Medical Laboratory Science Section, Department of Health Technology & Informatics, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region, Kowloon.
Photodiagnosis Photodyn Ther. 2012 Sep;9(3):243-55. doi: 10.1016/j.pdpdt.2011.12.005. Epub 2012 Jan 28.
This study employed a doxorubicin resistant (MES-SA-Dx5) human uterine sarcoma cell line and its counterpart (MES-SA), to elucidate the efficacy of aminolevulinic acid-hexylester (hexyl-ALA) mediated PDT at molecular and transcriptional levels.
Hexyl-ALA generated protoporphyrin IX in both cells were determined by molecular probes using Confocal Laser Scanning Microscopy. The hexyl-ALA-PDT induced signal transduction proteins and mode of cell death were quantitated by CASE ELISA assays and DAPI staining. The modulation of hTERT mRNA expression and telomerase activity were investigated by TaqMan real-time PCR and ELISA respectively. Hexyl-ALA-PDT mediated cell migratory effect was determined by wound-healing assay.
The results demonstrated that mitochondria were the major target of hexyl-ALA. At LD(30), hexyl-ALA-PDT significantly provoked an up-regulation of phosphorylated p38MAPK and JNK proteins in both cells. Hexyl-ALA-PDT down-regulated hTERT (a catalytic subunit of telomerase) mRNA expression and showed a strong correlation with diminished telomerase activity in both cells (MES-SA: r(2) = 0.9932; MES-SA-Dx5: r(2) = 0.9775). The suppression of cell migratory effect in both cells was obtained after hexyl-ALA-PDT. Further, 50% and 30% of apoptotic cells were attained at LD(50), for wild-type and drug resistant cells respectively. Unlike the wild-type, a higher PDT dose was crucial to induce apoptosis in the drug resistant cells.
Our study provides the first evidence that p38MAPK and JNK kinases played a vital role in triggering hexyl-ALA-PDT-induced apoptosis, down-regulated hTERT mRNA expression and telomerase activity in both proposed cells. In vivo studies are worth examining for the benefit of clinical applications in drug resistant cancers and PDT development.
本研究采用多柔比星耐药（MES-SA-Dx5）人子宫肉瘤细胞系及其相应的（MES-SA），以阐明氨基酮戊酸己酯（己基-ALA）介导的 PDT 在分子和转录水平上的疗效。
通过共聚焦激光扫描显微镜用分子探针确定两种细胞中生成的原卟啉 IX。通过 CASE ELISA 测定和 DAPI 染色定量测定己基-ALA-PDT 诱导的信号转导蛋白和细胞死亡方式。通过 TaqMan 实时 PCR 和 ELISA 分别研究 hTERT mRNA 表达和端粒酶活性的调节。通过划痕愈合试验测定己基-ALA-PDT 介导的细胞迁移效应。
结果表明，线粒体是己基-ALA 的主要靶标。在 LD(30)时，己基-ALA-PDT 显着引发两种细胞中磷酸化 p38MAPK 和 JNK 蛋白的上调。己基-ALA-PDT 下调 hTERT（端粒酶的催化亚基）mRNA 表达，并与两种细胞中降低的端粒酶活性呈强相关（MES-SA：r(2) = 0.9932；MES-SA-Dx5：r(2) = 0.9775）。两种细胞的细胞迁移效应在己基-ALA-PDT 后均受到抑制。此外，在 LD(50)时，野生型和耐药细胞分别获得 50%和 30%的凋亡细胞。与野生型不同，较高的 PDT 剂量对于诱导耐药细胞凋亡至关重要。
本研究首次提供证据表明，p38MAPK 和 JNK 激酶在触发己基-ALA-PDT 诱导的凋亡、下调两种细胞中 hTERT mRNA 表达和端粒酶活性中起关键作用。值得进行体内研究，以促进耐药性癌症和 PDT 发展的临床应用。
