, and cells administered with 8-bromo-cAMP increased pro-DYN levels and the release of opioid peptides ^[127][254].

Proprotein convertase 2 and pro-enkephalin have been reported in human pheochromocytomas ^[128][255]. MET has been observed in human pheochromocytomas, and nicotine promoted the release of the peptide from cultured pheochromocytoma cells ^[129][130][256,257]. LEU was also observed in pheochromocytomas ^[130][257]. MET and LEU levels differed in extramedullary and medullary tumors: the MET to LEU ratio was higher in extramedullary than in medullary pheochromocytomas ^[131][258].

Beta-END has been reported in pheochromocytomas ^[130][132][257,259], and the release of the peptide has also been demonstrated ^[133][260].

The rat pheochromocytoma PC12 cell line expresses the pro-DYN gene and releases DYN ^[43][171]. The presence of DYN, but not alpha-neo-END, has been reported in human pheochromocytomas ^[130][134][257,261]; however, another study has shown the presence of both DYN and alpha-neo-END in these tumors and, in addition, it was demonstrated that nicotine favored the release of both peptides from pheochromocytomas ^[135][262]. In another study, MET, LEU, beta-END, and DYN were reported in all the pheochromocytomas studied in which the concentration of enkephalins was higher than that of DYN, and the DYN level was higher than that reported for beta-END ^[130][257]. DYN A_1-17 was the major component observed in pheochromocytomas, whereas DYN A_1-13 and DYN A_1-12 were minor components in these tumors ^[136][137][263,264].

MET level was increased in prolactin-releasing human pituitary adenomas ^[138][265].

Beta-END was observed in a pituitary adenoma ^[139][266], and the peptide was released from human pituitary cancer cells ^[140][267]. The presence of beta-END has been reported in clinically silent pituitary corticotroph adenomas ^[141][268], and beta-END and beta-END_1-27 have been found in extracts of pituitary melanotroph tumors transplanted subcutaneously in mice ^[142][269]. W7, a calmodulin inhibitor, potentiated beta-END release promoted by 8-BcrAMP from the mouse anterior pituitary AtT-20 cancer cell line ^[143][270]. Cerebrospinal fluid beta-END levels increased after resectioning an adrenocorticotropic hormone-secreting pituitary adenoma; however, the MET level was not altered ^[144][271].

The expression of opioid receptors has been described in prostate cancer cells and tissues ^[145][272]. Zeta-opioid receptor mRNA was expressed in all the prostate cancer cell lines studied, kappa-opioid receptors in only two cell lines (VCaP, LNCaP), and no expression was observed for mu- and delta-opioid mRNA receptors ^[145][272]. Compared with other prostate cancer cell lines, LNCaP (an androgen-sensitive cell) showed a higher expression of kappa- and zeta-opioid receptors and a synthetic androgen (R1881) repressed mRNA of both receptors ^[145][272]. Moreover, zeta-opioid receptors showed a higher expression in prostate cancer tissues than in normal ones, and this expression was elevated in aggressive and undifferentiated prostate cancer tissues ^[145][272]. A high mu-opioid receptor expression has been associated with poorer survival in patients with prostate cancer ^[146][273].

DAGO ([D-Ala², N-Me-Phe⁴-Gly-ol] enkephalin), DADLE ([D-Ala2, D-Leu5] enkephalin), and DSLET ([D-Ser², Leu⁵] enkephalin) blocked the proliferation on human prostate cancer cell lines (PC3, DU145, LNCaP); this effect was counteracted with the opioid antagonist diprenorphine ^[147][274].

Beta-END and LEU have been reported in prostatic carcinomas ^[148][275]. Rats with transplanted neurons expressing beta-END into the hypothalamic paraventricular nucleus showed a protective effect against prostate cancer development; an increased NK cell cytolytic action in the spleen and peripheral blood mononuclear cells; a decreased level of inflammatory cytokines (tumor necrosis factor-alpha) in plasma, and a higher level of anti-inflammatory cytokines (interferon-gamma) in plasma ^[149][276]. This observation indicates that the release of beta-END from the hypothalamic transplanted neurons counteracts the inflammatory mechanisms and increases the immune system’s response.

DYN A, DYN A_1-13, and DYN A_1-7 promote the growth of the DU145 prostatic carcinoma cell line ^[150][277]. Naloxone blocked the effect mediated by DYN A but increased the growth of tumor cells at a high concentration (10⁻⁷ M). Electroacupuncture counteracted bone-cancer-promoted hyperalgesia in a rat model that received AT-3.1 prostate cancer cells into the tibia ^[151][278]. Electroacupuncture blocked DYN and pre-pro-DYN mRNA upregulation, and the administration of anti-DYN A_1-17 antisera also blocked hyperalgesia ^[151][278]. In this sense, an upregulation of DYN A has been reported in the spinal dorsal horn, and the release of the peptide has been related to spontaneous pain in a mouse model of neuropathic cancer pain ^[152][279]. Moreover, in a murine model of cancer-pain, the number of immunoreactive neurons containing DYN was increased in the spinal cord (ipsilateral to the limb containing the tumor) ^[153][280].

MET blocked the proliferation of human renal cancer cells (Caki-2) ^[154][281].

MET has been reported in human retinoblastoma ^[155][282].

Beta-END-binding sites have been reported in the human retinoblastoma McA and Y79 cell lines ^[156][283].

Pro-DYN mRNA and its derived peptides have been observed in the R2C rat Leydig tumor cell line ^[157][284].

MET has been reported in a thymic carcinoid ^[158][285].

Beta-END binds to non-opioid binding sites expressed in thymoma cells ^[159][286], and beta-END has been observed in an oncocytic carcinoid tumor of the thymus ^[160][287].

A released dibasic cleaving peptidase that converts DYNs (e.g., DYN A_1-12, DYN A_1-9, proDYN B) into LEU-Arg⁶ has been obtained from the medium of EL-4 mouse thymoma cells ^[161][288].

Human anaplastic thyroid cancer cells (KAT-18) express MET and the opioid growth factor receptor; MET blocked cell replication, the opioid antagonist naltrexone promoted cell growth, and anti-MET antibodies counteracted the inhibitory action mediated by MET ^[162][289].

Opioid peptides (e.g., beta-END, alpha-neo-END) derived from the three opioid precursors have been reported in human thyroid medullary carcinomas ^[163][11], and the release of beta-END has been demonstrated from cultured medullary thyroid carcinoma cells ^[164][290].