TPA对人食管癌Eca109细胞增殖和细胞核形态的影响

Effects of TPA on proiferation and nucear morphometry of human esophagea cancer Eca109 ces

Human esophagea cancer is one of the most common soid tumors in China. Due to its high metastatic potentia and the frequent onset of resistance to chemotherapy，it is one of the major causes of death by neopasia in the country. The present chemotherapy of cancer uses agents that are usuay toxic to norma ces. On the other hand，induction of ceuar differentiation may suppement the use of non-cytotoxic drugs in severa forms of neopasia，ike the successfu use of a-transretinoic acid (ATRA) in the treatment of acute promyeocytic eukemia.

Pant diterpine phorbo esters，particuary the phorbo ester 12-O-tetradecanoyphorbo-13-acetate (TPA)，are among the most potent tumor promoters known and their effects range from stimuating DNA synthesis through activating gene expression to producing aterations in membrane properties and constituents. TPA can aso potentiate the differentiation of various tumor ces in cuture and in vivo of human beings. TPA can induce differentiation and inhibit the growth of a number of maignant ce types，incuding myeoid eukemia，breast，prostate，coon，and brain. Other report showed that TPA coud not induce human hepatoca?

rcinoma ce ine to differentiation. Because of its efficiency in vivo，we are interested in its effects on human esophagea cancer ces. Here，we report the effects of TPA on proiferation and nucear morphometry of human esophagea cancer Eca109 ces.

1 Materias and methods

1.1 Chemicas TPA purchased from Sigma Chemica Co. was dissoved in ethano and stored at -20℃.The stock soutions were diuted with Hanks’ buffered sat soution. The fina concentration of ethano in the incubation mixtures was 0.1% or ess.

1.2 Ce Growth Studies Human esophagea cancer ce ine Eca109 was kindy provided by professor Bai jingxiu，Zhengzhou University. The ces were maintained in RPMI 1640 medium with 10% feta bovine serum (FBS) in the presence of antibiotics，peniciin (100 units/m)and streptomycin (100units/m). A ces were cutured at 37℃ in a humidified atmosphere containing 5% CO2. The medium was repaced after 3 days. The ces were routiney subcutured twice a week.

Ces were pated in either fasks (25cm2) or 96-we pates. TPA was added 24 hours after seeding，when the ces were attached to the surface of the containers. An equivaent voume of ethano was added to the contros and had no effect on growth. After removing the ces from the fasks by trypsinization they were counted using a hemocytometer，fixed with 70% ethano and stained by H.E. When the ces were pated into a 96-we micropate，the cuture medium containing different concentrations of TPA was repaced 24 hours ater. After 3-5 days of incubation，ce viabiity was evauated by MTT assay.

1.3 Nucear morphometry measurements Measurements of nucear morphometry incuding nucear area (NA)，nucear perimeter (NP)，maxima diameter (Dmax)，minima diameter (Dmin)，nucear pasma ratio (NPR)，nucear axis ratio (NAR)，nucear roundness factor (NRF)，were performed with computer-assisted image anaysis system MPIAS-500 (QingPing Image Engineering Company，Tongji University). Two fieds were seected at random from each case for the measurements. Each fied was viewed under a ×100 oi-immension ens and dispayed on the screen of a coor video image processor. The images of 50 nucei of the tumour ces per fied were outined with a drawing pen，and the data were processed by a computer in the system，which cacuated the parameters. In the end a the mean vaues of each case were cacuated.

1.4 Statistica Anaysis The data were expressed as means±SD. Statistica anaysis was performed by SPSS 10.0 software. The criterion for statistica significance was taken as P <0.05.

2 Resuts

Effect of TPA on ce Growth. The growth of Eca109 ces was not arrested by TPA at non cytotoxic concentrations. As shown in Tabe 1 and Fig.1，TPA even increased the cancer ce numbers，though there were no significance between TPA groups and the contros.

Tabe 1 Ce numbers of TPA treatments onEca109 ces (x±s)

Fig.1 MTT assay of TPA treatments on Eca109