ABSTRACT
An important molecular target for cancer therapy is the possible reactivation of tumor suppressor genes that have been silenced by promoter methylation. It was observed that the treatment of an adenocarcinoma cervical cancer cell line, HeLa with 20 μg/ml of the ethanolic extract of Withania somnifera for 6 days resulted in demethylation of promoter of RARβ2 gene. However, treatment with Ocimum sanctum and Azadirachta indica (20μg/ml) did not cause the reversal of hypermethylation after 6 days of treatment. This is the first report to show the reversal of hypermethylation of RARβ2 gene by Withania somnifera extract in a cervical cancer cell line.
ABSTRACT
Objective:To investigate the relationship between cervical lesion development and histone acetylation that regulates RAR-β2 expression. Methods:Immunohistochemistry and Western blot analysis were performed to detect AcH3, RAR-β2, and involu-crin expression in normal cervical tissues as well as in tissues with cervical intraepithelial neoplasia (CIN)Ⅱ-Ⅲand squamous cell cer-vical carcinoma. The relationship among histone acetylation level, RAR-β2 expression, and cervical lesion severity were analyzed. Re-sults:AcH3, RAR-β2, and involucrin expression were reduced or absent with the progression of cervical lesions;significant differences were noted between the groups (P<0.05). Histone acetylation level and RAR-β2 expression were positively correlated (r=0.797, P<0.05). AcH3 and RAR-β2 expression, which were both associated with the cervical lesions, were negatively correlated [r=-0.547(AcH3), r=-0.585(RAR-β2), P<0.05]. Conclusion:Histone acetylation modification is associated with the regulation of RAR-β2 expression. This pro-cess is also likely to participate in the carcinogenesis of cervical carcinoma.
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Objective To observe the expression of RAR-β gene in SiHa, HeLa,C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promotor region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.
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Objective: To observe the expression of RAR-β gene in SiHa, HeLa, C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results: The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion: The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promoter region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.
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PURPOSE: Response to radioiodine therapy for thyroid cancer is related to the loss of sodium-iodine symporter protein caused by dedifferentiation of thyroid cancer cells. So we aimed to study mRNA expression of CD97, dedifferentiation marker, and sodium-iodine symporter after retinoic acid treatment according to retinoids receptor status. METHODS: Thyroid cancer cell lines; ARO, FRO, NPA, TPO, and FTC133 were prepared. 5µM of all trans retinoic acid were administered to each cell lines and then expression of m RNA for retinoids receptors (RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ), CD97, and Sodium-Iodine symporter by RT-PCR. RESULTS: RARs and RXRs were differently expressed in each cell line. After retinoic acid treatment, relative density of retinoic acid receptor mRNA were increased by time dependently in each cell line except TPO cell line. Expression of CD97 also was decreased in every cell lines (P<0.001). Retinoic acid increased expression of sodium-iodine symporter only in FTC133 cell line (P<0.001), and TSH or forskolin did not enhance NIS expression by retinoic acid. RARβ and RXRγ were expressed only in FTC 133cell line before treatment. Induction of sodium-iodine symporter by retinoids disappeared after RARβ specific antagonist LE135 or pan RXR antagonist PA452 administration. CONCLUSION: Retinoic acid reduced expression of CD97 in five thyroid cancer cell lines. However, retinoic acid could restore sodium-iodine symporter expression in only FTC 133 cell line specifically containing RARβ and RXRγ. Restoration of sodium-iodine symporter expression by retinoic acid is related to RARβ and RXRγ expression.