| Objective: At present malignant tumor has become a major cause which influences people's living quality and threatens people's health severely. So the urgent affairs are studying the pathogenesis of tumor and developing effective anti-tumor drugs which can help the sufferers racked with cancer improve their living quality and prolong their life span. In the past researchers summed up the pathogenesis of tumor in two aspects. One was the activation of oncogene which made cells proliferate limitlessly. The other was the block of the cellular differentiation which made immature cells multiply in large amount. Recently researchers find the maladjustment of apoptosis is also one of the important mechanisms of cancerization. Even someone thinks that cancer cells originate from cells which should but can't happen apoptosis normally. With the development of study, researchers recognize that apoptosis is a cellular voluntary death controlled by gene and different from necrosis in morphological and biochemical characters. Killing tumor cells by inducing apoptosis can protect the normal tissues from intensive inflammatory reaction. Therefore researchers pay more attention to the task of developing drugs which can induce apoptosis of tumor cells. Arsenic trioxide is the main component of white arsenic, which is know as the traditional Chinese medicine, arsenic base. low dose's arsenic trioxide has already carried in Chinese traditional medicine history. Recent studies show that arsenic trioxide can inhibit proliferation and induce differentiation or apoptosis of many kinds of carcinoma cells. Those characters make arsenic trioxide possess good applying prospects as a new anti-tumor drug. Cisplatin was clinically common anti-carcinogen, both domestic and abroad correlation literature reported cisplatin have got comprehensive resist tumor action, it can cure a great variety of cancer of late stage, such as ovarian cancer and lung cancer. Breast cancer is the most common malignant tumor in our country. So more and more studies appear which aim at the prevention and cure of breast cancer which has become a popular topic in the field of cancer research. Therefore, this study was designed to investigate the anti-tumor effect of arsenic trioxide combining with cisplatin on xenografted tumor growth of human breast cancer cell line MCF-7 in nude mice, and explore the mechanism of their interaction.Methods: MCF-7 cells were cultured in RPMI1640 medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100ug/ml streptomycinin in an atmosphere of 5% CO2 at 37℃. The logarithmically growing MCF-7 cells were digested by 0.25% typsin and 0.02% EDTA. 1 To built BALB/C nude mice xenograft model: Human breast cancer cells(MCF-7 cells) were implanted under the skin of female nude mice to generate tumors and measured xenograft's volume. After the tumors grew to a definite size, the mice were randomly divided into four groups: control group, arsenic trioxide group, cisplatin group, and the combination group of arsenic trioxide and cisplatin.2 To observe Morphological changes through light microscopy: The apoptosis-related morphological changes of xenografted tumor induced by arsenic trioxide and cisplatin for 2 weeks were analysis. One part tissue was place in 10% formalin. After 24 hours they were taken out to make the dyeing of HE and observed. The side effects were observed with HE staining of organs.3 To analyse Morphology changes through transmission electron microscopy: Xenografted tumor treated with 3mg/kg arsenic trioxide and cisplatin for 2 weeks were harvested, and were divided into pieces of 1mm×1mm×1mm, and pre-fixed with 4% glutaraldehyde at 4℃. Xenografted tumor were postfixed for 1h with 1% osmium tetroxide, then dehydrated through a graded ethanol series, and embedded in Epon812.The ultrastructure of cells were analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate. 4 To examine cell apoptosis and cell cycle through flow cytometry: Xenografted tumor were treated with 3mg/kg arsenic trioxide and cisplatin for 2 weeks. Experimental group and control group tissue were harvested and divided into 2mm×2mm×2mm ,then washed twice with PBS and fixed with ice cold 70% ethanol at 4℃.And then cells were resuspended in 0.5ml propedium iodide(PI)/Rnase A solution. Cells were incubated in the dark at room temperature for 30min. The fluorescence emission of stained cells were measured with a flow cytometer. Date were analyzed with Multipcycle software. 5 To observe Survivin mRNA expression through Reverse transcription polymerase chain reaction (RT-PCR): total tissue RNA was extracted with TRIZOL reagent according to the manufactures instructions. Reverse transcription reaction and amplification were carried out. PCR products were electropheresed on 8% polyacrylmide gels(1×TBE running buffer) and stained by ethidiu-m bromide visualized by gels imaging system(BIO-PROFIL,VL company, France). The fluorescence intensity ofβ-actin fragments served as the criterion for the fragments. 6 To analyse expression of Survivin proteins through flow cytometry: Cells were stained by indirect immunofluorescence labing method. A histogram plot of FITC-fluorescence intensity (in logarithmic fluorescence intensity) versus counts has been shown by flow cytometry. Fluorescence index was used to analysis the expression of Survivin proteins.Result: 1 The subcutaneous tumor model in nude mice was successfully established. 2 Microscopic examination showed that there was a small area of cytoclasis and signs of cell apoptosis in the center of tumors of the combination group of arsenic trioxide and cisplatin (comparing with arsenic trioxide group,cisplatin group,control group).3 Under transmission electron microscope, drug combination caused typical apoptotic changes, including characteristic chromatin condensation, nuclear shrinkage, nuclear cleavage, and the cytoplasm was vacuolated. The membrane of the cell was complete. But arsenic trioxide group and cisplatin group did not so a typical result: the cytoplasm has a great deal of rough endoplasmic reticulum, mitochondria engorgement, crista disappeared.4 The analysis of celluar DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups. That was a typical apoptotic peak, which was not showed in the graph of control group. After xenografted tumor was treated with 3mg/kg arsenic trioxide and cisplatin for 2 weeks, the apoptosis rates of arsenic trioxide group,cisplatin group and the combination group were(26.4±3.27)%,(21.9±2.66)%,(36.9±3.28)% respectively, which were significantly higher than control(5.4±0.06)%. And the apoptosis rate in the combination group of arsenic trioxide and cisplatin was markedly higher than the other two groups. The quantity of the combination group cells in G0/G1 phase increased but that in S phase decreased. The combination group most MCF-7 cells were blocked at G0/G1 phase [from(51.3±3.31)% of arsenic trioxide group,(48.5±3.37)% of cisplatin group to(75.9±4.25)% of the combination group, respectively]. Expressions of Survivin protein was decreased (fluorescence index were 0.65 of arsenic trioxide group,0.70 of cisplatin group and 0.40 of the combination group, respectively). 5 The result of RT-PCR showed that expression of Survivin mRNA was decreased. The expression of Survivin mRNA in the combination group was markedly lower than other groups. Arsenic trioxide group compared with cisplatin group, the difference wasn't significant.Conclusion: 1 Arsenic trioxide and cisplatin can inhibit proliferation and induce apoptosis of xenografted tumor cells. Arsenic trioxide of low concentration combining with cisplatin of low concentration may obviously increase the anti-tumor effect in compare to using arsenic trioxide or cisplatin in high concentration respectively. 2 The mechanisms of interaction are maybe due to that` arsenic trioxide and cisplatin may reduce the expression of Survivin .
|
PDF Full Text Request