| Backgroud and objectiveLung cancer is one of the most common malignant tumor in human beings, while its morbility and mortality still continue to rise, especial ly in the people aboved 40-year-old. The male to female ratio is about 5:1. Smoking has been recognized as risk factor for lung cancer. Other risk factors include air pollution, tuberculosis, HPV or other viral infections, dichloro ether or chloromethyl ether, chromium, chromate, mustard gas, tar, coal combustion products, mineral oil, chlorinated ethylene, radiation and so on.Cancer Epidemiology data shew that the total five-year survival rate of lung cancer is 14%,which has become the first cause of death in malignant tumors. And its incidence in most countries continued to rise year by year, however, the mortality and prognosis has not markedly improved.The development of tumors in the process of evolution are regulated by many factors. People have come to realize that cancer is a disease where the body environment are disordered. Cells are the basic components of the human body environment. They continue to accept the signals from the upstream or downstream environment, and response or make integration to them.Celastrol is drawn from the traditional Chinese medicines Tripterygium. Its molecular weight is 450.61 and its molecular formula is C29H38O4. It is exerted cytotoxic effects in broad-spectrum anti-cancer. In present,more and more investigations were shown that celastrol could induce many tumor cells apoptosis derived from human. In recent years, many domestic and international studies have shown celastrol a variety of in vitro induced apoptosis of human tumor cell, but the detailed mechanism of apoptosis induction has not been reported.Purpose1. The influence of celastrol on human lung cancer cell proliferation of A549;2. The influence of celastrol on human lung adenocarcinoma A549 cell apoptosis;3. The Mechanism of celastrol on human lung adenocarcinoma A549 cells induced apoptosis;4.The influence of celastrol human lung adenocarcinoma A549 cells PI3K/Akt signaling pathway.Materials and methods1.MTTA549 cells of 1×106/ml per well were in oculated into 24 well plates and then pre-cultured at 37℃under the atmosphere of 5% CO2. In the tested group the final concentrations of 0.5μmol/L, 1.0μmol/L,1.4μmol/L,1.6μmol/L,1.8μmol/L,2. 0μmol/L,2.2μmol/L, 2.4μmol/L,2.6μmol/L,3.0μmol/L,4.0μmol/L Celastrol were given respectively while another group with no celastrol was used as control. All the A549 cells with or without treatment of Celastrol were cultured for 24h under the same conditions as above and then the cells were collected. Aboved A549 cells tested with celastrol was cultured for Oh,36h and 48 h respectively, and then the cells was collected. All the collected cells were washed with PBS twice and then resuspened.The concentration of the cell suspensions was regulated to 1×106ml. MTT assay was performed to determine cell inhibition ratio.2. Cell apoptosisUse the ordinary light microscope and the Hochest 33258 Staining to observe the morphological changes of A549 cells which are dealed with Celastrol. Cell apoptosis are observed by AnnexinV/PI flow cytometry.Take the same concentration of different time celastrol (Oh,36h,48h) of the A549 cell suspension 100ul,then add AnnexinV, PI 5ul respectively, the processed cells are dealed with by flow cytometry.3. RT-PCR, Western Blot gene and protein detectionReal-time quantitative RT-PCR, and Western blot method to detect the changes of those A549 cells which are dealed with by celastrol with 2.2umol/L after Oh,36h,48h, including PI3K, Akt, Bcl-2, Bax gene and protein changes. Specifically:RT-PCR:first through the NCBI database into the GenBank database.The person BCL-2, BAX, PI3KCD, Akt2 gene sequences are provided by this database. Then use the application software Primer Premier 5.0 to design PCR primers. When it refers to RT-PCR process, the total A549 cell RNA treated by Celastrol from different groups are extracted using Trizol Reagent firstly;then use the kit to complete reverse transcription of RNA to cDNA process; Finally, the SYBR Green mix reagent boxes, the upstream and downstream primers, cDNA, ddH20 added to the react ion sys tem on the machine. Use the fully automatic fluorescence quantitative PCR instrument (SLAN) to analyze the data, the amplification curve and the melting curve are drawed at the same time. Counting out the CT value to get the amplifying multiple.Western Blot:firstly use the PMSF lysis buffer containing with cocktail to get the total protein extraction of A549 cells which are processed by Celastrol in different groups; then use SDS-PAGE gel electrophoresis to divide the proteins into different bands according to molecular size; Since then, the purpose of the required protein bands are transferred to PVDF membrane; using immunity method to get chemiluminescence, then get them developed in the dark room.4. Caspase-3 activity detectionBy Caspase-3 activity detection kit. The kit provides a pNA standard dilution with diluted 0,10,20,50,100 and 200 umol/L which were measured after a standard pNA standard curve. The different groups of A549 cells processed by Celastrol were measured after adding Caspase-3 enzymatic activity.The main results of this study were as follows1. Celastrol could induce NSCLC A549 cells apoptosis.A549 cells's growth was inhibited significantly when treated by celastrol, and the proliferation rate decreased, too. A549 cells gradually becomes oval by the spindle cells, smaller size Reduction of cytoplasmic shrinkage, cell transparency decreased, and can see a lot of bubble-like cell changes in the phenomenon, some cells from the adherent state, as suspension cells, and normal spindle cells are significantly reduced. The A549 cells apoptosis is in a dose-and time-dependent manner, and the IC50 value of 2.2umol/L.2. Hochest33258 found celastrol can lead to apoptosis in A549 cells,48h is more obvious than 36h; Annexin V/PI flow cytometry experiments were found that using 2.2umol/L celastrol to deal with A549 cells after 36h and 48h, apoptosis can be observed clearly in both groups, with significant difference between the control group.3. RT-PCR and Western Blot results show that the A549 cells, after be treated by celastrol 36h,48h later, the Bcl-2 gene and corresponding protein levels decreased, while Bax mRNA and protein expression related to the level did not change significantly, but the resulting Bax/Bcl-2 ratio has been increased. The expression of PI3KCD and Akt2 gene, with the corresponding PI3K and p-Akt protein are higher than the control group. The changes are also in a time-dependent manner.4. The activity of Caspase-3 in A549 cells was significantly higher (P<0.05) when induced by Celastrol.Its intensity was enhanced with prolonged medication, and it present a time-dependent manner.The main conclusion of this study was as follows1. Celastrol can inhibit A549 cell growth, and with the drug concentration increased, Celastrol on A549 cell growth inhibition became more clearly. When A549 cells were stimulated by different concentrations of Celastrol after 36h and 48h, the IC50 values were close to 2.2umol/L. 2. Celastrol can induced apoptosis in A549 cells, and with the time extension, it was gradually dominated by late apoptosis.3. Celastrol induced A549 cells' apoptosis are related to the Bcl-2 family. The apoptosis mechanism was possibly through changing the ratio of Bax/Bcl-2, then ultimately affect mitochondrial function.4. Celastrol cann't stop PI3K/Akt signal transduction pathways, nevertheless, the PI3K and Akt2 genes, the PI3K and p-Akt protein expression increased, suggesting that with the treating of celastrol, PI3K/Akt signal transduction pathway may not directly induce A549 cells' apoptosis. |
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