IL-36α suppresses proliferation of ovarian cancer cells

Abstract

Interleukin-36α (IL-36α), also formerly known as IL-1F6, is pertaining to IL-1 family members that has been shown to play an important pro-inflammatory role in chronic immune disorders. However, the role IL-36α in the setting of cancer remains unknown. Here, in our study, to investigate the clinical relevance of IL-36α in ovarian cancer, clinicopathological significance as well as expression level of IL-36α were analyzed in epithelial ovarian cancer clinical tissues and paired normal control. To explore the biological role of IL-36α in vitro in epithelial ovarian cancer cells, both overexpression and knockdown of IL-36α were performed. Based on the successful re-expression and silencing of IL-36α, proliferation, migration, and invasion were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound-healing, and Transwell assays, respectively. To further confirm the effect over proliferation in vivo, nude mice xenografted with epithelial ovarian cancer cells whose endogenous IL-36α was stably upregulated or downregulated were employed. It was found that IL-36α was shown to be markedly downregulated in epithelial ovarian cancer tissues relative to paired normal control and that reduced IL-36α expression was significantly associated with poor overall prognosis. In addition, IL-36α was observed to be able to suppress the growth of epithelial ovarian cancer cells both in vivo and in vitro. Taken together, IL-36α was displayed to be able to suppress the growth of epithelial ovarian cancer cells in our setting, which is suggestive of its druggable potential in curing the epithelial ovarian cancer and that upregulation of IL-36α was found to be capable of inhibiting the growth of epithelial ovarian cancer cells.

Introduction

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer worldwide. ¹ Although advanced treatment including integrated surgery and chemoradiotherapy have been applied, ² less than 30% of the patients can survive 5 years after diagnosis. ³ Regardless of the type of treatment, repeated therapies favor chemoresistance allowing for tumor survival and progression and force patients to undergo several lines of chemotherapy with poor prognosis and severe side effects. ² In this setting, given the chemoresistance that was readily caused by first-line of chemoreagents clinically used, there is urgent need for alternative treatment to improve clinical outcome of patients especially with advanced EOC.

Tumor-promoting inflammation has been established as a hallmark of cancer. Several cytokines have been reported to be able to play either diagnostic ⁴ or prognostic ⁵ or therapeutic^6,7 role in the background of cancer. ⁸ Therefore, cancer immunotherapy tries to stimulate the immune system to destroy tumor cells avoiding the chemoresistance^9,10 caused by first-line chemoreagents commonly used in clinic to a large extent. Interleukin-36 (IL-36), pertaining to IL-1 family,^11,12 has been extensively reported in the psoriatic ¹³ or pulmonary disorders. Three subtypes of IL-36—IL-36α, β, and γ—have been identified. ¹⁴ Nevertheless, there has been no report regarding the association between IL-36, whichever subtypes mentioned above, and cancer, with the exception of only one recent study ¹⁵ that was carried out in fibrosarcoma mouse model reporting that IL-36 can suppress tumor progression, suggesting IL-36 could be tumor suppressor. In our previous unpublished works, IL-36α was found to be significantly downregulated in serum of patients with EOC relative to normal controls, which leads to the implication that IL-36α might play a preventive role in EOC. The role IL-36α in EOC, however, remains to be studied given no relevant report available regarding IL-36α in the setting of ovarian cancer.

In consideration of the unknown role played by IL-36α in EOC, IL-36α subtype was picked up as a cytokine of interest based on our previous observation in EOC. To investigate the clinical correlation of IL-36α in EOC, clinicopathological significance of IL-36α expression was statistically analyzed. To explore the biological role IL-36α plays in the proliferation, migration, and invasion of EOC cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound-healing, and Transwell assays were used, respectively. To further confirm the influence over proliferation in vivo, nude mice xenografted with EOC cells whose endogenous IL-36α was stably upregulated or downregulated were employed. It was observed that IL-36α was shown to be remarkably reduced in EOC tissues in comparison with paired normal control, reduced IL-36α expression was displayed to be significantly associated with poor overall prognosis of patients with EOC, and IL-36α was presented to be pronouncedly capable of suppressing the growth of EOC cells both in vitro and in vivo, suggesting that IL-36α could be a promising therapeutic target in the therapy of EOC.

Materials and methods

Clinical tissues

This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Tissue microarray used for immunostaining analysis of IL-36α was purchased from Shanghai Outdo Biotech Co. Ltd (Shanghai, China). The tissue microarray consisted of 90 cases of EOC and paired adjacent normal control. Staging and grading was assessed in accordance with the World Health Organization classification and grading system (2014 version). None of the patients received chemoradiotherapy before undergoing oocytectomy. Informed consents were obtained from all the subjects involved, as claimed by the Shanghai Outdo Biotech company. In addition, 70 cases of sera from patients with EOC and 70 cases of sera from female healthy control were collected at the Department of Gynecology, the First Affiliated Hospital of Zhengzhou University, after obtaining the written informed consents from the patients involved.

Cell culture

The human EOC cell lines OV2008, JHOS-3, JHOS-2, ES-2, OVCAR-3, and SKOV-3 were all purchased from American Type Culture Collection (Rockville, MD, USA). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin in a 5% CO₂ humidified incubator at 37°C, unless otherwise stated.

Construction and transfection

For overexpression experiment, eukaryotic vector harboring full-length sequence of open reading frame (ORF) of IL-36α (NC_0000002.12) was cloned and subcloned into blank vector pCMV6-GFP, details are presented in Supplementary Figure 1. For knockdown experiment, small interfering RNA (siRNA) sequences against homo IL-36α (NC_000002.12) were designed and synthesized by GenePharma (Shanghai, China). The sequences are listed in Supplementary Table 1. For a vector-based RNAi approach, a double-stranded short hairpin RNA (shRNA) was cloned into the BamHI-Hind III sites of the pRNAT-U6.1/neo-cGFP vector (GenScript; Piscataway, NJ, USA). The detailed construction of shRNA-IL-36α is presented in Supplementary Figure 2. ShRNA-IL-36α-2 vector and shRNA-scramble were transfected using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA). After fluorescence-activated cell sorting for green fluorescent protein expressing cells, the transfectants were pooled and cultured in 1000 µg/mL of G418 (Sigma-Aldrich, St. Louis, MO, USA) and monitored under fluorescence microscopy.

Cell proliferation assay

MTT (Sigma-Aldrich) spectrophotometric dye assay was used to observe and compare cell proliferation ability. OV2008 and SKOV-3 cells were plated on 96-well plates at a density of 4 × 10³ cells per well. After seeding, the cell proliferation was assessed. Cells were incubated for 4 h in 20 µL MTT at 37°C. The color was developed by incubating the cells in 150 µL dimethyl sulfoxide (DMSO); the absorbance value was detected at 490 nm wavelength. The data were obtained from three independent experiments.

Cell migration assay

Cell migration ability was calculated by wound-healing assay. OV2008 and SKOV-3 cells were plated on six-well plate at a concentration of 5 × 10⁵ cells/well and allowed to form a confluent monolayer for 24 h. After transfection, the monolayer was scratched with a sterile pipette tip (10 µL), washed with serum-free medium to remove floated and detached cells, and photographed (at 0 and 48 h) by fluorescent inversion fluorescence microscope (Olympus, Tokyo, Japan).

Cell invasion assay

The invasion assay was performed using Transwell 24-well dishes with a pore size of 8 µm (Corning, NY, USA). Approximately, 5 × 10⁴ OV2008 and SKOV-3 cells in 200 µL of RPMI-1640 serum-free medium were placed in the upper chamber, and 300 µL of medium containing 50% serum-free RPMI-1640 was placed in the lower chamber. The cells were incubated for 24 h at 37°C in 5% CO₂, fixed in methanol for 15 min, and stained with 0.1% crystal violet in phosphate-buffered saline (PBS) for 15 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each clone was plated in triplicate in each experiment and each experiment was repeated at least three times.

Immunohistochemistry

Immunohistochemical staining was carried out using heat-induced epitope retrieval, an avidin–biotin complex method. The rabbit anti-IL-36α antibody (Catalog Number: TA326665; OriGene Technologies, Inc., Rockville, MD, USA) was diluted in the ratio of 1:100. The sections were evaluated by light microscopy, and cellular localization of the protein and immunostaining level in each section were assessed by two pathologists. The staining patterns were scored as follows: negative, weak positive staining (+), moderate positive (++), and strong positive (+++) according to the immunostaining intensity. Both moderate and strong positive expression were categorized into high expression, whereas negative and weak staining were categorized into low expression.

Western blotting

After 72 h of transfection, OV2008 and SKOV-3 cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer (BioTeke, Beijing, China) and 50 µg of cellular protein was subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation. Proteins were then transferred to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA) and blots were probed with rabbit polyclonal antibodies against IL-36α (dilution at 1:1000; Catalog Number: TA326665; OriGene Technologies, Inc.) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778; Santa Cruz, CA, USA). GAPDH was used as loading control and the blots were visualized with chemiluminescence with SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and images were captured with a Bio-Rad camera system (Bio-Rad, Hercules, CA, USA).

Enzyme-linked immunosorbent assay

Sera from patients with EOC before surgery and female healthy controls were stored at −80°□ until use. IL-36α in sera was detected strictly following the enzyme-linked immunosorbent assay (ELISA) kit instructions (Catalog Number: ab178008; Abcam, Cambridge, UK). The plates provided in the test kits were pre-coated with the antibody specific to IL-36α. Standard controls or samples were then added to wells with a bioin-conjugated polyclonal antibody preparation specific for IL-36α. Measurements were conducted at 450 nm by the microplate reader immediately (Bio-Rad).

Xenografted nude mice model

Nude mice experiment was approved by Animal Ethics Committee of The First Affiliated Hospital of Zhengzhou University and relevant performance was carried out strictly in accordance with the protocol and requirements given by the Animal Ethics Committee of Zhengzhou University. In total, 16 female BALB/c-nu mice of 3 weeks old were prepared for SKOV-3 cells implantation. All animals were maintained in a sterile environment on a daily 12-h light/12-h dark cycle, and the 16 mice were grouped into 2, with 8 in each group. SKOV-3 cells transfected with pCMV6-IL-36α as well as pCMV6-vector were subjected to screening using antibiotics (G418; 1000 ng/mL), followed by subcutaneous injection (1 × 10⁶/mouse) into the flanks of the nude mice. After 5 weeks, all the 16 mice were euthanized. Tumor lesions dissected were harvested and weighted. Tumor volume (TV) was calculated weekly for 5 weeks according to the formula: TV (mm³) = length × width² × 0.5.

Statistical analysis

The data were expressed as mean ± standard error of the mean (SEM). For correlation between IL-36α staining scores and clinicopathological variables, Fisher’s exact or chi-square test analysis was used, whereas two-tailed independent sample Student’s t test was used for continuous data that was normal-distribution. Mann–Whitney’s non-parametric U test was performed for the study on sera. Kaplan–Meier survival curve was plotted for the analysis of overall prognosis with log-rank test. All the statistical analysis was carried out with SPSS 17.0, and statistical figures were made using the Graphpad Prism 5.0 version (La Jolla, CA, USA). Values were expressed as mean ± SEM. All results were considered as significant when p < 0.05.

Results

IL-36α was shown to be significantly downregulated in clinical tissues as well as peripheral serum of EOC compared with paired normal control

In our previous work, IL-36α was found to be significantly downregulated in the peripheral serum of patients with EOC in comparison with female healthy normal control (Supplementary Figure 3) using ELISA, which leads to the implication that IL-36α might play a preventive role in EOC. However, no report available regarding IL-36α in the background of EOC. The observation we have made, together with one relevant report concerning IL-36α performed in fibrosarcoma mice model, indicates that IL-36α might play a tumor-suppressing role. To investigate the expression level of IL-36α in clinical tissue samples from EOC and normal control tissues, we carried out immunohistochemistry using tissue microarray that consisted of 90 cases of EOC and paired normal control tissues. Considering that the three subtypes of IL-36—IL-36α, β, and γ—have similarity in amino acid sequence, to avoid the non-specific immunostaining and ensuing false positive when immunoscoring, we performed pre-test to evaluate the specificity of the primary antibody against IL-36α using antigen pre-adsorption method as reported previously. ¹⁶ It was shown that the primary antibody used was able to specifically recognize IL-36α that was to be detected (Supplementary Figure 4a). On the basis of assurance that antibody against IL-36α was adequately specific, we set out to conduct immunohistochemistry. It can be seen that immunostaining of IL-36α was mainly both membranous and cytoplasmic (Figure 1). IL-36α expression was heterogeneous on the tissue of EOC and paired normal control with its expression being moderate and weak positive. Overall, the expression of IL-36α was pronouncedly downregulated in EOC relative to paired normal control tissues (Table 1). The result obtained by immunostaining on tissues was entirely compatible with that from peripheral serum with ELISA method, suggesting that IL-36α might play tumor-suppressing role in EOC.

Figure 1.

IL-36α was reduced in EOC tissues in comparison with paired normal control tissues. The clinicopathological morphology of the enrolled EOC tissue samples includes endometrioid carcinoma of ovarian cancer, serous carcinoma of ovarian carcinoma, mucinous carcinoma, and mixed type of ovarian cancer. Representative figure of each case mentioned was shown here. In addition, the immunoscoring of each case was also presented. One plus (+) stands for weak positive immunostaining, two plus (++) means moderate positive immunostaining, and three plus (+++) represents strong positive immunostaining of IL-36α (magnification fold: 200×; the scale bar: 50 µm).

Table 1.

Clinicopathological significance of IL-36α expression in EOC.

Parameters	Cases (n = 90)	IL-36α expression		χ2	p value
		High (++, +++)	low (−, +)
EOC	90	38	52	14.100	0.000
normal control	90	63	27
Age (years)
<50	42	14	28	2.551	0.136
≥50	48	24	24
T classification
T1 + 2	34	22	12	11.323	0.001
T3 + 4	56	16	40
N classification
N0	29	20	9	12.544	0.001
N1–3	61	18	43
Clinical stage
I + II	43	28	15	17.691	0.000
III + IV	47	10	37
Metastasis
Yes	60	19	41	8.221	0.006
No	30	19	11
Tumor volume (cm3)
<3	19	11	8	6.195	0.045
3–6	29	15	14
>6	42	12	30
Gross classification
Mucinous	18	8	10	7.137	0.068
Serous	46	11	35
Endometrioid	14	8	6
Mixed subtypes	12	6	6

IL-36: Interleukin-36α; epithelial ovarian cancer.

Here, mixed subtypes include transitional cell carcinoma and clear-cell carcinoma subtypes. On account of rather limited cases, we used the term mixed to represent the two specific subtypes.

Reduced IL-36α expression was displayed to be pronouncedly associated with tumor progression and prognosis of patients with EOC

To understand the clinicopathological significance of IL-36α expression, Cross-table statistical approach was used to analyze the clinical relevance of its expression. IL-36α was shown to be pronouncedly downregulated in EOC in comparison with paired normal control (Table 1). Furthermore, downregulated IL-36α expression was found to be remarkably inversely associated with lymph node metastases, tumor progression, and tumor size (Table 1), suggesting that IL-36α could suppress the tumor progression of EOC in vivo. No significant correlation was observed between IL-36α expression and other clinicopathological parameters, including demographical factor and gross morphology of EOC. Moreover, to observe whether or not there was association between IL-36α expression level and overall prognosis, Kaplan–Meier survival curves were plotted. It was found that there was significant difference in overall prognosis between patients with high expression of IL-36α and patients with low expression of IL-36α (Figure 2). Exactly, IL-36α expression was observed to be negatively associated with overall prognosis. The results obtained through clinicopathological analysis indicate that IL-36α could prevent the tumor progression of patients with EOC.

Figure 2.

Reduced IL-36α expression was significantly negatively associated with favorable overall prognosis. Survival curve was plotted using Kaplan–Meier survival analysis. Of the total 90 cases enrolled, patients with high expression (immunoscoring: ++ and +++) of IL-36α were 38 cases and patients with low expression (immunoscoring: – and +) were 52 cases; a significant difference in overall prognosis was observed between them (p = 0.031) using log-rank statistical analysis in SPSS 17.0 version.

IL-36α was presented to be markedly able to suppress the growth, migration, and invasion of EOC cells in vitro

Having discovered the trend that IL-36α could suppress the tumor progression of EOC in vivo on clinical tissue level, we, next, investigated the possible biological roles of IL-36α in vitro in the proliferation, migration, and invasion of EOC cells. Among the six different kinds of EOC cell lines representing different tumor stage and grade we enrolled, basal expression level of IL-36α was first detected before subjected to functional analysis. It was shown that endogenous IL-36α was highest for OV2008 cell line, whereas SKOV-3 cell line had the lowest basal IL-36α among the six different kinds of cell lines (Figure 3(a)). To clarify the basal IL-36α expression level in vitro cell line in our experimental setting, we selected OV2008 and SKOV-3, the two extreme cases, in the following in the functional analysis of IL-36α. Given the highest basal level of IL-36α in OV2008, IL-36α was transiently knocked down using specific siRNA against IL-36α (Figure 3(b)). While as for SKOV-3, on the contrary, whose basal level of IL-36α was lowest in our setting was transiently overexpressed by transfection with eukaryotic expression vector harboring full-length complementary DNA (cDNA) of IL-36α from human (Figure 3(c)). On the strength of successful silence or overexpression of IL-36α, we subsequently evaluated the proliferative, migratory, and invasive variation of OV2008 and SKOV-3 by MTT, wound-healing, and Transwell assays, respectively. It can be seen that in OV2008 cells, silencing of IL-36α was found to be markedly able to promote growth, migration, and invasion relative to control group. In contrast, proliferation, migration, and invasion were significantly inhibited in SKOV-3 in which IL-36α was re-expressed compared with control group (Figure 3(d)–3(h)), suggesting that IL-36α was capable of suppressing the growth, migration, and invasion after it being overexpressed. Furthermore, to observe whether or not recombinant IL-36α has the same effect over growth, migration, and invasion as done by transfection with siRNA or overexpression vectors, both OV2008 and SKOV-3 cells after administration with recombinant IL-36α were repeated with MTT, wound-healing, and Transwell assays in parallel. It was discovered that proliferation, migration, and invasion of both OV2008 and SKOV-3 were pronouncedly inhibited in the presence of recombinant IL-36α in a dose-dependent way (data not shown) in comparison with control group, suggesting that recombinant IL-36α can also make significance difference on the suppression of growth and motility of EOC cells.

Figure 3.

IL-36α was found to be able to suppress the growth, migration, and invasion of EOC cells in vitro. (a) Basal expression level of IL-36α in a panel of six different kinds of EOC cell lines from EOC with different tumor stages. (b) Silencing effect of specific small interfering RNA (siRNA) against IL-36α after transient transfection into OV2008 cells. It can be seen that siRNA termed 2 against IL-36α was the most significant of all. Thus, siRNA-IL36a-2 was selected to construct the ensuing shRNA vector used for nude mice model experiment. (c) Overexpression of IL36a was detected after transient transfection into SKOV-3 cells whose basal IL-36α was lowest of all in our setting. (d) Proliferation assay of SKOV-3 cells after IL-36α being overexpressed versus control. (e) Proliferation of OV2008 was analyzed after stable knockdown of IL-36α using shRNA vector. (f) Quantitative assay of Transwell experiments in SKOV-3 and OV2008 cells. (g) Qualitative assay of wound-healing of SKOV-3 and OV2008 cells whose IL-36α was upregulated or downregulated, respectively (scale bar: 100 µm). (h) Quantitative assay of wound-healing experiment (two-tailed independent sample Student’s t test was employed; *p < 0.05; **p < 0.01; ***p < 0.001 vs control group; all experiments were done in independent triplicate and representative figures were shown here).

IL-36α was shown to be capable of significantly suppress the proliferation of EOC in vivo

To further confirm the effect exerted by IL-36α over proliferation of EOC cells in vivo, nude mice were xenografted with SKOV-3 whose endogenous IL-36α was stably upregulated successfully. It was shown that tumor volume of nude mice xenografted with transgenic SKOV-3 whose IL-36α was stably overexpressed was significantly smaller versus control group (Figure 4), demonstrating that IL-36α can suppress the growth of EOC cells in vivo.

Figure 4.

IL-36α can suppress the proliferation of EOC cells in vivo in nude mice. In total, 16 athymic nude mice were randomly grouped into two, with each consisting of eight mice. Experimental group was defined as mice xenografted with SKOV-3 cells whose IL-36α was stably overexpressed after transfected SKOV-3 cells being antibiotically screened; by contrast, control group was defined as mice xenografted with SKOV-3 cells whose IL-36α was only transfected with blank vector, as control. The tumor volume of tumor lesions dissected from mice at fifth week after inoculation with tumor cells presented no significant difference between experimental and control group, using two-tailed independent sample t test.

Discussion

This is the first report on IL-36α expression and clinicopathological correlation of IL-36α expression in EOC to the best of our knowledge. IL-36α was shown to be significantly reduced in EOC compared with paired normal control both in clinical tissue and peripheral sera. Reduced IL-36α was observed to be remarkably negatively associated with tumor size and progression. Compared with patients with low IL-36α expression, patients with high IL-36α expression were found to have favorable overall prognosis. Our study, for the first time reported the biological roles IL-36α played in proliferation and motility of EOC cells in vitro and showed that IL-36α can suppress, compared to the control group, the proliferation both in vivo and in vitro. These results demonstrate the direct tumor-suppressing role of IL-36α in EOC, which might provide a promising therapeutic target for patients with EOC.

In consideration of the great resistance that can be caused by first-line chemoreagents clinically employed in clinics, immunotherapy that could circumvent the resistance in the chemotherapy of cancer deserves to be explored.^9,10 In our previous work, to make clear the differential cytokines between patients with EOC versus healthy controls, the differential cytokines were screened using antibody microchip technique revealing that IL-36α was significantly downregulated in sera from patients with EOC compared with healthy control (unpublished). Given the paucity of data regarding IL-36α in cancer, IL-36α was picked up as a cytokine of interest in the following functional analysis. Till now, most of the published studies regarding the association of IL-36α in the pathogenesis of disease came from either the psoriatic ¹³ or pulmonary literature. ¹⁷ Nevertheless, no relevant report concerning IL-36α has been available in the background of cancer with the exception one recent study ¹⁵ performed in fibrosarcoma reporting that IL-36α could suppress the progression of fibrosarcoma. As given above, the role IL-36α in EOC remains elusive that deserves to be investigated further. Based on the expression level of IL-36α in EOC, we fairly postulated that IL-36α may be able to suppress the tumor progression in EOC, as similarly reported in fibrosarcoma by Solahaye-Kahnamouii et al. ¹⁵ To test the postulation, we extended the experiment from clinical tissue level to EOC cell line level. IL-36α was observed to be markedly capable of suppressing the growth and motility of EOC cells in two different kinds of EOC cell lines through artificial upregulation or downregulation of cDNA of IL-36α. The result obtained on cell line level was entirely consistent with observation on clinical tissue level that IL-36α was displayed to be pronouncedly downregulated in EOC tissues versus normal control and that reduced IL-36α expression was shown to be significantly inversely associated with tumor size and progression. However, caution is needed when interpreting the data because of relatively small number of patients enrolled. To observe whether or not the IL-36α could have the same effect on protein level, recombinant human-origin IL-36α was administrated to cell culture media. It was found that recombinant IL-36α can have the same suppressing effects over proliferation and motility of EOC cells as it had on messenger RNA (mRNA) level, indicating that IL-36α can directly suppress the growth and motility of EOC cells in vitro. How does IL-36α suppress the growth and motility of EOC cells remains unclear which left to be further studied. Nevertheless, recent mechanistic studies from cell lines and mouse model trying to disclose how IL-36α works in gut inflammation, ¹⁸ skin diseases,^13,19 and lung diseases. ²⁰ In these studies, IL-36α was reported to be able to activate mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway. ²¹ Activated MAPK (including phosphorylated extracellular signal–regulated protein kinases 1 and 2 (p-ERK1/2), ²² p-p38, ²³ and phosphorylated C-jun n-terminal kinase (p-JNK ²⁴ )) signal pathway has been reported to be capable of playing an anti-oncogenic role in cancers, which could in part account for IL-36α-mediated suppression of the growth of EOC cells. In our study, we have failed to detect the variation of MAPK signal pathway, further basic investigation is therefore needed to evaluate whether it is through the activation of MAPK pathway that IL-36α can suppress growth of EOC cells.

Despite scooping of IL-36α in EOC, there are several limitations that need to be acknowledged. First, our observations were made in limited clinical tissues as well as only in two kinds of EOC cell lines; further studies need to be warranted in large tissue samples and cell lines. Second, the molecular mechanism through which IL-36α works in EOC remains unknown, which deserves to be further investigated. Third, to make sure whether tumor-suppressing role of IL-36α was generic or not, future studies need to be extended in other different types of cancers. Therefore, direct extrapolation of functional phenotype of IL-36α from our setting to other different subtypes of IL-36 family should be approached with caution in the absence of evidence provided.

In conclusion, we reported for the first time that IL-36α expression was displayed to be reduced in EOC tissues as well as sera versus healthy controls and that reduced IL-36α expression was found to be significantly inversely associated with tumor size and progression and overall poor prognosis. Furthermore, we have identified the tumor-suppressing role of IL-36α in EOC cells in vitro. Our findings demonstrate that IL-36α could be used as promising therapeutic target in the curing patients with EOC.