Development of new therapy for pancreas cancer using PPAR γ-ligand targeting hTERT.

为探讨PPARγ配体对胰腺癌细胞的细胞毒作用，我们用不同浓度的15-脱氧-前列腺素J_2(15d-Δ_2)与MIAPaCa-2细胞共同培养72 h，并测定活细胞数。15D-PGJ_2呈剂量依赖性地减少活细胞数。经15d-PGJ2处理的细胞在20μM中存活的细胞比例降至未处理细胞的20%，然后我们检测了15d-PGJ2处理的MIAPaCa-2细胞中hTERT mRNA和蛋白的表达水平。鉴于已有报道转录因子Sp1、c-Myc和雌激素受体(ER)促进hTERT基因的转录，我们用EMSA检测了这三种分子与hTERT启动子的DNA结合活性。15d-PGJ_2对DNA结合活性的下调作用仅见于内质网。接下来，我们研究了15d-PGJ_2对ER表达的影响。虽然MIAPaCa-2细胞只表达ERβ，但ERβ的表达水平不受15d-PGJ2的影响。提示ERβ中丝氨酸残基的磷酸化可能增强了ERα的结合活性。我们检测了15d-pGJ_2处理的细胞中磷酸化ERβ的表达。15d-pGJ_2下调了磷酸化ERβ的表达。此外，我们还研究了可能使ERβ中的丝氨酸残基磷酸化的MAP激酶的活性。通过磷酸化ERK的表达来评估MAPK活性。提示15d-PGJ2通过抑制ERβ的磷酸化而下调端粒酶逆转录酶，从而抑制胰腺癌细胞的生长。本研究提示PPARγ配体有可能成为治疗胰腺癌的新药物。

To investigate the cytotoxic effect of PPAR γ-ligand in pancreas cancer cells, we cultured MIAPaCa-2 cells with various concentrations of 15-deoxy-Δ^<12,14>-prostaglandin J_2 (15d-PGJ_2) for 72 h and determined viable cells number. 15d-PGJ_2 decreased the viable cell number in a dose-dependent manner. Specifically, the proportion of viable cells in 20 μM of 15d-PGJ_2-treated cells decreased to 20% of untreated cells.We then determined the expression level of hTERT mRNA and protein in 15d-PGJ_2-treated MIAPaCa-2 cells. 15d-PGJ_2 strongly inhibited both hTERT mRNA and protein expression.Since it has been reported that transcription factor Sp1, c-Myc and estrogen receptor (ER) promote transcription of hTERT gene, we examined DNA binding activity of these three molecules to the hTERT promoter in 15d-PGJ_2-treated cells using EMSA. Down-regulation of DNA binding activity by 15d-PGJ_2 was observed only in ER. We next investigated the effect of 15d-PGJ_2 on the expression of ER. Although MIAPaCa-2 cells expressed only ERβ, expression level of ERβ was not affected by 15d-PGJ_2.There is a possibility that phosphorylation of serine residues in ERβ enhances its DNA binding activity as well as ERα. We assessed expression of phosphorylated ERβ in 15d-PGJ_2-treated cells. Phosphorylated ERβ was down-regulated by 15d-PGJ_2.In addition, we investigated the activity of MAP kinase which is speculated to phosphorylate the serine residues in ERβ. MAP kinase activity was assessed by expression of phosphorylated ERK. 15d-PGJ_2 reduced phosphorylated ERK expression.These results revealed 15d-PGJ_2 inhibit the growth of pancreas cancer cells by down-regulation of hTERT via inhibiting the phosphorylation of ERβ. This study indicates the possibility that PPAR γ-ligand become a new agent for treatment of pancreas cancer.