| Objects: As is well known that topoisomerase II exist in cells ubiquitously and play an essential role in DNA replication, transcription, chromosome formation and separation of sister chromosome. This enzyme express in a higher level in proliferating cells, especially in malignant tumor cells. So, it has been an ideal target for antitumor drugs. Both clinical findings from clinical chemotherapy to patients with oral and maxillofacial malignant tumor and in vitro chemosensitivity testing with MTT method showed that the antitumor effect of teniposide (VM-26), a kind of topoisomerase II poison, are more significant than those of other chemotherapeutic drugs like cisplatin (CDDP). Present study is designed to identify quantitatively the effect of VM-26 both in vitro and in vivo and furthermore, to explore the underlying mechanism, and the findings will be used to indicate the usage of this drug in clinical practice.Material and Methods: Human tongue squamous cell carcinoma cell line (Tca8113 cell) and cell strain with high metastatic ability to lung induced from human adenoid cystic carcinoma (ACC-M cell) were used as subjects in this study. We firstly tested quantitatively the inhibition rate of tumor cell proliferation by VM-26 using MTT testing, with CDDP as control drug: Tca8113 cells and ACC-M cells were inoculated in 96 wells micro culture plate at a concentration of 5 104/well and 4 104/well and cultured for 24 h.Then, VM-26 of 0.1 g/ml, 0.5 g/mK 1.5 g/ml, 5.0 g/ml, 15 g/ml, 45 g/ml and CDDP of 0.1ng/ml, 0.3 g/ml, 1 g/ml, 3 g/mk 9 g/ml were added in culture medium. Cells were continued to be cultured for 1 d, 2 d, 3 d, 4 d, 5 d. Supernatants were aspirated and MTT was added in each well after culture for certain time. DMSO was added in to dissolve crystals and the ODs of these solutions were tested using UV spectrometer. 36 nu/nuBALB/C nude mice, aged 5 weeks, fed in thespecific pathogen free (SPF) condition, were divided randomly into 2 groups, 18 mice in each group. Mice in one group were inoculated with xenografts of Tca8113 cells of 8 mm3 in size, in the right armpit region and mice in the other group were inoculated with xenografts of ACC-M cells, 8 mm3 in size, in the left armpit region. 7 days later, each group was divided into 3 subgroups: control group, group treated with VM-26 and group treated with CDDP, 6 mice in each group. Mice in each subgroup were injected intraperitoneally with normal saline, VM-26 of 10mg/kg and CDDP of 5mg/kg respectively, once every 3 days and total 3 times. The volumes of tumors and general conditions of mice were recorded every 3 days. Tca8113 cells were selected as subject in further study on mechanism of VM-26 killing tumor cells. EB/AO fluorescent staining reagent were added in cells culture medium having been treated with VM-26 of 0.1 g/ml, 0.5 g/ml, 1.5 g/ml, 5.0 g/ml, 15 g/ml and 45 g/ml for 1 d, 2 d, 3 d, 4 d and 5 d, and cells were observed using fluorescent microscope. On the other hand, such cells were collected, fixed with 2% glutaraldehyde in PBS, dehydrated, embeded, chiped, double stained with lead citrate and uranyl acetate, observed its apoptosis using TEM and recorded in photographs. Tca8113 cells having been treated with VM-26 of 0.15 g/ml, 1.5 g/ml, 5.0 g/ml and 15 g/ml for 24 h, were collected and washed with PBS, fixed with cold 70% ethanol in PBS, washed in PBS again, treated with RNase to eliminate RNA and stained with PI in dark. Then, cells were filtered through 200-pore nylon membrane and collected using FCM. Cells distribution in cell cycle was analysed using Mod Fit software. Cells treated as above were stained using reagents in Annexin V kit, and acquired using FCM to test their apoptosis rate. Cells having been treated with VM-26 of 0.15 g/ml and 5.0 g/ml for 12 h, 24 h, 36 h, 48 h, and 72 h, were tested cell cycle distribution and apoptosis with FCM as above. This experiment was repeated in triplicate and the average values were acquired.Results: Findings from MTT assay showed VM-26 and CDDP inhibit the proliferations of tumor cells both i... |
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