Biphasic effect of androgens on prostate cancer cells and its correlation with androgen receptor coactivator dopa decarboxylase.

The aim of this study was to explore the mechanism underlying the dual effect of androgen on prostate cancer cells and further explore its correlation with dopa decarboxylase (DDC), an androgen receptor (AR) coactivator and a traditional neuroendocrine differentiation (NED) marker. Cell proliferation and cycling after treatment with synthetic nonmetabolizable androgen R1881 was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method and flow cytometry. Differential gene expression was analyzed by cDNA microarrays. DDC expression during the dual effect of R1881 was further explored with microarray, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and enzyme activity assays. Proliferation of LNCaP cells was inhibited by 1 nM R1881 but stimulated by 0.1 nM R1881. Compared with the untreated cells, 320 (2.26%; 170 up-regulated, 150 down-regulated) and 4608 (32.65%; 2046 up-regulated, 2562 down-regulated) genes were found to be expressed differentially in the 1 nM and 0.1 nM R1881-treated cells, respectively. The results were partially confirmed by RT-PCR and Western blot. The DDC gene was down-regulated in the 1 nM R1881-treated cells and up-regulated in 0.1 nM R1881- and 30 nM hydroxyflutamide-treated cells. The enzymatic activity of DDC in the latter 2 groups was also strengthened. Meanwhile, the NED markers CgA and synaptophysin were not affected by these AR activators. R1881 had a dose-dependent biphasic effect on LNCaP cell proliferation. AR coactivator DDC was respectively down- and up-regulated in high and low concentrations of R1881. DDC up-regulation by exogenous AR activators is not accompanied by up-regulation of definitive NED markers.

Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism.

AIM: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. METHODS: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips. RESULTS: After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. CONCLUSION: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

[Antisense oligonucleotide targeting survivin induces apoptosis of renal clear-cell carcinoma cells and enhances their sensitivity to epirubicin in vitro].

OBJECTIVE: To investigate the effect of antisense oligonucleotide (ASODN) targeting survivin on the apoptosis and proliferation of renal cancer cell line 786-O and enhancement of its sensitivity to epirubicin. METHODS: ASODN targeting survivin was designed and constructed. Cultured cells were divided into 6 groups: control group, liposome group, sense oligonucleotide (SODN) group, 600 nmol/L ASODN group, and 600 nmol/L ASODN combined with epirubicin group. After transfected for 24 h, cultured cells were harvested to carry on the next tests. Cell morphological changes were examined by transmission electron microscopy. Survivin protein was detected by immunohistochemical method. Apoptosis index (AI) and proliferation index (PI) were examined by flow cytometry. RESULTS: Morphological abnormalities of cells were observed in ASODN transfected groups. Expression of survivin in ASODN groups were significantly decreased compared with that in the control group, liposomes group and SODN group. AI of ASODN groups was significantly higher than that in other groups. PI of ASODN groups was significantly lower than that in other groups. The PI of ASODN combined with epirubicin group was (35.7 +/- 1.67)%, but (9.3 +/- 0.34)% or (8.5 +/- 0.21)% in liposomes group or SODN group that had combined with epirubicin. The ASODN group achieved the strongest effects to enhance apoptosis in comparison with control group (P < 0.05), while SODN did not cause statistically significant change (P > 0.05). CONCLUSION: The expression of survivin protein in the renal clear cell carcinoma cell line 786-O is downregulated by survivin ASODN. ASODN targeting survivin induces apoptosis and inhibits proliferation of 786-O cells. Inhibition of survivin enhances sensitivity of 786-O to epirubicin.

[The biological characteristics of beta glucuronidase cDNA transfected renal cancer cell line GRC 1/betaG].

AIM: To establish a renal carcinoma cell line which can highly express beta-glucuronidase(betaG), and to observe the biological characteristics of the transfected cells. METHODS: Recombinant eukaryotic expression vector pcDNA3.1-betaG was constructed. It was transfected into renal cancer cells GRC-1 via liposome. The transcription and expression of betaG gene were detected by dot blot and Western blot. The biological characteristics of the betaG gene transfected cells was observed under light microscope, transmission electron microscope and flow cytometry. RESULTS: Dot blot and Western blot detection confirmed that the betaG gene had been stably integrated into the genomic DNA of the GRC-1 cells and was highly expressed. Transmission electron microscope observation showed that the lysosomes and endoplasmic reticulum were abundant, the number of microvili and process was significantly increased in the transfected cells, but growth condition and cell cycle of GRC-1 cells had no notable difference before and after transfection. CONCLUSION: A renal carcinoma cell line that can highly express betaG gene was established, which lays the foundation for further study on gene therapy.

[The relationships of PSA, FPSAR, clinical and pathological stage in patients with prostate cancer].

OBJECTIVES: To investigate the relationship between clinical and pathological stage, serum prostate specific antigen (PSA) concentration and free-to-total PSA ratio (FPSAR) in patients with prostate cancer. METHODS: Clinical and pathological stage were determined on the basis of pathological examination and clinic material in 42 prostate cancer patients treated by prostatectomy. PSA and FPSAR were measured before the operation. Spearman rank correlation was applied to evaluate the relationship between clinical and pathological stage, serum PSA concentration and FPSAR. RESULTS: Serum PSA concentration was significantly positively correlated with pathological stage(P < 0.05) but not correlated with clinical stage (P > 0.05) in prostate cancer patients. FPSAR was significantly correlated with pathological stage and negatively correlated with clinical stage in prostate cancer patients (P < 0.05). CONCLUSIONS: FPSAR is a more powerful predictor of clinical stage, pathological stage and prognosis than PSA.