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Clinical Cancer Research Vol. 6, 1563-1573, April 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Identification of Receptor-selective Retinoids That Are Potent Inhibitors of the Growth of Human Head and Neck Squamous Cell Carcinoma Cells1

Shi-Yong Sun2, Ping Yue, Li Mao, Marcia I. Dawson, Braham Shroot, William W. Lamph, Richard A. Heyman, Roshantha A. S. Chandraratna, Koichi Shudo, Waun K. Hong and Reuben Lotan

Department of Thoracic/Head and Neck Medical Oncology [S-Y. S., P. Y., L. M., W. K. H., R. L.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; Retinoid Program, SRI International, Menlo Park, California 94025 [M. I. D.]; Galderma Research and Development, 06905 Sophia Antipolis, France [B. S.]; Cell Biology, Ligand Pharmaceuticals Inc., San Diego, California 92121 [W. W. L., R. A. H.]; Retinoid Research, Allergan, Irvine, California 92623 [R. A. S. C.]; and Faculty of Pharmaceutical Sciences, Tokyo University, Tokyo 113, Japan [K. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids modulate the growth and differentiation of cancer cells presumably by activating gene transcription via the nuclear retinoic acid receptor (RAR) {alpha}, ß, and {gamma} and retinoid X receptor (RXR) {alpha}, ß, and {gamma}. We analyzed the effects of 38 RAR-selective and RXR-selective retinoids on the proliferation of 10 human head and neck squamous cell carcinoma (HNSCC) cell lines. All of these cell lines expressed constitutively all of the receptor subtypes except RARß, which was detected in only two of them. Most of the RAR-selective retinoids inhibited the growth of HNSCC cells to varying degrees, whereas the RXR-selective retinoids showed very weak or no inhibitory effects. Three RAR antagonists suppressed growth inhibition by RAR-selective agonists, as well as by RAR/RXR panagonists such as 9-cis-retinoic acid. Combinations of RXR-selective and RAR-selective retinoids exhibited additive growth-inhibitory effects. Furthermore, we found that CD437, the most potent growth-inhibitory retinoid induced apoptosis and up-regulated the expression of several apoptosis-related genes in HNSCC cells. These results indicate that: (a) retinoid receptors are involved in the growth-inhibitory effects of retinoids; (b) RXR-RAR heterodimers rather than RXR-RXR homodimer are the major mediators of growth inhibition by retinoids in HNSCC cells; and (c) induction of apoptosis can account for one mechanism by which retinoids such as CD437 inhibit the growth of HNSCC cells. Finally, these studies identified several synthetic retinoids, which are much more effective than the natural RAs and can be good candidates for chemoprevention and therapy of head and neck cancers.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over 90% human head and neck cancers—which include cancers of the oral cavity, pharynx, and larynx—are squamous cell carcinomas (1) . It has been estimated that there will be 31,400 new cases and 12,300 deaths from head and neck cancers in the United States in 1999 (2) . HNSCCs3 are generally treated by radiation therapy and surgery. However, the 5-year relative survival for all stages is 53% and has not improved much over the last few decades despite adjuvant chemotherapy. Therefore, the morbidity and mortality from head and neck cancer remains a significant problem. Clearly, new approaches for the prevention and treatment of head and neck cancers must be extensively explored. A recent promising approach is based on the use of retinoids.

Retinoids, which include retinol (vitamin A) and its structurally or functionally related analogues, exert profound effects on the growth, maturation, and differentiation of many cell types both in vivo and in vitro (3) . Several studies have demonstrated that retinoids suppress the proliferation of HNSCC cells in monolayer cultures, inhibit the formation of HNSCC colonies in semisolid agar, and decrease the growth of HNSCC multicellular spheroids (4 , 5) . In addition, retinoids suppress the squamous differentiation of HNSCC cells (6) . More recently, the synthetic retinoid N-(4-hydroxylphenyl)retinamide (4HPR) has been found to induce apoptosis in human HNSCC cells (7) .

Retinoids have been shown to suppress oral premalignant lesions (e.g., leukoplakia) and to decrease the incidence of second primary tumors in patients who had been treated earlier for primary head and neck cancers (8) . However, the long-term use and the realization of the full potential of the few retinoids that have been tested as chemopreventive agents were hampered by their undesirable systemic side effects including teratogenicity (8) . Therefore, the identification and development of new retinoids with a more favorable therapeutic index and with reduced side effects and teratogenic risk has being pursued intensively.

Strong evidence exists to support a major role for nuclear retinoid receptors, which are members of the steroid hormone-receptor gene superfamily, in mediating the effects of retinoids on gene expression and, thereby, in altering the growth and differentiation of both normal and tumor cells. The two distinct classes of nuclear retinoid receptors are termed RARs and RXRs, each of which has three distinct subtypes {alpha}, ß, and {gamma} (9) . The RARs bind ATRA and 9-cis-RA, whereas the RXRs bind 9-cis-RA selectively. These receptors form RXR-RAR heterodimers or RXR-RXR homodimers, which, respectively, bind consensus DNA sequences or response elements named RAREs and RXREs, located within the regulatory regions of retinoid-regulated genes (9) .

In addition to ATRA and 9-cis-RA, the RARs and RXRs bind a variety of synthetic retinoids that possess various degrees of receptor selectivity both among the RAR subtypes ({alpha}, ß, and {gamma}), and between RARs and RXRs (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) . The availability of new receptor-selective retinoids has raised the possibility that some of them will be more useful than the natural retinoids in the prevention and treatment of human HNSCC. Therefore, we have investigated in this study the effects of 38 synthetic retinoids, with different RAR- or RXR-selectivities, on the growth of 10 human HNSCC cell lines. In addition, we analyzed the expression patterns of different RARs and RXRs in these cell lines to find out whether a correlation exists between growth inhibition and receptor expression status in the cells. Finally, we examined the effects of CD437, the most potent growth-inhibitory retinoid, on the induction of apoptosis and expression of several apoptosis-related genes in HNSCC cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture.
The 13 human HNSCC cell lines described in Table 1Citation (24, 25, 26, 27, 28) were grown in monolayer culture in a 1:1 (v/v) mixture of DMEM and Ham’s F12 medium containing 5% regular fetal bovine serum and antibiotics at 37°C in a humidified atmosphere composed of 95% air and 5% CO2.


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  		Table 1 The HNSCC cell lines used in this study

 
Retinoids.
ATRA, Am80, and TTNN were obtained from Dr. W. Bollag (F. Hoffmann-La Roche, Basel, Switzerland). CD270, CD271, CD336 (Am580), CD437, CD666, CD2314, CD2325, CD2366, and CD2665 were synthesized by Centre Internationale de Recherche Dermatologique/Galderma (Sophia Antipolis, France; Refs. 11 , 12 , 17 ). SR3985, SR11203, SR11217, SR11234, SR11236, SR11238, SR11246, SR11254, SR11363, and SR11364 were synthesized by the group of Dr. M. I. Dawson (SRI International, Menlo Park, CA; Refs. 10 , 13 , 15 , 16 ). LG1069 was synthesized by Ligand Pharmaceuticals (San Diego, CA; Refs. 21 ). Ch55, Am555S, TD550, LE540, HX600, Re80, and LE135 were synthesized by the group of Dr. K. Shudo (Tokyo University, Tokyo, Japan; Refs. 18, 19, 20 ). AGN193312, AGN193174, AGN193273, AGN190521, AGN193109, and AGN193078 were synthesized by Allergan (Irvine, CA; Ref. 22 ). The chemical structures and receptor affinity or transactivation activity of these retinoids were reported previously and summarized by us recently (23) . The acyclic retinoid all-trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid (E5166) was provided by Dr. Y. Moto (Gifu University School of Medicine, Gifu, Japan). Retinoids were dissolved in DMSO at a concentration of 10 mM and were stored under N2 in the dark at -80°C. Stock solutions were diluted to the appropriate concentrations with growth medium just prior to use.

Cell Treatment with Retinoids and Determination of Growth Inhibition.
Cells were seeded at densities ranging from 3 x 103 to 6 x 103 cells per well in 96-well tissue culture plates. After 24 h, cells were treated with different concentrations of retinoids. Control cultures received the same amount of DMSO (0.01–0.1%) as did the treated cultures. Cells were treated again with fresh retinoids in fresh medium on day 4 in fresh medium. On day 7, cell numbers were estimated by using the SRB assay as described in detail previously (23) . The percentage of growth inhibition was calculated by using the equation: % growth inhibition = (1 - At/Ac) x 100, where At and Ac represent the absorbance in treated and control cultures, respectively. IC50, the drug concentration causing a 50% cell growth inhibition, was determined by interpolation from the dose-response curve.

DNA Fragmentation Assay.
Cells were plated on 10-cm diameter dishes 1 day before treatment. After a 24-h treatment with retinoids, cells were harvested by trypsinization and were counted. Cell Death Detection ELISAPlus kit (Boehringer Mannheim, Indianapolis, IN) was used according to the manufacturer’s protocol to detect cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) occurring during apoptosis. In addition, APO-DIRECT TUNEL kit (Phoenix Flow Systems, Inc., San Diego, CA) was used following the manufacturer’s protocol to determine DNA fragments with 3'-hydroxyl ends.

Reporter Plasmids, Transient Transfection, and Luciferase Assay.
The COL-AP-1-LUC reporter plasmid, which contains the luciferase gene controlled by a promoter fragment of the collagenase gene (-74 to -63) harboring a consensus AP-1 binding site (TGAGTCA) connected to herpes simplex virus thymidine kinase (TK) promoter, was obtained from Dr. J. Kurie (The University of Texas M. D. Anderson Cancer Center, Houston, TX). pAP1-Luc reporter plasmid, which contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to seven repeats of AP-1 binding sites, was purchased from Stratagene (La Jolla, CA). pCH110 plasmid encoding ß-galactosidase (ß-gal) was purchased from Pharmacia Biotech (Piscataway, NJ). These plasmids were purified with QIAGEN/Filter Plasmid Maxi Kit (QIAGEN, Chatsworth, CA). Cells were seeded in 24-well plates and cotransfected with AP-1 reporter plasmid and pCH110 plasmid using FuGene 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) following the manufacturer’s protocol. Luciferase activity was determined using Luciferase Assay System (Promega, Madison, WI) using a luminometer. Relative luciferase activity was normalized with respect to ß-galactosidase activity, which was measured as described previously (29)

RNA Purification and Northern blotting.
Total RNA preparation and Northern blotting were essentially described previously (23) . pSG5 expression vectors containing RAR{alpha} (30) , -ß (31) , and -{gamma} (32) cDNA were obtained from Dr. P. Chambon (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, Strasbourg, France). Human RXR{alpha} and murine RXRß and -{gamma} cDNA (33) were obtained from Dr. R. Evans (The Salk Institute, La Jolla, CA). GAPDH cDNA was purchased from Ambion (Austin, TX). pSVc-Myc-1 plasmid containing mouse c-Myc cDNA was obtained from Dr. P. Chiao (University of Texas M. D. Anderson Cancer Center, Houston, TX). GST-CIP1 plasmid containing human p21WAF1 cDNA was obtained from the American Type Culture Collection (Rockville, MD). pCR-Killer-Race-6 plasmid containing human Killer/DR5 cDNA was provided by Dr. W. S. El-Deiry (University of Pennsylvania School of Medicine, Philadelphia, PA). Human Bax cDNA in pSFV-neo vector was provided by Dr. S. J. Korsmeyer (Washington University School of Medicine, Saint Louis, MO).

Detection of p53 Mutation.
p53 mutations were detected by PCR amplification and sequencing as follows: genomic DNA was amplified by PCR using 20 ng of genomic DNA. Primer sets used for detecting deletions of p53 exon 4 to exon 9 are 4S (sense primer), 5'-TTCACTTGTGCCCTGACTT-3', and 9AS (antisense primer), 5'-CTGGAAACTTTCCACTTGAT-3'. Thermal cycling was performed in a temperature cycler (Hybaid; Omnigene, Woodbridge, NJ) in 500-µl plastic tubes for one initial cycle of denaturation at 95°C for 2 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 1.5 min, extension at 70°C for 1 min, and a final step at 70°C for 5 min. PCR products were purified and used as DNA templates for direct sequencing analysis. About 30 ng of DNA template was used for each sequencing analysis, with sequencing primers covering exons 4–9. Sequencing primers labeled with [{gamma}-33P]ATP and DNA templates were amplified by PCR for 30 cycles using AmpliCycle sequencing kit (Perkin-Elmer, Branchburg, NJ) according to the manufacturer’s protocol. Each amplified product (3 µl) was run on a 6% long-range gel (FMC BioProducts, Rockland, ME) and exposed to film. Each mutation identified was confirmed by a repeat sequence analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of RAR-Selective Retinoids on the Growth of Human HNSCC Cells.
The 10 HNSCC cell lines used in this study are presented in Table 1Citation . Fig. 1Citation shows representative concentration-dependent inhibition by different RAR-selective retinoids of UMSCC22B cells. Retinoids with RAR{gamma}- or RARß/{gamma}-selectivity, except for SR11364 and TTNN (Fig. 1, B and C)Citation , exerted greater growth-inhibitory effects than those with either RAR{alpha}- or RARß-selectivity (Fig. 1A).Citation The growth-inhibitory patterns of AGN193078, AGN193273, AGN190521, SR3985, and Ch55 (<1 µM) were different from those of CD437, CD270, CD271, CD666, and CD2325, which showed a clear dose-dependent activity (Fig. 1B).Citation The increase in concentration <=10 µM for the former retinoids was not accompanied by a corresponding increase in growth inhibition because their effect reached a plateau of 50–70% growth inhibition at lower concentrations (Fig. 1C).Citation



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  		Fig. 1. Growth-inhibitory effects of different RAR-selective retinoids on UMSCC22B cells. Cells were seeded at a density of 5 x 103 cells per well in 96-well culture plates. Cell treatment with retinoids and the determination of cell numbers were described in detail in "Materials and Methods." Each point, the mean of four replicate determinations. SDs were less than 10% of the mean and not included in the graph. A, RAR{alpha}- or ß-selective retinoids with weak growth-inhibitory effects; B, RAR{gamma}- or ß/{gamma}-selective retinoids with potent growth-inhibitory effects; C, RAR{gamma}-, ß/{gamma}-, or {alpha}/ß/{gamma}-selective retinoids with potent growth-inhibitory effects.

 
Similar experiments with other cell lines were used to calculate the IC50 values that are summarized in Table 2Citation . All of the RAR-selective retinoids inhibited the growth of the cells to varying degrees. RAR{alpha}/ß/{gamma}-selective Ch55, RARß/{gamma}-selective CD271, and RAR{gamma}-selective CD437 and CD2325 inhibited the growth of all of the 10 cell lines. CD437 was the most active among them with an IC50 < 0.3 µM. RAR{gamma}-selective SR11364, RARß/{gamma}-selective CD2665, and RARß-selective LE540 showed similar activity patterns, but their potencies were relatively low (IC50, >1 µM). CD2665 and LE540, the antagonists, showed weaker activity than SR11364 (IC50, >2 µM), which suggests that these retinoids behave as partial agonists at high concentrations. In addition, some RAR-selective retinoids (such as CD336, Am80, AGN193312, CD666, SR11254, SR11363, AGN193078, CD270, SR3985, AGN193273, ATRA, AGN190521, Re80, and 9-cis-RA) inhibited cell growth in only a few of the 10 cell lines (UMSCC 22A, UMSCC 22B, and 183A). The rest of the RAR-selective retinoids Am555S, CD2314, AGN193174, TTNN, CD2366, and AGN193109 exhibited either very weak or no activity in cell growth inhibition. UMSCC22A, UMSCC22B, and 183A cells responded to most of the tested retinoids, whereas UMSCC17A, UMSCC17B, UMSCC38, 1483, and TR146 were resistant to many of the retinoids.


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  		Table 2 Effects of different synthetic nuclear retinoid receptor-selective retinoids on the growth of human HNSCC cells

 
Growth Inhibition by RXR-Selective or Anti-AP-1 Compounds in HNSCC Cells.
Both natural RAs ATRA and 9-cis-RA inhibited the growth of only a few cells, such as UMSCC22A, UMSCC22B, and MDA886Ln (Table 2)Citation . Table 2Citation indicates that almost all of the RXR-selective compounds such as SR11203, SR11217, SR11234, SR11236, SR11246, HX600, and LG1069 exhibited very weak or no inhibitory effects on the growth of all of the HNSCC cell lines. These results indicated that the activation of the putative RXR-RXR pathway by RXR-selective retinoids was not effective in growth inhibition. SR11238 is a retinoid with reported anti-AP-1 activity but no RAR or RXR transactivation ability (15) . It had no growth-inhibitory effects on our cell lines. SR11217, a strong RXR-selective compound with some anti-AP-1 activity (15) , also was inactive in inhibiting the growth of most cell lines. 9-cis-RA, with both RAR- and RXR-binding activity, behaved almost the same as ATRA, a RAR-selective ligand.

Because SR11238 failed to inhibit any of the 10 HNSCC cell lines in our study, we were interested in determining whether this retinoid has anti-AP-1 activity in HNSCC cells. By transient transfection of two different AP-1 reporter vectors into UMSCC22B cells, we found that this retinoid indeed transrepressed AP-1 activity at high concentrations (1 to 10 µM). However, SR11238 was less potent than ATRA in transrepressing AP-1 activity (Fig. 2)Citation



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  		Fig. 2. AP-1 transrepression by SR11238 and ATRA in UMSCC22B cells. Cells were seeded at a density of 9 x 104 cells/well in 24-well plates and transfected 2 days later with the indicated AP-1 reporter plasmid plus pCH110 plasmid. Six h after transfection, the cells were treated with the indicated concentrations of retinoids for 24 h. The cells were then lysed and used for luciferase activity assay as described in "Materials and Methods." Columns, means of triplicate determinations; bars, SD.

 
Suppressive Effects of RAR-specific Antagonists on the Growth-inhibitory Effects of Retinoids in HNSCC Cells.
The results above showing that RAR-selective retinoids are much more active than RXR-selective ones in inhibiting the growth of HNSCC cells suggested that an RXR-RAR-mediated, rather than an RXR-RXR-mediated, pathway plays an important role in the growth inhibition of HNSCC cells. We used UMSCC22B cells to determine whether the growth-inhibitory effect of RAR-selective retinoids could be suppressed by RAR-specific antagonists. The results presented in Fig. 3Citation indicate that the growth-inhibitory effects of all of the RAR-selective retinoids used in this study could be blocked by RAR-specific antagonists, especially by AGN193109. Both CD2366 and CD2665 showed partial antagonism of ATRA and 9-cis-RA, whereas AGN193109 almost completely abolished the growth-inhibitory effects of ATRA and 9-cis-RA. Similar results were also observed in UMSCC22A cells (data not shown).



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  		Fig. 3. Suppressive effects of RAR-selective antagonists CD2366 (A), CD2665 (B), and AGN193109 (C) on the growth-inhibitory effects of some retinoids in UMSCC22B cells. Cells were seeded at densities of 3 x 103 to 5 x 103 cells per well in 96-well culture plates 1 day before treatment. The cells were treated with various retinoids (10-7 M) alone and with retinoids (10-7 M) plus antagonists (10-6 M) on the 2nd day. After 4 days of treatment, cell numbers were determined using the SRB assay as described in "Materials and Methods." Columns, means of four replicate determinations; bars, SD.

 
Growth-inhibitory Effects of RXR-selective Retinoids Combined with RAR-Agonist.
Previously, we (34) and others (35) have shown that a combination of a RAR-selective and a RXR-selective retinoid was more potent than each agent alone in certain tumor cells. Therefore, we investigated effects of RXR-selective retinoids in combination with RAR-selective retinoids on UMSCC22B cells. The combination of RAR{alpha}-selective CD336, RARß-selective CD2314, and RAR{gamma}-selective SR11254 with RXR-selective agents, such as SR11203, SR11234, SR11236, and SR11246, resulted in either additive or more than additive growth-inhibitory effects in UMSCC22B cells (Fig. 4)Citation .



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  		Fig. 4. Effects of RAR-selective retinoid CD336 (A), CD2314 (B), or SR11254 (C) in combination with different RXR-selective retinoids on the growth of UMSCC22B cells. Experimental conditions were the same as those described in Fig. 3Citation . The concentrations for CD336-, CD2314-, SR11254-, and RXR-selective retinoids were 10-8 M, 5 x 10-7 M, 10-8 M, and 10-6 M, respectively. Columns, means of four replicate determinations; bars, SD.

 
Differential Expression of RARs and RXRs in HNSCC Cells.
The differences in responsiveness to retinoids among the 10 HNSCC cell lines could have been associated with the basal constitutive expression status of RARs and RXRs. To examine this possibility, we analyzed the constitutive expression of RAR and RXR mRNAs in these cells by Northern blotting. All of the cell lines expressed RAR{alpha} and -{gamma} and RXR{alpha}, -ß, and -{gamma} constitutively, whereas RARß was detectable only in cell lines 183A and MDA886Ln (Fig. 5)Citation . The expression levels of RAR{alpha} and -{gamma} varied among the cell lines. Of the 10 cell lines, UMSCC22B and 183A expressed the highest level of RAR{alpha} mRNA, whereas UMSCC22A, UMSCC38, and MDA886Ln expressed relatively lower levels of RAR{alpha}. The expression of RAR{gamma} was highest in UMSCC22B and lower in the TR146 and MDA886Ln cells. The levels of RXR{alpha}, -ß, and -{gamma} mRNAs were almost the same in all of the 10 HNSCC cell lines except for a lower level of RXR{alpha} in UMSCC17A and 17B cells.



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  		Fig. 5. Constitutive expression of RAR and RXR mRNA in 10 HNSCC cell lines. Total RNA (20 µg) was subjected to electrophoresis in an agarose gel and blotted to a nylon membrane. The procedures for total RNA purification and Northern blotting are described in "Materials and Methods."

 
Induction of Apoptosis by CD437 in Human HNSCC Cell Lines.
Because CD437 was the most potent growth-inhibitory retinoid, we examined its effects on apoptosis in various HNSCC cell lines. After a 24 h-treatment, 1 µM CD437 induced apoptosis in the majority of cell lines albeit with different potencies. Among the 10 cell lines, MDA886Ln, SqCC/Y1, and UMSCC17B were the most sensitive to apoptosis induction by CD437, whereas 1483, 183A, and UMSCC10B showed almost no apoptosis after treatment with CD437 (Fig. 6)Citation .



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  		Fig. 6. Effects of CD437 on growth inhibition (A) and induction of DNA fragmentation (B and C) in different HNSCC cell lines. The indicated cell lines were exposed to 1 µM CD437 for 24 h. The growth inhibition was estimated by SRB assay, and DNA fragmentation was measured by ELISA and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling), respectively, as described in "Materials and Methods." Columns, means of four replicates (A) or triplicate (B and C) determinations; bars, SD.

 
Differential Modulation of the Expression of Apoptosis-related Genes by CD437 in Different HNSCC Cell Lines.
To identify genes that might be involved in CD437-induced apoptosis in HNSCC cells, we examined the effects of this retinoid on the expression of several apoptosis-related genes in the 10 HNSCC cell lines. Depending on the cell line, CD437 showed differential patterns of modulation of the expression of the apoptosis-related genes including Killer/DR5, c-Myc, Bax, and p21WAF1 (Fig. 7)Citation . For example, among the 10 cell lines, Killer/DR5 expression was induced in five cell lines (UMSCC11B, UMSCC14B, UMSCC17B, MDA886Ln, and sqCC/Y1); c-Myc expression was increased in three cell lines (UMSCC11B, MDA886Ln, and SqCC/Y1); Bax expression was weakly induced in three cell lines (UMSCC17B, MDA886Ln, and SqCC/Y1); and the expression of p21WAF1 was induced in seven cell lines (UMSCC10B, UMSCC11B, UMSCC14B, UMSCC17B, MDA886Ln, 1483, SqCC/Y1, and TR146). It is worth mentioning that CD437 induced the expression of all of the these genes in the cell line 886 and SqCC/Y1 [which were the most sensitive to CD437-induced apoptosis (Fig. 6)Citation ] but not in cell lines 1483 and 183A [which were the most resistant to apoptosis induction by CD437 (Fig. 6)Citation ]. In the CD437-sensitive UMSCC17B cell line, CD437 increased the expression of the majority of the genes except for c-Myc. These results suggest that different genes may be involved in CD437-induced apoptosis in HNSCC cells and that the contribution and the importance of subsets of these genes for CD437-induced apoptosis in HNSCC cells depend on the cell line.



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  		Fig. 7. Differential modulation of the expression of apoptosis-related genes by CD437 in different HNSCC cell lines. The cells were treated with 1 µM CD437 for 15 h. Twenty µg of total RNA were subjected to electrophoresis in an agarose gel and blotted to a nylon membrane. The procedures for total RNA purification and Northern blotting were described in "Materials and Methods." W, wild-type; M, mutant.

 
Detection of p53 Mutations in Human HNSCC Cell Lines and Their Relationship to Response to CD437.
To be able to evaluate whether cell sensitivity to CD437-induced apoptosis is related to p53 status, we analyzed p53 mutations in these cell lines by a combination of PCR amplification and sequencing. p53 mutations were detected in 5 of 10 cell lines including a polymorphism (Table 1)Citation . No mutation in p53 gene was detected in UMSCC17B, UMSCC22B, MDA886Ln, 1483, and TR146 cells. 183A had a polymorphism from codon 261 AGG to AGA. Therefore, this cell line is expected to have a functional p53. A comparison of p53 status of the cells and their sensitivity to CD437 or other retinoids shows no straightforward association between these two parameters. Four (MDA886Ln, UMSCC17B, UMSCC22B, and TR146) of 6 cell lines with functional p53 responded well to CD437-induced apoptosis, whereas only 2 (SqCC/Y1 and UMSCC14B) of 4 cell lines with p53 mutation underwent apoptosis after a 24-h treatment. MDA886Ln cells carrying wild-type p53 were the most sensitive to induction of apoptosis by CD437, whereas SqCC/Y1 cells with mutant p53 were also very sensitive to CD437-induced apoptosis. 1483 and 183A cells having functional p53 but were resistant to CD437 treatment (Fig. 6)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nuclear retinoid receptors are thought to mediate most of the effects of retinoids on gene expression that are associated with the modulation of cell growth and differentiation (3 , 9) . Over the last few years, new retinoids that exhibit preferential binding to, or transactivation of, individual RARs or RXRs have been synthesized (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) . The availability of receptor-selective retinoids has raised the possibility that some of them may be more useful for cancer chemoprevention or therapy than the natural RAs because of increased potency and decreased side effects. We found that most RAR-selective retinoids were active in inhibiting the growth of HNSCC cell lines. The ability of RAR-specific antagonists to block the growth-inhibitory effects of many RAR-selective retinoids indicated that RARs mediate the effects of the agonistic retinoids in HNSCC cells. RARs require heterodimerization with RXRs for effective DNA binding and transactivation of transcription, whereas RXRs can form either RXR/RXR homodimers or RXR/RAR heterodimers in the presence of 9-cis-RA (9 , 36, 37, 38, 39) . Because RXR/RAR heterodimers and RXR/RXR homodimers activate RARE and RXRE, respectively, they mediate two distinct pathways of response to retinoids. In our study, the RXR-selective retinoids failed to inhibit cell growth, whereas most RAR-selective retinoids were active, although they exhibited a range of potencies. These results are similar to reports that several RXR-selective retinoids were ineffective in inducing differentiation of myeloid leukemia cells (40) and breast carcinoma cells (41) .

It has been shown that the binding of ligand to RAR in an RXR/RAR heterodimer is sufficient to activate transcription via RARß RARE, whereas binding of ligand to RXR is not sufficient to activate RARE in human keratinocytes (42) . Nonetheless, it has been found by us in cervical carcinoma cells (34) and by others in embryonal carcinoma cells (35) that a combination of a RAR-selective and a RXR-selective retinoids can result in enhanced efficacy relative to each retinoid alone. In HNSCC cells, we also found that a combination of retinoids with distinct selectivity exerts additive or more than additive effects, presumably by activating the RAREs more efficiently than the RAR-selective retinoid alone (34) because the occupancy of ligand-binding sites of both RXR and RAR in a heterodimer can enhance the binding of a coactivator (e.g., SRC-1; Ref. 43 ).

Although the binding of retinoids with their receptors seems to be a prerequisite for their biological activities, we found no correlation between the receptor subtype-binding activity and the growth-inhibitory effects of retinoids. For example, the RAR{gamma}-selective retinoids CD437 and CD2325 were the most potent retinoids among all of the retinoids tested; in contrast, other RAR{gamma}-selective retinoids were not as active; SR11364 was less potent, whereas CD666, SR11254, and SR11363 were effective in only a few HNSCC cell lines. Certain retinoids with different subtype-selectivity exhibited similar biological activities, which suggested that different retinoid receptor subtypes may mediate growth inhibition.

The 10 HNSCC cell lines exhibited different sensitivities to growth-inhibitory effects of retinoids. This difference did not seem to be associated with the status of retinoid receptors in these cells. The cells differed mainly in the expression of RARß, which was detected in only 2 of the 10 cell lines. Nonetheless, cells that lacked RARß expression did not seem to be less sensitive to retinoids compared with cells that did express this receptor. This result suggests that: (a) RARß may not be required for mediating the growth-inhibitory effects of retinoids; or (b) the other RARs (i.e., {alpha} or {gamma}) can compensate for the loss of RARß.

There were considerable differences in sensitivity to retinoids among the HNSCC cell lines. These differences could not be explained on the basis of the receptor status because the most sensitive cells (UMSCC22B) had the same pattern of receptor expression as the most resistant cells (UMSCC17A and TR146). Furthermore, the differences in sensitivity were not associated with the histology of the tumors from which the cells were derived or their in vitro squamous differentiation characteristics. Interestingly, in the two cases for which we had cell lines derived from a primary HNSCC and from a metastatic lesions in the same patient, the metastatic cells were more sensitive to many retinoids than the cells from the primary tumor, which suggests that tumor progression did not diminish retinoid signaling unlike what was observed in human lung cancer cells (44) . What, then, could account for the differences in responsiveness of the HNSCC cell lines to retinoids? It has been reported recently that transcriptional activation by retinoid receptors depends on the release of bound corepressor and recruitment of a coactivator to the RXR/RAR heterodimer (9 , 45, 46, 47) . Thus, it is possible that the different HNSCC cell lines differ in the expression of the cofactors.

AP-1 is a protein complex comprised of c-Jun and c-Fos; it mediates mitogenic signals, and its activation is associated with increased cell proliferation (48) . One way that RARs regulate the expression of certain genes is thought to involve transrepression of AP-1 activity (49) . Recent studies suggest that this effect may also mediate retinoid-induced growth inhibition (14 , 15 , 49) . Our study has shown, however, that none of the 10 HNSCC lines used by us responded to SR11238, which has been reported to exhibit anti-AP-1 activity without activating RARE-mediated transcription (15) . One explanation for this result is that, if anti-AP-1 activity is important for growth inhibition, this retinoid does not have anti-AP-1 activity in HNSCC cells. However, we found that SR11238 does have anti-AP-1 activity in HNSCC cells. It is noteworthy that this retinoid exerted anti-AP-1 activity only at high concentrations (above 1 µM). Because ATRA exhibited potent growth-inhibitory effects and was a potent AP-1 inhibitor in HNSCC, especially in UMSCC22B cells, we suggest that the weak growth-inhibitory effects of SR11238 may be related to its weak anti-AP-1 activity in HNSCC cells.

Our previous study showed that human lung cancer cells responded to only a few of these retinoids, i.e., CD437, CD2325, CD271, and SR11364, in regard to the growth inhibition (23) . In the present study, we found that human HNSCC cells, in general, responded better than lung cancer cells to these retinoids. Some cell lines such as UMSCC22A, UMSCC22B, and 183A were sensitive to many retinoids including ATRA, whereas some cell lines such as UMSCC17A, UMSCC17B, and TR146 were resistant to most of the retinoids. However, the retinoids CD437, CD2325, CD271, and SR11364 were actually effective against all of the tested cell lines, as we found in human lung cancer cells. Recently, more attention has been given to CD437, the most potent retinoid identified thus far in inhibiting the growth of human lung cancer (23) and HNSCC (present study) cells. CD437 has been demonstrated to exert potent apoptosis-inducing activity in a variety of cancer cells both in vitro (50, 51, 52, 53, 54, 55) and in vivo (54) . Therefore, CD437 and related retinoids may represent potential candidates for the prevention and treatment of human HNSCC cancers.

In this study, we also observed that CD437 induced apoptosis in the majority of HNSCC cell lines albeit with different potencies. This result indicates that induction of apoptosis can be one mechanism by which CD437 inhibits the growth of HNSCC cells. Our previous studies demonstrated that wild-type p53 enhances cell susceptibility to CD437-induecd apoptosis in human lung cancer cells (50 , 56) . In this study, we did not find a clear association between cell sensitivity to CD437-induced apoptosis and p53 status. The association between cell sensitivity to CD437-induced apoptosis and p53 status in human HNSCC cells was not as strong as that in human lung cancer cells (56) . However, we cannot exclude the role of p53 in mediating CD437-induced apoptosis in some cell lines such as UMSCC17B and MDA886Ln. We do not have a clear understanding about why the cell lines 1483 and 183A, which have functional p53, were resistant to CD437 treatment. One possibility is that defects in p53 downstream-signaling pathway may exist in these two cell lines, which may compromise cell responsiveness to CD437-induced apoptosis if p53 plays any role in CD437-induced apoptosis in HNSCC cells.

The involvement of some apoptosis-related genes such as Killer/DR5 (50 , 56) , Bax (50 , 53 , 56) , p21WAF1 (50 , 53 , 56) , and c-Myc (57) in mediating CD437-induced growth arrest and apoptosis in several type of cancer cells has been suggested. In the present study, we found that all of these genes could be associated with CD437-induced apoptosis in most of the HNSCC cell lines, although their contribution and importance varied among different cell lines. The up-regulation of the expression of all these genes by CD437 was observed in the cell lines MDA886Ln and SqCC/Y1, which were the most sensitive to CD437-induced apoptosis, but not in the cell lines 183A and 1483, which were the most resistant to CD437 treatment. In another sensitive cell line, UMSCC17B, CD437 also induced the expression of these genes except for c-Myc. In other cell lines such as UMSCC11B, UMSCC14B, UMSCC22B, and TR146, the importance or involvement of the genes in CD437-induced apoptosis may vary.

Killer/DR5 was reported to be a p53-regulated gene (58) . Recently, p53-indepenednt regulation of the Killer/DR5 expression by tumor necrosis factor-{alpha} and methyl methanesulfonate was also observed (59) . Our previous study showed that CD437 up-regulated the expression of Killer/DR5 via p53-dependent mechanism in human lung cancer cells (50 , 56) . In the present study, we found that CD437 could induce the expression of Killer/DR5 in the cell lines either with wild-type p53 (UMSCC17B and MDA886Ln) or with mutant p53 (UMSCC11B, UMSCC14B, and SqCC/Y1), which indicated that CD437 can induce the expression of Killer/DR5 via either p53-dependent or -independent mechanism in human HNSCC cells. This is the first observation that CD437 exerts p53-independent induction of Killer/DR5 in human cancer cells.

In conclusion, we have demonstrated that several synthetic retinoids are more potent than natural RA in several HNSCC cell lines and that retinoid receptors mediate the growth-inhibitory effects of these retinoids probably via RXR/RAR heterodimer activation. In addition, we found that the most potent growth-inhibitory retinoid, CD437, induces apoptosis in the majority of HNSCC cell lines, which involves up-regulation of multiple apoptosis-related genes including Killer/DR5, Bax, p21WAF1, and c-Myc.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by USPHS Grants PO1 CA52051 from National Cancer Institute and P50 DE11906 from NIDCR (to R. L.) and PO1 CA51993 (to M. I. D.) from National Cancer Institute. W. K. H is an American Cancer Society Clinical Research Professor. Back

2 To whom requests for reprints should be addressed, at Department of Thoracic/Head and Neck Medical Oncology, Box 108, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, TX 77030. Phone: (713) 745-5062; Fax: (713) 794-0209; Email: ssun{at}notes.mdacc.tmc.edu Back

3 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; RA, retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; RARE, RA response element; RXRE, retinoid X response element; ATRA, all-trans-RA; SRB, sulforhodamine B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 9/21/99; revised 12/27/99; accepted 1/ 6/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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H.-J. Kim and R. Lotan
Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting
Cancer Res., April 1, 2004; 64(7): 2439 - 2448.
[Abstract] [Full Text] [PDF]

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 Clin. Cancer Res.Home page
E. M. Youssef, D. Lotan, J.-P. Issa, K. Wakasa, Y.-H. Fan, L. Mao, K. Hassan, L. Feng, J. J. Lee, S. M. Lippman, et al.
Hypermethylation of the Retinoic Acid Receptor-{beta}2 Gene in Head and Neck Carcinogenesis
Clin. Cancer Res., March 1, 2004; 10(5): 1733 - 1742.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
K.-H. Chun, D. M. Benbrook, K. D. Berlin, W. K. Hong, and R. Lotan
The Synthetic Heteroarotinoid SHetA2 Induces Apoptosis in Squamous Carcinoma Cells through a Receptor-independent and Mitochondria-dependent Pathway
Cancer Res., July 1, 2003; 63(13): 3826 - 3832.
[Abstract] [Full Text] [PDF]

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