Cancer cell-derived IL-8 induces monocytic THP1 cells to secrete IL-8 via the mitogen-activated protein kinase pathway
Aberrant activity of transcription factors in oral squamous cell carcinoma (OSCC) results in the spontaneous secretion of various cytokines and chemokines. Among them, IL-8, owing to its angiogenic activity, promotes the growth of OSCCs. In the present study, we examined the role of IL-8 secreted by OSCCs, on the angiogenic activity of monocytic THP1 cells. Culture supernatant (Ca-sup) augmented IL-8 secretion by THP1 cells, which was found to be significantly reduced following the removal Ca9-22-derived IL-8 from the Ca-sup. IL-8 induction was regulated at the transcriptional level, because real-time PCR demonstrated the augmented IL-8 messenger RNA (mRNA) expression. We further performed the luciferase assay using the 5′-untranslated region of IL-8 gene. Contradictory to our speculations, luciferase activity was not augmented by Ca-sup stimulation. NF-κB-independent IL-8 induction was further confirmed by pre-treating THP1 cells with NF-κB-specific inhibitors. To elucidate the signaling pathway, THP1 was pre-treated with MEK inhibitors. The results demonstrated that pre-treatment of cells with MEK inhibitor drastically reduced IL-8 levels, suggesting the role of MEK. Moreover, Ca-sup was found to increase ERK1/2 phosphorylation in a time-dependent manner. These results indicated that OSCC-derived IL-8 appears to activate angiogenic activity in monocytes within the tumor microenvironment via the mitogen-activated protein kinase (MAPK) pathway.
KeywordsOral squamous cell carcinoma Interleukin-8 Macrophage Mitogen-activated protein kinase
Oral squamous cell carcinoma (OSCC) has the highest incidence rates among malignant tumors of the head and neck , and is one of the six most common cancers in the world. Some of the aberrant gene and protein expressions observed in OSCC are dependent on the deregulated activities of transcription factors, such as NF-κB . NF-κB activity is known to increase gradually from premalignant lesions to invasive cancer, indicating their importance during the early stages of carcinogenesis [2, 3, 4, 5]. Interference with NF-κB activity leads to a remarkable reduction in the number of cytokines and chemokines, including IL-2, IL-6, and IL-8 . IL-8, one of the most relevant factors for the growth of OSCC, belongs to the CXC chemokine family  and induces angiogenesis . It is produced by several different types of cells, including the OSCCs , and binds with high affinity to the cognate receptors CXCR1 and CXCR2, thereby facilitating the proliferation, migration, and invasion of these cells .
The complex interplay between tumor cells and the tumor microenvironment plays a pivotal role in carcinogenesis and cancer progression . The tumor microenvironment consists of various stromal cells, including activated endothelial cells, tumor-associated macrophages (TAMs), fibroblasts, and bone marrow-derived cells . Most studies have reported that infiltrating TAMs are associated with cancer progression [12, 13, 14, 15]. TAMs might be recruited from the peripheral blood by chemokines and positioned within the tumor stroma. Activation of TAMs may lead to their differentiation into the M1 or M2 types. Although the expression of IL-8 receptors on these macrophages has been reported [16, 17], not much is known about the effect of IL-8 on these cells.
The aim of this study was to evaluate the effect of IL-8, secreted by OSCC, on the angiogenic activity of the macrophages. IL-8 derived from cancer cells can augment the secretion of IL-8 by the monocytic cell line THP1, through the mitogen-activated protein kinase (MAPK) pathway. The results from this study suggest that cancer-derived IL-8 can induce the angiogenic activity of macrophages in the tumor stroma.
Materials and methods
The THP1 and Ca9-22 (OSCC) cells were maintained in RPMI1640 medium supplemented with 10 % fetal calf serum (FCS), 50 μg/ml streptomycin, and 50 U/ml penicillin (10 % FCS-RPMI).
The Ca-sup and THP1-derived culture supernatants were cleared by centrifugation and subjected to ELISA. IL-8 concentration was measured by DuoSet ELISA Development System (R&D Systems, Tokyo, Japan); absorbance was measured on a Model 3550 Microplate Reader (Bio-Rad, Tokyo, Japan).
The Ca-sup was rotated with or without 400 ng of anti-IL-8 antibody (Ab) (R&D System) for 18 h at 4 °C. After incubation, 10 μl of protein G-sepharose (GE Healthcare, Tokyo, Japan) was added to the samples and rotated for another 2 h. The samples were centrifuged for 1 min at 4 °C following which the supernatants were collected into new tubes. IL-8 concentration was measured by ELISA. The resultants were used to stimulate THP1 cells as described above.
The 5′-untranslated region (5′-UTR) of the human IL-8 gene spanning from −1 to −133 was amplified by PCR using genomic DNA obtained from the human intestinal adenocarcinoma cell line HT-29. This fragment was subcloned to the BamHI and HindIII sites of pGL4-basic vector (Promega, Tokyo, Japan) and designated as wild type (wt). A reporter plasmid lacking the NF-κB binding site (ΔκB) was constructed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Tokyo, Japan) with wt as a template.
THP1 cells were washed twice with OPTI-MEM (Life Technologies, Tokyo, Japan) and transfected with 500 ng of the reporter plasmids (wt or ΔκB) using the Lipofectamine (1 μl/well) and Plus Reagent (1 μl/well) (Life Technologies, Tokyo, Japan). After 3 h, the cells were washed with 10 % FCS-RPMI1640 and cultured for another 3 h. At the end of culture, the concentration of the cells was adjusted to 2 × 106/ml using fresh medium. The cells (250 μl) were then plated on to a 48-well plate and stimulated with or without 25 μl of Ca-sup for 3 h. Subsequently, the cells were collected and washed with PBS. Cell lysates were collected following lysis with 1× passive lysis buffer (Promega, Tokyo, Japan). Transfection efficiency was normalized to the renilla luciferase activity by co-transfection with pRL/CMV vector (Promega). Both firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) with Lumat LB9507 luminometer (Berthold, Bad Wildbad, Germany).
THP1 cells were pre-treated with or without various concentrations of specific NF-κB inhibitors such as l-1-4′-tosylamino-phenylethyl-chloromethyl ketone (TPCK) (Sigma-Aldrich, Tokyo, Japan), MEK inhibitor U0126 (Promega), or JNK inhibitor SP600125 (Promega) for 1 h. After treatment, the cell were washed and further incubated with Ca-sup for 20 h. The culture supernatants were collected and subjected to IL-8 ELISA.
THP1 cells were stimulated with or without Ca-sup for 1, 2, or 3 h. After stimulation, the cells were washed twice, with ice-cold PBS, and lysed with 100 μl of cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 % Triton X-100). Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad), and 100 μg of total protein was subjected to 10 % SDS-PAGE. Western blotting was performed as described previously . Primary Abs against total ERK1/2 (×200) (Santa Cruz, San Diego, CA, USA), phosphorylated ERK1/2 (×200) (Santa Cruz), and GAPDH (×10,000) (Santa Cruz) were diluted with 1 % BSA-PBST (0.1 % Tween-20/PBS). The secondary goat anti-mouse IgG (H+L) (Jackson Immuno Research, West Grove, PA, USA) and goat anti-rabbit IgG (H+L) (Jackson Immuno Research) Abs were diluted to ×10,000 with 1 % BSA-PBST.
One-way ANOVA and Student t test were used to evaluate correlations between different groups. P values below 0.05 were regarded as significant. All data were analyzed using SPSS software version 22 (IBM, Tokyo, Japan).
Ca9-22 secreted IL-8
IL-8 accumulation in the culture supernatant of the Ca9-22 cells was found to increase in a time-dependent fashion. IL-8 secretion was first detected after 6 h (39 pg/ml) of culture and reached to 125 pg/ml after 24 h (Fig. 1).
Ca9-22-derived IL-8 can augment IL-8 secretion by THP1 cells
Non-stimulated THP1 cell supernatant (Fig. 2a) and Ca-sup (Fig. 1) contained low levels of IL-8 (69 and 39 pg/ml), respectively, whereas IL-8 concentration in the supernatant of the THP1 cells induced by Ca-sup was found to be as high as 1.9 ng/ml (Fig. 2a). Therefore, we speculated that Ca9-22-derived IL-8 may have augmented the secretion of IL-8 in THP1 cells. To examine this possibility, IL-8 was removed from the Ca-sup by immunoprecipitation, and the concentration was measured by ELISA. Treatment with anti-IL-8 Ab significantly reduced IL-8 concentrations to 2.4 pg/ml in the Ca-sup (IL-8 concentration in control supernatant was 180 pg/ml) (Fig. 2b).
THP1 cells were then stimulated with the IL-8-deprived and IL-8-undeprived Ca-sup. Although IL-8 concentrations in cells treated with the undeprived supernatant did not change significantly (2.2 ng/ml), IL-8 concentration in cells treated with IL-8-deprived supernatants was drastically lowered (0.1 ng/ml) (Fig. 2c). These results indicated that IL-8 secreted from Ca9-22 cells directly augmented IL-8 secretion in the THP1 cells.
IL-8 secretion is augmented at the transcriptional level
NF-κB-independent IL-8 secretion
The findings were further confirmed by the luciferase assay. The 5′-UTR of the IL-8 gene, shown in Fig. 4b , contains one NF-κB binding site. Using this construct as a template, we generated a mutant lacking the NF-κB binding site (ΔκB). The two then were subjected to the luciferase assay, which revealed no augmentation in luciferase activity by Ca-sup in both constructs (Fig. 4c), thereby indicating that NF-κB is not intrinsically important for IL-8 induction in THP1 cells.
MEK-dependent induction of IL-8
The phosphorylation status of ERK in the THP1 cells was evaluated by Western blotting, following the stimulation of the cells with or without Ca-sup for 0, 1, 2, and 3 h. Although the total amount of ERK1/2 was not changed throughout the stimulation (irrespective of Ca-sup treatment), augmentation of phosphorylation was clearly detected after 3 h of stimulation (Fig. 5c, right panel), indicating MEK-dependent induction of IL-8 in the THP1 cells.
Aberrant activity of transcription factors such as NF-κB or AP-1, in OSCCs, has resulted in the augmented expression of several different cytokines and chemokines . The production of these factors by the cancer cells has proven to be beneficial for them. Cancer cells react to hypoxic conditions by synthesizing new vasculature and secreting several factors that contribute to angiogenesis. IL-8 is secreted by several cancer cells and facilitates angiogenesis by inducing the branching of pre-existing blood vessels . Nevertheless, among the different types of cells within the tumor stroma, the TAMs have received considerable attention lately . The complex interplay between tumor cells and the stroma contribute significantly to cancer growth.
In malignant melanoma, the mutual interaction of melanoma cells and macrophages was reported to enhance the angiogenic potential of both cell types . The importance of cancer cell-macrophage interaction, for angiogenesis, has also been reported in cervical carcinomas . In spite of these reports, experimental evidence demonstrating the augmented production of IL-8 by macrophages driven by tumor-derived IL-8 has never been reported. In the present study, Ca9-22 was shown to secrete IL-8 spontaneously, and this secreted IL-8 was found to induce IL-8 secretion in the THP1 monocytic cells.
The IL-8 receptors CXCR1 and CXCR2 belong to the seven-transmembrane domain family of G-proteins , which is involved in several downstream signaling pathways mediated by various mediators . In a previous report, we have demonstrated the importance of the transcription factor NF-κB, on IL-8 induction in OSCCs . Based on these results, we speculated that the NF-κB-specific inhibitor TPCK might inhibit the augmented secretion of IL-8 in THP1 cells; on the contrary, IL-8 secretion was not affected by TPCK. This fact was further confirmed by the luciferase assay. Furthermore, the detection of IL-8 mRNA by real-time PCR, which peaked after 6 h of stimulation with Ca-sup, indicated that IL-8 induction was controlled at the transcriptional level. The transcriptional induction of both VEGF and β-FGF was also demonstrated.
The construct used in this study contained the 5′-UTR of the IL-8 gene spanning from −1 to −133, which is sufficient for transcriptional regulation of the gene . The findings from our study indicated that regions further upstream might contribute to IL-8-dependent, IL-8 secretion in the THP1 cells. IL-8 gene induction is regulated by multiple signaling pathways ; for instance, in mesenchymal stem cells, IL-8 can induce the expression of vascular endothelial growth factor through the Akt and ERK pathways . In the present study, pre-treatment of THP1 cells with the ERK1/2 inhibitor significantly reduced IL-8 secretion. Furthermore, apparent phosphorylation of ERK1/2 occurred after Ca-sup stimulation, leading us to speculate that IL-8 induction mechanisms in the THP1 cells are very different from those in the OSCCs. Intrinsic signaling via the MAPK pathways appears to play a significant role in augmenting the production of IL-8 in the THP1 cells.
The expression of cognate IL-8 receptors in OSCC has been demonstrated , and the role of OSCC-derived IL-8, on the migration and invasion of the cancer cells themselves, has also been extensively examined. Expression levels of IL-8 correlate with metastatic potential  and vascularity [27, 28, 29]. Moreover, elevated serum levels of IL-8 have been reported in OSCC , and cancer-derived IL-8 contributes to the recruitment of TAMs to the cancer stroma . Taken together, these reports suggest the correlation of IL-8 with cancer activity. In the present study, we have demonstrated that cancer-derived IL-8 enhanced the angiogenic potential of TAM. Considering the importance of tumor-stromal cell interaction in cancer growth and metastasis, it is safe to assume that tumor cells have the ability to adjust the microenvironment in their favor.
This work was supported by a grant of Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), 2010–2014 (S1001024); MEXT-supported program for the Strategic Research Foundation at Private Universities, 2013–2018; Sato Fund, Uemura Fund, and Grant from the Dental Research Center Nihon University School of Dentistry; and Nihon University Multidisciplinary Research Grant for 2015.
- 2.Ondrey FG, Dong G, Sunwoo J, Chen Z, Wolf JS, Crowl-Bancroft CV, et al. Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Mol Carcinog. 1999;26:119–29.CrossRefPubMedGoogle Scholar
- 25.Hou Y, Ryu CH, Jun JA, Kim SM, Jeong CH, Jeun SS. IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor. Cell Biol Int. 2014;38:1050–9.Google Scholar