Free
Perioperative Medicine  |   April 2012
Overexpression of the μ-Opioid Receptor in Human Non-Small Cell Lung Cancer Promotes Akt and mTOR Activation, Tumor Growth, and Metastasis
Author Affiliations & Notes
  • Frances E. Lennon, Ph.D.
    *
  • Tamara Mirzapoiazova, M.D., Ph.D.
  • Bolot Mambetsariev, Ph.D.
  • Ravi Salgia, M.D., Ph.D.
  • Jonathan Moss, M.D., Ph.D.
    §
  • Patrick A. Singleton, Ph.D.
  • *Postdoctoral Fellow, Department of Medicine, Section of Pulmonary and Critical Care, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois. Research Associate, Department of Medicine, Section of Pulmonary and Critical Care, Pritzker School of Medicine, The University of Chicago. Professor, Department of Medicine, Section of Hematology/Oncology, Pritzker School of Medicine, The University of Chicago. §Professor, Department of Medicine, Department of Anesthesia and Critical Care, Pritzker School of Medicine, The University of Chicago. Assistant Professor, Department of Medicine, Section of Pulmonary and Critical Care, and Department of Anesthesia and Critical Care, Pritzker School of Medicine, The University of Chicago.
Article Information
Perioperative Medicine / Pain Medicine / Respiratory System
Perioperative Medicine   |   April 2012
Overexpression of the μ-Opioid Receptor in Human Non-Small Cell Lung Cancer Promotes Akt and mTOR Activation, Tumor Growth, and Metastasis
Anesthesiology 4 2012, Vol.116, 857-867. doi:10.1097/ALN.0b013e31824babe2
Anesthesiology 4 2012, Vol.116, 857-867. doi:10.1097/ALN.0b013e31824babe2
What We Already Know about This Topic
  • Recent epidemiologic studies indicate a positive association between perioperative opioid administration and tumor progression, but the mechanisms are unclear

  • Previous studies show up-regulation of μ-opioid receptors in specific cancer cells including non-small cell lung cancer

What This Article Tells Us That Is New
  • Overexpression of μ-opioid receptors in a human non-small cell lung cancer cell line increased in vitro  and in vivo  measures of tumor growth and metastasis

  • These findings further support the role of μ-opioid receptor activation in tumor progression, and suggest both therapeutic and diagnostic opportunities

THERE is currently much debate in the literature regarding the role of anesthesia and analgesia in the recurrence and metastasis rate of numerous malignancies.1  3 Several retrospective studies have shown a reduced incidence of cancer recurrence after regional anesthesia with reduced doses of opioids after surgery for breast, prostate, and colon cancer and melanoma, although other studies have failed to detect any significant differences.4  7 Several hypotheses have emerged to explain the differences in recurrence rates, including immunosuppressive effects and direct effects on tumor cell growth.8  10 Our research has focused on the μ-opioid receptor (MOR) and its role in directly regulating tumor growth and metastasis.11 
The opioid receptors are divided into three major subgroups, μ, κ, and δ and are members of the G-protein coupled receptor superfamily. MOR is the main target for opiates such as morphine, fentanyl, and heroin. MOR is responsible for mediating the clinically beneficial effects of opioids, including analgesia, but also mediates the secondary side effects such as addiction, respiratory depression, and constipation.12,13 Endogenous MOR ligands include endomorphin-1 and -2, β-endorphin, enkephalins, and dynorphin A.14 MOR is expressed in both the central nervous system and peripheral tissues. The exact mechanisms governing MOR expression are unclear but a number of OPRM  transcription factors have been identified, including AP-1, NF-κB and STAT-6.15 OPRM  is also subject to epigenetic regulation including promoter methylation and histone modification.15,16 The human MOR gene OPRM1  is 236 kb and consists of 11 exons, which give rise to at least 17 splice variants. MOR1 is the most abundant transcript and consists of exons 1, 2, 3, and 4 and gives rise to a protein with a molecular weight of approximately 44 kDa.13 Acute opioid activation can lead to phosphorylation of the receptor (although this may be ligand dependent), inhibition of adenylate cyclase activity, and activation of mitogen-activated protein kinase, phosphotidylinositol 3-kinase, and phospholipase C.17 After activation, MOR may undergo endocytosis and be recycled. The rate and mechanisms of receptor densensitization and endocytosis are believed to be important in the development of opioid tolerance and addiction and are an area of ongoing investigation.18 It should be noted that there are a large number of agonist ligands for MOR, each of which may induce different cellular and physiologic outcomes.13,18 
Previous studies have also reported on the role of MOR in angiogenesis, tumor cell growth, and metastatic spread.9,10 A number of reports have indicated that both endogenous opioids (endomorphin-1,-2) and those used clinically (morphine) can stimulate angiogenesis both in vitro  and in vivo  .19  21 Morphine has also been reported to stimulate tumor growth and metastasis, although the results vary and may be dependent on the cell type and the model used.12 However, an early report by Zagon and McLaughlin reported that the μ-opioid antagonist, naltrexone, inhibited the growth of metastatic murine neuroblastoma, resulting in increased survival times.22 In our own work, we have previously shown that methylnaltrexone, a peripheral MOR antagonist, can inhibit angiogenesis and potentiate the effects of mTOR inhibitors in vivo  .23  25 
Effective therapeutic strategies for lung cancer, the leading cause of cancer-associated mortality worldwide, are extremely limited, exemplifying the need for early diagnosis and novel therapeutic interventions.26,27 We have previously reported that the MOR is up-regulated in several types of human non-small cell lung cancer (NSCLC).11 However, the functional consequences of MOR overexpression in NSCLC are poorly defined. In this study, we investigated the effects of stable overexpression of MOR1 on Akt and mTOR signaling pathways and the downstream functional consequences in human NSCLC cells. Here we present evidence of a role for MOR in the regulation of NSCLC tumor cell proliferation, migration, and invasion in the absence of exogenous opioid stimulation. Further, through the use of tumor xenograft models and the MOR peripheral antagonist methylnaltrexone, our results suggest a possible therapeutic role for MOR antagonism on cancer growth and metastasis that merits further research.
Materials and Methods
Cell Culture and Reagents
Human NSCLC cell lines A549, SKLU-1, H1703, H358, H1993, H661, SW1573, H522, H226, H1437, H1838, H1975, H2170, and noncancerous BEAS-2B were obtained from ATCC (Walkersville, MD) and cultured in Roswell Park Memorial Institute complete medium (Cambrex, East Rutherford, NJ) at 37°C in a humidified atmosphere of 5% CO2, 95% air, with passages 6–10 used for experimentation. Unless otherwise specified, reagents were obtained from Sigma Chemical Company (St. Louis, MO). Methylnaltrexone bromide or methylnaltrexone was purchased from Mallinckrodt Specialty Chemicals (Phillipsburg, NJ). Temsirolimus was acquired through Wyeth Pharmaceuticals (Madison, NJ). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad (Richmond, CA) and Immobilon-P transfer membrane was purchased from Millipore Corporation (Bedford, MA). Rabbit anti-pSer473Akt, rabbit anti-pThr308Akt, rabbit anti-Akt, rabbit anti-pThr389mTOR, rabbit anti-epidermal growth factor receptor, and rabbit anti-mTOR antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Rabbit anti-pTyr1173epidermal growth factor receptor antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Akt Inhibitor XIII was purchased from EMD Biosciences (Gibbstown, NJ). Mouse anti-human nestin antibody that does not react with mouse or rat nestin (clone 10C2) was purchased from Millipore Corporation. Mouse anti-β-actin antibody was purchased from Sigma Chemical Company. Secondary horseradish peroxidase-labeled antibodies were purchased from Amersham Biosciences (Piscataway, NJ).
Immunoblotting
Immunoblotting was performed as we have previously described.11,23 Cellular materials from treated or untreated human NSCLC cells were incubated with lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1% Triton X-100, 0.1% SDS, 0.4 mM Na3VO4, 40 mM NaF, 50 μM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem protease inhibitor mixture 3). The samples were then run on SDS-PAGE in 4–15% polyacrylamide gels, transferred onto Immobilon® membranes, and developed with specific primary and secondary antibodies. Visualization of immunoreactive bands was achieved using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). In some instances, immunoreactive bands were quantitated using computer-assisted densitometry.
Stable Vector Control and MOR1 Overexpression in NSCLC Cells
The pCMV6-XL5 human MOR1 overexpression vector and vector control (pCMV6-AC-GFP) were purchased from Origene (Rockville, MD). H358 cells were transfected with small hairpin RNA (shRNA) using FuGENE HD®as the transfection reagent (Roche Applied Sciences, Indianapolis, IN) according to the protocol provided by Roche as we have previously described.11,23 Cells (approximately 40% confluent) are serum-starved for 1 h followed by incubation with shRNA for 6 h in serum-free media. Serum-containing media was then added (10% serum final concentration) for 42 h and G418 selection reagent was added. Overexpression was confirmed by immunoblot analysis with anti-MOR antibody (GeneTex, San Antonio, TX).
NSCLC Cell Proliferation Assay
Measurement of in vitro  NSCLC cell growth was performed as we have previously described.11,23 Stable vector control or MOR1 overexpressing H358 cells (5 × 103cells/well) were incubated with 0.2 ml serum-free media containing either vehicle (control), 1 μM Akt Inhibitor XIII, 10 nM temsirolimus or 100 nM methylnaltrexone for 72 h at 37°C in 5%CO2/95% air in 96-well culture plates. The in vitro  cell proliferation assay was analyzed by measuring increases in cell number using the CellTiter96® MTS assay (Promega, Madison, WI) and read at 492 nm. Each assay was set up in triplicate and repeated at least five times.
NSCLC Cell Migration Assay
Measurement of in vitro  NSCLC cell migration was performed as we have previously described.11,23 Twenty-four transwell units with 8 μM pore size (Millipore Corporation) were used for monitoring in vitro  cell migration as we have previously described.11 Stable vector control or MOR1 overexpressing H358 cells (approximately 1 × 104cells/well) were incubated with 0.2 ml serum-free media containing either vehicle (control), 1 μM Akt Inhibitor XIII, 10 nM temsirolimus, or 100 nM methylnaltrexone were plated with various treatments (methylnaltrexone, control shRNA, MOR shRNA) to the upper chamber and media with serum was added to the lower chamber. Cells were allowed to migrate through the pores for 18 h. Cells from the upper and lower chamber were quantitated using the CellTiter96TMMTS assay (Promega, San Luis Obispo, CA) and read at 492 nm. Percent migration was defined as the number of cells in the lower chamber divided by the number of cells in both the upper and lower chamber. Each assay was set up in triplicate and repeated at least five times.
NSCLC Cell Invasion Assay
Measurement of in vitro  NSCLC cell invasion was performed as we have previously described.11,23 Twenty-four transwell units with 8 μM pore size coated with Matrigel (QCM ECMatrix Cell Invasion Assay kit, Millipore, Billerica, MA)) were used for monitoring in vitro  cell invasion as we have previously described.11 Stable vector control or MOR1 overexpressing H358 cells (approximately 1 × 104cells/well) were incubated with 0.2 ml serum-free media containing either vehicle (control), 1 μM Akt Inhibitor XIII, 10 nM temsirolimus, or 100 nM methylnaltrexone were plated with various treatments (methylnaltrexone, control shRNA, MOR shRNA) to the upper chamber and media with serum was added to the lower chamber. Cells were allowed to invade through the Matrigel and pores for 18 h. Cells from the upper and lower chamber were quantitated using the CellTiter96TMMTS assay (Promega, San Luis Obispo, CA) and read at 492 nm. Percent invasion was defined as the number of cells in the lower chamber divided by the number of cells in both the upper and lower chamber. Each assay was set up in triplicate and repeated at least five times.
Transendothelial Extravasation Assay
The ability of NSCLC cells to invade through a layer of endothelial cells was quantified using transendothelial monolayer resistance measurements using an electrical substrate-impedence sensing system (Applied Biophysics, Troy, NY) as previously described.28  30 Briefly, human pulmonary microvascular endothelial cells were grown to confluence on gold-plated microelectrodes connected to a phase-sensitive, lock-in amplifier. Stable vector control or MOR1 overexpressing H358 cells (5 × 103cells/well) untreated or treated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus, or 100 nM methylnaltrexone or 5% serum media-only control were added to the confluent endothelial monolayers on the electrodes. The electrical substrate-impedence sensing system allows for continuous measurement of the endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in transendothelial monolayer resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of NSCLC cells. Resistance readings were normalized relative to an undisturbed confluent endothelial monolayer.
Human NSCLC Xenograft Studies in Nude Mice
All animal procedures were carried out in accordance with the guidelines provided by the Institutional Animal Care and Use Committee of the University of Chicago (Chicago, Illinois). All mice were 8- to 12-week-old males obtained from Harlan Laboratories (Indianapolis, IN). At the end of each experiment, lungs and primary tumors were collected, fixed in formalin, and embedded in paraffin or solubilized in extraction buffer for immunoblot analysis. 1.0 × 106stable vector control or MOR1 overexpressing H358 cells were mixed with Matrigel supplemented with 100 ng/ml epidermal growth factor (conditions we previously established to produce optimal H358 tumor growth) and injected subcutaneously into the flank of Ncr-nude mice. Tumor nodules were measured regularly for 31 days using calipers, and tumor volume VT(mm3) was calculated using the ellipsoid formula A2× B ×π/6, where A represents the smaller diameter.31 
Hematoxylin and Eosin Staining of Mouse Lungs
To characterize the morphologic changes due to metastasis from primary tumors growing in mouse hind flank, lungs from vector control or MOR1 overexpressing H358 hind flank-injected mice were formalin fixed and 5 μm paraffin section were subjected to hematoxylin and eosin histostaining. The sections of entire lungs were photographed using a Leica Axioscope (Leica, Bannockburn, IL) and analyzed. Slide images were processed using ImageJ (National Institutes of Health, Bethesda, MD) software.
Quantification of Lung Metastasis
To characterize metastasis from primary tumors growing in mouse hind flank, lungs from vector control or MOR1 overexpressing H358 hind flank-injected mice were solubilized in extraction buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1% Triton X-100, 0.2% SDS, 0.4 mM Na3VO4, 40 mM NaF, 50 μM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem protease inhibitor mixture 3) with sonication. The resulting material was run on SDS-PAGE in 4–15% polyacrylamide gels, transferred onto Immobilon® membranes, and developed with specific primary and secondary antibodies. An anti-human nestin antibody (clone 10C2, Millipore) that does not react with mouse or rat nestin was used for quantitation of H358 cell metastasis to the lung.
Statistical Analysis
Data are expressed as means ± SD. Two-way analysis of variance and Student-Newman-Keuls tests were used for data analysis with a P  value less than 0.05 considered statistically significant. All statistical analyses were performed using the program SPSS 17.0 for Windows (SPSS, Chicago, IL).
Results
NSCLC, which accounts for approximately 80% of all lung cancers, is heterogeneous disease composed of several types including adenocarcinoma, bronchioloalveolar carcinoma, squamous cell carcinoma, adenosquamous carcinoma, and large cell carcinoma.32,33 We determined the relative levels of MOR expression in human NSCLC cell lines representing these various types and observed a fivefold to tenfold increase in MOR expression is most cell lines relative to control noncancerous BEAS-2B cells (fig. 1). These results are consistent with our previously published data indicating the MOR is up-regulated in lung tissue from patients with NSCLC.11 To test the functional significance of this MOR up-regulation, we generated stable vector control and MOR1 (the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpressing human H358 BAC cells. We chose the human H358 bronchioloalveolar carcinoma cell line to study MOR overexpression based on its low basal metastatic potential.35 Our results indicate that stable transfection of vector control plasmid in H358 cells does not change the levels of MOR expression (fig. 2). However, we observe robust MOR immunoreactivity in stable overexpressing MOR H358 cells. Because there is an intimate relationship between the MOR and the epidermal growth factor receptor often resulting in transactivation of these receptors in HEK293 cells,36 astrocytes,37 and H2009 human NSCLC cells,38 we analyzed the effect of MOR overexpression on epidermal growth factor receptor expression and activation. Our results indicate MOR overexpression in H358 NSCLC cells did not change basal epidermal growth factor receptor expression or activation (pTyr1173epidermal growth factor receptor phosphorylation)38 (fig. 2A). Phase-contrast images of stable MOR overexpressing, but not control or stable vector control H358 cells, had elongated cellular projections suggestive of a migratory phenotype (fig. 2B). We therefore focused our consequent studies on comparing stable vector control and MOR overexpressing H358 functional analysis.
Fig. 1. The μ-opioid receptor (MOR) is overexpressed in several human non-small cell lung cancer cell lines. Immunoblot analysis using anti-MOR (A  ) and anti-actin (B  ) antibodies of cell lysates from human adenosquamous carcinoma (A549), adenocarcinoma (SKLU-1, H1703, H1993, H522, H1437, H1838, H1975), bronchioloalveolar carcinoma (H358, SW1573), large cell carcinoma (H661), squamous cell carcinoma (H226, H2170), and noncancerous BEAS-2B cells.
Image Not Available
Fig. 1. The μ-opioid receptor (MOR) is overexpressed in several human non-small cell lung cancer cell lines. Immunoblot analysis using anti-MOR (A  ) and anti-actin (B  ) antibodies of cell lysates from human adenosquamous carcinoma (A549), adenocarcinoma (SKLU-1, H1703, H1993, H522, H1437, H1838, H1975), bronchioloalveolar carcinoma (H358, SW1573), large cell carcinoma (H661), squamous cell carcinoma (H226, H2170), and noncancerous BEAS-2B cells.
×
Fig. 2. Characterization of MOR1 overexpressing human H358 non-small cell lung cancer cells. (A  ) Control (nontransfected, C), stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-μ-opioid receptor (a), anti-epidermal growth factor receptor (b), anti-pTyr1173epidermal growth factor receptor (c), and anti-actin (2-day) antibodies. (B  ) Phase-contrast images of H358 control (nontransfected), stable VC, or stable MOR1 O/E cells. The arrows  indicate elongated cellular projections suggestive of a migratory phenotype.
Image Not Available
Fig. 2. Characterization of MOR1 overexpressing human H358 non-small cell lung cancer cells. (A  ) Control (nontransfected, C), stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-μ-opioid receptor (a), anti-epidermal growth factor receptor (b), anti-pTyr1173epidermal growth factor receptor (c), and anti-actin (2-day) antibodies. (B  ) Phase-contrast images of H358 control (nontransfected), stable VC, or stable MOR1 O/E cells. The arrows  indicate elongated cellular projections suggestive of a migratory phenotype.
×
Interestingly, we observed that overexpression of MOR induced activation (phosphorylation) of the serine/threonine kinases Akt and mTOR, which are implicated in cancer progression39 (fig. 3). Specifically, we observed increased basal serine and to a lesser extent threonine phosphorylation of Akt and serine phosphorylation of mTOR in MOR overexpressing H358 cells. Akt and mTOR are implicated in numerous cancer cell functions, including proliferation and invasion.40,41 We next tested the contributions of Akt and mTOR to in vitro  NSCLC proliferation, migration, and invasion, which are reflective of in vivo  tumor growth. Our results indicate that MOR overexpressing NSCLC cells have an approximately45% increase in basal proliferation in serum-free media (fig. 4). Addition of 1 μM Akt Inhibitor XIII, a cell-permeable allosteric inhibitor of Akt 1/2, inhibited vector control cell proliferation by approximately 20% and MOR overexpressing cell proliferation by approximately 50%. In addition, the mTOR complex 1 inhibitor, temsirolimus,42 decreased vector control cell proliferation byapproximately 30% and MOR overexpressing NSCLC proliferation by approximately 60%. These data indicate the importance of Akt and mTOR in these processes and suggest a potential increased sensitivity to Akt and mTOR inhibition in MOR overexpressing NSCLC cells.43 Further, addition of 100 nM of the peripheral MOR antagonist, methylnaltrexone, exhibited a potent inhibition of basal proliferation in vector control (approximately 40%) and MOR overexpressing (approximately 60%) cells. We observe a similar trend in our migration and invasion assays in which MOR overexpression increases activity that is inhibited by Akt inhibitor, mTOR inhibitor, and the peripheral MOR antagonist methylnaltrexone (fig. 4-B, C).
Fig. 3. Overexpression of MOR1 in human H358 non-small cell lung cancer cells increases Akt and mTOR Activation. (A  ) Stable vector control (VC) and μ-opioid receptor 1 (MOR1) (the most abundant MOR transcript that consists of exons 1, 2, 3 and 4)34 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-pSer473Akt (a), anti-pThr308Akt (b), anti-Akt (c), anti-pSer2448mTOR (d), anti-mTOR (e), and anti-actin (f) antibodies. (B  ) Graphic representation of immunoblot quantitation of the ratio of phosphoprotein to total protein (pSer473Akt/total Akt, pThr308Akt/total Akt and pSer2448mTOR/total mTOR), n = 3 per group. The asterisks  indicate a statistically significant (P  < 0.05) difference between vector control and MOR1 overexpression.
Image Not Available
Fig. 3. Overexpression of MOR1 in human H358 non-small cell lung cancer cells increases Akt and mTOR Activation. (A  ) Stable vector control (VC) and μ-opioid receptor 1 (MOR1) (the most abundant MOR transcript that consists of exons 1, 2, 3 and 4)34 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-pSer473Akt (a), anti-pThr308Akt (b), anti-Akt (c), anti-pSer2448mTOR (d), anti-mTOR (e), and anti-actin (f) antibodies. (B  ) Graphic representation of immunoblot quantitation of the ratio of phosphoprotein to total protein (pSer473Akt/total Akt, pThr308Akt/total Akt and pSer2448mTOR/total mTOR), n = 3 per group. The asterisks  indicate a statistically significant (P  < 0.05) difference between vector control and MOR1 overexpression.
×
Fig. 4. MOR1 Overexpression increases proliferation, migration, and invasion and enhances sensitivity to Akt and mTOR inhibitors in human H358 non-small cell lung cancer cells. (A–C  ) Graphic representations of the percent proliferation (A  ), migration (B  ), or invasion (C  ) of stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cells either untreated (control) or treated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor), or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) as described in the Methods section. Proliferation, migration, or invasion of untreated vector control H358 cells was set at 100%. There is a statistically significant difference (P  < 0.05) in proliferation, migration, and invasion between basal VC versus  MOR1 O/E H358 cells. The asterisks  indicate a statistically significant difference (P  < 0.05) between treatment and corresponding control groups.
Image Not Available
Fig. 4. MOR1 Overexpression increases proliferation, migration, and invasion and enhances sensitivity to Akt and mTOR inhibitors in human H358 non-small cell lung cancer cells. (A–C  ) Graphic representations of the percent proliferation (A  ), migration (B  ), or invasion (C  ) of stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cells either untreated (control) or treated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor), or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) as described in the Methods section. Proliferation, migration, or invasion of untreated vector control H358 cells was set at 100%. There is a statistically significant difference (P  < 0.05) in proliferation, migration, and invasion between basal VC versus  MOR1 O/E H358 cells. The asterisks  indicate a statistically significant difference (P  < 0.05) between treatment and corresponding control groups.
×
In order for many tumor cells to metastasize, they need to breach the endothelium to enter the bloodstream. We tested the in vitro  ability of vector control and MOR overexpressing H358 cells to disrupt a confluent human pulmonary microvascular endothelial cell monolayer using an electrical substrate-impedance sensing system.28  30 This system continuously measures endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of the NSCLC cells. Figure 5indicates MOR overexpressing H358 cells have increased extravasation properties compared with vector control cells, which becomes apparent approximately 7 h after NSCLC cell addition to the confluent endothelial monolayer. These effects are inhibited by Akt Inhibitor XIII, temsirolimus, and methylnaltrexone with MOR cells exhibiting increased inhibitor sensitivity (fig. 5B), results similar to in vitro  proliferation, migration, and invasion.
Fig. 5. MOR1 overexpression increases transendothelial extravasation of human H358 non-small cell lung cancer (NSCLC) cells. (A  ) Graphic representation of the ability of vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpressing (O/E) H358 cells to disrupt a confluent human pulmonary microvascular endothelial cell monolayer using an electrical substrate-impedance sensing system.10  12 This system continuously measures endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of the NSCLC cells. MOR1 O/E H358 cells have increased extravasation properties compared with vector control cells which becomes apparent approximately 7 h after NSCLC cell addition to the confluent endothelial monolayer with n = 3 per condition and error bars = SD (B  ) Quantitation of the percent extravasation of VC and MOR1 O/E H358 cells pretreated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor) or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) for 1 h before addition to confluent human pulmonary microvascular endothelial monolayers. The graph represents values calculated from the endothelial resistance 7 h after H358 cell addition with n = 3 per condition.
Image Not Available
Fig. 5. MOR1 overexpression increases transendothelial extravasation of human H358 non-small cell lung cancer (NSCLC) cells. (A  ) Graphic representation of the ability of vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpressing (O/E) H358 cells to disrupt a confluent human pulmonary microvascular endothelial cell monolayer using an electrical substrate-impedance sensing system.10  12 This system continuously measures endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of the NSCLC cells. MOR1 O/E H358 cells have increased extravasation properties compared with vector control cells which becomes apparent approximately 7 h after NSCLC cell addition to the confluent endothelial monolayer with n = 3 per condition and error bars = SD (B  ) Quantitation of the percent extravasation of VC and MOR1 O/E H358 cells pretreated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor) or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) for 1 h before addition to confluent human pulmonary microvascular endothelial monolayers. The graph represents values calculated from the endothelial resistance 7 h after H358 cell addition with n = 3 per condition.
×
We next translated our in vitro  results to an in vivo  human NSCLC xenograft model. Vector control and MOR overexpressing H358 cells were injected into the hind flank of nude mice and tumor volumes were measured using calipers for 31 days (fig. 6) as described in the Methods section. MOR overexpressing H358 cells had an approximately 2.5 fold increase in primary tumor growth rate compared with vector control cells (fig. 6B), suggesting a role of MOR overexpression in the proliferative properties of NSCLC.
Fig. 6. Overexpression of MOR1 increases primary tumor growth rates in human non-small cell lung cancer xenograft models. (A  ) Representative graph indicating the change in tumor volume versus  time in days of vector control (VC) or μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice. (B  ) Graphic representation of normalized tumor growth rates from VC and MOR1 overexpressing (O/E) H358 hind flank tumors in nude mice as depicted in A  with a statistically significant difference (P  < 0.05) between groups and n = 4 per group.
Image Not Available
Fig. 6. Overexpression of MOR1 increases primary tumor growth rates in human non-small cell lung cancer xenograft models. (A  ) Representative graph indicating the change in tumor volume versus  time in days of vector control (VC) or μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice. (B  ) Graphic representation of normalized tumor growth rates from VC and MOR1 overexpressing (O/E) H358 hind flank tumors in nude mice as depicted in A  with a statistically significant difference (P  < 0.05) between groups and n = 4 per group.
×
Considering the differential growth rates of the primary tumors in nude mice, we next examined the metastatic potential of vector control and MOR overexpressing H358 primary flank tumors. Human bronchioloalveolar carcinoma tends to have low metastatic potential.44 However, as time progresses and/or mutations arise, the invasive properties of bronchioloalveolar carcinoma can increase.45,46 The histologic analysis of lungs from nude mice with hind flank tumors from vector control H358 cells revealed minimal cellular infiltration with normal alveolar morphology (fig. 7). In contrast, lungs from nude mice with MOR overexpressing H358 hind flank tumors exhibited areas of atypical cellular density, dysplastic morphology, and disrupted alveolar epithelium. Because these areas did not exhibit well-demarcated tumors, we quantitated lung metastasis using an anti-human nestin antibody that does not react with mouse.47  50 Figure 8indicates robust nestin immunoreactivity in the lung homogenates of MOR overexpressing, but not vector control, mice. Further, there is increased MOR expression in lung homogenates from mice with MOR overexpressing H358 hind flank tumors. Quantitation of nestin immunoreactivity revealed an approximately 20-fold increase in lung metastasis from MOR overexpressing primary tumors (fig. 8B).
Fig. 7. Histochemical analysis of lungs from vector control and MOR1 overexpressing nude mouse hind flank tumors. Representative hematoxylin and eosin stained lung sections from vector control and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice illustrated at 1×, 2.5×, and 20×. The vector control lung sections exhibit normal morphology with little cellular infiltration. In contrast, the MOR1 overexpressing lung sections lungs exhibit areas of atypical cellular density, dysplastic morphology, and disrupted alveolar epithelium (arrows  ).
Image Not Available
Fig. 7. Histochemical analysis of lungs from vector control and MOR1 overexpressing nude mouse hind flank tumors. Representative hematoxylin and eosin stained lung sections from vector control and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice illustrated at 1×, 2.5×, and 20×. The vector control lung sections exhibit normal morphology with little cellular infiltration. In contrast, the MOR1 overexpressing lung sections lungs exhibit areas of atypical cellular density, dysplastic morphology, and disrupted alveolar epithelium (arrows  ).
×
Fig. 8. Overexpression of MOR1 increases lung metastasis in human non-small cell lung cancer (NSCLC) xenograft models. (A  ) Representative immunoblot analysis of human H358 NSCLC cells alone, control nude mouse lung homogenate, lung homogenate from nude mouse with vector control H358 hind flank tumor or lung homogenates from nude mice with μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors using anti-nestin (a), anti-MOR (b), or anti-actin (c) antibodies. (B  ) Graphic representation of lung metastasis from vector control or MOR1 overexpressing H358 hind flank tumors in nude mice using quantitation of nestin immunoreactivity as depicted in A  . There is a statistically significant difference (P  < 0.05) between groups with n = 4 per group.
Image Not Available
Fig. 8. Overexpression of MOR1 increases lung metastasis in human non-small cell lung cancer (NSCLC) xenograft models. (A  ) Representative immunoblot analysis of human H358 NSCLC cells alone, control nude mouse lung homogenate, lung homogenate from nude mouse with vector control H358 hind flank tumor or lung homogenates from nude mice with μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors using anti-nestin (a), anti-MOR (b), or anti-actin (c) antibodies. (B  ) Graphic representation of lung metastasis from vector control or MOR1 overexpressing H358 hind flank tumors in nude mice using quantitation of nestin immunoreactivity as depicted in A  . There is a statistically significant difference (P  < 0.05) between groups with n = 4 per group.
×
Discussion
Based on the recent interest of potential effects of anesthesia and analgesia regimens on the recurrence and metastatic potential of various cancers and our previously published data indicating MOR is increased in lung cancer,1,11,51 this study investigated the functional effects of MOR overexpression in human NSCLC cells both in vitro  and in vivo  . We observed that MOR1 (the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpression in H358 human NSCLC cells increased proliferation, migration, invasion, and transendothelial migration and activation of two serine/threonine kinases implicated in cancer progression, Akt and mTOR. Consistent with this kinase activation, MOR overexpressing H358 cells exhibited increased sensitivity to Akt and mTOR inhibitors in our in vitro  cellular assays. In addition, overexpression of MOR in H358 cells increased primary tumor growth rates and lung metastasis in human NSCLC xenografts. Taken as a whole, our data suggest that MOR upregulation promotes lung cancer progression potentially through Akt and mTOR-regulated pathways.
Our results are consistent with other studies indicating a MOR antagonist can inhibit various parameters of cancer progression.22,52,53 Specifically, Zagon et al.  reported that daily subcutaneous injection of 0.1 mg/kg naltrexone inhibited tumor appearance and increased survival time in A/Jax mice inoculated with S20Y neuroblastoma cells.22,52 Further, naltrexone inhibited the incidence and multiplicity of 7,12-dimethylbenz[α]anthracene-induced rat mammary tumors.53 In addition, incubation of 100 nM naloxone inhibited MCF-7 breast cancer cell proliferation.54 
Exogenous opioids have been reported to either inhibit or enhance cancer growth and angiogenesis depending on the concentration and model used.9,10,12 A study by Gupta et al.  demonstrated stimulatory effect of morphine on tumor growth and angiogenesis.21 This group found that clinically relevant doses of morphine led to increased tumor volumes and increased tumor vascularization. Although we did not add exogenous opioids in our study, a role for endogenous opioids cannot be ruled out. The endogenous MOR ligands, endomorphin-1 and endomorphin-2, increased angiogenesis and endothelial cell proliferation, migration, and adhesion in vitro  , effects that were reversed by the MOR antagonist naltrexone.20 Further, β-endorphin has been implicated in malignant melanoma progression.55 
Our observations that MOR overexpression increases Akt and mTOR activation can have clinical significance because these molecules are targets for anticancer drugs.39 We have previously demonstrated that methylnaltrexone inhibits vascular endothelial growth factor-induced Akt activation and that methylnaltrexone acts synergistically with temsirolimus on inhibition of angiogenesis both in vitro  and in vivo  .23,24 In this study, we demonstrate that temsirolimus inhibits NSCLC proliferation and transendothelial extravasation in NSCLC cells with MOR overexpression conferring increased sensitivity to these drugs. Temsirolimus, currently used to treat renal cell carcinoma, exerts its action by binding to the intracellular protein, FKBP12, and inhibiting mTOR complex 1 formation.56  58 However, mTOR can still form a complex with other proteins including SIN1 and Rictor (mTOR complex 2), leading to investigation of drugs that can directly inhibit the mTOR kinase.59  61 The mTOR complex 2 serine phosphorylates Akt and is involved in actin cytoskeletal regulation, proliferation, and cell survival.62  64 In contrast, PI3 kinase activation of PDK1 induces threonine phosphorylation of Akt.65  67 Interestingly, our results indicate MOR overexpression activates serine phosphorylation of Akt to a greater extent than threonine phosphorylation, suggesting a potentially greater role for mTOR in this process. However, we cannot rule out the contributions of other kinases in Akt activation.66,67 
We have recently reported that silencing of MOR in lung cancer cells (Lewis lung carcinoma) reduces tumor growth and metastasis in mouse models.11 These findings are in agreement with the current study indicating overexpression of MOR in human NSCLC increases tumor growth and metastasis. In addition, our previous studies indicate the peripheral MOR antagonist, methylnaltrexone, inhibits Lewis lung carcinoma growth both in vitro  and in vivo  .11 We have extended these findings by demonstrating that methylnaltrexone inhibits human NSCLC cell proliferation and transendothelial extravasation. Although our studies used methylnaltrexone because it can be coadministered with opiates without reversing analgesia and is often used clinically in patients with advanced cancer, the effects appear to extend more generally to the class of MOR antagonists. Naloxone and naltrexone can inhibit opioid-induced angiogenesis.21,68,69 In addition, naltrexone is being studied as a possible adjuvant therapy for pancreatic cancer and as a treatment for solid metastatic tumors.70,71 
Given our previously published data indicating MOR is increased in lung cancer,11 we undertook a series of in vitro  and in vivo  experiments to examine the direct effect of MOR overexpression in lung cancer progression using human NSCLC cells. We hypothesized that overexpression and activation of MOR might be a plausible explanation for the differences in recurrence rates observed in the epidemiologic studies.1  6 This is supported by our results indicating that MOR overexpression increases the proliferative and metastatic properties of human NSCLC cells. Importantly, our observations that MOR overexpression activates Akt and mTOR suggest a potential therapeutic advantage using combinational MOR, Akt and/or mTOR inhibitors in the treatment of NSCLC. Taken together, our data suggest a possible direct effect of MOR overexpression on lung cancer progression, and provides a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists that merits further evaluation.
References
Bovill JG: Surgery for cancer: Does anesthesia matter? Anesth Analg 2010; 110:1524–6
Snyder GL, Greenberg S: Effect of anaesthetic technique and other perioperative factors on cancer recurrence. Br J Anaesth 2010; 105:106–15
Yeager MP, Rosenkranz KM: Cancer recurrence after surgery: A role for regional anesthesia? Reg Anesth Pain Med 2010; 35:483–4
Biki B, Mascha E, Moriarty DC, Fitzpatrick JM, Sessler DI, Buggy DJ: Anesthetic technique for radical prostatectomy surgery affects cancer recurrence: A retrospective analysis. ANESTHESIOLOGY 2008; 109:180–7
Exadaktylos AK, Buggy DJ, Moriarty DC, Mascha E, Sessler DI: Can anesthetic technique for primary breast cancer surgery affect recurrence or metastasis? ANESTHESIOLOGY 2006; 105:660–4
Christopherson R, James KE, Tableman M, Marshall P, Johnson FE: Long-term survival after colon cancer surgery: A variation associated with choice of anesthesia. Anesth Analg 2008; 107:325–32
Myles PS, Peyton P, Silbert B, Hunt J, Rigg JR, Sessler DI: Perioperative epidural analgesia for major abdominal surgery for cancer and recurrence-free survival: Randomised trial. BMJ 2011; 342:d1491
Tavare AN, Perry NJ, Benzonana LL, Takata M, Ma D: Cancer recurrence after surgery: Direct and indirect effects of anesthetic agents*. Int J Cancer 2012; 130:1237–50
Afsharimani B, Cabot PJ, Parat MO: Morphine use in cancer surgery. Front Pharmacol 2011; 2:46
Afsharimani B, Cabot P, Parat MO: Morphine and tumor growth and metastasis. Cancer Metastasis Rev 2011; 30:225–38
Mathew B, Lennon FE, Siegler J, Mirzapoiazova T, Mambetsariev N, Sammani S, Gerhold LM, LaRiviere PJ, Chen CT, Garcia JG, Salgia R, Moss J, Singleton PA: The novel role of the mu opioid receptor in lung cancer progression: A laboratory investigation. Anesth Analg 2011; 112:558–67
Gach K, Wyrebska A, Fichna J, Janecka A: The role of morphine in regulation of cancer cell growth. Naunyn Schmiedebergs Arch Pharmacol 2011; 384:221–30
Kasai S, Ikeda K: Pharmacogenomics of the human μ-opioid receptor. Pharmacogenomics 2011; 12:1305–20
Trescot AM, Datta S, Lee M, Hansen H: Opioid pharmacology. Pain Physician 2008; 11:S133–53
Wei LN, Loh HH: Transcriptional and epigenetic regulation of opioid receptor genes: Present and future. Annu Rev Pharmacol Toxicol 2011; 51:75–97
Hwang CK, Kim CS, Kim do K, Law PY, Wei LN, Loh HH: Up-regulation of the mu-opioid receptor gene is mediated through chromatin remodeling and transcriptional factors in differentiated neuronal cells. Mol Pharmacol 2010; 78:58–68
Chen YL, Law PY, Loh HH: The other side of the opioid story: Modulation of cell growth and survival signaling. Curr Med Chem 2008; 15:772–8
Martini L, Whistler JL: The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence. Curr Opin Neurobiol 2007; 17:556–64
Leo S, Nuydens R, Meert TF: Opioid-induced proliferation of vascular endothelial cells. J Pain Res 2009; 2:59–66
Dai X, Song HJ, Cui SG, Wang T, Liu Q, Wang R: The stimulative effects of endogenous opioids on endothelial cell proliferation, migration and angiogenesis in vitro. Eur J Pharmacol 2010; 628:42–50
Gupta K, Kshirsagar S, Chang L, Schwartz R, Law PY, Yee D, Hebbel RP: Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth. Cancer Res 2002; 62:4491–8
Zagon IS, McLaughlin PJ: Opioid antagonists inhibit the growth of metastatic murine neuroblastoma. Cancer Lett 1983; 21:89–94
Singleton PA, Mambetsariev N, Lennon FE, Mathew B, Siegler JH, Moreno-Vinasco L, Salgia R, Moss J, Garcia JG: Methylnaltrexone potentiates the anti-angiogenic effects of mTOR inhibitors. J Angiogenes Res 2010; 2:5
Singleton PA, Garcia JG, Moss J: Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis. Mol Cancer Ther 2008; 7:1669–79
Singleton PA, Lingen MW, Fekete MJ, Garcia JG, Moss J: Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: Role of receptor transactivation. Microvasc Res 2006; 72:3–11
Spiro SG, Tanner NT, Silvestri GA, Janes SM, Lim E, Vansteenkiste JF, Pirker R: Lung cancer: Progress in diagnosis, staging and therapy. Respirology 2010; 15:44–50
Stinchcombe TE, Bogart J, Veeramachaneni NK, Kratzke R, Govindan R: Annual review of advances in non-small cell lung cancer research: A report for the year 2010. J Thorac Oncol 2011; 6:1443–50
Tarantola M, Marel AK, Sunnick E, Adam H, Wegener J, Janshoff A: Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis. Integr Biol (Camb) 2010; 2:139–50
Chen J, Ye L, Zhang L, Jiang WG: Placenta growth factor, PLGF, influences the motility of lung cancer cells, the role of Rho associated kinase, Rock1. J Cell Biochem 2008; 105:313–20
Keese CR, Bhawe K, Wegener J, Giaever I. Real-time impedance assay to follow the invasive activities of metastatic cells in culture. Biotechniques 2002; 33:842–44–50, 846–50, 848–50
Hoffman RM, Yang M: Whole-body imaging with fluorescent proteins. Nat Protoc 2006; 1:1429–38
Maione P, Rossi A, Sacco PC, Bareschino MA, Schettino C, Gridelli C: Advances in chemotherapy in advanced non-small-cell lung cancer. Expert Opin Pharmacother 2010; 11:2997–3007
Beland MD, Wasser EJ, Mayo-Smith WW, Dupuy DE: Primary non-small cell lung cancer: Review of frequency, location, and time of recurrence after radiofrequency ablation. Radiology 2010; 254:301–7
Xu J, Xu M, Hurd YL, Pasternak GW, Pan YX: Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene. J Neurochem 2009; 108:962–72
Onn A, Isobe T, Itasaka S, Wu W, O'Reilly MS, Ki Hong W, Fidler IJ, Herbst RS: Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin Cancer Res2003; 9:5532–9
Chen Y, Long H, Wu Z, Jiang X, Ma L: EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation. Mol Biol Cell 2008; 19:2973–83
Belcheva MM, Tan Y, Heaton VM, Clark AL, Coscia CJ: Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: Implications for mitogen-activated protein kinase signaling. Mol Pharmacol 2003; 64:1391–1401
Fujioka N, Nguyen J, Chen C, Li Y, Pasrija T, Niehans G, Johnson KN, Gupta V, Kratzke RA, Gupta K: Morphine-induced epidermal growth factor pathway activation in non-small cell lung cancer. Anesth Analg 2011; 113:1353–64
Wu P, Hu YZ: Pi3K/akt/mTOR pathway inhibitors in cancer: A perspective on clinical progress. Curr Med Chem 2010; 17:4326–41
Chen JS, Wang Q, Fu XH, Huang XH, Chen XL, Cao LQ, Chen LZ, Tan HX, Li W, Bi J, Zhang LJ: Involvement of PI3k/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP-9. Hepatol Res 2009; 39:177–86
Zhao N, Guo Y, Zhang M, Lin L, Zheng Z: Akt-mTOR signaling is involved in Notch-1-mediated glioma cell survival and proliferation. Oncol Rep 2010; 23:1443–7
Malizzia LJ, Hsu A: Temsirolimus, an mTOR inhibitor for treatment of patients with advanced renal cell carcinoma. Clin J Oncol Nurs 2008; 12:639–46
Altomare DA, Wang HQ, Skele KL, De Rienzo A, Klein-Szanto AJ, Godwin AK, Testa JR: AKTand mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene 2004; 23:5853–7
Arenberg D: Bronchioloalveolar carcinoma. Semin Respir Crit Care Med 2011; 32:52–61
Levy BP, Drilon A, Makarian L, Patel AA, Grossbard ML: Systemic approaches for multifocal bronchioloalveolar carcinoma: Is there an appropriate target? Oncology 2010; 24:888–98, 900
Toonkel RL, Borczuk AC, Powell CA: Tgf-beta signaling pathway in lung adenocarcinoma invasion. J Thorac Oncol 2010; 5:153–7
Chen Z, Wang T, Luo H, Lai Y, Yang X, Li F, Lei Y, Su C, Zhang X, Lahn BT, Xiang AP: Expression of nestin in lymph node metastasis and lymphangiogenesis in non-small cell lung cancer patients. Hum Pathol 2010; 41:737–44
Janikova M, Skarda J, Dziechciarkova M, Radova L, Chmelova J, Krejci V, Sedlakova E, Zapletalova J, Langova K, Klein J, Grygarkova I, Kolek V: Identification of CD133+/nestin+ putative cancer stem cells in non-small cell lung cancer. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2010; 154:321–26
Krupkova O Jr, Loja T, Zambo I, Veselska R: Nestin expression in human tumors and tumor cell lines. Neoplasma 2010; 57:291–98
Ryuge S, Sato Y, Wang GQ, Matsumoto T, Jiang SX, Katono K, Inoue H, Satoh Y, Masuda N: Prognostic significance of nestin expression in resected non-small cell lung cancer. Chest 2011; 139:862–9
Singleton PA, Moss J: Effect of perioperative opioids on cancer recurrence: A hypothesis. Future Oncol 2010; 6:1237–42
Zagon IS, McLaughlin PJ: Naltrexone modulates tumor response in mice with neuroblastoma. Science 1983; 221:671–73
Koo KL, Tejwani GA, Abou-Issa H: Relative efficacy of the opioid antagonist, naltrexone, on the initiation and promotion phases of rat mammary carcinogenesis. Anticancer Res 1996; 16:1893–8
Farooqui M, Geng ZH, Stephenson EJ, Zaveri N, Yee D, Gupta K: Naloxone acts as an antagonist of estrogen receptor activity in MCF-7 cells. Mol Cancer Ther 2006; 5:611–20
Boehncke S, Hardt K, Schadendorf D, Henschler R, Boehncke WH, Duthey B: Endogenous -opioid peptides modulate immune response towards malignant melanoma. Exp Dermatol 2011; 20:24–8
Bhatia S, Thompson JA: Temsirolimus in patients with advanced renal cell carcinoma: An overview. Adv Ther 2009; 26:55–67
Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, Staroslawska E, Sosman J, McDermott D, Bodrogi I, Kovacevic Z, Lesovoy V, Schmidt-Wolf IG, Barbarash O, Gokmen E, O'Toole T, Lustgarten S, Moore L, Motzer RJ, Global ARCC Trial: Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 2007; 356:2271–81
Figlin RA: Mechanisms of disease: Survival benefit of temsirolimus validates a role for mTOR in the management of advanced RCC. Nat Clin Pract Oncol 2008; 5:601–9
Garcia JA, Danielpour D: Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies. Mol Cancer Ther 2008; 7:1347–54
Faivre S, Kroemer G, Raymond E: Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006; 5:671–88
Brachmann S, Fritsch C, Maira SM, Garca-Echeverra C: PI3K and mTOR inhibitors: A new generation of targeted anticancer agents. Curr Opin Cell Biol 2009; 21:194–8
Jacinto E, Loewith R, Schmidt A, Lin S, Regg MA, Hall A, Hall MN: Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004; 6:1122–8
Fasolo A, Sessa C: mTOR inhibitors in the treatment of cancer. Expert Opin Investig Drugs 2008; 17:1717–34
Abraham RT, Gibbons JJ: The mammalian target of rapamycin signaling pathway: Twists and turns in the road to cancer therapy. Clin Cancer Res 2007; 13:3109–14
Manning BD, Cantley LC: AKT/PKB signaling: Navigating downstream. Cell 2007; 129:1261–74
Frost RA, Lang CH: Protein kinase B/akt: A nexus of growth factor and cytokine signaling in determining muscle mass. J Appl Physiol 2007; 103:378–87
Du K, Tsichlis PN: Regulation of the Akt kinase by interacting proteins. Oncogene 2005; 24:7401–09
Donahue RN, McLaughlin PJ, Zagon IS: Low-dose naltrexone suppresses ovarian cancer and exhibits enhanced inhibition in combination with cisplatin. Exp Biol Med (Maywood) 2011; 236:883–95
Donahue RN, McLaughlin PJ, Zagon IS: The opioid growth factor (OGF) and low dose naltrexone (LDN) suppress human ovarian cancer progression in mice. Gynecol Oncol 2011; 122:382–8
Berkson BM, Rubin DM, Berkson AJ: Revisiting the ALA/N (alpha-lipoic acid/low-dose naltrexone) protocol for people with metastatic and nonmetastatic pancreatic cancer: A report of 3 new cases. Integr Cancer Ther 2009; 8:416–22
Lissoni P, Malugani F, Malysheva O, Kozlov V, Laudon M, Conti A, Maestroni G: Neuroimmunotherapy of untreatable metastatic solid tumors with subcutaneous low-dose interleukin-2, melatonin and naltrexone: Modulation of interleukin-2-induced antitumor immunity by blocking the opioid system. Neuro Endocrinol Lett 2002; 23:341–4
Fig. 1. The μ-opioid receptor (MOR) is overexpressed in several human non-small cell lung cancer cell lines. Immunoblot analysis using anti-MOR (A  ) and anti-actin (B  ) antibodies of cell lysates from human adenosquamous carcinoma (A549), adenocarcinoma (SKLU-1, H1703, H1993, H522, H1437, H1838, H1975), bronchioloalveolar carcinoma (H358, SW1573), large cell carcinoma (H661), squamous cell carcinoma (H226, H2170), and noncancerous BEAS-2B cells.
Image Not Available
Fig. 1. The μ-opioid receptor (MOR) is overexpressed in several human non-small cell lung cancer cell lines. Immunoblot analysis using anti-MOR (A  ) and anti-actin (B  ) antibodies of cell lysates from human adenosquamous carcinoma (A549), adenocarcinoma (SKLU-1, H1703, H1993, H522, H1437, H1838, H1975), bronchioloalveolar carcinoma (H358, SW1573), large cell carcinoma (H661), squamous cell carcinoma (H226, H2170), and noncancerous BEAS-2B cells.
×
Fig. 2. Characterization of MOR1 overexpressing human H358 non-small cell lung cancer cells. (A  ) Control (nontransfected, C), stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-μ-opioid receptor (a), anti-epidermal growth factor receptor (b), anti-pTyr1173epidermal growth factor receptor (c), and anti-actin (2-day) antibodies. (B  ) Phase-contrast images of H358 control (nontransfected), stable VC, or stable MOR1 O/E cells. The arrows  indicate elongated cellular projections suggestive of a migratory phenotype.
Image Not Available
Fig. 2. Characterization of MOR1 overexpressing human H358 non-small cell lung cancer cells. (A  ) Control (nontransfected, C), stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-μ-opioid receptor (a), anti-epidermal growth factor receptor (b), anti-pTyr1173epidermal growth factor receptor (c), and anti-actin (2-day) antibodies. (B  ) Phase-contrast images of H358 control (nontransfected), stable VC, or stable MOR1 O/E cells. The arrows  indicate elongated cellular projections suggestive of a migratory phenotype.
×
Fig. 3. Overexpression of MOR1 in human H358 non-small cell lung cancer cells increases Akt and mTOR Activation. (A  ) Stable vector control (VC) and μ-opioid receptor 1 (MOR1) (the most abundant MOR transcript that consists of exons 1, 2, 3 and 4)34 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-pSer473Akt (a), anti-pThr308Akt (b), anti-Akt (c), anti-pSer2448mTOR (d), anti-mTOR (e), and anti-actin (f) antibodies. (B  ) Graphic representation of immunoblot quantitation of the ratio of phosphoprotein to total protein (pSer473Akt/total Akt, pThr308Akt/total Akt and pSer2448mTOR/total mTOR), n = 3 per group. The asterisks  indicate a statistically significant (P  < 0.05) difference between vector control and MOR1 overexpression.
Image Not Available
Fig. 3. Overexpression of MOR1 in human H358 non-small cell lung cancer cells increases Akt and mTOR Activation. (A  ) Stable vector control (VC) and μ-opioid receptor 1 (MOR1) (the most abundant MOR transcript that consists of exons 1, 2, 3 and 4)34 overexpressing (O/E) H358 cell lines were generated, cell lysates obtained and immunoblotted with anti-pSer473Akt (a), anti-pThr308Akt (b), anti-Akt (c), anti-pSer2448mTOR (d), anti-mTOR (e), and anti-actin (f) antibodies. (B  ) Graphic representation of immunoblot quantitation of the ratio of phosphoprotein to total protein (pSer473Akt/total Akt, pThr308Akt/total Akt and pSer2448mTOR/total mTOR), n = 3 per group. The asterisks  indicate a statistically significant (P  < 0.05) difference between vector control and MOR1 overexpression.
×
Fig. 4. MOR1 Overexpression increases proliferation, migration, and invasion and enhances sensitivity to Akt and mTOR inhibitors in human H358 non-small cell lung cancer cells. (A–C  ) Graphic representations of the percent proliferation (A  ), migration (B  ), or invasion (C  ) of stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cells either untreated (control) or treated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor), or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) as described in the Methods section. Proliferation, migration, or invasion of untreated vector control H358 cells was set at 100%. There is a statistically significant difference (P  < 0.05) in proliferation, migration, and invasion between basal VC versus  MOR1 O/E H358 cells. The asterisks  indicate a statistically significant difference (P  < 0.05) between treatment and corresponding control groups.
Image Not Available
Fig. 4. MOR1 Overexpression increases proliferation, migration, and invasion and enhances sensitivity to Akt and mTOR inhibitors in human H358 non-small cell lung cancer cells. (A–C  ) Graphic representations of the percent proliferation (A  ), migration (B  ), or invasion (C  ) of stable vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing (O/E) H358 cells either untreated (control) or treated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor), or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) as described in the Methods section. Proliferation, migration, or invasion of untreated vector control H358 cells was set at 100%. There is a statistically significant difference (P  < 0.05) in proliferation, migration, and invasion between basal VC versus  MOR1 O/E H358 cells. The asterisks  indicate a statistically significant difference (P  < 0.05) between treatment and corresponding control groups.
×
Fig. 5. MOR1 overexpression increases transendothelial extravasation of human H358 non-small cell lung cancer (NSCLC) cells. (A  ) Graphic representation of the ability of vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpressing (O/E) H358 cells to disrupt a confluent human pulmonary microvascular endothelial cell monolayer using an electrical substrate-impedance sensing system.10  12 This system continuously measures endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of the NSCLC cells. MOR1 O/E H358 cells have increased extravasation properties compared with vector control cells which becomes apparent approximately 7 h after NSCLC cell addition to the confluent endothelial monolayer with n = 3 per condition and error bars = SD (B  ) Quantitation of the percent extravasation of VC and MOR1 O/E H358 cells pretreated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor) or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) for 1 h before addition to confluent human pulmonary microvascular endothelial monolayers. The graph represents values calculated from the endothelial resistance 7 h after H358 cell addition with n = 3 per condition.
Image Not Available
Fig. 5. MOR1 overexpression increases transendothelial extravasation of human H358 non-small cell lung cancer (NSCLC) cells. (A  ) Graphic representation of the ability of vector control (VC) and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 4)34 overexpressing (O/E) H358 cells to disrupt a confluent human pulmonary microvascular endothelial cell monolayer using an electrical substrate-impedance sensing system.10  12 This system continuously measures endothelial monolayer resistance as the H358 cells attach and begin to invade into the monolayer. A decrease in resistance indicates a disrupted endothelial monolayer barrier via  transendothelial extravasation of the NSCLC cells. MOR1 O/E H358 cells have increased extravasation properties compared with vector control cells which becomes apparent approximately 7 h after NSCLC cell addition to the confluent endothelial monolayer with n = 3 per condition and error bars = SD (B  ) Quantitation of the percent extravasation of VC and MOR1 O/E H358 cells pretreated with 1 μM Akt Inhibitor XIII, 10 nM temsirolimus (mTOR complex 1 inhibitor) or 100 nM methylnaltrexone (MNTX, peripheral MOR antagonist) for 1 h before addition to confluent human pulmonary microvascular endothelial monolayers. The graph represents values calculated from the endothelial resistance 7 h after H358 cell addition with n = 3 per condition.
×
Fig. 6. Overexpression of MOR1 increases primary tumor growth rates in human non-small cell lung cancer xenograft models. (A  ) Representative graph indicating the change in tumor volume versus  time in days of vector control (VC) or μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice. (B  ) Graphic representation of normalized tumor growth rates from VC and MOR1 overexpressing (O/E) H358 hind flank tumors in nude mice as depicted in A  with a statistically significant difference (P  < 0.05) between groups and n = 4 per group.
Image Not Available
Fig. 6. Overexpression of MOR1 increases primary tumor growth rates in human non-small cell lung cancer xenograft models. (A  ) Representative graph indicating the change in tumor volume versus  time in days of vector control (VC) or μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice. (B  ) Graphic representation of normalized tumor growth rates from VC and MOR1 overexpressing (O/E) H358 hind flank tumors in nude mice as depicted in A  with a statistically significant difference (P  < 0.05) between groups and n = 4 per group.
×
Fig. 7. Histochemical analysis of lungs from vector control and MOR1 overexpressing nude mouse hind flank tumors. Representative hematoxylin and eosin stained lung sections from vector control and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice illustrated at 1×, 2.5×, and 20×. The vector control lung sections exhibit normal morphology with little cellular infiltration. In contrast, the MOR1 overexpressing lung sections lungs exhibit areas of atypical cellular density, dysplastic morphology, and disrupted alveolar epithelium (arrows  ).
Image Not Available
Fig. 7. Histochemical analysis of lungs from vector control and MOR1 overexpressing nude mouse hind flank tumors. Representative hematoxylin and eosin stained lung sections from vector control and μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors in nude mice illustrated at 1×, 2.5×, and 20×. The vector control lung sections exhibit normal morphology with little cellular infiltration. In contrast, the MOR1 overexpressing lung sections lungs exhibit areas of atypical cellular density, dysplastic morphology, and disrupted alveolar epithelium (arrows  ).
×
Fig. 8. Overexpression of MOR1 increases lung metastasis in human non-small cell lung cancer (NSCLC) xenograft models. (A  ) Representative immunoblot analysis of human H358 NSCLC cells alone, control nude mouse lung homogenate, lung homogenate from nude mouse with vector control H358 hind flank tumor or lung homogenates from nude mice with μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors using anti-nestin (a), anti-MOR (b), or anti-actin (c) antibodies. (B  ) Graphic representation of lung metastasis from vector control or MOR1 overexpressing H358 hind flank tumors in nude mice using quantitation of nestin immunoreactivity as depicted in A  . There is a statistically significant difference (P  < 0.05) between groups with n = 4 per group.
Image Not Available
Fig. 8. Overexpression of MOR1 increases lung metastasis in human non-small cell lung cancer (NSCLC) xenograft models. (A  ) Representative immunoblot analysis of human H358 NSCLC cells alone, control nude mouse lung homogenate, lung homogenate from nude mouse with vector control H358 hind flank tumor or lung homogenates from nude mice with μ-opioid receptor 1 (MOR1, the most abundant MOR transcript that consists of exons 1, 2, 3, and 434) overexpressing H358 hind flank tumors using anti-nestin (a), anti-MOR (b), or anti-actin (c) antibodies. (B  ) Graphic representation of lung metastasis from vector control or MOR1 overexpressing H358 hind flank tumors in nude mice using quantitation of nestin immunoreactivity as depicted in A  . There is a statistically significant difference (P  < 0.05) between groups with n = 4 per group.
×