| background:In the America, prostate cancer has become the highest incident malignant tumor of adult male, and is the second reason in resulted in the death of male patients with cancer malignant tumor in most western countries. There are more than 29000 men die of prostate cancer in USA 2013. n our country, patients with prostate cancer incidence rate showed a rising trend year by year. With the continuous progress of science and technology, screening and diagnostic level corresponding to a rapid increase,85% of the early prostate cancer patients can be found early. The patient is lucky for the desease can be completely cured. In this period. the patient can be treated with radiotherapy or radical operation therapy, in theory the desease can be completely cured. On the contrary, many patients have not been founded early, These advanced prostate cancer patients lose the chance of having an operation or radiotherapy treatment and then turn to endocrine therapy. The main methods of endocrine therapy at present is the application of androgen receptor antagonist drugs, such as bicalutamide, flutamide, At first, prostate cancer is more sensitive to endocrine therapy. After a period, this part of hormone dependent prostate cancer patients most be transformed into hormone refractory prostate cancer (CRPC), once become the latter, patient prognosis become transformation, median survival time, average only 16 to 18 months. The study of CRPC patients have never stopped, but the median survival time of such patients doesn’t get a lot of improvement.At present the formation mechanism of CRPC is still unclear, is a hot spots of prostate cancer research. More and more attention has been paid on mitochondria in this transformation. Mitochondria, contained their own genome, is the most basic of organelles, and also relatively extremely complex life need to survive. Most of the basic biochemical reactions such as:synthesis of energy production,apoptosis, amino acids and lipids occurs in mitochondria. For that, scientists have reason to suspect that the mitochondrial dysfunction or disorders can cause tumor loes the sensitive to anti-androgen drug treatment, and increase the degree of malignancy and invasion of tumor. Having been proved mitochondrial DNA (mtDNA) mutations in prostate cancer, such as:point mutation, segment deletion and DNA depletion can accelerate tumor development and metastasis. In recent years, some studys found that mtDNA mutations can cause hormone dependent prostate cancer transformation to become androgen independent prostate cancer in vitro. A research found that reduce the copies of 10-100 mtDNA can cause intracellular oxygen consumption decreased, the internal environment of the whole cell by hypoxia state to high oxygen condition, and further activate the mevalonate pathway and proto oncogene such as:Ras, ERK, AKT, NF-Kb and JNK pathways. The mutation of mitochondrial DNA affect proliferation of cells and can cause latter cell show a malignant phenotype.PurposeThis pilot study, using effective concentrations of androgen-receptor antagonist R-bicalutamide for long-term in vitro cultivating of androgen sensitive prostate cancer cell line R3327-H, successfully culture of R3327-H resistant R-subline of bicalutamide (R3327-Rbic) by the ultimate. The detection of R3327-Rbic cell morphology keeps invasive. The R3327-H and R3327-Rbic cells were cultured using gene chip technology, studied the effect of R-bicalutamide on two kinds of cells. AR, mitochondrial DNA gene mutation of the androgen receptor, two during cell proliferation changes and the regulation of mitochondrial protein expression of Drp-1 were detected using realtime RT-PCR technology, and western-blot technology. The results obtained verify gene sequencing, clarifies the R3327-H prostate cancer cell stransformed from the hormone sensitive to hormone refractory for the mutation of mtDNA, AR, has been clear about the expression of oxidative respiratory chain related protein in this process. The experiment established the animal model of R-bicalutamide refractory prostate cancer cell line R3327-Rbic, further mechanism of androgen sensitive cell line R3327-H to produce drug tolerance under the effect of bicalutamide is studyed, so as to the future for the research of CRPC has laid a theoretical foundation, provides the cell lines and animal experimental basis for further treatment of CRPC patients, to provide convenient conditions for the treatment of CRPC.Methodpart 1:the role of R-bicalutamide on cancer cell lines R3327-H and R3327-Rbic and R3327-Rbic cell’s feather:1) Changes of R3327-Rbic cell morphology using electron microscope to observe the effect of R-bicalutamide on R3327-H formed.2) Concentration of R-bicalutamide is increased to 20μmol/l,then used the thiazolyl blue method to study the inhibitory effect on 2 cell lines, and observe the structure changes with the concentration change,render two cells generated according to the inhibition rate curve3) Using flow cell cycle to research cell instrument type of two cell lines (R3327-H and R3327-Rbic) under the action of of bicalutamide4) Detecte invasive ability using Transwell R-experiments on R3327-H and R3327-Rbic of two tumor cell line after acted by bicalutamide.part 2:Study on the mechanism of R3327-Rbic cells to R-bicalutamide tolerance1) Application of real time fluorescence quantitative PCR technique(q-PCR technique) to detecte changes of mtDNA content of mitochondrial of R3327-Rbic R- cells after the action of bicalutamide in two important functional region of D-Loop and CO2.2) Application of realtime PCR technology to detect AR and mitochondrial respiratory chain related 12s,16S ribosomal RNA, MT-ND2, expression of MT-ND4 and MT-ND6, cytochrome B, ATP6 polypeptide mRNA.3) Sequence of complete gene rats using Illumina Sequencing program, data obtained is joined to the gene pool, gene sequencing was performed on R3327-Rbic cell to find gene mutation, compared with R3327-H cell.4) Western-blot technique for detecting R- bicalutamide effect on expression of AR and Drp-1 protein of two kinds of cell proliferation in the whole process, as the resulting verification gene sequencing results.part 3:Study on establishment of R-bicalutamide tolerance in mouse models of prostate cancer R3327-Rbic1) Application of Martigel and other auxiliary means in Copenhagen rats in vivo to establish animal model t of two kinds of cells of R3327-H and R3327-Rbic cells.2) The animal model of tumor are divided into 4 groups equally as follows: R3327-H, R3327-Rbic, R3327-H\Rbic,R3327-Rbic\Rbic, to study tolerance to bicalutamide and research cell tolerance model in R-bicalutamide under the effect of tumor size.3) Collecting tumors of two kinds of animal model, using TDT mediated dUTP nick end labeling method (TUNEL) to compare the difference of the expression of apoptosis level of two kinds of cells in vivo.Resultpart 1:the role of R-bicalutamide on cancer cell lines R3327-H and R3327-Rbic and R3327-Rbic cell’s feather:1) The cell growth of R3327-H cells were significantly inhibited after 5 days affected by R-bicalutamide. This situation lasted for 24 weeks, then cells began to copy. Cell separated from the new cells line is called R3327-Rbic cells, this cell grows faster compared with R3327-H, the doubling time cut from 50 hours to 25 hours. Two kinds of cells all had a typical cell morphology, the characteristics of nuclear, clearly visible to the nuclear envelope, there are multiple nucleolar chromosome structure. At high magnification, the latter can show a subtle change in mitochondrial morphology, including tubular morphology and mitochondrial cristae.2) Used MTT test to analysis the influence on the two cell linces with the concentration of bicalutamide increased. The survival curve showed that the cell survival rate, R3327 showed a concentration dependent inhibition in R-bicalutamide solution 0.02μmol/l concentration, 0.7μmol/l is chected to be the half inhibition concentration. However, the influence of drug R- bicalutamide influence on R3327-Rbic is weak.Cell growth curve reached only 20 mol/l in the case of the concentration did not decline. This shows that the treatment of R- bicalutamide doses have lost their regulatory role for the proliferation of R3327-Rbic cells, that is to say, the cell line has not sensitive to R-bicalutamide.3) Compared with the control group, measured by flow cytometry in two kinds of cell cycle showed, R3327-Hcells in G1 phase, the ratio reached 61%, while most R3327-Rbic cells in S phase, the proportion can reach 28%,which indicates that R-bicalutamide inhibited R3327-H group cell proliferation, cell cycle restriction in G1 phasc,and the the R3327-Rbic cells were mostly located in the active proliferation phase S, illustrate the inhibitory effect of R3327-Rbic cells on R-of bicalutamide has no reaction.4) The number of cell of penetrating the membrane detected by Transwell assay,this ability can reflect tumor cell invasion ability. Found in 400 times at high magnification by two groups of cells membrane cell detection, normal cell penetrating number is (40.2 ± 6.23), R3327-H group were (25.2 ± 7.55), R3327-Rbic group were(32.2 ± 4.73). Experimental results we collected show that even after R-bicalutamide treatment, but the in vitro invasive ability of cells of R3327-Rbic group were not significantly affected, while cell penetrating ability greatly decreased,, invasiveness ability is significantly weakened in R3327-H group.part 2Study on the mechanism of R3327-Rbic cells to R-bicalutamide tolerance1) By the method of qPCR supercoiling sensitive to observe the content of bicalutamide in mtDNA of the two cells and the effect of expression of mtDNA associated protein. The results show that two kinds of contents of D-Loop and CO2mtDNA cells and the template in mtDNA difference, were maintained at low levels, suggesting that R-bicalutamide affect very little in mtDNA structure. However, the detection of D-Loop and CO2 gene in the preheating in the template copy number can be found after R3327-Rbic cells were lower than that of R3327-H, which indicates the number of R3327-Rbic cells after treated by R-bicalutamide was highly decreased.2) Realtime RT-PCR experiments were used to detect changes on respiratory chain complex of a series of genes. The result is very clear that R3327-Rbic cells of mRNA levels than R3327-H cells decreased. The expression of 12s gene decreased greatly, while the expression of ND6 gene decreased most. AR gene expression in R3327-Rbic cells relative to R3327-H cells is 1.8, while the R3327-H cells increased significantly in expression after R-bicalutamide treated Drp-1, over expression of R3327-Rbic cells, and the expression of Drp-1 of the R3327-Rbic after bicalutamide treated almost no change.3) Using IlluMina gene sequencing to detect of R3327 cell mitochondrial DNA and AR platform, and the genome analysis of mitochondrial DNA R3327-Rbic cells compared with the observation results, gene mutation. Results ATP6 T8993 mutation in mitochondrial DNA, mutation of T877A in AR.4) Through the weston-blot method to detect two kinds of cells in the androgen receptor (AR) level and the expression of Drp-1 protein. The level of the AR is increase the expression in R3327-Rbic group cells than R3327-H group of cells, but Drp-1 level was lower than that in R3327-H group.part 3Study on establishment of R-bicalutamide tolerance in mouse models of prostate cancer R3327-Rbic1) Two kinds of cell lines were planted dorsal in Copenhagen mice in vivo, establish tumor animal model of two kinds cells. R3327-H group were inoculated with tumor formation ratio of 80%(8/10), R3327-Rbic were inoculated with tumor proportion 70% (7/10 only).2) Two cell lines R3327-Rbic and R3327-H animal model were founded natural processes and compared the tumor size of the two cells under the effect of bicalutamide. Besides the R3327-H \ Rbic group the other three groups in tumor size had no significant difference (P>0.05), and the R3327-H\ Rbic group tumor size compared with the former three groups is obviously smaller, the difference was significant (P<0.05).3) Collecting the tumor specimens and use TDT mediated dUTP nick end labeling method (TUNEL) to compare the difference of the expression of two kinds of cells in vivo apoptosis level. The experimental results show that:under fluorescence microscope by different amount of each specimen cells after DAPI staining had no statistical significance. The positive rate of apoptosis in other three groups:R3327-H group, R3327-Rbic group and R3327-Rbic\ Rbic group had no significant difference, the positive rate of apoptosis of R3327-H cells in R3327-Rbic group is significantly higher than the other three groups. Conclusion1) Study on the related mechanism of R-bicalutamide on cancer cell lines R3327-H and R3327-Rbic prostate by MTT test and flow cytometry in vitro findings that R-bicalutamide has lost control of R3327-Rbic proliferation, cell is not sensitive to R-bicalutamide, Transwell proved that the invasive ability of R3327-Rbic was largely unaffected to the effect of R-bicalutamide.2) RT-PCR study showed that the R-bicalutamide for structure of two groups of cell mtDNA is insignificant, but the content of mtDNA of R3327-Rbic cells was decreased in total. A variety of experimental methods can level than group R3327-H cells increased AR expression validation group R3327-Rbic cells, but also the level of Drp-1 is lower than that in R3327-H group, the specific reason is to be further studied. ATP6 T8993 mutation in mitochondrial DNA, mutation of T877A in AR, which proved that mtDNA and AR mutations in genes involved inhormone independent prostate cancer formation process.3) Study on establishment of prostate cancer R3327-Rbic models tolerance to R-bicalutamide in mouse discovery that R3327-Rbic cells can be successfully established tumor animal model, and the model continues to maintain tolerance to R- bicalutamide, not regulated by this drug. |
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