As mentioned previously, metastatic cells are the major cause of cancer deaths; they may lie dormant for many years before becoming active. In a series of experiments, we tested the hypothesis that the lytic peptide-βCG conjugates are also able to seek out and destroy metastases and dormant cells derived from primary breast and prostate cancer tumors. Estrogen independent, MDA-MB-435S cells were stably transfected by lipofection with the plasmid PRC/CMV-luc containing the Photinus pyralis luciferase gene and an antibody resistance gene. The stably transfected MDA-MB-435S.luc cells were selected for by 400 m g/ml G418 and the clones with the highest expression of the luciferase gene were characterized for their LH/CG binding capacities. Transfection with the luc gene did not affect the ability of the cells to bind LH/CG, nor the ability of Hecate-βCG to kill MDA-MB-435S cells.
Nude mice were injected with MDA-MB-435S.luc cells in a Matrigel suspension, as described above. Luciferase assays were used to quantitate the number of metastatic cells in homogenates of lymph nodes, bones, livers, lungs, ovaries and spleens of treated animals. On day 36 after tumor inoculation the mice were allotted to 3 treatment groups as follows: (1) Saline treated controls; (2) Phor14-βCG (12 mg/kg); and (3) Hecate-βCG (8 mg/kg). Primary tumors were removed surgically and all animals were injected once a week for 3 weeks beginning on day 43. All animals were necropsied on day 63. At necropsy, tumor, body and organ weights were determined and the tumors, peripheral organs, bones and lymph nodes were collected, homogenized and luciferase activity in the homogenates was measured. Luciferase positive cells in each organ represented metastatic cells. The results of this experiment are shown in Figs. 9 and 10. Both treatments (Hecate-βCG and Phor14-βCG given once a week for 3 weeks) caused highly significant decreases in metastatic cells in bones (Fig. 9) and lymph glands (Fig. 10). Similar reductions of metastatic cells were found in livers, lungs, ovaries, uteri and spleens of both Phor14-βCG and Hecate-βCG treated mice. In subsequent experiments, Phor21-βCG(ala) proved to be effective in producing highly significant reductions in metastatic cells in all of these same organs at doses as low as 0.2 mg/kg. Since these experiments were conducted with mice from which the primary tumors were removed prior to treatment, it is evident that the lytic peptide-βCG conjugates have the ability to seek out and destroy metastases already established in peripheral organs and tissues; the destruction of metastases and disseminated cells is independent of the presence of the primary tumor.
Similar results have been obtained in experiments in which the ability of the lytic peptide-βCG conjugates to destroy metastases of prostate cancer cells was studied. In these experiments, PC-3 luc cells were injected with Matrigel into the interscapular area of nude male mice and, beginning 35 days after tumor induction the mice were treated with single (1x/wk) or multiple (3x/wk) injections of 0.02, 0.2, 2.5 or 10 mg/kg of Phor14-βCG(ala) for 3 weeks. The results (Fig. 11) showed that the numbers of metastatic cells in lymph nodes were markedly reduced by all dose levels of Phor14--βCG(ala); multiple injections were more effective than single injections at all dose levels (P < 0.002).
In order to assess the extent to which these results with human prostate cancer cell metastases may have been influenced by the presence of the primary tumor; a second experiment was conducted in which the primary tumors were surgically removed before the beginning of treatment. As was the case for breast cancer cells in the absence of tumors, all dose levels of Phor14-βCG(ala) caused marked reductions in lymph node and other metastases. Multiple injections were more effective than single injections (P < 0.05).
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