First Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Mod Pathol. 2011 Jun;24(6):876-84. doi: 10.1038/modpathol.2011.10. Epub 2011 Feb 11.
Ca2+ is a chemopreventive agent for colon cancer. Ion transport systems are often altered in human cancer. The aim of this study was to clarify the alterations of calcium-sensing receptor (CASR), a member of the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed the expression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues by RT-PCR and immunostaining. In addition, we analyzed methylation of the CASR promoter by using bisulfite sequence analysis and methylation-specific PCR. CASR mRNA and protein expression was significantly downregulated in most of the cancer cell lines. CpG islands were densely methylated in cancer cell lines with reduced CASR mRNA expression. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, and/or a histone deacetylase inhibitor, trichostatin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression in CASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca2+ on the cytotoxic response to 5-fluorouracil. CASR expression was observed in normal colonic epithelial cells and was retained in most adenoma tissues. CASR mRNA and protein expression was significantly downregulated in cancer tissues. There was an inverse relationship between CASR expression and degree of differentiation. Immunohistochemical CASR staining was reduced more predominantly in less-differentiated cancer tissues and/or in cancer cells at the invasive front, where nuclear/cytoplasmic β-catenin was often localized. CASR methylation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatic tissues and was significantly correlated with reduced CASR expression. CASR methylation was also detected in 32% of advanced adenoma tissues but was detected in only 9% of adenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylation seems to occur at an early stage and progress in colorectal carcinogenesis. The results suggest that epigenetic inactivation of CASR has an important role in colorectal carcinogenesis.
钙离子是结肠癌的化学预防剂。离子转运系统在人类癌症中经常发生改变。本研究的目的是阐明钙敏感受体（CASR）在结直肠癌变中的改变，CASR 是 G 蛋白偶联受体家族的成员。我们通过 RT-PCR 和免疫染色分析了 CASR 在结直肠癌细胞系和癌组织及腺瘤组织中的表达。此外，我们还通过亚硫酸氢盐测序分析和甲基化特异性 PCR 分析了 CASR 启动子的甲基化。在大多数癌细胞系中，CASR mRNA 和蛋白表达显著下调。在 CASR mRNA 表达降低的癌细胞系中，CpG 岛高度甲基化。用去甲基化剂 5-氮杂-2'-脱氧胞苷和/或组蛋白去乙酰化酶抑制剂曲古抑菌素 A 处理可恢复癌细胞系中的 CASR 表达。在未甲基化的 HCT-8 细胞中破坏 CASR 表达可阻断 Ca2+对 5-氟尿嘧啶细胞毒性反应的增强作用。在正常结肠上皮细胞中观察到 CASR 表达，并在大多数腺瘤组织中保留。在癌组织中，CASR 表达显著下调。CASR 表达与分化程度呈负相关。免疫组化 CASR 染色在分化程度较低的癌组织和/或在核/细胞质 β-连环蛋白常定位的侵袭前沿的癌细胞中减少更为明显。在 69%的结直肠癌组织和 90%的淋巴结转移组织中检测到 CASR 甲基化，与 CASR 表达下调显著相关。在 32%的高级腺瘤组织中也检测到 CASR 甲基化，但在 9%的腺瘤组织中未检测到，在增生性息肉组织中未检测到。CASR 甲基化似乎发生在结直肠癌变的早期，并进展。结果表明，CASR 的表观遗传失活在结直肠癌变中起重要作用。
