食管鳞癌细胞分化状态对内源性光敏剂PpIX产量的影响及对PDT的应答

Effects of differentiation of human esophageal cancer cells on 5-aminolevulinic acid-induced endogenous protopophyrin IX production and photodynamic therapy

目的:研究食管癌细胞的分化状态对ALA产生的内源性光敏剂PpIX累积水平的影响,及对PDT的应答敏感程度.方法:以高分化食管鳞癌细胞KYSE-450和低分化食管鳞癌细胞KYSE-70为研究对象,利用荧光分光光度法测定不同浓度ALA处理后细胞内PpIX的浓度;分别使用不同剂量的红光和蓝光对细胞进行照射处理,MTS法测定PDT对细胞的光毒毒性;TdT-流式细胞法测定PDT诱导的凋亡水平,并观察凋亡细胞的胞核形态.结果:高分化KYSE-450细胞比低分化KYSE-70细胞具有更强的PpIX合成或累积水平,两者PpIX绝对浓度值在高于0.5mmol/LALA时差异显著(315.2±88.3vs156.9±79.2,P<0.05);虽然高分化细胞PpIX产量高于低分化细胞,但无论是红光PDT还是蓝光PDT,其敏感性均明显差于低分化细胞,光剂量为3.6J/cm2(红光)和0.16J/cm2(蓝光)时,两者细胞存活率有极显著差异(88.0±7.4vs25.2±4.9,红光;97.4±7.3vs40.8±4.2,蓝光;P<0.01);KYSE-450凋亡率(12.5%)亦低于KYSE-70(23.2%),细胞主要以坏死方式为主.结论:食管鳞癌细胞的分化影响了内源性光敏剂PpIX的细胞内水平;PDT效应并不单纯依赖于细胞内的光敏剂含量,细胞的生物学特征如分化状态也影响了PDT的效果;高分化细胞部分通过抑制凋亡抵制了PDT作用.

AIM： To explore the effects of differentiation on 5-aminolevulinic acid （5-ALA）-derived endogenous protopophyrin IX （PpIX） production and photodynamic therapy （PDT） in human esophageal cancer. METHODS： Well differentiated KYSE-450 and poorly differentiated KYSE-70 cell lines were used in this study. Intracellular PplX production after incubation with various concentrations of ALA was quantified with spectrofluorometry. Moreover, Cell survival after ALA-PDT with different light doses of red light （〉 600 nm） and blue light （450 nm） was determined by MTS assay. The apoptosis of the cells after ALA-PDT were detected by flow cytometry with TdT staining as well as fluorescent microscopy with Hoechst 33342 staining. RESULTS： KYSE-450 cells had a higher PpIX production than KYSE-70 did, and a significant difference was found when the ALA concentration was reached above 0.5 mmol/L （315.2 ± 88.3 vs 156.9 79.2, P 〈 0.05）. However, the sensitivity of the well-differentiated cells to ALA-PDT was lower than that of the poorly-differentiated ones. Cell viability assay showed a significant difference between KYSE-450 and KYSE-70 cells when the red light of 3.6 J/cm^2 and blue light of 0.16 J/cm^2 were employed （red light： 88.0 ± 7.4% vs 25.2 ± 4.9%, P 〈 0.01; blue light： 97.4% ±7.3 vs 40.8 ± 4.2%, P 〈 0.01）. The apoptotic rate of KYSE-450 cells （12.5%） was lower than that of KYSE-70 cells （23.2%）, and necrosis was the main death style for both kinds of cells. CONCLUSION： The differentiation of esophageal cancer cells can affect the synthesis and accumulation of ALA-induced endogenous PpIX. The efficacy of ALA-PDT don＇t just depended on the amount of intracellular PpIX, and the well- differentiated cells with more PpIX production are more resistant to ALA-PDT than the poorly- differentiated cells with less PpIX production.