Morphine and Tumor Growth and Metastasis

Cancer Metastasis Rev DOI 10.1007/s10555-011-9285-0

NON-THEMATIC REVIEW

Morphine and tumor growth and metastasis

Banafsheh Afsharimani & Peter Cabot & Marie-Odile Parat

# Springer Science+Business Media, LLC 2011

Abstract Morphine is an analgesic widely used to alleviate 1 Introduction cancer pain. In addition, the perioperative management of pain in cancer surgery patients most often includes opioids. Opioids are widely used in the pain management of cancer However, there are reports that these drugs may alter cancer patients, and not surprisingly, interest in the possibility that recurrence or metastasis. Several mechanisms have been these may alter the course of cancer is not recent [1]. proposed, such as the modulation of the immune response However, there has been recent awareness regarding the use or cellular pathways that control the survival and migratory of opioids in cancer surgery patients based on a combina- behavior of cancer cells. The published literature, however, tion of (1) multiple articles from the 1990s indicating that presents some discrepancies, with reports suggesting that morphine has immunosuppressant properties which can opioids may either promote or prevent the spread of cancer. promote cancer, but that suppressing pain alleviates the It is of great importance to determine whether opioids, in surgical stress and thus might be protective against tumor particular the most widely used, morphine, may increase metastasis; (2) more recent cellular and biological in vitro the risk of metastasis when used in cancer surgery. This and in vivo studies exploring pathways by which opioids review examines the available data on the effects of may directly or indirectly promote or prevent cancer, morphine which influence cancer metastasis or recurrence, including recent literature on morphine and angiogenesis; including immunomodulation, tumor cell aggressiveness, and (3) recent retrospective clinical studies and ongoing and angiogenesis, with special emphasis on recently prospective trials testing whether regional anesthesia and published clinical and laboratory based studies. We further analgesia offers protection against metastasis or recurrence discuss the parameters that may explain the difference when compared to general anesthesia with opioid analgesia. between reports on the effects of morphine on cancer. This review attempts to clarify the positive and negative effects of opioids, more specifically morphine, on tumor Keywords Morphine . Opioids . Invasion . Metastasis growth and metastasis which are mediated by a combination of direct effects on tumor cell proliferation and invasion and indirect effects on processes that are key to tumor development, such as immunity, inflammation, or angiogenesis.

: : * B. Afsharimani P. Cabot M.-O. Parat ( ) 2 Direct effect of morphine on tumor cells University of Queensland School of Pharmacy, 20 Cornwall Street, Woolloongabba QLD 4012, Australia 2.1 Opioids affect tumor cell proliferation and apoptosis e-mail: [email protected] The three major types of receptors to which endogenous M.-O. Parat — μ κ δ Department of Outcomes Research, Cleveland Clinic, and exogenous opioids bind the -, -, and -opioid Cleveland, OH, USA receptors—have been identified not only in peripheral Cancer Metastasis Rev sensory neurons and the CNS but also in various other cell anticancer effects at high doses (1–10 mM) by inhibiting types and, in cancer cells, via detection of the corresponding the growth of several cancer cell lines in vitro and mRNA [2], the protein [3, 4], or the aptitude of the cells to inhibiting TNF-α expression [18, 19]. Nitric oxide or bind [5–8] and/or respond to opioids. Opioids directly reactive oxygen species (ROS) have direct and indirect regulate the growth of normal and neoplastic cells by tumoricidal effects, and morphine stimulates their produc- modulating cell proliferation and/or apoptosis, which gives tion and release, which could also account for some of the these drugs the theoretical capability to regulate tumor anti-tumor effects of morphine [13]. growth and metastasis. However, reports are discrepant, In contrast, the anti-apoptotic actions of morphine in depending on the cell type studied and the dose of morphine tumor cells subjected to pro-apoptotic stimuli have been or other opioid used. Whether direct effects of morphine on reported [20, 21]. Similarly, morphine-induced increased tumor cells matter in vivo and whether the final outcome of proliferation was documented in K562 leukemia cells [22]. morphine administration to a patient would be the inhibition It is likely that this variability could be accounted for by or promotion of malignant cell proliferation and survival is cell-specific activity. However, the majority of published still debatable. reports point to the ability of exogenous opioids to prevent Several reports have documented a pro-apoptotic effect rather than promote the survival and proliferation of tumor of opioids on cancer cells in vitro at nanomolar [6], cells. micromolar [9], or millimolar [10, 11] concentrations either by quantifying apoptotic cells [12]orbymeasuring 2.2 Opioids alter tumor cell invasion hallmarks of apoptosis such as the cleavage of pro- apoptotic caspases or the release of cytochrome c from Tumor cell invasion requires cellular processes such as the mitochondria [10, 11]. Pathways proposed to be adhesion, extracellular matrix degradation, and cell migra- involved in the effect of opioids on apoptosis include the tion. Studies have indicated that opioids can modulate all of activation of the anti-apoptotic kinase Akt, activation of c- these processes. The adhesion of cells to the extracellular Jun N-terminal kinase, generation of reactive oxygen matrix components collagen type I and fibronectin was species, generation of nitric oxide, increased expression of reduced by micromolar concentrations of morphine, and so pro-apoptotic Bim, and decreased expression of anti- was adhesion to type IV collagen, while adhesion to apoptotic Bcl-2, as well as p53- and NFκB-mediated laminin or basement membrane was unaltered. The cells pathways [9–11, 13]. P53 is a key player in the regulation used in this study were non-cancer cells engineered to of apoptosis, and it has been shown to be involved in express functional μ-opioid receptor. Mechanistic insight morphine-induced apoptosis via (1) morphine-induced indicated reduced phosphorylation of kinases involved in stabilization of p53 and (2) morphine-induced apoptosis in focal adhesions [23]. Adhesion of colon cancer cells to cells with normal p53, but lack of an effect in cells with collagen type IV was reduced as well in cells pretreated mutant dominant-negative p53 [14]. NFκB is a transcrip- with morphine in another study [24]. tion factor which, besides controlling the immune response The migratory behavior of different cell types responds to stress, regulates several genes associated with apoptosis differently to morphine: decreased for immune cells in a and angiogenesis in various tissues. It is suggested that number of studies as described in our immune suppression morphine can block NFκB directly by modulation of NO paragraph, decreased or increased for endothelial cells as release [13]. described in our angiogenesis section, increased for micro- The involvement of the opioid receptor(s) in the anti- glial cells [25], decreased in colon carcinoma cells [24], and proliferative effect of morphine is not always clear, as unchanged in multiple cancer cell lines [26]. shown by the reports assembled in Table 1.Atnano- Invasion relies on matrix digestion by enzymes secreted molar concentrations, morphine and other opioid agonists by the cancer cells themselves or by stromal cells in were shown to inhibit the proliferation of a breast cancer response to cancer cells. Matrix metalloproteases and cell line that had κ-andδ-opioid but not μ-opioid urokinase type plasminogen activator are known to play a receptors, and the authors hypothesized that morphine key role in this process. Morphine inhibits matrix metal- interacted with receptor systems other than opioid loprotease (MMP)-2 and MMP-9 expression [24, 27] but receptors [6]. The somatostatin receptor system has been enhances uPA production [4, 28] by breast and colon cancer proposed as a potential candidate, and the interaction of cells. Invasion into reconstituted basement membrane opioid signaling with nicotine or estrogen signaling in the (Matrigel) was shown to be decreased in colon cancer cells regulation of the proliferation of tumor cells has also been [24] but unchanged in a number of cancer cell lines tested suggested [5, 15, 16]. An effect of opioids on the in another study [26]. cytoskeleton could also mediate their antiproliferative Except for the documented increase in uPA, the in vitro action [17]. Morphine has been shown to possess data therefore indicate that by acting directly on the tumor Cancer Metastasis Rev cells, morphine may reduce their invasive potential. It Opioids are now known to modulate the immune should be noted that the direct effect of morphine on tumor response by a combination of central and peripheral cells may not be as important in promoting or preventing mechanisms [44]. Signals of central origin may be relayed tumor growth and metastasis as the effect of morphine on through (1) the hypothalamic–pituitary–adrenal (HPA) axis, the stroma, whose involvement in regulating the cancer resulting in the production of glucocorticoids which are growth is receiving increasing attention. immunosuppressive, and (2) the sympathetic nervous system eliciting the release of biologic amines into lymphoid organs, which also reduce immunocompetence 3 Indirect effects of morphine on tumor growth [45]. Studies have documented that corticosteroids only and metastasis partially mediate morphine immunosuppression [46–48]. It has been suggested that the HPA axis is involved in the 3.1 Opioids are immunosuppressive immunosuppression by chronic opioid exposure, while the sympathetic nervous system mediates the immunosuppres- The vast majority of reports indicate that morphine and sion induced by acute opioid administration [49]. A other exogenous opioids are immunosuppressive. The receptor-mediated effect has been proposed based on the immune system plays a key role in the control of tumor finding that morphine immunosuppression is abolished in formation and metastasis. Tumor cells express non-self- mice genetically deprived of the μ-opioid receptor [50]. antigens and are subjected to killing by activated T and NK In addition, withdrawal from morphine in dependent mice cells and to the cytotoxic action of cytokines such as has also been shown to induce marked immunosupression interferon gamma [29, 30]. Mice lacking various compo- [51]. nents of the immunosurveillance system are more suscep- It should be noted that although the immunosuppressive tible to tumorigenesis [29, 30]. effect of opioids is widely documented, there are a number Moreover, after cancer cells have escaped immunosur- of studies showing a favorable immunomodulatory effect of veillance and a tumor develops, innate and adaptive perioperative morphine administration on surgical stress immune cells are also present in the tumor microenviron- effects and metastasis promotion (Table 2), presumably due ment where they can promote or fight tumor development to the relief of pain and the attenuation of the pain [30, 31]. Acute and chronic administration of exogenous component of surgical stress. Given the immune suppres- opioids including the one most commonly used—morphine sion intrinsic to morphine, alleviating the postoperative —inhibits components of the cellular and humoral immune pain via other means is proposed as an alternative to offer function such as antibody production, NK cell activity, better protection than morphine against surgical stress- cytokine expression, blood lymphocyte proliferative induced immune suppression and tumor promotion. Using responses to mitogen, and phagocytic activity [32–35]. alternate opioids such as tramadol or the partial μ-agonist While many studies have tested the immunosuppressive buprenorphine [35, 39] or anti-inflammatory drugs [52–54] effects of morphine in vitro and in vivo in rodents and has been suggested to be beneficial following findings in humans [32, 33, 35, 36], other opioids have been shown to either animal or clinical studies. In addition, utilizing also possess immunosuppressive effects, e.g., therapeutic regional anesthesia and analgesia is another option that agents such as fentanyl [37–39], or pharmacological tools has attracted a lot of recent attention, as discussed later in such as subtype-specific opioid receptor agonists U50 this review. Results from studies testing the effect of 488H, deltorphin II, or DPDPE [40]. morphine on the immune response and tumor burden are Opioid receptors have been identified in cells of the assembled in Table 2. immune system such as polymorphonuclear leukocytes, macrophages, T lymphocytes, splenocytes, macrophage- 3.2 Opioids and inflammation are closely interrelated like, and T cell-like cell lines [33]. Opioids added directly to a number of cell types of the immune system in vitro Inflammation, including its subclinical forms, is thought to alter their protein expression profile [41] and their function: mediate the tumorigenic effect of most cancer risk factors, reduced chemotaxis and phagocytosis by macrophages, including tobacco, obesity, infections, and old age [30]. reduced B-cell proliferation, and suppression of antibody Inflammation further modulates the host immune response formation [42]. However, a number of studies have failed to to tumors [30], and tumor development is accompanied by find a direct effect of morphine in vitro on NK cell activity. an inflammatory response which creates a pro-tumorigenic In addition, in contrast to exogenous opioids, an increase in microenvironment [30]. Opioids regulate the expression of natural killer cell activity in response to endogenous inflammatory cytokines and their receptors, and immune opioids has been documented [43], which was inhibited cells under the influence of cytokines can release endoge- by naloxone. nous opioids at sites of inflammation [33, 55, 56]. It has Cancer Metastasis Rev

Table 1 Effect of morphine on tumor cell growth and survival

Study model Morphine dosage Effect on cell growth Proposed mechanism Reference and duration of and survival administration

MCF-7 human breast 10−8–10−7 M for 24 h Decrease in cell Activation of μ- and κ-opioid [16] carcinoma cells proliferation receptors on the tumor cells; reversed by naloxone; dependent on presence of oestrogens Multiple lung cancer cells 10−9–10−7 M for 24 h Decrease in cell proliferation Activation of μ- κ-, and δ-opioid [5] receptors on tumor cells Effect of morphine inhibited by nicotine EL-4 leukemia cells, P388, MM-46 350 μM to 2.8 mM for Inhibition of cell growth with – [98] and Meth-A cells 48/72 h different IC50 values for different cell lines K562 leukemia cells 10−9–10−6 M for 24 h Increased proliferation of – [22] leukemia cells Lung cancer cells NCI-Hi 57 (non- 10−8–10−7 M for 24 h Increased apoptosis Activation of opioids receptors [99] small cell), NCI-N4i 7 and NCI- and reduction in PKC activity H146 (small cell) T47D human breast cancer cells 10−12–10−6 for 4 days Dose-dependent inhibition of Interaction of morphine with the [15] cell proliferation somatostatinergic system (IC50 = 1.08 nM) T47D human breast cancer cells 1–100 nM for 5 days Dose-dependent inhibition of Effect of morphine independent from [6] cell proliferation μ-opioid receptors Human prostate cancer cells 10−10 to 10−9 M for 4– Inhibition of tumor cell Activation of κ-opioid receptors or [7] (LNCaP, DU145, and PC3) 5 days proliferation effect of morphine not mediated by opioid binding sites depending on the cell line tested PC9 (lung cancer cells) KATO III Incubation with high Morphine inhibits tumor cell Inhibition of NFκB activation; [18, 19] (stomach cancer cells) HL-60 pro- concentrations of growth in a dose-dependent suppression of TNF-α expression myelocytic leukemia cells Neuro- morphine (mM manner blastoma cells U251 glioma cells range) for 3–4 days SEKI melanoma cells SKNO-1 leukemia cells Human oral tumor cells (HSC-2 and 10−5–5×10−4 M Cytotoxicity of morphine – [100] HSG) Colon carcinoma cells 26-L5 00–0.35 μM for 24 h Unchanged cell growth – [24] MCF-7, MDA-MB231 breast carci- 10−5–2×10−3 M for Reduction in tumor cell Naloxone-independent p53 [14] noma cells HT-29 colon cancer 24 h proliferation at low dose, stabilization and subsequent cells induction of apoptosis at increase in pro-apoptotic factors high doses Lower effect on p21, Bax, Fas death receptor HT-29 cells SC injection in nude mice of: MCF- IP 10– Reduction in tumor size for 7, MDA-MB231 breast carcinoma 30 mg kg−1 day−1 MCF-7, MDA-MB231 cells HT-29 colon cancer cells for 3 weeks breast cancer cells but not (resulting in 0.9– HT-29 colon cancer cells 60 μM circulating concentration) SH-SY5Y neuroblastoma cells 10−7–10−5 M for 24 h Protects against serum μ-opioid receptor activates a Gi/0 [20] withdrawal-induced cell linked, PI3Kinase/Akt pathway death HT-29 colon cancer, MIA PaCa-2 10−6 M for 2, 5 and Increase in tumor cell Pro-apoptotic effect of morphine [12] pancreatic cancer CAL-27 squa- 7 days apoptosis Increase in cell partially reversed by naloxone mous cell carcinoma of the head necrosis and neck Jurkat cells (leukemia) 10−5 or 3 × 10−5 M for Increase in apoptosis Activation of FADD/p53, anti- [9] 36 h apoptotic PI3K/Akt and NF-κB pathways Neuroblastoma cells (SH-SY5Y) Morphine (50– Inhibition of doxorubicin- Inhibition of ROS and mitochondrial [21] 200 μM) was added induced apoptosis cytochrome c release by morphine 1 h before Inhibition of NFkB activation doxorubicin treatment (48 h) Cancer Metastasis Rev

Table 1 (continued)

Study model Morphine dosage Effect on cell growth Proposed mechanism Reference and duration of and survival administration

Colon cancer HT-29 cells Morphine 1 nM to No significant change in cell – [4] 1 μM for 24 h proliferation MCF-7 and MDA-MB231 breast 500 μM to 2 mM Increase in tumor cell Decreased activation of Akt [10] cancer cells apoptosis Increased cleavage of caspase 8 Inhibition of morphine-induced cell death by β-arrestin MCF7 breast cancer cells 10−8–10−4 M No change in cell numbers at – [28] morphine for 2, 3, any concentration or time and 5 days assayed Neuroblastoma cells (SH-SY5Y) 0.5–4 mM for 48 h Dose-dependent increase in Activation of c-Jun N-terminal kinase [11] apoptosis and increase in reactive oxygen species upregulation of pro-apototic protein Bim and downregulation of anti-apoptotic protein Bcl-2 increase in cytochrome c release and caspase-3 and −9 activation Lewis lung carcinoma cells 1 nM for 24 h Increased proliferation μ-opioid receptor dependent [3]

been suggested that the κ- and μ-opioid receptors play [65]. Accordingly, morphine enhanced endothelial cell opposing roles, with the κ-opioid receptor activation proliferation and tube formation in vitro and increased in inducing an anti-inflammatory response while the μ- vivo blood vessel formation in a Matrigel plug assay [60] opioid receptor favors a pro-inflammatory response [57]. and in tumor xenographs [60, 66]. Morphine further However, the picture is far more complex. Morphine has improved wound healing, possibly via an angiogenesis- been shown to prevent inflammation-induced angiogenesis mediated mechanism [67]. The opioid antagonist in vivo [58]. The modulation of inflammation may therefore methylnaltrexone was shown to prevent VEGF-induced provide mechanistic insights into how morphine and other angiogenesis in an in vivo Matrigel plug assay [68]. In opioids influence the development of cancer. vitro pro-angiogenic effects are also reported with physiological concentrations of the endogenous opioids 3.3 Morphine modulates angiogenesis endomorphin-1 and endomorphin-2, and deltorphin 1 [69]. Of note is that some of the pro-angiogenic effects of Recent publications point to a role of morphine in morphine are not reversed by naloxone [60], but morphine- angiogenesis (Table 3). Endothelial cells express opioid induced tumor growth and angiogenesis are prevented by receptor(s) [59, 60] and respond to opiates by increasing co-administration of the cyclooxygenase-2 COX-2 inhibitor intracellular calcium concentration and nitric oxide produc- celecoxib [70]. tion [59–62]. In addition to vascular permeability, nitric One mechanism through which the VEGF pathway oxide regulates endothelial cell proliferation, migration, and increases endothelial cell migration involves adhesion protease release, all of which are important for angiogen- molecules, especially intercellular adhesion molecule 1 or esis. Clinically relevant concentrations of morphine result ICAM-1: VEGF stimulation of endothelial cells increases in the activation of mitogen-activated protein kinase/ ICAM-1 expression [71], VEGF-induced endothelial cell extracellular signal-regulated kinase MAPK/ERK and pro- migration is decreased by a ICAM-1 blocking antibody survival Akt in cultured endothelial cells [60]. In vitro, [71], and ICAM-1-deficient endothelial cells exhibit re- morphine was shown to induce endothelial cell proliferation duced eNOS activation, NO bioavailability, and migration and migration via the transactivation of growth factor [72]. ICAM-1 is reported to not only increase endothelial receptors that play a key role in migration and survival of cell migration [72] but also to mediate the recruitment of these cells, including the vascular endothelial growth factor endothelial progenitor cells, which participate in angiogen- (VEGF) receptor 2 [63, 64] or the platelet-derived growth esis [73]. Interestingly, endothelial cells exposed to μ- factor (PDGF)-beta receptor [63]. In a non-endothelial cell agonists upregulate ICAM-1 [74]. Taken together, these system, morphine was further shown to transactivate the data suggest that morphine transactivation of VEGF receptor (FGFR1) of another growth factor playing a key receptor 2 may result in an increased endothelial cell role in angiogenesis, namely fibroblast growth factor (FGF) motility mediated by nitric oxide and ICAM-1. Cancer Metastasis Rev

Table 2 Effect of morphine-induced immunomodulation on tumor growth and metastasis

Study Morphine dosage and Tumor type Effect on Immunomodulatory Proposed Reference model duration of exposure tumor effect mechanism

In vivo SC injection (20 mg/kg) Colon Reduced incidence – Natural killer cell [101] (rats) 1 day before and 2 days adenocarcinoma and weight of cytotoxicity is after tumor cell cells injected hepatic metastases increased 24 h after inoculation into cecal vein morphine administration (rebound after initial suppression) In vivo Daily SC injection of SC growth of Increase in the weight of Spleen and thymus atrophy Macrophage and T cell [98] (mice) 10 mg/kg for 10 days, solid tumors: solid tumors Reduction in T cell and B inactivation through starting 24 h after tumor EL-4 leukemia cell mitogen-induced MOR Modulation of cell inoculation cells and Sarco- proliferation and re- hormone release ma 180 carci- sponse to antigens (prolactin, growth noma. hormone or insulin) IP growth of Decrease in the survival through MOR ascites tumor rate of mice cells: P388 leukemia and Meth-A fibro- sarcoma In vivo SC injection of 15 mg/kg Injection of Significant increase in NK cytotoxicity Surgical stress depresses [52] (rats) —4 times in the syngeneic colon hepatic tumor burden suppressed NK cell cytotoxicity perioperative 24 h adenocarcinoma which is made worse cells in by morphine sulfate ileocecal vein+ standardized surgical stress In vivo IP injection 5 mg/kg Tail vein injection Attenuation of the surgery- The pain, which [102] (rats) 30 min before surgery, of MADB106 induced increase in re- increases tumor SC slow-release 5 mg/kg mammary tention of malignant cells retention, is prevented immediately after surgery adenocarcinoma in lungs by morphine and again at time of cells 5 h after tumor cell inoculation laparotomy In vivo IP injection 10 mg/kg Tail vein injection Attenuation of the surgery- ?—The surgery induced NK cells play a critical [103] (rats) 30 min before surgery, of MADB106 induced increase in re- decrease in NK cell role in morphine’s SC slow-release 5 mg/kg mammary tention of malignant cells cytotoxicity is not altered attenuation of surgery immediately after surgery adenocarcinoma in lungs by morphine. However induced increase in and after tumor cell in- cells 4 h after morphine’s protective tumor cell retention oculation laparotomy effects require the presence of NK cells In vivo IP injection 8 mg/kg IV injection of Attenuation of surgery- Attenuation of surgery- The pain, which is an [104] (rats) 30 min before surgery MADB106 induced increase in re- induced cortisol surge important component and/or SC slow-release mammary tention of malignant cells of surgical stress, is 4 mg/kg immediately af- adenocarcinoma in lungs by all timings of attenuated by ter surgery and/or 2 mg/ cells 4 h after morphine administration. morphine kg at the time of tumor laparotomy Preoperative morphine cell inoculation the most efficient In vitro 0.3–300 ng/ml for 5 days T cell leukemia Enhanced cell death in Stimulation of the Direct stimulation of [105] during co-culture or re- cells (MT-2) MT-2 malignant cells ex- cytotoxic T cells activity cytotoxic T cell peated addition of 3 ng/ induced by posed to the sensitized Increase in the number of generation at the onset ml to CM for 5 days HTLV-1 (hu- cytotoxic T cells treated IFN gamma- producing of sensitization phase man leukemia with morphine CD8+ T cells NK regulation by virus) morphine is mediated by central action 3.2 pg/ml to 32 μg/ml for T cell leukemia No significant difference in No effect on NK activity 16 h and daily addition cells (MT-2) the number of dead cells of 3 ng/ml induced by when exposed to 6 to CM for 5 days HTLV-1 (hu- different mononuclear man leukemia cell samples treated or virus) not treated with morphine In vivo IP injection 10 mg/kg Tail vein injection Systemic morphine No significant effect on Reduction of [85] (rats) immediately before of MADB106 attenuated the surgery- immune defenses by neuroendocrine stress laparotomy mammary induced increase in re- systemic morphine was response to surgery by Spinal blockade with adenocarcinoma tention of malignant cells measured alleviating pain Cancer Metastasis Rev

Table 2 (continued)

Study Morphine dosage and Tumor type Effect on Immunomodulatory Proposed Reference model duration of exposure tumor effect mechanism

50 μg bupivacaine+ cells 4–5 h after in lungs by 50–62% but morphine 10 μg laparotomy not the pulmonary metastasis (quantified 3 weeks after inoculation) In vivo SC injection of 5 and Orthotopic B16- significant reduction in Hypothesized but not Morphine provides [106] (mice) 10 mg/kg daily for BL6 melanoma tumor growth and lung shown cancer pain relief 6 days starting 16 days inoculated in metastasis post tumor inoculation the paw In vivo 10 mg/kg immediately Tail vein injection Morphine increased lung The elevation of Increase in serum [39] (rats) after laparotomy and 5 h of MADB106 tumor metastasis in the corticosterone and the corticosterone level by later. mammary absence of surgery. reduction in NK cell HPA activation causes adenocarcinoma Morphine failed to activity 6 h post surgery immunosuppression cells 5 h after prevent the surgery- match the increased laparotomy induced increase in lung tumor numbers at tumor number 3 weeks 3 weeks post-surgery

In contrast to all the literature reporting the pro- experiments often use endothelial cells of human origin, angiogenic effect of morphine, there is a body of evidence while in vivo angiogenesis experiments use rodents. indicating that morphine is detrimental to endothelial cells Analysis of the opioid receptor(s) present on endothelial and to neovascularization. An early study showed reduced cells demonstrated sensitivity to opiate alkaloids but angiogenesis in the chicken chorioallantoic membrane insensitivity to opioid peptides (a profile characteristic of (CAM) assay in the presence of morphine [75], which the μ3-opioid receptor) in both rat and human endothelial was later corroborated by the opioid receptor-dependent cells [59]. The concentrations of morphine used in the reduced angiogenesis shown in the CAM assay using the experiments showing anti-angiogenic properties were re- endogenous opioid [Met5]-enkephalin [76]. High concen- portedly high—up to 100 μM and even 1 mM in vitro and trations of morphine are toxic to endothelial cells in vitro up to doses of 20–30 mg kg−1 day−1 in mice. Concen- [62, 77] and result in oxidative stress-mediated endothelium trations of 1 mM morphine are reportedly toxic to several dysfunction in vivo [77]. Accordingly, morphine reduced cell types in vitro. The relatively short half-life of morphine wound healing and mobilization of endothelial progenitor in mice would suggest that studies with intermittent cells in mice and decreased the formation of capillaries administration of morphine could provide different kinetics in vitro as well as in an in vivo Matrigel plug assay [58, of receptor exposure to circulating levels of morphine 78, 79]. In the context of ischemia, where neovasculariza- compared to studies using minipump-controlled release. tion is beneficial via the development of collateral Furthermore, mice process morphine differently from circulation, morphine has been shown to prevent humans and generate mostly morphine-3-glucuronide, hypoxia-induced VEGF production in vitro and in vivo, which is inactive on the MOR, whereas humans generate and thus presumably angiogenesis [80, 81]. These results both active M6G and inactive M3G. Justifying a morphine were confirmed using a mouse tumor angiogenesis assay dose administered to mice based on a human relevant dose where the effect of morphine was abolished by the adjusted for body weight is therefore irrelevant. pharmacological or molecular ablation of the μ-opioid receptor [82]. Morphine was also shown to indirectly 3.4 Perioperative opioids in tumor surgery patients inhibit angiogenesis in in vivo models via decreased inflammation [58, 79]. Finally, naltrexone alone has been While they appear to be an essential component in the pain suggested to increase angiogenesis in vivo in two separate management of terminal cancer patients, opioids, and in models, indicating the potential tonic inhibition exerted by particular morphine, are also used perioperatively in endogenous opioids [76, 83]. patients undergoing solid tumor resection. In this context, At present, whether morphine would impact angiogen- the potential immunosuppressive and pro-angiogenic prop- esis when administered to cancer patients is still unclear. erties of morphine have raised concerns. Several considerations about experimental conditions can It is well known that in non-cancer surgery patients, explain the discrepancy between results from different regional analgesia prevents the postoperative drop of groups on the effect of morphine on angiogenesis. In vitro immune response compared to opioid analgesia. Epidural Cancer Metastasis Rev

Table 3 Effect of opioids on tumor angiogenesis

Type of Type Morphine dosage and Study model Effect on Proposed Reference opioid of duration of exposure angiogenesis mechanism (s) study

Morphine In 24–96 h 5, 10, Chick embryo Angiostatic effects (lower Not reversed by Naloxone [75] sulfate vivo 50 μg (peak angiostatic chorioallantoic doses (5 μg) caused faster effect (53%) with 5 μg membrane and more intense effect than at 72 h) higher doses (10 μg/50 μg) β-endorphine In 24–96 h 5, 10, 15 μg Chick embryo Time- and dose-dependent Stimulation of IFN production vivo chorioallantoic angiostatic effect (peak at by LGL/NK cells Inhibition membrane 10 μg) of PG-E2 as a pro-angiogenic factor [Met5]- In 5 μg per disk Chick embryo Decreased number of blood Action reversed by naloxone; [76] enkephalin vivo chorioallantoic vessels and total vessel Methylnaltrexone alone membrane length increases angiogenesis, suggesting tonic inhibition by endogenous opioid(s) Morphine In 1.5–150 nM for 24 h Mouse heart Inhibition of hypoxia-induced Inhibition of HIF-1α Reversed [80] vitro microvascular VEGF expression in both by naloxone endothelial cells types of ECs Inhibition of Human PI3kinase and PKC- medi- umbilical vein ated activation of the VEGF endothelial cells promoter Morphine In 1 μM for 24–48 h Human dermal Increased vascular endothelial Morphine elicits NO release, [60] vitro microvascular cell proliferation and MAPK/ERK signaling endothelial cells Enhancement of endothelial Morphine inhibits EC tube formation on Matrigel apoptosis and activates In Morphine (10 μM) Matrigel plug Increased neovascularization survival promoting Akt The vivo containing Matrigel assay in mice pro-angiogenic effect of implanted into mice morphine is not reversed by 0.714 mg kg−1 day−1 for MCF-7 cell breast Stimulation of tumor naloxone in the Matrigel plug first 15 days and then tumor xenograft angiogenesis and tumor assay but is reversed by 1.43 mg kg−1 day−1 in mice growth naloxone in the tumor angiogenesis assay Morphine; In Topical cream: morphine, Open wounds in Enhanced wound healing; Upregulation of eNOS and [67] Fentanyl; vivo 1.5 mg/g fentanyl, 5 rats with Increased neovascularisation iNOS in the wound; Hydromorphone mg/g, hydromorphone, ischemic skin in wound tissue upregulation of VEGF 0.2 mg/g, flap receptor Flk1 Morphine; In 0.1 μM in the migration Human dermal Increased proliferation and VEGF receptor transactivation [64] morphine-6- vitro assay 0.1 μM for 24 h in microvascular migration of endothelial reversed by methylnaltrexone glucuronide; the proliferation assay EC cells DAMGO Morphine In 0.01–10 μM for 48 h Mouse retinal Increased proliferation and Transactivation of VEGF [63] vitro endothelial cells survival of EC receptor Flk1 and PDGF-β receptor by morphine; subsequent MAPK/ERK and Akt phosphorylation Morphine In 0.714 mg kg−1 day−1 for SCK mammary Increased tumor Morphine upregulates COX-2 [70] vivo 7 days then carcinoma angiogenesis and tumor and PGE2 levels 1mgkg−1 day−1 for 7 implanted SC growth and metastasis more days into right hind thigh Morphine In 20 mg/kg IP for 2 weeks Excisional wound Impaired angiogenesis Increased superoxide [78] vivo injury and production by endothelial Matrigel assays cells; reduced endothelial in mice progenitor cell mobilization In 10−8–10−4 M for 24 h human umbilical Reduced capillary tube vitro vein endothelial formation cells Endomorphin-1; In 1, 10, 100 nmol/disk during Chick embryo Dose-dependent stimulation Reversed by naloxone [107] Endomorphin- vivo 48 h chorioallantoic of angiogenesis Stimulation of the release of 2; Deltorphin I membrane pro-angiogenic factors such as NO and IL-8; transactivation of growth factor receptors; Protecting endothelial cells from oxidative stress Morphine In Incubation with 1–106 nM Human umbilical Increased apoptosis of Stimulation of NOS; ROS [62] Cancer Metastasis Rev

Table 3 (continued)

Type of Type Morphine dosage and Study model Effect on Proposed Reference opioid of duration of exposure angiogenesis mechanism (s) study

vitro morphine for 48 h vein endothelial endothelial cells significant release; NFκB activation cells at 105 nM morphine and higher Naltrexone In SC injections Experimental Naltrexone increased the Naltrexone may increase liver [83] vivo 20 mg kg−1 day−1 cholestasis number of microvessels in glutathione concentration and model in rats cholestatic but not sham inhibition of apoptosis animals induced by endogenous opioids. Morphine In 0.714 mg kg−1 day−1 by s. Ehrlich ascites Stimulation of tumor – [66] vivo c. injection for 7 days, tumor cell angiogenesis starting 5 days post tumor xenografts inoculation subcutaneously implanted in mice Morphine In SC implantation of 75 mg Mice implanted Decreased tumor growth Mu-opioid receptor mediated [82] vivo slow-release morphine with lung and angiogenesis Morphine reduces hypoxia- pellets, then IP carcinoma cells induced VEGF secretion by 20 mg kg−1 day−1 mixed with cancer cells days 7–14, then IP Matrigel VEGF 30 mg kg−1 day−1 - Matrigel plug days 15–21 assay Morphine In IP injection of 6.25 mg/kg Excisional Delayed wound healing Reduced macrophage and [79] vivo every 6 h for 24 h and sc cutaneous Impaired inflammation- neutrophil recruitment to the implantation of 75-mg wound and induced angiogenesis wound in the presence of slow-release pellet subcutaneously morphine. The effect is μ- implanted opioid receptor mediated Matrigel plugs in mice Morphine In SC implantation of 75- mg SC implanted Decrease in Reduced macrophage-derived [58] vivo slow-release pellets Matrigel plugs lipopolysaccharide-induced HIF-1α and VEGF in re- in mice angiogenesis sponse to LPS Reduced recruitment of neutrophils and macrophages in the Matrigel plug Endomorphin-1; In Micromolar concentrations Human umbilical Dose-dependent increase in Opioid receptor activation [69] Endomorphin-2; vitro vein endothelial endothelial cell leading to stimulation of the Deltorphin I cells proliferation, migration, release of pro-angiogenic adhesion and tube formation factors such as NO and IL-8 Transactivation of growth factor receptors

analgesia (bupivacaine+fentanyl) has been documented to This hypothesis, supported by animal studies [85, 86], attenuate the postoperative suppression of lymphocyte has led to retrospective studies recently carried out to proliferation and to reduce the pro-inflammatory cytokine compare recurrence-free survival in patients subjected to response when compared to opioid analgesia after abdom- general anesthesia and opioid analgesia and patients inal surgery [84]. undergoing surgery with regional anesthesia/analgesia. There is growing interest in the hypothesis that using They showed that regional anesthesia/analgesia signifi- regional anesthesia and analgesia (RAA) in cancer surgery cantly reduced the recurrence or metastasis in breast [87] patients may reduce the rate of recurrence or metastasis. and prostate [88] cancer surgery patients and enhanced This is relevant to our topic because one of the mechanisms survival in colon cancer patients that did not have by which RAA may be protective is that it prevents or metastases at the time of surgery [89]. In contrast, epidural reduces the need for intra- and postoperative morphine use. analgesia for perioperative pain control was found to offer Other proposed mechanisms include preserved postopera- no advantage for cancer recurrence after colorectal surgery tive immunity by attenuation of the metabolic, neuroendo- in a separate study [90], and secondary analysis of records crine, and cytokine stress response to surgery and reduced from patients randomized to general anesthesia alone or use of general anesthetics. combined general/epidural anesthesia failed to confirm a Cancer Metastasis Rev

Fig. 1 Effect of morphine on tumor growth and metastasis. a Morphine has a dual effect on tumor cell proliferation and survival either by activation of different opioid receptors on the cell surface or via opioid receptor-independent pathways. b Both pro- and anti-migratory effects have been reported for morphine. Morphine affects migration by modulating chemo- attractants and ECM proteolytic enzymes such as MMPs and uPA expressed by either the tumor or stroma cells. c Mor- phine modulates angiogenesis through angiogenic factors, endothelial cell proliferation and migration. d Morphine positive or negative regulatory effect on proteases may affect tumor cell extravasation. e Tumor cell invasion and growth in the secondary organ can also be affected by morphine as in the tissue surrounding the primary tumor

difference in prostate cancer biochemical recurrence shown to promote cancer cell proliferation without between epidural and control groups [91]. Clearly, a affecting cancer cell migration when compared to the definitive answer will require prospective, large random- serum of patients undergoing RAA [96]. ized trials such as [92]; five such studies are currently Furthermore, while the data from the prospective studies recruiting patients (clinicaltrials.gov using the search will provide clinically important results, they will not words cancer recurrence regional anesthesia). However, specifically outline the part that morphine plays in the recurrence data and thus interpretation of the results are different anesthesia and analgesia regimens tested. Such not expected for several years. In the meantime, prospec- data are currently unavailable, and attempts to determine tive studies have attempted to elucidate the mechanism whether opioids promote the development of new tumors in through which regional anesthesia/analgesia might be patients via retrospective studies have not shown significant protective toward cancer recurrence or metastasis in difference [97]. patients. RAA was confirmed to inhibit the surgical stress response, as indicated by lower plasma glucose, cortisol, and C-reactive protein in RAA patients com- 4 Conclusions pared to general anesthesia/morphine analgesia—but RAA did not seem to affect the postoperative percent Opioids and the most widely used—morphine—have change in pro-angiogenic factors VEGF and prostaglan- proven direct and indirect effects that can affect the din E2 [93]. In contrast, regional anesthesia/analgesia course of cancer; therefore, the question of their use in decreased circulating TGF-β and IL-1β and attenuated the cancer patients at a crucial time of the disease develop- postoperative increase in lymphangiogenic VEGF-C and ment is legitimate if appropriate pain management in pro-invasion, pro-metastatic, and pro-angiogenic MMP- alternatives are available. At present, it is impossible to 9[94, 95]. Lastly, in an ex vivo experiment, the serum conclude from the existing data whether altered tumor from general anesthesia/morphine analgesia patients was cell proliferation and invasion, inflammation, angiogen- Cancer Metastasis Rev esis, and immune response (Fig. 1)willresultinharmor cell death through Akt and caspase-8 pathways. Neoplasma, 56 – benefit. This is due to the complexity of cellular pathways (2), 108 113. 11. Lin, X., Wang, Y. J., Li, Q., Hou, Y. Y., Hong, M. H., Cao, Y. 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