IL-17a Prompts Human Tumor Cells Migration and Akt, Erk, P38 Phosphorylation in Vitro

Rapid Communication

Austin Pharmacol Pharm. 2016; 1(1): 1003.

IL-17a Prompts Human Tumor Cells Migration and Akt, Erk, P38 Phosphorylation in Vitro

Gehring MP¹*, Pereira TCB², Zanin R¹, Bogo M² and Morrone FB¹

¹Laboratory of Applied Pharmacology, PUCRS, Brazil

²Laboratory of Molecular Biology and Genomics, PUCRS, Brazil

*Corresponding author: Marina Petersen Gehring, Laboratory of Applied Pharmacology, Graduate Program in Cellular and Molecular Biology, PUCRS, Partenon- Porto Alegre, RS, Brazil

Received: August 03, 2016; Accepted: August 18, 2016; Published: August 22, 2016


Changes of interleukin levels have been associated with advanced stages of several cancer types. Increased levels of cytokines induce tumorigenesis, being a poor prognostic marker for malignant disease. Chronic inflammation is a pathological feature of cancer and interleukin-17 (IL-17A) is an inflammatory cytokine with diverse functions in host defense. This study aimed to clarify the direct role of IL-17 on tumor cells. Several human tumors cells from glioma, bladder and esophagus were first analyzed for IL-17 receptor (IL-17RA) expression through qRT-PCR. The cells were then treated with different concentrations of IL-17, ranging from 10 pg/mL to 100 ng/mL, and evaluated for IL-17 ability to modify tumor cell proliferation and viability through cell counting and MTT assay. Cell migration capacity and intracellular signaling pathways activated by IL-17 were assessed by wound healing assay and flow cytometry, respectively. IL-17RA mRNA expression was detected on the cell lines tested, but the incubation of IL-17 did not alter tumor cell viability or proliferation. However, the incubation of IL-17 was able to promote tumor cell migration and AKT, ERK and P38 phosphorylation in vitro. Our data show, for the first time, the ability of IL-17RA/IL-17 to induce migration of human tumor cell lines in vitro, possibly via activation of MAP kinases and AKT.

Keywords: IL-17A; IL-17RA; Glioma; Bladder tumor; Esophageal tumor; Cell migration


Interleukin-17 (IL-17A) is classified as a pro-inflammatory cytokine and it is shown to be elevated in several types of cancer [1]. Data regarding the role of IL-17 on tumor development are not consensual. It is important to highlight that exogenously delivered IL-17 might display different effects in comparison to endogenous IL- 17 [2]. Recently, researches on this field point out the idea that IL-17 promotes tumor growth, especially through the activation of IL-6- STAT3 cascade, an oncogenic transcription factor that up regulates pro-survival and pro-angiogenic genes [1-4]. In contrast, other studies suggest that IL-17 may protect against tumors, by promoting immune system-mediated tumor rejection [1,5,6].

After binding to its receptor (IL-17/R), intracellular IL-17 signaling includes transcription factor NF-κB [7,8], which induces the coordinated expression of several inflammatory genes causing the perpetuation of the inflammatory response [8]; PI3K, MAPKs, JNK, ERK and p38 are clearly involved in IL-17-induced responses [7,8].

Studies about the role of IL-17 in cancer are still very few regarding to different types of cancer such as bladder, brain and esophageal cancer. In relation to gliomas, Hu et al. 2011, described that IL-17 expression may play an important role in tumorigenesis and progression, since they observed high mRNA-positive ratios of IL-17 in glioma tissues, but not in tissues subjected to trauma [3]. For bladder cancer, the tumor was reduced in IL-17-/- mice, indicating a role of this cytokine in promoting tumor growth through IL-6/ STAT3 pathway [4]. Another study suggests that IL-17 may play an important role in the recruitment and infiltration of antitumor immune cells in early stages of bladder cancer [5]. In esophageal squamous cell carcinoma (ESCC), IL-17 led to the production of inflammatory chemokines (CXCL9, CXCL10 and CCL2, CCL20), which are associated with the migration of T cells, NK cells, and DCs, respectively. In addition, IL-17 enhanced the cytotoxic effects of NK cells against tumor cells [6]. The present study aimed to clarify the direct action of IL-17 in a series of solid tumor human cells, from glioma, esophageal carcinoma and bladder cancer.

Materials and Methods

Cell lines and cell culture

GL261 mouse glioma cells, M059J and U138 human glioma cells and T24 human bladder transitional cell carcinoma cells were obtained from ATCC (Rockville, Maryland, USA). OE-21 human esophageal squamous cell carcinoma was a gift from INCA (Rio de Janeiro, Brazil). M059J and U138 were cultured in DMEM/10% FBS and OE-21 and T24 were cultured in RPMI/10% FBS at a temperature of 37°C, a minimum relative humidity of 95%, and an atmosphere of 5% CO2 in air.

Analysis of IL-17RA mRNA expression

Cells (2 x 105) were collected and processed as previously described [9]. Briefly, total RNA was isolated using Trizol LS reagent (Invitrogen) and cDNA species synthesized with ImProm- II™ Reverse Transcription System (Promega). Quantitative PCR, using SYBR Green I as intercalating dye, was performed for IL-17RA (F: 5’-GCCCTGGACAGGTTCCGGGACTG-3’; R: 5’-CCCCTCCTCTGCGGCGAGCAC-3’) and for 18S and β-actin as reference genes. Relative mRNA levels were determined using 2-ΔΔCT method including individual efficiency calculated per sample using LinReg 11.0 Software

Cell viability and proliferation

For measuring cell viability and proliferation, we performed MTT assay and cell counting, respectively, as previously described [9]. Cells were treated with IL-17A (10, 20, 50, 100 pg/mL and 10, 20, 100 ng/mL) for 24 h.

Wound-healing migration assay

Cells were seeded in medium containing 10% FBS at 3x105 cells per well in 24-well plates. In order to minimize cell proliferation, cell cultures were grown to 80-90% confluence and deprived of serum medium for 18 h. The objective was to obtain a reproducible measurement of the migration of the wound edge towards the wound space with minimum cell proliferation, but without loss of cell viability over the experimental period. Wounds were made by sterile pipette tips and remaining cells were washed twice with CMF to eliminate detached cells and it was added DMEM/RPMI deprived serum medium. Cells were treated with IL-17A (10 ng/ml) and then incubated at 37°C. After 48 h, migrating cells at the wound front were photographed by Olympus inverted microscope IX71 (Tokyo, Japan) with a magnification×100 and compared. The cell-free area was correlated with tumor cell ability to migrate into the scratch. TScratch software was used to calculate the cell-free area. Three independent experiments were performed.

Intracellular signaling pathways activated by IL-17

In order to analyze IL-17 intracellular signaling activation, tumor cells were treated with IL-17A (10 ng/ml) for 1, 15 and 30 min and then processed according to the manufacturer’s instructions using anti-AKT, anti-p38 MAPK and anti-ERK 1/2 antibodies (BD Phosflow, BD Biosciences). Cells were analyzed on FACSCanto II Flow Cytometer (BD Biosciences) and the results were analyzed using FlowJo Software (Tree Star).

Statistical analysis

Data were analyzed by one-way analysis of variance (one-way ANOVA), followed by Tukey post-hoc test, using GraphPad Software version 5.0 (San Diego, CA, USA.). p values <0.05 were taken as statistically significant.

Results and Discussion

Human glioma, esophageal and bladder tumor cells express IL-17 receptor (IL-17RA) but their viability and proliferation are unaltered by exogenous IL-17A

In this study, we demonstrated, for the first time, that M059J and U138 human glioma cells, 0E-21 human esophageal cancer cells and T24 human bladder cancer cells express IL-17A receptor (IL- 17RA) (Figure 1A). A previous study has already shown the IL-17RA expression on GL261 cells [10]. When we tested the effects of different concentrations of IL-17 on these tumor cell lines, the exogenous IL- 17 had no direct effect on the tumor cells proliferation or viability in vitro (Figure 1B and Figure 1C). These results are in agreement with studies performed with GL261 mouse glioma cell line [10] and human hepatocarcinoma cell lines [11]. Another study showed that recombinant IL-17 protein or retroviral transduction of IL-17 gene into tumors did not affect in vitro proliferation, but in vivo–-IL-17- transfected cells grew more rapidly when compared with controls [12].

Citation:Gehring MP, Pereira TCB, Zanin R, Bogo M and Morrone FB. IL-17a Prompts Human Tumor Cells Migration and Akt, Erk, P38 Phosphorylation in Vitro. Austin Pharmacol Pharm. 2016; 1(1): 1003.