alitretinoin and Carcinoma--Squamous-Cell

To study the effects of 9-cis-retinoic acid (9-cis-RA) on cell cycle and expression of cyclin D1 and cdk4 in lung cancer cells.. 9-cis-RA (1 x 10(-6) mol.L-1) was used to treat lung cancer cells for 24 h; Flow cytometry (FCM) was used to detect the percent of G0/G1 phase and S phase cells of three groups including blank control, DMSO control and 9-cis-RA groups; RT-PCR was used to analyze the expression changes of cyclin D1 and cdk4 before and after treatment with 9-cis-RA in lung cancer cells.. The percent of G0/G1 phase cells of 9-cis-RA groups was significantly higher than that of the control groups (P < 0.01 or P < 0.05) and the percent of S phase cells of 9-cis-RA groups was lower than that of the control groups (P < 0.01 or P < 0.05); the expression of cyclin D1 of PG, SPC-A1 and L78 cells was decreased (P < 0.01) and the expression of cdk4 of PG, A549 and L78 cells was also decreased (P < 0.01) after treatment with 9-cis-RA.. Most of the proliferation and the expression of cyclin D1 and cdk4 of PG, A549, SPC-A1 and L78 were inhibited by 9-cis-RA.

Topics: Adenocarcinoma; Alitretinoin; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; G1 Phase; Humans; Lung Neoplasms; Proto-Oncogene Proteins; Resting Phase, Cell Cycle; S Phase; Tretinoin

Peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimerises with retinoid X receptor alpha (RXRalpha) and is thought to be a novel therapeutic target for human malignancies. We evaluated the ability of troglitazone (TRO) alone or in combination with 9-cis retinoic acid (9CRA), ligands of PPARgamma and RXRalpha, respectively, to inhibit the growth of oesophageal squamous cell carcinoma (OSCC). All 10 tested OSCC cell lines of a KYSE series expressed PPARgamma and RXRalpha at both the mRNA and protein levels. In four tested cell lines, TRO inhibited growth, and a synergistic effect was observed with simultaneous 9CRA application. In KYSE 270 cells, a luciferase reporter assay showed that the simultaneous application of TRO and 9CRA to the cells increased the relative luciferase activity approximately 20-fold compared with the controls without TRO or 9CRA application. In this cell line, flow cytometry demonstrated that combined treatment with TRO and 9CRA greatly increased the sub-G1 phase, and Hoechst 33342/propidium iodide (PI) staining showed that apoptotic cell death was mainly induced through ligand treatment. In addition, implanted tumours in nude mice showed significant inhibition of tumour growth when treated with TRO. These results suggest that the PPARgamma/RXRalpha heterodimer may be a new therapeutic target for OSCC.

Topics: Alitretinoin; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cell Division; Chromans; Drug Evaluation; Esophageal Neoplasms; Humans; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Transcription Factors; Tretinoin; Troglitazone; Tumor Cells, Cultured

Expression of retinoic acid receptor beta (RARbeta) is reported to be absent or down-regulated in oral squamous cell carcinomas. Recently, we found that the growth-inhibitory effect of 9-cis-retinoic acid (9CRA) on oral squamous cell carcinoma may depend on the expression levels of endogenous RARbeta. In order to clarify the role of RARbeta in growth and differentiation, we transfected RARbeta expression vector into oral squamous carcinoma cell lines, HSC-4 and Ho-1-N-1. Both RARbeta-transfected cell lines displayed growth inhibition. Moreover, RARbeta-transfected clones underwent morphological changes, and RARbeta-transfected HSC-4 clones underwent apoptosis even in the absence of 9CRA treatment. In contrast, RARbeta-transfected Ho-1-N-1 clones exhibited cell cycle arrest without undergoing apoptosis initially; however, apoptosis was induced in these cells after 6 days of 9CRA treatment. RARalpha and RARgamma expression was reduced at both the protein and mRNA levels in RARbeta transfectants, whereas the expression of retinoid X receptor alpha (RXRalpha) was not altered. RARb transfectants exhibited alterations in the levels of cell cycle-associated proteins, histone acetyltransferase (HAT) and apoptosis-associated proteins. After 6 days of 9CRA treatment, RARbeta transfectants overexpressed Waf1 / Cip1 / Sdi1 / p21, Kip1 / p27, chk1, p300 / CBP, BAX, Bak, Apaf 1, caspase 3 and caspase 9. Conversely, E2F1, cdc25B and HDAC1 were down-regulated in these transfectants. In addition, histone H4 acetylation was induced in RARb transfectants. These findings suggest that histone acetylation mediated by histone acetyltransferase and p300 / CBP may play a role in the growth arrest and apoptosis induced by RARbeta transfection in oral squamous cell carcinoma.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Humans; Mouth Neoplasms; Neoplasm Proteins; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) has been shown to be effective in suppressing premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck. However, the precise mechanisms of these effects are still uncertain. In the present study, we examined the effect of 9-cis-RA on the growth of six oral cancer cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-N-1 and Ho-1-u-1). In addition, the relationship among growth and differentiation of tumor cells, RA responsiveness and the expression of nuclear retinoic acid receptors were also investigated. Among the six cell lines examined, five (HSC-2, HSC-3, HSC-4, Ca9-22 and Ho-1-u-1) displayed growth inhibition after treatment with 1x10(-6) M 9-cis-RA, while Ho-1-N-1 cells were resistant to 9-cis-RA. The expression level of RARbeta in 9-cis-RA resistant Ho-1-N-1 cells was very low in comparison with the sensitive cell lines. On the other hand, all of the six the cell lines expressed RARalpha, RARgamma, and RXRalpha at various levels. 9-cis-RA induced accumulation of cell population in G1 phase in HSC-3 cells on the 6th day of the treatment, followed by a marked reduction in the levels of hyperphosphorylated pRB, whereas p53 level was not altered. Interestingly, 9-cis-RA induced transiently the expression of p21(Waf1/Cip1), p27(Kip1), p300, CBP, BAX, Bak and bcl-2 proteins, respectively. This effect was associated with reduction of cyclin D1, cdk4 and CDK-activating kinase (cyclin H and cdk7) protein in HSC-3 cells. These results suggest that the growth inhibitory effect of 9-cis-RA on oral squamous cell carcinoma may depend on the expression levels of RARs, especially RARbeta proteins and RXRalpha proteins, and that 9-cis-RA may provide a powerful therapeutic agent for head and neck cancers.

Topics: Alitretinoin; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Differentiation; Cell Division; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, cdc; Humans; Mouth Neoplasms; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

We recently demonstrated that the constitutive expression of cyclooxygenase (COX)-2 protein and prostaglandin E(2) (PGE(2)) biosynthesis were significantly enhanced in human skin epidermal cancer cells and that cancer cell growth was effectively inhibited by the suppression of COX-2 expression by transfection with COX-2 antisense oligonucleotide. The purpose of this study was to search for agents which suppress COX-2 expression and examine their effects on cell growth. Since retinoids and antioxidants have been used for chemoprevention of cancers in several tissues, the effects of these agents on COX-2 expression and PGE(2) biosynthesis in skin squamous carcinoma cells were investigated. Treatment with a retinoid (9-cis-retinoic acid (9-cis-RA)) or an antioxidant (pyrrolidinedithiocarbamate (PDTC)) suppressed COX-2 expression and PGE(2) biosynthesis in a concentration-dependent manner. Cell growth was significantly inhibited by 9-cis-RA and PDTC. These results suggest that 9-cis-RA and PDTC may be useful in preventing skin cancer growth and that COX-2 is involved in their protective effects on skin carcinogenesis.

Topics: Acetylcysteine; Alitretinoin; Antineoplastic Agents; Antioxidants; Blotting, Western; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; Isoenzymes; Keratinocytes; Membrane Proteins; Oligonucleotides, Antisense; Prostaglandin-Endoperoxide Synthases; Pyrrolidines; Retinoids; Skin Neoplasms; Thiocarbamates; Time Factors; Transfection; Tretinoin; Tumor Cells, Cultured

A class of less toxic retinoids, called heteroarotinoids, was evaluated for their molecular mechanism of growth inhibition of two head and neck squamous cell carcinoma (HNSCC) cell lines SCC-2 and SCC-38. A series of 14 heteroarotinoids were screened for growth inhibition activity in vitro. The two most active compounds, one that contained an oxygen heteroatom (6) and the other a sulfur heteroatom (16), were evaluated in a xenograph model of tumor establishment in nude mice. Five days after subcutaneous injection of 10(7) SCC-38 cells, groups of 5 nu/nu mice were gavaged daily (5 days/week for 4 weeks) with 20 mg/kg/day of all-trans-retinoic acid (t-RA, 1), 10 mg/kg/day of 6, 10 mg/kg/day of 16, or sesame oil. After a few days, the dose of t-RA (1) was decreased to 10 mg/kg/day to alleviate the side effects of eczema and bone fracture. No significant toxic effects were observed in the heteroarotinoid groups. All three retinoids caused a statistically significant reduction in tumor size as determined by the Student t-test (P < 0. 05). Complete tumor regression was noted in 3 of 5 mice treated with t-RA (1), 4 of 5 mice treated with 16, 1 of 5 mice treated with 6, and 1 of 5 mice treated with sesame oil. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine that the expression levels of RARalpha, RXRalpha, and RXRbeta were similar in the two cell lines, while RARbeta expression was higher in SCC-2 over SCC-38, and RARgamma expression was higher in SCC-38 over SCC-2. Receptor cotransfection assays in CV-1 cells demonstrated that 16 was a potent activator of both RAR and RXR receptors, while 6 was selective for the RXR receptors. Transient cotransfection assays in CV-1 cells using an AP-1 responsive reporter plasmid demonstrated that t-RA (1), 6, and 16 each inhibited AP-1-driven transcription in this cell line. In conclusion, the growth inhibition activity of the RXR-selective 6 and the more potent growth inhibition activity of the RAR/RXR pan-agonist 16 implicate both RARs and RXRs in the molecular mechanism of retinoid growth inhibition. Moreover, the chemoprevention activity and the lack of toxicity of heteroarotinoids demonstrate their clinical potential in head and neck cancer chemoprevention.

Topics: Animals; Antineoplastic Agents; Benzamides; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Squamous Cell; Cell Division; Drug Screening Assays, Antitumor; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Nuclear retinoic acid receptor beta(RARbeta) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARbeta expression in such cells. To investigate the role of RARbeta in HNSCC growth and differentiation, we transfected a retroviral RARbeta2 expression vector (LNSbeta) into HNSCC SqCC/Y1 cells, which do not express endogenous RARbeta but do express RARalpha, RARgamma, and retinoid X receptors. Transfected clones expressing RARbeta2 mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10(-10) M ATRA in four of five LNSbeta clones, whereas it was not suppressed in LNSX cells even by 10(-6) M ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSbeta clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSbeta-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale necarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARbeta mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.

Topics: 3T3 Cells; Alitretinoin; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Drug Resistance; Head and Neck Neoplasms; Humans; Mice; Naphthalenes; Neoplasm Proteins; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; RNA, Neoplasm; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids are a group of vitamin A analogues that have shown promise as chemopreventive and therapeutic agents in many types of malignancy and have been entered in clinical trials with some successful results. To better understand the mechanism that mediates retinoid action and the anti-proliferative effects, we treated 7 human oral squamous-cell carcinoma (SCC) cell lines (FADU, HEp-2, CCL-17, SCC-9, SCC-15, SCC-25 and HN-212) with 10(-6) M of all-trans retinoic acid (ATRA), 9-cis and 13-cis retinoic acid (RA) in continuous for different periods of time. We assessed the extent of growth inhibition, the stability of the anti-proliferative effect and the mRNA expression levels (by RT-PCR) of RA receptors (RARs), retinoid X receptors alpha (RXR alpha) and cytosolic RA-binding proteins (CRBP I and CRABP II) in treated cells compared with controls. The data obtained showed that all 3 RAs were able to inhibit the cellular growth of the tested cell lines, although to a different extent. The cis compounds were able to inhibit the proliferation of all cell lines, whereas ATRA was ineffective in inhibiting the proliferation of the CCL-17 cell line, which was naturally resistant to ATRA concentrations in the range between 10(-5) and 10(-6) M. All inhibitory effects were completely reversible since all cell lines restored their normal growth proliferation within few days after drug removal. RT-PCR analysis of the receptor and cell binding protein status of control and treated cells showed a good correlation between growth inhibition and induction of, or increase in, the expression levels of RAR beta in RA-treated cells. No differences were observed in RAR alpha and RXR alpha mRNA expression levels between control and treated cells. CRBP I, CRABP II and RAR gamma mRNA levels increased in some treated cell lines but not in all.

Topics: Alitretinoin; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Isotretinoin; Mouth Neoplasms; Neoplasm Proteins; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.

Topics: Alitretinoin; Animals; Antineoplastic Combined Chemotherapy Protocols; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cisplatin; Female; Humans; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transplantation, Heterologous; Tretinoin