Cancer Prevention Laboratory

PROOF OF CONCEPT

To test the validity of our hypothesis we compared the toxicity in vitro of the Hecate-βCG conjugate in cells from the Chinese Hamster Ovary (CHO), which do not express the CG (LH) receptor, and CHO cells that were transfected with the rat CG (LH) receptor gene. Cell death was measured by Trypan blue staining or by measuring the release of lactate dehydrogenase into the culture medium. As may be seen in Figure 3, Hecate -βCG caused more cell death at all concentrations in the cells transfected with the CG/LH receptor than in the wild type CHO cells which lack the receptor. In contrast, Hecate-βCG was relatively ineffective in killing the wild type CHO cells, and no dose response was seen. This result laid the foundation for numerous in vitro and in vivo experiments and to development of a new class of drugs that specifically target prostate, breast, ovarian and testicular cancers and their metastases.

In 2001, we reported that Hecate-βCG and Phor14-βCG selectively and in a dose dependent manner each destroyed both androgen dependent and independent prostate cancer cells in vitro. Cytotoxicity was dependent on the number of LH/CG receptor sites expressed by the cells. Similar results were obtained with breast cancer cells (2003). Cytotoxicity was dependent on the number of LH/CG receptor sites present. The correlation coefficient (R²) between toxicity (EC 50 ) of Hecate-βCG and LH receptor binding capacity (F mol/10⁶ cells) in 7 different cells lines, including breast cancer cells MDA-MB-435S and MCF-7, prostate cancer cells BRF41T, DU145, PC-3, LNCAP, and CHO cells transfected with rat LH receptor gene was 0.709.

Removal of steroids from the culture media reduced sensitivity of PC-3 cells to the drug, and addition of FSH, or estradiol, each of which is known to upregulate LH/CG receptors, restored the sensitivity of the cells to the drug. Administration of tamoxifen, a competitive inhibitor of estradiol, prevented estradiol from restoring the sensitivity of the cells to the drug (Fig. 4).

Fig. 3. Chinese hamster ovary (CHO) cells transfected with LH/CG receptors are killed at lower concentrations and in greater numbers by Hecate-βCG than wild type CHO cells, which lack the LH/CG receptors.

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Fig. 4. Pretreatment with Estradiol or FSH restore the sensitivity of PC-3 prostate cancer cells to Phor14-βCG after extraction of steroids from the medium by charcoal. Tamoxifen, a competitive inhibitor of estrogen, blocks the effect of estrogen treatment. Charcoal extracted medium vs complete medium, P < 0.0002; charcoal treated medium vs Estradiol (5 nM), P < 0.001; Tam = Tamoxifen; FSH = follicle stimulating hormone.

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