School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, West Bengal, India.
Micron. 2010 Apr;41(3):247-56. doi: 10.1016/j.micron.2009.10.008. Epub 2009 Nov 10.
Analysis of changes in cancer cell morphology and cytoskeletal element induced by external stimuli is focus of current cancer chemotherapeutic studies. Cancer cell cytoskeleton is complex network of interwoven protein fibers composed of microtubules, microfilaments and intermediate filaments. These interwoven protein fibers are responsible for maintaining cell morphology, movement, adhesion and transmembrane signal transmission. In this study, morphological and cytoskeletal changes induced by AEE788 and/or Celecoxib on colon cancer cell HCT 15 were analyzed using advanced microscopic techniques. Cell proliferation assay was used for determining IC(50) of AEE788 and/or Celecoxib on HCT 15. Confocal microscopic analysis of AEE788 and/or Celecoxib treated HCT 15 was performed using Rhodamine-Phalloidin (actin stain) and Hoechst 33342 (nuclear stain). Atomic force (AFM) and scanning electron microscopic (SEM) studies were also performed to analyze cell morphology and cell wall extension (filopodia and lamellipodia). In addition, quantitative analysis of morphological parameters was studied using cellular image processing technique. This is the first report that combination of AEE788 and Celecoxib additively increase growth inhibition and cell death on human colon cancer cell HCT 15 as estimated by cell proliferation assay. Morphological analysis of AEE788 or Celecoxib treated HCT 15 cell for 24h have not revealed significant change in morphology under phase contrast microscopy. But, slight morphological changes were observed in combination (AEE788+Celecoxib) treated HCT 15 for 24h. In contrast, high resolution confocal laser fluorescence and atomic force microscopic studies have revealed cell shrinkage, disorganized actin filament and, loss of filopodia and lamellipodia. These changes were more prominent in combination of AEE788 and Celecoxib treated HCT 15 than either drug alone. These results may suggest antiproliferative and antimetastatic activity of AEE788 and/or Celecoxib. Quantitative analysis of morphological parameters using cellular image processing technique have shown decrease in mean area, perimeter, compactness and eccentricity of combination drug treated cells than either drug alone. These results further support the confocal and AFM study. Scanning electron microscopic study of AEE788 and/or Celecoxib treated HCT 15 has also shown morphological changes and loss of filopodia and lamellipodia. In conclusion, this investigation of morphological and cytoskeletal changes using advanced microscopic techniques present a significant foundation for evaluating anticancer activity of a drug and form a new strategy for evaluating effect of AEE788 and/or Celecoxib on colon cancer.
分析细胞形态变化和细胞骨架元素引起的外部刺激是目前癌症化学治疗研究的焦点。癌细胞骨架是由微管、微丝和中间丝组成的相互交织的蛋白纤维复杂网络。这些相互交织的蛋白纤维负责维持细胞形态、运动、黏附和跨膜信号转导。在这项研究中，使用先进的显微镜技术分析了 AEE788 和/或塞来昔布对结肠癌细胞 HCT15 的形态和细胞骨架变化。细胞增殖测定法用于测定 AEE788 和/或塞来昔布对 HCT15 的 IC50。用罗丹明鬼笔环肽（肌动蛋白染色）和 Hoechst33342（核染色）对 AEE788 和/或塞来昔布处理的 HCT15 进行共焦显微镜分析。还进行了原子力（AFM）和扫描电子显微镜（SEM）研究，以分析细胞形态和细胞壁延伸（丝状伪足和片状伪足）。此外，使用细胞图像处理技术对形态参数进行了定量分析。这是第一项报告，表明 AEE788 和塞来昔布联合使用可通过细胞增殖测定法在人结肠癌细胞 HCT15 上相加增加生长抑制和细胞死亡。在相差显微镜下，24 小时 AEE788 或塞来昔布处理的 HCT15 细胞的形态分析没有显示出明显的形态变化。但是，在联合（AEE788+Celecoxib）处理的 HCT15 中观察到轻微的形态变化，24 小时。相比之下，高分辨率共焦激光荧光和原子力显微镜研究显示细胞收缩、肌动蛋白丝紊乱以及丝状伪足和片状伪足丧失。在 AEE788 和塞来昔布联合处理的 HCT15 中，这些变化比单独使用任何一种药物都更为明显。这些结果可能表明 AEE788 和/或塞来昔布具有抗增殖和抗转移活性。使用细胞图像处理技术对形态参数进行定量分析表明，与单独使用任何一种药物相比，联合药物处理的细胞的平均面积、周长、紧密度和偏心率均减小。这些结果进一步支持共焦和 AFM 研究。AEE788 和/或塞来昔布处理的 HCT15 的扫描电子显微镜研究也显示出形态变化和丝状伪足和片状伪足丧失。总之，使用先进的显微镜技术对形态和细胞骨架变化的研究为评估药物的抗癌活性提供了重要基础，并为评估 AEE788 和/或塞来昔布对结肠癌的作用提供了新的策略。
