1 INTRODUCTION

Patients with oral squamous cell carcinomas (OSCCs) that have spread to the regional lymph nodes have a poor 5-year survival rate compared with patients without metastasis [1]. The spread to regional lymph nodes in the neck²⁾, which can occur soon after the development of OSCC, is facilitated by rich lymphatic drainage from of the oral cavity [3]. A number of molecules have been implicated in the metastasis of OSCC^4),5).

Chemokines are small soluble molecules (8 – 14kDa) secreted by different types of cells, which can mediated the directional movement of cells and chemotaxis by binding to G-protein coupled seven-span transmembrane receptors (GPCRs) on the plasma membrane of target cells⁶⁾. Also, binding of chemokines to their receptors causes attraction of different types of blood leukocytes to sites of infection and inflammation⁷⁾.

At present, more than 50 chemokines and 20 chemokine receptors have been found. Among these chemokines and their receptors, the stromal cell-derived factor 1 (SDF-1 also called CXCL12) / CXCR4 axis has been demonstrated to be involved in the lymph node or distant metastasis of several types of cancer, including prostate cancer⁸⁾, kidney cancer ⁹⁾, neuroblastoma¹⁰⁾, breast cancer⁶⁾, and melanoma¹¹⁾. In addition, our previous studies have demonstrated that among OSCC patient tissue samples, a CXCR4-positive group demonstrated significantly higher PCNA LI than did the CXCR4-negative group¹²⁾.

Chemokines usually bind to multiple receptors and the same receptor may bind more than one chemokine. However, one exception to this rule has been accepted for many years ; the SDF-1 binds exclusively to CXCR4, and has CXCR4 as its only receptor^13),14). However, several reports recently provided evidence that SDF-1 also binds to another seven-transmembrane span receptor called CXCR7, sharing this receptor with another chemokine family member called interferon-inducible T-cell chemoattractant (I-TAC)^15),16). This finding broke the long-standing assumption that CXCR4 was the only receptor specific to SDF-1. Therefore, downstream cell function, which has been previously attributed to CXCR4, may also result from a CXCR7-mediated effect.

Over the past several years, it has been found that CXCR7 signaling promotes cancer cell survival through an antiapoptotic mechanism^17),18).

Miao et al., have demonstrated that expression of CXCR7 on breast and lung cancer cells positively correlates with their proliferation, vascularization, and metastatic potential. While CXCR7-transduced murine human breast or lung cancer cell lines grow larger tumors in immunodeficient mice, down-regulation of CXCR7 expression by siRNA resulted in formation of smaller tumors. CXCR7 overexpressing breast cancer cells also showed higher seeding efficiency in murine lungs in vivo¹⁹⁾. In another study, CXCR7 was found to be highly expressed on human prostate cancer cells. According to this study, CXCR7 regulates cell proliferation most likely because of enhanced cell survival, adhesion, and chemotaxis²⁰⁾.

Thus similarly to CXCR4, CXCR7 may promote expansion and metastasis of certain tumor types and could be a potential prognostic factor and a target for developing new anti-cancer and anti-metastatic drugs.

However, there have been no reports on the effect of transient down-regulation of CXCR7 by siRNA in oral cancer cells. And further, research is needed to evaluate the role of CXCR7 in OSCC cells. In the current study, we investigated the effects of CXCR7-downregulation on in vitro proliferation and migration of OSCC cells.

Ⅱ MATERIALS and METHODS

This study primarily made use of a published cell line (obtained from Dr. Lee’s Laboratory²¹⁾ and is exempt from assessment by Institutional Review Board of Seoul National University School of Dentistry.

1 CELL CULTURES

SCC-4, SCC-9, SCC-15 and SCC-25 human OSCC cells, purchased from American Tissue Cell Collection (Manassas, VA, USA) were cultured in DMEM:F12 mixture. HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-N-1 and HO-1-U-1 cell lines, purchased from Japanese Collection of Research Bioresources Cell Bank were cultured in RPMI 1640. KOSCC-11, KOSCC-25B, and KOSCC-33A human OSCC cells established from tumors obtained from Korean cancer patients, and named Korean oral squamous cell carcinoma cell line (KOSCC), were cultured in DMEM.

These 13 cell lines were cultured in media supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1% antibiotics antimycotic solution (100 U/ml penicillin and 100 ㎍/ml streptomycin; Gibco). The cell cultures were maintained at 37℃ in a humidified atmosphere of 95% air and 5% CO₂.

2 siRNA TRANSFECTION OF CXCR7

KOSCC25B cells were cultured in a 6-well plate (1 × 10⁶ cells per well) in 10% FBS containing medium. After overnight incubation, cells were transfected with Hs_CMKOR1_6 Flexitube siRNA (Qiagen, Hilden, Germany) at a concentration of 50nM, using Hiperfect transfection reagent (6 ㎕/ml, Qiagen) according to the manufacturer’s instructions. Transfected cells were named si.KOSCC25B. And negative control siRNA transfected KOSCC25B cells were used as control.

3 RT-PCR AND SEMI-QUANTITATIVE PCR ANALYSIS

After 24, 48, and 72h of transfection, we evaluated CXCR7 down-regulation by semi-quantitative PCR analysis.

The mRNA was purified from the cells using Trizol reagent (Invitrogen, Calsbad, CA, USA) according to the manufacturer’s recommended protocol. Two micrograms of RNA was converted into cDNA using random primers and reverse transcriptase. cDNA was PCR amplified in 30 cycles each consisting of 94℃ for 30 s, 60℃ for 50 s, 72℃ for 50 s. The primer pairs for CXCR7 and GAPDH were as follows ; CXCR7 forward, 5”- AAG AAG ATG GTA CGC CGT GTC GTC TGC ATC CTG –3’ ; CXCR7 reverse, 5”–CTG CTG TGC TTC TCC TGG TCA CTG GAC GCC GAG -3”; GAPDH forward, 5”- GAA GGT GAA GGT CGG AGT C-3”; GAPDH reverse, 5”- CAA AGT TGT CAT GGA TGA CC -3”.

PCR products were electrophoresed on 1.5% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence.

4 CELL PROLIFERATION ASSYAS

To determine the proliferation rate, KOSCC25B cells were seeded on 12-well plates (BD Falcon, Franklin Lakes, NJ, USA) at 3 × 10⁴ cells per well in DMEM with or without 10% FBS. After overnight of incubation, the si.CXCR7 cells were transfected with CXCR7 siRNA or si.Con cells were transfected with negative control siRNA.

And then, after 1, 2, and 3days the cells were trypsinized and stained with 0.4% Trypan Blue (Gibco). The total cell number and the proportion of dead cells were counted by hemocytometer. Cell death was determined by the presence of cytoplasmic Trypan Blue. This assay was performed in triplicate in each experiment, and each experiment was repeated three times.

5 IN VITRO MIGRATION ASSAY

A total of 1 × 10⁵ KOSCC25B cells were seeded in the upper compartment (8 μm pore size) in DMEM medium with or without 10% FBS. After overnight incubation, si.CXCR7 cells or si.Con cells were transfected using CXCR7 siRNA or negative control si.RNA as above.

Then, after 24, 48h incubation, the cells on the upper surface of the filter were wiped off with a cotton swab, and the remaining cells were stained with the Diff-Quick stain set (Sysmex, Kobe, Japan). Using a microscope at × 100 magnification, migration was quantified by counting the number of cells that migrated through the pores to the lower side of the filter. This assay was performed in triplicate in each experiment, and each experiment was repeated 3 times.

6 STATISTICAL ANALYSIS

Statistical significance was determined by unpaired Student’s t test with a threshold of P < 0.05. The correlation coefficient was calculated by using SPSS 12.0.

Ⅲ RSULTS

1 SCREENING OF ORAL SQUAMOUS CELL CARCINOMA CELL LINES

We screened 13 OSCC cell lines in order to obtain a cell line with high expression cell line of CXCR7. Of these 13 cell lines, SCC4, HSC-2, KOSCC11 and KOSCC33A showed markedly low CXCR7 levels as detected by semi-quantitative PCR analysis. Except for these 4 cell lines, the cell lines showed high CXCR7 expression levels (Fig.1).

Thus, of the KOSCC cell line series, the only cell line with high level expression was KOSCC25B, this cell line was chosen for the present study.

2 CONFIRMATION OF CXCR7 KNOCKDOWN BY siRNA

We assessed siRNA mediated CXCR7 down-regulation by semi-quantitative PCR analysis. As shown in Fig. 2A, CXCR7 mRNA levels were reduced in si.KOSCC25B cells, as compared with siRNA control transfected KOSCC25B cells. The CXCR7 mRNA level had decreased by 48h after transfection with siRNA. At 72h after transfection, the CXCR7 mRNA level had recovered. GAPDH was used as an internal control to monitor whether RNA isolation and RT-PCR were reliable. As shown in Fig. 2B, GAPDH was detected in both transfected and siRNA control transfected samples, as expected.

Therefore, CXCR7 expression was down-regulated at the mRNA level in si.KOSCC25B cells in the first 48h.

3 ANTI-PROLIFERATIC EFFECTS OF CXCR7- DOWNREGULATION

To determine the effect of CXCR7 down-regulation on proliferation of si.KOSCC-25B, the cell numbers were counted by hemocytometer. In serum-free media condition, si.KOSCC25B cells proliferated at 88.21%, 73.03% and 75.38% at 1, 2, and 3 days after transfection, respectively, compared with control. Additionally, in the presence of 10% FBS, si.KOSCC25B cells proliferated at 82.34%, 68.97%, and 55.45% at 1, 2 and 3days after transfection, respectively (P < 0.05). Thus, we established that si.KOSCC25B cells grew significantly slower than the siRNA control transfected KOSCC-25B cells (Fig 3).

These data indicated that the down-regulation of CXCR7 has an anti-proliferative effect on OSCC cells.

4 REDUCED MIGRATION DUE TO CXCR7- DOWNREGULATION

Due to the important role of the CXCR7/SDF-1 axis in cancer metastasis, we investigated whether CXCR7-downregulation decreased migration of OSCC cells. As shown in Fig. 4, CXCR7 down-regulation decreased KOSCC25B cell migration. At 24 h and 48 h after siRNA transfection, si.KOSCC25B decreased to 73% and 64%, compared to siRNA control transfected cells, respectively, in serum-starved media (P < 0.000). In the presence of 10% FBS, si.KOCC25B migration decreased to 35% and 22% at 24 h and 48 h, respectively, compared to the control cells (P < 0.003).

Therefore, these data indicated that the down-regulation of CXCR7 decreased migration both in the presence and absence of FBS.

Ⅳ DISCUSSION

It is well-known that chemokines interact with GPCRs to activate down-stream signaling pathways that enhance cancer cell growth, migratory behavior, and cell survival [22, 23], however, the role of the chemokine receptor, CXCR7, in cancer has not been elucidated fully. In this study, we show that CXCR7 is involved in the proliferation and migration of OSCC cells.

SDF-1 is a member of the CXC subfamily of chemokines and is expressed in a variety of tissues, including lung, liver, bone marrow and lymph nodes^24)-26). SDF-1 exerts its functions through binding to its receptor, CXCR4, which is present on the cell surface and is a seven-transmembrane span GPCR²⁷⁾. SDF-1 plays a role in a number of important physiological processes, including leukocyte trafficking and vasculogenesis^25),28). More importantly, SDF-1 plays a crucial role in the process of invasion and metastasis of tumor cells 29). SDF-1 stimulates proliferation, dissociation, migration, and invasion in a wide variety of tumor cells, including breast cancer cells, pancreatic cancer cells and HCC cells ^29),30),31). Moreover, expression of the SDF-1 receptor, CXCR4, in clinical samples is associated with lymph node metastasis of OSCC^32),33).

A recent study has shown that chemokine receptor CXCR7 can also bind to SDF-1, and it has thus been identified as a second receptor for SDF-1¹⁵⁾. CXCR7 is expressed in many different tissues, including neurons, immune cells, and endothelial cells; receptor-mediated signaling can occur by binding either of its 2 known ligands, CXCL11 or CXCL12^34)-37).

Similar to what is known about CXCR4, recent reports have indicated that CXCR7 promotes cancer cell survival through anti-apoptotic mechanisms^34),38). Therefore, downstream cell functions, which have been previously attributed to CXCR4 may also result from CXCR7-mediated signaling. In the present study, we investigated the effect of CXCR7-down-regulation on cell proliferation and migration. Down-regulation of CXCR7 induced anti-proliferative and anti-migration effects in KOSCC25B cells. To our knowledge, this the first description of siRNA mediated CXCR7-knockdown in oral cancer cells.

Recent studies in breast cancer cells indicated that downregulation of CXCR4 by siRNA inhibits invasion in vitro³⁹⁾ and metastasis and growth in vivo^40),41). In addition, we previously investigated CXCR4 signaling in oral cancer cells and observed effects on cell proliferation, migration, and invasion¹³⁾.

Thus, it was plausible that, similar to CXCR4, CXCR7 may promote proliferation and migration of OSCC cells. Our results support this proposal, and indicate that CXCR7 could be both a potential prognostic factor and a target for developing new anti-cancer and anti-metastatic drugs.