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Anti-tumor effect of vitamin A and D on head and neck squamous cell carcinoma
Auris Nasus Larynx 30 (2003) 403 /412 www.elsevier.com/locate/anl
Anti-tumor effect of vitamin A and D on head and neck squamous cell carcinoma Kenichi Satake a,*, Emi Takagi a, Akiko Ishii a, Yasumasa Kato a,b, Yukari Imagawa a, Yuu Kimura a, Mamoru Tsukuda a a
Department of Otolaryngology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan b Department of Biochemistry and Molecular Biology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan Received 27 January 2003; received in revised form 10 April 2003; accepted 16 May 2003
Abstract Objectives : Vitamin A and D3 have a very strong differentiation induction effect. Study design: We examined the anti tumor effect on head and neck squamous cell carcinoma (HNSCC) by treatment with several vitamins having strong differentiation induction effects in vitro. Methods: We used KB cell that an oral floor squamous cell carcinoma, vitamins as all-trans retinoic acid (ATRA), 4[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), 1a,25(OH)2D3 (calcitriol) and 22-oxa-1,25-(OH)2D3 (OCT). We determined receptors of vitamin A and D3 using RT-PCR. Furthermore, we investigated the proliferation of tumor cells in concentration dependency using [3H]TdR uptake method, apoptosis and apoptosis related factors using TUNEL method and realtime PCR, cell cycle changes using flow cytometry, changing of the sensitivity of using MTT method, cytokine production and the angiogenesis factor using ELISA, by treatment with these vitamins. Results: The deficit of RAR-b was found in the KB cell. Each vitamin suppressed the cell proliferation, induced apoptosis, and cell cycle arrest, upregulated sensitivity of the chemotherapeutics drugs and downregulated several angiogenesis factors and an apoptotic factor; survivin . Conclusions: These results support the idea that vitamin A, D3 and their derivatives are useful for preventing and/or treating patients with HNSCC. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: All-trans Retinoic acid; TAC-101; Calcitriol; OCT; Head and neck squamous cell carcinoma
1. Introduction Research on all-trans retinoic acid (ATRA) has been progressing rapidly since the report on the differentiation induction effect on the mouse teratoma cell line (F9) by Strick et al. [1] along with the human promyelocytic leukemia cell line (HL-60) by Breitman et al. [2]. The complementary DNA (cDNA) cloning of the retinoic acid receptor-a (RAR-a) [3,4], the receptor within a nucleus described in 1987, and the RAR-b, RAR-g and cDNA were investigated subsequently. In 1988 the cDNA of the vitamin D receptor (VDR) was cloned [5], and the VDR was determined to be a receptor of 1a, 25 (OH) 2D3 (calcitriol) with the receptor within the nucleus as a ligand. RAR and VDR are
* Corresponding author. Tel.: /81-45-787-2687; fax: /81-45-7832580. E-mail address: [email protected] (K. Satake).
molecules that belong to a steroid hormone receptor super family found in the action mechanism and are ligand dependent transcription control proteins of the complement system. For acute promyelocytic leukemia, ATRA and calcitriol show strong differentiation induction actions and ATRA is especially useful for therapy. The antitumor effect of ATRA from the point of view of differentiation induction action is helpful even in solid tumors and head and neck squamous cell carcinoma (HNSCC) [6 /8]. The research on other derivatives is continuously being reported [9 /11]. Some of the antitumor mechanisms of these vitamins are still unclear, although one of them is confirmed to inhibit cell proliferation, angiogenesis and enhances apoptosis. Apoptosis is an important mechanism in cancer chemotherapy [12]. One of the functions of the p53 tumor suppressor gene is the induction of apoptosis by G1 arrest [13,14]. The p21 protein which inhibits cyclin D kinase (cdk) makes, also, induces apoptosis [15 /17].
0385-8146/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0385-8146(03)00091-9
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The bax is known to affect the induction of apoptosis, and the mechanism is antagonistic to bcl-2 [18], one of the inhibitor of apoptosis protein (IAP) family. Survivin is a part of the IAP family, and was not detected in adult normal tissue but in survivin expressed in cancer [19]. These proteins involved in apoptosis were also investigated in this study. It is also known that chemotherapeutic drugs act on the cell cycle of tumor cells [11]. We investigated the actions that each vitamin has an ability on cell cycle change and an effect on the sensitivity to chemotherapeutic drugs. Cis -diamine dichloro platinum (CDDP) and 5-fluorouracil (5-FU) as chemotherapeutic drugs which have been commonly applied for HNSCC were used in this study [20]. Recent studies have revealed that antiangiogenic therapy inhibit the progression of recurrence and metastasis. The inhibitors of angiogenesis, such as ATRA, several retinoids, and cox-2 inhibitor, have been reported to exhibit antiangiogenic and antimetastatic activities [21,22]. Interleukin-8 (IL-8) is associated with inflammation and angiogenesis, and its increased expression has been implicated in a variety of malignancies [23]. Vascular endothelial growth factor (VEGF), known as a typical angiogenesis factor, is a unique angiogenic dimeric glycoprotein. Basic fibroblast growth factor (bFGF), which belongs to a family of heparin-binding growth factors, and is a multifunctional protein, which plays a key role in tumor angiogenesis [24]. Platelet-derived growth factor (PDGF) was reported to be a vascular permeability factor [25]. ATRA is the final active substance of vitamin A, and 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a vitamin A derivative [21,26,27]. As vitaminD3, we used calcitriol, which is the final active substance of VitaminD3. And its derivative substance 22-oxa-1,25-dihydroxyvitamin D3 (OCT) was also studied [28]. In terms of these vitamins, we have examined antitumor mechanisms and the effects on HNSCC cell line.
2. Materials and methods
2.1. Cell culture KB cells [29], a human oral floor carcinoma cell line, were grown in RPMI-1640 medium (Life Technologies Inc., Tokyo, Japan) supplemented with 10% fetal calf serum (FCS), 2 mM glutamin, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 8C in a 5% CO2 atmosphere.
2.2. Materials ATRA was purchased from Wako Pure Chemical Industries Ltd. OCT was synthesized by Chugai Pharmaceutical Co., Ltd. Calcitriol was purchased from Wako Pure Chemical Industries Ltd. TAC-101 and 5FU were synthesized by Taiho Pharmaceutical Co., Ltd. ATRA was dissolved in DMSO at a concentration of 1 mM and stored in the dark at /80 8C. TAC-101, calcitriol and OCT were dissolved in DMSO at a concentration of 10 mM and stored in the dark at / 20 8C. CDDP was purchased from Nihonkayaku Co., Ltd. 2.3. Reverse transcription PCR Total cellular RNA was extracted using ISOGEN reagent according to the manufacture’s recommendations (Nippon Gene Inc.) The first strand complementary DNA (cDNA) was synthesized with 1 mg of total RNA using SUPERSCRIPTTM First-Strand Synthesis System for RT-PCR and was employed according to the manufacturer’s protocol (Life Technologies Inc.). The primer sequences that reported by Xu et al. were as shown on Table 1[30]. The PCR amplification was performed at a constant temperature of 94 8C with denaturation for 30 s, and annealing at 56 8C (b-actin, RAR-a and VDR), 52 8C (RAR-b and g) for 1 min, and an extention to 72 8C for 1 min, using Ex TaqTM (Takara Shuzo Co., Ltd., Shiga, Japan). Equal volumes of the PCR product from each sample were subjected to electrophoresis on a 10% polyacrylamid gel, stained with ethidium bromide, and photographed. 2.4. Cell treatment with vitamins and determination of cell growth KB cells were seeded at densities of 1 /104 cell per well in 96-well tissue culture plates. After 24 h, the cells were treated with different concentrations of vitamins. The concentration was 1 and 10 mM of ATRA, 0.1 and 1 mM TAC-101, calcitriol and OCT. The control cultures received no stimulation. Following the 48 h incubation, the cells were pulsed for 4 h with 0.25 mCi/well [3H]TdR (Amersham, Amersham, Buckinghamshire, UK) before radioactivity measurements. The percentage of cell growth was calculated by using the equation: % tumor growth/the absorbance in treated (At)/the absorbance in control (Ac) /100. At and Ac represent the absorbance in treated and control cultures, respectively. 2.5. TUNEL assay After 1 /105 of KB cells were seeded on the Lab-Tek chamber slide (Nalge Nunc International) and treated with 1 mM ATRA, TAC-101, calcitriol and OCT. The
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Table 1 RT-PCR primer sequences specifications
control KB cells were not treated. TUNEL assay was performed with Apoptosis in situ Detection Kit Wako according to the manufacturer’s protocol (Wako Pure Chemical Industries Ltd.) and the apoptotic cells were photographed. 2.6. Quantitative apoptotic factor using real-time PCR Total RNA was extracted from KB cells that had been treated with 1 mM each of vitamins for 48 h and were not treated as controls, by the acid quanidinium thiocyanate /phenol/chloroform extraction method using RNAzolB (Sawady, Tokyo, Japan), and was collected from the precipitate in ethanol. cDNA was synthesized with a survivin 466/485 primer (5?-AGA GGC CTC AAT CCA TGG CA-3?) and a glyceraldehydes-3-phosphate dehydrogenase (G3PDH), as an internal control, exon 8 primer (5?-CTT GAT GTC ATC ATA TTT GGC AGG-3?), we synthesized the
cDNA of p53 , p21 and bax with a random primer. The PCR reaction mixture was prepared using a TaqMan Universal Master Mix (PE Applied Biosystems, CA, USA). The primer and probe were designed by SRL Co., Ltd. Tokyo Japan (Table 2). This primer for survivin did not detect survivin-beta mRNA. Each PCR reaction was made at 50 cycles (95 8C for 30 s, 60 8C for 40 s, 72 8C for 30 s) using a real-time PCR system (ABI PRISM 7700 Sequence Detection System: PE Applied Biosystems). The PCR product of each mRNA was purified using a High pure PCR product purification kit (Roche Molecular Biochemicals Diagnostic, IN, USA) and was directly sequenced using the Thermosequenase Cy5 Dye Terminator kit (Amersham Pharmacia Biotech, Uppsala, Sweden) with an automated laser fluorescence DNA sequencer ALF express (Amersham Pharmacia Biotech). The sequence was finally compared with each target mRNA sequence.
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Table 2 Real-time PCR primer and probe sequences specifications
2.7. Cell cycle analysis One mM ATRA, TAC-101, calcitriol and OCT were added to 1 /106 of KB cells. The control was not treated. After 24 and 48 h, the medium was removed and
the cell monolayers were washed twice with cold phosphate-buffered saline (PBS). The cells were then trypsinized and the cell pellets were collected. The cells were again washed twice with PBS, fixed in cold methanol, and rewashed with PBS to remove methanol.
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24 h later CDDP as 0.1, 1.0, 10.0 mg/ml and 5-FU as 0.1, 1.0, 50.0 mg/ml were added. The Tetracolor ONE (Seikagaku Co., Ltd. Tokyo Japan) as 10 ml reacted in addition in each well, and 48 h later and the absorbance of 450 /500 nm was harvested using the NJ-2100 (Nippon InterMed Co., Ltd.). 2.9. ELISA for IL-8, VEGF, PDG and bFGF
Fig. 1. cDNA was synthesized with 1 mg of total RNA. The PCR amplification was performed consisting of denaturation at 94 8C for 30 s, annealing for 1 min (temperature was different at each primer), and extention at 72 8C for 1 min, using Ex TaqTM (Takara Shuzo Co., Ltd.). Equal volumes of the PCR product from each sample were subjected to electrophoresis on a 10% polyacrylamid gel, stained with ethidium bromide, and photographed.
They were suspended in 500 ml of PBS and then digested with 20 mg/ml RNase at 37 8C for 30 min and chilled on ice for 10 min. The cellular DNA was then stained with 50 mg/ml propidium iodide and incubated for 1 h at room temperature in the dark. The cell cycle distribution was analyzed by flow cytometry using a Becton Dickinson FACScan.
1 /106 of KB cells were seeded in a 10-cm plastic dish (Falcon). The supernatants of cells were treated with 1 ml of 1 mM of ATRA, TAC-101 and 1 mM calcitriol and OCT. After 48 h, they were determined to be at a concentration of IL-8, VEGF, PDGF and bFGF using the Quantine ELISA kit (R&D System, Mineapolis, MN). The protein concentration of these cytokine and factors was determined by absorbance compared with the standard curve and analyzed by NJ-2100.
3. Results 3.1. Nuclear receptors The expression of RARs and VDR was determined in KB cells using RT-PCR of total cellular RNA (Fig. 1). KB cells expressed RAR-a, RAR-g, and VDR. But RAR-b was not expressed in KB cells. 3.2. Growth inhibition
2.8. Changing the sensitivity to CDDP and 5-FU by treatment with ATRA and calcitriol KB cells, with the addition of 1 mM ATRA, 1 mM calcitriol and combinations of them were incubated for 1 week. 1 /104 of cells were seeded into 96 hole plates and
The proliferation of tumor cells by treatment with vitamins was determined by uptaking with [3H]TdR. The proliferation of KB cells was inhibited by the vitamin treatment. The vitamins also induced concentration dependency and conspicuously reinforced it (Fig. 2). 3.3. Apoptosis Apoptotic cells were stained by the TUNEL method. The expression of the apoptotic cell was confirmed in all the vitamin treatments (Fig. 3). Many expressions of the apoptotic cells were observed in all treated groups. 3.4. Apoptosis factors
Fig. 2. 1 /104 cells per well in 96-well cultured for 24 h the cells were treated with different concentrations of vitamins for 48 h and were pulsed for 4 h with 0.25 mCi/well [3H]TdR (Amersham), was detected using radioactivity. The percentage of cell growth was calculated by using the equation: % tumor growth/At/Ac/100, where At and Ac represent the absorbance in treated and control cultures, respectively.
We quantitated for mRNA of p53 , p21 , bax and survivin using real-time PCR. There were no clear changes in these mRNA of p53, p21 and bax, even when KB cells were treated with vitamins. But mRNA of survivin clearly decreased by treatment with all vitamins (Fig. 4). TAC-101 had the most effect on suppressing survivn expression. Calcitriol has the same suppression effect on the expression of survivin . ATRA
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Fig. 3. After 1/105 of KB cells were seeded in the chamber slide and treated with 1 mM ATRA (A), TAC-101 (B), calcitriol (C) and OCT (D). TUNEL assay was performed and apoptotic cells were photographed ( /40). (Scale bars/50 mm).
and OCT, however, have fewer effects than TAC-101 and calcitriol. 3.5. Cell cycle Cell cycle was analyzed by flow cytometry. By treatment with vitamins, the tendency of the cells of the G0/G1 phase increased and at the same time the cells of the S phase decreased (Fig. 5). Also, the accumulation to the G0/G1 phase of the cell strengthened and the reaction time increased. The cells of G2/M phase decreased by treatment with ATRA and calcitriol, but the cells increased in the G2/M phase by treatment with TAC-101. 3.6. Changing of sensitivity to CDDP and 5-FU by treatment with ATRA and calcitriol The decline of the 50% inhibitory concentration of CDDP was confirmed in the ATRA and calcitriol treatment group before CDDP exposable in comparison with a control group. This effect appears more pronounced in calcitriol than in ATRA. On the other hand, the decline of the 50% inhibitory concentration was not confirmed by the addition of 5-FU (Fig. 6).
3.7. Cytokin and angiogenesis factors By the ELISA method, the cytokin and angiogenesis factors, were determined. Concentration of each factor in the cell culture supernatant was as shown in Fig. 7. VEGF decreased after each vitamin treatment. IL-8 had no changing by treatment with vitamin (data not shown). On the other hand bFGF could not be detected (data not shown). We think that it falls below the measurement possibility value. It is apparent that PDGF decreases by treatment with each vitamin.
4. Discussion This is the first detailed study of the effects of vitamins on HNSCC and their nuclear receptor expression of cultured HNSCC in vitro. The expression of RAR-b of HNSCC was suppressed in the nuclear receptors. RAR-b seems to be a useful intermediate marker in retinoid trials in several studies of RAR-b reported by Xu et al. who suggested that the decreased expression of RAR-b may be associated with HNSCC development [30,31]. Cells that have no expression of RAR-b were transfected with RAR-b gene, and the
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Fig. 4. Total RNA was extracted from KB cells that were treated with 1 mM each vitamins for 48 h and were not treated as control, by the acid quanidinium thiocyanate /phenol /chloroform extraction method using RNAzolB. cDNA synthesized with a survivin 466 /485 primer and for p53 , p21 and bax , cDNA were synthesized with a random primer. The PCR reaction mixture was prepared using a TaqMan Universal Master Mix. This primer for survivin did not detect a survivin -beta mRNA. Each PCR reaction was made at 50 cycles (95 8C for 30 s, 60 8C for 40 s, 72 8C for 30 s) using a real-time PCR system. The PCR product of each mRNA was purified using a High pure PCR product purification kit and were directly sequenced using the Thermosequenase Cy5 Dye Terminator kit with an automated laser fluorescence DNA sequencer ALF express. The sequence was finally compared with each target mRNA sequence.
effect of treatment with retinoids was increased on the cell with expression of RAR-b [32,33]. In this study KB cell has not expressed RAR-b, but ATRA and TAC-101 inhibit the growth of tumor cells. This indicates that the presence of RAR-b is not involved with inhibitory effects of cell growth by retinoids [34]. When ATRA and TAC-101 are compared, the cell proliferation control of TAC-101 is much more effective than that of ATRA. This result shows that TAC-101 has a strong differentiation induction effect than ATRA although TAC-101 is a ligand of only RAR-a. Recently, it has been reported that the expression of RAR-b is associated with the inhibition of the keratinization in HNSCC cells [35], and methylation of the RAR-b p2 promoter is one mechanism that silences the RAR-b gene in lung carcinoma [36], which may be similar to HNSCC. KB cells have VDR and the proliferation of KB cells is suppressed by treatment with each vitaminD3. Among the differential induction effects on HNSCC by treatment with vitamins, one of the most important mechanisms is apoptosis [12]. The expression of TUNEL positive cells increased by treatments with each vitamin, compared with group without treatment. We suggest on the basis of these results that the each vitamin contributes to the enhancement of apoptosis.
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The p53 cancer suppressor gene induces apoptosis by G0/G1 arrest and promotes the expression of p21. The bax antagonizes bcl -2 and induces apoptosis through caspase-3 activation. In terms of p53 , p21 , bax expressions, there was no change of each expression at the mRNA level. Survivin , one of IAP, is expressed in all of the cancer tissue, although not in normal adult tissue. Apoptosis is inhibited by survivin expressed in the G2/M phase. The expression of survivin at the mRNA level was suppressed by treatment with vitamins. Among the groups treated with TAC-101, ATRA and vitaminD3, the expression of survivin was greatly inhibited in the TAC-101 treated group. Shin et al. reported that survivin is a direct inhibitor of caspase-3 and -7 [37]. We did not investigate these enzymes but our result is indication of these vitamins downregulate survivin, and this phenomenon induces apoptosis. In this study our findings gives suggestion that cells of the G0/G1 phase increased and that its S phase decreased by treatment with ATRA, calcitriol and OCT. Calcitriol especially has a strong influence. But the group treated with TAC-101 increased the G2/M phase. The cells were gathered in the G0/G1 phase by treatment with vitamins (in other words, G0/G1 arrest). Oridate et al. reported that cells treated with retinoids have induction of apoptosis in HNSCC [10]. Also, Light et al. reported that vitamin D3 acted on the squamous cell carcinomas of mice and noted an increase in a wellknown G1 phase [20]. Our results indicated the same findings in HNSCC cells. Our results show a sensitivity to CDDP was enhanced by treatment with ATRA and Calcitriol. But the sensitivity of 5-FU did not increase. Generally it is thought that CDDP shows most antitumor activity in the cell of the G1 phase [38]. Also the sensitivity of CDDP increased when treated with vitamin D as reported by Light et al. [20]. This phenomenon is thought to be the result of the increase in tumor cells in the G1 phase as a result of each vitamin treatment. Calcitriol has the effect of stopping the G0/G1 phase. The combination therapy of these vitamins makes greater changes to the sensitivity of CDDP. However, the sensitivity to 5-FU did not increase, as the mechanism of action by 5-FU is not cell cycle dependent. Recently antiangiogenic therapy has been applied in HNSCC and other solid tumors. Some studies have pointed out that antiangiogenic therapy can inhibit the progression and recurrence of metastatic disease, which depends on adequate vasculature and, therefore, on angiogenesis. In this study, we have investigated IL-8, VEGF, bFGF and PDGF production by treatment with vitamins in HNSCC [39,40]. Our results show that VEGF and PDGF production decreased as a result of the treatments. The results suggest that these vitamins have an antiangiogenic effect. Sauter et al. reported that VEGF is a marker of invasion and metastasis [41], and
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Fig. 5. KB cells left untreated, was treated with 1 mM ATRA, TAC-101, calcitriol and OCT. The control was not treated. After 24 and 48 h, the cells were stained with 50 mg/ml of propidium iodide and analyzed with a FACScan flow cytometry.
VEGF expression has been described as of prognostic significance by Salven et al. in HNSCC [42]. bFGF were not detected and IL-8 had no changing by each vitamin treatment. bFGF and IL-8 are a proangiogenesis factor and depends on cell line.
In this study we examined antitumor effects on HNSCC. In summary, treatments with each vitamin inhibited the proliferation of tumor cells. Apoptosis, cell cycle arrest and the down regulation of survivin were induced. It is evident that each vitamin controlled the
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evident that each vitamin controlled the expression of growth factors of the tumor cells.
Acknowledgements We thank Taiho Pharmaceutical Co., Ltd. and Chugai Pharmaceutical Co., Ltd. for their contribution of materials.
Fig. 6. KB cells, after add 1 mM ATRA, 1 mM calcitriol and each combination were incubated for 1 week, and 1/104 of cells were seeded into each 96 of hole plates and 24 h later were added CDDP as 0.1, 1.0, 10.0 mg/ml and 5-FU as 0.1, 1.0, 50.0 mg/ml. The Tetracolor ONE as 10 ml are caused reacted in addition to each well 48 h later and was harvested by using MTT method.
Fig. 7. 1/106 of KB cells were seeded in a 10-cm plastic dish. The supernatants of cells that were treated with 1 ml of 1 mM of ATRA, TAC-101, calcitriol and OCT. After 48 h, were determined using the commercial Quantine ELISA kit for IL-8, VEGF, PDGF and bFGF. The protein concentration of these cytokin and factor were determined by absorbance comparison to the standard curve and analyzed by NJ2100.
expression of growth factors of the tumor cells. Today, clinical trials for cancer chemoprevention are beginning in HNSCC. However, these vitamins combined with sensitive chemotherapeutic drug might be more useful to suppress recurrence and metastasis. It is indispensable to examine combination therapies along with other chemotherapeutic drugs and radiotherapy [43].
5. Conclusion In this study we examined antitumor effects on s. In summary, treatments with each vitamin inhibited the proliferation of tumor cells. Apoptosis, cell cycle arrest and the down regulation of survivin were induced. It is
References [1] Strickland S, Mahdavi V. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 1978;15:393 /403. [2] Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci USA 1980;77:2936 /40. [3] Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987;330:444 /50. [4] Giguere V, Ong ES, Segui P, Evans RM. Identification of a receptor for the morphogen retinoic acid. Nature 1987;330:624 /9. [5] Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, et al. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 1988;85:3294 /8. [6] Sacks PG, Oke V, Amos B, Vasey T, Lotan R. Modulation of growth, differentiation and glycoprotein synthesis by beta-alltrans retinoic acid in a multicellular tumor spheroid model for squamous carcinoma of the head and neck. Int J Cancer 1989;44:926 /33. [7] Zou CP, Clifford JL, Xu XC, Sacks PG, Chambon P, Hong WK, et al. Modulation by retinoic acid (RA) of squamous cell differentiation, cellular RA-binding proteins, and nuclear RA receptors in human head and neck squamous cell carcinoma cell lines. Cancer Res 1994;54:5479 /87. [8] Jetten AM, Kim JS, Sacks PG, Rearick JI, Lotan D, Hong WK, et al. Inhibition of growth and squamous-cell differentiation markers in cultured human head and neck squamous carcinoma cells by beta-all-trans retinoic acid. Int J Cancer 1990;45:195 /202. [9] Lippman SM, Benner SE, Hong WK. Cancer chemoprevention. J Clin Oncol 1994;12:851 /73. [10] Oridate N, Lotan D, Xu XC, Hong WK, Lotan R. Differential induction of apoptosis by all-trans -retinoic acid and N -(4hydroxyphenyl)retinamide in human head and neck squamous cell carcinoma cell lines. Clin Cancer Res 1996;2:855 /63. [11] Diaz GD, Paraskeva C, Thomas MG, Binderup L, Hague A. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Res 2000;60:2304 /12. [12] Makin G, Hickman JA. Apoptosis and cancer chemotherapy. Cell Tissue Res 2000;301:143 /52. [13] Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, et al. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989;244:217 /21. [14] Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, et al. Mutations in the p53 gene occur in diverse human tumour types. Nature 1989;342:705 /8. [15] Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701 /4.
412
K. Satake et al. / Auris Nasus Larynx 30 (2003) 403 /412
[16] Gu Y, Turck CW, Morgan DO. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 1993;366:707 /10. [17] el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817 /25. [18] Reed JC, Miyashita T, Takayama S, Wang HG, Sato T, Krajewski S, et al. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem 1996;60:23 /32. [19] Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet 1998;351:882 /3. [20] Light BW, Yu WD, McElwain MC, Russell DM, Trump DL, Johnson CS. Potentiation of cisplatin antitumor activity using a vitamin D analogue in a murine squamous cell carcinoma model system. Cancer Res 1997;57:3759 /64. [21] Murakami K, Sakukawa R, Sano M, Hashimoto A, Shibata J, Yamada Y, et al. Inhibition of angiogenesis and intrahepatic growth of colon cancer by TAC-101. Clin Cancer Res 1999;5:2304 /10. [22] Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798 /801. [23] Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993;362:841 / 4. [24] Slavin J. Fibroblast growth factors: at the heart of angiogenesis. Cell Biol Int 1995;19:431 /44. [25] Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989;246:1309 /12. [26] Sakukawa R, Murakami K, Ikeda T, Yamada Y, Saiki I. Effect of 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) on the liver metastasis of colon 26-L5 carcinoma cells. Oncol Res 1998;10:287 /93. [27] Fujimoto K, Hosotani R, Doi R, Wada M, Lee JU, Koshiba T, et al. Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid derivative, TAC-101, in human pancreaticcancer cells. Int J Cancer 1999;81:637 /44. [28] Endo K, Ichikawa F, Uchiyama Y, Katsumata K, Ohkawa H, Kumaki K, et al. Evidence for the uptake of a vitamin D analogue (OCT) by a human carcinoma and its effect of suppressing the transcription of parathyroid hormone-related peptide gene in vivo. J Biol Chem 1994;269:32693 /9. [29] Eagle H. Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc Soc Exp Biol Med 1955;89:362 /4. [30] Xu XC, Liu X, Tahara E, Lippman SM, Lotan R. Expression and up-regulation of retinoic acid receptor-beta is associated with retinoid sensitivity and colony formation in esophageal cancer cell lines. Cancer Res 1999;59:2477 /83.
[31] Xu XC, Ro JY, Lee JS, Shin DM, Hong WK, Lotan R. Differential expression of nuclear retinoid receptors in normal, premalignant, and malignant head and neck tissues. Cancer Res 1994;54:3580 /7. [32] Castillo L, Milano G, Santini J, Demard F, Pierrefite V. Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method. Clin Cancer Res 1997;3:2137 /42. [33] Crowe DL. Retinoic acid receptor beta induces terminal differentiation of squamous cell carcinoma lines in the absence of cyclin-dependent kinase inhibitor expression. Cancer Res 1998;58:142 /8. [34] Wan H, Oridate N, Lotan D, Hong WK, Lotan R. Overexpression of retinoic acid receptor beta in head and neck squamous cell carcinoma cells increases their sensitivity to retinoid-induced suppression of squamous differentiation by retinoids. Cancer Res 1999;59:3518 /26. [35] Zou CP, Hong WK, Lotan R. Expression of retinoic acid receptor beta is associated with inhibition of keratinization in human head and neck squamous carcinoma cells. Differentiation 1999;64:123 / 32. [36] Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, et al. Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst 2000;92:1303 /7. [37] Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, et al. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 2001;40:1117 /23. [38] Donaldson KL, Goolsby GL, Wahl AF. Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle. Int J Cancer 1994;57:847 /55. [39] Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith CW, et al. Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer. Clin Cancer Res 1999;5:1369 /79. [40] Eisma RJ, Spiro JD, Kreutzer DL. Role of angiogenic factors: coexpression of interleukin-8 and vascular endothelial growth factor in patients with head and neck squamous carcinoma. Laryngoscope 1999;109:687 /93. [41] Sauter ER, Nesbit M, Watson JC, Klein-Szanto A, Litwin S, Herlyn M. Vascular endothelial growth factor is a marker of tumor invasion and metastasis in squamous cell carcinomas of the head and neck. Clin Cancer Res 1999;5:775 /82. [42] Salven P, Heikkila P, Anttonen A, Kajanti M, Joensuu H. Vascular endothelial growth factor in squamous cell head and neck carcinoma: expression and prognostic significance. Mod Pathol 1997;10:1128 /33. [43] Arnold A. Moving promising research findings to the clinic: methodological issues in the design and conduct of clinical trials of retinoids. Int J Cancer 1997;70:467 /9.
Anti-tumor effect of vitamin A and D on head and neck squamous cell carcinoma Kenichi Satake a,*, Emi Takagi a, Akiko Ishii a, Yasumasa Kato a,b, Yukari Imagawa a, Yuu Kimura a, Mamoru Tsukuda a a
Department of Otolaryngology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan b Department of Biochemistry and Molecular Biology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan Received 27 January 2003; received in revised form 10 April 2003; accepted 16 May 2003
Abstract Objectives : Vitamin A and D3 have a very strong differentiation induction effect. Study design: We examined the anti tumor effect on head and neck squamous cell carcinoma (HNSCC) by treatment with several vitamins having strong differentiation induction effects in vitro. Methods: We used KB cell that an oral floor squamous cell carcinoma, vitamins as all-trans retinoic acid (ATRA), 4[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), 1a,25(OH)2D3 (calcitriol) and 22-oxa-1,25-(OH)2D3 (OCT). We determined receptors of vitamin A and D3 using RT-PCR. Furthermore, we investigated the proliferation of tumor cells in concentration dependency using [3H]TdR uptake method, apoptosis and apoptosis related factors using TUNEL method and realtime PCR, cell cycle changes using flow cytometry, changing of the sensitivity of using MTT method, cytokine production and the angiogenesis factor using ELISA, by treatment with these vitamins. Results: The deficit of RAR-b was found in the KB cell. Each vitamin suppressed the cell proliferation, induced apoptosis, and cell cycle arrest, upregulated sensitivity of the chemotherapeutics drugs and downregulated several angiogenesis factors and an apoptotic factor; survivin . Conclusions: These results support the idea that vitamin A, D3 and their derivatives are useful for preventing and/or treating patients with HNSCC. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: All-trans Retinoic acid; TAC-101; Calcitriol; OCT; Head and neck squamous cell carcinoma
1. Introduction Research on all-trans retinoic acid (ATRA) has been progressing rapidly since the report on the differentiation induction effect on the mouse teratoma cell line (F9) by Strick et al. [1] along with the human promyelocytic leukemia cell line (HL-60) by Breitman et al. [2]. The complementary DNA (cDNA) cloning of the retinoic acid receptor-a (RAR-a) [3,4], the receptor within a nucleus described in 1987, and the RAR-b, RAR-g and cDNA were investigated subsequently. In 1988 the cDNA of the vitamin D receptor (VDR) was cloned [5], and the VDR was determined to be a receptor of 1a, 25 (OH) 2D3 (calcitriol) with the receptor within the nucleus as a ligand. RAR and VDR are
* Corresponding author. Tel.: /81-45-787-2687; fax: /81-45-7832580. E-mail address: [email protected] (K. Satake).
molecules that belong to a steroid hormone receptor super family found in the action mechanism and are ligand dependent transcription control proteins of the complement system. For acute promyelocytic leukemia, ATRA and calcitriol show strong differentiation induction actions and ATRA is especially useful for therapy. The antitumor effect of ATRA from the point of view of differentiation induction action is helpful even in solid tumors and head and neck squamous cell carcinoma (HNSCC) [6 /8]. The research on other derivatives is continuously being reported [9 /11]. Some of the antitumor mechanisms of these vitamins are still unclear, although one of them is confirmed to inhibit cell proliferation, angiogenesis and enhances apoptosis. Apoptosis is an important mechanism in cancer chemotherapy [12]. One of the functions of the p53 tumor suppressor gene is the induction of apoptosis by G1 arrest [13,14]. The p21 protein which inhibits cyclin D kinase (cdk) makes, also, induces apoptosis [15 /17].
0385-8146/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0385-8146(03)00091-9
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The bax is known to affect the induction of apoptosis, and the mechanism is antagonistic to bcl-2 [18], one of the inhibitor of apoptosis protein (IAP) family. Survivin is a part of the IAP family, and was not detected in adult normal tissue but in survivin expressed in cancer [19]. These proteins involved in apoptosis were also investigated in this study. It is also known that chemotherapeutic drugs act on the cell cycle of tumor cells [11]. We investigated the actions that each vitamin has an ability on cell cycle change and an effect on the sensitivity to chemotherapeutic drugs. Cis -diamine dichloro platinum (CDDP) and 5-fluorouracil (5-FU) as chemotherapeutic drugs which have been commonly applied for HNSCC were used in this study [20]. Recent studies have revealed that antiangiogenic therapy inhibit the progression of recurrence and metastasis. The inhibitors of angiogenesis, such as ATRA, several retinoids, and cox-2 inhibitor, have been reported to exhibit antiangiogenic and antimetastatic activities [21,22]. Interleukin-8 (IL-8) is associated with inflammation and angiogenesis, and its increased expression has been implicated in a variety of malignancies [23]. Vascular endothelial growth factor (VEGF), known as a typical angiogenesis factor, is a unique angiogenic dimeric glycoprotein. Basic fibroblast growth factor (bFGF), which belongs to a family of heparin-binding growth factors, and is a multifunctional protein, which plays a key role in tumor angiogenesis [24]. Platelet-derived growth factor (PDGF) was reported to be a vascular permeability factor [25]. ATRA is the final active substance of vitamin A, and 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101) is a vitamin A derivative [21,26,27]. As vitaminD3, we used calcitriol, which is the final active substance of VitaminD3. And its derivative substance 22-oxa-1,25-dihydroxyvitamin D3 (OCT) was also studied [28]. In terms of these vitamins, we have examined antitumor mechanisms and the effects on HNSCC cell line.
2. Materials and methods
2.1. Cell culture KB cells [29], a human oral floor carcinoma cell line, were grown in RPMI-1640 medium (Life Technologies Inc., Tokyo, Japan) supplemented with 10% fetal calf serum (FCS), 2 mM glutamin, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 8C in a 5% CO2 atmosphere.
2.2. Materials ATRA was purchased from Wako Pure Chemical Industries Ltd. OCT was synthesized by Chugai Pharmaceutical Co., Ltd. Calcitriol was purchased from Wako Pure Chemical Industries Ltd. TAC-101 and 5FU were synthesized by Taiho Pharmaceutical Co., Ltd. ATRA was dissolved in DMSO at a concentration of 1 mM and stored in the dark at /80 8C. TAC-101, calcitriol and OCT were dissolved in DMSO at a concentration of 10 mM and stored in the dark at / 20 8C. CDDP was purchased from Nihonkayaku Co., Ltd. 2.3. Reverse transcription PCR Total cellular RNA was extracted using ISOGEN reagent according to the manufacture’s recommendations (Nippon Gene Inc.) The first strand complementary DNA (cDNA) was synthesized with 1 mg of total RNA using SUPERSCRIPTTM First-Strand Synthesis System for RT-PCR and was employed according to the manufacturer’s protocol (Life Technologies Inc.). The primer sequences that reported by Xu et al. were as shown on Table 1[30]. The PCR amplification was performed at a constant temperature of 94 8C with denaturation for 30 s, and annealing at 56 8C (b-actin, RAR-a and VDR), 52 8C (RAR-b and g) for 1 min, and an extention to 72 8C for 1 min, using Ex TaqTM (Takara Shuzo Co., Ltd., Shiga, Japan). Equal volumes of the PCR product from each sample were subjected to electrophoresis on a 10% polyacrylamid gel, stained with ethidium bromide, and photographed. 2.4. Cell treatment with vitamins and determination of cell growth KB cells were seeded at densities of 1 /104 cell per well in 96-well tissue culture plates. After 24 h, the cells were treated with different concentrations of vitamins. The concentration was 1 and 10 mM of ATRA, 0.1 and 1 mM TAC-101, calcitriol and OCT. The control cultures received no stimulation. Following the 48 h incubation, the cells were pulsed for 4 h with 0.25 mCi/well [3H]TdR (Amersham, Amersham, Buckinghamshire, UK) before radioactivity measurements. The percentage of cell growth was calculated by using the equation: % tumor growth/the absorbance in treated (At)/the absorbance in control (Ac) /100. At and Ac represent the absorbance in treated and control cultures, respectively. 2.5. TUNEL assay After 1 /105 of KB cells were seeded on the Lab-Tek chamber slide (Nalge Nunc International) and treated with 1 mM ATRA, TAC-101, calcitriol and OCT. The
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Table 1 RT-PCR primer sequences specifications
control KB cells were not treated. TUNEL assay was performed with Apoptosis in situ Detection Kit Wako according to the manufacturer’s protocol (Wako Pure Chemical Industries Ltd.) and the apoptotic cells were photographed. 2.6. Quantitative apoptotic factor using real-time PCR Total RNA was extracted from KB cells that had been treated with 1 mM each of vitamins for 48 h and were not treated as controls, by the acid quanidinium thiocyanate /phenol/chloroform extraction method using RNAzolB (Sawady, Tokyo, Japan), and was collected from the precipitate in ethanol. cDNA was synthesized with a survivin 466/485 primer (5?-AGA GGC CTC AAT CCA TGG CA-3?) and a glyceraldehydes-3-phosphate dehydrogenase (G3PDH), as an internal control, exon 8 primer (5?-CTT GAT GTC ATC ATA TTT GGC AGG-3?), we synthesized the
cDNA of p53 , p21 and bax with a random primer. The PCR reaction mixture was prepared using a TaqMan Universal Master Mix (PE Applied Biosystems, CA, USA). The primer and probe were designed by SRL Co., Ltd. Tokyo Japan (Table 2). This primer for survivin did not detect survivin-beta mRNA. Each PCR reaction was made at 50 cycles (95 8C for 30 s, 60 8C for 40 s, 72 8C for 30 s) using a real-time PCR system (ABI PRISM 7700 Sequence Detection System: PE Applied Biosystems). The PCR product of each mRNA was purified using a High pure PCR product purification kit (Roche Molecular Biochemicals Diagnostic, IN, USA) and was directly sequenced using the Thermosequenase Cy5 Dye Terminator kit (Amersham Pharmacia Biotech, Uppsala, Sweden) with an automated laser fluorescence DNA sequencer ALF express (Amersham Pharmacia Biotech). The sequence was finally compared with each target mRNA sequence.
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Table 2 Real-time PCR primer and probe sequences specifications
2.7. Cell cycle analysis One mM ATRA, TAC-101, calcitriol and OCT were added to 1 /106 of KB cells. The control was not treated. After 24 and 48 h, the medium was removed and
the cell monolayers were washed twice with cold phosphate-buffered saline (PBS). The cells were then trypsinized and the cell pellets were collected. The cells were again washed twice with PBS, fixed in cold methanol, and rewashed with PBS to remove methanol.
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24 h later CDDP as 0.1, 1.0, 10.0 mg/ml and 5-FU as 0.1, 1.0, 50.0 mg/ml were added. The Tetracolor ONE (Seikagaku Co., Ltd. Tokyo Japan) as 10 ml reacted in addition in each well, and 48 h later and the absorbance of 450 /500 nm was harvested using the NJ-2100 (Nippon InterMed Co., Ltd.). 2.9. ELISA for IL-8, VEGF, PDG and bFGF
Fig. 1. cDNA was synthesized with 1 mg of total RNA. The PCR amplification was performed consisting of denaturation at 94 8C for 30 s, annealing for 1 min (temperature was different at each primer), and extention at 72 8C for 1 min, using Ex TaqTM (Takara Shuzo Co., Ltd.). Equal volumes of the PCR product from each sample were subjected to electrophoresis on a 10% polyacrylamid gel, stained with ethidium bromide, and photographed.
They were suspended in 500 ml of PBS and then digested with 20 mg/ml RNase at 37 8C for 30 min and chilled on ice for 10 min. The cellular DNA was then stained with 50 mg/ml propidium iodide and incubated for 1 h at room temperature in the dark. The cell cycle distribution was analyzed by flow cytometry using a Becton Dickinson FACScan.
1 /106 of KB cells were seeded in a 10-cm plastic dish (Falcon). The supernatants of cells were treated with 1 ml of 1 mM of ATRA, TAC-101 and 1 mM calcitriol and OCT. After 48 h, they were determined to be at a concentration of IL-8, VEGF, PDGF and bFGF using the Quantine ELISA kit (R&D System, Mineapolis, MN). The protein concentration of these cytokine and factors was determined by absorbance compared with the standard curve and analyzed by NJ-2100.
3. Results 3.1. Nuclear receptors The expression of RARs and VDR was determined in KB cells using RT-PCR of total cellular RNA (Fig. 1). KB cells expressed RAR-a, RAR-g, and VDR. But RAR-b was not expressed in KB cells. 3.2. Growth inhibition
2.8. Changing the sensitivity to CDDP and 5-FU by treatment with ATRA and calcitriol KB cells, with the addition of 1 mM ATRA, 1 mM calcitriol and combinations of them were incubated for 1 week. 1 /104 of cells were seeded into 96 hole plates and
The proliferation of tumor cells by treatment with vitamins was determined by uptaking with [3H]TdR. The proliferation of KB cells was inhibited by the vitamin treatment. The vitamins also induced concentration dependency and conspicuously reinforced it (Fig. 2). 3.3. Apoptosis Apoptotic cells were stained by the TUNEL method. The expression of the apoptotic cell was confirmed in all the vitamin treatments (Fig. 3). Many expressions of the apoptotic cells were observed in all treated groups. 3.4. Apoptosis factors
Fig. 2. 1 /104 cells per well in 96-well cultured for 24 h the cells were treated with different concentrations of vitamins for 48 h and were pulsed for 4 h with 0.25 mCi/well [3H]TdR (Amersham), was detected using radioactivity. The percentage of cell growth was calculated by using the equation: % tumor growth/At/Ac/100, where At and Ac represent the absorbance in treated and control cultures, respectively.
We quantitated for mRNA of p53 , p21 , bax and survivin using real-time PCR. There were no clear changes in these mRNA of p53, p21 and bax, even when KB cells were treated with vitamins. But mRNA of survivin clearly decreased by treatment with all vitamins (Fig. 4). TAC-101 had the most effect on suppressing survivn expression. Calcitriol has the same suppression effect on the expression of survivin . ATRA
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Fig. 3. After 1/105 of KB cells were seeded in the chamber slide and treated with 1 mM ATRA (A), TAC-101 (B), calcitriol (C) and OCT (D). TUNEL assay was performed and apoptotic cells were photographed ( /40). (Scale bars/50 mm).
and OCT, however, have fewer effects than TAC-101 and calcitriol. 3.5. Cell cycle Cell cycle was analyzed by flow cytometry. By treatment with vitamins, the tendency of the cells of the G0/G1 phase increased and at the same time the cells of the S phase decreased (Fig. 5). Also, the accumulation to the G0/G1 phase of the cell strengthened and the reaction time increased. The cells of G2/M phase decreased by treatment with ATRA and calcitriol, but the cells increased in the G2/M phase by treatment with TAC-101. 3.6. Changing of sensitivity to CDDP and 5-FU by treatment with ATRA and calcitriol The decline of the 50% inhibitory concentration of CDDP was confirmed in the ATRA and calcitriol treatment group before CDDP exposable in comparison with a control group. This effect appears more pronounced in calcitriol than in ATRA. On the other hand, the decline of the 50% inhibitory concentration was not confirmed by the addition of 5-FU (Fig. 6).
3.7. Cytokin and angiogenesis factors By the ELISA method, the cytokin and angiogenesis factors, were determined. Concentration of each factor in the cell culture supernatant was as shown in Fig. 7. VEGF decreased after each vitamin treatment. IL-8 had no changing by treatment with vitamin (data not shown). On the other hand bFGF could not be detected (data not shown). We think that it falls below the measurement possibility value. It is apparent that PDGF decreases by treatment with each vitamin.
4. Discussion This is the first detailed study of the effects of vitamins on HNSCC and their nuclear receptor expression of cultured HNSCC in vitro. The expression of RAR-b of HNSCC was suppressed in the nuclear receptors. RAR-b seems to be a useful intermediate marker in retinoid trials in several studies of RAR-b reported by Xu et al. who suggested that the decreased expression of RAR-b may be associated with HNSCC development [30,31]. Cells that have no expression of RAR-b were transfected with RAR-b gene, and the
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Fig. 4. Total RNA was extracted from KB cells that were treated with 1 mM each vitamins for 48 h and were not treated as control, by the acid quanidinium thiocyanate /phenol /chloroform extraction method using RNAzolB. cDNA synthesized with a survivin 466 /485 primer and for p53 , p21 and bax , cDNA were synthesized with a random primer. The PCR reaction mixture was prepared using a TaqMan Universal Master Mix. This primer for survivin did not detect a survivin -beta mRNA. Each PCR reaction was made at 50 cycles (95 8C for 30 s, 60 8C for 40 s, 72 8C for 30 s) using a real-time PCR system. The PCR product of each mRNA was purified using a High pure PCR product purification kit and were directly sequenced using the Thermosequenase Cy5 Dye Terminator kit with an automated laser fluorescence DNA sequencer ALF express. The sequence was finally compared with each target mRNA sequence.
effect of treatment with retinoids was increased on the cell with expression of RAR-b [32,33]. In this study KB cell has not expressed RAR-b, but ATRA and TAC-101 inhibit the growth of tumor cells. This indicates that the presence of RAR-b is not involved with inhibitory effects of cell growth by retinoids [34]. When ATRA and TAC-101 are compared, the cell proliferation control of TAC-101 is much more effective than that of ATRA. This result shows that TAC-101 has a strong differentiation induction effect than ATRA although TAC-101 is a ligand of only RAR-a. Recently, it has been reported that the expression of RAR-b is associated with the inhibition of the keratinization in HNSCC cells [35], and methylation of the RAR-b p2 promoter is one mechanism that silences the RAR-b gene in lung carcinoma [36], which may be similar to HNSCC. KB cells have VDR and the proliferation of KB cells is suppressed by treatment with each vitaminD3. Among the differential induction effects on HNSCC by treatment with vitamins, one of the most important mechanisms is apoptosis [12]. The expression of TUNEL positive cells increased by treatments with each vitamin, compared with group without treatment. We suggest on the basis of these results that the each vitamin contributes to the enhancement of apoptosis.
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The p53 cancer suppressor gene induces apoptosis by G0/G1 arrest and promotes the expression of p21. The bax antagonizes bcl -2 and induces apoptosis through caspase-3 activation. In terms of p53 , p21 , bax expressions, there was no change of each expression at the mRNA level. Survivin , one of IAP, is expressed in all of the cancer tissue, although not in normal adult tissue. Apoptosis is inhibited by survivin expressed in the G2/M phase. The expression of survivin at the mRNA level was suppressed by treatment with vitamins. Among the groups treated with TAC-101, ATRA and vitaminD3, the expression of survivin was greatly inhibited in the TAC-101 treated group. Shin et al. reported that survivin is a direct inhibitor of caspase-3 and -7 [37]. We did not investigate these enzymes but our result is indication of these vitamins downregulate survivin, and this phenomenon induces apoptosis. In this study our findings gives suggestion that cells of the G0/G1 phase increased and that its S phase decreased by treatment with ATRA, calcitriol and OCT. Calcitriol especially has a strong influence. But the group treated with TAC-101 increased the G2/M phase. The cells were gathered in the G0/G1 phase by treatment with vitamins (in other words, G0/G1 arrest). Oridate et al. reported that cells treated with retinoids have induction of apoptosis in HNSCC [10]. Also, Light et al. reported that vitamin D3 acted on the squamous cell carcinomas of mice and noted an increase in a wellknown G1 phase [20]. Our results indicated the same findings in HNSCC cells. Our results show a sensitivity to CDDP was enhanced by treatment with ATRA and Calcitriol. But the sensitivity of 5-FU did not increase. Generally it is thought that CDDP shows most antitumor activity in the cell of the G1 phase [38]. Also the sensitivity of CDDP increased when treated with vitamin D as reported by Light et al. [20]. This phenomenon is thought to be the result of the increase in tumor cells in the G1 phase as a result of each vitamin treatment. Calcitriol has the effect of stopping the G0/G1 phase. The combination therapy of these vitamins makes greater changes to the sensitivity of CDDP. However, the sensitivity to 5-FU did not increase, as the mechanism of action by 5-FU is not cell cycle dependent. Recently antiangiogenic therapy has been applied in HNSCC and other solid tumors. Some studies have pointed out that antiangiogenic therapy can inhibit the progression and recurrence of metastatic disease, which depends on adequate vasculature and, therefore, on angiogenesis. In this study, we have investigated IL-8, VEGF, bFGF and PDGF production by treatment with vitamins in HNSCC [39,40]. Our results show that VEGF and PDGF production decreased as a result of the treatments. The results suggest that these vitamins have an antiangiogenic effect. Sauter et al. reported that VEGF is a marker of invasion and metastasis [41], and
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Fig. 5. KB cells left untreated, was treated with 1 mM ATRA, TAC-101, calcitriol and OCT. The control was not treated. After 24 and 48 h, the cells were stained with 50 mg/ml of propidium iodide and analyzed with a FACScan flow cytometry.
VEGF expression has been described as of prognostic significance by Salven et al. in HNSCC [42]. bFGF were not detected and IL-8 had no changing by each vitamin treatment. bFGF and IL-8 are a proangiogenesis factor and depends on cell line.
In this study we examined antitumor effects on HNSCC. In summary, treatments with each vitamin inhibited the proliferation of tumor cells. Apoptosis, cell cycle arrest and the down regulation of survivin were induced. It is evident that each vitamin controlled the
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evident that each vitamin controlled the expression of growth factors of the tumor cells.
Acknowledgements We thank Taiho Pharmaceutical Co., Ltd. and Chugai Pharmaceutical Co., Ltd. for their contribution of materials.
Fig. 6. KB cells, after add 1 mM ATRA, 1 mM calcitriol and each combination were incubated for 1 week, and 1/104 of cells were seeded into each 96 of hole plates and 24 h later were added CDDP as 0.1, 1.0, 10.0 mg/ml and 5-FU as 0.1, 1.0, 50.0 mg/ml. The Tetracolor ONE as 10 ml are caused reacted in addition to each well 48 h later and was harvested by using MTT method.
Fig. 7. 1/106 of KB cells were seeded in a 10-cm plastic dish. The supernatants of cells that were treated with 1 ml of 1 mM of ATRA, TAC-101, calcitriol and OCT. After 48 h, were determined using the commercial Quantine ELISA kit for IL-8, VEGF, PDGF and bFGF. The protein concentration of these cytokin and factor were determined by absorbance comparison to the standard curve and analyzed by NJ2100.
expression of growth factors of the tumor cells. Today, clinical trials for cancer chemoprevention are beginning in HNSCC. However, these vitamins combined with sensitive chemotherapeutic drug might be more useful to suppress recurrence and metastasis. It is indispensable to examine combination therapies along with other chemotherapeutic drugs and radiotherapy [43].
5. Conclusion In this study we examined antitumor effects on s. In summary, treatments with each vitamin inhibited the proliferation of tumor cells. Apoptosis, cell cycle arrest and the down regulation of survivin were induced. It is
References [1] Strickland S, Mahdavi V. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 1978;15:393 /403. [2] Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci USA 1980;77:2936 /40. [3] Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987;330:444 /50. [4] Giguere V, Ong ES, Segui P, Evans RM. Identification of a receptor for the morphogen retinoic acid. Nature 1987;330:624 /9. [5] Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, et al. Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 1988;85:3294 /8. [6] Sacks PG, Oke V, Amos B, Vasey T, Lotan R. Modulation of growth, differentiation and glycoprotein synthesis by beta-alltrans retinoic acid in a multicellular tumor spheroid model for squamous carcinoma of the head and neck. Int J Cancer 1989;44:926 /33. [7] Zou CP, Clifford JL, Xu XC, Sacks PG, Chambon P, Hong WK, et al. Modulation by retinoic acid (RA) of squamous cell differentiation, cellular RA-binding proteins, and nuclear RA receptors in human head and neck squamous cell carcinoma cell lines. Cancer Res 1994;54:5479 /87. [8] Jetten AM, Kim JS, Sacks PG, Rearick JI, Lotan D, Hong WK, et al. Inhibition of growth and squamous-cell differentiation markers in cultured human head and neck squamous carcinoma cells by beta-all-trans retinoic acid. Int J Cancer 1990;45:195 /202. [9] Lippman SM, Benner SE, Hong WK. Cancer chemoprevention. J Clin Oncol 1994;12:851 /73. [10] Oridate N, Lotan D, Xu XC, Hong WK, Lotan R. Differential induction of apoptosis by all-trans -retinoic acid and N -(4hydroxyphenyl)retinamide in human head and neck squamous cell carcinoma cell lines. Clin Cancer Res 1996;2:855 /63. [11] Diaz GD, Paraskeva C, Thomas MG, Binderup L, Hague A. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Res 2000;60:2304 /12. [12] Makin G, Hickman JA. Apoptosis and cancer chemotherapy. Cell Tissue Res 2000;301:143 /52. [13] Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, et al. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989;244:217 /21. [14] Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, et al. Mutations in the p53 gene occur in diverse human tumour types. Nature 1989;342:705 /8. [15] Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701 /4.
412
K. Satake et al. / Auris Nasus Larynx 30 (2003) 403 /412
[16] Gu Y, Turck CW, Morgan DO. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 1993;366:707 /10. [17] el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817 /25. [18] Reed JC, Miyashita T, Takayama S, Wang HG, Sato T, Krajewski S, et al. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem 1996;60:23 /32. [19] Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet 1998;351:882 /3. [20] Light BW, Yu WD, McElwain MC, Russell DM, Trump DL, Johnson CS. Potentiation of cisplatin antitumor activity using a vitamin D analogue in a murine squamous cell carcinoma model system. Cancer Res 1997;57:3759 /64. [21] Murakami K, Sakukawa R, Sano M, Hashimoto A, Shibata J, Yamada Y, et al. Inhibition of angiogenesis and intrahepatic growth of colon cancer by TAC-101. Clin Cancer Res 1999;5:2304 /10. [22] Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798 /801. [23] Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993;362:841 / 4. [24] Slavin J. Fibroblast growth factors: at the heart of angiogenesis. Cell Biol Int 1995;19:431 /44. [25] Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 1989;246:1309 /12. [26] Sakukawa R, Murakami K, Ikeda T, Yamada Y, Saiki I. Effect of 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) on the liver metastasis of colon 26-L5 carcinoma cells. Oncol Res 1998;10:287 /93. [27] Fujimoto K, Hosotani R, Doi R, Wada M, Lee JU, Koshiba T, et al. Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid derivative, TAC-101, in human pancreaticcancer cells. Int J Cancer 1999;81:637 /44. [28] Endo K, Ichikawa F, Uchiyama Y, Katsumata K, Ohkawa H, Kumaki K, et al. Evidence for the uptake of a vitamin D analogue (OCT) by a human carcinoma and its effect of suppressing the transcription of parathyroid hormone-related peptide gene in vivo. J Biol Chem 1994;269:32693 /9. [29] Eagle H. Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc Soc Exp Biol Med 1955;89:362 /4. [30] Xu XC, Liu X, Tahara E, Lippman SM, Lotan R. Expression and up-regulation of retinoic acid receptor-beta is associated with retinoid sensitivity and colony formation in esophageal cancer cell lines. Cancer Res 1999;59:2477 /83.
[31] Xu XC, Ro JY, Lee JS, Shin DM, Hong WK, Lotan R. Differential expression of nuclear retinoid receptors in normal, premalignant, and malignant head and neck tissues. Cancer Res 1994;54:3580 /7. [32] Castillo L, Milano G, Santini J, Demard F, Pierrefite V. Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method. Clin Cancer Res 1997;3:2137 /42. [33] Crowe DL. Retinoic acid receptor beta induces terminal differentiation of squamous cell carcinoma lines in the absence of cyclin-dependent kinase inhibitor expression. Cancer Res 1998;58:142 /8. [34] Wan H, Oridate N, Lotan D, Hong WK, Lotan R. Overexpression of retinoic acid receptor beta in head and neck squamous cell carcinoma cells increases their sensitivity to retinoid-induced suppression of squamous differentiation by retinoids. Cancer Res 1999;59:3518 /26. [35] Zou CP, Hong WK, Lotan R. Expression of retinoic acid receptor beta is associated with inhibition of keratinization in human head and neck squamous carcinoma cells. Differentiation 1999;64:123 / 32. [36] Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, et al. Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst 2000;92:1303 /7. [37] Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, et al. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 2001;40:1117 /23. [38] Donaldson KL, Goolsby GL, Wahl AF. Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle. Int J Cancer 1994;57:847 /55. [39] Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith CW, et al. Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer. Clin Cancer Res 1999;5:1369 /79. [40] Eisma RJ, Spiro JD, Kreutzer DL. Role of angiogenic factors: coexpression of interleukin-8 and vascular endothelial growth factor in patients with head and neck squamous carcinoma. Laryngoscope 1999;109:687 /93. [41] Sauter ER, Nesbit M, Watson JC, Klein-Szanto A, Litwin S, Herlyn M. Vascular endothelial growth factor is a marker of tumor invasion and metastasis in squamous cell carcinomas of the head and neck. Clin Cancer Res 1999;5:775 /82. [42] Salven P, Heikkila P, Anttonen A, Kajanti M, Joensuu H. Vascular endothelial growth factor in squamous cell head and neck carcinoma: expression and prognostic significance. Mod Pathol 1997;10:1128 /33. [43] Arnold A. Moving promising research findings to the clinic: methodological issues in the design and conduct of clinical trials of retinoids. Int J Cancer 1997;70:467 /9.