Establishment of Models for Studying Prostate Cancer Cell Progression

Because PCA is initially dependent on As for its growth, hormonal therapy (A deprivation), pioneered by Charles Huggins 60 years ago (20) using castration or more recently anti-androgens, has been a standard PCA therapy. Although over 70% of patients may benefit from this therapy, PCA recurs in most of these patients in one to three years as tumors that do not need A for growth (A-independent). For lack of effective therapy, patients die from this A-independent PCA.

We believe that a better understanding of the process of PCA cell progression is very important for establishing better methods for PCA treatment. To investigate PCA progression, we established a model system (Figure 2) using a clone derived from the human PCA cell line, LNCaP, whose growth was dependent on the presence of nanomolar concentrations of testosterone, 5a-DHT or 17 P-hydroxy-17-methyl-estra-4,9,11 -trien-3 -one (R1881) (21-23). The original cancer cell (named LNCaP 104-S) population was cultured through weekly passages and after about 40-70 passages in A-depleted culture medium, these cells progressed to A-independent cancer cells that we named LNCaP 104-R1 cells. These cells can grow well in culture without A. After continuous culture of 104-R1 cells in A-depleted medium for 60-120 additional passages, these 104-R1 cells were transformed into faster-growing cells, named 104-R2 cells. This transition of A-dependent 104-S cells to A-independent 104-R1 and 104-R2 cells is accompanied by dramatically increased AR expression apparently without new mutations in the ligand binding domain of AR gene. Despite an increase in cellular levels of AR, we found that the growth of both 104-R1 and 104-R2 cells in culture are suppressed by physiological concentrations of A. In

Cupcuoiin Alizarin

Figure 1. Structures of natural inhibitors of 5a-reductases.

Cupcuoiin Alizarin

Figure 1. Structures of natural inhibitors of 5a-reductases.

the presence of a high A concentration (>20 nM), some 104-R1, but not 104-R2 cells, can revert back to A-dependent cells (104-R1Ad) that behave like 104-S cells.

Recently, we found an alternative method to generate A-repressed cells from 104-S cells by using the anti-androgen, Casodex (bicalutamide), to suppress the growth of 104-S cells and isolate cells that can grow in the presence of anti-androgen. These cells, called CDXR cells, behave like 104-R2 cells and can not revert back to A-dependent cells. These A-independent cells also contain high levels of AR and their growth is suppressed by low A concentrations (<1 nM). When CDXR cells were cultured further in the presence of A, we were able to isolate A-insensitive cells, named IS cells. IS cells express very low levels of AR and are not stimulated or suppressed by A in the culture medium (Figure 2). These cells resemble human prostate PC-3 cells, since they do not have AR and can grow in the absence or presence of A.

Reversible Irreversible

A-dependent A-independent A-insensitive (A-repressed)

"CDXR-

CDX Androgen

Androgeu f Androgen 1 Androgen (-)

A nil androgen i Anliandrogen f Aiitiaiidrogeni-h

Phytotherapy—Green Tea EGCG

Figure 2. Four stages of PCA progression and treatment.

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