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Research article
IL-17 affects progression, metastasis, and recurrence of laryngeal cancer via the inhibition of apoptosis through activation of the PI3K/AKT/FAS/FASL pathways
Jianming Yang
Second Affiliated Hospital of Anhui Medical University
Corresponding Author
ORCiD: 0000-0002-2823-7339
Abstract
Background. Cytokines play important roles in development and prognosis of laryngeal cancer (LC). Interleukin-17 (IL-17) from a distinct subset of CD4 + T-cells may significantly induce cancer-elicited inflammation to prevent cancer cells from immune surveillance. Methods. The expression levels of IL-17 were examined among 60 patients with LC. Immunofluorescence co-localization experiments were performed to verify the localization of IL-17 and FAS/FASL in Hep-2 and Tu212 cells. IL-17 was silenced for expression in LC cell lines by siRNA techniques for determination of the role of IL-17 in LC. Results. In our LC patients, cytokines were dysregulated in LC tissues compared with normal tissues. We found that IL-17 was overexpressed in a cohort of 60 LC tumors paired with non-tumor tissues. Moreover, high IL-17 expression was significantly associated with advanced T category, late clinical stage, differentiation, lymph node metastasis, and disease recurrence. In addition, the time-course expression of FAS and FASL was observed after stimulation and treatment with IL-17 stimulator. Finally, in vitro experiments demonstrated that IL-17 functioned as an oncogene by inhibiting the apoptosis of LC cells via the PI3K/AKT/FAS/FASL pathways. Conclusions. Taken together, our findings for the first time demonstrate the role of IL-17 as a tumor promoter and a pro-metastatic factor in LC, indicating that IL-17 may have an oncogenic role and serve as a potential prognostic biomarker and therapeutic target in LC.
Keywords
IL-17, apoptosis, recurrence, metastasis, laryngeal cancer, PI3K/AKT/FAS/FASL pathway, biomarkers, prognosis
Figures
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Background
The development of laryngeal carcinoma (LC) has increased recently and is the second most common malignant tumor of the head and neck. It is the sixth most common tumor worldwide, with a 5-year survival rate of approximately 50% [1]. Although advances in treatment including surgery, in combination with radiotherapy and chemotherapy, adjuvant combination chemotherapy and molecular targeting are emerging as more effective therapeutic options for advanced LC [2, 3].
IL–17 (IL–17A) is one of members in IL–17 family, including IL–17B, IL–17C, IL–17D, IL–17E (IL–25), and IL–17F. IL–17F had the highest degree of homology to IL–17A in IL–17 family. T-helper cell 17 secreting cells (Th17) is the main source of IL–17, while other cell types also develop this cytokine, such as group 3 innate lymphoid cells (ILC3), δγT cells, natural killer (NK) cells, etc [4–6].Some evidence suggests that IL–17 is a key proinflammatory cytokine for induction of cytokines and chemokines secretion by other different cell types, among which mesenchymal cells and myeloid cells can recruit monocytes and neutrophils for inflammation [7, 8]. In addition, much evidence demonstrated that IL–17 markedly causes tumor growth and angiogenesis, indicating IL–17 play a role in tumor promotion [9]. IL–17 activated the Src/PI3K/Akt/nuclear factor-κB (NF-κB), MAPK, Stat3, and COX–2 pathways, which play significant roles in tumorigenesis, angiogenesis and metastasis [10, 11]. Studies showed that IL–17 was overexpressed in some human tumors, such as cervical cancer, breast cancer, gastric cancer, colorectal cancer, etc [12–18].
Fas, a type I membrane protein, transmits a suicide signal to the cell, binds to its ligand (FasL) or anti-Fas antibodies, and leads to caspase 8-dependent cell death [19, 20]. The PI3K/AKT pathway play an important role in regulation of cellular processes, which control cell size/growth, proliferation, survival, glucose metabolism, genome stability, and neo-vascularization. It was demonstrated that Forkhead box O3 (also known as FOXO3) encoded by the FOXO3 inhibited the FasL gene promoter [21, 22]. Furthermore, Akt-mediated phosphorylation of FOXO3 more likely favors cellular survival via enhancing the retention of FOXO3 in the cytoplasm [23, 24].
Given the roles of IL–17 in transducing multiple signals in cells, IL–17 also activates Src to promote cancer development [6]. For example, PI3K/Akt signaling pathway is one of major Src-activating pathways and may block the FAS-associated death domain protein to inhibit cellular apoptosis [25, 26]. Thus it is likely that IL–17 exerts its function through the Fas and Fasl signaling pathways [27, 28].To date, little is known about the roles of IL–17 in LC, and the regulation of IL–17 in LC have not been fully investigated. This study was aimed to characterize its function in LC both in vitro and in vivo.
Methods
Study patients
A total of 60 patients (ages 22 to 88 years old, 35 female cases, and 25 male cases), who were pathologically diagnosed with LC from January 2014 to December 2017 from our hospital, were included in this study. The patients’ tumor tissues were collected from several groups with different degree of differentiation. All these patients received surgery only and were confirmed with LC by pathological examination at the Second Hospital of Anhui Medical University. All patients in this study met the criteria of the World Health Organization (WHO) Histological Classification of Tumors of the Gallbladder (2003). Tumor staging was assessed according to the TNM staging guidelines by International Union Against Cancer (UICC). The signed written consents were obtained from each patient and the study was approved by the institutional research board of ethical committee at the Second Hospital of Anhui Medical University.
Quantitative real-time PCR
Total RNA from LC tissues and LC cells was isolated using TRIzol reagent. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan) and Mir-X™ miRNA first-strand synthesis (Takara) were used to synthesize cDNA from mRNA and miR, respectively. The qRT-PCR was performed using the iQ5 Real Time PCR System (Bio-Rad, Hercules, CA, USA) with the SYBR Premix Ex Taq™ (Takara, Japan). The cycle threshold value was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold. The cycle threshold value of each product was determined and normalized against that of the internal control. All measurements were performed in triplicate. Data were analyzed using the 2 -ΔΔCT method (Table 1)..
Cell culture
In vitro studies were performed on an immortalized human laryngeal epidermoid carcinoma cells (Hep–2) and human laryngeal carcinoma cells (Tu212) obtained from the American Type Culture Collection. Recombinant Human IL–17A (200–17) was the product of Peprotech Company (Peprotech, Hamburg, Germany). Z-IETD-FMK was obtained from Abcam (Cambridge, MA, USA). The PI3K inhibitor, Wortmannin, was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All cells were incubated at 37°C with 5% CO 2 in DMEM containing 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, and 5000 IU/mL penicillin/5000 g/mL streptomycin for 1 to 2 days before starting experiments.
Cell transfection
Hep–2 cells and Tu212 cells were cultured in 60-mm plates or 100-mm plates (for western blotting analysis) without antibiotics overnight. When the cells reached 70–80% confluency, they were transiently transfected with IL–17 siRNA and scramble siRNA (Genepharma, Shanghai, China) using Lipofectamine RNAiMAX (add the source) according to the manufacturer’s recommendations.
Flow cytometry
The cells at the logarithmic phase were inoculated into fresh culture medium and cultured in a 37°C and 5% CO2 incubator for 48 hours. Then the cells were digested with trypsin to obtain a cell suspension, which was centrifuged at 1000 rpm for 5 minutes to allow the collection of the cells. After the cells were washed with pre-cooled PBS twice, 1 mL of PBS was used to re-suspend the cell suspension that was divided into 2 tubes (0.5 mL/tube), both of which were centrifuged in order to remove the supernatant. One of the tubes was re-suspended using 500 μL of binding buffer with cell cycle kit (Beyotime Institute of Biotechnology, Shanghai, China). Next, 1 mL of pre-cooled 70% ethyl alcohol was added to fix the cells at 4°C for 24 hours, the cell suspension was centrifuged to move the fixation solution, washed in pre-cooled PBS twice, centrifuged to move the supernatant and added with propidium iodide (PI). Finally, the cell cycle was determined using a flow cytometer. The other tube of cell suspension was re-suspended with 500 μL of binding buffer from the cell apoptosis kit (Beyotime Institute of Biotechnology, Shanghai, China), mixed with 5 μL of Annexin V-FIFC and, cultured at 2 ~ 8°C for 15 min in the dark. Then another 15 μL of Annexin V-FIFC was added, the suspension was cultured again at 2 ~ 8°C for 15 min in the dark. Within 2 hours, the cell cycle was determined using a flow cytometer.
Immunofluorescence co-localization assay
For fluorescence staining, cells were fixed with 40 g/L formaldehyde, permeabilized with 0.1% Triton X–100 in PBS, and blocked with 1% BSA in PBS for 30 min, followed by incubation overnight at 4oC with both anti-flag and anti-RhoC antibodies. The cells were washed three times with PBS for 5 min, incubated with DyLight™ 488 conjugated Goat anti-Mouse IgG along with DyLight™ 549 conjugated Goat anti-Rabbit IgG for 30 min, and then nuclear stained with 1 mg/L 4, 6-diamidino–2-phenylindole (DAPI, Roche, Germany). The fluorescence images were acquired with an Olympus FV1000 confocal microscope (Olympus, Japan) using a 100 × oil immersion objective.
TUNEL
Apoptosis within populations of transplanted cells was detected using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. One day after transplantation, the apoptosis of transplanted ADMSCs was detected in the peri-infarct zone using a TUNEL assay kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were counted in one brain tissue section of each animal (n = 5 per group). The number of double staining-positive (red and green fluorescence) cells was counted in a minimum of 10 microscopic fields based on their nuclear morphology, and dark color was quantified using a 40× objective and Image-Pro image analysis software.
Western Blotting
Laryngeal carcinoma tissue and paracancerous tissues samples were excided in operation and flash-frozen at –80oC until use. In order to prepare lysates, frozen laryngeal carcinoma tissues were minced with eye scissors on ice. Then they were homogenized in lysis buffer (1% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), 1 % Triton X–100, 1 %NP–40, pH 7.5, 5 mmol/l EDTA, 50 mmol/l tris, 1μg/ml leupeptin, 10μg/ml aprotinin, and 1 mmol/l PMSF) and centrifuged at 12,000 rpm at 4oC for 20 min to collect the supernatant. Cell cultures for immunoblot were lysed with sodium lauryl sulfate loading buffer and stored at –80 oC until use. The collected supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after determining the protein concentration with the Bradford assay (Bio-Rad). After the separated proteins were transferred to polyvinylidene difluoride filter (PVDF) membranes, the membranes were blocked with 1% (w/v) bovine serum albumin (BSA) for 2 h and incubated with primary antibodies against IL–17 (anti-rabbit, 1:1,000; Abcam), IL–17R (anti-mouse, 1:1,000; Sigma), FAS (anti-rabbit, 1:1,000; Cell Signaling), FASL (anti-rabbit, 1:1,000; Cell Signaling), and β-actin (anti-rabbit, 1:1,000, Abgent) at 4oC overnight. At last, the membranes were incubated with second antibody at 37 oC for 2 h. Bound proteins were scanned by using Chemi Doc XRS (Hercules, CA).
Statistical analysis
The data was expressed as the mean ± standard deviation. Values in tables and figures were given as means and standard deviation of the mean if not otherwise indicated. The analysis of variance (ANOVA) and Student’s t-test were used in the SPSS software to determine significant differences between groups. A χ2 test was used to evaluate the associations between clinicopathologic variables and IL–17 protein expression. The correlation between the mRNA levels of IL–17 and FAS and FASL in LC tissue was analyzed with Pearson’s correlation analysis. Kaplan-Meier survival analysis and log-rank tests were used to calculate survival curves. The Cox proportional hazard regression model was used to estimate overall survival. The values of p less than 0.05 were considered to be statistically significant. The data were analyzed using the SPSS 20.0 statistical software program (version 20.0; IBM Corporation, Armonk, NY, USA).
Results
Association of increased IL–17 expression with tumor progression in LC patients
As shown in Fig. 1A, the Western blotting in different pathological grades of LC tissues showed that the expression levels of IL–17and IL–17 protein increased significantly with increasing severity of LC. These findings were consistent with P-AKT, and P-PI3K levels of protein expression, while the expression of FAS and FASL in the LC tissues decreased significantly. The expression of PI3K and AKT had no significant expression changes compared with paracancerous tissues of normal control group (Fig. 1B).. In hematoxylin and eosin assay (Fig. 1C), paracancerous tissues demonstrate keratosis and superficially invasive LSCC with pushing invasive tumor. Additionally, front and band-like dense inflammatory infiltrate (Fig. 1C-a),, high differentiation group tissue demonstrate tumor infiltrating lymphocytes in nonkeratinizing LSCC (Fig. 1C-b),, medium differentiation tissues show keratinizing invasive LC with more intense lymphocytic host response (Fig. 1C-c), superficially invasive keratinizing LC of the poorly differentiation tissues demonstrate minimal lymphocytic host response (Fig. 1C-d), The hematoxylin and eosin assay demonstrate more lymphocytic infiltration, with the decrease of degree of differentiation in LC. Moreover, the mRNA expression levels of IL–17、IL–17R、FAS, and FASL were determined by qRT-PCR by different pathological grades of LC tissues in poorly differentiated, medium differentiated, and high differentiated groups, respectively. The mRNA expression of IL–17、IL–17R was significantly increased in LC tissues, while FAS and FASL decreased markedly with the decrease of degree of differentiation (Fig. 2A)..
Correlation analysis of IL–17 and FAS and FASL expression
Pearson’s correlation analysis showed that the expression of IL–17 mRNA had a significant negative correlation with the expression of FAS and FASL mRNA levels，the higher the IL–17 expression, the lower the FAS and FASL expression, and the more severe the LC (r = –0.82 for FAS, r = –0.847 for FASL, and all P < 0.01, Fig. 2B)..
Associations between IL–17 protein expression and clinical parameters of patients with LC
The expression of IL–17 in LC tissues was associated with TNM stage, T stage, lymph node metastasis (LNM), and differentiation (all P < 0.05). The results showed that the elevated IL–17 expressions was observed in the patients with LNM, low degree of differentiation, late overall TNM stage, and advanced T3/T4 stage, while no significant associations of IL–17 expression with patients’ age, gender, and tumor sites (all P > 0.05) (Table 2)..
Associations between IL–17 protein expression and recurrence of LC patients
The IL–17 protein expression distributions and associated survival among the patients are shown in Table 3. Among 60 LC patients, 15 cases were found to have recurrence or metastasis with a median of 3 years of follow up. The low IL–17 protein expression was significantly associated with better disease-free survival than the high expression of IL–17 protein (log-rank P = 0.022, Fig. 2C).. Cox regression analysis was performed to adjust for other important confounders, including age, sex, tumor size, stage, LNM, and differentiation (Table 3). After adjustment for these confounders, the patients with high expression of IL–17 had an approximately 3-fold increased risk of disease recurrence (HR, 3.29, 95% CI, 2.73–8.75) than the patients with the low expression of IL–17, indicating that IL–17 expression was an independent factor for disease influence of the patients with LC.
IL–17 induces FAS and FASL expression via activation of PI3K/AKT pathways
To determine whether IL–17 and FAS/FASL proteins are distributed in the same cellular location, we performed immunofluorescence co-localization experiments with a confocal laser scanning microscope to confirm the localization of IL–17 and FAS/FASL in Hep–2 and Tu212 cells. Our confocal microscope analysis revealed that the IL–17 fusion protein co-localized with the FAS/FASL fusion protein in the membrane of Hep–2 and Tu212 cells (Fig. 3).. In vitro recombinant Human IL–17A (200–17) significantly promoted expression of IL–17 in LC tissues, which enhanced FAS and FASL expression in LC cell lines after a 6h treatment, while Wortmannin simultaneously abrogated the increase of the phosphorylated PI3K. The time-course for expression of FAS and FASL in LC cell lines with stimulation by 200–17(50 ng /ml) following pre-treatment with Wortmannin (10µM/L) was observed. In Fig. 4A, we found that Wortmannin simultaneously abrogated the decrease of FAS and FASL induced by 200–17, and the expression of FAS and FASL increased and peaked at 6h and 8h after stimulation with 200–17 following pre-treatment with Wortmannin in Hep–2 cells. Furthermore, Wortmannin promoted 200–17-elicited downregulation of phosphorylated FOXO3 protein expression. The similar findings were observed in Tu212 cells (Fig. 4B).. Therefore, these results suggested that the activation of PI3K/AKT pathways may play an important role in IL–17-mediated reduction of FAS and FASL expression.
IL–17 inhibited apoptosis through the FAS/FASL Pathway in Hep–2 and Tu212 cells
To confirm whether the effects of IL–17 were FAS/FASL-dependent, we performed the assays with treatment by Z-IETD-FMK, which is a specific inhibitor of caspase–8. The caspase–8 phosphorylation was inhibited by using Z-IETD-FMK. The protein expression levels of cleavage of caspase–8 and cleavage of caspase–3 were evaluated after treatment with 40 µM of Z-IETD-FMK both with and without siRNA-IL–17. As shown in Fig. 5A, The Z-IETD-FMK treatment abrogated the siRNA-IL–17-induced promoting of cleavage of caspase–8 and cleavage of caspase–3 protein expression in Hep–2 cells. Additionally, cleavage of caspase–8 and cleavage of caspase–3 protein expression was decreased markedly by Z-IETD-FMK treatment in Scramble group, while cleavage of caspase–8/3 did not show lower level in combination with siRNA-IL–17.The similar findings were observed in Tu212 cells7(Fig. 5B).. These results show that the effects of IL–17 may inhibit apoptosis through the FAS/FASL Pathway.
siRNA silencing IL–17 gene expression promotes cell apoptosis
To determine the role of IL–17 in LC, we silenced IL–17 gene expression in LC cell lines using siRNA techniquesto evaluate the effects of IL–17 on apoptosis in both Hep–2 and Tu212 cells by flow cytometry. The RT-PCR results revealed that there was an approximately 63% decrease of IL–17 mRNA in the stable cell line of LC cells transfected with siRNA compared with that in the controls. In addition, siRNA led to an approximately 73% reduction of IL–17 protein expression by Western blot as shown in Fig. 6A. Furthermore, we found that siRNA-IL–17 promoted Hep–2 cell apoptosis (15.0%±2.78 compared to 8.20%±3.57 in controls). Similarly, siRNA-IL–17 promoted Tu212 cell apoptosis (12.90% ± 4.58 compared to 8.50% ± 2.37 in controls) as shown in Fig. 6B. In addition, the apoptotic cells were detected by the TUNEL assay, siRNA-IL–17 pretreatment dramatically increased the number of apoptotic cells,staining showed that the number of apoptotic the siRNA-IL–17 group was 3.19 times higher than that of apoptotic controls group (1323.02 ± 278.71 vs. 415.06 ± 68.79, p<0.01) as shown in Fig 6C and Fig 6D. These results show that silenced IL–17 expression may be involved in promoting LC cell apoptosis.
siRNA-IL–17 regulated PI3K/AKT/FAS/FASL pathways in Hep–2 cells and Tu212 cells
IL–17 may mediate apoptosis of LC cells via activation of downstream PI3K/AKT/FAS/FASL pathways. To further examine the mechanism by which siRNA-IL–17 induces apoptosis, we detected the protein levels of PI3K、P-PI3K、AKT、P-AKT、FAS and FASL by Western blot in siRNA-IL–17 transfected LC cell line and controls. P-PI3K and P-AKT activity was significantly inhibited by IL–17 knockdown in Hep–2 cells and Tu212 cells, while FAS and FASL were markedly elevated in the IL–17 siRNA group compared with those in the controls as shown inFig. 7. These results indicate that silencing of IL–17 expression mediated apoptosis via the PI3K/AKT/FAS/FASL pathways in LC cells.
Discussion
LC is one of the most common malignant head and neck tumors, accounting for 1–2.5% of all malignancies throughout the body [29, 30]. With the advance in treatment and management of LC, the prognosis has significantly improved, while the survival of this tumor remains poor, with a 5-year overall survival less than 60%. In particular, the prognosis for advanced stage and recurrent tumors of this disease are still poor after treatment by surgery and the chemoradiotherapy becomes the major choice for treatment. So far, the efficacy of chemoradiotherapy still remains limited [31, 32]. Our clinical data on patients showed that cytokines are dysregulated in LC tissues as compared with normal tissues. Our previous study also found that well-differentiated tumor tissue had high mRNA expression levels of IL–12Rβ2 and INF-γ, whereas moderately and poorly differentiated tumor tissues had low levels of these markers [33]. COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients [34].The results from this current study may support the theory that IL–17 depletion may diminish LC cell mitogenic signaling and promote apoptosis. Targeting IL–17 may therefore be a therapeutically useful way to treat LC.
In this study, we explored the effects of IL–17 on apoptosis of LC cells and the role that IL–17 acts as a biomarker in the recurrence of this disease, metastasis and prognosis of LC patients. The tumor microenvironment is infiltrated by a wide array of cells from both adaptive and innate immune systems, and hematoxylin and eosin assays demonstrate more lymphocytic infiltration, with the decrease of degree of differentiation in laryngeal carcinoma. We found that IL–17 expression was increased in different pathological grades of LC tissues compared with paracancer tissues of normal controls in both levels of mRNAs and proteins. We found that the expression of IL–17 and IL–17R mRNA in the tumor group was significantly higher than that in the control group. These findings were consistent with those levels of protein expression. The induced levels of expressions of IL–17、IL–17R、P-AKT and P-PI3K protein significantly increased with increasing severity of LC. While, the expression of FAS and FASL in the LC tissues decreased significantly. Previous studies reported that the patients with early stage laryngeal carcinoma exhibited a lower level of IL17 mRNA expression than those with advanced stages, and cancer tissues exhibited a significantly higher level of IL17 mRNA expression than pericarcinoma tissues [35, 36]. Overexpression of both IL17 and IL17R was observed in tumors compared with that in adjacent tissues [37, 38]. Furthermore, our results showed that IL–17 and IL–17R expression in LC tissue was correlated with tumor size, TNM stage, degree of differentiation in LC, and LNM. Our results may help demonstrate that expression of IL–17 was a significantly independent factor that may contribute to the recurrence of LC patients after adjustment with other significant confounders including tumor size and TNM stage [39–41]. A previous study also has showed that increased IL–17 expression has correlation with the poor overall survival in different other types of cancers [42].
FAS and FASL are members of the tumor necrosis factor (TNF)-receptors and TNF family, respectively. The ligation of FAS with FASL results in the activation of a caspase cascade that initiates apoptosis [43–45]. With the aggravation of LC, the expression of FAS and FASL in LC tissues markedly decreased, suggesting a restraining in FAS/FASL pathways due to accelerated apoptosis of LC cells. We found that mRNA levels of FAS and FASL increased in cancer tissues compared to control with increasing severity of LC, which is not consistent with the protein expression. Several reports have shown that mRNA and protein is not always strictly linear, but has a more intrinsic and complex dependence. Different regulatory mechanisms (such as synthesis and degradation rates) may play roles in both the synthesized mRNA and the synthesized protein [46–48]. In this study, our data showed that IL–17 expression had a positive correlation with the expression of FAS and FASL (r = 0.820, P < 0.01). A previous study highlighted IL–17 to be a potential prognostic marker for patients with respiratory, digestive and other system cancers [14]. In addition, IL–17 may be significantly correlated with the differentiation and angiogenesis in the development of LC as the expression of CXCL9, CXCL10, and IL–17 mRNAs in the skin in FAS- and FASL-deficient mice was decreased [28, 37, 38]. In this study IL–17 fusion protein co-localized with the FAS/FASL fusion protein in the membrane of Hep–2 and Tu212 cells. IL–17 activates PI3K/Akt signaling pathway which may block the FAS-associated death domain protein, which inhibits the cellular apoptosis pathway by improving the phosphorylation of FOXO3a [49, 50]. Recombinant Human IL–17A (200–17) had a growth stimulating effect on IL–17 [51, 52]. Wortmannin covalently modifies PI3K and is a potent and specific PI3K inhibitor [53–55]. Our results showed that the expression of FAS and FASL increased after stimulation with the 200–17, an IL–17 stimulator, following pre-treatment with Wortmannin in Hep–2 cells and Tu212 cells. Furthermore, Wortmannin promoted 200–17-elicited downregulation of P-FOXO3 protein expression. Among cell death receptors, the Fas/FasL system provides an important apoptotic mechanism [53, 56]. Fas-mediated apoptosis following death receptor stimulation, leads to cleavage of procaspase–8，subsequent activation of a downstream caspase, such as caspase–3, which induces apoptosis [57, 58]. When cells were treated with siRNA-IL–17, Z-IETD-FMK resulted in a greater decrease in cleavage of caspase–8 and cleavage of caspase–3 protein expression, compared with treatment with siRNA-IL–17 alone.These results suggested that IL–17 stimulation might restrain apoptosis in vitro by inhibiting FAS/FASL pathways via activating PI3K/AKT pathways.
Accumulating studies have demonstrated that IL–17 acts as a tumor suppressor and promotes cell proliferation, migration, and invasion [59, 60]. However, little has been reported about the mechanism by which IL–17 is involved in the progression of LC. To further investigate the role of IL–17 in mitogenic effects on LC cells, in the present study we silenced endogenous IL–17 by siRNA to determine the effect of endogenous IL–17 in LC cells. We found that downregulation of IL–17 expression inhibited PI3K/AKT pathway, which in turn significantly suppressed the activation of FAS/FASL pathway, leading to reduced apoptosis of LC cells.Our current finding might indicate that silenced IL–17 expression significantly promote LC cell apoptosis via the PI3K/AKT/FAS/FASL pathway.
Conclusions
In summary, the present investigation provides further evidence for the involvement of IL–17 in the progression of LC. Aberrant expression of IL–17 contributes to the pathogenesis of LC. Interfering the expression of IL–17 at the posttranscriptional level could inhibit the apoptosis of LC cell lines via the activation of FAS/FASL pathway, suggesting that IL–17 can be targeted for therapeutic intervention against LC. Further studies are required to elucidate the mechanisms and diverse signaling networks of IL–17-related in LC.
Declarations
Ethics approval and consent to participate: The signed written consents were obtained from each patient and the study was approved by the institutional research board of ethical committee at theSecond Hospital of Anhui Medical University.
Consent for publication: Not applicable.
Availability of data and material: All data generated or analyzed during this study are included in this published article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Conflict of Interest Statement: The authors declare that they have no competing interests.
Funding: This his research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Author’s contributions: YS, MY, JZ, YS, and YT carried out the majority of the experiment, data analysis, and wrote the manuscript. They were all helped by HL, JZ, YL, and JY. All authors reviewed and approved the final manuscript.
Acknowledgements: None.
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Tables
Table 1. Primer sequences of IL-17、IL-17R、FAS、FASL, and GAPDH
|
Primers used |
Forward |
Reverse |
|
IL-17 |
5'-GACCCTTCACCCCTCACC-3' |
5'-TTATGGATCATGCCCACAAG-3' |
|
IL-17R |
5′-GG GATTACA GGCGTGAGCCA-3′ |
5′-GCGGTCTGGTTATCGTCTAT-3′ |
|
FAS |
5´-GGTGCTGTCTCTCTATGCCT CTGGA-3´ |
5´-GGTGCTGTCTCTCTATGCCT CT GGA-3´ |
|
FASL |
5´-AAGGACCTCCAGCATCACTGTGTCA-3´ |
5´-CCTTCAGAGCCCGCAGCT TC CACG T-3´ |
|
GAPDH |
5'- AAAGTCCGCCATTTTGCCACT-3' |
5'-CCAAATCGTTAGCGCTCCTT-3' |
Table 2. Correlations of IL-17 expression with clinical characteristics in patients with LC
|
Clinical characteristics |
n |
IL-17 expression |
P |
|
|
Gender |
|
|
|
0.280 |
|
|
Male |
35 |
3.89 ± 3.28 |
|
|
|
Female |
25 |
4.28 ± 3.42 |
|
|
Age |
|
|
|
0.321 |
|
|
< 50 |
22 |
5.48 ± 2.74 |
|
|
|
≥ 50 |
38 |
4.29 ± 3.37 |
|
|
TNM stage |
I/II |
27 |
4.82 ± 0.53 |
0.007 |
|
|
III/IV |
33 |
5.36 ± 3.24 |
|
|
Degree of differentiation |
|
|
|
0.009 |
|
|
Middle/high |
42 |
4.58 ± 3.27 |
|
|
|
Low |
18 |
6.17 ± 3.37 |
|
|
LNM |
|
|
|
< 0.0001 |
|
|
Yes |
38 |
5.63 ± 3.16 |
|
|
|
No |
22 |
3.15 ± 2.37 |
|
|
Tumor site |
|
|
|
0.285 |
|
|
Glottis |
26 |
3.47 ± 2.31 |
|
|
|
Supraglottis |
20 |
2.25 ± 3.43 |
|
|
|
Subglottis |
4 |
3.54 ± 3.22 |
|
|
T classification |
Tis |
8 |
2.04 ± 4.13 |
0.013 |
|
|
T1/T2 |
23 |
3.61 ± 4.13 |
|
|
|
T3/T4 |
29 |
4.75 ± 2.36 |
|
Table 3. Association of IL-17 expression with disease recurrence among the patients with LC (N = 60)
|
IL-17 expression |
Recurrence number/all patients’ number |
aHR1 (95% CI) |
P |
|
Low2 |
8/38 |
1.00 |
|
|
High |
7/22 |
3.29 (2.73-8.75) |
0.028 |
1Hazard ratio adjusted for age, sex, tumor size, stage, LNM, and differentiation.
2Reference group (The median of expression as cut off point in patient without recurrence).
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Research article
IL-17 affects progression, metastasis, and recurrence of laryngeal cancer via the inhibition of apoptosis through activation of the PI3K/AKT/FAS/FASL pathways
Jianming Yang
Second Affiliated Hospital of Anhui Medical University
Corresponding Author
ORCiD: 0000-0002-2823-7339
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Abstract
Background. Cytokines play important roles in development and prognosis of laryngeal cancer (LC). Interleukin-17 (IL-17) from a distinct subset of CD4 + T-cells may significantly induce cancer-elicited inflammation to prevent cancer cells from immune surveillance. Methods. The expression levels of IL-17 were examined among 60 patients with LC. Immunofluorescence co-localization experiments were performed to verify the localization of IL-17 and FAS/FASL in Hep-2 and Tu212 cells. IL-17 was silenced for expression in LC cell lines by siRNA techniques for determination of the role of IL-17 in LC. Results. In our LC patients, cytokines were dysregulated in LC tissues compared with normal tissues. We found that IL-17 was overexpressed in a cohort of 60 LC tumors paired with non-tumor tissues. Moreover, high IL-17 expression was significantly associated with advanced T category, late clinical stage, differentiation, lymph node metastasis, and disease recurrence. In addition, the time-course expression of FAS and FASL was observed after stimulation and treatment with IL-17 stimulator. Finally, in vitro experiments demonstrated that IL-17 functioned as an oncogene by inhibiting the apoptosis of LC cells via the PI3K/AKT/FAS/FASL pathways. Conclusions. Taken together, our findings for the first time demonstrate the role of IL-17 as a tumor promoter and a pro-metastatic factor in LC, indicating that IL-17 may have an oncogenic role and serve as a potential prognostic biomarker and therapeutic target in LC.
Figures
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Background
The development of laryngeal carcinoma (LC) has increased recently and is the second most common malignant tumor of the head and neck. It is the sixth most common tumor worldwide, with a 5-year survival rate of approximately 50% [1]. Although advances in treatment including surgery, in combination with radiotherapy and chemotherapy, adjuvant combination chemotherapy and molecular targeting are emerging as more effective therapeutic options for advanced LC [2, 3].
IL–17 (IL–17A) is one of members in IL–17 family, including IL–17B, IL–17C, IL–17D, IL–17E (IL–25), and IL–17F. IL–17F had the highest degree of homology to IL–17A in IL–17 family. T-helper cell 17 secreting cells (Th17) is the main source of IL–17, while other cell types also develop this cytokine, such as group 3 innate lymphoid cells (ILC3), δγT cells, natural killer (NK) cells, etc [4–6].Some evidence suggests that IL–17 is a key proinflammatory cytokine for induction of cytokines and chemokines secretion by other different cell types, among which mesenchymal cells and myeloid cells can recruit monocytes and neutrophils for inflammation [7, 8]. In addition, much evidence demonstrated that IL–17 markedly causes tumor growth and angiogenesis, indicating IL–17 play a role in tumor promotion [9]. IL–17 activated the Src/PI3K/Akt/nuclear factor-κB (NF-κB), MAPK, Stat3, and COX–2 pathways, which play significant roles in tumorigenesis, angiogenesis and metastasis [10, 11]. Studies showed that IL–17 was overexpressed in some human tumors, such as cervical cancer, breast cancer, gastric cancer, colorectal cancer, etc [12–18].
Fas, a type I membrane protein, transmits a suicide signal to the cell, binds to its ligand (FasL) or anti-Fas antibodies, and leads to caspase 8-dependent cell death [19, 20]. The PI3K/AKT pathway play an important role in regulation of cellular processes, which control cell size/growth, proliferation, survival, glucose metabolism, genome stability, and neo-vascularization. It was demonstrated that Forkhead box O3 (also known as FOXO3) encoded by the FOXO3 inhibited the FasL gene promoter [21, 22]. Furthermore, Akt-mediated phosphorylation of FOXO3 more likely favors cellular survival via enhancing the retention of FOXO3 in the cytoplasm [23, 24].
Given the roles of IL–17 in transducing multiple signals in cells, IL–17 also activates Src to promote cancer development [6]. For example, PI3K/Akt signaling pathway is one of major Src-activating pathways and may block the FAS-associated death domain protein to inhibit cellular apoptosis [25, 26]. Thus it is likely that IL–17 exerts its function through the Fas and Fasl signaling pathways [27, 28].To date, little is known about the roles of IL–17 in LC, and the regulation of IL–17 in LC have not been fully investigated. This study was aimed to characterize its function in LC both in vitro and in vivo.
Methods
Study patients
A total of 60 patients (ages 22 to 88 years old, 35 female cases, and 25 male cases), who were pathologically diagnosed with LC from January 2014 to December 2017 from our hospital, were included in this study. The patients’ tumor tissues were collected from several groups with different degree of differentiation. All these patients received surgery only and were confirmed with LC by pathological examination at the Second Hospital of Anhui Medical University. All patients in this study met the criteria of the World Health Organization (WHO) Histological Classification of Tumors of the Gallbladder (2003). Tumor staging was assessed according to the TNM staging guidelines by International Union Against Cancer (UICC). The signed written consents were obtained from each patient and the study was approved by the institutional research board of ethical committee at the Second Hospital of Anhui Medical University.
Quantitative real-time PCR
Total RNA from LC tissues and LC cells was isolated using TRIzol reagent. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan) and Mir-X™ miRNA first-strand synthesis (Takara) were used to synthesize cDNA from mRNA and miR, respectively. The qRT-PCR was performed using the iQ5 Real Time PCR System (Bio-Rad, Hercules, CA, USA) with the SYBR Premix Ex Taq™ (Takara, Japan). The cycle threshold value was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold. The cycle threshold value of each product was determined and normalized against that of the internal control. All measurements were performed in triplicate. Data were analyzed using the 2 -ΔΔCT method (Table 1)..
Cell culture
In vitro studies were performed on an immortalized human laryngeal epidermoid carcinoma cells (Hep–2) and human laryngeal carcinoma cells (Tu212) obtained from the American Type Culture Collection. Recombinant Human IL–17A (200–17) was the product of Peprotech Company (Peprotech, Hamburg, Germany). Z-IETD-FMK was obtained from Abcam (Cambridge, MA, USA). The PI3K inhibitor, Wortmannin, was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All cells were incubated at 37°C with 5% CO 2 in DMEM containing 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, and 5000 IU/mL penicillin/5000 g/mL streptomycin for 1 to 2 days before starting experiments.
Cell transfection
Hep–2 cells and Tu212 cells were cultured in 60-mm plates or 100-mm plates (for western blotting analysis) without antibiotics overnight. When the cells reached 70–80% confluency, they were transiently transfected with IL–17 siRNA and scramble siRNA (Genepharma, Shanghai, China) using Lipofectamine RNAiMAX (add the source) according to the manufacturer’s recommendations.
Flow cytometry
The cells at the logarithmic phase were inoculated into fresh culture medium and cultured in a 37°C and 5% CO2 incubator for 48 hours. Then the cells were digested with trypsin to obtain a cell suspension, which was centrifuged at 1000 rpm for 5 minutes to allow the collection of the cells. After the cells were washed with pre-cooled PBS twice, 1 mL of PBS was used to re-suspend the cell suspension that was divided into 2 tubes (0.5 mL/tube), both of which were centrifuged in order to remove the supernatant. One of the tubes was re-suspended using 500 μL of binding buffer with cell cycle kit (Beyotime Institute of Biotechnology, Shanghai, China). Next, 1 mL of pre-cooled 70% ethyl alcohol was added to fix the cells at 4°C for 24 hours, the cell suspension was centrifuged to move the fixation solution, washed in pre-cooled PBS twice, centrifuged to move the supernatant and added with propidium iodide (PI). Finally, the cell cycle was determined using a flow cytometer. The other tube of cell suspension was re-suspended with 500 μL of binding buffer from the cell apoptosis kit (Beyotime Institute of Biotechnology, Shanghai, China), mixed with 5 μL of Annexin V-FIFC and, cultured at 2 ~ 8°C for 15 min in the dark. Then another 15 μL of Annexin V-FIFC was added, the suspension was cultured again at 2 ~ 8°C for 15 min in the dark. Within 2 hours, the cell cycle was determined using a flow cytometer.
Immunofluorescence co-localization assay
For fluorescence staining, cells were fixed with 40 g/L formaldehyde, permeabilized with 0.1% Triton X–100 in PBS, and blocked with 1% BSA in PBS for 30 min, followed by incubation overnight at 4oC with both anti-flag and anti-RhoC antibodies. The cells were washed three times with PBS for 5 min, incubated with DyLight™ 488 conjugated Goat anti-Mouse IgG along with DyLight™ 549 conjugated Goat anti-Rabbit IgG for 30 min, and then nuclear stained with 1 mg/L 4, 6-diamidino–2-phenylindole (DAPI, Roche, Germany). The fluorescence images were acquired with an Olympus FV1000 confocal microscope (Olympus, Japan) using a 100 × oil immersion objective.
TUNEL
Apoptosis within populations of transplanted cells was detected using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. One day after transplantation, the apoptosis of transplanted ADMSCs was detected in the peri-infarct zone using a TUNEL assay kit according to the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were counted in one brain tissue section of each animal (n = 5 per group). The number of double staining-positive (red and green fluorescence) cells was counted in a minimum of 10 microscopic fields based on their nuclear morphology, and dark color was quantified using a 40× objective and Image-Pro image analysis software.
Western Blotting
Laryngeal carcinoma tissue and paracancerous tissues samples were excided in operation and flash-frozen at –80oC until use. In order to prepare lysates, frozen laryngeal carcinoma tissues were minced with eye scissors on ice. Then they were homogenized in lysis buffer (1% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), 1 % Triton X–100, 1 %NP–40, pH 7.5, 5 mmol/l EDTA, 50 mmol/l tris, 1μg/ml leupeptin, 10μg/ml aprotinin, and 1 mmol/l PMSF) and centrifuged at 12,000 rpm at 4oC for 20 min to collect the supernatant. Cell cultures for immunoblot were lysed with sodium lauryl sulfate loading buffer and stored at –80 oC until use. The collected supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after determining the protein concentration with the Bradford assay (Bio-Rad). After the separated proteins were transferred to polyvinylidene difluoride filter (PVDF) membranes, the membranes were blocked with 1% (w/v) bovine serum albumin (BSA) for 2 h and incubated with primary antibodies against IL–17 (anti-rabbit, 1:1,000; Abcam), IL–17R (anti-mouse, 1:1,000; Sigma), FAS (anti-rabbit, 1:1,000; Cell Signaling), FASL (anti-rabbit, 1:1,000; Cell Signaling), and β-actin (anti-rabbit, 1:1,000, Abgent) at 4oC overnight. At last, the membranes were incubated with second antibody at 37 oC for 2 h. Bound proteins were scanned by using Chemi Doc XRS (Hercules, CA).
Statistical analysis
The data was expressed as the mean ± standard deviation. Values in tables and figures were given as means and standard deviation of the mean if not otherwise indicated. The analysis of variance (ANOVA) and Student’s t-test were used in the SPSS software to determine significant differences between groups. A χ2 test was used to evaluate the associations between clinicopathologic variables and IL–17 protein expression. The correlation between the mRNA levels of IL–17 and FAS and FASL in LC tissue was analyzed with Pearson’s correlation analysis. Kaplan-Meier survival analysis and log-rank tests were used to calculate survival curves. The Cox proportional hazard regression model was used to estimate overall survival. The values of p less than 0.05 were considered to be statistically significant. The data were analyzed using the SPSS 20.0 statistical software program (version 20.0; IBM Corporation, Armonk, NY, USA).
Results
Association of increased IL–17 expression with tumor progression in LC patients
As shown in Fig. 1A, the Western blotting in different pathological grades of LC tissues showed that the expression levels of IL–17and IL–17 protein increased significantly with increasing severity of LC. These findings were consistent with P-AKT, and P-PI3K levels of protein expression, while the expression of FAS and FASL in the LC tissues decreased significantly. The expression of PI3K and AKT had no significant expression changes compared with paracancerous tissues of normal control group (Fig. 1B).. In hematoxylin and eosin assay (Fig. 1C), paracancerous tissues demonstrate keratosis and superficially invasive LSCC with pushing invasive tumor. Additionally, front and band-like dense inflammatory infiltrate (Fig. 1C-a),, high differentiation group tissue demonstrate tumor infiltrating lymphocytes in nonkeratinizing LSCC (Fig. 1C-b),, medium differentiation tissues show keratinizing invasive LC with more intense lymphocytic host response (Fig. 1C-c), superficially invasive keratinizing LC of the poorly differentiation tissues demonstrate minimal lymphocytic host response (Fig. 1C-d), The hematoxylin and eosin assay demonstrate more lymphocytic infiltration, with the decrease of degree of differentiation in LC. Moreover, the mRNA expression levels of IL–17、IL–17R、FAS, and FASL were determined by qRT-PCR by different pathological grades of LC tissues in poorly differentiated, medium differentiated, and high differentiated groups, respectively. The mRNA expression of IL–17、IL–17R was significantly increased in LC tissues, while FAS and FASL decreased markedly with the decrease of degree of differentiation (Fig. 2A)..
Correlation analysis of IL–17 and FAS and FASL expression
Pearson’s correlation analysis showed that the expression of IL–17 mRNA had a significant negative correlation with the expression of FAS and FASL mRNA levels，the higher the IL–17 expression, the lower the FAS and FASL expression, and the more severe the LC (r = –0.82 for FAS, r = –0.847 for FASL, and all P < 0.01, Fig. 2B)..
Associations between IL–17 protein expression and clinical parameters of patients with LC
The expression of IL–17 in LC tissues was associated with TNM stage, T stage, lymph node metastasis (LNM), and differentiation (all P < 0.05). The results showed that the elevated IL–17 expressions was observed in the patients with LNM, low degree of differentiation, late overall TNM stage, and advanced T3/T4 stage, while no significant associations of IL–17 expression with patients’ age, gender, and tumor sites (all P > 0.05) (Table 2)..
Associations between IL–17 protein expression and recurrence of LC patients
The IL–17 protein expression distributions and associated survival among the patients are shown in Table 3. Among 60 LC patients, 15 cases were found to have recurrence or metastasis with a median of 3 years of follow up. The low IL–17 protein expression was significantly associated with better disease-free survival than the high expression of IL–17 protein (log-rank P = 0.022, Fig. 2C).. Cox regression analysis was performed to adjust for other important confounders, including age, sex, tumor size, stage, LNM, and differentiation (Table 3). After adjustment for these confounders, the patients with high expression of IL–17 had an approximately 3-fold increased risk of disease recurrence (HR, 3.29, 95% CI, 2.73–8.75) than the patients with the low expression of IL–17, indicating that IL–17 expression was an independent factor for disease influence of the patients with LC.
IL–17 induces FAS and FASL expression via activation of PI3K/AKT pathways
To determine whether IL–17 and FAS/FASL proteins are distributed in the same cellular location, we performed immunofluorescence co-localization experiments with a confocal laser scanning microscope to confirm the localization of IL–17 and FAS/FASL in Hep–2 and Tu212 cells. Our confocal microscope analysis revealed that the IL–17 fusion protein co-localized with the FAS/FASL fusion protein in the membrane of Hep–2 and Tu212 cells (Fig. 3).. In vitro recombinant Human IL–17A (200–17) significantly promoted expression of IL–17 in LC tissues, which enhanced FAS and FASL expression in LC cell lines after a 6h treatment, while Wortmannin simultaneously abrogated the increase of the phosphorylated PI3K. The time-course for expression of FAS and FASL in LC cell lines with stimulation by 200–17(50 ng /ml) following pre-treatment with Wortmannin (10µM/L) was observed. In Fig. 4A, we found that Wortmannin simultaneously abrogated the decrease of FAS and FASL induced by 200–17, and the expression of FAS and FASL increased and peaked at 6h and 8h after stimulation with 200–17 following pre-treatment with Wortmannin in Hep–2 cells. Furthermore, Wortmannin promoted 200–17-elicited downregulation of phosphorylated FOXO3 protein expression. The similar findings were observed in Tu212 cells (Fig. 4B).. Therefore, these results suggested that the activation of PI3K/AKT pathways may play an important role in IL–17-mediated reduction of FAS and FASL expression.
IL–17 inhibited apoptosis through the FAS/FASL Pathway in Hep–2 and Tu212 cells
To confirm whether the effects of IL–17 were FAS/FASL-dependent, we performed the assays with treatment by Z-IETD-FMK, which is a specific inhibitor of caspase–8. The caspase–8 phosphorylation was inhibited by using Z-IETD-FMK. The protein expression levels of cleavage of caspase–8 and cleavage of caspase–3 were evaluated after treatment with 40 µM of Z-IETD-FMK both with and without siRNA-IL–17. As shown in Fig. 5A, The Z-IETD-FMK treatment abrogated the siRNA-IL–17-induced promoting of cleavage of caspase–8 and cleavage of caspase–3 protein expression in Hep–2 cells. Additionally, cleavage of caspase–8 and cleavage of caspase–3 protein expression was decreased markedly by Z-IETD-FMK treatment in Scramble group, while cleavage of caspase–8/3 did not show lower level in combination with siRNA-IL–17.The similar findings were observed in Tu212 cells7(Fig. 5B).. These results show that the effects of IL–17 may inhibit apoptosis through the FAS/FASL Pathway.
siRNA silencing IL–17 gene expression promotes cell apoptosis
To determine the role of IL–17 in LC, we silenced IL–17 gene expression in LC cell lines using siRNA techniquesto evaluate the effects of IL–17 on apoptosis in both Hep–2 and Tu212 cells by flow cytometry. The RT-PCR results revealed that there was an approximately 63% decrease of IL–17 mRNA in the stable cell line of LC cells transfected with siRNA compared with that in the controls. In addition, siRNA led to an approximately 73% reduction of IL–17 protein expression by Western blot as shown in Fig. 6A. Furthermore, we found that siRNA-IL–17 promoted Hep–2 cell apoptosis (15.0%±2.78 compared to 8.20%±3.57 in controls). Similarly, siRNA-IL–17 promoted Tu212 cell apoptosis (12.90% ± 4.58 compared to 8.50% ± 2.37 in controls) as shown in Fig. 6B. In addition, the apoptotic cells were detected by the TUNEL assay, siRNA-IL–17 pretreatment dramatically increased the number of apoptotic cells,staining showed that the number of apoptotic the siRNA-IL–17 group was 3.19 times higher than that of apoptotic controls group (1323.02 ± 278.71 vs. 415.06 ± 68.79, p<0.01) as shown in Fig 6C and Fig 6D. These results show that silenced IL–17 expression may be involved in promoting LC cell apoptosis.
siRNA-IL–17 regulated PI3K/AKT/FAS/FASL pathways in Hep–2 cells and Tu212 cells
IL–17 may mediate apoptosis of LC cells via activation of downstream PI3K/AKT/FAS/FASL pathways. To further examine the mechanism by which siRNA-IL–17 induces apoptosis, we detected the protein levels of PI3K、P-PI3K、AKT、P-AKT、FAS and FASL by Western blot in siRNA-IL–17 transfected LC cell line and controls. P-PI3K and P-AKT activity was significantly inhibited by IL–17 knockdown in Hep–2 cells and Tu212 cells, while FAS and FASL were markedly elevated in the IL–17 siRNA group compared with those in the controls as shown inFig. 7. These results indicate that silencing of IL–17 expression mediated apoptosis via the PI3K/AKT/FAS/FASL pathways in LC cells.
Discussion
LC is one of the most common malignant head and neck tumors, accounting for 1–2.5% of all malignancies throughout the body [29, 30]. With the advance in treatment and management of LC, the prognosis has significantly improved, while the survival of this tumor remains poor, with a 5-year overall survival less than 60%. In particular, the prognosis for advanced stage and recurrent tumors of this disease are still poor after treatment by surgery and the chemoradiotherapy becomes the major choice for treatment. So far, the efficacy of chemoradiotherapy still remains limited [31, 32]. Our clinical data on patients showed that cytokines are dysregulated in LC tissues as compared with normal tissues. Our previous study also found that well-differentiated tumor tissue had high mRNA expression levels of IL–12Rβ2 and INF-γ, whereas moderately and poorly differentiated tumor tissues had low levels of these markers [33]. COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients [34].The results from this current study may support the theory that IL–17 depletion may diminish LC cell mitogenic signaling and promote apoptosis. Targeting IL–17 may therefore be a therapeutically useful way to treat LC.
In this study, we explored the effects of IL–17 on apoptosis of LC cells and the role that IL–17 acts as a biomarker in the recurrence of this disease, metastasis and prognosis of LC patients. The tumor microenvironment is infiltrated by a wide array of cells from both adaptive and innate immune systems, and hematoxylin and eosin assays demonstrate more lymphocytic infiltration, with the decrease of degree of differentiation in laryngeal carcinoma. We found that IL–17 expression was increased in different pathological grades of LC tissues compared with paracancer tissues of normal controls in both levels of mRNAs and proteins. We found that the expression of IL–17 and IL–17R mRNA in the tumor group was significantly higher than that in the control group. These findings were consistent with those levels of protein expression. The induced levels of expressions of IL–17、IL–17R、P-AKT and P-PI3K protein significantly increased with increasing severity of LC. While, the expression of FAS and FASL in the LC tissues decreased significantly. Previous studies reported that the patients with early stage laryngeal carcinoma exhibited a lower level of IL17 mRNA expression than those with advanced stages, and cancer tissues exhibited a significantly higher level of IL17 mRNA expression than pericarcinoma tissues [35, 36]. Overexpression of both IL17 and IL17R was observed in tumors compared with that in adjacent tissues [37, 38]. Furthermore, our results showed that IL–17 and IL–17R expression in LC tissue was correlated with tumor size, TNM stage, degree of differentiation in LC, and LNM. Our results may help demonstrate that expression of IL–17 was a significantly independent factor that may contribute to the recurrence of LC patients after adjustment with other significant confounders including tumor size and TNM stage [39–41]. A previous study also has showed that increased IL–17 expression has correlation with the poor overall survival in different other types of cancers [42].
FAS and FASL are members of the tumor necrosis factor (TNF)-receptors and TNF family, respectively. The ligation of FAS with FASL results in the activation of a caspase cascade that initiates apoptosis [43–45]. With the aggravation of LC, the expression of FAS and FASL in LC tissues markedly decreased, suggesting a restraining in FAS/FASL pathways due to accelerated apoptosis of LC cells. We found that mRNA levels of FAS and FASL increased in cancer tissues compared to control with increasing severity of LC, which is not consistent with the protein expression. Several reports have shown that mRNA and protein is not always strictly linear, but has a more intrinsic and complex dependence. Different regulatory mechanisms (such as synthesis and degradation rates) may play roles in both the synthesized mRNA and the synthesized protein [46–48]. In this study, our data showed that IL–17 expression had a positive correlation with the expression of FAS and FASL (r = 0.820, P < 0.01). A previous study highlighted IL–17 to be a potential prognostic marker for patients with respiratory, digestive and other system cancers [14]. In addition, IL–17 may be significantly correlated with the differentiation and angiogenesis in the development of LC as the expression of CXCL9, CXCL10, and IL–17 mRNAs in the skin in FAS- and FASL-deficient mice was decreased [28, 37, 38]. In this study IL–17 fusion protein co-localized with the FAS/FASL fusion protein in the membrane of Hep–2 and Tu212 cells. IL–17 activates PI3K/Akt signaling pathway which may block the FAS-associated death domain protein, which inhibits the cellular apoptosis pathway by improving the phosphorylation of FOXO3a [49, 50]. Recombinant Human IL–17A (200–17) had a growth stimulating effect on IL–17 [51, 52]. Wortmannin covalently modifies PI3K and is a potent and specific PI3K inhibitor [53–55]. Our results showed that the expression of FAS and FASL increased after stimulation with the 200–17, an IL–17 stimulator, following pre-treatment with Wortmannin in Hep–2 cells and Tu212 cells. Furthermore, Wortmannin promoted 200–17-elicited downregulation of P-FOXO3 protein expression. Among cell death receptors, the Fas/FasL system provides an important apoptotic mechanism [53, 56]. Fas-mediated apoptosis following death receptor stimulation, leads to cleavage of procaspase–8，subsequent activation of a downstream caspase, such as caspase–3, which induces apoptosis [57, 58]. When cells were treated with siRNA-IL–17, Z-IETD-FMK resulted in a greater decrease in cleavage of caspase–8 and cleavage of caspase–3 protein expression, compared with treatment with siRNA-IL–17 alone.These results suggested that IL–17 stimulation might restrain apoptosis in vitro by inhibiting FAS/FASL pathways via activating PI3K/AKT pathways.
Accumulating studies have demonstrated that IL–17 acts as a tumor suppressor and promotes cell proliferation, migration, and invasion [59, 60]. However, little has been reported about the mechanism by which IL–17 is involved in the progression of LC. To further investigate the role of IL–17 in mitogenic effects on LC cells, in the present study we silenced endogenous IL–17 by siRNA to determine the effect of endogenous IL–17 in LC cells. We found that downregulation of IL–17 expression inhibited PI3K/AKT pathway, which in turn significantly suppressed the activation of FAS/FASL pathway, leading to reduced apoptosis of LC cells.Our current finding might indicate that silenced IL–17 expression significantly promote LC cell apoptosis via the PI3K/AKT/FAS/FASL pathway.
Conclusions
In summary, the present investigation provides further evidence for the involvement of IL–17 in the progression of LC. Aberrant expression of IL–17 contributes to the pathogenesis of LC. Interfering the expression of IL–17 at the posttranscriptional level could inhibit the apoptosis of LC cell lines via the activation of FAS/FASL pathway, suggesting that IL–17 can be targeted for therapeutic intervention against LC. Further studies are required to elucidate the mechanisms and diverse signaling networks of IL–17-related in LC.
Declarations
Ethics approval and consent to participate: The signed written consents were obtained from each patient and the study was approved by the institutional research board of ethical committee at theSecond Hospital of Anhui Medical University.
Consent for publication: Not applicable.
Availability of data and material: All data generated or analyzed during this study are included in this published article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Conflict of Interest Statement: The authors declare that they have no competing interests.
Funding: This his research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Author’s contributions: YS, MY, JZ, YS, and YT carried out the majority of the experiment, data analysis, and wrote the manuscript. They were all helped by HL, JZ, YL, and JY. All authors reviewed and approved the final manuscript.
Acknowledgements: None.
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Tables
Table 1. Primer sequences of IL-17、IL-17R、FAS、FASL, and GAPDH
|
Primers used |
Forward |
Reverse |
|
IL-17 |
5'-GACCCTTCACCCCTCACC-3' |
5'-TTATGGATCATGCCCACAAG-3' |
|
IL-17R |
5′-GG GATTACA GGCGTGAGCCA-3′ |
5′-GCGGTCTGGTTATCGTCTAT-3′ |
|
FAS |
5´-GGTGCTGTCTCTCTATGCCT CTGGA-3´ |
5´-GGTGCTGTCTCTCTATGCCT CT GGA-3´ |
|
FASL |
5´-AAGGACCTCCAGCATCACTGTGTCA-3´ |
5´-CCTTCAGAGCCCGCAGCT TC CACG T-3´ |
|
GAPDH |
5'- AAAGTCCGCCATTTTGCCACT-3' |
5'-CCAAATCGTTAGCGCTCCTT-3' |
Table 2. Correlations of IL-17 expression with clinical characteristics in patients with LC
|
Clinical characteristics |
n |
IL-17 expression |
P |
|
|
Gender |
|
|
|
0.280 |
|
|
Male |
35 |
3.89 ± 3.28 |
|
|
|
Female |
25 |
4.28 ± 3.42 |
|
|
Age |
|
|
|
0.321 |
|
|
< 50 |
22 |
5.48 ± 2.74 |
|
|
|
≥ 50 |
38 |
4.29 ± 3.37 |
|
|
TNM stage |
I/II |
27 |
4.82 ± 0.53 |
0.007 |
|
|
III/IV |
33 |
5.36 ± 3.24 |
|
|
Degree of differentiation |
|
|
|
0.009 |
|
|
Middle/high |
42 |
4.58 ± 3.27 |
|
|
|
Low |
18 |
6.17 ± 3.37 |
|
|
LNM |
|
|
|
< 0.0001 |
|
|
Yes |
38 |
5.63 ± 3.16 |
|
|
|
No |
22 |
3.15 ± 2.37 |
|
|
Tumor site |
|
|
|
0.285 |
|
|
Glottis |
26 |
3.47 ± 2.31 |
|
|
|
Supraglottis |
20 |
2.25 ± 3.43 |
|
|
|
Subglottis |
4 |
3.54 ± 3.22 |
|
|
T classification |
Tis |
8 |
2.04 ± 4.13 |
0.013 |
|
|
T1/T2 |
23 |
3.61 ± 4.13 |
|
|
|
T3/T4 |
29 |
4.75 ± 2.36 |
|
Table 3. Association of IL-17 expression with disease recurrence among the patients with LC (N = 60)
|
IL-17 expression |
Recurrence number/all patients’ number |
aHR1 (95% CI) |
P |
|
Low2 |
8/38 |
1.00 |
|
|
High |
7/22 |
3.29 (2.73-8.75) |
0.028 |
1Hazard ratio adjusted for age, sex, tumor size, stage, LNM, and differentiation.
2Reference group (The median of expression as cut off point in patient without recurrence).
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