- Open Access
Vesnarinone downregulates CXCR4 expression via upregulation of Krüppel-like factor 2 in oral cancer cells
Molecular Cancer volume 8, Article number: 62 (2009)
We have demonstrated that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC). Chemotherapy is a powerful tool for the treatment of oral cancer, including oral SCC; however, the effects of chemotherapeutic agents on the expression of CXCR4 are unknown. In this study, we examined the expression of CXCR4 associated with the chemotherapeutic agents in oral cancer cells.
The expression of CXCR4 was examined using 3 different chemotherapeutic agents; 5-fluorouracil, cisplatin, and vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2-(1H)-quinolinone) in B88, a line of oral cancer cells that exhibits high levels of CXCR4 and lymph node metastatic potential. Of the 3 chemotherapeutic agents that we examined, only vesnarinone downregulated the expression of CXCR4 at the mRNA as well as the protein level. Vesnarinone significantly inhibited lymph node metastasis in tumor-bearing nude mice. Moreover, vesnarinone markedly inhibited 2.7-kb human CXCR4 promoter activity, and we identified the transcription factor, Krüppel-like factor 2 (KLF2), as a novel vesnarinone-responsive molecule, which was bound to the CXCR4 promoter at positions -300 to -167 relative to the transcription start site. The forced-expression of KLF2 led to the downregulation of CXCR4 mRNA and impaired CXCR4 promoter activity. The use of siRNA against KLF2 led to an upregulation of CXCR4 mRNA.
These Results indicate that vesnarinone downregulates CXCR4 via the upregulation of KLF2 in oral cancer.
Worldwide, oral cancer, excluding pharyngeal cancer, represents 2.65% of all human malignancies . Most oral cancer is histopathologically diagnosed as SCC. Oral cancers, including OSCC are characterized by a high degree of local invasion and a high rate of metastases to the cervical lymph nodes. In particular, it has been demonstrated that lymph node metastasis directly affects the prognosis of patients with OSCC as a results of extracapsular spread and induces the secondary distant metastasis [2, 3]. Lymph node metastasis is frequently detected upon the first visit to a hospital, but it also appears during initial treatments that include radiotherapy and chemotherapy. Lymph node metastasis has been primarily treated by surgical resection, although pre- or post-operative chemotherapy and/or pre- or post-operative radiotherapy have been reported to be useful for achieving a favorable course [4, 5]. Despite the evidence accumulated to date, there is no known molecular target for anti-metastatic therapy against nodal involvement patients with oral cancer.
Chemokines are a large family of small (7–15 kDa), structurally related, heparin-binding proteins that have been identified as attractants of different types of blood leukocytes to sites of infection and inflammation . They are produced locally in various tissues and act on leukocytes via selective membrane-bound G protein-coupled receptors, the two major subfamilies of which are CCR and CXCR. Among these chemokines and their receptors, the SDF-1 (also referred to as CXCL12)/CXCR4 system has been shown to be involved in both lymph node and distant metastases of several types of cancer [7–12]. We have also demonstrated that lymph node metastatic OSCC cells specifically express functional CXCR4, and the SDF-1/CXCR4 system is involved in lymph node metastatic processes in OSCC [13, 14]. Moreover, we recently revealed that the expression of CXCR4 in OSCC at the primary site was significantly associated with lymph node metastasis, mode of invasion, tumor recurrence, and patient prognosis . Recently, we have demonstrated that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system .
Considering the importance of CXCR4 in a variety of cancers, attempts have been made to blockade this molecule as a therapeutic target. For example, the administration of CXCR4-neutralizing antibody into tumor-bearing mice by Müller and colleagues inhibited lymph node and lung metastasis in a breast cancer metastasis model . Furthermore, CXCR4 has been shown to function as a co-receptor for T cell line-tropic HIV-1 entry ; and for this reason, there has been considerable interest in the clinical potential of CXCR4 inhibitors such as bicyclam AMD3100 , horseshoe crab-derived peptide T22 , and the non-peptide TAK-779  for inhibiting HIV infection. However, the clinical application of such inhibitors must be approached with caution, in light of the importance of the CXCR4 pathway in normal lymphohematopoiesis . Moreover, for the effective treatment of cancer, compounds with growth inhibitory effects as well as anti-metastatic effects are desirable. Therefore, if conventional chemotherapeutic agents that exert inhibitory effects on CXCR4 are identified, it is expected that anti-metastatic therapy could be performed more safely and effectively. However, the molecular mechanism(s) of diverse chemotherapeutic agents against CXCR4 expression are not yet fully understood. Thus, in this study, we examined the transcriptional regulation of CXCR4 by chemotherapeutic agents in an oral cancer cell line, B88, which expresses high levels of CXCR4, and which exhibits lymph node metastatic potential in vivo.
Reagents and materials
For in vitro experiments, vesnarinone (OPC-8212; Otsuka Pharmaceutical Company, Tokyo, Japan) was dissolved in DMSO at concentration of 10 mg/ml as the first stock solution, and the first stock solution was diluted with the complete culture medium . For in vivo experiment, vesnarinone was suspended in 0.5% CMC (Daiichi seiyakukogyo, Kyoto, Japan) . 5-FU (Sigma, St. Louis, MO) and CDDP (Sigma) was dissolved in DMSO at concentration of 1 mg/ml, and diluted with the complete culture medium at 10 μg/ml. Several CXCR4 promoter luciferase construct was kindly provided by Drs. Peter Staller and Wilhelm Krek (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland). KLF2 expression vectors (pcDNA3-KLF2, pcDNA3-Δ KLF2, and pGEX-4T-1-KLF2) were generous gifts from Drs. Sucharita Banerjee and Mukesh K Jain (Cardiovascular Division, Brigham and Women's Hospital, Boston, MA).
BALB/c nude mice were purchased from CLEA Japan (Osaka, Japan). The mice were maintained under pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experimentation of Tokushima University. The experiments were initiated when the mice were 8 weeks of age and were performed as described previously . The experimental chemotherapy by use of vesnarinone was performed as described previously with slight modification . Briefly, B88 cells constitutively expressing firefly luciferase (B88-luc) were orthotopically inoculated into masseter muscle of nude mice. One week later, the mice were treated by the daily oral administration of 0.5% CMC (5 ml/kg) for a vehicle or 0.2 ml of 10 mg/ml vesnarinone (50 mg/kg). The tumor volume was estimated by measuring tumor size and using the following formula: tumor volume = 1/2 × L × W2, where L and W represent the largest diameter, and the smallest diameter, respectively. The presence or absence of lymph node and distant metastases was confirmed by the H&E staining.
Cells and cell culture
High pulmonary metastases clone ACC-M was selected from cell line ACC-2 that was derived from surgically excised primary tumor tissue from a histologically diagnosed patient with adenoid cystic carcinoma of the palate . ACC-M and B88 cells  were maintained in RPMI-1640 and DMEM, respectively, supplemented with 10% fetal calf serum (FCS), 100 μg/ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. ACC-M cell lines were supplied friendly from Drs. Weiliu Qiu and Wantao Che (Laboratory of Tumor Biology, Ninth People's Hospital, Shanghai Jiao Tong University, School of Stamotology).
After stimulation of the cells with or without chemotherapeutic agents, the preparation of total RNA and reverse transcription were performed as described previously . In quantitative PCR, CXCR4 and KLF2, CXCR7 or GAPDH mRNA were detected with QuantiTect™ Gene Expression Assays (Hs-CXCR4; Qiagen, Hilden, Germany) and Taqman™ Gene Expression Assay (Applied Biosystems, Foster City, CA), respectively. Gene-specific products were measured continuously by an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) during 40 cycles. Experiments were performed at least three times.
Flow cytometric analysis
Logarithmically growing oral cancer cells were trypsinized and fixed in 4% (w/v) paraformaldehyde on ice for 10 min. The cells were washed and incubated with anti-human CXCR4 mAb (dilution 1:100, 12G5; BioSource International, Inc. Camarillo, CA) or mouse IgG isotype control (SouthernBiotech, Birmingham, CA) for 30 min at room temperature. After being washed twice with PBS, the cells were incubated with PE-labeled goat anti-mouse IgG (Serotec, Sapporo, Japan) for 30 min at room temperature and analyzed with an EPICS flow cytometer (Coulter, San Jose, CA).
In vitro cell migration assay
The in vitro migration of B88 cells was evaluated using the Transwell (CORNING, Corning, NY) as described previously . In some experiments, cells were incubated with vesnarinone (50 μg/ml) or were transfected with KLF2 expression vectors before the addition of cells in the upper chamber.
Construction of CXCR4 promoter-luciferase reporter plasmids
Several truncated promoter constructs (Figure 1) were generated by restriction digestion of pGL2-CXCR4 (-832/+86) with Kpn I and Mlu I for pGL2-CXCR4 (-300/+86), with Kpn I and Sac II for pGL2-CXCR4 (-29/+86), or with Kpn I and Xho I for pGL2-CXCR4 (0). The digested fragments were treated with T4 DNA polymerase (Takara Biomedicals, Kusatsu, Japan) and self-ligated with T4 DNA ligase (Takara Biomedicals). pGL2-CXCR4 (-167/+86) and pGL2-CXCR4 (-72/+86) were generated by PCR, using CXCR4-UP (5'-GGGGTACCCAAACTTAGGAAATGCCTCTGGGA-3') and CXCR4-DN1 (5'-GGGGTACCAGCGGCGCATGCGCCGCGCTCGGAGCG-3'), and CXCR4-UP and CXCR4-DN2 (5'-CCGCTCGAGCCTCCATGGTAACCGCTGGT-3'), respectively.
Transcriptional regulation of CXCR4 promoter by vesnarinone
B88 or ACC-M cells (5 × 105 cells/dish) were co-transfected with 5 μg of CXCR4-luciferase reporter vectors and 0.5 μg of pcDNA3 (Invitrogen, Carlsbad, CA) by electroporation (140 V, 99 msec, 1 pulse), using ELECTRO SQUARE PORATOR T820 (BTX Inc., San Diego, CA). After transfection, stable transfectants were isolated and the cells were treated with or without 50 μg/ml of vesnarinone as indicated. Then, the cells were lysed with cell culture lysis reagent (Pikkagene, Toyo Ink, Tokyo, Japan), and luciferase activity was measured by a Lumat LB9507 (BERTHOLD TECHNOLOGIES, Wildbad, Germany) using a luciferase assay kit (Toyo Ink) according to the manufacture's instruction. The luciferase activity was normalized by protein quantitation. Each experiment was repeated at least twice.
Total RNA for cDNA microarray analysis was extracted from B88 cells treated with or without vesnarinone for 48 h. cDNA microarray was performed and analyzed in Hokkaido System Science (Sapporo, Japan) using a human 1A Oligo Array (Agilent Technologies, Palo Alto, CA).
Chromatin immunoprecipitation (ChIP) assay
B88 cells were treated with or without vesnarinone for 48 h, or were transfected with pcDNA3 or pcDNA3-KLF2. ChIP assay was performed by using a ChIP assay kit according to the manufacture's instruction (upstate, Lake Placid, NY). Briefly, equal aliquots of isolated chromatin DNA were subjected to immunoprecipitation with a rabbit KLF2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The DNA fragments associated with specific immunoprecipitates were purified and used as templates for the PCR in order to amplify the CXCR4 promoter sequences containing a CACCC/KLF binding site (-401 to -142). The primers used were 5'-CAGCAAGTCACTCCC-3' and 5'-GGAGAGGTGCGCGGC-3'.
Electrophoretic mobility-shift assay (EMSA)
Biotinylated double stranded oligonucleotides of CXCR4 promoter sequence at positions -204 to -175 containing the CACCC site (5'-ACTCACTACCGACCACCC GCAAACAGCAGG-3') or a mutated CACCC site (5'-ACTCACTACCGACTGTTT GCAAACAGCAGG-3') were synthesized in Hokkaido System Science. After transformation of glutathione S-transferase (GST) or GST-KLF2 plasmid vectors into BL21 (DE3) competent cells (Merck, Darmstadt, Germany), recombinant proteins were extracted, and were purified with glutathione sepharose column (Amersham Pharmacia Biotech.). EMSA was performed using LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL) according to the manufacture's instruction.
siRNA against KLF2 was synthesized in B-Bridge international (Mountain View, CA). siRNA cocktails against KLF2 or negative control siRNA (B-Bridge International) were transfected into B88 cells, using Lipofectamine 2000 reagent (Invitrogen) at the final concentration of 20 nM or 200 nM. After 48-h incubation, total RNA was extracted and quantitative RT-PCR was performed.
Statistical differences between the means for the different groups were evaluated with StatView 4.5 (Abacus Concepts, Berkeley, CA) using one-way ANOVA, with the level of significance at P < 0.05. All experiments were repeated two to three times, and similar Results were obtained.
Expression of CXCR4 induced by the chemotherapeutic agents
In complete medium or DMSO, the expression of CXCR4 mRNA was transiently downregulated after the medium was changed, but mRNA expression was gradually upregulated and recovered to the basal level at 24 h (Figure 2a). Initially, two conventional cytocidal chemotherapeutic agents for treating oral cancer, namely 5-FU and CDDP, were assessed, but the expression of CXCR4 mRNA was not altered with these treatments. Therefore, we tested the effect of a novel cytostatic chemotherapeutic agent, vesnarinone . It was revealed that vesnarinone markedly downregulated the expression of CXCR4 mRNA and protein, as compared to the effects of DMSO, 5-FU, or CDDP in B88 cells (Figure 2a and 2b). Downregulation of CXCR4 mRNA by treatment with vesnarinone was also detected in oral cancer cells, ACC-M, in a time dependent manner (Figure 2c). Moreover, we also found the reduced expression of CXCR4 protein by the treatment with vesnarinone, as compared to the effects of 5-FU and CDDP both in ACC-M cells and in Hela cells (Additional File 1). Next, we examined the effect of vesnarinone on the expression of CXCR7, which was recently identified as a chemokine receptor that binds chemokines CXCL11 and SDF-1 . However, analyses of cDNA microarray and quantitative RT-PCR revealed that vesnarinone was not found to change the expression of CXCR7 (data not shown).
Effects of vesnarinone on the migration and lymph node metastasis of B88 cells
In order to investigate the functional role played by CXCR4 downregulation with vesnarinone, an in vitro migration assay was performed. SDF-1 was found to enhance the migration of B88 cells, which was in contrast significantly inhibited by treatment with vesnarinone (Figure 3a). In order to further clarify the role of vesnarinone on lymph node metastasis, experimental chemotherapy was carried out. Vesnarinone significantly inhibited the size and weight of the metastatic lymph nodes in the orthotopic inoculation of B88 cells to nude mice (Figure 3b, c and 3d). Furthermore, when treated with vesnarinone, B88 cells constitutively expressing firefly luciferase showed significantly impaired luciferase activity in the metastatic lymph nodes (Figure 3e).
Transcriptional regulation of the CXCR4 gene by vesnarinone
Since vesnarinone appeared to inhibit the lymph node metastasis by downregulating CXCR4, we investigated in greater detail the transcriptional regulation of CXCR4 by vesnarinone. A luciferase reporter vector driven by the 2.7-kb CXCR4 promoter region was transfected into B88 cells, and the stable transfectants were isolated. As shown in Additional File 2a, luciferase activities of the CXCR4 promoter, when treated with vesnarinone, were significantly lower than those of the promoter treated with DMSO vehicle alone. In order to further clarify the transcriptional regulatory region activated by vesnarinone in the CXCR4 promoter, we isolated several B88-transfectants with truncated CXCR4 promoter-luciferase constructs, and in those treated with vesnarinone, we observed an inhibition of luciferase activity driven downstream of the truncated CXCR4 promoters up to position -300 from the transcription starting site (Additional File 2b and 2c). However, the luciferase activity driven upstream of position -167 was not significantly impaired by treatment with vesnarinone (Additional File 2d, e, f and Figure 1). These results indicate that the vesnarinone-regulatory element on the CXCR4 promoter was located at positions -300 to -167 from the transcription starting site (Figure 1).
Identification of vesnarinone-regulatory element on the CXCR4 promoter region
Positions -300 to -167 contained several canonical transcription factor binding sites such as E-box, NF-κB, nuclear factor of activated T cells, and Krüppel-like factor (KLF). Therefore, we performed a microarray analysis to identify the vesnarinone-regulatory element on the CXCR4 promoter. Numerous molecules were upregulated or downregulated by treatment with vesnarinone, but the gene related to the vesnarinone-regulatory element and that with the highest upregulation detected was KLF2. To verify whether or not KLF2 binds to vesnarinone-regulatory element, we next performed a ChIP assay. After treatment with vesnarinone, KLF2 preferentially bound to vesnarinone-regulatory element (Figure 4a). Moreover, forced expression of KLF2 also led to binding to vesnarinone-regulatory element in the absence of vesnarinone stimulation, which indicated that KLF2 might regulate the expression of CXCR4 (Figure 4a). Next, we carried out an EMSA in order to confirm the binding of KLF2 to vesnarinone-regulatory element. Recombinant KLF2 effectively bound to the CXCR4 promoter sequence at positions -204 to -175 containing the CACCC site; however, the binding was not inhibited by the addition of excess volume of mutant probe (Figure 4b).
Effects of KLF2 on the downregulation of CXCR4
Next, we confirmed the upregulation of KLF2 by treatment with vesnarinone. Vesnarinone was found to upregulate KLF2 mRNA in a time-dependent manner both in B88 cells (Figure 5a) and in ACC-M (Figure 2c) cells. Therefore, we investigated the role played by KLF2 in the downregulation of CXCR4. Forced expression of KLF2 downregulated CXCR4 mRNA, but forced expression of ΔKLF2, with a deleted DNA binding domain, did not lead to such downregulation (Figure 5b). Moreover, forced expression of KLF2 also attenuated the luciferase activity driven by the CXCR4 promoter (-300/+76) both in B88 cells (Figure 5c) and in ACC-M cells (Additional File 3). Next, we examined the functional effects of KLF2 on the cell migration induced by SDF-1. In B88 cells, the migration induced by SDF-1 was significantly inhibited by the forced expression of KLF2, but this was not observed in the case of either pcDNA3 or ΔKLF2 (Figure 5d).
Effects of silencing KLF2 on the expression of CXCR4
Since the overexpression of KLF2 induced the downregulation of CXCR4, we next attempted to examine the expression of CXCR4 using KLF2 knock-down and siRNA approaches. The introduction of the siRNA against KLF2 exhibited a 50% decrease in the KLF2 mRNA levels (Figure 6a), which in turn markedly upregulated CXCR4 mRNA (Figure 6b).
Vesnarinone, a synthetic quinolinone derivative with inotropic effects, was originally developed to treat cardiac failure  and has been clinically used for the treatment of patients with chronic congestive heart failure . Vesnarinone was not developed as a targeted agent; however, recent studies have revealed that vesnarinone is a unique anti-proliferation [22, 23], differentiation-inducing  and apoptosis inducing [30, 31] agent against several human malignancies, including leukemia and several types of solid tumors. Furthermore, clinical studies have shown that vesnarinone potentiates the effects of conventional cytotoxic chemotherapy and radiotherapy [32–34]. We have been studying the molecular mechanism of the growth inhibitory effects of vesnarinone against the head and neck cancer cells, and we demonstrated that vesnarinone induces the expression of various kinds of genes such as p21waf1  and transforming growth factor β stimulated clone (tsc)-22 . A previous phase study revealed that two partial responses against metastatic lesions in a patient with metastatic breast cancer involving lymph nodes, liver, and bone, and in a patient with metastatic non-small-cell lung cancer involving the brain and lymph node, when the patients were treated with vesnarinone and gemcitabine . These clinical observations suggested the anti-metastatic activity of vesnarinone; however, the molecular mechanism of the anti-metastatic action of vesnarinone remained unclarified in those studies. Vesnarinone was shown to exhibit anti-proliferating effects at a dose of 200 mg/kg against a human adenoid squamous carcinoma-forming cell line grown in nude mice . In order to specifically investigate the anti-metastatic effects of vesnarinone, we administrated a lower dose of vesnarinone (50 mg/kg) to tumor-bearing nude mice. As expected, vesnarinone did not inhibit tumor size in the masseter muscle (data not shown), but it did inhibit the lymph node metastasis. These results indicated that although vesnarinone specifically inhibits lymph node metastasis, this does not occur by inhibition of lymph node cell growth.
In the present study, we demonstrated the novel transcriptional regulation of CXCR4 via the transcription factor KLF2 in vesnarinone-treated oral cancer cells. It has been shown that the expression of CXCR4 is transcriptionally upregulated by several transcription factors such as c-Myc , NF-κB  and hypoxia inducible factor (HIF) , and is downregulated by Yin-Yang 1 . In addition, cytokine signaling induced by granulocyte colony-stimulating factor , interferon gamma , and tumor necrosis factor-alpha  have also been reported to regulate CXCR4 expression. Among these transcription factors and cytokine signaling, vesnarinone-regulatory element located in the region between -300 and -167 from the transcriptional starting site contained one canonical NF-κB binding site. On the other hand, Manna and co-workers reported that vesnarinone suppresses the TNF-induced activation of NF-κB . Thus, we initially expected to find that NF-κB is a vesnarinone-inducible transcriptional regulator for CXCR4. However, our site-directed mutagenesis study showed that vesnarinone did alter promoter activity in samples with either a mutated NF-κB site or that of samples with a wild-type NF-κB site (data not shown]. Moreover, our microarray analysis revealed that treatment with vesnarinone did not alter NF-κB subunit p50 and p65 mRNA levels. Thus, we concluded that NF-κB is not a vesnarinone-inducible CXCR4 regulator.
KLF2 [i.e., lung Krüppel-like factor (LKLF)], was originally identified through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe; and KLF2 is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen . KLF2 is required to program the quiescent state of T cells via the downregulation of c-Myc . Moreover, it has been demonstrated that KLF2 regulates diverse biological activities, such as adipogenesis , inflammation , cell growth , apoptosis , tumorigenesis , and angiogenesis . In these processes, KLF2 upregulates p21waf1  and cytosolic phospholipase A2 alpha , and downregulates several types of molecules, including peroxisome proliferator-activated receptor-gamma , vascular cell adhesion molecule-1 , E-selectin , WEE1 , and vascular endothelial growth factor receptor 2 . KLF2 is grouped with KLF1 and KLF4, which are characterized by amino-terminal acidic activation domains, inhibitory regions adjacent to the zinc fingers, and conserved nuclear localization signals, and which function both as activators as well as repressors . However, abundant evidence suggested that KLF2 dominantly functions as a repressor. In the present study, KLF2 was also found to negatively regulate CXCR4 expression, most likely via the binding of a canonical CACCC motif. Since other KLF families are able to recognize this motif, CXCR4 may be downregulated by other KLF families. However, of the 13 members of the KLF family examined in the present microarray analysis (data not shown), only KLF2 mRNA was upregulated by 48-h treatment with vesnarinone. Thus, as regards the regulation of CXCR4 expression by vesnarinone, it is expected that KLF2 is the most important regulator among the member of KLF family that putatively binds to this CACCC motif.
In the present study, the mechanism of transcriptional upregulation of KLF2 by vesnarinone was still unclear. We have previously demonstrated that vesnarinone directly activates p21waf1 gene promoter via Sp1 sites in a human salivary gland cancer cell line . Since the 5'-flanking region of the KLF2 gene manifests typical features of GC-rich promoter with multiple transcription factor Sp1 binding sites , KLF2 might be also upregulated by vesnarinone via the activation of Sp1.
CXCR4 functions as a coreceptor for T cell line-tropic HIV-1 entry. Previously, vesnarinone was found to inhibit the replication of HIV-1 in a peripheral blood lymphocyte model via the inhibition of cytokine production . Although vesnarinone was not developed as a targeted agent against CXCR4, it may have other targets against the metastasis of oral cancer. However, this effect might have been the result of the impaired expression of CXCR4 in peripheral blood lymphocytes due to treatment with vesnarinone. The findings of the present study suggest that vesnarinone might be useful as an anti-metastatic agent for patients with CXCR4-related oral cancer. Moreover, our clinical observations suggest that whereas vesnarinone does not exert dramatic anti-tumor effects, it does induce tumor differentiation and dormancy, effects previously shown to prolong the lives of patients with head and neck cancers [32, 33]. Due to agranulocytosis and mild anti-tumor effects, care must be taken to administer vesnarinone as the sole agent as a first-line therapy to patients with OSCC. However, a combination of vesnarinone with conventional chemotherapy or radiotherapy might be used as a novel second- or third-line anti-metastatic intervention in patients with CXCR4-related OSCC.
Our results indicated that vesnarinone downregulates CXCR4 expression via the upregulation of KLF2 in oral cancer.
oral squamous cell carcinoma
stromal cell-derived factor-1; vesnarinone; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2-(1H)-quinolinone
Dulbecco's modified Eagle medium
glyceraldehyde 3-phosphate dehydrogenase.
Parkin DM, Bray F, Ferlay J, Pisani P: Estimating the world cancer burden: Globocan 2000. Int J Cancer. 2001, 94: 153-156. 10.1002/ijc.1440
Johnson JT, Barnes EL, Myers EN, Schramm VL, Borochovitz D, Sigler BA: The extracapsular spread of tumors in cervical node metastasis. Arch Otolaryngol. 1981, 107: 725-729.
Decroix Y, Ghossein NA: Experience of the Curie Institute in treatment of cancer of the mobile tongue: II. Management of the neck nodes. Cancer. 1981, 47: 503-508. 10.1002/1097-0142(19810201)47:3<503::AID-CNCR2820470313>3.0.CO;2-1
Johnson JT, Wagner RL, Myers EN: A long-term assessment of adjuvant chemotherapy on outcome of patients with extracapsular spread of cervical metastases from squamous carcinoma of the head and neck. Cancer. 1996, 77: 181-185. 10.1002/(SICI)1097-0142(19960101)77:1<181::AID-CNCR29>3.0.CO;2-0
Labadie RF, Yarbrough WG, Weissler MC, Pillsbury HC, Mukherji SK: Nodal volume reduction after concurrent chemo- and radiotherapy: correlation between initial CT and histopathologic findings. Am J Neuroradiol. 2000, 21: 310-314.
Rossi D, Zlotnik A: The biology of chemokines and their receptors. Annu Rev Immunol. 2000, 18: 217-242. 10.1146/annurev.immunol.18.1.217
Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, Verástegui E, Zlotnik A: Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001, 410: 50-56. 10.1038/35065016
Scotton CJ, Wilson JL, Milliken D, Stamp G, Balkwill FR: Epithelial cancer cell migration: a role for chemokine receptors?. Cancer Res. 2001, 61: 4961-4965.
Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman NS, McCauley LK: Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res. 2002, 62: 1832-1837.
Schrader AJ, Lechner O, Templin M, Dittmar KE, Machtens S, Mengel M, Probst-Kepper M, Franzke A, Wollensak W, Gatzlaff P, Atzpodien J, Buer J, Lauber J: CXCR4/CXCL12 expression and signalling in kidney cancer. Br J Cancer. 2002, 86: 1250-1256. 10.1038/sj.bjc.6600221
Zhou Y, Larsen PH, Hao C, Yong VW: CXCR4 is a major chemokine receptor on glioma cells and mediates their survival. J Biol Chem. 2002, 277: 49481-49487. 10.1074/jbc.M206222200
Kijima T, Maulik G, Ma PC, Tibaldi EV, Turner RE, Rollins B, Sattler M, Johnson BE, Salgia R: Regulation of cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cancer cells. Cancer Res. 2002, 62: 6304-6311.
Uchida D, Begum NM, Ammar A, Nakashiro K, Kawamata H, Tateishi Y, Hamakawa H, Yoshida H, Sato M: Possible role of stromal cell-derived factor-1/CXCR4 signaling on lymph-node metastasis of oral squamous cell carcinoma. Exp Cell Res. 2003, 290: 289-302. 10.1016/S0014-4827(03)00344-6
Uchida D, Begum NM, Tomizuka Y, Bando T, Almofti A, Yoshida H, Sato M: Acquisition of lymph node, but not distant metastatic potentials, by the over-expression of CXCR4 in human oral squamous cell carcinoma cells. Lab Invest. 2004, 84: 1538-1546. 10.1038/labinvest.3700190
Ammar A, Uchida D, Begum NM, Tomizuka Y, Iga H, Yoshida H, Sato M: The clinicopathological significance of the expression of CXCR4 protein in oral squamous cell carcinoma. Int J Oncol. 2004, 25: 65-71.
Uchida D, Onoue T, Tomizuka Y, Begum NM, Miwa Y, Yoshida H, Sato M: Involvement of an autocrine stromal cell-derived factor-1/CXCR4 system on the distant metastasis of human oral squamous cell carcinoma. Mol Cancer Res. 2007, 5 (7): 685-694. 10.1158/1541-7786.MCR-06-0368
Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA: The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996, 382: 829-833. 10.1038/382829a0
De Clercq E: The bicyclam AMD3100 story. Nat Rev Drug Discov. 2003, 2: 581-587. 10.1038/nrd1134
Murakami T, Nakajima T, Koyanagi Y, Tachibana K, Fujii N, Tamamura H, Yoshida N, Waki M, Matsumoto A, Yoshie O, Kishimoto T, Yamamoto N, Nagasawa T: A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection. J Exp Med. 1997, 186: 1389-1393. 10.1084/jem.186.8.1389
Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, Shiraishi M, Aramaki Y, Okonogi K, Ogawa Y, Meguro K, Fujino M: A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Nat Acad Sci USA. 1999, 96: 5698-5703. 10.1073/pnas.96.10.5698
Moore MA: The role of chemoattraction in cancer metastases. Bioessays. 2001, 23: 674-676. 10.1002/bies.1095
Kawamata H, Nakashiro K, Uchida D, Hino S, Omotehara F, Yoshida H, Sato M: Induction of TSC-22 by treatment with a new anti-cancer drug, vesnarinone, in a human salivary gland cancer cell. Br J Cancer. 1998, 77: 71-78.
Sato M, Harada K, Bando T, Shirakami T, Nakashiro K, Yoshida H, Nakai S, Kawai K, Adachi M: Characteristics of antitumor activity of 3, 4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]- 2(1H)-quinolinone (vesnarinone) against a human adenoid squamous carcinoma-forming cell line grown in athymic nude mice. Cancer Lett. 1995, 91: 1-9. 10.1016/0304-3835(95)03713-7
Kawamata H, Nakashiro K, Uchida D, Hino S, Harada K, Yoshida H, Sato M: Possible contribution of active MMP2 to lymph-node metastasis and secreted cathepsin L to bone invasion of newly established human oral-squamous-cancer cell lines. Int J Cancer. 1997, 70: 120-127. 10.1002/(SICI)1097-0215(19970106)70:1<120::AID-IJC18>3.0.CO;2-P
Guan XF, Qiu WL, He RG, Zhou XJ: Selection of adenoid cystic carcinoma cell clone highly metastatic to the lung: an experimental study. Int J Oral Maxillofac Surg. 1997, 26: 116-119. 10.1016/S0901-5027(05)80830-6
Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ: A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006, 203: 2201-2213. 10.1084/jem.20052144
Asanoi H, Sasayama S, Iuchi K, Kameyama T: Acute hemodynamic effects of a new inotropic agent (OPC-8212) in patients with congestive heart failure. J Am Coll Cardiol. 1987, 9: 865-871.
Cohn JN, Goldstein SO, Greenberg BH, Lorell BH, Bourge RC, Jaski BE, Gottlieb SO, McGrew F, DeMets DL, White BG: A dose-dependent increase in mortality with vesnarinone among patients with severe heart failure. Vesnarinone Trial Investigators. N Engl J Med. 1998, 339: 1810-1816. 10.1056/NEJM199812173392503
Sato S, Tomoyasu S, Okabe-Kado J, Hozumi M, Tsuruoka N, Nakai S, Adachi M, Honma Y: Induction of differentiation and enhancement of vincristine sensitivity of human erythroleukemia HEL cells by vesnarinone, a positive inotropic agent. Exp Hematol. 1996, 24: 37-42.
Isaka K, Fujito A, Sagawa Y, Yudate T, Nishi H, Ito H, Takayama M: Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3, 4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone). Oncol Rep. 2002, 9: 1299-1305.
Khalaf W, Maina C, Byers J, Harvey W: Interferon-alpha 2β and vesnarinone influence levels of tumor necrosis factor-alpha, apoptosis, or interleukin 6 in ESKOL, a hairy cell leukemic cell line. A potential cytokine and oncogene relationship regulating apoptosis is suggested. Leuk Res. 2002, 26: 169-177. 10.1016/S0145-2126(01)00100-X
Sato M, Harada K, Yura Y, Bando T, Azuma M, Kawamata H, Iga H, Yoshida H: Induction of tumour differentiation and apoptosis and LeY antigen expression in treatment with differentiation-inducing agent, vesnarinone, of a patient with salivary adenoid cystic carcinoma. Apoptosis. 1997, 2: 106-113.
Sato M, Harada K, Yura Y, Azuma M, Kawamata H, Iga H, Tsujimoto H, Yoshida H, Adachi M: The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma. Apoptosis. 1997, 2: 313-318. 10.1023/A:1026493205097
Patnaik A, Rowinsky EK, Tammara BK, Hidalgo M, Drengler RL, Garner AM, Siu LL, Hammond LA, Felton SA, Mallikaarjun S, Von Hoff DD, Eckhardt SG: Phase I and pharmacokinetic study of the differentiating agent vesnarinone in combination with gemcitabine in patients with advanced cancer. J Clin Oncol. 2000, 18: 3974-3985.
Omotehara F, Kawamata H, Uchida D, Hino S, Nakashiro K, Fujimori T: Vesnarinone, a differentiation inducing drug, directly activates p21(waf1) gene promoter via Sp1 sites in a human salivary gland cancer cell line. Br J Cancer. 2002, 87: 1042-1046. 10.1038/sj.bjc.6600592
Moriuchi M, Moriuchi H, Margolis DM, Fauci AS: USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity of CXCR4, a coreceptor for HIV-1 entry. J Immunol. 1999, 162: 5986-5992.
Helbig G, Christopherson KW, Bhat-Nakshatri P, Kumar S, Kishimoto H, Miller KD, Broxmeyer HE, Nakshatri H: NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J Biol Chem. 2003, 278: 21631-21638. 10.1074/jbc.M300609200
Staller P, Sulitkova J, Lisztwan J, Moch H, Oakeley EJ, Krek W: Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature. 2003, 425: 307-311. 10.1038/nature01874
Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T: G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol. 2002, 3: 687-694. 10.1038/ni813
Katayama A, Ogino T, Bandoh N, Nonaka S, Harabuchi Y: Expression of CXCR4 and its down-regulation by IFN-gamma in head and neck squamous cell carcinoma. Clin Cancer Res. 2005, 11: 2937-2946. 10.1158/1078-0432.CCR-04-1470
Kulbe H, Hagemann T, Szlosarek PW, Balkwill FR, Wilson JL: The inflammatory cytokine tumor necrosis factor-alpha regulates chemokine receptor expression on ovarian cancer cells. Cancer Res. 2005, 65: 10355-10362. 10.1158/0008-5472.CAN-05-0957
Manna SK, Aggarwal BB: Vesnarinone suppresses TNF-induced activation of NF-kappa B, c-Jun kinase, and apoptosis. J Immunol. 2000, 164: 5815-5825.
Anderson KP, Kern CB, Crable SC, Lingrel JB: Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family. Mol Cell Biol. 1995, 15: 5957-5965.
Buckley AF, Kuo CT, Leiden JM: Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc-dependent pathway. Nat Immunol. 2001, 2: 698-704. 10.1038/90633
Banerjee SS, Feinberg MW, Watanabe M, Gray S, Haspel RL, Denkinger DJ, Kawahara R, Hauner H, Jain MK: The Krüppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis. J Biol Chem. 2003, 278: 2581-2584. 10.1074/jbc.M210859200
SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, García-Cardeña G, Jain MK: KLF2 is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med. 2004, 199: 1305-1315. 10.1084/jem.20031132
Wu J, Lingrel JB: KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene. 2004, 23: 8088-8096. 10.1038/sj.onc.1207996
Wang F, Zhu Y, Huang Y, McAvoy S, Johnson WB, Cheung TH, Chung TK, Lo KW, Yim SF, Yu MM, Ngan HY, Wong YF: Transcriptional repression of WEE1 by Krüppel-like factor 2 is involved in DNA damage-induced apoptosis. Oncogene. 2005, 24: 3875-3885. 10.1038/sj.onc.1208546
Wick MJ, Blaine S, Van Putten V, Saavedra M, Nemenoff RA: Lung Krüppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A2 alpha promoter. Biochem J. 2005, 387: 239-246. 10.1042/BJ20041458
Bhattacharya R, Senbanerjee S, Lin Z, Mir S, Hamik A, Wang P, Mukherjee P, Mukhopadhyay D, Jain MK: Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Krüppel-like factor KLF2. J Biol Chem. 2005, 280: 28848-28851. 10.1074/jbc.C500200200
Lomberk G, Urrutia R: The family feud: turning off Sp1 by Sp1-like KLF proteins. Biochem J. 2005, 392: 1-11. 10.1042/BJ20051234
Maruyama I, Maruyama Y, Nakajima T, Kitajima I, Osame M, Zhao JQ, Chen IS, Nakai S, Ikeda M, Yabu-uchi Y, Adachi M: Vesnarinone inhibits production of HIV-1 in cultured cells. Biochem Biophys Res Commun. 1993, 195: 1264-1271. 10.1006/bbrc.1993.2180
We thank Dr. Toyoki Mori (Director of Research Institute of Pharmacological & Therapeutical Development, Otsuka Pharmaceutical Company, Tokushima, Japan) for providing vesnarinone. This study was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.
The authors declare that they have no competing interests.
DU designed experiments, drafted manuscript and performed siRNA knock-down. TO performed luciferase assay and nude mice experiments. NMB performed bacterial culture, recombination of plasmid and purification of the plasmid. YT performed real-time PCR and flow cytometry. NK was responsible for cell cultures and participated in PCR analysis and data collection. TT and HN participated in discussion and manuscript preparation. YM conceived the study and revised the manuscript. All authors read and approved the final version of the manuscript.
Electronic supplementary material
Additional file 1: . ACC-M or Hela cells treated with or without chemotherapeutic agents were incubated with mouse IgG isotype control or with anti-CXCR4 monoclonal antibody. Then the cells were incubated with PE-labeled goat anti-mouse IgG and analyzed by flow cytometry in order to determine the expression of CXCR4 protein. The white zones show the cells treated with vesnarinone stained by mouse IgG isotype control. The black zones show cells treated with vesnarinone, and gray zones show the cells treated with 5-FU (upper) or CDDP (lower), respectively. The black zones and gray zones were stained by anti-CXCR4 monoclonal antibody. (PDF 34 KB)
Additional file 2: Inhibition of luciferase activity in the presence of vesnarinone. Several CXCR4-luciferase constructs were transfected into B88 cells, which were treated with or without vesnarinone, and then luciferase activities were measured at the indicated time points. (a) -2632/+86, (b) -832/+86, (c) -300/+86, (d) -167/+86, (e) -72/+86, (f) -29/+86. (PDF 38 KB)
Additional file 3: . Stable transfectants of CXCR4 promoter-luciferase construct (-300/+86) were transfected with pcDNA3 or KLF2 expression vectors, and then luciferase activity was measured at the indicated time points. *, Significant as determined by one-way ANOVA (vs. pcDNA3, P < 0.05). (PDF 9 KB)
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Uchida, D., Onoue, T., Begum, N. et al. Vesnarinone downregulates CXCR4 expression via upregulation of Krüppel-like factor 2 in oral cancer cells. Mol Cancer 8, 62 (2009). https://doi.org/10.1186/1476-4598-8-62
- Oral Cancer
- Oral Squamous Cell Carcinoma
- CXCR4 Expression
- Oral Cancer Cell