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The effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells lines
Gene 718 (2019) 144030
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Research paper
The effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells lines
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Uğur Özgürbüza, , Sema Gencürb, Feyzan Özdal Kurt (PhD)c, Murat Özkalkanlıa, H. Seda Vatansever (MD, PhD)b,d İzmir Atatürk Research and Training Hospital, Dept. of Anesthesiology and Reanimation, Turkey Manisa Celal Bayar University, Dept. of Histology and Embryology, Turkey c Manisa Celal Bayar University, Faculty of Science & Letters, Dept. of Biology, Turkey d Near East University, Experimental Research Center of Health (DESAM), Mersin 10, Turkey a
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Opioids Tramadol Cell culture Colon cancer
Opioids are widely used in the treatment of cancer related pain. They mainly exert their effects on opioid receptors. The most common opioid in the treatment of pain is morphine. Previous studies show that they may have effects on cancer cell behavior. These may include apoptosis, angiogenesis, invasion, inflammation and immune reactions. Tramadol, also an opioid is widely used in the treatment of cancer pain and is not well studied in cancer behavior. We aimed to investigate the effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells. We used Colo320 (ATCC, CCL-220), Colo741 (ECACC, 93052621) and HCT116 (ATCC, CCL-247) colon cancer cell lines. CD133 was considered colon cancer stem cell marker and used to sort CD133+ and CD133− cells by magnetic cell sorting. MTT (mitochondria-targeted therapeutics) technique was used to detect tramadol's cytotoxic effect on cells in the study groups. Cells were treated with 1 mg/kg, 1.5 mg/kg and 2 mg/kg tramadol for 24 h at 37 °C and 5% CO2.Caspase-3, Ki-67, Bcl-2 and VGEF distributions were performed using indirect immunoperoxidase staining for immunohistochemical analysis. The study showed that tramadol has triggering effect on apoptosis in Colo320 colon cancer stem cells.
1. Introduction Opioids are considered the “gold standard” for the management of severe pain associated with cancer. They mainly act directly on the central nervous system and exert their effects on various opioid receptors. Morphine may also be responsible for some side effects (addiction, tolerance, respiratory depression, immunosuppression, and constipation). It's been debated that opioids have some effects on tumor development and metastasis (Afshamani et al., 2011; Zhang et al., 2018). Morphine has both inhibitor and promotary effects on cancer growth. There are some complex mechanisms which morphine affects tumor cells. The proposed mechanisms of action on tumor growth for morphine are apoptosis, angiogenesis, invasion, inflammation and immune reactions. Signaling pathways may also be involved in secondary effects of morphine. Tramadol, mainly acting on central nervous system, serotonergic and noradrenergic nociception. Its metabolite Odesmethyltramadol acts on the μ-opioid receptor (McCartney and Choi, 2017). Tramadol is used to treat both acute and chronic pain, especially
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in the postoperative period for cancer patients. While morphine is widely studied in tumor growth there are limited data for tramadol effects on tumor growth. There is some literature that investigates the effects of tramadol on angiogenesis and immunoreactivity. However, none of the studies investigated the effects of tramadol on cancer stem cells (Karaman et al., 2017; Kovelowski et al., 1998). The identification of stem cells may have a significant effect on research, prevention, and therapy. The aim of the present study is to investigate the effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells. 2. Material and method 2.1. Colo320, Colo741 and HCT116 Cell Culture In the present study, Colo320 (ATCC, CCL-220), Colo741 (ECACC, 93052621) and HCT116 (ATCC, CCL-247) colon cancer cell lines were used. Colo320, Colo741 and HCT116 cells were cultured in RPMI-1640
Corresponding author. E-mail address: [email protected] (U. Özgürbüz).
https://doi.org/10.1016/j.gene.2019.144030 Received 24 January 2019; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 04 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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were cultured in 96 well culture plate. Every well was included 3 × 103 cells. And they were incubated at 37 °C and 5% CO2 for 24 h. The cells were treated with 1 mg/kg, 1.5 mg/kg and 2 mg/kg tramadol for 24 h at 37 °C and 5% CO2. For the control group, every cell line was treated only with culture medium. The culture was applicate three consecutive times. At the end of incubation, PBS solution which included 5 mg/mL MTT solution (Glentham Life Sciences, 471OVO) was diluted at 1:10 and added each well and incubated for 4 h at 37 °C. Following incubation, 50 μl dimethylsulphoxide (DMSO, Sigma, D2650) was added and measurements were performed at 450–690 nm absorbance spectrophotometry (BioTek Instruments Inc., ELX800UV, USA).
medium which supplemented 10% fetal bovine serum (FBS, Capricorn scientific, FBS-HI-12B), 1% penicillin-streptomycine (Gibco™, 15140122) and 1% L-glutamine (Gibco™, A2916801). The cell lines were divided into study groups after becoming 80% confluent and other cells were stocked in freezing medium [FBS:DMSO (Dimethyl sulfoxide, Sigma-Aldrich, D8418) = 9:1] at −80 °C. 2.2. Isolation of cancer stem cells CD133 was considered colon cancer stem cell marker and used to sort CD133+ and CD133- cells by magnetic cell sorting (MACS). The CD133 immune magnetic bead separation kit was used (Miltenyi Biotech). The confluent of Colo320, Colo741 and HCT116 cells were incubated for 8–10 min with 0.5% Trypsin-EDTA solution (Biochrom, L2133) for digest and they were centrifuged for 5 min at 1000 rpm. The supernatants were discarded and the cells were collected re-suspending with 60 μl phosphate buffer solution containing 0.5% BSA and 0.08% EDTA (PBS-BE). The cells were then incubated CD133IgGs (Miltenyi Biotech) on ice for 15 min. They were then centrifuged with 1 ml PBSBEat 1000 rpm for 5 min and cells were resuspended with 500 μl PBSBE. MiniMACs system was placed in a sterile container and miniMACS magnet was fixed on the MACS multistand and washed with 500 μl PBSBE. The cell suspension for each cell lines was poured into the column reservoir, and unlabeled nonmagnetic cells (CD133-) were collected flushing the column into 50 ml tube. The column was separated from multistand and washed with PBS-BE another 50 ml tube for collecting of labeled magnetic cells (CD133+). BothCD133(+) and CD133(−) cells from each cell lines were centrifuged at 1000 rpm for 5 min, the supernatants were removed and cells were cultured in different flasks that contained DMEM 10% fetalbovine serum (FBS, Capricorn scientific,FBS-HI-12B),1% L-glutamine (Gibco™, A2916801) and %1 penicillin-streptomycine (Gibco™, 15140122) at 37 °C temperature in 5% CO2 incubators until becoming 80% confluent. After then they were passage into study groups and remaining cells were stocked in freezing medium (FBS:DMSO = 9:1) at −80 °C.
2.5. Immunohistochemical analysis Caspase-3, Ki-67, Bcl-2 and VGEF distributions were performed using indirect immunoperoxidase staining. CD133(+) and CD133(−) cells from Colo320, Colo741 and HCT116 colon carcinoma cell lines (5 × 103 cells in each well) were incubated with 2 mg/kg tramadol for 24 h at37°C incubator with humidified atmosphere of 5% CO2. They were washed with sterile PBS (Lonza, BE17-516F) solution, and fixed with 4% paraformaldehyde (pH 7.4 - Merck, TP704404-415) at room temperature for 30 min. Following fixation, PBS washing was performed 3 times in 15 min intervals. For permeability purpose, cells were treated with 0,1% Triton®-X-100 (AppliChem, A4977-0100) on ice for 15 min and washed with PBS three times in five minute intervals. Cells were incubated in 3% hydrogen peroxide (H2O2, Merck, K31355100 303) for 10 min and PBS washing was performed for three times at 5 minute intervals. The cells were blocked in blocking solution (Novex Life Technologies, 859,043)for 1 h at room temperature and then incubated with anti-caspase-3 (GeneTex, GTX78090), anti-Ki-67 (NeoMarkers, RB-081-A1), anti-Bcl-2 (Delta Biolabs, DB001) and antiVGEF (Santa Cruz Biotechnology, SC-7269) primary antibodies(150 μl) for overnight at +4 °C. The cells were washed next morning three times with PBS in 5 minute intervals. Biotinylated secondary antibody and streptavidin-horsedish peroxidase (Novex Life Technologies, 859043) 30 min for every incubation. The cells were washed with PBS three times for 5 min between two solutions. Cells were stained for 5 min with diaminobenzidine (DAB, Millipore IHC Select®, 71898) in order to visualize immunoreactivity. The cells were washed with distilled water then stained with Mayer's hematoxylin for 1 min and washed with distilled water again and mounted with medium (Spring Bioscience, DMM-125). Light microscope (BX43, Olympus) was used for examination and photography. Positive and negative control staining were performed for detection of specify of immunoreactivity during immunocytochemistry. Experiments were performed three times for every group and evaluations were performed by two independent researchers. Immunoreactivities were evaluated Image Pro Plus 5.1 analyses program according to brown precipitate area measure in each slide from each groups.
2.3. Characterization of cancer stem cells Indirect immunocytochemistry technique was applied to characterize CD133(+) cancer stem cells which were obtained from three different colon cancer cell lines. The Colo320, Colo741 and HCT116 CD133(+) and CD133(−) cancer cells were fixed in 4% paraformaldehyde (pH:7.4, Merck, TP704404-415) for 30 min at room temperature. Then, they were washed with PBS and incubated with 3% hydrogen peroxide (H2O2, Merck, K31355100 303) for 30 min. All CD133(+) and CD133(−) cancer cells were re-washed with PBS and they were incubated with blocking solution (Novex Life Technologies, 859043) for 1 h at room temperature. The cells were then incubated for overnight at +4 °C with anti-CD133 primary antibody (1:50, St John's Labs, STJ20168). After washing with PBS three times, they were stained with biotinylated secondary antibody and streptavidin-horsedish peroxidase (Novex Life Technologies, 859043) 30 min for every incubation. The cells were washed with PBS three times for 5 min between two solutions. Cells were stained for 5 min with diaminobenzidine (DAB, Millipore IHC Select®, 71898) in order to visualize immunoreactivity. The cells were washed with distilled water then stained with Mayer's hematoxylin for 1 min and washed with distilled water again and mounted with medium (Spring Bioscience, DMM-125). PBS containing 0.1% BSA (Bovine Serum Albumin) was added instead of primary antibody for negative control.
2.6. Statistical analysis Intensity of antibodies were compared using the InStat Statistical Analyses Software (Graphpad Software, La Jolla, CA) values were given mean ± standard deviation (SD) and P values were < 0.05 considered significant. 3. Results 3.1. Cell culture In the present study, we cultured three different colon cancer cells, including Colo320 (primary colon carcinoma cells), Colo741 (metastatic colon adenocarcinoma cells) and HCT116 (primary colon adenocarcinoma cells). Colo320 cells were semi adhesive, rounded and refractile morphology (Fig. 1A). Colo741 cells have fibroblast-like cell
2.4. Analysis of tramadols cytotoxic effect on cells MTT (mitochondria-targeted therapeutics) technique was used to detect tramadol's cytotoxic effect on cells in the study groups. After becoming confluent cellular lines in CD133(+) and CD133(−) cells 2
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COLO320
A
COLO741
HCT116
C
B
Fig. 1. Colo320 (A), Colo741 (B) and HCT116 (C) cells' morphology on 5th culture day. Scale Bars: 100 μm.
A
B
C
D
E
F
Fig. 2. CD133(+) (A–C) and CD133 (−) (D–F) cells morphology on 5th day of culture that were obtained from Colo320 (A, D), Colo741 (B, E) and HCT116 (C, F) cells. Scale Bars: 100 μm.
A
B
C
D
E
F
Fig. 3. CD 133 immunoreactivity of the CD133(+) (A–C) and CD133 (−) (D–F) cells that were obtained from Colo320 (A, D), Colo741 (B, E) and HCT116 (C, F) cells Scale Bars: 10 μm.
immunoreactivity was negative or weak in CD133(−) cells of Colo320, Colo741 and HCT116 (Fig. 3D, E, F).
type and adherent cells (Fig. 1B). HCT116 cells have also fusiform shape and adherent properties during culture (Fig. 1C). CD133(+) cell were separated easily with magnetic activated cell sorting system and they were cultured separately from CD133(−) cells (Fig. 2). After dissociation of CD133+ and CD 133- cells were well-defined morphology and become confluent at first week of the culture (Fig. 2). Colo320, Colo741 and HCT166 CD133(+) cells were stained positively with immunohistochemistry and the purity of CD133(+)was detectable brown staining in all type of cell lines (Fig. 3A, B, C), CD133
3.2. Determination of cytotoxic effect of tramadol with MTT assay The cytotoxic effect of Tramadol on CD133(+) and CD133(−) Colo320, Colo741 and HCT116 cells was evaluated with MTT assay, therefore the cells were treated with tramadol at concentrations 1 mg/ kg, 1.5 mg/kg and 2 mg/kg for 24 h as shown Fig. 4, the IC50-value on 3
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0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 COLO 320 CD133(+)
COLO 320 CD133(-)
CULTURE MEDIUM
COLO 741 CD133(+) CONTROL
COLO 741 CD133(-) 1 mg/kg
HCT 116 CD133(+) 1,5 mg/kg
HCT 116 CD133(-) 2 mg/kg
Fig. 4. MTT analysis of CD133(+) and CD133 (−) cells that were obtained from tramadol administration to Colo320, Colo741 and HCT116 cells.
cells (Fig. 5I, J, K, L). Ki-67 immunoreactivity was 22% moderately positive in Colo320 CD133(+) cells and strongly positive cells were detected in control group of Colo320 CD133(+) cells (Fig. 6A, C). Ki-67 immunoreactivity was weak or moderate in control and tramadol group in CD133(−) Colo320 cells and there was no difference between groups (Fig. 6B, D). Ki-67 immunoreactivity was increased following tramadol administration in both Colo741 CD133(+) and CD133(−) cells compared with control group of Colo741 CD133(+) and CD133(−) cells (Fig. 6E, F, G, H). Ki-67 immunoreactivity was observed in HCT116 cells both in control and tramadol treated groups and there were rarely Ki-67 negative cells in tramadol group of HCT116 cells (Fig. 6I, J, K, L); however, Ki-67 expression was less affected when compared with Colo320 group. It was observed that Bcl-2 immunoreactivity was less positive in Colo320, Colo741 and HCT116 CD133(+) cells compared with control group (Fig. 7A, C, E, G, I, K). Bcl-2 expression was similar in CD133 (−)
all type of cells was calculated to be 2 mg/kg.
3.3. Induction of apoptosis, proliferation and angiogenesis in CD133(+) and CD133(−) cells by tramadol treatment To check whether tramadol has a role in the apoptosis of cancer stem cells (CD133+ cells), distribution of caspase-3 was evaluated using indirect immunocytochemistry. Caspase-3 immunoreactivity was higher in Colo320 CD133(+) cells when compared with control group and HCT 116 cells after tramadol administration (Fig. 5A, C, I, K). In CD133(−) cells, caspase-3 immunoreactivity was present in Colo320, Colo741 and HCT116 cells but it was less then CD133(+) cells following tramadol administration (Fig. 5E, F, G, H). In HCT116 cells, caspase-3 immunoreactivity was moderate and strong positive in CD133(+) and CD133(−) cells following tramadol administration. It was moderately positive in control group of HCT116 cells and there were rarely strong positive cells in CD133(+) and CD133(−) HCT116
TRAMADOL(+) CD133(+)
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CONTROL
CD133(-)
B
CD133(+)
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CD133(-)
D Colo320
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G
H Colo741
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L HTC116
Fig. 5. Caspase-3 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L) CD133(+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm. 4
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TRAMADOL(+) CD133(+)
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CONTROL
CD133(-)
B
CD133(+)
C
CD133(-)
D Colo320
E
F
G
H Colo741
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L HTC116
Fig. 6. Ki-67 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L). CD133 (+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm.
Colo320 CD133(−)and HCT CD133(+) cells after tramadol administration compared with control groups (Graphic 1). Bcl-2 expression was similar HCT199 cells in both control and tramadol groups, it was increased in Colo320 CD133(+) and Colo 741 CD133(−) cells after tramadol administration (Graphic 1). VEGF expression was similar in all groups (Graphic 1).
Colo320 cells both in tramadol and control groups. Bcl-2 expression was less in HCT116 CD133(−) tramadol group then control group (Fig. 7B, D, F, G, I, K). VEGF immunoreactivity was more in Colo320 and Colo741 CD133(+) cells compared to CD133(−) Colo320 and Colo741 cells following tramadol administration. In HCT116 cells there was no difference between CD133(+) andCD133(−) cells and VEGF expression was weak (Fig. 8). All immunocytochemical analyses were evaluated Image ProPlus 5.1 program and staining area in control and tramadol groups were given in Table 1, comparation of the values after statistically were given Graphic 1. After this analyses,caspase-3 immunoreactivity was increased in both CD133(+) and CD133(−) cells from all groups after tramadol administration (Table 1, Graphic 1). Ki-67 expression was also similar in Colo320 CD133(+) cells, Colo741 CD133(+), CD133(−) cells and HCT199 CD133(−) cells, however, it was more detectable
4. Discussion The main finding of this study is tramadol's triggering effect on apoptosis in Colo320 colon cancer stem cells. The effect on apoptosis is proposed by the increase in caspase-3 activity, the decrease in bcl-2 and ki-67 immunoreactivity in Colo320 CD133(+) cells. However, in Colo320 CD133(−) cells caspase-3 immunoreactivity was less then Colo320 CD133(+) cells. The difference of caspase-3 immunoreactivity between control group and Colo320 CD133(−) was minimum. These
TRAMADOL(+) CD133(+)
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CONTROL
CD133(-)
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CD133(+)
CD133(-)
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C
Colo320
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H Colo741
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Fig. 7. bcl-2 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L). CD133 (+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm. 5
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TRAMADOL(+) CD133(+)
A
and δ opioid receptors. They also found that opioid agonists inhibit cell proliferation in a dose dependent manner. Hatsukari et al. showed that morphine caused apoptosis in breast cancer cells (Hatsukari et al., 2007). In that study they compared lung cancer cells with breast cancer cells. They concluded that different types of tumor response to morphine may occur regarding apoptosis. On the other hand, Iglesias et al. showed that morphine inhibits apoptosis in neuroblastoma cells (Igleisas et al., 2003). They stated that endogenous opioids may play a neurotrophic role during development in neuronal cells. These findings are similar with our results in different type of colorectal cancer cells regarding tumor development and apoptosis. Angiogenesis plays major role in cancer mechanisms, cancer micro environment and crucial for cancer spread and continuity. Our findings suggest that VEGF expression was not different in HCT116 CD133(+) and CD133(−) cells after tramadol. However, VEGF expression in Colo 320 and Colo741 CD133(+) were increased compared to control group which suggests that tramadol may cause vascular formation. VEGF signaling pathway plays a major role in tumor angiogenesis and VEGF is the most potent angiogenic growth factor. It plays major role for cancer growth. Morphine can influence angiogenesis for successful development of tumor. Different concentrations of morphine have different effects on angiogenesis. Pasi et al. observed reduction of blood vessel proliferation (Pasi et al., 1991). They concluded that involvement of morphine may affect proliferation of vascular endothelial cells compared with control group in chicken chorioallantoic membrane model. At lower concentrations different opioids have antiangiogenetic effects. Karaman et al. concluded that tramadol has some anti-angiogenic effects at 10 μM concentration compared with 1 and 0.1 μM (Karaman et al., 2017). They also observed that codeine significantly inhibited angiogenesis at 10 μM and morphine is more potent anti-angiogenic in a chorioallantoic membrane model. There are some studies that show effects of tramadol on tumor cell behavior. Xia et al. showed that tramadol inhibits proliferation, migration and invasion in breast cancer cells up to 2 μM dose. The main pathway was α2-adrenoceptor signaling (Xia et al., 2016a). In another study Xia et al. demonstrated that tramadol regulates proliferation, migration and invasion in lung cancer cells. They also concluded that this effect was due to elevation of Phosphatase and tensin homolog and inactivation of phosphoinositide 3-kinase/protein kinase B signaling (Xia et al., 2016b). However, some studies found that morphine may promote angiogenesis in different models. Gupta et al. showed that morphine stimulates endothelial proliferation and survival at medically relevant concentrations of morphine in a breast cancer model. This effect was observed both invivo and invitro (Gupta et al., 2002). Farooqi et al. demonstrated that chronic morphine therapy induced angiogenesis in a
CD133(-)
B Colo320
C
D Colo741
E
F HTC116
Fig. 8. VEGF distribution in Colo320 (A, B), Colo741 (C, D) and HCT116 (E, F). CD133 (+) (A, C, E) and CD133(−) (B, D, F) cells after tramadol administration. Scale: 100 μm.
findings suggest that tramadol triggers apoptosis in Colo320 CD133(+) cells. The increase in caspase-3 immunoreactivity in HCT116 CD133(+) cells was minimum. This may suggest that tramadol may have different effects on individuals who have different types of colon cancer. Tramadol decreased Ki-67 and bcl-2 expressions in HCT116 CD133(+) and CD133(−) cells. The decrease in bcl-2 expression may cause cancer cell viability. The similarity of bcl-2 expression in Colo741 CD133(−) and Colo741 CD133(+) cells may cause stronger survival signal. This may contribute to cancer cells metastasis to new tissues. Opioids have double-sided effects on tumor regulation and development. Mechanisms include metastasis, angiogenesis, immunosuppression and inflammation. Most of the studies included morphine in various doses and concentrations. In the present study we preferred tramadol which is also used widely in cancer pain treatment and exerts its clinical effect on opioid receptors. Opioid receptors are considered to be involved in cancer cell behavior. Hatzoglou et al. demonstrated that morphine receptor agonists inhibit the proliferation of breast cancer (Hatzoglou et al., 1996). They concluded that morphine and other receptor agonists inhibit the proliferation of breast cancer cell through κ
Table 1 Immunocytochemical analyses of caspase-3, Ki-67, Bcl-2 and VEGF in control and tramadol groups were given mean ± SD values after Image ProPlus 5.1 analyses. Colo320
Control Caspase-3 Ki-67 Bcl-2 Tramadol Caspase-3 Ki-67 Bcl-2 VEGF
Colo741
HCT116
CD133(+)
CD133(−)
CD133(+)
CD133(−)
CD133(+)
CD133(−)
2230.4 ± 14.71 2525.24 ± 35.02 1993.07 ± 4.34
1371.17 ± 29.93 1527.7 ± 17,96 2414.34 ± 20.27
1878.08 ± 25.57 2312.34 ± 17.45 1943.16 ± 12.95
1608.27 ± 11.70 2514.48 ± 20.48 668.09 ± 11.44
1566.62 ± 30.58 620.20 ± 11.60 2306.24 ± 8.83
2352.25 ± 22.98 2526.19 ± 37,03 2133.1 ± 15.70
2500.06 ± 0.08 2147.99 ± 18.36 2543.66 ± 12.25 2402.07 ± 2.92
2502.30 ± 3.25 2212.41 ± 17.54 1247.08 ± 18.50 2204.09 ± 5.78
2318.47 ± 26.12 2547.44 ± 13.35 1754.08 ± 14,26 2544.15 ± 5.87
2433.57 ± 31.92 2210.08 ± 14.26 2546.09 ± 8.61 2533.16 ± 4.47
1944.22 ± 17.28 2506.0 ± 8.49 2100.2 ± 0.28 1028.10 ± 11.45
1326.07 ± 31.21 2213.10 ± 15.69 2424.17 ± 11.56 1904.09 ± 5.79
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Caspase-3
A 3000
> p>0.05
2500
>
p>0.05
>
> >
> 2000
1500
1000
500
0 Colo320 CD133(+) Colo320 CD133(-) Colo741 CD133(+) Colo741 CD133(-) HCT116 CD133(+) HCT116 CD133(-)
CONTROL
TRAMADOL
>
>
Graphic 1. After statistically analyses of caspase-3 (A), Ki-67 (B), Bcl-2 (C) and VEGF (D) values in control and tramadol groups were given. The non-significant (P > 0.05) comparations were indicated on the graphics. Rest of the values were compared with each other, they were significant (P < 0.05).
breast cancer model in A/J mice (Farooqui et al., 2007). They concluded that chronic use of morphine stimulates cyclooxygenase-2 (COX2) which leads to increased tumor angiogenesis, growth, metastasis and mortality. Chen et al. demonstrated that morphine stimulates angiogenesis in mouse retinal endothelial cells (Chen et al., 2006). Also, Mathew et al. showed that μ receptor regulates tumor growth and metastasis in lung carcinoma. They stated that μ receptor agonists reduced metastasis and silenced Lewis lung carcinoma (Mathew et al., 2011). Our findings are comparable with the literature regarding tumor angiogenesis in various types of colorectal cancer cells. Our findings show that tramadol effected cell behavior in different types of colorectal cancer cells. Tramadol may affect cancer cell viability and metastasis. Our results supported that the effects of tramadol on cancer microenvironments will be need further studies.
Chen, C., Farooqui, M., Gupta, K., 2006. Morphine stimulates vascular endothelial growth factor-like signaling in mouse retinal endothelial cells. Curr. Neurovasc. Res. 3, 171–180. Farooqui, M., Li, Y., Rogers, T., Poonawala, T., Griffin, R.J., Song, C.W., Gupta, K., 2007. COX-2 inhibitor celecoxib prevents chronic morphine induced promotion of angiogenesis, tumour growth, metastasis and mortality, without compromising analgesia. Br. J. Cancer 97, 1523–1531. Gupta, K., Kshirsagar, S., Chang, L., Schwartz, R., 2002. Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth. Cancer Res. 62, 4491–4498. Hatsukari, I., Hitosugi, N., Ohno, R., Hashimito, K., 2007. İnduction of apoptosis by morphine in human tumor cell lines in vitro. Anticancer Res. 27, 857–864. Hatzoglou, A., Bakageorgou, E., Castanas, E., 1996. The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors. Eur. J. Pharmacol. 296, 199–207. Igleisas, M., Segura, M.F., Comella, J., Olmos, G., 2003. μ-Opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase. Neuropharmacology 44, 482–492. Karaman, H., Tüfek, A., Karaman, E., Tokgöz, O., 2017. Opioids inhibit angiogenesis in a chorioallantoic membrane model. Pain Physician 20, SE11–SE21. Kovelowski, C.J., Raffa, R.B., Porreca, F., 1998. Tramadol and its enantiomers differentially supress c-fos-like immunoreactivity in rat brain and spinal cord following acute noxiusstimulus. Eur. J. Pain 2, 211–219. Mathew, B., Lennon, F.E., Siegler, J., et al., 2011. Novel role of the mu opioid receptor in lung cancer progression a laboratory study. Anesth. Analg. 112, 558–567. McCartney, Colin J.L., Choi, S., 2017. Analgesic adjuvants in the peripheral nervous system. In: Hadjic, A. (Ed.), Hadzic's Textbook of Regional Anesthesia and Acute Pain Management. Mc Graw Hill Education, pp. 150. Pasi, A., Qu, B.X., Steiner, R., Senn, H.J., Bar, W., Messiha, F.S., 1991. Angiogenesis modulation with opioids. Gen. Pharmacol. 22, 1077–1079. Xia, M., Tong, J.H., Zhou, Z.Q., Duan, M.L., 2016a. Tramadol inhibits proliferation, migration and invasion via alpha 2 adrenoceptor signaling in breast cancer cells. Eur. Rev. Med. Pharmacol. Sci. 20, 157–165. Xia, M., Tong, J.H., Ji NN Duan, M.L., Xu, J.G., 2016b. Tramadol regulates proliferation, migration and invasion via PTEN/PI3K/AKT signaling in lung adenocarcinoma cells. Eur. Rev. Med. Pharmacol. Sci. 20, 2573–2580. Zhang, X.Y., Liang, Y.X., Yan, Y., Dai, Z., Chu, H.C., 2018. Morphine: double-faced roles in the regulation of tumor development. ClinTransl Oncol 20, 808–814.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that there is no conflict of interests. References Afshamani, B., Cabot, P., Parat, M.O., 2011. Morphine and tumor growth and metastasis. Cancer Metastasis Rev. 30, 225–238.
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Research paper
The effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells lines
T
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Uğur Özgürbüza, , Sema Gencürb, Feyzan Özdal Kurt (PhD)c, Murat Özkalkanlıa, H. Seda Vatansever (MD, PhD)b,d İzmir Atatürk Research and Training Hospital, Dept. of Anesthesiology and Reanimation, Turkey Manisa Celal Bayar University, Dept. of Histology and Embryology, Turkey c Manisa Celal Bayar University, Faculty of Science & Letters, Dept. of Biology, Turkey d Near East University, Experimental Research Center of Health (DESAM), Mersin 10, Turkey a
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Opioids Tramadol Cell culture Colon cancer
Opioids are widely used in the treatment of cancer related pain. They mainly exert their effects on opioid receptors. The most common opioid in the treatment of pain is morphine. Previous studies show that they may have effects on cancer cell behavior. These may include apoptosis, angiogenesis, invasion, inflammation and immune reactions. Tramadol, also an opioid is widely used in the treatment of cancer pain and is not well studied in cancer behavior. We aimed to investigate the effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells. We used Colo320 (ATCC, CCL-220), Colo741 (ECACC, 93052621) and HCT116 (ATCC, CCL-247) colon cancer cell lines. CD133 was considered colon cancer stem cell marker and used to sort CD133+ and CD133− cells by magnetic cell sorting. MTT (mitochondria-targeted therapeutics) technique was used to detect tramadol's cytotoxic effect on cells in the study groups. Cells were treated with 1 mg/kg, 1.5 mg/kg and 2 mg/kg tramadol for 24 h at 37 °C and 5% CO2.Caspase-3, Ki-67, Bcl-2 and VGEF distributions were performed using indirect immunoperoxidase staining for immunohistochemical analysis. The study showed that tramadol has triggering effect on apoptosis in Colo320 colon cancer stem cells.
1. Introduction Opioids are considered the “gold standard” for the management of severe pain associated with cancer. They mainly act directly on the central nervous system and exert their effects on various opioid receptors. Morphine may also be responsible for some side effects (addiction, tolerance, respiratory depression, immunosuppression, and constipation). It's been debated that opioids have some effects on tumor development and metastasis (Afshamani et al., 2011; Zhang et al., 2018). Morphine has both inhibitor and promotary effects on cancer growth. There are some complex mechanisms which morphine affects tumor cells. The proposed mechanisms of action on tumor growth for morphine are apoptosis, angiogenesis, invasion, inflammation and immune reactions. Signaling pathways may also be involved in secondary effects of morphine. Tramadol, mainly acting on central nervous system, serotonergic and noradrenergic nociception. Its metabolite Odesmethyltramadol acts on the μ-opioid receptor (McCartney and Choi, 2017). Tramadol is used to treat both acute and chronic pain, especially
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in the postoperative period for cancer patients. While morphine is widely studied in tumor growth there are limited data for tramadol effects on tumor growth. There is some literature that investigates the effects of tramadol on angiogenesis and immunoreactivity. However, none of the studies investigated the effects of tramadol on cancer stem cells (Karaman et al., 2017; Kovelowski et al., 1998). The identification of stem cells may have a significant effect on research, prevention, and therapy. The aim of the present study is to investigate the effects of tramadol on cancer stem cells and metabolic changes in colon carcinoma cells. 2. Material and method 2.1. Colo320, Colo741 and HCT116 Cell Culture In the present study, Colo320 (ATCC, CCL-220), Colo741 (ECACC, 93052621) and HCT116 (ATCC, CCL-247) colon cancer cell lines were used. Colo320, Colo741 and HCT116 cells were cultured in RPMI-1640
Corresponding author. E-mail address: [email protected] (U. Özgürbüz).
https://doi.org/10.1016/j.gene.2019.144030 Received 24 January 2019; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 04 August 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.
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were cultured in 96 well culture plate. Every well was included 3 × 103 cells. And they were incubated at 37 °C and 5% CO2 for 24 h. The cells were treated with 1 mg/kg, 1.5 mg/kg and 2 mg/kg tramadol for 24 h at 37 °C and 5% CO2. For the control group, every cell line was treated only with culture medium. The culture was applicate three consecutive times. At the end of incubation, PBS solution which included 5 mg/mL MTT solution (Glentham Life Sciences, 471OVO) was diluted at 1:10 and added each well and incubated for 4 h at 37 °C. Following incubation, 50 μl dimethylsulphoxide (DMSO, Sigma, D2650) was added and measurements were performed at 450–690 nm absorbance spectrophotometry (BioTek Instruments Inc., ELX800UV, USA).
medium which supplemented 10% fetal bovine serum (FBS, Capricorn scientific, FBS-HI-12B), 1% penicillin-streptomycine (Gibco™, 15140122) and 1% L-glutamine (Gibco™, A2916801). The cell lines were divided into study groups after becoming 80% confluent and other cells were stocked in freezing medium [FBS:DMSO (Dimethyl sulfoxide, Sigma-Aldrich, D8418) = 9:1] at −80 °C. 2.2. Isolation of cancer stem cells CD133 was considered colon cancer stem cell marker and used to sort CD133+ and CD133- cells by magnetic cell sorting (MACS). The CD133 immune magnetic bead separation kit was used (Miltenyi Biotech). The confluent of Colo320, Colo741 and HCT116 cells were incubated for 8–10 min with 0.5% Trypsin-EDTA solution (Biochrom, L2133) for digest and they were centrifuged for 5 min at 1000 rpm. The supernatants were discarded and the cells were collected re-suspending with 60 μl phosphate buffer solution containing 0.5% BSA and 0.08% EDTA (PBS-BE). The cells were then incubated CD133IgGs (Miltenyi Biotech) on ice for 15 min. They were then centrifuged with 1 ml PBSBEat 1000 rpm for 5 min and cells were resuspended with 500 μl PBSBE. MiniMACs system was placed in a sterile container and miniMACS magnet was fixed on the MACS multistand and washed with 500 μl PBSBE. The cell suspension for each cell lines was poured into the column reservoir, and unlabeled nonmagnetic cells (CD133-) were collected flushing the column into 50 ml tube. The column was separated from multistand and washed with PBS-BE another 50 ml tube for collecting of labeled magnetic cells (CD133+). BothCD133(+) and CD133(−) cells from each cell lines were centrifuged at 1000 rpm for 5 min, the supernatants were removed and cells were cultured in different flasks that contained DMEM 10% fetalbovine serum (FBS, Capricorn scientific,FBS-HI-12B),1% L-glutamine (Gibco™, A2916801) and %1 penicillin-streptomycine (Gibco™, 15140122) at 37 °C temperature in 5% CO2 incubators until becoming 80% confluent. After then they were passage into study groups and remaining cells were stocked in freezing medium (FBS:DMSO = 9:1) at −80 °C.
2.5. Immunohistochemical analysis Caspase-3, Ki-67, Bcl-2 and VGEF distributions were performed using indirect immunoperoxidase staining. CD133(+) and CD133(−) cells from Colo320, Colo741 and HCT116 colon carcinoma cell lines (5 × 103 cells in each well) were incubated with 2 mg/kg tramadol for 24 h at37°C incubator with humidified atmosphere of 5% CO2. They were washed with sterile PBS (Lonza, BE17-516F) solution, and fixed with 4% paraformaldehyde (pH 7.4 - Merck, TP704404-415) at room temperature for 30 min. Following fixation, PBS washing was performed 3 times in 15 min intervals. For permeability purpose, cells were treated with 0,1% Triton®-X-100 (AppliChem, A4977-0100) on ice for 15 min and washed with PBS three times in five minute intervals. Cells were incubated in 3% hydrogen peroxide (H2O2, Merck, K31355100 303) for 10 min and PBS washing was performed for three times at 5 minute intervals. The cells were blocked in blocking solution (Novex Life Technologies, 859,043)for 1 h at room temperature and then incubated with anti-caspase-3 (GeneTex, GTX78090), anti-Ki-67 (NeoMarkers, RB-081-A1), anti-Bcl-2 (Delta Biolabs, DB001) and antiVGEF (Santa Cruz Biotechnology, SC-7269) primary antibodies(150 μl) for overnight at +4 °C. The cells were washed next morning three times with PBS in 5 minute intervals. Biotinylated secondary antibody and streptavidin-horsedish peroxidase (Novex Life Technologies, 859043) 30 min for every incubation. The cells were washed with PBS three times for 5 min between two solutions. Cells were stained for 5 min with diaminobenzidine (DAB, Millipore IHC Select®, 71898) in order to visualize immunoreactivity. The cells were washed with distilled water then stained with Mayer's hematoxylin for 1 min and washed with distilled water again and mounted with medium (Spring Bioscience, DMM-125). Light microscope (BX43, Olympus) was used for examination and photography. Positive and negative control staining were performed for detection of specify of immunoreactivity during immunocytochemistry. Experiments were performed three times for every group and evaluations were performed by two independent researchers. Immunoreactivities were evaluated Image Pro Plus 5.1 analyses program according to brown precipitate area measure in each slide from each groups.
2.3. Characterization of cancer stem cells Indirect immunocytochemistry technique was applied to characterize CD133(+) cancer stem cells which were obtained from three different colon cancer cell lines. The Colo320, Colo741 and HCT116 CD133(+) and CD133(−) cancer cells were fixed in 4% paraformaldehyde (pH:7.4, Merck, TP704404-415) for 30 min at room temperature. Then, they were washed with PBS and incubated with 3% hydrogen peroxide (H2O2, Merck, K31355100 303) for 30 min. All CD133(+) and CD133(−) cancer cells were re-washed with PBS and they were incubated with blocking solution (Novex Life Technologies, 859043) for 1 h at room temperature. The cells were then incubated for overnight at +4 °C with anti-CD133 primary antibody (1:50, St John's Labs, STJ20168). After washing with PBS three times, they were stained with biotinylated secondary antibody and streptavidin-horsedish peroxidase (Novex Life Technologies, 859043) 30 min for every incubation. The cells were washed with PBS three times for 5 min between two solutions. Cells were stained for 5 min with diaminobenzidine (DAB, Millipore IHC Select®, 71898) in order to visualize immunoreactivity. The cells were washed with distilled water then stained with Mayer's hematoxylin for 1 min and washed with distilled water again and mounted with medium (Spring Bioscience, DMM-125). PBS containing 0.1% BSA (Bovine Serum Albumin) was added instead of primary antibody for negative control.
2.6. Statistical analysis Intensity of antibodies were compared using the InStat Statistical Analyses Software (Graphpad Software, La Jolla, CA) values were given mean ± standard deviation (SD) and P values were < 0.05 considered significant. 3. Results 3.1. Cell culture In the present study, we cultured three different colon cancer cells, including Colo320 (primary colon carcinoma cells), Colo741 (metastatic colon adenocarcinoma cells) and HCT116 (primary colon adenocarcinoma cells). Colo320 cells were semi adhesive, rounded and refractile morphology (Fig. 1A). Colo741 cells have fibroblast-like cell
2.4. Analysis of tramadols cytotoxic effect on cells MTT (mitochondria-targeted therapeutics) technique was used to detect tramadol's cytotoxic effect on cells in the study groups. After becoming confluent cellular lines in CD133(+) and CD133(−) cells 2
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COLO320
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COLO741
HCT116
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Fig. 1. Colo320 (A), Colo741 (B) and HCT116 (C) cells' morphology on 5th culture day. Scale Bars: 100 μm.
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B
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D
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Fig. 2. CD133(+) (A–C) and CD133 (−) (D–F) cells morphology on 5th day of culture that were obtained from Colo320 (A, D), Colo741 (B, E) and HCT116 (C, F) cells. Scale Bars: 100 μm.
A
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Fig. 3. CD 133 immunoreactivity of the CD133(+) (A–C) and CD133 (−) (D–F) cells that were obtained from Colo320 (A, D), Colo741 (B, E) and HCT116 (C, F) cells Scale Bars: 10 μm.
immunoreactivity was negative or weak in CD133(−) cells of Colo320, Colo741 and HCT116 (Fig. 3D, E, F).
type and adherent cells (Fig. 1B). HCT116 cells have also fusiform shape and adherent properties during culture (Fig. 1C). CD133(+) cell were separated easily with magnetic activated cell sorting system and they were cultured separately from CD133(−) cells (Fig. 2). After dissociation of CD133+ and CD 133- cells were well-defined morphology and become confluent at first week of the culture (Fig. 2). Colo320, Colo741 and HCT166 CD133(+) cells were stained positively with immunohistochemistry and the purity of CD133(+)was detectable brown staining in all type of cell lines (Fig. 3A, B, C), CD133
3.2. Determination of cytotoxic effect of tramadol with MTT assay The cytotoxic effect of Tramadol on CD133(+) and CD133(−) Colo320, Colo741 and HCT116 cells was evaluated with MTT assay, therefore the cells were treated with tramadol at concentrations 1 mg/ kg, 1.5 mg/kg and 2 mg/kg for 24 h as shown Fig. 4, the IC50-value on 3
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0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 COLO 320 CD133(+)
COLO 320 CD133(-)
CULTURE MEDIUM
COLO 741 CD133(+) CONTROL
COLO 741 CD133(-) 1 mg/kg
HCT 116 CD133(+) 1,5 mg/kg
HCT 116 CD133(-) 2 mg/kg
Fig. 4. MTT analysis of CD133(+) and CD133 (−) cells that were obtained from tramadol administration to Colo320, Colo741 and HCT116 cells.
cells (Fig. 5I, J, K, L). Ki-67 immunoreactivity was 22% moderately positive in Colo320 CD133(+) cells and strongly positive cells were detected in control group of Colo320 CD133(+) cells (Fig. 6A, C). Ki-67 immunoreactivity was weak or moderate in control and tramadol group in CD133(−) Colo320 cells and there was no difference between groups (Fig. 6B, D). Ki-67 immunoreactivity was increased following tramadol administration in both Colo741 CD133(+) and CD133(−) cells compared with control group of Colo741 CD133(+) and CD133(−) cells (Fig. 6E, F, G, H). Ki-67 immunoreactivity was observed in HCT116 cells both in control and tramadol treated groups and there were rarely Ki-67 negative cells in tramadol group of HCT116 cells (Fig. 6I, J, K, L); however, Ki-67 expression was less affected when compared with Colo320 group. It was observed that Bcl-2 immunoreactivity was less positive in Colo320, Colo741 and HCT116 CD133(+) cells compared with control group (Fig. 7A, C, E, G, I, K). Bcl-2 expression was similar in CD133 (−)
all type of cells was calculated to be 2 mg/kg.
3.3. Induction of apoptosis, proliferation and angiogenesis in CD133(+) and CD133(−) cells by tramadol treatment To check whether tramadol has a role in the apoptosis of cancer stem cells (CD133+ cells), distribution of caspase-3 was evaluated using indirect immunocytochemistry. Caspase-3 immunoreactivity was higher in Colo320 CD133(+) cells when compared with control group and HCT 116 cells after tramadol administration (Fig. 5A, C, I, K). In CD133(−) cells, caspase-3 immunoreactivity was present in Colo320, Colo741 and HCT116 cells but it was less then CD133(+) cells following tramadol administration (Fig. 5E, F, G, H). In HCT116 cells, caspase-3 immunoreactivity was moderate and strong positive in CD133(+) and CD133(−) cells following tramadol administration. It was moderately positive in control group of HCT116 cells and there were rarely strong positive cells in CD133(+) and CD133(−) HCT116
TRAMADOL(+) CD133(+)
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CONTROL
CD133(-)
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CD133(+)
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CD133(-)
D Colo320
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Fig. 5. Caspase-3 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L) CD133(+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm. 4
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TRAMADOL(+) CD133(+)
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CONTROL
CD133(-)
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CD133(+)
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CD133(-)
D Colo320
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H Colo741
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Fig. 6. Ki-67 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L). CD133 (+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm.
Colo320 CD133(−)and HCT CD133(+) cells after tramadol administration compared with control groups (Graphic 1). Bcl-2 expression was similar HCT199 cells in both control and tramadol groups, it was increased in Colo320 CD133(+) and Colo 741 CD133(−) cells after tramadol administration (Graphic 1). VEGF expression was similar in all groups (Graphic 1).
Colo320 cells both in tramadol and control groups. Bcl-2 expression was less in HCT116 CD133(−) tramadol group then control group (Fig. 7B, D, F, G, I, K). VEGF immunoreactivity was more in Colo320 and Colo741 CD133(+) cells compared to CD133(−) Colo320 and Colo741 cells following tramadol administration. In HCT116 cells there was no difference between CD133(+) andCD133(−) cells and VEGF expression was weak (Fig. 8). All immunocytochemical analyses were evaluated Image ProPlus 5.1 program and staining area in control and tramadol groups were given in Table 1, comparation of the values after statistically were given Graphic 1. After this analyses,caspase-3 immunoreactivity was increased in both CD133(+) and CD133(−) cells from all groups after tramadol administration (Table 1, Graphic 1). Ki-67 expression was also similar in Colo320 CD133(+) cells, Colo741 CD133(+), CD133(−) cells and HCT199 CD133(−) cells, however, it was more detectable
4. Discussion The main finding of this study is tramadol's triggering effect on apoptosis in Colo320 colon cancer stem cells. The effect on apoptosis is proposed by the increase in caspase-3 activity, the decrease in bcl-2 and ki-67 immunoreactivity in Colo320 CD133(+) cells. However, in Colo320 CD133(−) cells caspase-3 immunoreactivity was less then Colo320 CD133(+) cells. The difference of caspase-3 immunoreactivity between control group and Colo320 CD133(−) was minimum. These
TRAMADOL(+) CD133(+)
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Colo320
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Fig. 7. bcl-2 distribution after tramadol administration (A, B, E, F, I, J) and control group (C, D, G, H, K, L) in Colo320 (A, B, C, D), Colo741 (E, F, G, H) and HCT116 (I, J, K, L). CD133 (+) (A, C, E, G, I, K) and CD133(−) (B, D, F, H, J, L) cells. Scale: 100 μm. 5
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TRAMADOL(+) CD133(+)
A
and δ opioid receptors. They also found that opioid agonists inhibit cell proliferation in a dose dependent manner. Hatsukari et al. showed that morphine caused apoptosis in breast cancer cells (Hatsukari et al., 2007). In that study they compared lung cancer cells with breast cancer cells. They concluded that different types of tumor response to morphine may occur regarding apoptosis. On the other hand, Iglesias et al. showed that morphine inhibits apoptosis in neuroblastoma cells (Igleisas et al., 2003). They stated that endogenous opioids may play a neurotrophic role during development in neuronal cells. These findings are similar with our results in different type of colorectal cancer cells regarding tumor development and apoptosis. Angiogenesis plays major role in cancer mechanisms, cancer micro environment and crucial for cancer spread and continuity. Our findings suggest that VEGF expression was not different in HCT116 CD133(+) and CD133(−) cells after tramadol. However, VEGF expression in Colo 320 and Colo741 CD133(+) were increased compared to control group which suggests that tramadol may cause vascular formation. VEGF signaling pathway plays a major role in tumor angiogenesis and VEGF is the most potent angiogenic growth factor. It plays major role for cancer growth. Morphine can influence angiogenesis for successful development of tumor. Different concentrations of morphine have different effects on angiogenesis. Pasi et al. observed reduction of blood vessel proliferation (Pasi et al., 1991). They concluded that involvement of morphine may affect proliferation of vascular endothelial cells compared with control group in chicken chorioallantoic membrane model. At lower concentrations different opioids have antiangiogenetic effects. Karaman et al. concluded that tramadol has some anti-angiogenic effects at 10 μM concentration compared with 1 and 0.1 μM (Karaman et al., 2017). They also observed that codeine significantly inhibited angiogenesis at 10 μM and morphine is more potent anti-angiogenic in a chorioallantoic membrane model. There are some studies that show effects of tramadol on tumor cell behavior. Xia et al. showed that tramadol inhibits proliferation, migration and invasion in breast cancer cells up to 2 μM dose. The main pathway was α2-adrenoceptor signaling (Xia et al., 2016a). In another study Xia et al. demonstrated that tramadol regulates proliferation, migration and invasion in lung cancer cells. They also concluded that this effect was due to elevation of Phosphatase and tensin homolog and inactivation of phosphoinositide 3-kinase/protein kinase B signaling (Xia et al., 2016b). However, some studies found that morphine may promote angiogenesis in different models. Gupta et al. showed that morphine stimulates endothelial proliferation and survival at medically relevant concentrations of morphine in a breast cancer model. This effect was observed both invivo and invitro (Gupta et al., 2002). Farooqi et al. demonstrated that chronic morphine therapy induced angiogenesis in a
CD133(-)
B Colo320
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D Colo741
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Fig. 8. VEGF distribution in Colo320 (A, B), Colo741 (C, D) and HCT116 (E, F). CD133 (+) (A, C, E) and CD133(−) (B, D, F) cells after tramadol administration. Scale: 100 μm.
findings suggest that tramadol triggers apoptosis in Colo320 CD133(+) cells. The increase in caspase-3 immunoreactivity in HCT116 CD133(+) cells was minimum. This may suggest that tramadol may have different effects on individuals who have different types of colon cancer. Tramadol decreased Ki-67 and bcl-2 expressions in HCT116 CD133(+) and CD133(−) cells. The decrease in bcl-2 expression may cause cancer cell viability. The similarity of bcl-2 expression in Colo741 CD133(−) and Colo741 CD133(+) cells may cause stronger survival signal. This may contribute to cancer cells metastasis to new tissues. Opioids have double-sided effects on tumor regulation and development. Mechanisms include metastasis, angiogenesis, immunosuppression and inflammation. Most of the studies included morphine in various doses and concentrations. In the present study we preferred tramadol which is also used widely in cancer pain treatment and exerts its clinical effect on opioid receptors. Opioid receptors are considered to be involved in cancer cell behavior. Hatzoglou et al. demonstrated that morphine receptor agonists inhibit the proliferation of breast cancer (Hatzoglou et al., 1996). They concluded that morphine and other receptor agonists inhibit the proliferation of breast cancer cell through κ
Table 1 Immunocytochemical analyses of caspase-3, Ki-67, Bcl-2 and VEGF in control and tramadol groups were given mean ± SD values after Image ProPlus 5.1 analyses. Colo320
Control Caspase-3 Ki-67 Bcl-2 Tramadol Caspase-3 Ki-67 Bcl-2 VEGF
Colo741
HCT116
CD133(+)
CD133(−)
CD133(+)
CD133(−)
CD133(+)
CD133(−)
2230.4 ± 14.71 2525.24 ± 35.02 1993.07 ± 4.34
1371.17 ± 29.93 1527.7 ± 17,96 2414.34 ± 20.27
1878.08 ± 25.57 2312.34 ± 17.45 1943.16 ± 12.95
1608.27 ± 11.70 2514.48 ± 20.48 668.09 ± 11.44
1566.62 ± 30.58 620.20 ± 11.60 2306.24 ± 8.83
2352.25 ± 22.98 2526.19 ± 37,03 2133.1 ± 15.70
2500.06 ± 0.08 2147.99 ± 18.36 2543.66 ± 12.25 2402.07 ± 2.92
2502.30 ± 3.25 2212.41 ± 17.54 1247.08 ± 18.50 2204.09 ± 5.78
2318.47 ± 26.12 2547.44 ± 13.35 1754.08 ± 14,26 2544.15 ± 5.87
2433.57 ± 31.92 2210.08 ± 14.26 2546.09 ± 8.61 2533.16 ± 4.47
1944.22 ± 17.28 2506.0 ± 8.49 2100.2 ± 0.28 1028.10 ± 11.45
1326.07 ± 31.21 2213.10 ± 15.69 2424.17 ± 11.56 1904.09 ± 5.79
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Caspase-3
A 3000
> p>0.05
2500
>
p>0.05
>
> >
> 2000
1500
1000
500
0 Colo320 CD133(+) Colo320 CD133(-) Colo741 CD133(+) Colo741 CD133(-) HCT116 CD133(+) HCT116 CD133(-)
CONTROL
TRAMADOL
>
>
Graphic 1. After statistically analyses of caspase-3 (A), Ki-67 (B), Bcl-2 (C) and VEGF (D) values in control and tramadol groups were given. The non-significant (P > 0.05) comparations were indicated on the graphics. Rest of the values were compared with each other, they were significant (P < 0.05).
breast cancer model in A/J mice (Farooqui et al., 2007). They concluded that chronic use of morphine stimulates cyclooxygenase-2 (COX2) which leads to increased tumor angiogenesis, growth, metastasis and mortality. Chen et al. demonstrated that morphine stimulates angiogenesis in mouse retinal endothelial cells (Chen et al., 2006). Also, Mathew et al. showed that μ receptor regulates tumor growth and metastasis in lung carcinoma. They stated that μ receptor agonists reduced metastasis and silenced Lewis lung carcinoma (Mathew et al., 2011). Our findings are comparable with the literature regarding tumor angiogenesis in various types of colorectal cancer cells. Our findings show that tramadol effected cell behavior in different types of colorectal cancer cells. Tramadol may affect cancer cell viability and metastasis. Our results supported that the effects of tramadol on cancer microenvironments will be need further studies.
Chen, C., Farooqui, M., Gupta, K., 2006. Morphine stimulates vascular endothelial growth factor-like signaling in mouse retinal endothelial cells. Curr. Neurovasc. Res. 3, 171–180. Farooqui, M., Li, Y., Rogers, T., Poonawala, T., Griffin, R.J., Song, C.W., Gupta, K., 2007. COX-2 inhibitor celecoxib prevents chronic morphine induced promotion of angiogenesis, tumour growth, metastasis and mortality, without compromising analgesia. Br. J. Cancer 97, 1523–1531. Gupta, K., Kshirsagar, S., Chang, L., Schwartz, R., 2002. Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth. Cancer Res. 62, 4491–4498. Hatsukari, I., Hitosugi, N., Ohno, R., Hashimito, K., 2007. İnduction of apoptosis by morphine in human tumor cell lines in vitro. Anticancer Res. 27, 857–864. Hatzoglou, A., Bakageorgou, E., Castanas, E., 1996. The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors. Eur. J. Pharmacol. 296, 199–207. Igleisas, M., Segura, M.F., Comella, J., Olmos, G., 2003. μ-Opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase. Neuropharmacology 44, 482–492. Karaman, H., Tüfek, A., Karaman, E., Tokgöz, O., 2017. Opioids inhibit angiogenesis in a chorioallantoic membrane model. Pain Physician 20, SE11–SE21. Kovelowski, C.J., Raffa, R.B., Porreca, F., 1998. Tramadol and its enantiomers differentially supress c-fos-like immunoreactivity in rat brain and spinal cord following acute noxiusstimulus. Eur. J. Pain 2, 211–219. Mathew, B., Lennon, F.E., Siegler, J., et al., 2011. Novel role of the mu opioid receptor in lung cancer progression a laboratory study. Anesth. Analg. 112, 558–567. McCartney, Colin J.L., Choi, S., 2017. Analgesic adjuvants in the peripheral nervous system. In: Hadjic, A. (Ed.), Hadzic's Textbook of Regional Anesthesia and Acute Pain Management. Mc Graw Hill Education, pp. 150. Pasi, A., Qu, B.X., Steiner, R., Senn, H.J., Bar, W., Messiha, F.S., 1991. Angiogenesis modulation with opioids. Gen. Pharmacol. 22, 1077–1079. Xia, M., Tong, J.H., Zhou, Z.Q., Duan, M.L., 2016a. Tramadol inhibits proliferation, migration and invasion via alpha 2 adrenoceptor signaling in breast cancer cells. Eur. Rev. Med. Pharmacol. Sci. 20, 157–165. Xia, M., Tong, J.H., Ji NN Duan, M.L., Xu, J.G., 2016b. Tramadol regulates proliferation, migration and invasion via PTEN/PI3K/AKT signaling in lung adenocarcinoma cells. Eur. Rev. Med. Pharmacol. Sci. 20, 2573–2580. Zhang, X.Y., Liang, Y.X., Yan, Y., Dai, Z., Chu, H.C., 2018. Morphine: double-faced roles in the regulation of tumor development. ClinTransl Oncol 20, 808–814.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that there is no conflict of interests. References Afshamani, B., Cabot, P., Parat, M.O., 2011. Morphine and tumor growth and metastasis. Cancer Metastasis Rev. 30, 225–238.
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