Androgen suppresses testicular cancer cell growth in vitro and in vivo

Abstract

Silencing of androgen receptor (AR)-meditated androgen signaling is thought to be associated with the development of testicular germ cell tumors (TGCTs). However, the role of the androgen/AR signal in TGCT development has not been investigated. In this study, we show that the androgen/AR signal suppressed the cell growth of seminomas (SEs), a type of TGCT, in vitro and in vivo. Growth of SE cells was suppressed by DHT treatment and reduction of androgen levels by surgical castration promoted cancer cell growth in an in vivo xenograft model. Tryptophan hydroxylase 1 (TPH1), the rate limit enzyme in serotonin synthesis, was one of the genes which expression was reduced in DHT-treated SE cells. TPH1 was highly expressed in SE cancer tissues compared with adjacent normal tissues. Activation of androgen/AR signaling in SE cells reduced the expression of TPH1 in SE cells, followed by the reduction of serotonin secretion in cell culture supernatant. These results suggested that silencing of androgen/AR signaling may cause initiation and progression of SE through increase in TPH1 gene expression level.

Keywords: androgen; androgen receptor; seminoma; testicular cancer; tryptophan hydroxylase 1.

Figure 1. AR expression in TGCT cell lines

A. mRNA expression levels of AR in four types of TGCT cells were examined by real-time quantitative RT-PCR. The expression of AR was normalized to the GAPDH. Data are presented as mean ± s.d. (n=2). B. AR protein levels in TGCT cell lines. Western blots were performed using whole cell lysates extracted from each cell type. The same results were reproduced for each experiment three times.

Figure 2. Suppression of TCam-2 cell proliferation by DHT treatment

A. TCam-2 cell proliferation was observed with or without DHT. 10,000 cells of TCam-2 were plated and cultured with DHT or without DHT (DHT: 10⁻⁷, 10⁻⁹ or 10⁻¹¹ M) for four days. The number of living cells was examined by measurement of absorbance (450 nm) at 1, 2 and 4 days after cell seeding. Data are presented as mean ± s.d. (n=3). B. Colony formation assays using TCam-2 cells. Cells were cultured with or without DHT (DHT: 10⁻⁷ M) for 2 weeks. Colony numbers in each dish (n = 5) were visually counted and presented using a column graph. Each experiment was performed three times.* p < 0.01.

Figure 3. Promotion of SE cell growth in castrated mice

A. and B. Castration or sham operation was performed in 5-week-old male SCID mice. TCam-2 cells were subcutaneously implanted on the right dorsal gluteal region of these mice. After 45 days, the tumors were resected (B). The average volumes of resected tumors in castrated mice (n = 8) and sham-operated mice (n = 8) are shown (A). * p < 0.05.

Figure 4. TPH1 was highly expressed in SE patients and down-regulated by DHT in SE cells

A. Using human SE tumor samples (Table S1), we examined the gene expression profile of these cells by Agilent Microarray technology. 1,006 genes (dark gray circle) exhibited a more than 2-fold increase in mRNA expression in tumor v.s. normal. On the other hand, microarray analysis was also performed using TCam-2 cells, which were treated with or without DHT (10⁻⁷M for 4 hr). 1,500 genes (light gray circle) showed a more than 2-fold decrease in DHT treated TCam-2 cells. B. TCam-2 cells were transfected with siAR for 3 days. 10⁻⁷M DHT or EtOH were added to the cultured medium for 24 hr until cell extraction. RNA expression of TPH1 in the TCam-2 cells was measured using quantitative RT-PCR. n = 2, *, p < 0.05.

Figure 5. Suppression of TCam-2 cell proliferation by TPH1 knockdown

A. Confirmation of TPH1 knockdown efficiency by quantitative RT-PCR. TPH1 siRNA or control siRNA was transfected to TCam-2 cells for 3 days. The mRNA levels of TPH1 were normalized to GAPDH. n = 2, *, p < 0.01. B. Serotonin levels in TCam-2 cell culture supernatants were exarmined by ELISA. For TPH1 knockdown, siRNA was transfected to TCam-2 cells for 3 days. DHT was added to the cultured medium at a concentration of 10⁻⁷ M for 24 hrs. C. TCam-2 cells were transfected with control siRNA or TPH1 siRNA for 3 days. After that, the number of living cells was examined by measurement of absorbance (450 nm). Data are presented as mean ± s.d. (n=3), *, p < 0.01.

Figure 6. Expression and functions of 5-HTs in TCam-2 cells

A. mRNA expression of six type of 5-HTs in TCam-2 were examined by quantitative RT-PCR. 10⁻⁷ M DHT or EtOH were added to the cultured medium for 24 hrs until cell extraction. mRNA levels of 5-HTs were normalized to GAPDH. n = 2, *, p < 0.01. B. mRNA expression of downstream genes of 5-HTs in TCam-2 cells were examined by quantitative RT-PCR. DHT (10–7 M) or Asenapine maleate (10–9 M) was added to the cultured medium for 24hr. C. Confirmation of 5-HT7 knockdown efficiency by quantitative RT-PCR. 5-HT7 siRNA or control siRNA was transfected to TCam-2 cells for 3 days. D. TCam-2 cells were trasnfected with control siRNA or 5-HT7 siRNA for 3 days. After that, the number of living cells was measured by determination of absorbance (450 nm). Data are presented as mean ± s.d. (n=3), *, p < 0.01.