Retinoic acid nuclear receptor α (RARα), a major role in mediating retinoids inhibition of growth in human breast carcinoma cells

Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each classes of these nuclear retinoid receptor is further subdivided into three species namely α, β and γ. Recent studies demonstrate that ER - positive HBC cell lines are sensitive and ER -negative cell lines are resistant to growth inhibitory effects of retinoic acid (RA). In this study we look at the expresion of RARs and RXRs in 6 HBC cell lines, we found only RARα mRNA level was strong correlated with ER - status. To further investigate the major role of RARα in retinoid - mediated inhibition of growth, we transfected RARα cDNA in two RA - resistant ER - negative HBC cell lines. Analyses of different clonal populations of RARα transfectants from each cell line revealed growth inhibition by retinoids. Our results demonstrates that RARα plays a major role in mediating retinoids inhibition of growth in HBC cells and adequate levels are required for such actions.     

Response of four human ovarian carcinoma cell lines to ALL-TRANS retinoic acid: Relationship with induction of differentiation and retinoic acid receptor expression

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR₃ and OVCCR₁ were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV₁ cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RARα was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RARβ was not detected in any of the cell lines, while RARγ (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV₁ cells.     

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [³H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and supershift assays using a DR2 (“direct repeat” 2) RA response element demonstrated DNA-binding of RARα, RARγ, RXRα and RXRβ in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRα1, TRα2, and TRβ. Northern-blot analysis showed low expression of RXRβ mRNA in FTC-133 and of TRα1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARα, RARβ, RARγ, RXRα, and RXRβ in the 4 cell lines and in human thyroid-carcinoma samples. RARβ mRNA was reduced in FTC-238 cells and RXRβ mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding. Int. J. Cancer 76:368–376, 1998.© 1998 Wiley-Liss, Inc.     

High expression of α-1-6 fucosyltransferase during rat hepatocarcinogenesis

α-1-6 Fucosylated α-fetoprotein (AFP) is known to be elevated in patients with primary hepatoma and has been suggested as being useful as an early indicator and predictor of the poor prognosis for hepatoma. Although GDP-L-fucosyl-N-acetyl-β-D-glucosaminide α-1-6 fucosyltransferase (α-1-6 FucT), is the key enzyme involved in α-1-6 fucosylation of AFP, when and how the expression of α-1-6 FucT is enhanced during hepatocarcinogenesis is unknown. Recently, we established a convenient assay method for this enzyme and were successful in the purification and cDNA cloning of α-1-6 FucT from human gastric cancer, as well as from porcine brain. In the present study, levels of α-1-6 FucT activity and mRNA expression have been determined during hepatocarcinogenesis in LEC rats which spontaneously develop hereditary hepatitis and hepatoma. The fetal liver contained the highest enzymatic activity, which tended to increase in inverse proportion to gestation. The enzymatic activity was significantly increased in hepatoma tissues as compared with uninvolved adjacent tissues. Northern-blot analysis revealed high expression of α-1-6 FucT mRNA in hepatoma tissues, whereas the expression was fairly low in normal, hepatitis and uninvolved adjacent liver tissues. While the fetal liver had the highest enzymatic activity, the expression of α-1-6 FucT mRNA was low, suggesting that another α-1-6 FucT is induced in fetal liver or that post-translational modification occurs. High expression of α-1-6 FucT was also observed in 3′-MeDAB-induced rat hepatomas and some rat hepatoma cell lines. Collectively, α-1-6 FucT was strongly enhanced from an early stage of hepatocarcinogenesis and was maintained at a high level in rat hepatomas. Int. J. Cancer 75:444–450, 1998. © 1998 Wiley-Liss, Inc.     

All-TRANS, 13-CIS and 9-CIS retinoic acids induce a fully reversible growth inhibition in HNSCC cell lines: Implications for in vivo retinoic acid use

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 α (RXRα) 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 RARβ in RA-treated cells. No differences were observed in RARα and RXRα mRNA expression levels between control and treated cells. CRBP I, CRABP II and RARγ mRNA levels increased in some treated cell lines but not in all. Int. J. Cancer, 70:194–200, 1997. © 1997 Wiley-Liss, Inc.     

维甲酸抑制雌激素受体阳性乳腺癌细胞生长机制的研究

目的探讨雌激素受体的表达对维甲酸抑制乳腺癌细胞生长的影响和ＲＡＲα表达的变化。方法雌激素受体阴性的乳腺癌细胞株ＭＤＡ－ＭＢ－２３１细胞,采用分子生物学质粒转染技术,将ＥＲ阴性的乳腺癌细胞ＭＤＡ－ＭＤ－２３１转染成ＥＲ阳性细胞。结果发现ＥＲ转染后的细胞不仅ＲＡＲα的基础表达升高,而且ＲＡ能明显抑制该细胞的生长。同时还发现,无论是已确立的雌激素受体阳性的乳腺癌细胞株,还是雌激素受体转染后的细胞,雌激素都能明显刺激ＲＡＲα的表达。结论雌激素通过ＥＲ上调ＲＡＲα量的表达,在维甲酸对乳腺癌细胞生长的抑制中起着很重要的作用。     

High expression levels of IKKα and IKKβ are necessary for the malignant properties of liver cancer

IKK‐NF‐κB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF‐κB signaling including IKKα, IKKβ, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c‐FLIP, Bcl‐xl, Stat3, Cip1 and Cip2 by real‐time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKα, IKKβ, RANK, Maspin, c‐FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKα and IKKβ were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC‐97L and MHCC‐97H in vitro, the numbers of BrdU positive cells were decreased in both IKKα and IKKβ knockdown cells. Levels of apoptosis were also investigated in IKKα and IKKβ knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKα and IKKβ in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl‐XL and c‐FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKα and IKKβ downregulation depends on extent of NF‐κB inhibition and the malignant nature of the HCC. We anticipate that IKK‐targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.     

Effect of ligands of nuclear hormone receptors on sodium/iodide symporter expression and activity in breast cancer cells

Iodide uptake by normal and cancerous thyroid cells is an active process mediated by the sodium/iodide symporter (NIS). Using quantitative real-time RT-PCR, we found that all 22 fresh human breast cancer samples had very low NIS expression similar to levels in untreated MCF-7 breast cancer cells. 9-cis retinoic acid (9-cis RA), a ligand for both retinoic acid receptor (RAR)/retinoic X receptor (RXR) heterodimers as well as RXR/RXR homodimers, markedly induced NIS mRNA expression in MCF-7 breast cancer cells in a dose- and time-dependent fashion, with maximal levels occurring at 12 h. All-trans retinoic acid, ATRA, a RAR specific ligand had a similar potency. Among eight breast cancer cell lines, three out of four estrogen receptor (ER)-positive and zero of four ER-negative cell lines responded to 9-cis RA by increasing their expression of NIS. Combining a RAR with a RXR selective ligand enhanced both NIS mRNA expression and iodide uptake in MCF-7 cells. Similarly, a ligand for proliferator-activated receptor (PPAR) when combined with 9-cis RA synergistically increased both NIS mRNA levels and iodide uptake in these MCF-7 cells. The iodide uptake was blocked by KClO₄. In conclusions, these findings suggest that selected combinations of NHR ligands should be examined in a limited trial to determine if their administration to patients allows the use of radioactive iodine for diagnosis and possibly treatment of metastatic breast cancer.     

Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression

We previously reported that fenretinide (4HPR) is effective against a human ovarian carcinoma xenografted in nude mice. The effects of 4HPR on ovarian tumors have been further studied in in vitro ovarian carcinoma cell lines A2780, IGROV-I, SW626 and OVCA432. A2780 was the most sensitive line: 50% growth inhibition was obtained after 3 days of exposure to 1 μM 4HPR, a pharmacologically achievable concentration, whereas approx. 10 μM 4HPR gave a similar inhibition in the other cell lines. All-trans retinoic acid (RA), at doses up to 10 μM, did not inhibit cell proliferation. Gel electrophoresis of DNA from either detached or attached A2780 cells treated with 4HPR revealed DNA ladders in detached cells. Apoptosis was also evidenced in detached 4HPR-treated cells by flow cytometry and microscopic observation. The difference in cell line sensitivity to the anti-proliferative effect of 4HPR was not related to drug uptake or efflux. Only A2780 cells, the most sensitive to 4HPR, expressed constitutive levels of RARβ; moreover, the levels of RARα and RARγ expression in these cells were higher than in the other cell lines. In A2780 cells, the association of an IC₂₀ of 4HPR to cisplatin resulted in a strong potentiation of the anti-proliferative effect. These data show (i) that 4HPR, in contrast to RA, has an anti-proliferative effect in human ovarian carcinoma cells which is related to induction of apoptosis and (ii) that among the tested lines, the most responsive to the drug expressed RARβ and the highest levels of RARα and RARγ. The results also suggest that 4HPR can potentiate the effects of cisplatin in ovarian carcinoma. © 1996 Wiley-Liss, Inc.