Urologic Oncology: Seminars and Original Investigations ] (2013) ∎∎∎–∎∎∎
Original article
Kidney cancer cells secrete IL-8 to activate Akt and promote migration of mesenchymal stem cells Bi Liang-kuan, M.D., Ph.D.a,1, Zhou Nan, M.D.b,1, Liu Cheng, M.D.a,1, Lu Fu-Ding, M.D.a, Lin Tian-Xin, M.D., Ph.D.a, Xuan Xu-Jun, M.D., Ph.D.a, Jiang Chun, M.D., Ph.D.a, Han Jin-Li, M.D., Ph.D.a, Huang Hai, M.D., Ph.D.a, Zhang Cai-Xia, M.D., Ph.D.a, Dong Wen, M.D.a, Liu Hao, M.D.a, Huang Jian, M.D., Ph.D.a, Xu Ke-Wei, M.D., Ph.D.a,* a
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China b Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Received 17 August 2013; received in revised form 21 October 2013; accepted 25 October 2013
Abstract Background: Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have the capability of homing to cancer cells. Thus, MSCs play an important role in the development, metastasis, and drug resistance of cancers. The mechanisms underlying the homing of MSCs in kidney cancer are still poorly understood. Methods: In the present study, enzyme-linked immunosorbent assay was used to measure the level of IL-8 in patients with kidney cancer and in the culture medium of kidney cancer cells. Immunofluorescence staining and reverse transcription polymerase chain reaction were utilized to explore the main receptor for IL-8 in MSCs. Transwell migration assay was performed to measure the migration ability of MSCs and Western blot test was performed to test the activation of signaling pathways. Results: The serum level of IL-8 was markedly increased in patients with kidney cancer, and 2 kidney cancer cell lines were found to secrete IL-8. MSCs had high expression of the IL-8 receptor (CXCR2). Blocking IL-8 or CXCR2 could decrease the migration ability of MSCs. IL-8 could significantly increase Akt phosphorylation in MSCs. Conclusions: Kidney cancer cells secrete IL-8 to activate the Akt signaling pathway via CXCR2 on MSCs, inducing the migration of MSCs, which may be one of the important mechanisms underlying the homing of MSCs in kidney cancer. r 2013 Elsevier Inc. All rights reserved. Keywords: Kidney cancer; Mesenchymal stem cell; Interleukin-8; Extracellular signal-regulated protein kinase; Akt
1. Introduction
This work was supported by National Natural Science Foundation of China (Nos. 81001138 and 81101519); Natural Science Foundation of Guangdong Province (Nos. 06021283, 10151008901000024, 10151008901000070, and S2011040003777); Science and Technology Development Program of Guangdong Province (Nos. 2008B030301078 and 2012B031800081); Young Teacher Foundation of Sun Yat-sen university(Nos.11ykpy33 and 12ykpy31); Yat-Sen Scholarship for Young Scientists (B.L.); Grant [2013] 163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Transcriptional Medicine of Guangzhou Bureau of Science and Information Technology. * Corresponding author. Tel.: þ86-20-8133-2336; fax: þ86-20-81332336. E-mail address: [email protected] (X. Ke-Wei). 1 These authors contributed equally to this work. 1078-1439/$ – see front matter r 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2013.10.018
Mesenchymal stem cells (MSCs) are multipotent stem cells that are derived mainly from the bone marrow and adipose tissues. Thus, MSCs are also known as bone marrow- or adipose-derived MSCs. MSCs are capable of multipotent differentiation. Studies have shown that MSCs can differentiate into osteoblasts, chondrocytes, fibroblasts, adipocytes, and other cell types [1]. MSCs have specific surface markers, expressing high levels of CD105, CD73, and CD90, but low levels of CD34, CD45, and CD11b. MSCs have been shown to have therapeutic effects on some diseases. A variety of studies have shown that MSCs could be used to treat myocardial infarction, spinal cord injury, and bone injury [2].
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MSCs are closely associated with cancers, and in addition, MSCs possess immunosuppressive properties. Studies have shown that MSCs can inhibit the function of cytotoxic T cells and dendritic cells, which leads to immunosuppression. This inhibition of immune function is thought to be one of the causes of tumor immune escape [3]. Moreover, studies have also revealed that MSCs have the capability of homing to breast cancer cells and can promote the development and metastasis of cancers [4]. Other cancers also are potential targets of MSCs, including renal cell carcinoma (RCC), a common malignancy of the urinary tract. Studies have demonstrated that MSCs can home to renal cancer cells in an animal model [5], but the specific mechanism is still unclear. The occurrence and development of cancers are closely related to the microenvironment. Cancer cells produce several cytokines that act on the surrounding interstitial cells and that may build a microenvironment for the growth and metastasis of cancer cells. IL-8, a cytokine that has been studied extensively, is an important chemokine that may exert chemotactic effects on numerous cells including MSCs [6]. Breast cancer cells secrete IL-8 [7], the level of which is closely related to the growth and metastasis of breast cancer cells [8]. Whether RCC can secrete IL-8 is unclear. The present study aimed to detect IL-8 in RCC and explore the regulatory effect of IL-8 on MSCs. Our findings may provide evidence for interaction between RCC and MSCs. 2. Materials and methods 2.1. General information Blood was collected from 20 patients with RCC and 20 healthy subjects. After admission, blood (5 ml) was collected before surgery and was centrifuged, and serum was collected for use. Patients with pathologically proven suprarenal epithelioma were recruited—8 women and 12 men, with a median age of 62 years (range: 42–77 y). No lymph node metastasis was found in any of the patients. Healthy subjects undergoing routine physical examination were recruited as controls—10 men and 10 women, with a median age of 60 years. 2.2. Materials RCC cell lines (786-O cells and ACHN cells) were purchased from American Type Culture Collection; IL-8 enzyme-linked immunosorbent assay (ELISA) kit (R&D systems); anti-IL-8 antibody, anti-CXCR1 antibody, antiCXCR2 antibody, wortmannin, Super ECL Plus hypersensitivity luminous fluid (Sigma); antibodies against phospho-Akt, Akt, and phospho-ERK (Cell Signaling); antibodies against extracellular signal-regulated kinase (ERK) and α-actin (Santa Cruz Biotechnology); dishes, plates, and kit for migration test (Corning), RPMI-1640,
Dulbecco's modified eagle medium—low glucose (DMEMLG) (Life technology), fetal bovine serum (FBS; Hyclone), phenylmethylsulfonyl fluoride, bovine serum albumin (Genview), polyvinylidene difluoride (PVDF) membrane (Millipore), and cell lysis buffer (our laboratory) were used in the present study. 2.3. Isolation of primary bone marrow stromal cells After being informed regarding the scientific contributions, possible risks, and complications and the corresponding prevention and treating measures for bone marrow aspirations, all of the healthy volunteers expressed approval and signed the informed consent. The bone marrow aspirations were all performed by skilled allied health professionals strictly according to the international standardized procedure for bone marrow aspirations. The bone marrow samples of the volunteers were diluted with DMEM (DMEM-LG) containing 10% FBS. The mononuclear cells were prepared by gradient centrifugation at 900g for 30 minutes on Percoll (Pharmacia Biotech, Uppsala, Sweden) of density 1.073 g/ml. The cells were washed, counted, seeded at 2 106 cells/cm2 in 25-cm2 flasks containing DMEM-LG supplemented with 10% FBS and cultured at 371C and 5% carbon dioxide. Medium was replaced and the cells in suspension were removed at 48 hours and every 3 or 4 days thereafter. Bone marrow stromal cells (BMSCs) were recovered using 0.25% trypsin-ethylenediaminetetraacetic acid and replated at a density of 5 103 to 6 103cells/cm2 surface area as passage 1 cell when the culture reached 90% confluency. BMSCs after the third subculture were used for described experiments. 2.4. Cell culture ACHN and 786-O cells were cultured at 371C with 5% CO2 atmosphere in RPMI-1640, which contained 10% FBS with 100-IU/ml penicillin and 100-mg/ml streptomycin. MSCs were cultured at 371C with 5% CO2 atmosphere in DMEM-LG, which contained 10% FBS with 100-IU/ml penicillin and 100-mg/ml streptomycin. 2.5. Enzyme-linked immunosorbent assay The sera from patients with RCC and controls and the culture medium from 786-O cells and ACHN cells were centrifuged, and the supernatant was collected and stored. Detection of IL-8 with an ELISA kit was performed according to the manufacturer's instructions. 2.6. Cell immunofluorescence staining MSCs with good growth were harvested by treatment with 0.25% trypsin, and a single cell suspension was prepared. MSCs were seeded onto coverslips in dishes and were maintained in an incubator with 5% CO2. When
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monolayer cells were observed, the coverslips were removed, washed twice with phosphate buffered saline (PBS), and fixed in 95% ethanol for 30 minutes. Then, the coverslips were placed in a staining box and were washed with PBS for 5 minutes. After drying, the coverslips were treated separately with antibodies against CXCR1 and CXCR2 (1:200) in a humidified box at 41C overnight. After washing thrice in PBS, the coverslips were dried and then incubated with luciferin-conjugated antibodies in a humidified box at 371C for 30 minutes. After washing twice in PBS (5 minutes each) and once in distilled water, coverslips were mounted with 50% glycerol and were observed under a fluorescence microscope. 2.7. Reverse transcription-polymerase chain reaction The complementary DNA (cDNA) sequences of GAPDH, CXCR1, and CXCR2 were obtained from NCBI database, and Primer Express software was employed to design primers: CXCR1: 5′-ACCATAGGAGGCCAACCCAAAATA-3′ (forward) and 5′-TCCATGCTGTGCCAAGAGTCA-3′ (reverse) and CXCR2: 5′-CTACCTTCC AGTTCCTCATTTTT-3′ (forward) and 5′-ACATTTACAAGTTGCAGTTTTCAGC-3′ (reverse). Total RNA was extracted with a total RNA kit, and then was used to reverse transcribe into cDNA according to manufacturer's instructions. cDNA was used as templates for amplification of CXCR1, CXCR2, and GAPDH. The polymerase chain reaction (PCR) products were resolved by agarose gel electrophoresis, followed by observation by imaging. 2.8. Detection of cell migration Cell migration test was performed according to the manufacturer's instructions. In brief, 100-μl matrigel was added to the upper chamber. Then, the Transwell chamber was incubated at 371C for 2 hours to allow the matrigel to solidify. MSCs with good growth were digested with trypsin followed by cell counting. These cells were washed and resuspended in serum-free DMEM. The cell density was adjusted to 5 105 cells/ml. Conditioned medium (CM) with or without anti-IL-8/anti-CXCR2 was added to the lower chamber, and 1 105 cells were added to the upper chamber. The Transwell chamber was then incubated at 371C for 12 and 24 hours. The upper chamber was obtained and fixed in 5% glutaraldehyde for 10 minutes and then in 1% crystal violet (in 2% ethanol) for 20 minutes. These cells were observed under a light microscope, photographed, and counted. 2.9. Western blot assays MSCs with good growth were harvested and the cell density was adjusted to 1 106 per ml. Cell suspension was added to 6-well plates (3 ml/well, 3 106 per well). Cells were maintained in serum-free medium overnight. In
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the IL-8 group, cells were treated with IL-8 for 15, 30, and 60 minutes; in the anti-CXCR2 group, cells were pretreated with anti-CXCR2 antibodies for 30 minutes and then with IL-8 for 15, 30, and 60 minutes. Then, these cells were washed twice with cold PBS and were transferred into centrifuge tubes followed by centrifugation at 1,800g for 4 minutes. The supernatant was removed. Cells were mixed with lysis buffer (80 μl) and were pipetted. The lysate was kept on ice for 15 minutes followed by centrifugation at 13,000 rpm for 10 minutes. The supernatant was collected (60 μl) and mixed with 4 sodium dodecyl sulfate (20 μl), followed by heating at 951C for 5 minutes. Proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then were transferred onto a PVDF membrane. The PVDF membrane was blocked in 5% nonfat milk in 1 tris-buffered saline–containing 0.05% Tween-20 at room temperature for 60 minutes. The membrane was incubated with the corresponding antibody (1:1,000) at 41C overnight (10 ml of 1 TBS and Tween20 (TBST), 0.5 g of bovine serum albumin, and 10 μL of 1mg/ml antibody). The membrane was washed with TBST and then was treated with horseradish peroxidase–conjugated goat antirabbit secondary antibody (1:5,000) at room temperature for 1 hour. After washing in TBST, visualization was performed with an ECL kit, and bands were observed with a gel image system. Similar procedures were employed to detect the expression of α-actin. The expression of target proteins was normalized to that of α-actin. 2.10. Statistical analysis Data are expressed as mean ⫾ standard deviation. Statistical analysis was performed with SPSS version 13.0. Comparisons were performed with t test. A value of P o 0.05 was considered statistically significant. 3. Results 3.1. IL-8 in RCC and kidney cancer cell lines It is well known that cancer is closely related to inflammation. Previous studies have indicated that IL-8, an important inflammatory cytokine, plays important roles in some malignancies including breast cancer, lung cancer, and colon cancer [10,11]. To explore the role of IL-8 in kidney cancer, the serum level of IL-8 was measured in patients with kidney cancer. As shown in Fig. 1A, ELISA demonstrated that the serum IL-8 level in patients with kidney cancer was significantly higher than that in controls. To explore whether the increase in IL-8 levels was due to secretion by kidney cancer cells, kidney cancer tissues were collected from 5 patients and were cultured in vitro. As shown in Fig. 1B, kidney cancer tissues secreted IL-8. Further investigation showed that IL-8 was detectable in 786-O cells and ACHN cells (2 RCC cell lines) after culture
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cancer cells (also known as CM). CM was used to stimulate MSCs, and the migration of MSCs was observed. As shown in Fig. 3A, the number of MSCs that migrated in the CM treatment group was significantly higher than the number that migrated in the untreated group. In the presence of antiIL-8 antibody, the number of MSCs that migrated was reduced markedly. This suggests that kidney cancer cells can promote MSC migration via secreting IL-8. The aforementioned findings indicate that MSCs express CXCR2. To confirm whether CXCR2 mediates the interaction between IL-8 and MSCs, we pretreated MSCs with anti-CXCR2 antibody and then with CM. As shown in Fig. 3B, the number of MSCs that migrated decreased dramatically after CXCR2 antibody pretreatment. This suggests that IL-8 binds to CXCR2 to stimulate the migration of MSCs. 3.4. IL-8 increases Akt phosphorylation in MSCs
Fig. 1. RCC secrets IL-8. (A) The level of IL-8 in the serum from control and RCC group was detected by ELISA. (B) The level of IL-8 in the medium from RCC tissue was detected by ELISA. (C and D) Renal carcinoma cell line 786-O and ACHN cells were cultured for indicated time, IL-8 levels were observed by ELISA.
for 24, 48, and 72 hours, and that IL-8 level in 786-O cells was higher than that in ACHN cells (Fig. 1C and D). On the basis of the aforementioned findings, we speculate that kidney cancer and RCC cells can secrete IL-8, suggesting that IL-8 plays an important role in kidney cancer. 3.2. High CXCR2 expression and low CXCR1 expression on MSCs Studies have shown that MSCs are closely related to cancer metastasis. MSCs can migrate into cancer tissues and promote the metastasis of cancer cells by altering the microenvironment in the cancer [12]. The surface receptors on MSCs are crucial molecules mediating the biological functions of MSCs. To investigate the effect of IL-8 on MSCs, these cells were subjected to immunofluorescence staining for CXCR1 and CXCR2. As shown in Fig. 2A, immunofluorescence demonstrated that MSCs had high CXCR2 expression but low CXCR1 expression. Reverse transcription-PCR further confirmed that CXCR2 is a major receptor on MSCs, but CXCR1 is expressed at a low level (Fig. 2B). 3.3. Kidney cancer cells secrete IL-8 to promote MSC migration To explore the interaction between kidney cancer cells and MSCs, we collected the culture medium from kidney
Akt is also known as protein kinase B. It is one of the important protein kinases in cells. The Akt signaling pathway is involved in the regulation of cellular biological behaviors, especially the migration of cells. Recent studies have revealed that activated Akt may stimulate the migration of MSCs [13]. To further explore the effect of IL-8 on MSCs, we treated MSCs with IL-8 and determined the expression of phosphorylated Akt. As shown in Fig. 4, IL-8 significantly increased the expression of phosphorylated Akt in MSCs, suggesting that IL-8 may activate the Akt signaling pathway in MSCs. At the same time, anti-CXCR2 antibody was employed to block CXCR2, and the expression of phosphorylated Akt was reduced markedly. Thus, we speculate that IL-8 secreted by kidney cancer cells may promote the migration of MSCs via the ERK and Akt signaling pathway. 3.5. IL-8 activates Akt to promote MSC migration To further explore the regulatory effect of Akt on the biological behavior of MSCs, we used an Akt inhibitor (wortmannin) to inhibit the activity of Akt. As shown in Fig. 5A, the Akt inhibitor dramatically reduced IL-8induced activation of Akt. Further investigation showed
Fig. 2. The expression of CXCR1 and CXCR2 in MSCs. The expression of CXCR1 and CXCR2 in MSCs was identified by immunofluorescence stain (A) and RT-PCR (B). RT-PCR ¼ reverse transcription-polymerase chain reaction. (Color version of figure is available online.)
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Fig. 3. RCC secreted IL-8 to promote MSC migration via CXCR2. (A) The secretion of IL-8 in the conditional medium from RCC cancer cells promoted MSC migration. (B) The secretion of IL-8 in the conditional medium promoted MSC migration via CXCR2. (Color version of figure is available online.)
that inhibition of Akt activity markedly suppressed the migration of MSCs (Fig. 5B). This suggests that IL-8 regulates the migration of MSCs via activation of Akt. 4. Discussion MSCs are multipotent adult stem cells that play important roles in the occurrence, development, metastasis, and drug resistance of cancers. Homing of MSCs to the cancer is a key step in the interaction between MSCs and cancer. The present study demonstrated that MSCs expressed high levels of CXCR2, and that kidney cancer secreted IL-8, which was able to promote the migration of MSCs via CXCR2. Molecular biological experiments demonstrated that IL-8 significantly increased the expression of phosphorylated ERK and Akt. ERK and Akt are 2 crucial kinases in the growth and migration of cells. Thus, we postulate that kidney cancer may secrete IL-8 to activate ERK and Akt in MSCs, which promotes the migration of MSCs. This may be an important mechanism underlying the homing of MSCs to kidney cancer. Generally, MSCs are found mainly in the bone marrow. Increasing evidence shows that inflammation following injury may induce the homing of MSCs to the injured site, but this homing of MSCs is not organ specific [14,15]. The homing of MSCs to the cancer is attributed to several soluble inflammation-related factors in the microenvironment of cancers [16]. These factors include growth factors (epidermal growth factor, fibroblast growth factor, plateletderived growth factor, and vascular endothelial growth
Fig. 4. IL-8 regulated the phosphorylation of Akt in MSCs. MSCs were stimulated by IL-8 with or without anti-CXCR2 for indicated time, and the phosphorylation level of Akt was observed by Western blot assay.
factor), chemokines (IL-8 and MCP-1) and cytokines (tumor necrosis factor-α and IL-6) [6]. The present study demonstrated that the serum level of IL-8 increased in patients with kidney cancer, and kidney cancer cell lines also secreted a large amount of IL-8. This suggests that kidney cancer may release IL-8 into the microenvironment of cancer to promote the migration of MSCs to the cancer, and IL-8 mediates the interaction between MSCs and kidney cancer. The mechanisms underlying the interaction between MSCs and cancers are complex. Studies have shown that MSCs after migrating to the cancer can promote angiogenesis, influence the formation of matrix, and secrete cytokines to promote the development of cancers [17,18]. In addition, MSCs possess immunosuppressive properties; coculture of MSCs and breast cancer cells can protect the breast cancer cells from immune clearance [19]. In our previous study, we also demonstrated that MSCs could inhibit inflammation and may exert a therapeutic effect on
Fig. 5. IL-8 promotes the migration of MSCs through the activation of Akt (A). MSCs were stimulated by IL-8 with or without Akt inhibitor for indicated time, and the phosphorylation level of Akt was observed by Western blot assay. (B) The migration of MSCs were observed when the MSCs were stimulated by conditional medium from RCC cancer cells with or without Akt inhibitor for 48 hours.
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infection-induced systemic inflammation [9]. The immunosuppressive effect of MSCs is nonspecific; however, the means by which the MSCs regulate the immune response in kidney cancer is still unclear. IL-8, also known as CXCL8, is a chemokine secreted by many types of cells. Its receptors include CXCR1 and CXCR2 [20]. CXCR1 has structure similar to that of CXCR2, and both belong to the G protein-coupled receptor family; however, both receptors have distinct biological activities and their expression is also different on cells [21]. The present study showed that MSCs had low CXCR1 expression but high CXCR2 expression. After blocking the CXCR2 receptor, the effect of IL-8 on MSCs was markedly compromised. This suggests that CXCR2 is a major receptor on MSCs and that IL-8 acts on MSCs mainly via CXCR2. Akt, a cytoplasmic serine/threonine protein kinase, is an important signaling pathway in cells, and it is involved in the regulation of cell growth and in cell proliferation and migration. Akt may become phosphorylated in response to a variety of exogenous stimuli, thus activating Akt to its functional (phosphorylated) state. IL-8 in cancer cells may phosphorylate Akt, which promotes the growth and metastasis of cancer cells [11]. Jeun and colleagues [22] found that stromal cell-derived factor-1 in the umbilical cord blood MSCs could promote the phosphorylation of Akt and ERK via CXCR4, which facilitates the migration of these stem cells. CXCR4 and CXCR2 belong to the G protein-coupled receptor family and have similarities in mediating intracellular signal transduction. The present study demonstrated that IL-8 could rapidly and significantly increase the expression of phosphorylated ERK and Akt, which remained at high levels; however, after blocking CXCR2, the expression of phosphorylated ERK and Akt was reduced markedly. This suggests that CXCR2 may initiate the phosphorylation of ERK and Akt, which might be the initial signal causing the migration of MSCs and exerting important regulatory effects on the biological behavior of MSCs. References [1] Kode. JA, Mukherjee S, Joglekar MV, Hardikar AA. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration. Cytotherapy 2009;11:377–91. [2] Augello A, De Bari C. The regulation of differentiation in mesenchymal stem cells. Hum Gene Ther 2010;21:1226–38. [3] Chen X, Armstrong MA, Li G. Mesenchymal stem cells in immunoregulation. Immunol Cell Biol 2006;84:413–21. [4] Loebinger MR, Kyrtatos PG, Turmaine M, Price AN, Pankhurst Q, M.F. Lythgoe, et al. Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles. Cancer Res 2009;69:8862–7.
[5] Gao P, Ding Q, Wu Z, Jiang H, Fang Z. Therapeutic potential of human mesenchymal stem cells producing IL-12 in a mouse xenograft model of renal cell carcinoma. Cancer Lett 2010;290: 157–66. [6] Yang X, Hou J, Han Z, Wang Y, Hao C, Wei L, et al. One cell, multiple roles: contribution of mesenchymal stem cells to tumor development in tumor microenvironment. Cell Biosci 2013;3:5. [7] Li C, Guo S, Shi T. Role of NF-κ-B activation in matrix metalloproteinase 9, vascular endothelial growth factor and interleukin 8 expression and secretion in human breast cancer cells. Cell Biochem Funct 2013;31:263–8. [8] Kim H, Choi JA, Park GS, Kim JH. BLT2 up-regulates interleukin-8 production and promotes the invasiveness of breast cancer cells. PLoS One 2012;7:e49186. [9] Bi LK, Tang B, Zhu B, Xie CL, Li SA, Lin TX, et al. Systemic delivery of IL-10 by bone marrow derived stromal cells has therapeutic benefits in sepsis therapy. Prog Biochem Biophys 2010;37:678–85. [10] Chavey C, Muehlbauer M, Bossard C, Freund A, Durand S, Jorgensen C, et al. Interleukin-8 expression is regulated by histone deacetylases through the nuclear factor-kappa B pathway in breast cancer. Mol Pharmacol 2008;74:1359–66. [11] Waugh DJJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res 2008;14:6735–41. [12] SA Bergfeld, DeClerck YA. Bone marrow-derived mesenchymal stem cells and the tumor microenvironment. Cancer Metastasis Rev 2010;29:249–61. [13] Bulj Z, Duchi S, Bevilacqua A, Gherardi A, Dozza B, Piccinini F, et al. Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells. Int J Oncol 2013;42:118–26. [14] Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33:145–52. [15] Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 2005;106:756–63. [16] Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F. Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther 2008;15:730–8. [17] Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A, et al. Origins of the tumor microenvironment: quantitative assessment of adiposederived and bone marrow-derived stroma. PLoS One 2012;7:e30563. [18] Pasquet M, Golzio M, Mery E, Rafii A, Benabbou N, Mirshahi P, et al. Hospicells (ascites-derived stromal cells) promote tumorigenicity and angiogenesis. Int J Cancer 2010;126:2090–101. [19] Patel SA, Meyer JR, Greco SJ, Corcoran KE, Bryan M, Rameshwar P. Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta. J Immunol 2010;184:5885–94. [20] Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neurol Oncol 2005;7:122–33. [21] Schraufstatter IU, Chung J, Burger M. IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways. Am J Physiol Lung Cell Mol Physiol 2001;280:L1094–103. [22] Ryu CH, Park SA, Kim SM, Lim JY, Jeong CH, Jun JA, et al. Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways. Biochem Biophys Res Commun 2010;398:105–10.
Original article
Kidney cancer cells secrete IL-8 to activate Akt and promote migration of mesenchymal stem cells Bi Liang-kuan, M.D., Ph.D.a,1, Zhou Nan, M.D.b,1, Liu Cheng, M.D.a,1, Lu Fu-Ding, M.D.a, Lin Tian-Xin, M.D., Ph.D.a, Xuan Xu-Jun, M.D., Ph.D.a, Jiang Chun, M.D., Ph.D.a, Han Jin-Li, M.D., Ph.D.a, Huang Hai, M.D., Ph.D.a, Zhang Cai-Xia, M.D., Ph.D.a, Dong Wen, M.D.a, Liu Hao, M.D.a, Huang Jian, M.D., Ph.D.a, Xu Ke-Wei, M.D., Ph.D.a,* a
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China b Department of Obstetrics and Gynecology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Received 17 August 2013; received in revised form 21 October 2013; accepted 25 October 2013
Abstract Background: Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have the capability of homing to cancer cells. Thus, MSCs play an important role in the development, metastasis, and drug resistance of cancers. The mechanisms underlying the homing of MSCs in kidney cancer are still poorly understood. Methods: In the present study, enzyme-linked immunosorbent assay was used to measure the level of IL-8 in patients with kidney cancer and in the culture medium of kidney cancer cells. Immunofluorescence staining and reverse transcription polymerase chain reaction were utilized to explore the main receptor for IL-8 in MSCs. Transwell migration assay was performed to measure the migration ability of MSCs and Western blot test was performed to test the activation of signaling pathways. Results: The serum level of IL-8 was markedly increased in patients with kidney cancer, and 2 kidney cancer cell lines were found to secrete IL-8. MSCs had high expression of the IL-8 receptor (CXCR2). Blocking IL-8 or CXCR2 could decrease the migration ability of MSCs. IL-8 could significantly increase Akt phosphorylation in MSCs. Conclusions: Kidney cancer cells secrete IL-8 to activate the Akt signaling pathway via CXCR2 on MSCs, inducing the migration of MSCs, which may be one of the important mechanisms underlying the homing of MSCs in kidney cancer. r 2013 Elsevier Inc. All rights reserved. Keywords: Kidney cancer; Mesenchymal stem cell; Interleukin-8; Extracellular signal-regulated protein kinase; Akt
1. Introduction
This work was supported by National Natural Science Foundation of China (Nos. 81001138 and 81101519); Natural Science Foundation of Guangdong Province (Nos. 06021283, 10151008901000024, 10151008901000070, and S2011040003777); Science and Technology Development Program of Guangdong Province (Nos. 2008B030301078 and 2012B031800081); Young Teacher Foundation of Sun Yat-sen university(Nos.11ykpy33 and 12ykpy31); Yat-Sen Scholarship for Young Scientists (B.L.); Grant [2013] 163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Transcriptional Medicine of Guangzhou Bureau of Science and Information Technology. * Corresponding author. Tel.: þ86-20-8133-2336; fax: þ86-20-81332336. E-mail address: [email protected] (X. Ke-Wei). 1 These authors contributed equally to this work. 1078-1439/$ – see front matter r 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2013.10.018
Mesenchymal stem cells (MSCs) are multipotent stem cells that are derived mainly from the bone marrow and adipose tissues. Thus, MSCs are also known as bone marrow- or adipose-derived MSCs. MSCs are capable of multipotent differentiation. Studies have shown that MSCs can differentiate into osteoblasts, chondrocytes, fibroblasts, adipocytes, and other cell types [1]. MSCs have specific surface markers, expressing high levels of CD105, CD73, and CD90, but low levels of CD34, CD45, and CD11b. MSCs have been shown to have therapeutic effects on some diseases. A variety of studies have shown that MSCs could be used to treat myocardial infarction, spinal cord injury, and bone injury [2].
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MSCs are closely associated with cancers, and in addition, MSCs possess immunosuppressive properties. Studies have shown that MSCs can inhibit the function of cytotoxic T cells and dendritic cells, which leads to immunosuppression. This inhibition of immune function is thought to be one of the causes of tumor immune escape [3]. Moreover, studies have also revealed that MSCs have the capability of homing to breast cancer cells and can promote the development and metastasis of cancers [4]. Other cancers also are potential targets of MSCs, including renal cell carcinoma (RCC), a common malignancy of the urinary tract. Studies have demonstrated that MSCs can home to renal cancer cells in an animal model [5], but the specific mechanism is still unclear. The occurrence and development of cancers are closely related to the microenvironment. Cancer cells produce several cytokines that act on the surrounding interstitial cells and that may build a microenvironment for the growth and metastasis of cancer cells. IL-8, a cytokine that has been studied extensively, is an important chemokine that may exert chemotactic effects on numerous cells including MSCs [6]. Breast cancer cells secrete IL-8 [7], the level of which is closely related to the growth and metastasis of breast cancer cells [8]. Whether RCC can secrete IL-8 is unclear. The present study aimed to detect IL-8 in RCC and explore the regulatory effect of IL-8 on MSCs. Our findings may provide evidence for interaction between RCC and MSCs. 2. Materials and methods 2.1. General information Blood was collected from 20 patients with RCC and 20 healthy subjects. After admission, blood (5 ml) was collected before surgery and was centrifuged, and serum was collected for use. Patients with pathologically proven suprarenal epithelioma were recruited—8 women and 12 men, with a median age of 62 years (range: 42–77 y). No lymph node metastasis was found in any of the patients. Healthy subjects undergoing routine physical examination were recruited as controls—10 men and 10 women, with a median age of 60 years. 2.2. Materials RCC cell lines (786-O cells and ACHN cells) were purchased from American Type Culture Collection; IL-8 enzyme-linked immunosorbent assay (ELISA) kit (R&D systems); anti-IL-8 antibody, anti-CXCR1 antibody, antiCXCR2 antibody, wortmannin, Super ECL Plus hypersensitivity luminous fluid (Sigma); antibodies against phospho-Akt, Akt, and phospho-ERK (Cell Signaling); antibodies against extracellular signal-regulated kinase (ERK) and α-actin (Santa Cruz Biotechnology); dishes, plates, and kit for migration test (Corning), RPMI-1640,
Dulbecco's modified eagle medium—low glucose (DMEMLG) (Life technology), fetal bovine serum (FBS; Hyclone), phenylmethylsulfonyl fluoride, bovine serum albumin (Genview), polyvinylidene difluoride (PVDF) membrane (Millipore), and cell lysis buffer (our laboratory) were used in the present study. 2.3. Isolation of primary bone marrow stromal cells After being informed regarding the scientific contributions, possible risks, and complications and the corresponding prevention and treating measures for bone marrow aspirations, all of the healthy volunteers expressed approval and signed the informed consent. The bone marrow aspirations were all performed by skilled allied health professionals strictly according to the international standardized procedure for bone marrow aspirations. The bone marrow samples of the volunteers were diluted with DMEM (DMEM-LG) containing 10% FBS. The mononuclear cells were prepared by gradient centrifugation at 900g for 30 minutes on Percoll (Pharmacia Biotech, Uppsala, Sweden) of density 1.073 g/ml. The cells were washed, counted, seeded at 2 106 cells/cm2 in 25-cm2 flasks containing DMEM-LG supplemented with 10% FBS and cultured at 371C and 5% carbon dioxide. Medium was replaced and the cells in suspension were removed at 48 hours and every 3 or 4 days thereafter. Bone marrow stromal cells (BMSCs) were recovered using 0.25% trypsin-ethylenediaminetetraacetic acid and replated at a density of 5 103 to 6 103cells/cm2 surface area as passage 1 cell when the culture reached 90% confluency. BMSCs after the third subculture were used for described experiments. 2.4. Cell culture ACHN and 786-O cells were cultured at 371C with 5% CO2 atmosphere in RPMI-1640, which contained 10% FBS with 100-IU/ml penicillin and 100-mg/ml streptomycin. MSCs were cultured at 371C with 5% CO2 atmosphere in DMEM-LG, which contained 10% FBS with 100-IU/ml penicillin and 100-mg/ml streptomycin. 2.5. Enzyme-linked immunosorbent assay The sera from patients with RCC and controls and the culture medium from 786-O cells and ACHN cells were centrifuged, and the supernatant was collected and stored. Detection of IL-8 with an ELISA kit was performed according to the manufacturer's instructions. 2.6. Cell immunofluorescence staining MSCs with good growth were harvested by treatment with 0.25% trypsin, and a single cell suspension was prepared. MSCs were seeded onto coverslips in dishes and were maintained in an incubator with 5% CO2. When
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monolayer cells were observed, the coverslips were removed, washed twice with phosphate buffered saline (PBS), and fixed in 95% ethanol for 30 minutes. Then, the coverslips were placed in a staining box and were washed with PBS for 5 minutes. After drying, the coverslips were treated separately with antibodies against CXCR1 and CXCR2 (1:200) in a humidified box at 41C overnight. After washing thrice in PBS, the coverslips were dried and then incubated with luciferin-conjugated antibodies in a humidified box at 371C for 30 minutes. After washing twice in PBS (5 minutes each) and once in distilled water, coverslips were mounted with 50% glycerol and were observed under a fluorescence microscope. 2.7. Reverse transcription-polymerase chain reaction The complementary DNA (cDNA) sequences of GAPDH, CXCR1, and CXCR2 were obtained from NCBI database, and Primer Express software was employed to design primers: CXCR1: 5′-ACCATAGGAGGCCAACCCAAAATA-3′ (forward) and 5′-TCCATGCTGTGCCAAGAGTCA-3′ (reverse) and CXCR2: 5′-CTACCTTCC AGTTCCTCATTTTT-3′ (forward) and 5′-ACATTTACAAGTTGCAGTTTTCAGC-3′ (reverse). Total RNA was extracted with a total RNA kit, and then was used to reverse transcribe into cDNA according to manufacturer's instructions. cDNA was used as templates for amplification of CXCR1, CXCR2, and GAPDH. The polymerase chain reaction (PCR) products were resolved by agarose gel electrophoresis, followed by observation by imaging. 2.8. Detection of cell migration Cell migration test was performed according to the manufacturer's instructions. In brief, 100-μl matrigel was added to the upper chamber. Then, the Transwell chamber was incubated at 371C for 2 hours to allow the matrigel to solidify. MSCs with good growth were digested with trypsin followed by cell counting. These cells were washed and resuspended in serum-free DMEM. The cell density was adjusted to 5 105 cells/ml. Conditioned medium (CM) with or without anti-IL-8/anti-CXCR2 was added to the lower chamber, and 1 105 cells were added to the upper chamber. The Transwell chamber was then incubated at 371C for 12 and 24 hours. The upper chamber was obtained and fixed in 5% glutaraldehyde for 10 minutes and then in 1% crystal violet (in 2% ethanol) for 20 minutes. These cells were observed under a light microscope, photographed, and counted. 2.9. Western blot assays MSCs with good growth were harvested and the cell density was adjusted to 1 106 per ml. Cell suspension was added to 6-well plates (3 ml/well, 3 106 per well). Cells were maintained in serum-free medium overnight. In
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the IL-8 group, cells were treated with IL-8 for 15, 30, and 60 minutes; in the anti-CXCR2 group, cells were pretreated with anti-CXCR2 antibodies for 30 minutes and then with IL-8 for 15, 30, and 60 minutes. Then, these cells were washed twice with cold PBS and were transferred into centrifuge tubes followed by centrifugation at 1,800g for 4 minutes. The supernatant was removed. Cells were mixed with lysis buffer (80 μl) and were pipetted. The lysate was kept on ice for 15 minutes followed by centrifugation at 13,000 rpm for 10 minutes. The supernatant was collected (60 μl) and mixed with 4 sodium dodecyl sulfate (20 μl), followed by heating at 951C for 5 minutes. Proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then were transferred onto a PVDF membrane. The PVDF membrane was blocked in 5% nonfat milk in 1 tris-buffered saline–containing 0.05% Tween-20 at room temperature for 60 minutes. The membrane was incubated with the corresponding antibody (1:1,000) at 41C overnight (10 ml of 1 TBS and Tween20 (TBST), 0.5 g of bovine serum albumin, and 10 μL of 1mg/ml antibody). The membrane was washed with TBST and then was treated with horseradish peroxidase–conjugated goat antirabbit secondary antibody (1:5,000) at room temperature for 1 hour. After washing in TBST, visualization was performed with an ECL kit, and bands were observed with a gel image system. Similar procedures were employed to detect the expression of α-actin. The expression of target proteins was normalized to that of α-actin. 2.10. Statistical analysis Data are expressed as mean ⫾ standard deviation. Statistical analysis was performed with SPSS version 13.0. Comparisons were performed with t test. A value of P o 0.05 was considered statistically significant. 3. Results 3.1. IL-8 in RCC and kidney cancer cell lines It is well known that cancer is closely related to inflammation. Previous studies have indicated that IL-8, an important inflammatory cytokine, plays important roles in some malignancies including breast cancer, lung cancer, and colon cancer [10,11]. To explore the role of IL-8 in kidney cancer, the serum level of IL-8 was measured in patients with kidney cancer. As shown in Fig. 1A, ELISA demonstrated that the serum IL-8 level in patients with kidney cancer was significantly higher than that in controls. To explore whether the increase in IL-8 levels was due to secretion by kidney cancer cells, kidney cancer tissues were collected from 5 patients and were cultured in vitro. As shown in Fig. 1B, kidney cancer tissues secreted IL-8. Further investigation showed that IL-8 was detectable in 786-O cells and ACHN cells (2 RCC cell lines) after culture
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cancer cells (also known as CM). CM was used to stimulate MSCs, and the migration of MSCs was observed. As shown in Fig. 3A, the number of MSCs that migrated in the CM treatment group was significantly higher than the number that migrated in the untreated group. In the presence of antiIL-8 antibody, the number of MSCs that migrated was reduced markedly. This suggests that kidney cancer cells can promote MSC migration via secreting IL-8. The aforementioned findings indicate that MSCs express CXCR2. To confirm whether CXCR2 mediates the interaction between IL-8 and MSCs, we pretreated MSCs with anti-CXCR2 antibody and then with CM. As shown in Fig. 3B, the number of MSCs that migrated decreased dramatically after CXCR2 antibody pretreatment. This suggests that IL-8 binds to CXCR2 to stimulate the migration of MSCs. 3.4. IL-8 increases Akt phosphorylation in MSCs
Fig. 1. RCC secrets IL-8. (A) The level of IL-8 in the serum from control and RCC group was detected by ELISA. (B) The level of IL-8 in the medium from RCC tissue was detected by ELISA. (C and D) Renal carcinoma cell line 786-O and ACHN cells were cultured for indicated time, IL-8 levels were observed by ELISA.
for 24, 48, and 72 hours, and that IL-8 level in 786-O cells was higher than that in ACHN cells (Fig. 1C and D). On the basis of the aforementioned findings, we speculate that kidney cancer and RCC cells can secrete IL-8, suggesting that IL-8 plays an important role in kidney cancer. 3.2. High CXCR2 expression and low CXCR1 expression on MSCs Studies have shown that MSCs are closely related to cancer metastasis. MSCs can migrate into cancer tissues and promote the metastasis of cancer cells by altering the microenvironment in the cancer [12]. The surface receptors on MSCs are crucial molecules mediating the biological functions of MSCs. To investigate the effect of IL-8 on MSCs, these cells were subjected to immunofluorescence staining for CXCR1 and CXCR2. As shown in Fig. 2A, immunofluorescence demonstrated that MSCs had high CXCR2 expression but low CXCR1 expression. Reverse transcription-PCR further confirmed that CXCR2 is a major receptor on MSCs, but CXCR1 is expressed at a low level (Fig. 2B). 3.3. Kidney cancer cells secrete IL-8 to promote MSC migration To explore the interaction between kidney cancer cells and MSCs, we collected the culture medium from kidney
Akt is also known as protein kinase B. It is one of the important protein kinases in cells. The Akt signaling pathway is involved in the regulation of cellular biological behaviors, especially the migration of cells. Recent studies have revealed that activated Akt may stimulate the migration of MSCs [13]. To further explore the effect of IL-8 on MSCs, we treated MSCs with IL-8 and determined the expression of phosphorylated Akt. As shown in Fig. 4, IL-8 significantly increased the expression of phosphorylated Akt in MSCs, suggesting that IL-8 may activate the Akt signaling pathway in MSCs. At the same time, anti-CXCR2 antibody was employed to block CXCR2, and the expression of phosphorylated Akt was reduced markedly. Thus, we speculate that IL-8 secreted by kidney cancer cells may promote the migration of MSCs via the ERK and Akt signaling pathway. 3.5. IL-8 activates Akt to promote MSC migration To further explore the regulatory effect of Akt on the biological behavior of MSCs, we used an Akt inhibitor (wortmannin) to inhibit the activity of Akt. As shown in Fig. 5A, the Akt inhibitor dramatically reduced IL-8induced activation of Akt. Further investigation showed
Fig. 2. The expression of CXCR1 and CXCR2 in MSCs. The expression of CXCR1 and CXCR2 in MSCs was identified by immunofluorescence stain (A) and RT-PCR (B). RT-PCR ¼ reverse transcription-polymerase chain reaction. (Color version of figure is available online.)
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Fig. 3. RCC secreted IL-8 to promote MSC migration via CXCR2. (A) The secretion of IL-8 in the conditional medium from RCC cancer cells promoted MSC migration. (B) The secretion of IL-8 in the conditional medium promoted MSC migration via CXCR2. (Color version of figure is available online.)
that inhibition of Akt activity markedly suppressed the migration of MSCs (Fig. 5B). This suggests that IL-8 regulates the migration of MSCs via activation of Akt. 4. Discussion MSCs are multipotent adult stem cells that play important roles in the occurrence, development, metastasis, and drug resistance of cancers. Homing of MSCs to the cancer is a key step in the interaction between MSCs and cancer. The present study demonstrated that MSCs expressed high levels of CXCR2, and that kidney cancer secreted IL-8, which was able to promote the migration of MSCs via CXCR2. Molecular biological experiments demonstrated that IL-8 significantly increased the expression of phosphorylated ERK and Akt. ERK and Akt are 2 crucial kinases in the growth and migration of cells. Thus, we postulate that kidney cancer may secrete IL-8 to activate ERK and Akt in MSCs, which promotes the migration of MSCs. This may be an important mechanism underlying the homing of MSCs to kidney cancer. Generally, MSCs are found mainly in the bone marrow. Increasing evidence shows that inflammation following injury may induce the homing of MSCs to the injured site, but this homing of MSCs is not organ specific [14,15]. The homing of MSCs to the cancer is attributed to several soluble inflammation-related factors in the microenvironment of cancers [16]. These factors include growth factors (epidermal growth factor, fibroblast growth factor, plateletderived growth factor, and vascular endothelial growth
Fig. 4. IL-8 regulated the phosphorylation of Akt in MSCs. MSCs were stimulated by IL-8 with or without anti-CXCR2 for indicated time, and the phosphorylation level of Akt was observed by Western blot assay.
factor), chemokines (IL-8 and MCP-1) and cytokines (tumor necrosis factor-α and IL-6) [6]. The present study demonstrated that the serum level of IL-8 increased in patients with kidney cancer, and kidney cancer cell lines also secreted a large amount of IL-8. This suggests that kidney cancer may release IL-8 into the microenvironment of cancer to promote the migration of MSCs to the cancer, and IL-8 mediates the interaction between MSCs and kidney cancer. The mechanisms underlying the interaction between MSCs and cancers are complex. Studies have shown that MSCs after migrating to the cancer can promote angiogenesis, influence the formation of matrix, and secrete cytokines to promote the development of cancers [17,18]. In addition, MSCs possess immunosuppressive properties; coculture of MSCs and breast cancer cells can protect the breast cancer cells from immune clearance [19]. In our previous study, we also demonstrated that MSCs could inhibit inflammation and may exert a therapeutic effect on
Fig. 5. IL-8 promotes the migration of MSCs through the activation of Akt (A). MSCs were stimulated by IL-8 with or without Akt inhibitor for indicated time, and the phosphorylation level of Akt was observed by Western blot assay. (B) The migration of MSCs were observed when the MSCs were stimulated by conditional medium from RCC cancer cells with or without Akt inhibitor for 48 hours.
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infection-induced systemic inflammation [9]. The immunosuppressive effect of MSCs is nonspecific; however, the means by which the MSCs regulate the immune response in kidney cancer is still unclear. IL-8, also known as CXCL8, is a chemokine secreted by many types of cells. Its receptors include CXCR1 and CXCR2 [20]. CXCR1 has structure similar to that of CXCR2, and both belong to the G protein-coupled receptor family; however, both receptors have distinct biological activities and their expression is also different on cells [21]. The present study showed that MSCs had low CXCR1 expression but high CXCR2 expression. After blocking the CXCR2 receptor, the effect of IL-8 on MSCs was markedly compromised. This suggests that CXCR2 is a major receptor on MSCs and that IL-8 acts on MSCs mainly via CXCR2. Akt, a cytoplasmic serine/threonine protein kinase, is an important signaling pathway in cells, and it is involved in the regulation of cell growth and in cell proliferation and migration. Akt may become phosphorylated in response to a variety of exogenous stimuli, thus activating Akt to its functional (phosphorylated) state. IL-8 in cancer cells may phosphorylate Akt, which promotes the growth and metastasis of cancer cells [11]. Jeun and colleagues [22] found that stromal cell-derived factor-1 in the umbilical cord blood MSCs could promote the phosphorylation of Akt and ERK via CXCR4, which facilitates the migration of these stem cells. CXCR4 and CXCR2 belong to the G protein-coupled receptor family and have similarities in mediating intracellular signal transduction. The present study demonstrated that IL-8 could rapidly and significantly increase the expression of phosphorylated ERK and Akt, which remained at high levels; however, after blocking CXCR2, the expression of phosphorylated ERK and Akt was reduced markedly. This suggests that CXCR2 may initiate the phosphorylation of ERK and Akt, which might be the initial signal causing the migration of MSCs and exerting important regulatory effects on the biological behavior of MSCs. References [1] Kode. JA, Mukherjee S, Joglekar MV, Hardikar AA. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration. Cytotherapy 2009;11:377–91. [2] Augello A, De Bari C. The regulation of differentiation in mesenchymal stem cells. Hum Gene Ther 2010;21:1226–38. [3] Chen X, Armstrong MA, Li G. Mesenchymal stem cells in immunoregulation. Immunol Cell Biol 2006;84:413–21. [4] Loebinger MR, Kyrtatos PG, Turmaine M, Price AN, Pankhurst Q, M.F. Lythgoe, et al. Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles. Cancer Res 2009;69:8862–7.
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