1. Introduction
The interleukin 12 (IL-12) family consists of at least four members of cytokines (IL-12, IL-23, IL-27 and IL-35) that play diverse roles in different subtypes of T lymphocytes, and is the only cytokine family that forms a functional α or β heterodimer [
1–
3]. The α subunit of IL-12 family includes p 19, p 28 and p 35 chains that are individually paired with the β subunit (p 40 or Ebi3) of IL-12 family. The combinations of the paired α/β chains for IL-12, IL-23, IL-27 and IL-35 are p 35/p 40, p 19/p 40, p 28/Ebi3 and p 35/Ebi3, respectively [
2,
3]. The above paired ligands can be specifically recognized by a shared array of heterodimerized receptors. Interleukin 12 (IL-12) signals IL-12Rβ1 and IL-12Rβ2 [
4,
5], whereas IL-23 signals IL-12Rβ1 and IL-23R [
6,
7]. Differently, IL-27 recognizes WSX-1 (IL-27R) and gp130 [
8], whereas IL-35 binds to gp130 and IL-12β2 [
9]. Although structurally related, the members of IL-12 family even conduct distinct immune functions. Both IL-12 and IL-23 can be released by the major antigen presenting cells (APCs) such as dendritic cells and macrophages,
etc., into the inflammatory sites upon microbial infection. A positive feedback loop is required for continuous IL-12 production by APCs as well as for the IFN-γ mediated T
H1 differentiation. Similarly, a positive feedback loop is also found in the regulation of IL-23 production by enhancing the IL-23R expression. Unlike IL-12, IL-23 does not mediate cell differentiation but can positively regulate IL-17- T
H17 cell pathway. In contrast, both IL-27 and IL-35 possess inhibitory properties to limit or counteract the cellular inflammatory responses.
The expression of IL-12 receptor family was initially thought to be restricted in immune cells. However, broader examinations on a variety of tissues indicated that the expressions of the members of IL-12/IL-12R family could be regulated in many non-immune tissues and cells including human cancers [
10]. Recent study shows that IL-23 regulates the proliferation versus inhibition of IL-23R positive lung cancer cells in a concentration dependent manner [
11]. However, in most instances, genetic defects, structural defects and/or the gene expression defects in IL-12/IL12R family members might be the causing issues for the development of a variety of human cancers such as esophageal cancer [
12], lung cancer [
10], acute myeloid leukemia [
13], oesophageal squamous cell carcinoma [
14] and human chronic B cell malignancies [
15]. Therefore, supplementing a functional IL-12/IL-12R family gene into the disease tissues might generate great therapeutic potential.
Mounting evidence has shown that some IL-12/IL12R family members possess anti- proliferation or pro-apoptotic functions. Airoldi
et al. first demonstrated that IL-12Rβ2 could function as a tumor suppressor gene in human chronic B cell lymphoproliferative disorders, and IL-12 treatment in IL-12R transfected B lymphoma cells significantly inhibited cell proliferation and reduced tumorigenesis in animal model [
15]. IL-12 induced and IL-12R mediated apoptosis has also been found in acute myeloid leukemia cells [
16] and ovarian carcinoma cells [
17]. IL-12 based tumor therapies have drawn greatly attention for the past 15 years and are apparently effective in prolonging the survival of cancer-bearing patients [
18]. The situation seems to be the same for the other members of IL-12 family such as IL-23 and IL-27 [
19].
We showed previously that spliced variants of IL-23R could generate defective IL-23R in various human tumor cell lines and different lung cancer tissues that might be a possible mechanism to account for the escape of immune surveillance in some human cancers [
10]. IL-12Rβ2 can function as a tumor suppressor gene and can induce apoptosis in cancer cells. Due to the functional and structural similarity between IL-23R and IL-12Rβ2, we speculate that human IL-23R may also negatively regulate cell proliferation and promotes cell apoptosis. Indeed, in this study, we demonstrate that over-expression of human IL-23R could markedly induced cell apoptosis in both 293ET and HeLa cells. Mechanistic studies demonstrate that the classical intrinsic pathways might be activated in responding to
IL-23R gene delivery.
IL-23R gene might have the great potential to be developed as a therapeutic target against human cancers.
3. Experimental Section
3.1. Cell Lines, Chemicals and Reagents
Human embryonic kidney cells expressing SV40T and EBNA1 (293ET) and human cervical cancer cell line (HeLa) were derived from the Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Mouse anti-Myc, rabbit anti-caspase 3, rabbit anti-STAT3, rabbit anti-p-STAT3 goat anti-Akt1/2, rabbit anti-p-Akt1/2/3, rabbit anti-ERK1, rabbit anti-p-ERK and mouse anti β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-PARP1 was purchased from Epitomics Inc. (Burlingame, CA, USA). Rabbit anti-Apaf1 antibody was purchased from Beijing Biosynthesis Biotechnology (Beijing, China). Rabbit anti-STAT1 and Rabbit anti-p-STAT1 were purchased from Bioworld Technology Inc. (Bioworld, CA, USA). The peroxidase-conjugated secondary antibodies were from Zhongshan Biotechnology (Beijing, China). The full length of human IL-23R cDNA was isolated from the total RNAs of PBMC [
10], and subcloned into the SalI/KpnI sites of pCMV-Myc vector to generate the eukaryotic expression vector pCMV-Myc-IL-23R whose sequence was subsequently confirmed by DNA sequencing analysis.
3.2. Cell Culture and Transfection
Cells were cultured in DMEM containing 10% fetal calf serum at 37 °C supplemented with 5% CO2. Transient transfection was performed using Vigofect (Vigorous Inc, Beijing, China) with the indicated plasmid DNAs. Cells seeded onto 6 well culture plates or 35 mm2 culture plates were received 0, 3 and 6 μg transfected pCMV-Myc-IL-23R DNAs, while cells plated onto 12 well culture plates were delivered with the doses of 0, 0.5 and 2 μg of pCMV-Myc-IL-23R DNAs. Then the transfected cells were continuously incubated for 48 h before harvesting.
3.3. Cell Proliferation Assay by Cell-Counting Kit-8 (CCK-8)
Cell proliferation was monitored by CCK-8 assay. Briefly, cells were cultured to a density of 5 × 104 cells/mL and then certain amount cells were released and transferred into a 96-well plate after 24, 48 or 72 h post-transfection. Cell-counting kit-8 (Dojindo, Kumamoto, Japan) solution was added into each well. After 2.5 h incubation, the absorbance at 450 nm was measured using a microplate reader.
3.4. Flow Cytometric Analysis
Cell apoptosis was detected by Annexin V-FITC apoptosis detection kit (Beijing RuiBang XingYe Science & Technology Co., LTD, Beijing, China). After 48 h transfection, cells were released and washed with pre-cooled 1× PBS. Then the cell pellet was resuspended with 50 μL binding buffer followed by adding 5 μL Annexin V-FITC and 5 μL Propidium Iodide (PI). Then the cell suspension was incubated away from light for 15 min at room temperature. Another 150 μL binding buffer was added into the mixture before flow cytometric analysis by Accuri C6 (BD Biosciences, San Jose, CA, USA).
3.5. Caspase 3/7 Assay
Caspase-3/7 activity was detected using Caspase-Glo 3/7 assay kit (Promega, Madison, WI, USA). After 48 h transfection, cells were released and washed with pre-cooled 1× PBS. Then cells were transferred into a 96-well plate and the same amount of Caspase-Glo 3/7 Reagent was added to each well. The mixture was incubated at room temperature for 1 h. The luminescence of each sample was measure in a plate-reading luminometer.
3.6. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNAs were extracted from the cultured cells with TRIzol (Invitrogen, Carlsbad, CA, USA). The primers used in the RT-PCR reactions were listed in
Table 1. One microgram of total RNAs was subjected to RT-PCR analysis using PrimeScript One Step RT-PCR Kit (Takara Biotechnology, Dalian, China). The reverse transcription was conducted at 42 °C for 35 min. After being denatured at 94 °C for 5 min, the reaction products were subsequently PCR amplified at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 40 s for 25 cycles before extension at 72 °C for 10 min. The level of
β-actin gene expression was served as internal control.
3.7. Western Blot Analysis
The transfected cells were lysed with gentle rotation in a lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 200 μM NaVO4, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 1 μM PMSF). Cell lysate for each sample was resolved on 12% SDS-PAGE followed by blotting onto Hybond nitrocelluar membrane (Amersham Biosciences, Freiburg, Germany). The transferred membrane was then probed with primary antibodies followed by relevant secondary antibodies conjugated to horseradish peroxidase. Detection was enhanced by chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3.8. Enzyme Linked Immunosorbent Assay (ELISA)
The secretion of IL-23 ligand was monitored by human interleukin 23 (IL-23) ELISA Kit (Beijing Gersion Biotechnology Co. Ltd, Beijing, China). Briefly, after 48 h transfection, 10 μL of culture supernatant together with 40 μL of sample diluent were added into the testing samples that were well pre-coated with hIL-23 antibody. Then, 100 μL of HRP-conjugated reagent were added into each well and incubated at 37 °C for 60 min. After aspirating and washing, 50 μL of chromogen A and B were added into each well and incubated away from light at 37 °C for 15 min. Finally, about 50 μL of stopping solution was added to each reaction before detection at 450 nm using a microtiter plate reader.
3.9. Statistical Analysis
All values were calculated as mean ± standard deviation (SD) from four independent experiments. The statistical difference between the assayed group and the standard group was subject to student’s t test (two-tailed, unpaired). The calculated difference was considered significant as the p value < 0.05 or < 0.01.
