Nalmefene attenuates malignant potential in colorectal cancer cell via inhibition of opioid receptor

Nalmefene attenuates malignant potential in colorectal cancer cell via inhibition of opioid receptor Abstract Morphine is postulated a risk factor in promoting tumor growth and metastasis during the preoperative period, and high glycolysis of tumor cells is proved to accelerate tumor progression. In this study, we investigated whether nalmefene, an opioid receptor inhibitor, could inhibit CT26 colon cancer cell growth through influencing cell glycolysis. CCK8 and transwell migration assays showed that nalmefene inhibited CT26 cells viability and migration in a concentration-dependent manner. Extracellular acidification rate and oxygen consumption rate showed that nalmefene inhibited glycolysis of CT26 cells. Moreover, western blot analysis and quantitative real-time PCR revealed that nalmefene decreased the expressions of enzymes related to glycolysis. Flow cytometry results revealed that intracellular calcium (Ca2+) level was changed by nalmefene, western blot analysis showed that nalmefene decreased calmodulin expression and calcium/calmodulin dependent protein kinases II (CaMK II) phosphorylation, thus inhibiting the serine/threonine kinase (AKT)-glycogen synthase kinase-3β (GSK-3β) pathway. Furthermore, the effects of KN93, an inhibitor of CaMK II, were similar to the effects of nalmefene, and the anti-tumor effect of nalmefene could be counteracted by morphine. In conclusion, the anti-tumor effect of nalmefene may be achieved by inhibiting opioid receptor and down-regulating calmodulin expression and CaMK II phosphorylation, thus inhibiting AKT-GSK-3β pathway and the glycolysis of CT26 cells. colorectal cancer, glycolysis, nalmefene Introduction Colorectal cancer is the most common gastrointestinal tumor and the third reason for cancer-related death globally [1]. Surgery is the main option for colorectal cancer treatment. However, surgery may influence the immune and neuroendocrine systems, and result in inadvertent seeding of tumor cells intraoperatively [2–5], thus, affecting the prognosis of cancer patients. Cancer cells uptake and convert glucose mainly to lactate, known as Warburg glycolysis [6,7]. This metabolic reprogramming provides cancer cells with metabolic intermediates and energy for rapid proliferation [8]. The transcription factor c-Myc induces the expressions of glycolytic genes, including glucose transporter 1 (GLUT1), hexokinase-II (HK2), and lactate dehydrogenase A (LDHA) [9–11], and plays an important role in the switch to glycolytic metabolism for cancer cells. Moreover, c-Myc synthesis is regulated by serine/threonine kinase (AKT)-glycogen synthase kinase-3β (GSK-3β) pathway [12,13]. Opioid-mediated opioid receptor activation can induce an epithelial mesenchymal transition (EMT), which can be attenuated by opioid receptors antagonist or opioid receptors silencing in cancer cells [14,15]. In clinically relevant doses, opioid receptor antagonists reduce tumor growth in some in vivo models [16–18]. Nalmefene, an opiate antagonist, which is reported to attenuate opiate-induced side effects perioperatively [19–23], may also be used in cancer patients. However, whether and how nalmefene inhibits the malignant potential of cancer cells is not fully understood. Therefore, in the present study, we explored whether and how nalmefene inhibits cancer cell malignant potential. Materials and Methods Cell culture and reagents Murine CT26 colon cancer cells were cultured in RPMI 1640 cell culture medium (Hyclone, Logan, USA) with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, USA) in an incubator with 5% CO2 at 37°C. KN93, an inhibitor of calcium/calmodulin dependent protein kinases II (CaMK II) was purchased from Selleck (Shanghai, China). Nalmefene was administered to cells in a concentration gradient (N1: 0.625 μg/l, N2: 0.25 μg/l, N3: 1 μg/l, and N4: 10 μg/l) for 10 h. Cell viability analysis Cell suspensions (3000 cells/well) were added into wells of the 96-well culture plate and cultured in the incubator. After corresponding treatments, 10 μl CCK-8 was added to each well and the cells were further cultured for 2 h. Finally, the optical density at 450 nm was detected using a microplate reader. The cell viability curve was determined by measuring the optical density of each well, and the results were the average of six wells. Transwell migration assay The transwell migration assay was performed using 24-well transwell permeable inserts containing polycarbonate membranes (Sigma-Aldrich, St Louis, USA). A total of 2 × 104 cells (600 μl from the cell suspension) were added to each well. The number of migrated cells was counted after 24 h of incubation. The non-migrated cells on the top side of the membranes were carefully wiped off using wet cotton swabs. Migrated cells on the membrane were fixed with cold paraformaldehyde for 30 min, air dried and washed three times and then stained for nuclei using crystal violet for 15 min, followed by at least three times wash with PBS to remove excess stain. The membranes were left to air dry. Quantification of cells was carried out by imaging 10 random ×10 high-power fields per membrane with an Olympus microscope (IX2-ILL100; Tokyo, Japan) and the number of migrated cells was calculated using Image J software. Extracellular acidification rate and oxygen consumption rate analysis Simultaneous multiparameter metabolic analysis of colorectal cancer cells was performed using the Seahorse XF24® extracellular flux analyzer (Seahorse Bioscience, Santa Clara, USA) according to the manufacturer’s instructions. Briefly, CT26 cells were seeded in a 96-well cell culture XF microplate at 2 × 104 cells per well and treated with drugs 24 h prior to assay. Cells were switched to Seahorse buffer (medium with phenol red containing 25 mM glucose, 2 mM sodium pyruvate, and 2 mM glutamine) 1 h prior to assay. Then, 25 μl of 10 mM glucose, 1 μM oligomycin, and 100 mM 2-deoxy-glucose (2-DG) were added to each well to measure extracellular acidification rate (ECAR). Each measurement cycle consisted of a mixing time of 3 min and a data acquisition time of 3 min for the XF96. The glucose stimulation indicates cell glycolysis rate and the oligomycin inhibition indicates cell glycolysis capacity. While, before and after added to oligomycin (1 μM), of the electron transport chain uncoupler FCCP (1 μM) and of specific inhibitors of the mitochondrial respiratory chain antimycin A/rotenone (0.5 μM) in order, the oxygen consumption rate (OCR) was analyzed. The ECAR and OCAR levels were calculated after normalization to cell number. Flow cytometry analysis Intracellular calcium (Ca2+) level was measured by using a Fluo-3 AM fluorescence kit (Beyotime, Haimen, China) according to the manufacturer’s instruction. A total of 2 × 106 cells were seeded into each well of six-well plates. After corresponding treatment, cells were washed with PBS (without CaCl2 and MgCl2), and then fluo-3 AM fluorescence probes (0.5 μM) were added to each well. Cells were incubated with the probes at 37°C in dark for 60 min, then washed with serum-free medium three times and prepared in 1 ml ice-cold PBS for flow cytometry analysis at 488 nm. The results were calculated relative to the control group. cDNA preparation and quantitative real-time PCR Total RNA was extracted using Trizol (TIANGEN, Shanghai, China) according to the manufacturer’s protocol. cDNA was synthesized by reverse transcription using a TaKaRa PrimeScript® RT regent kit (TaKaRa, Dalian, China) with gDNA eraser. The expressions of target genes and β-actin (as a house-keeping gene) were determined by quantitative real-time PCR. The prime sequences for β-actin were 5′-CTGTCCCTGTATGCCTCTG-3′ (forward) and 5′-ATGTCACGCACGATTTCC-3′ (reverse); for GLUT1 were 5′-TTGTTGTAGAGCGAGCTGGACGAC-3′ (forward) and 5′-GGCCACGATGCTCAGATAGGACA-3′ (reverse); for HK2 were 5′-CCTTGCTGAAGGAAGCCATTCGC-3′ (forward) and 5′-ACGCATCTCCTCCATGTAGCAGG-3′ (reverse); for LDHA were 5′-ACATGGCGACTCCAGTGTGCCTGT-3′ (forward) and 5′-GAGGCCAATGGCCCAGGATGTGTA-3′ (reverse). Protein extraction and western blot analysis Whole cell extracts were prepared using the cell lysis buffer (Cell Signaling Technology, Beverly, USA). Protein concentration was measured by using BCA protein assay reagent (Pierce, Rockford, USA). Equal amounts of proteins were separated by SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS)-Tween 20 (TBS-T) at room temperature for 1 h, then incubated with corresponding primary antibody overnight at 4°C. After being incubated with corresponding secondary antibody, the membranes were washed three times with TBS-T, and detected using an enhanced chemiluminescence (ECL) reagent kit (Millipore Corporation, Billerica, USA) with a LAS-4000 mini CCD camera (GE Healthcare, Wisconsin, USA). The primary antibodies used were as follows: monoclonal antibody against β-actin (catalog sc-70,319; Santa Cruz Biotechnology, Santa Cruz, USA), calmodulin (catalog ab45689; Abcam, Cambridge, UK), phospho-AKT (Thr308, catalog 13,038; Cell Signaling Technology), AKT (catalog 2920; Cell Signaling Technology), phospho-GSK 3β (Thr390, catalog 3548; Cell Signaling Technology), GSK 3β (catalog 40,989; Cell Signaling Technology), c-Myc (catalog 10,828-1-AP; Proteintech Group, Chicago, USA), phospho-CaMKII (Thr286, catalog 12,716; Cell Signaling Technology), CaMKII (catalog ab134041; Abcam), GLUT1 (catalog 21,829-1-AP; Proteintech Group), HK2 (catalog 22,029-1-AP; Proteintech Group), and LDHA (catalog 19,987-1-AP; Proteintech Group). Immunofluorescence analysis Cells were plated in six-well petri dishes with cover slips at 1 × 104 cells/well and cultured at 37°C in humidified 5% CO2 overnight. Then cells were harvested and fixed with 4% paraformaldehyde for 20 min at room temperature, and washed with phosphate buffered saline (PBS) five times. After being permeabilized with 0.1% Triton X-100 for 10 min, cells were washed and incubated with primary polyclonal antibody against calmodulin overnight at 4°C. After wash with PBS, cells were incubated with HRP-conjugated secondary antibody for 2 h at room temperature in the dark. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Negative controls were conducted in all cases without primary antibody. Photomicrographs were captured using Leica TCS SP5II microscope at the same light intensity. Five fields were randomly selected from each section and imaged. The mean density (MD) of each sample was calculated using IPP6 (Image-Pro Plus 6) software. The cumulative optical density of the red portion is divided by the area to obtain the optical density of the average red portion (MD) according to the different picture conditions to set the appropriate parameters. Statistical analysis Results were obtained from five independent experiments and expressed as the mean ± SD. N represents the number of repeated experiments using different cell cultures. Statistical significance between conditions was assessed by two-tailed Student’s t-test. A value of P < 0.05 was considered significant. Results Nalmefene reduced viability and migration of CT26 cells Compared with the control group, nalmefene inhibited CT26 cells viability (Fig. 1A) and migration (Fig. 1B,C) in a concentration-dependent manner. Incubation of cells with 1 μg/l nalmefene for 10 h induced a significant inhibition of cell viability and migration. Figure 1. View largeDownload slide Nalmefene reduces viability and migration of CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then cell viability and migration were measured. (A) Nalmefene inhibited CT26 viability in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cell viability. (B,C) Nalmefene reduced CT26 migration in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cells migration (n = 5, *P < 0.05 vs. control group). Figure 1. View largeDownload slide Nalmefene reduces viability and migration of CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then cell viability and migration were measured. (A) Nalmefene inhibited CT26 viability in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cell viability. (B,C) Nalmefene reduced CT26 migration in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cells migration (n = 5, *P < 0.05 vs. control group). Nalmefene inhibits glycolysis and AKT/GSK-3β pathway in CT26 cells As accelerated glycolysis is a hallmark of cancer cells, we first examined the effect of nalmefene on lactate production. It was found that nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner (Fig. 2A). Then nalmefene-treated cells showed a decreased ECAR, confirming its anti-glycolytic properties. When mitochondrial respiration was suppressed by the adenosine tri-phosphate (ATP) synthase inhibitor oligomycin, glycolysis remained nevertheless at low levels in nalmefene-treated cells, as reflected by the dose-dependent reduction in glycolytic reserve (Fig. 2B). To characterize the effects of nalmefene on mitochondrial respiration, the oxygen consumption rate (OCR) was evaluated. Nalmefene had no effect on the basal OCR, while it enhanced the spare capacity of OCR when the electron transport chain uncoupler carbonilcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was injected (Fig. 2C). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (Fig. 2C). Finally, a series of enzymes related to glycolysis, such as GLUT1, HK2, and LDHA were examined. Nalmefene decreased the expressions of GLUT1, HK2, and LDHA at both mRNA and protein levels (Fig. 2D,E). Figure 2. View largeDownload slide Nalmefene inhibits AKT-GSK-3β pathway and glycolysis in CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then ECAR, OCR, and enzymes related to glycolysis were measured. (A) Compared with control group, nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner. (B) Nalmefene-treated cells showed decreased initial ECAR (left), and lower glycolytic reserve in dose-dependent manners (right). (C) Nalmefene did not modify the basal OCR (left), but enhanced the spare capacity of mitochondrial respiration (middle). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (right). (D). Nalmefene decreased the mRNA expressions of GLUT1, HK2, and LDHA. (E) Nalmefene inhibited the protein expressions of GLUT1, HK2, and LDHA (n = 5, *P < 0.05 vs. control group). Figure 2. View largeDownload slide Nalmefene inhibits AKT-GSK-3β pathway and glycolysis in CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then ECAR, OCR, and enzymes related to glycolysis were measured. (A) Compared with control group, nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner. (B) Nalmefene-treated cells showed decreased initial ECAR (left), and lower glycolytic reserve in dose-dependent manners (right). (C) Nalmefene did not modify the basal OCR (left), but enhanced the spare capacity of mitochondrial respiration (middle). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (right). (D). Nalmefene decreased the mRNA expressions of GLUT1, HK2, and LDHA. (E) Nalmefene inhibited the protein expressions of GLUT1, HK2, and LDHA (n = 5, *P < 0.05 vs. control group). Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on intracellular Ca2+ level Nalmefene decreased the expression of calmodulin and phosphorylation of CaMKII (Fig. 3A). To determine whether nalmefene affects intracellular Ca2+ level, flow cytometry analysis was carried out. Results showed that nalmefene had no effect on cellular Ca2+ level (Fig. 3B,C). Figure 3. View largeDownload slide Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on cellular Ca2+concentration CT26 cells were incubated with different concentrations of nalmefene for 10 h, then the expression of calmodulin, phosphorylation of CaMKII and cellular Ca2+ levels were measured. (A) The expression of calmodulin and the phosphorylation of CaMKII were inhibited by nalmefene. (B,C) Nalmefene had no effect on intracellular Ca2+ level (n = 5, *P < 0.05 vs. control group). Figure 3. View largeDownload slide Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on cellular Ca2+concentration CT26 cells were incubated with different concentrations of nalmefene for 10 h, then the expression of calmodulin, phosphorylation of CaMKII and cellular Ca2+ levels were measured. (A) The expression of calmodulin and the phosphorylation of CaMKII were inhibited by nalmefene. (B,C) Nalmefene had no effect on intracellular Ca2+ level (n = 5, *P < 0.05 vs. control group). Nalmefene inhibits calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression via inhibition of morphine receptors To illustrate the mechanism by which nalmefene inhibits glycolysis in CT26 cells, morphine, and KN93 were used. Compared with the control group, nalmefene inhibited calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression (Fig. 4A), which could be reversed by morphine. Furthermore, KN93 inhibited CaMK II phosphorylation, AKT/GSK-3β pathway and c-Myc expression, which was similar to the effect of nalmefene (Fig. 4A). Under the fluorescent microscope, the expression of calmodulin with red light was observed in cytoplasm (Fig. 4B). Compared with the control group, the fluorescence density of calmodulin was decreased when treated with nalmefene, but increased when treated with morphine. Furthermore, KN93 had no effect on calmodulin expression (Fig 4B,C). Figure 4. View largeDownload slide Nalmefene inhibits calmodulin expression and CaMK II phosphorylation via inhibition of morphine receptors (A) Compared with control group, nalmefene inhibited CaM expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression, which could be reversed by morphine. Moreover, morphine increased calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression. KN93 inhibited the phosphorylation of CaMK II, AKT/GSK-3β pathway and c-Myc expression, but had no effect on CaM expression. (B,C) Confocal microscopy images of the effects of nalmefene, KN93 and morphine on the expression of calmodulin (red) in CT26 cells, the nuclei were stained with DAPI (blue) (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). Figure 4. View largeDownload slide Nalmefene inhibits calmodulin expression and CaMK II phosphorylation via inhibition of morphine receptors (A) Compared with control group, nalmefene inhibited CaM expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression, which could be reversed by morphine. Moreover, morphine increased calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression. KN93 inhibited the phosphorylation of CaMK II, AKT/GSK-3β pathway and c-Myc expression, but had no effect on CaM expression. (B,C) Confocal microscopy images of the effects of nalmefene, KN93 and morphine on the expression of calmodulin (red) in CT26 cells, the nuclei were stained with DAPI (blue) (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). Nalmefene attenuates glycolysis in CT26 cells via inhibiting the morphine receptor-calmodulin-CaMK II pathway As nalmefene could inhibit calmodulin expression and CaMK II phosphorylation, we determined whether nalmefene could thereby alter the tumor cell metabolism by the following methods. ECAR results showed that, compared with the control group, nalmefene inhibited lactate production, which was similar to the effect of KN93 (Fig. 5A). Moreover, the effect of nalmefene could be reversed by morphine (Fig. 5A). In OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine (Fig. 5B). Accordingly, nalmefene inhibited the expressions of glycolytic enzymes at both mRNA and protein levels, which was similar to the effect of KN93 (Fig. 5C,D). Furthermore, morphine increased the expressions of glycolytic enzymes at mRNA and protein levels in CT26 cells (Fig. 5C,D). Figure 5. View largeDownload slide Nalmefene attenuates glycolysis in CT26 cells via inhibiting opiod receptor-calmodulin-CaMK II pathway (A) ECAR were measured in CT26 cells. Compared with the control group, nalmefene and KN93 could inhibit initial lactate production (left); nalmefene and KN93 attenuated the glycolytic reserve (right). (B) In the OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine. Morphine could inhibit mitochondrial respiration of cancer cells. (C) Compared with the control group, the mRNA expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Compared with the control group, the protein expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine. Figure 5. View largeDownload slide Nalmefene attenuates glycolysis in CT26 cells via inhibiting opiod receptor-calmodulin-CaMK II pathway (A) ECAR were measured in CT26 cells. Compared with the control group, nalmefene and KN93 could inhibit initial lactate production (left); nalmefene and KN93 attenuated the glycolytic reserve (right). (B) In the OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine. Morphine could inhibit mitochondrial respiration of cancer cells. (C) Compared with the control group, the mRNA expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Compared with the control group, the protein expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine. Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway Compared with the control group, nalmefene inhibited CT26 cells viability (Fig. 6A) and migration (Fig. 6B,C), which was similar to the effect of KN93 (Fig. 6A–C). Moreover, the effect of nalmefene could be reversed by morphine (Fig. 6). Figure 6. View largeDownload slide Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway (A) Nalmefene inhibited CT26 cells viability, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine augmented CT26 cell viability. (B,C) Nalmefene reduced CT26 migration, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine increased cell migration (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Speculative scheme for the mechanism by which nalmefene inhibits CT26 cell viability and migration. Nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, cell migration, and cell viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. Figure 6. View largeDownload slide Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway (A) Nalmefene inhibited CT26 cells viability, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine augmented CT26 cell viability. (B,C) Nalmefene reduced CT26 migration, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine increased cell migration (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Speculative scheme for the mechanism by which nalmefene inhibits CT26 cell viability and migration. Nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, cell migration, and cell viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. Discussion In the present study, we found that nalmefene could inhibit CT26 cell viability and migration. The anti-tumor effect of nalmefene may be achieved by down-regulation of calmodulin expression and CaMK II phosphorylation, leading to the inhibition of AKT-GSK-3β pathway, decrease in the expressions of c-Myc and glycolytic enzymes, and attenuation of CT26 cell glycolysis. Cancer cell metabolism is performed as an incremental uptake and transform of glucose to lactate via glycolysis [6,7]. Changes in metabolism play an important role in cancer cell proliferation and growth [8,24]. c-Myc is an important regulator of cancer cell metabolism. When activated, c-Myc induces the expressions of glycolytic enzymes, including LDHA, HK2, and GLUT1 [9–11]. It was reported that GSK-3β was involved in c-Myc proteolysis [25,26]. Moreover, GSK-3β activity is modulated by the phosphorylation status of Ser9. AKT is a key protein kinase that regulates GSK-3β phosphorylation at Ser9. AKT attenuates GSK-3β activity via upregulation of GSK-3β phosphorylation at Ser9. The present study indicated that nalmefene could inhibit the AKT-GSK-3β pathway, down-regulate the expressions of c-Myc and glycolytic enzymes, attenuate CT26 glycolysis, thus inhibiting CT26 cell viability and migration. Moreover, KN93, an inhibitor of CaMK II, inhibited AKT-GSK-3β pathway, down-regulated c-Myc in CT26 cells, which was similar to the effect of nalmefene. It was reported that KN93 could inhibit AKT phosphorylation and attenuate cell migration in pancreatic cancer cells [27], suggesting that CaMK II is in the upstream of AKT-GSK-3β pathway. CaMKs are multifunctional serine/threonine kinases whose activities are modulated by Ca2+ signaling [28] and calmodulin expression [29]. Calmodulin is an important Ca2+ sensor which possess four Ca2+ binding motifs [30]. When bound with Ca2+, calmodulin exhibits conformational change which facilitates its binding to a variety of target proteins, through which Ca2+-sensitivity is expressed in a variety of cell biological functions [30]. It was reported that several cancer cells express high levels of CaMK II [31–34]. CaMK II modulates the phosphorylation of a mass of proteins [35,36] and plays a key role in modulating cancer cell proliferation, differentiation and survival. In the present study, we showed that nalmefene attenuated intracellular Ca2+ levels in CT26 cells. However, the expression of calmodulin was down-regulated by nalmefene. These data indicate that nalmefene inhibits CaMK II phosphorylation via inhibition of calmodulin expression. Nalmefene is a new opioid antagonist with a duration of action ~8–9 h [37]. Its efficacy has been shown in the reversal of opioid-induced sedation and opioid overdose in adults and children [19–22]. Prophylactic administration of nalmefene significantly attenuated morphine-induced pruritus and emesis [23]. Previous studies have indicated that opioid receptors are expressed on the surface of cancer cells [15], and our pre-experiments also proved that these receptors are expressed on the surface of CT26 cells. In this study, we demonstrated that nalmefene could inhibit calmodulin expression and CaMK II phosphorylation in CT26 cells, which could be reversed by opioid receptor activator, morphine. However, in a previous study, nalmefene was not shown to have effect on cell growth, apoptosis in cancer cells [38]. We speculate that the discrepancies might be resulted from different nalmefene dose, different route and duration of administration, and difference type of cancer cells were used. It has also been reported that treatment with opioid receptor antagonist is associated with increased survival in patients with advanced cancer [39]. These studies are quite consistent with our study, which indicated that opioid receptor antagonists possess anti-tumor effect. However, some limitations should be noted in this study. First, the present study was carried out only in vitro, and the anti-tumor effect of nalmefene should be further confirmed by studies using in vivo systems. Second, we only used murine colon cancer cell line in the present study. Human colon cancer cell lines should be employed in future studies. Third, the exact mechanism by which nalmefene inhibits calmodulin expression should be further explored. In summary, we have demonstrated that nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits the AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, migration, and viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. Funding This work is supported by the grants from the Shanghai Charity Cancer Research Center (No. HYXH1407) and Shanghai Outstanding Academic Leaders Plan (No. KW1602). Acknowledgments We would like to thank Wang Zhen (Fudan University Shanghai Cancer Center, Shanghai, China) for experimental and equipment support. References 1 Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin  2017, 67: 7– 30. Google Scholar CrossRef Search ADS PubMed  2 Boomsma MF, Garssen B, Slot E, Berbee M, Berkhof J, Meezenbroek Ede J, Slieker W, et al.  . Breast cancer surgery-induced immunomodulation. J Surg Oncol Suppl  2010, 102: 640– 648. Google Scholar CrossRef Search ADS   3 Camara O, Kavallaris A, Noschel H, Rengsberger M, Jorke C, Pachmann K. Seeding of epithelial cells into circulation during surgery for breast cancer: the fate of malignant and benign mobilized cells. World J Surg Oncol  2006, 4: 67. 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Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

Nalmefene attenuates malignant potential in colorectal cancer cell via inhibition of opioid receptor

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Abstract

Abstract Morphine is postulated a risk factor in promoting tumor growth and metastasis during the preoperative period, and high glycolysis of tumor cells is proved to accelerate tumor progression. In this study, we investigated whether nalmefene, an opioid receptor inhibitor, could inhibit CT26 colon cancer cell growth through influencing cell glycolysis. CCK8 and transwell migration assays showed that nalmefene inhibited CT26 cells viability and migration in a concentration-dependent manner. Extracellular acidification rate and oxygen consumption rate showed that nalmefene inhibited glycolysis of CT26 cells. Moreover, western blot analysis and quantitative real-time PCR revealed that nalmefene decreased the expressions of enzymes related to glycolysis. Flow cytometry results revealed that intracellular calcium (Ca2+) level was changed by nalmefene, western blot analysis showed that nalmefene decreased calmodulin expression and calcium/calmodulin dependent protein kinases II (CaMK II) phosphorylation, thus inhibiting the serine/threonine kinase (AKT)-glycogen synthase kinase-3β (GSK-3β) pathway. Furthermore, the effects of KN93, an inhibitor of CaMK II, were similar to the effects of nalmefene, and the anti-tumor effect of nalmefene could be counteracted by morphine. In conclusion, the anti-tumor effect of nalmefene may be achieved by inhibiting opioid receptor and down-regulating calmodulin expression and CaMK II phosphorylation, thus inhibiting AKT-GSK-3β pathway and the glycolysis of CT26 cells. colorectal cancer, glycolysis, nalmefene Introduction Colorectal cancer is the most common gastrointestinal tumor and the third reason for cancer-related death globally [1]. Surgery is the main option for colorectal cancer treatment. However, surgery may influence the immune and neuroendocrine systems, and result in inadvertent seeding of tumor cells intraoperatively [2–5], thus, affecting the prognosis of cancer patients. Cancer cells uptake and convert glucose mainly to lactate, known as Warburg glycolysis [6,7]. This metabolic reprogramming provides cancer cells with metabolic intermediates and energy for rapid proliferation [8]. The transcription factor c-Myc induces the expressions of glycolytic genes, including glucose transporter 1 (GLUT1), hexokinase-II (HK2), and lactate dehydrogenase A (LDHA) [9–11], and plays an important role in the switch to glycolytic metabolism for cancer cells. Moreover, c-Myc synthesis is regulated by serine/threonine kinase (AKT)-glycogen synthase kinase-3β (GSK-3β) pathway [12,13]. Opioid-mediated opioid receptor activation can induce an epithelial mesenchymal transition (EMT), which can be attenuated by opioid receptors antagonist or opioid receptors silencing in cancer cells [14,15]. In clinically relevant doses, opioid receptor antagonists reduce tumor growth in some in vivo models [16–18]. Nalmefene, an opiate antagonist, which is reported to attenuate opiate-induced side effects perioperatively [19–23], may also be used in cancer patients. However, whether and how nalmefene inhibits the malignant potential of cancer cells is not fully understood. Therefore, in the present study, we explored whether and how nalmefene inhibits cancer cell malignant potential. Materials and Methods Cell culture and reagents Murine CT26 colon cancer cells were cultured in RPMI 1640 cell culture medium (Hyclone, Logan, USA) with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, USA) in an incubator with 5% CO2 at 37°C. KN93, an inhibitor of calcium/calmodulin dependent protein kinases II (CaMK II) was purchased from Selleck (Shanghai, China). Nalmefene was administered to cells in a concentration gradient (N1: 0.625 μg/l, N2: 0.25 μg/l, N3: 1 μg/l, and N4: 10 μg/l) for 10 h. Cell viability analysis Cell suspensions (3000 cells/well) were added into wells of the 96-well culture plate and cultured in the incubator. After corresponding treatments, 10 μl CCK-8 was added to each well and the cells were further cultured for 2 h. Finally, the optical density at 450 nm was detected using a microplate reader. The cell viability curve was determined by measuring the optical density of each well, and the results were the average of six wells. Transwell migration assay The transwell migration assay was performed using 24-well transwell permeable inserts containing polycarbonate membranes (Sigma-Aldrich, St Louis, USA). A total of 2 × 104 cells (600 μl from the cell suspension) were added to each well. The number of migrated cells was counted after 24 h of incubation. The non-migrated cells on the top side of the membranes were carefully wiped off using wet cotton swabs. Migrated cells on the membrane were fixed with cold paraformaldehyde for 30 min, air dried and washed three times and then stained for nuclei using crystal violet for 15 min, followed by at least three times wash with PBS to remove excess stain. The membranes were left to air dry. Quantification of cells was carried out by imaging 10 random ×10 high-power fields per membrane with an Olympus microscope (IX2-ILL100; Tokyo, Japan) and the number of migrated cells was calculated using Image J software. Extracellular acidification rate and oxygen consumption rate analysis Simultaneous multiparameter metabolic analysis of colorectal cancer cells was performed using the Seahorse XF24® extracellular flux analyzer (Seahorse Bioscience, Santa Clara, USA) according to the manufacturer’s instructions. Briefly, CT26 cells were seeded in a 96-well cell culture XF microplate at 2 × 104 cells per well and treated with drugs 24 h prior to assay. Cells were switched to Seahorse buffer (medium with phenol red containing 25 mM glucose, 2 mM sodium pyruvate, and 2 mM glutamine) 1 h prior to assay. Then, 25 μl of 10 mM glucose, 1 μM oligomycin, and 100 mM 2-deoxy-glucose (2-DG) were added to each well to measure extracellular acidification rate (ECAR). Each measurement cycle consisted of a mixing time of 3 min and a data acquisition time of 3 min for the XF96. The glucose stimulation indicates cell glycolysis rate and the oligomycin inhibition indicates cell glycolysis capacity. While, before and after added to oligomycin (1 μM), of the electron transport chain uncoupler FCCP (1 μM) and of specific inhibitors of the mitochondrial respiratory chain antimycin A/rotenone (0.5 μM) in order, the oxygen consumption rate (OCR) was analyzed. The ECAR and OCAR levels were calculated after normalization to cell number. Flow cytometry analysis Intracellular calcium (Ca2+) level was measured by using a Fluo-3 AM fluorescence kit (Beyotime, Haimen, China) according to the manufacturer’s instruction. A total of 2 × 106 cells were seeded into each well of six-well plates. After corresponding treatment, cells were washed with PBS (without CaCl2 and MgCl2), and then fluo-3 AM fluorescence probes (0.5 μM) were added to each well. Cells were incubated with the probes at 37°C in dark for 60 min, then washed with serum-free medium three times and prepared in 1 ml ice-cold PBS for flow cytometry analysis at 488 nm. The results were calculated relative to the control group. cDNA preparation and quantitative real-time PCR Total RNA was extracted using Trizol (TIANGEN, Shanghai, China) according to the manufacturer’s protocol. cDNA was synthesized by reverse transcription using a TaKaRa PrimeScript® RT regent kit (TaKaRa, Dalian, China) with gDNA eraser. The expressions of target genes and β-actin (as a house-keeping gene) were determined by quantitative real-time PCR. The prime sequences for β-actin were 5′-CTGTCCCTGTATGCCTCTG-3′ (forward) and 5′-ATGTCACGCACGATTTCC-3′ (reverse); for GLUT1 were 5′-TTGTTGTAGAGCGAGCTGGACGAC-3′ (forward) and 5′-GGCCACGATGCTCAGATAGGACA-3′ (reverse); for HK2 were 5′-CCTTGCTGAAGGAAGCCATTCGC-3′ (forward) and 5′-ACGCATCTCCTCCATGTAGCAGG-3′ (reverse); for LDHA were 5′-ACATGGCGACTCCAGTGTGCCTGT-3′ (forward) and 5′-GAGGCCAATGGCCCAGGATGTGTA-3′ (reverse). Protein extraction and western blot analysis Whole cell extracts were prepared using the cell lysis buffer (Cell Signaling Technology, Beverly, USA). Protein concentration was measured by using BCA protein assay reagent (Pierce, Rockford, USA). Equal amounts of proteins were separated by SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline (TBS)-Tween 20 (TBS-T) at room temperature for 1 h, then incubated with corresponding primary antibody overnight at 4°C. After being incubated with corresponding secondary antibody, the membranes were washed three times with TBS-T, and detected using an enhanced chemiluminescence (ECL) reagent kit (Millipore Corporation, Billerica, USA) with a LAS-4000 mini CCD camera (GE Healthcare, Wisconsin, USA). The primary antibodies used were as follows: monoclonal antibody against β-actin (catalog sc-70,319; Santa Cruz Biotechnology, Santa Cruz, USA), calmodulin (catalog ab45689; Abcam, Cambridge, UK), phospho-AKT (Thr308, catalog 13,038; Cell Signaling Technology), AKT (catalog 2920; Cell Signaling Technology), phospho-GSK 3β (Thr390, catalog 3548; Cell Signaling Technology), GSK 3β (catalog 40,989; Cell Signaling Technology), c-Myc (catalog 10,828-1-AP; Proteintech Group, Chicago, USA), phospho-CaMKII (Thr286, catalog 12,716; Cell Signaling Technology), CaMKII (catalog ab134041; Abcam), GLUT1 (catalog 21,829-1-AP; Proteintech Group), HK2 (catalog 22,029-1-AP; Proteintech Group), and LDHA (catalog 19,987-1-AP; Proteintech Group). Immunofluorescence analysis Cells were plated in six-well petri dishes with cover slips at 1 × 104 cells/well and cultured at 37°C in humidified 5% CO2 overnight. Then cells were harvested and fixed with 4% paraformaldehyde for 20 min at room temperature, and washed with phosphate buffered saline (PBS) five times. After being permeabilized with 0.1% Triton X-100 for 10 min, cells were washed and incubated with primary polyclonal antibody against calmodulin overnight at 4°C. After wash with PBS, cells were incubated with HRP-conjugated secondary antibody for 2 h at room temperature in the dark. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Negative controls were conducted in all cases without primary antibody. Photomicrographs were captured using Leica TCS SP5II microscope at the same light intensity. Five fields were randomly selected from each section and imaged. The mean density (MD) of each sample was calculated using IPP6 (Image-Pro Plus 6) software. The cumulative optical density of the red portion is divided by the area to obtain the optical density of the average red portion (MD) according to the different picture conditions to set the appropriate parameters. Statistical analysis Results were obtained from five independent experiments and expressed as the mean ± SD. N represents the number of repeated experiments using different cell cultures. Statistical significance between conditions was assessed by two-tailed Student’s t-test. A value of P < 0.05 was considered significant. Results Nalmefene reduced viability and migration of CT26 cells Compared with the control group, nalmefene inhibited CT26 cells viability (Fig. 1A) and migration (Fig. 1B,C) in a concentration-dependent manner. Incubation of cells with 1 μg/l nalmefene for 10 h induced a significant inhibition of cell viability and migration. Figure 1. View largeDownload slide Nalmefene reduces viability and migration of CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then cell viability and migration were measured. (A) Nalmefene inhibited CT26 viability in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cell viability. (B,C) Nalmefene reduced CT26 migration in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cells migration (n = 5, *P < 0.05 vs. control group). Figure 1. View largeDownload slide Nalmefene reduces viability and migration of CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then cell viability and migration were measured. (A) Nalmefene inhibited CT26 viability in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cell viability. (B,C) Nalmefene reduced CT26 migration in a concentration-dependent manner. Compared with control group, 1 μg/l nalmefene significantly decreased cells migration (n = 5, *P < 0.05 vs. control group). Nalmefene inhibits glycolysis and AKT/GSK-3β pathway in CT26 cells As accelerated glycolysis is a hallmark of cancer cells, we first examined the effect of nalmefene on lactate production. It was found that nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner (Fig. 2A). Then nalmefene-treated cells showed a decreased ECAR, confirming its anti-glycolytic properties. When mitochondrial respiration was suppressed by the adenosine tri-phosphate (ATP) synthase inhibitor oligomycin, glycolysis remained nevertheless at low levels in nalmefene-treated cells, as reflected by the dose-dependent reduction in glycolytic reserve (Fig. 2B). To characterize the effects of nalmefene on mitochondrial respiration, the oxygen consumption rate (OCR) was evaluated. Nalmefene had no effect on the basal OCR, while it enhanced the spare capacity of OCR when the electron transport chain uncoupler carbonilcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was injected (Fig. 2C). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (Fig. 2C). Finally, a series of enzymes related to glycolysis, such as GLUT1, HK2, and LDHA were examined. Nalmefene decreased the expressions of GLUT1, HK2, and LDHA at both mRNA and protein levels (Fig. 2D,E). Figure 2. View largeDownload slide Nalmefene inhibits AKT-GSK-3β pathway and glycolysis in CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then ECAR, OCR, and enzymes related to glycolysis were measured. (A) Compared with control group, nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner. (B) Nalmefene-treated cells showed decreased initial ECAR (left), and lower glycolytic reserve in dose-dependent manners (right). (C) Nalmefene did not modify the basal OCR (left), but enhanced the spare capacity of mitochondrial respiration (middle). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (right). (D). Nalmefene decreased the mRNA expressions of GLUT1, HK2, and LDHA. (E) Nalmefene inhibited the protein expressions of GLUT1, HK2, and LDHA (n = 5, *P < 0.05 vs. control group). Figure 2. View largeDownload slide Nalmefene inhibits AKT-GSK-3β pathway and glycolysis in CT26 cells CT26 cells were incubated with different concentrations of nalmefene for 10 h, then ECAR, OCR, and enzymes related to glycolysis were measured. (A) Compared with control group, nalmefene attenuated the phosphorylation of AKT and GSK-3β, and augmented the expressions of GSK-3β and c-Myc in a concentration-dependent manner. (B) Nalmefene-treated cells showed decreased initial ECAR (left), and lower glycolytic reserve in dose-dependent manners (right). (C) Nalmefene did not modify the basal OCR (left), but enhanced the spare capacity of mitochondrial respiration (middle). And the OCR-linked ATP generation was simultaneously increased by nalmefene treatment (right). (D). Nalmefene decreased the mRNA expressions of GLUT1, HK2, and LDHA. (E) Nalmefene inhibited the protein expressions of GLUT1, HK2, and LDHA (n = 5, *P < 0.05 vs. control group). Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on intracellular Ca2+ level Nalmefene decreased the expression of calmodulin and phosphorylation of CaMKII (Fig. 3A). To determine whether nalmefene affects intracellular Ca2+ level, flow cytometry analysis was carried out. Results showed that nalmefene had no effect on cellular Ca2+ level (Fig. 3B,C). Figure 3. View largeDownload slide Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on cellular Ca2+concentration CT26 cells were incubated with different concentrations of nalmefene for 10 h, then the expression of calmodulin, phosphorylation of CaMKII and cellular Ca2+ levels were measured. (A) The expression of calmodulin and the phosphorylation of CaMKII were inhibited by nalmefene. (B,C) Nalmefene had no effect on intracellular Ca2+ level (n = 5, *P < 0.05 vs. control group). Figure 3. View largeDownload slide Nalmefene reduces calmodulin expression and CaMKII phosphorylation with no effect on cellular Ca2+concentration CT26 cells were incubated with different concentrations of nalmefene for 10 h, then the expression of calmodulin, phosphorylation of CaMKII and cellular Ca2+ levels were measured. (A) The expression of calmodulin and the phosphorylation of CaMKII were inhibited by nalmefene. (B,C) Nalmefene had no effect on intracellular Ca2+ level (n = 5, *P < 0.05 vs. control group). Nalmefene inhibits calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression via inhibition of morphine receptors To illustrate the mechanism by which nalmefene inhibits glycolysis in CT26 cells, morphine, and KN93 were used. Compared with the control group, nalmefene inhibited calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression (Fig. 4A), which could be reversed by morphine. Furthermore, KN93 inhibited CaMK II phosphorylation, AKT/GSK-3β pathway and c-Myc expression, which was similar to the effect of nalmefene (Fig. 4A). Under the fluorescent microscope, the expression of calmodulin with red light was observed in cytoplasm (Fig. 4B). Compared with the control group, the fluorescence density of calmodulin was decreased when treated with nalmefene, but increased when treated with morphine. Furthermore, KN93 had no effect on calmodulin expression (Fig 4B,C). Figure 4. View largeDownload slide Nalmefene inhibits calmodulin expression and CaMK II phosphorylation via inhibition of morphine receptors (A) Compared with control group, nalmefene inhibited CaM expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression, which could be reversed by morphine. Moreover, morphine increased calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression. KN93 inhibited the phosphorylation of CaMK II, AKT/GSK-3β pathway and c-Myc expression, but had no effect on CaM expression. (B,C) Confocal microscopy images of the effects of nalmefene, KN93 and morphine on the expression of calmodulin (red) in CT26 cells, the nuclei were stained with DAPI (blue) (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). Figure 4. View largeDownload slide Nalmefene inhibits calmodulin expression and CaMK II phosphorylation via inhibition of morphine receptors (A) Compared with control group, nalmefene inhibited CaM expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression, which could be reversed by morphine. Moreover, morphine increased calmodulin expression, CaMK II phosphorylation, AKT/GSK-3β pathway, and c-Myc expression. KN93 inhibited the phosphorylation of CaMK II, AKT/GSK-3β pathway and c-Myc expression, but had no effect on CaM expression. (B,C) Confocal microscopy images of the effects of nalmefene, KN93 and morphine on the expression of calmodulin (red) in CT26 cells, the nuclei were stained with DAPI (blue) (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). Nalmefene attenuates glycolysis in CT26 cells via inhibiting the morphine receptor-calmodulin-CaMK II pathway As nalmefene could inhibit calmodulin expression and CaMK II phosphorylation, we determined whether nalmefene could thereby alter the tumor cell metabolism by the following methods. ECAR results showed that, compared with the control group, nalmefene inhibited lactate production, which was similar to the effect of KN93 (Fig. 5A). Moreover, the effect of nalmefene could be reversed by morphine (Fig. 5A). In OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine (Fig. 5B). Accordingly, nalmefene inhibited the expressions of glycolytic enzymes at both mRNA and protein levels, which was similar to the effect of KN93 (Fig. 5C,D). Furthermore, morphine increased the expressions of glycolytic enzymes at mRNA and protein levels in CT26 cells (Fig. 5C,D). Figure 5. View largeDownload slide Nalmefene attenuates glycolysis in CT26 cells via inhibiting opiod receptor-calmodulin-CaMK II pathway (A) ECAR were measured in CT26 cells. Compared with the control group, nalmefene and KN93 could inhibit initial lactate production (left); nalmefene and KN93 attenuated the glycolytic reserve (right). (B) In the OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine. Morphine could inhibit mitochondrial respiration of cancer cells. (C) Compared with the control group, the mRNA expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Compared with the control group, the protein expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine. Figure 5. View largeDownload slide Nalmefene attenuates glycolysis in CT26 cells via inhibiting opiod receptor-calmodulin-CaMK II pathway (A) ECAR were measured in CT26 cells. Compared with the control group, nalmefene and KN93 could inhibit initial lactate production (left); nalmefene and KN93 attenuated the glycolytic reserve (right). (B) In the OCAR test, the spare capacity of mitochondrial respiration was enhanced by nalmefene and KN93, and the effect was reversed by morphine. Morphine could inhibit mitochondrial respiration of cancer cells. (C) Compared with the control group, the mRNA expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Compared with the control group, the protein expressions of GLUT1, HK2 and LDHA were inhibited by nalmefene and KN93, and increased by morphine. Moreover, the effect of nalmefene was reversed by morphine. Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway Compared with the control group, nalmefene inhibited CT26 cells viability (Fig. 6A) and migration (Fig. 6B,C), which was similar to the effect of KN93 (Fig. 6A–C). Moreover, the effect of nalmefene could be reversed by morphine (Fig. 6). Figure 6. View largeDownload slide Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway (A) Nalmefene inhibited CT26 cells viability, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine augmented CT26 cell viability. (B,C) Nalmefene reduced CT26 migration, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine increased cell migration (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Speculative scheme for the mechanism by which nalmefene inhibits CT26 cell viability and migration. Nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, cell migration, and cell viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. Figure 6. View largeDownload slide Nalmefene inhibits CT26 cell viability and migration via inhibition of morphine receptor-CaMK II pathway (A) Nalmefene inhibited CT26 cells viability, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine augmented CT26 cell viability. (B,C) Nalmefene reduced CT26 migration, which was similar to KN93. Moreover, the effect of nalmefene was reversed by morphine. Furthermore, morphine increased cell migration (n = 5, *P < 0.05 vs. control group; #P < 0.05 vs. nalmefene group). (D) Speculative scheme for the mechanism by which nalmefene inhibits CT26 cell viability and migration. Nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, cell migration, and cell viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. Discussion In the present study, we found that nalmefene could inhibit CT26 cell viability and migration. The anti-tumor effect of nalmefene may be achieved by down-regulation of calmodulin expression and CaMK II phosphorylation, leading to the inhibition of AKT-GSK-3β pathway, decrease in the expressions of c-Myc and glycolytic enzymes, and attenuation of CT26 cell glycolysis. Cancer cell metabolism is performed as an incremental uptake and transform of glucose to lactate via glycolysis [6,7]. Changes in metabolism play an important role in cancer cell proliferation and growth [8,24]. c-Myc is an important regulator of cancer cell metabolism. When activated, c-Myc induces the expressions of glycolytic enzymes, including LDHA, HK2, and GLUT1 [9–11]. It was reported that GSK-3β was involved in c-Myc proteolysis [25,26]. Moreover, GSK-3β activity is modulated by the phosphorylation status of Ser9. AKT is a key protein kinase that regulates GSK-3β phosphorylation at Ser9. AKT attenuates GSK-3β activity via upregulation of GSK-3β phosphorylation at Ser9. The present study indicated that nalmefene could inhibit the AKT-GSK-3β pathway, down-regulate the expressions of c-Myc and glycolytic enzymes, attenuate CT26 glycolysis, thus inhibiting CT26 cell viability and migration. Moreover, KN93, an inhibitor of CaMK II, inhibited AKT-GSK-3β pathway, down-regulated c-Myc in CT26 cells, which was similar to the effect of nalmefene. It was reported that KN93 could inhibit AKT phosphorylation and attenuate cell migration in pancreatic cancer cells [27], suggesting that CaMK II is in the upstream of AKT-GSK-3β pathway. CaMKs are multifunctional serine/threonine kinases whose activities are modulated by Ca2+ signaling [28] and calmodulin expression [29]. Calmodulin is an important Ca2+ sensor which possess four Ca2+ binding motifs [30]. When bound with Ca2+, calmodulin exhibits conformational change which facilitates its binding to a variety of target proteins, through which Ca2+-sensitivity is expressed in a variety of cell biological functions [30]. It was reported that several cancer cells express high levels of CaMK II [31–34]. CaMK II modulates the phosphorylation of a mass of proteins [35,36] and plays a key role in modulating cancer cell proliferation, differentiation and survival. In the present study, we showed that nalmefene attenuated intracellular Ca2+ levels in CT26 cells. However, the expression of calmodulin was down-regulated by nalmefene. These data indicate that nalmefene inhibits CaMK II phosphorylation via inhibition of calmodulin expression. Nalmefene is a new opioid antagonist with a duration of action ~8–9 h [37]. Its efficacy has been shown in the reversal of opioid-induced sedation and opioid overdose in adults and children [19–22]. Prophylactic administration of nalmefene significantly attenuated morphine-induced pruritus and emesis [23]. Previous studies have indicated that opioid receptors are expressed on the surface of cancer cells [15], and our pre-experiments also proved that these receptors are expressed on the surface of CT26 cells. In this study, we demonstrated that nalmefene could inhibit calmodulin expression and CaMK II phosphorylation in CT26 cells, which could be reversed by opioid receptor activator, morphine. However, in a previous study, nalmefene was not shown to have effect on cell growth, apoptosis in cancer cells [38]. We speculate that the discrepancies might be resulted from different nalmefene dose, different route and duration of administration, and difference type of cancer cells were used. It has also been reported that treatment with opioid receptor antagonist is associated with increased survival in patients with advanced cancer [39]. These studies are quite consistent with our study, which indicated that opioid receptor antagonists possess anti-tumor effect. However, some limitations should be noted in this study. First, the present study was carried out only in vitro, and the anti-tumor effect of nalmefene should be further confirmed by studies using in vivo systems. Second, we only used murine colon cancer cell line in the present study. Human colon cancer cell lines should be employed in future studies. Third, the exact mechanism by which nalmefene inhibits calmodulin expression should be further explored. In summary, we have demonstrated that nalmefene attenuates calmodulin expression and CaMK II phosphorylation, inhibits the AKT-GSK-3β pathway, down-regulates the expressions of c-Myc and glycolytic enzymes, and eventually inhibits CT26 cell glycolysis, migration, and viability. The anti-tumor effect of nalmefene may be achieved via inhibition of opioids receptor. 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Acta Biochimica et Biophysica SinicaOxford University Press

Published: Feb 1, 2018

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