| Although most prostate cancer began with androgen dependent phenotype, and they were sensitive to endocrinal therapy at first, they transit to androgen independent phenotype after 12-18 months of continuous anti-androgen treatment, which is insensitive to endocrinal therapy of any kind. Then the disease progresses, metastases accur, and patients die soon. Aim:In our research, we treated LNCaP cell with artificial androgen R1881 and androgen receptor antagonist flutamide respecitively to study their exact effect on prostate cancer cells and their correlated molecular mechanism. After continuous in vitro culturing, we succeeded in establishing a flutamide insensitive subline of LNCaP, LNCaP-flu. Then we studied the molecular changes of LNCaP-flu with cDNA microarray and reverse transcriptase-polymerase chain reaction. Thus clarified the exact effect of androgen and flutamide on prostate cancer cells, the molecular changes of flutamide insensitivity, and established flutamide insensitive prostate cancer cell line and animal model. Method: 1. Effect of artificial androgen R1881 on LNCaP cells and its correslatedmolecular mechanism:a) Studied the effect of R1881 on LNCaP with MTT assay, and drewgrowth curve and inhibition rate curve.b) Studied cell cycle changes induced by R1881 of different concentrations with flowcytometer.c) Observe the morphological changes after R1881 treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of R1881's dual effect on LNCaP cells by gene chips, and confirmed the results by RT-PCR.2. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Studied the effect of flutamide on LNCaP with MTT assay, and drew growth curve and inhibition rate curve.b) Studied cell cycle changes induced by flutamide of different concentrations with flowcytometer.c) Observe the morphological changes after flutamide treatment by phase contrast microscope and electronic microscope.d) Studied the molecular mechanism of flutamide's inhibitive effect on LNCaP cell by gene chips, and confirmed the results by RT-PCR.3. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-fiu, and study of its correlated molecular mechanism.a) Established a flutamide insensitive subline of LNCaP,LNCaP-flu, after continuous in vitro culture of LNCaP cells in presence of flutamide.b) Evaluated the expression of prostate specific antigen in the supernatant of cells with radio-immunity to clarify flutamide's effect on LNCaP cell PSA secretion.c) Studied the molecular mechanism of flutamide resistance of LNCaP-flu cell with gene chips, and confirmed the results by RT-PCR.d) Tried to find AR mutation by cloning and sequencing AR cDNA.4. Established LNCaP cell implant carcinoma in SCID mice with matrigel. And measured tumor volume and PSA expression.Results:5. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) R1881 has a dual effect on LNCaP cell. 10-9M and above could inhibit its proliferation, while 10-10M and below could stimulate it. There is not noticeable morphological changes in this process.b) Microscope morphology: Flutamide exerted a prolific effect on LNCaP cells primarily. Then more than 15 days later, after around 5 generations, the effect reversed. The LNCaP cells were inhibited by flutamide. The cells turned thinner and lost satiety. R1881 doesn't have obvious effect on LNCaP cells morphologically.c) Molecular mechanisms of R1881's dual effect: cDNA microarray revealed that after LNCaP had been inhibited by 10-9M R1881, there were 320 genes expressed differently. 170 were up-regulated, 150 were down-regulated. Among them, there are 2 genes may be classified as androgen receptor coactivator, viz. C4orfl and DDC. After LNCaP had been stimulated by 10-10M R1881, there were 4608 genes expressed differently. 2046 were up-regulated, 2562 were down-regulated. Among them, 8 genes may be classified as androgen receptor coactivator, viz. FHL2, NCOR1, SVIL, GRIP1, PIAS3, ACTN2, TBLR1, NCOA2. RT-PCR further proved all these results.6. Effect of artificial anti-androgen flutamide on LNCaP cells and its correslated molecular mechanism:a) Effect of flutamide on LNCaP cells: Flutamide may stimulate LNCaP cell proliferation at different concentrations. However, its effect could still not match with that of R1881's. After 5 generations of continuous in vitro culturing, its effect reversed. The inhibitive effect turns more obvious with the exposure time prolonging and the drug concentration rising. And a series of morphologic changes accompany all these.b) Molecular mechanisms of cell flutamide inhibition:cDNA microarrayrevealed that after LNCaP had been inhibited by flutamide, there were 326 genes expressed differently. 97 were up-regulated, 219 were down-regulated. Among them, 8 genes may be correlated with cell cycle, viz. CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2. And RT-PCR confirmed the up-regulation of all these 8 genes.7. Establishment of flutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a flutamide insensitive subline of LNCaP,LNCaP-flu, which could grow steadily in presence of 10-7M flutamide.LNCaP-flu. And morphologic observation, cell growth test, PSA secretion, cell cycle assay and transwell in vitro invasive ability test proved that all cellular characters of LNCaP-flu are not susceptible to flutamide.b) Molecular mechanisms of flutamide insensitivity: cDNA microarray revealed that after LNCaP had been insensitive to flutamide, there were 2428 genes expressed differently. 1234 were up-regulated, 1194 were down-regulated. Among them, 5 genes may be correlated with androgen receptor function, viz. NCOR1,NCOA2,NCOA4 ,NC0A6 and DDC. And RT-PCR confirmed the up- and down-regulation of all these 5 genes.c) AR changes in flutamide resistance transition: In the process of flutamide resistance transition ,there is no AR expression changes. After cloning and sequencing , we found a new mutation of AR, ie. A3747G.8. Establishment of LNCaP cell Implant carcinoma in SCID mice: In the SCID mice, 75% (15/20) and 60% (12/20) of LNCaP and LNCaP-flu implants respecitively grew up to solid tumor at last. Immunohistochermstry revealed that there were androgen receptor expression in all the implant tumors, while there were not any PSA expression. There were no significant difference between LNCaP,LNCaP-flu and LNCaP-flu/flu group cells in tumor weight and volume (P>0.05) . However, tumors from LNCaP/flu cells were much smaller (P<0.05) . Conclusion:1. Effect of artificial androgen R1881 on LNCaP cells and its correslated molecular mechanism:a) The artificial androgen R1881 have a dual effect on LNCaP cell. 10-9M and above could inhibit LNCaP cell proliferation, while 10-10M and below could stimulate it.b) The down-regulation of androgen receptor coregulator DDC and C4orfl may play a role in the inhibition induced by 10-9M R1881; while the down-regulation of ,FHL2,NCOR1,SVIL, GRIP1,PIAS3 and the up-regulation of ACTN2, TBLR1, NCOA2 may play a role in the prolific effect induced by 10-10M R1881.2. Effect of artificial anti-androgen fiutamide on LNCaP cells and its correslated molecular mechanism:a) Fiutamide has dual effect on LNCaP cell. It could stimulate it at relative low concentration, or relative short time. But when the time prolonged or concentration rose, the effect was reversed.b) The inhibitive effect of fiutamide on LNCaP cells may be the up-regulation of CDC10, NRAS, BTG1, Weel hu, CLK3, DKPZP564A122, CDKN1A, BTG2, which may affect downstream cell cycle related genes, and thus block the cell cycle at the G1/S checkpoint.3. Establishment of fiutamide insensitive LNCaP cell subline,LNCaP-flu, and study of its correlated molecular mechanism.a) Succeeded in establishing a fiutamide insensitive prostate cancer, LNCaP-flu.b) Androgen receptor related genes, such as NCOR1, NCOA2, NCOA4 , NCOA6 and DDC may be important in the transition of fiutamide sensitivity.
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