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【摘要】 目的 研究佛波酯 (12-O-tetradecanoylphorbol-13-acetate，TPA) 对人食管癌细胞的影响。方法 细胞增殖试验采用MTT法和细胞计数的方法，细胞核形态的测量采用计算机辅助的图像分析系统，分别测量计算细胞核面积(NA)、核周长(NP)、最大直径(Dmax)、 最小直径(Dmin)、核浆比(NPA)、核轴比(NAR)、核形状因子(NRF)等。结果 TPA没有抑制人食管癌Eca109细胞增殖和改变细胞核形态。结论 TPA对人食管癌Eca109细胞可能没有促分化作用。
【关键词】 佛波酯； 食管癌细胞； 细胞核形态
Effects of TPA on proliferation and nuclear morphometry of human esophageal cancer Eca109 cells
GUO Yu-zhong,JIA Lin-jing,GAO Na,et al.Laboratory for Cell Biology，Medical College，Zhengzhou University,Zhengzhou 450052,China
【Abstract】 Objectives To investigate the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on human esophageal cancer Eca109 cells. Methods The sensitivity of TPA to Eca109 cells was determined with MTT assay and counting cell numbers. After the cells were stained with HE，the measurements of nuclear morphometry were performed with computer-assisted image analysis system. Results TPA did not inhibit the cells proliferation and decrease the nuclear size. Conclusions TPA may not induce Eca109 cells to differentiate.
【Key words】 12-O-tetradecanoylphorbol-13-acetate (TPA); esophageal cancer cell line; nuclear morphometry
Human esophageal cancer is one of the most common solid tumors in China. Due to its high metastatic potential and the frequent onset of resistance to chemotherapy，it is one of the major causes of death by neoplasia in the country. The present chemotherapy of cancer uses agents that are usually toxic to normal cells. On the other hand，induction of cellular differentiation may supplement the use of non-cytotoxic drugs in several forms of neoplasia，like the successful use of all-transretinoic acid (ATRA) in the treatment of acute promyelocytic leukemia.
Plant diterpine phorbol esters，particularly the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)，are among the most potent tumor promoters known and their effects range from stimulating DNA synthesis through activating gene expression to producing alterations in membrane properties and constituents. TPA can also potentiate the differentiation of various tumor cells in culture and in vivo of human beings. TPA can induce differentiation and inhibit the growth of a number of malignant cell types，including myeloid leukemia［1］，breast［2］，prostate［3］，colon［4］，and brain［5］. Other report showed that TPA could not induce human hepatocarcinoma cell line to differentiation［6］. Because of its efficiency in vivo［7］，we are interested in its effects on human esophageal cancer cells. Here，we report the effects of TPA on proliferation and nuclear morphometry of human esophageal cancer Eca109 cells.
1 Materials and methods
1.1 Chemicals TPA purchased from Sigma Chemical Co. was dissolved in ethanol and stored at -20℃.The stock solutions were diluted with Hanks’ buffered salt solution. The final concentration of ethanol in the incubation mixtures was 0.1% or less.
1.2 Cell Growth Studies Human esophageal cancer cell line Eca109 was kindly provided by professor Bai jingxiu，Zhengzhou University. The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) in the presence of antibiotics，penicillin (100 units/ml)and streptomycin (100units/ml). All cells were cultured at 37℃ in a humidified atmosphere containing 5% CO2. The medium was replaced after 3 days. The cells were routinely subcultured twice a week.
Cells were plated in either flasks (25cm2) or 96-well plates. TPA was added 24 hours after seeding，when the cells were attached to the surface of the containers. An equivalent volume of ethanol was added to the controls and had no effect on growth. After removing the cells from the flasks by trypsinization they were counted using a hemocytometer，fixed with 70% ethanol and stained by H.E. When the cells were plated into a 96-well microplate，the culture medium containing different concentrations of TPA was replaced 24 hours later. After 3-5 days of incubation，cell viability was evaluated by MTT assay.
1.3 Nuclear morphometry measurements Measurements of nuclear morphometry including nuclear area (NA)，nuclear perimeter (NP)，maximal diameter (Dmax)，minimal diameter (Dmin)，nuclear plasma ratio (NPR)，nuclear axis ratio (NAR)，nuclear roundness factor (NRF)，were performed with computer-assisted image analysis system MPIAS-500 (QingPing Image Engineering Company，Tongji University). Two fields were selected at random from each case for the measurements. Each field was viewed under a ×100 oil-immension lens and displayed on the screen of a color video image processor. The images of 50 nuclei of the tumour cells per field were outlined with a drawing pen，and the data were processed by a computer in the system，which calculated the parameters. In the end all the mean values of each case were calculated.
1.4 Statistical Analysis The data were expressed as means±SD. Statistical analysis was performed by SPSS 10.0 software. The criterion for statistical significance was taken as P <0.05.
2 Results
Effect of TPA on cell Growth. The growth of Eca109 cells was not arrested by TPA at non cytotoxic concentrations. As shown in Table 1 and Fig.1，TPA even increased the cancer cell numbers，though there were no significance between TPA groups and the controls.The nuclear features were measured on microphotographs using a commercially available software. Table 2 summarizes the results. TPA might decrease the cells size but no significant difference with the control group.
3 Discussion
Our results show that Eca109 human esophageal cancer cell is a cell type which does not respond to exposure to TPA by growth arrest (Table 1 and Fig.1). A marked change in cell morphology was not observed. On comparison of the different human cell systems in which phorbol esters cause growth arrest，the behavior of the Eca109 cells appears to resemble that of SMMC7221 human hapatoma cells［6］ and HT29 human colon cancer cells［8］. Even in T cell leukemia TPA could induce Jurkat and Molt3 cells to differentiate but could not induce CCRF-CEM and CCRF-HSB2 cells to differentiate. The reasons are not known.
Morphometric analysis has been used successfully as a diagnostic and prognostic criterion in patients with malignancies［9］.Many investigators have sought an accurate and reproducible parameter that will allow accurate prediction of adjunctive immunotherapy or chemotherapy when appropriate［10］.These morphometric studies have provided valuable information. We used quantitative morphometry to investigate the shape difference of neoplastic nuclei from esophageal cancer cells with or without treatment of TPA. TPA did not clearly change the shape of the cells nuclei，but it did change other cells’ nuclei which were found in our laboratory (data not shown). This result is possibly consistent with that of TPA on cells proliferation. When TPA induces tumor cells to differentiation，it has antiproliferation function. If tumor cells proliferate actively，differentiation may stop at least temporarily. The reasons that TPA did not influence the proliferation and nuclear morphometry of esophageal cancer cells are possibly connected to the diversity and cellular distributions of protein kinase C (PKC) on the cells. Protein kinase C (PKC) is a family of phospholipid-dependent serine-threonine kinases that play important roles in the signal transduction and in the regulation of cell growth and differentiation［11］.At least eleven isoforms of PKC have been isolated so far，showing diversity in their structures，cellular distributions，and biological functions［12］.PKC is also the receptor for the potent tumor-promoting phorbol esters，which can substitute for DAG in PKC activation. Reports also showed that different PKC subtype was involved in the differentiation of different tumor cells during TPA treatments［13］. Therefore，the distributions and the activity of PKC in Eca109 cells might be different with other tumor cells and have different signal transduction systems. So TPA might have different effects on different tumor cells. Further studies about the distributions and active status of PKC subtypes on human esophageal cancer cells may not only provide useful information on selection effective differentiation-inducers but also contribute to elucidation of the molecular mechanism involved in their differentiation and tumorigenicity.
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作者单位： 450052 河南郑州，郑州大学医学院细胞生物学研究室
(编辑：悦 铭)