Life Sciences 115 (2014) 8–14

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Review article

Melatonin as a proteasome inhibitor. Is there any clinical evidence? Jerry Vriend a,⁎, Russel J. Reiter b a b

Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

a r t i c l e

i n f o

Article history: Received 15 July 2014 Accepted 27 August 2014 Available online 16 September 2014 Chemical compounds studied in this article: Melatonin (PubChem CID: 896) Bortezomib (PubChem CID: 387447) Keywords: Melatonin Proteasome Apoptosis Bax Bim Bcl-2 Tumor suppressor p53 Nuclear factor kappa beta Caspase 3 Caspase 9 TRAIL

a b s t r a c t Proteasome inhibitors and melatonin are both intimately involved in the regulation of major signal transduction proteins including p53, cyclin p27, transcription factor NF-κB, apoptotic factors Bax and Bim, caspase 3, caspase 9, anti-apoptotic factor Bcl-2, TRAIL, NRF2 and transcription factor beta-catenin. The fact that these factors are shared targets of the proteasome inhibitor bortezomib and melatonin suggests the working hypothesis that melatonin is a proteasome inhibitor. Supporting this hypothesis is the fact that melatonin shares with bortezomib a selective pro-apoptotic action in cancer cells. Furthermore, both bortezomib and melatonin increase the sensitivity of human glioma cells to TRAIL-induced apoptosis. Direct evidence for melatonin inhibition of the proteasome was recently found in human renal cancer cells. We raise the issue whether melatonin should be investigated in combination with proteasome inhibitors to reduce toxicity, to reduce drug resistance, and to enhance efficacy. This may be particularly valid for hematological malignancies in which proteasome inhibitors have been shown to be useful. Further studies are necessary to determine whether the actions of melatonin on cellular signaling pathways are due to a direct inhibitory effect on the catalytic core of the proteasome, due to an inhibitory action on the regulatory particle of the proteasome, or due to an indirect effect of melatonin on phosphorylation of signal transducing factors. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . Shared targets for proteasome inhibitors and melatonin NF-κB . . . . . . . . . . . . . . . . . . . . . . . Cell cycle regulation . . . . . . . . . . . . . . . . . Tumor suppressor factor p53 . . . . . . . . . . . . . VEGF . . . . . . . . . . . . . . . . . . . . . . . Apoptotic factors and the apoptosome . . . . . . . . Apoptosis: the apoptosome . . . . . . . . . . . Apoptosis: caspase-9, caspase-3 and AIF . . . . . Apoptosis: the TRAIL connection . . . . . . . . . Beta-catenin . . . . . . . . . . . . . . . . . . . . Melatonin and intracellular redox status . . . . . . . Phosphorylation of the proteasome — role for melatonin? Summary and conclusions . . . . . . . . . . . . . . Author contributions . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Human Anatomy and Cell Science, University of Manitoba, 745 Bannatyne Avenue, Winnipeg R3E 0W3, Manitoba, Canada. Tel.: +1 204 789 3732, +1 204 668 2871. E-mail address: [email protected] (J. Vriend).

http://dx.doi.org/10.1016/j.lfs.2014.08.024 0024-3205/© 2014 Elsevier Inc. All rights reserved.

J. Vriend, R.J. Reiter / Life Sciences 115 (2014) 8–14

Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Some of the major proteins of significance in cancer susceptibility include the tumor suppressor factor, p53, cell cycle regulator, p27, transcription factor NF-κB, anti-apoptotic factor Bcl-2, and the pro-apoptotic factor Bax. Cellular levels of these proteins are controlled by the ubiquitin–proteasome system and are targets of the proteasome inhibitor, bortezomib (Chen et al., 2011; Fuchs, 2013). Each of these proteins has also been reported to be influenced by the naturally-occurring indole, melatonin (Fig. 1). The increasing number of proteins reported as regulated by both the proteasome and by melatonin suggests the hypothesis that melatonin acts as an inhibitor of a component of the ubiquitin–proteasome system (Vriend and Reiter, 2014). Herein, we review the signal transduction proteins whose levels are modulated both by the ubiquitin–proteasome system and by melatonin, and we discuss mechanisms by which melatonin could interact with the ubiquitin– proteasome system in cancer cells. The use of proteasome inhibitors in treating some hematological disorders and cancers raises the question of whether melatonin should be added to drug regimens used to treat specific malignancies that are sensitive to proteasome inhibitors.

Shared targets for proteasome inhibitors and melatonin Several major targets of proteasome inhibitors were identified followed by the approval of bortezomib by the US Food and Drug Administration in 2004 for treatment of multiple myeloma. The effects of proteasome inhibitors on signal transduction proteins have been regularly reviewed (e.g. Adams et al., 1999; Chen et al., 2011; Crawford et al., 2011; Kisselev et al., 2013; Wu and Shi, 2013). Thus, treatment with proteasome inhibitors increases tumor suppressor protein p53, increases the cell cycle regulator p27, inhibits levels of the transcription factors NF-κB and beta-catenin, enhances apoptosis, inhibits angiogenesis and inhibits DNA repair. Herein, we provide documentation that

Fig. 1. Is melatonin a proteasome inhibitor? p53 — tumor suppressor protein; Akt — protein kinase B; NF-κB — nuclear factor kappa beta, a transcription factor; Nrf2 — nuclear factor-like 2, a transcription factor related to response to oxidative stress; Bax — Bcl-2associated X protein; Bim — a protein that regulates apoptosis; Bak — another apoptotic factor of the Bcl-2 family; VEGF — vascular endothelial growth factor; caspase 3, a protein mediator of apoptosis; and Apaf — apoptosis protease activating factor, a component of the apoptosome.

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melatonin administration influences the activity of the major targets of the proteasome inhibitor, bortezomib. NF-κB NF-κB as a transcription factor stimulates the expression of a number of genes related to oxidative stress, the immune response, cytokine production and apoptosis (Crawford et al., 2011). It is regulated in a complex manner by the ubiquitin–proteasome system. Degradation of the NF-κB inhibitor, IκK, by the proteasome results in activation of NF-κB (Traenckner et al., 1994; Chen, 2005; Gilmore, 2006; Brasier, 2006; Perkins, 2007; Skaug et al., 2009). The proteasome inhibitor bortezomib is considered an inhibitor of NF-κB (Wu and Shi, 2013; Traenckner et al., 1994) through its effect on IκK. This mechanism is complicated, however, by the fact that degradation of NF-κB itself is regulated by the proteasome and that NF-κB can be activated by more than one signaling pathway, the canonical pathway and the noncanonical pathway (Fuchs, 2013). Furthermore, a second major factor regulating the activity of NF-κB is phosphorylation (Balistreri el al., 2013). Baldwin (2001) has made the case that inhibition of NF-κB is clinically useful in selected cancers, including lymphomas and leukemias, through an effect on apoptosis. The use of bortezomib in multiple myeloma therapy is based partly on its effects on NF-κB (Fuchs, 2013) (Fig. 2). More recently, Wu and Shi (2013) have reviewed developments regarding the use of proteasome inhibitors which suppress various types of cancer through their effect on NF-κB. There are many reports that melatonin inhibits NF-κB activity (Natarajan et al., 1995; Chuang et al., 1996; Gilad et al., 1998; Bruck et al., 2004; Li et al., 2005; Huang et al., 2008; Jung et al., 2009; Choi et al., 2011; Bekyarova et al., 2012; Qin et al., 2012; Shi et al., 2012; Min et al., 2012) These reports would be consistent with the view that

Fig. 2. Role of NF-κB in bortezomib and melatonin-induced apoptosis. Both bortezomib and melatonin inhibit NF-κB. Inhibiting NF-κB will increase apoptosis by removing an inhibitory effect on the caspase group of enzymes.

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melatonin is a proteasome inhibitor, but could also be interpreted as evidence that melatonin is a natural NF-κB inhibitor. These inhibitory actions of melatonin on NF-κB may contribute to its anti-inflammatory and its pro-apoptotic (Fig. 2) effects in certain types of cancer cells (Kim et al., 2012; Zha et al., 2012; Uguz et al., 2012; Mauriz et al., 2013). Understanding the mechanisms controlling NF-κB is important in dealing with drug resistance to the proteasome inhibitor bortezomib (Buac et al., 2013). Shen et al. (2013) have pointed out that modifying drug combinations that better inhibit NF-κB may be useful in overcoming bortezomib resistance in multiple myeloma. The reports of melatonin inhibition of NF-κB suggest that melatonin should be investigated clinically, alone and in combination with the proteasome inhibitor bortezomib. Baldwin (2001) suggested that NF-κB inhibition in some leukemias and lymphomas in which this transcription factor plays a significant role could be an important contribution to treatment of these cancers. In Hodgkin's disease, activation of NF-κB is reportedly a requirement for survival of tumor cells (Bargou et al., 1997). Another pathological condition in which inhibition of NF-κB by a proteasome inhibitor was recently investigated is diabetic neuropathy (Aghdam and Sheibani, 2013). Melatonin also has been reported as having a protective effect in diabetic neuropathy (Negi et al., 2011) through its inhibitory action on NF-κB. Proteasome dysfunction, possibly due to deregulation of NF-κB, has also been implicated in atherosclerosis (Marfella et al., 2007). Likewise, melatonin is known to have a protective action against atherosclerosis via NF-κB signaling (Favero et al., 2013; Hu et al., 2013). An advantage of using melatonin is that it can be administered orally and that it appears to have no major side effects. Moreover, melatonin would be useful since its addition with the proteasome inhibitor would lead to the reduction of toxic doses of other components of the therapeutic cocktail (Reiter et al., 2002). For example, multiple myeloma is treated with thalidomide to induce apoptosis (Mitsiades et al., 2002). Since melatonin also induces apoptosis in cancer cells (Fig. 2), and is not toxic, theoretically, it would be useful in management of multiple myeloma. Cell cycle regulation The cell cycle regulator p27 is regulated by proteasomal degradation (Pagano et al., 1995). It is upregulated by proteasome inhibition (Hussain et al., 2009), thereby blocking the normal progression of the cell cycle as well as the abnormal proliferation of cancer cells. In prostate epithelial cells, p27 is also upregulated by melatonin (Shiu et al., 2013). According to these investigators, the effect on p27 turnover is related to melatonin inhibition of NF-κB signaling. They argue that melatonin administration would be useful in prostate cancer prevention and therapy. The proteasome inhibitor bortezomib has also been studied in androgen-refractory prostate cancer cells by Manna et al. (2013). These investigators found elevated NF-κB activity in prostate cells. In prostate cancer cells treated with bortezomib the authors noted a reduction in NF-κB but an augmented expression of cytokine IL-8, an indicator of poor prognosis. There are at least two non-cancer animal models in which melatonin depresses tissue levels of IL-8 levels (Zhong et al., 2013; Ganguly and Swarnakar, 2012). Theoretically, then, the combination of bortezomib and melatonin should yield a better response of prostate cancer cells than to bortezomib alone. Tumor suppressor factor p53 The tumor suppressor factor p53 is often reduced in cancer cells, facilitating tumor growth and drug regimen resistance. A major mechanism for reducing p53 is proteasomal degradation involving the E3 ligase, MDM2 (Momand et al., 1998). The proteasome inhibitor bortezomib results in accumulation of p53 in various types of cancer cells (Williams and McConkey, 2003; Lopes et al., 1997; Batchelor et al., 2009) including multiple myeloma, melanoma and chronic

lymphocytic leukemia. Based on several recent investigations the MDM2–p53 interaction potentially provides an important locus for anticancer therapy (Ciechanover, 2013; Naq et al., 2014). The p53 upregulated modulator of apoptosis (PUMA) is a proapoptotic protein that is required for p53 tumor suppression in some models (Hermann et al., 2004). Its degradation is proteasome dependent (Gomez-Lazaro et al., 2005). Melatonin administration increases cellular levels of p53 in several cancer models. It upregulates p53 (and downregulates SIRT) in osteosarcoma cells (Cheng et al., 2013), increases cellular p53 levels in breast cancer cells (Sanchez-Barcelo et al., 2012), in prostate cells (Kim and Yoo, 2010) and in the hepatocarcinoma, HepG2 (Martin-Renedo et al., 2008). Melatonin stimulates the p53/MDM2 ratio in breast cancer cells (Proietti et al., 2011; Bizzarri et al., 2013). There is also a report that melatonin administration upregulates PUMA expression in renal cancer cells treated with melatonin and the diterpene, kahweol (Um et al., 2011). If melatonin is a general cellular regulator of p53/MDM2 it could be a useful adjunct to current treatment of diseases such as multiple myeloma and chronic lymphocytic leukemia. VEGF Vascular endothelial growth factor (VEGF) is an important factor in solid tumor angiogenesis. Extracellular ubiquitin increases expression of VEGF (Steagall et al., 2013). Vlachostergios and Papandreou (2013) provided evidence that, in glioma, angiogenic NF-κB signaling is involved in the mechanism by which VEGF influences the growth of this malignant tumor. Melatonin has been shown to have anti-angiogenic actions in endothelial cells in culture (Alvarez-Garcia et al., 2013), an effect associated with inhibition of VEGF. Melatonin was also found to reduce VEGF in human breast cancer cells. In mice, melatonin limited growth of estrogen-responsive breast cancer cells in culture (Hill and Blask, 1988). Furthermore, melatonin was reported to inhibit angiogenesis and VEGF in a mouse adenocarcinoma tumor (Kim et al., 2013). Melatonin thus has the potential of contributing to therapeutic regimens used to treat VEGF-dependent tumors. Apoptotic factors and the apoptosome Regulation of apoptosis is an important cellular reaction in cancer cells. It is one of the mechanisms for the anti-tumor effects of the proteasome inhibitor bortezomib. Based on a review by Sainz et al. (2003) the process of bortezomib-induced apoptosis is described as initiated by inhibiting the destruction of pro-apoptotic proteins (such as Bax) by the proteasome. A recent review described the major targets of proteasome inhibitors in cancer therapy (Crawford et al., 2011). These targets include the transcription factor NF-κB, the tumor suppressor p53, cell cycle regulator p27, vascular endothelial growth factor VEGF, pro-apoptotic factor Bax, anti-apoptotic factors Bcl-2 and IAP, and the oncogene transcription factor FoxM1. In the previous paragraphs we have documented that melatonin modulates cellular levels of NF-κB, p53, p27 and VEGF, acting very much like a proteasome inhibitor. An effective proteasome inhibitor would be predicted also to stimulate apoptosis in cancer cells; furthermore, this augmentation would have a certain amount of selectivity (Crawford et al., 2011). The proapoptotic effects of melatonin in cancer cells have recently been reviewed by Bizzarri et al. (2013). These investigators summarized the evidence that documented melatonin-induced apoptosis in hematological malignancies, in breast cancer cells, in prostate cancer cells and in gastrointestinal cancers. One similarity that melatonin shares with proteasome inhibitors in terms of apoptosis is that the pro-apoptotic action of both these drugs are more effective in cancer cells than in normal cells. Indeed, melatonin has a well-known anti-apoptotic effect in normal cells (e.g., Sainz et al., 2003). Similarly, Crawford et al. (2011)

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reported that leukemic cells were more sensitive to proteasome inhibitors than normal cells. These investigators pointed out that what makes tumor cells more vulnerable to proteasome inhibitors is the increased protein synthesis which occurs in proliferating cancer cells. This reasoning could explain the vulnerability of certain cancer cells to melatonin as well. Based on data showing that solid tumors are less sensitive to proteasome inhibitors than hematological malignances, we can predict that if melatonin is a proteasome inhibitor it would be more effective in diseases such as multiple myeloma and mantle cell leukemia (which are susceptible to bortezomib) than in solid tumors. Likewise, if melatonin really is a proteasome inhibitor it should influence the activities of other factors regulated by the proteasome including Bax, Bcl-2, IAP, and the FOX transcription factors, caspases and Akt. FoxO proteins are transcription factors which contribute to cellular growth and differentiation, modulate the cell cycle and tumor suppressor pathways (Zhang et al., 2011). Cellular activity of these proteins is regulated by phosphorylation and ubiquitination (Fu and Tindall, 2008). In hepatocellular carcinoma, FoxO3a is reported to induce proapoptotic genes (Carbajo-Pescador et al., 2014). Melatonin treatment was found to induce apoptosis in hepatoblastoma cells (HepG2 cells), but not in normal primary human hepatocytes (Carbajo-Pescador et al., 2013). These investigators attributed the melatonin-induced apoptosis in these cells to activation of FoxO3a by reducing its phosphorylation and facilitating its nuclear translocation. Akt (protein kinase B) is a protein which stimulates phosphorylation of FoxO proteins thereby preventing nuclear translocation and resulting in degradation by the proteasome (Matsuzaki et al., 2003). It is activated (by phosphorylation) by the second messenger PI3K. Zhang et al. (2011) suggested that the PI3K/Akt/FoxO pathway is an important locus for cancer therapeutic agents. Since Akt acts as a cell survival signal, inhibition of Akt will contribute to proapoptotic effects of therapeutic agents (Franke et al., 2003; Fuchs, 2013). Fuchs (2013) reported that the PI3K/Akt/mTor pathway is one of the two most important signaling pathways targeted in therapy in multiple myeloma by bortezomib. Since melatonin inhibits this pathway (Wang et al., 2012a; Bizzarri et al., 2013), theoretically, it would also be useful in multiple myeloma (and other cancers with elevated Akt activation), where the challenge is to overcome resistance to current drugs. The second pathway important in multiple myeloma, according to Fuchs (2013), is the ras/raf/MEK/ERK pathway. It is activated by melatonin in mesenchymal stem cells (Radio et al., 2006), but according to Bizzarri et al. (2013) melatonin has a pro-apoptotic effect in rat hepatoma cells by downregulating ERK phosphorylation. Bax is a pro-apoptotic protein. It is degraded by the proteasome (Li and Dou, 2000). Its cellular concentrations are upregulated (or stabilized) by bortezomib (Nayak et al., 2013). Melatonin also upregulates Bax during melatonin-induced apoptosis (Bizzarri et al., 2013). In human renal cancer cells melatonin increases expression of the apoptotic protein Bim (Park et al., 2014). These investigators also demonstrated that the upregulation of Bim was associated with inhibition of proteasome activity. Bcl-2 (B cell lymphoma-2) is an anti-apoptotic protein. Moreover, it inhibits DNA repair mechanisms (Laulier and Lopez, 2012). It is reduced by the proteasome inhibitor bortezomib (Mitsiades et al., 2002; Pei et al., 2003). In human leukemic cells, melatonin was found to promote apoptosis (Trubiani et al., 2005; Sanchez-Hidalgo et al., 2012). Both these groups of investigators reported that melatonin-induced apoptosis was associated with down-regulation of Bcl-2. Rodriguez et al. (2013) noted that hematological tumors, such as RAMOS cells, were particularly sensitive to the apoptotic actions of melatonin. A proteasome inhibitor such as bortezomib would, of course, be more effective in hematological tumors than in solid tumors. The RAMOS cell line as such is also sensitive to the apoptotic actions of bortezomib (Deng et al., 2013).

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Apoptosis: the apoptosome The apoptosome has a ring shaped molecular structure that activates enzymes involved in cell death. One of the major units of this structure is the Apaf-1 molecule (Yuan and Akey, 2013). Apaf-1 expression is stimulated by bortezomib in hepatoma cells (Calvaruso et al., 2007). While melatonin enhances apoptosis of hepatoma cells (Fan et al., 2013), the effect of melatonin on Apaf-1 expression in these cells has not been tested. Apaf-1 expression is stimulated by melatonin in breast cancer cells (Wang et al., 2012b) raising the question of whether melatonin stimulates the formation of the apoptosome. Apaf-1 is reportedly required for the apoptotic action of bortezomib in T-cell leukemic cells (Ottosson-Wadlund et al., 2013). These results suggest that melatonin might be a useful adjunct to bortezomib treatment in promoting cell death in bortezomib-sensitive tumors and malignancies. Apoptosis: caspase-9, caspase-3 and AIF Bortezomib-induced apoptosis in multiple myeloma cells is related to activation of caspase-9 and caspase-3 (Hildeshima et al., 2003; Laubach et al., 2009). In a pancreatic cell line, melatonin also was reported to stimulate apoptosis and to activate caspase-9 and caspase-3 (Jaworek and Leja-Szpak, 2013). Caspase-3 and caspase-9 are likewise stimulated in breast cancer cells treated with melatonin (Wang et al., 2012a) and in human malignant lymphoid cells (Sanchez-Hidalgo et al., 2012). These results again document the similarity of actions of a proteasome inhibitor and melatonin. Furthermore, both bortezomib and melatonin interact with the pro-apoptotic factor AIF (apoptosis inducing factor) (Calvaruso et al., 2007; Cucina et al., 2009) in various cancer cells. Apoptosis: the TRAIL connection The tumor necrosis-related apoptosis-inducing ligand (TRAIL) has been studied for its apoptosis-inducing effects in a variety of tumor cells. TRAIL-induced apoptosis is selective for cancer cells. It apparently does not induce apoptosis in normal cells. The proteasome inhibitor bortezomib sensitizes a variety of tumor cells to TRAIL, including malignant human glioma cells (de Wilt et al., 2013; Jane et al., 2011). In this model the increase in sensitivity is associated with inhibition of NF-κΒ. Melatonin also increases the sensitivity of human malignant glioma cells to TRAIL-induced apoptosis (Martin et al., 2010). In this respect melatonin again acts similar to the proteasome inhibitor. Bortezomib has been shown to increase sensitivity to TRAIL-induced apoptosis in multiple myeloma cells (Mitsiades et al., 2001). The prediction that melatonin increases sensitivity to TRAIL-induced apoptosis in multiple myeloma cells has not been tested. It should be noted that TRAIL is at least partly regulated by calcium dependent calmodulin kinase II (CAMKII) (Fujikawa et al., 2009). This enzyme, which reportedly copurifies with the Rpt6 subunit of proteasomes (see Fig. 3) in the brain (Bingol et al., 2010), is inhibited by melatonin (Benitez-King et al., 1996). Beta-catenin Beta-catenin is another protein whose cellular levels are controlled by the ubiquitin–proteasome system (Aberle et al., 1997). It functions as a transcription factor but also as a cell adhesion molecule. It is involved in development of a number of solid tumors. It protects neurons from disorders associated with misfolded proteins (Jeong and Park, 2013). Melatonin has been reported to activate beta-catenin in osteoblastic cells (Park et al., 2011) and to increase beta-catenin levels in the brain of aged mice (Gutierrez-Cuesta et al., 2008), but to decrease beta-catenin levels in breast cancer cells (Mao et al., 2012).

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Phosphorylation of the proteasome — role for melatonin?

Fig. 3. Melatonin and the proteasome regulate apoptotic proteins. Rpn — structural components of the regulatory particle; Rpt1-6, a series of ATPase enzymes in the base of the regulatory particle. α1–7, subunits of the alpha rings of the proteasome, β1–7, subunits of the beta rings of the proteasome. The regulatory particle is involved in deubiquitination and translocation of proteins. The β1, β2 and β5 subunits are the catalytic components in the core particle of the proteasome. There is some that evidence that melatonin inhibits proteasome activity. Melatonin could inhibit the activity of the regulatory particle or inhibit directly the catalytic core of the proteasome.

Melatonin and intracellular redox status While some of the actions of melatonin involve classical membrane receptors, some are mediated by its ability to scavenge free radicals. As such the N\C_O structure in the side chain is considered to be an important functional group for this action (Tan et al., 2002). This structure, the amide group, a component of bortezomib (Kreidenweiss et al., 2008), is found adjacent to the pharmacophore. Lu and Wang (2013) have presented a model of the interaction of bortezomib with the B5 subunit of the beta ring of the proteasome (see Fig. 3). According to this model three amino acids of the proteasome (Ala49, Ala50, Thr21) interact with bortezomib via a hydrogen-bonding network. Structurally melatonin appears to have the components for a similar hydrogen-bonding interaction. However, currently there is no direct evidence that melatonin interacts with the B5 subunit of the proteasome. Melatonin also alters the intracellular redox status. This, according to Rodriguez et al. (2013), is important in the response of cancer cells to high concentrations of melatonin. Several investigators have suggested that S-glutathiolation, due to its effect on cellular redox status, modulates protein ubiquitination and protects proteins from degradation (Biswas et al., 2006; Bandyopadhyay et al., 1998; Jahngen-Hodge et al., 1997). Melatonin reportedly reduces glutathione levels in cells that undergo apoptosis (Rodriguez et al., 2013). The role of melatonin as an antioxidant has been well documented. Melatonin increases the activity of superoxide dismutase (SOD) (Reiter et al., 1999), glutathione peroxidase (Rodriguez et al., 2004), hemoxygenase 1 (Wang et al., 2009), nicotinamide adenine dinucleotide phosphate (NADPH) and quinone oxidoreductase (Wang et al., 2012b). A recent report provides evidence for involvement of the NRF2-ARE (NRF2-antioxidant response element) signaling pathway in melatonin-induced increase in these enzymes (Ding et al., 2014). Since NRF2 is degraded by the proteasome via a ubiquitin dependent process (Chapple et al., 2012), the melatonin-induced increase in antioxidant enzymes could be explained by melatonin as an inhibitor of the proteasome.

One mechanism for controlling proteasomes is phosphorylation of its subunits (116). Sha et al. (2011) provided evidence that phosphorylation of proteasome subunits enhances the activity of the proteasome. Proteasome subunits modified by phosphorylation include the Rpt2 and Rpt6 ATPases located in the Rpt ring in the base of the regulatory particle (see Fig. 3). The ATPases in this ring regulate such activities as protein substrate unfolding, gate opening, and translocation of proteins into the proteasome (Lasker et al., 2012; Xie, 2010). Rpt6 (aliases PCMC5, Sug1, Trip1) is phosphorylated by CAMKII (Jarome et al., 2013; Djakovic et al., 2009, 2012). CAMKII, as noted above, has been reported to copurify with the Rpt6 enzyme (Bingol et al., 2010). CAMKII is inhibited by melatonin in hepatocellular carcinoma cells in vitro (Carocci et al., 2013). Although melatonin signaling is not the same in cancerous cells and in non cancerous cells (Luchetti et al., 2010), these studies suggested a role for melatonin in regulating the proteasome of cancer cells. Indeed, the first report of a direct inhibitory effect of melatonin on the chymotryptic activity of the proteasome was recently reported by Park et al. (2014) in a study of melatonin treatment of human renal cancer cells. Summary and conclusions Melatonin and the proteasome inhibitor bortezomib share many cell signaling pathways, suggesting the hypothesis that melatonin is a proteasome inhibitor. The reactions of normal and cancer cells to proteasome inhibitors are not the same; some cancer cells are more sensitive to the actions of a proteasome inhibitor than normal cells; some cancer cells are also more sensitive to the antiproliferative and pro-apoptotic effects of melatonin than normal cells. Both bortezomib and melatonin inhibit the activity of NF-κB. Both bortezomib and melatonin stimulate apoptosis in cancer cells. Both bortezomib and melatonin increase the sensitivity to TRAIL-induced apoptosis in human malignant gliomas. While it has not been demonstrated that melatonin has a direct effect on the proteasome similar to that of bortezomib, nevertheless it should be tested more thoroughly for its antiproliferative and proapoptotic effects in tumors sensitive to proteasome inhibitors. Melatonin administration could be useful as an adjunct to proteasome inhibition therapy for several reasons. It could reduce the toxicity of these drugs and in some cases might be useful in preventing drug resistance. It is predicted that melatonin administration would be most effective in cancers and malignancies which have already been shown to be sensitive to proteasome inhibitors. Clinical trials of melatonin administration in these types of cancer appear to be warranted. Author contributions Both authors contributed to the article. J Vriend wrote the original draft. Conflict of interest statement The authors report no conflict of interest.

Acknowledgments J Vriend acknowledges the University of Manitoba for granting him a sabbatical leave to work on this project. The authors received no funding for this project. References Aberle H, Bauer A, Stappert J, Kispert A, Kemler R. Beta-catenin is a target for the ubiquitin–proteasome pathway. EMBO J 1997;16:3797–804. Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lararus DD, et al. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 1999; 59:2615–22.

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