Tamoxifen Citrate

Tamoxifen displays antitumor effect due to its antiestrogenic activity (ER). Values for the apparent affinity of Tamoxifen for the ER range between 30 and 0.01% of that obtained for estradiol, dependent on different ER source (species), protein concentration and condition used for assay. Binding of Tamoxifen to ER further leads to inhibition expression of estrogen-regulated genes, including growth factors and angiogenic factors secreted by the tumor that may stimulate growth by autocrine or paracrine mechanisms. Tamoxifen also directly induces programmed cell death. ^[1] Tamoxifen produces an inhibitory effect on MCF-7 cell [³H]thymidine incorporation and DNA polymerase activity as well as causing a reduction in DNA content of cultures and cell numbers. This inhibitory effect of Tamoxifen on MCF-7 cell growth can be readily reversed by addition of estradiol to the culture medium. 2 and 6 μM Tamoxifen reduces the proportion of cells in S phase and increases the number of cells in G1. At 10 μM, Tamoxifen causes cell death within 48 hr. ^[2] Tamoxifen inhibits MCF-7 growth with IC50 of ~10 nM after 10 days treatment. Tamoxifen inhibits plasminogen activator activity of MCF-7, and suppresses estradiol-stimulation of plasminogen activator activity. Tamoxifen also evokes minimal increases in cellular progesterone receptor levels. ^[3] Tamoxifen is able to inhibit the growth of prostate cancer cell PC3, PC3-M, and DU145 with IC50 ranged from 5.5-10 μM, which is related to its inhibition of protein kinase C and induction of p21(waf1/cip1). ^[4]

Tamoxifen administration to rapidly growing, estradiol-stimulated MCF-7 xenografts results in a dose-dependent retardation or cessation of tumor growth by significantly decreasing tumor cell proliferation in tumor. Tamoxifen treatment results in a slowing of tumor growth (tumor doubling time, 12 days), a significant increase in tumor potential doubling time (T_pot) (6.6 days), and a decrease in labeling index (%LI) (to 8%) by 23 days posttreatment, compared with untreated mice which shows a volume doubling time of 5 days, a T_pot of 2.3 days, and a %LI of 23%. ^[5] Tamoxifen has not only antiestrogenic but also estrogenic properties depending on the species, tissue, and gene. Tamoxifen displays favorable effects on bone and serum lipid concentrations and stimulation endometrium. ^[1]

• Competitive binding assays

  Cells are harvested from 150-sq cm T-flasks, and cytosol is prepared at a protein concentration of approximately 2 mg/mL in phosphate buffer. Aliquots of this 180,000 ×g supernatant are then incubated with various concentrations of Tamoxifen and 2.5 nM [³H]estradiol for 16 hr at 0-4 ℃. The free steroids are absorbed by dextran-charcoal [l0 μL of 0.5% Dextran C-5% Norite A in TE buffer] for 1 hr at 0 ℃, and aliquots are counted after centrifugation at 800 ×g, 30 min. The relative binding ability of each competitor is taken as the ratio of the concentration of radioinert estradiol/competitor required to inhibit one-half of the specific [³H]estradiol binding, with the affinity of estradiol set at 100%.

• Cell lines

  Human breast cancer cells MCF-7

• Concentrations

  ~1 μM

• Incubation Time

  10 days

• Method

  MCF-7 cells are seeded into T-25 flasks (1.5×10⁵ cells/flask) and grown for 2 days in the MEM supplemented with 10 mM HEPES buffer, gentamicin (50 μg/mL), penicillin (100 units/mL), streptomycin (0.1 mg/mL), bovine insulin (6 ng/mL), hydrocortisone (3.75 ng/mL), and 5% calf serum that has been treated with dextran-coated charcoal for 45 min at 55 ℃ to remove endogenous hormones. The medium is then changed to MEM supplemented as described above, except that it contains 2% charcoal dextran-treated calf serum and various concentrations of Tamoxifen. At the end of incubation, cell numbers are counted.

• Animal Models

  Human breast carcinoma xenografts MCF-7

• Dosages

  2 cm Tamoxifen capsules

• Administration

  Subcutaneous implantation