Research Of Gene Methylation During The Androgen-independent Transition Process Of The LNCaP Cell Line

BackgroundProstate cancer is the most common male malignancy in the United States and in other European countries. It is also the second leading cause of male mortality, following lung cancer. Prostate cancer accounts for28%of all malignancies in developed countries. Although the morbidity and mortality of prostate cancer in China are much lower than those in Europe, the United States, and other developed countries, the incidence of prostate cancer in our country has shown a significant trend of growth with the aging of the population and the increasing popularity of PSA screening. To date, the incidence of prostate cancer in China ranks third among male urinary and reproductive system malignancies. Prostate cancer has become a primary threat to the health of the country’s aging men. In China, majority of the patients often miss out on surgical treatment because their first diagnosis is advanced prostate cancer. Androgen deprivation therapy (surgery or medicine), combined with androgen blockage therapy and radiation therapy, is widely used for the treatment of prostate cancer. In the Chinese prostate cancer clinical guidelines (2011), several therapies for advanced prostate cancer have been established, such as anti-androgen monotherapy (AAM), maximal androgen blockade therapy (MAB) and intermittent hormonal therapy (IHT). The conditions of most patients who undergo the therapies are alleviated during median remission of14to30months. Most of their statuses transform from androgen-dependent prostate cancer (ADPC) to androgen-independent prostate cancer (AIPC), or even to hormone-refractory prostate cancer (HRPC). The clinical manifestations indicate that when the PSA level in the serum progressively increases, the prostate cancer cells begin to re-proliferate. No effective treatment exists for ADPC/HRPC, which is prone to bone metastases and high mortality. Prostate cancer has become a major disease that affects the health of elderly men. Related investigations for AIPC mechanisms and treatment have become research hotspots in recent years.In addition to mutations and deletions, studies have confirmed that abnormal hypermethylation of the gene promoter of the CpG island is the third inactivation mechanism of tumor suppressor genes. The abnormal hypermethylation of tumor suppressor genes could directly or indirectly affect gene transcription through expression suppression. Abnormal hypomethylation of proto-oncogenes activates the expression of the latter, which increases chromosomal instability and leads to abnormal expression of transposons and gene expression, malignant transformation of cells, and ultimately, tumorigenesis. Methylation of DNA refers to the process through which a methyl group from SAM is transferred to the CpG cytosine5□carbon atom with the action of DNMT and forms 5-methylcytosine.DNMT are enzymes that have a key function in DNA methylation. DNMT1is the enzyme that is primarily responsible for methylation in vivo. It can methylate the corresponding portion of progeny DNA under the guidance of a parental DNA methylation template strand. In addition, DNMT1is the main methylation modification in the normal process of DNA replication. DNMT and tumor occurrence, as well as their development, are closely linked.Glutathione S-transferase (GST) is a dimeric isoenzyme that comprises one of the most important phase II metabolic biotransformation enzymes in the body. It is also one of the three enzymatic super families that participate in detoxification. GST is a potent catalyst in reduced glutathione with electrophilic compounds that bind during intracellular detoxification. GSTs can be divided into a,11, π, σ,θ, κ,ζ, and Ω subtypes. Among these subtypes, GST-π is the most closely related to tumorigenesis. GSTP1is the encoding gene of GST-π. It is located at the position of chromosome Hql3, which contains seven exons and six introns. Numerous studies have shown that in prostate cancer cells, the methylation of the promoter region of GSTP1gene could result in low expression or deletion expression of GSTP1. The inactivation of GSTP1gene function has an important participation in the development of prostate cancer. 5-Aza-deoxycytidine (5-Aza-CdR) is deoxyribonucleotide analog azacitidine, a type of DNMT inhibitor that falls under the category of nucleosides. In DNA replication,5-Aza-CdR can be incorporated into DNA and recognized by the DNA methyl transferase enzyme through covalent combination with cysteine residues on the mercapto group of DNMT, leading to inactivation of the latter. Throughout the process, the DNMT activity in the cell and the high methylation rate of the gene promoter region could be reduced, and part of the inactivation of gene expression could be restored.Research Objectives:Based on the above premise, we chose DNMT and GSTP1as the breakthrough points of our study. First, we removed the androgen and added the exogenous anti-androgen into the culture medium of androgen-dependent prostate cancer cell line LNCaP. Through long-term subculture, we induced and established the androgen-independent prostate cell line LNCaP-F60. We used this scheme to simulate the process that androgen-dependent prostate cancer undergoes androgen deprivation therapy to androgen-independent prostate cancer. Second, we conducted biological identification of the androgen-independent prostate cancer cell line LNCaP-F60, followed by detection of DNMT and GSTP1expressions and gene methylation. Our goal was to identify and prove the role and significance of DNMT and GSTP1during the transition from androgen-dependent prostate cancer to androgen-independent prostate cancer. Third, we applied DNMT inhibitor5-Aza-CdR on the androgen-independent prostate cancer cell line LNCaP-F60, and then we again performed biological identification, detection of DNMT and GSTP1expressions, and gene methylation of the prostate cancer cell line LNCaP-F60+5-Aza-CdR. We aimed to investigate the detailed effect and mechanism of DNMT inhibitor on the androgen-independent prostate cancer cell line LNCaP-F60to possibly provide a new method or idea for clinical treatment of hormone-independent prostate cancer.Research contents:1. Androgen-dependent LNCaP prostate cancer cells were cultured in the medium without hormone. Appropriate anti-androgen was added. Through long-term subculture, the androgen-dependent prostate cancer cell line LNCaP was induced into androgen-independent prostate cancer cell line LNCaP-F60.2. Biological identification and detection of cell invasiveness were conducted in the prostate cancer cell lines LNCaP and LNCaP-F60.3. DNMT activity, DNMT1expression, methylation rate of GSTP1, and expression of GSTP1were detected in normal prostate epithelial cells, LNCaP cells, and LNCaP-F60cells separately.4. We determined the optimal concentration and action time of5-Aza-CdR, as well as its effect on the androgen-independent prostate cancer cell line LNCaP-F60, through detection of apoptosis and cell proliferation activity. Again, we examined the androgen-independent prostate cancer cell LNCaP-F60with optimal concentration and action time of5-Aza-CdR through biological identification, detection of cell invasiveness, DNMT activity, DNMT1expression, methylation rate of GSTP1, and expression of GSTP1.Results:1. We successfully induced and established the androgen-independent prostate cancer cell line LNCaP-F60through long-term subculture. We used activated charcoal/dextran-treated fetal bovine serum and phenol red-free RPMI1640culture medium. We added a low concentration of flutamide to simulate the cellular environment of prostate cancer patients who underwent androgen deprivation therapy. With the continuation of subculture, the flutamide concentration gradually increased. After subculturing for60generations, the cells gradually adapted to the external cellular environment. At that time, the prostate cancer cell subline LNCaP-F60was established.2. We cultured two cell lines (LNCaP, LNCaP-F60) under the same medium and duration, and found that the LNCaP-F60cells showed stronger growth ability than the LNCaP cells. Flutamide or DHT could not significantly inhibit or stimulate LNCaP-F60cell growth. Compared with LNCaP cells, LNCaP-F60cells exhibited a more invasive trend. 3. In the three groups (RWPE-1, LNCaP, and LNCaP-F60cells), the level of DNMT activity, DNMT1expression, and methylation rate of GSTP1increased, but the GSTP1expression level decreased.4. After the preliminary experiments, we found that the optimal concentration of5-Aza-CdR was10μM and the optimal action time of5-Aza-CdR was2days. The androgen-independent prostate cancer cell LNCaP-F60with optimal concentration and action time of5-Aza-CdR was examined for androgen-dependent partial restoration and cell invasiveness reduction. Meanwhile, we found that DNMT activity, DNMT1expression, and GSTP1methylation rate decreased, whereas GSTP1expression recovered to some degree.Conclusion:1. In castrate hormone culture environment and under flutamide blocking condition, LNCaP cells gradually changed in cell morphology and biological behavior after long-term subculture. During the subculture, LNCaP cell growth for androgen dependence decreased. Therefore, we established a cell model of the androgen-independent prostate cancer cell line LNCaP-F60to simulate the prostate cancer transition process from androgen dependent to androgen independent. The cell line LNCaP-F60was apparently different from the parent cell line LNCaP in terms of androgen response and cell growth characteristics, providing us a good cytology model for research on androgen-independent prostate cancer pathogenesis and mechanism.2. During the development of prostate cancer, DNMT activity, DNMT1expression, and methylation of GSTP1had enhancing or positive regulation functions. With increase in their expression level, prostate cancer progressed. The expression of GSTP1showed the opposite effect. With the progression of prostate cancer, the expression level of GSTP1decreased.3.5-Aza-CdR could partially restore androgen response and inhibit cell invasion of the androgen-independent prostate cancer cell LNCaP-F60. Its mechanism may involve the inhibition of DNMT activity.5-Aza-CdR inhibited the expression of DNMT1to reduce the methylation rate of GSTP1. When GSTP1gene function was reactivated, the expression of GSTP1was restored.