Encouraged by these favorable results in in vitro studies, we began to test the lytic peptide-βCG conjugates in vivo, using human breast, prostate and ovarian cell xenografts in nude mice. This test system is widely used in cancer studies because nude mice have impaired immune systems, and do not reject the xenografts. In a typical experiment, athymic Hsd-nu female nude mice were subcutaneously injected with 1x106 MDA-MB-435S human breast cancer cells suspended in PBS and Matrigel. Lytic peptide-βCG compounds were injected through the tail veins, beginning 14 days after tumor inoculation. Treatments were administered once a week for three weeks or for six weeks. Complete necropsies were performed 7 days after the last treatment, at which time body weights, tumor weights, and organ weights were recorded. Tumors and other tissues were fixed for subsequent histological studies. All three lytic peptide conjugates (Hecate-βCG, Phor14-βCG and Phor21-βCG(ala)) and the LHRH-Hecate proved to be remarkably effective in reducing tumor volumes and tumor weights (Fig. 5).
These results of experiments with human breast cancer xenografts are best illustrated by a single experiment in which we compared the effects of lytic peptides alone, lytic peptides conjugated with βCG or LHRH, and lytic peptides conjugated with non-specific peptides. The effects of the following treatments on primary tumor weights in nude mice bearing MDA-MB-435S xenografts were studied: (1) Saline; (2) Phor21 alone (8 mg/kg); (3) Hecate alone (8 mg/kg); (4) Phor21-MSH (0.08 mg/kg); (5) Phor21-TRDL (0.08 mg/kg); (6) Phor21-βCG(ala) (0.08 mg/kg); (7) Phor21-βCG(ala) (0.008 mg/kg); (8) LHRH-Hecate (8 mg/kg); and (9) Hecate-βCG (8 mg/kg) (Fig. 5). Phor21-MSH is a conjugate of Phor21 and a truncated a melanocyte stimulating hormone (MEHFRWGKP). Phor21-TRDL is a conjugate of Phor21 and a synthetic peptide corresponding to amino acid residues 34 to 37 of the β subunit of follicle stimulating hormone (FSH). TRDL binds to FSH receptors and has been reported to cause prolonged estrus and hasten puberty in mice. In this experiment which was designed to determine if the treatments caused an actual regression of the tumors, as compared to merely inhibiting growth, an additional group of tumor-bearing animals was killed at the beginning of the treatment period to provide baseline data. Data for each group are plotted as changes from the baseline; bars above the baseline represent tumor growth, while bars below the baseline represent tumor regression (Fig. 6).
As may be seen in Fig. 6, Phor21 alone, Hecate alone, and Phor21-MSH all failed to inhibit tumor growth, compared to saline treated controls (P > 0.05). Phor21 conjugated with βCG(ala) at doses of 0.08 and 0.008 mg/kg, Hecate-βCG and LHRH-Hecate all caused significant (P < 0.05) tumor regression. Phor21-TRDL reduced tumor growth compared with saline treated control animals (P < 0.05), but did not cause tumor regression. In contrast, to all other treatments, Phor21-TRDL caused severe damage to the liver, kidneys and adrenal cortices.
These data clearly illustrate that the LH/CG and LHRH conjugates of Hecate and Phor21 inhibit growth and cause regression of primary breast cancer tumors. Neither the lytic peptides alone, nor a lytic peptide (Phor21) conjugated to a non-CG-targeting peptide (truncated MSH) was effective in reducing tumor growth. The increased potency of Phor21-βCG(ala) (0.008 mg/kg body weight) compared to Hecate-βCG and LHRH-Hecate (8 mg/kg body weight) is remarkable.
The tumor tissue of animals treated with Hecate-βCG or Phor21-βCG(ala) consisted mainly of necrotic cells and fluid (Fig. 7). In contrast, the tumors of untreated animals consisted of sheets of cells with large hyperchromatic nuclei and prominent nucleoli; many mitotic figures were seen. The tumors of the treated mice were pale but contained intact, patent vessels within the neoplastic tissue. Metastatic tumor cell aggregates were seen in the lymph nodes of the saline treated control animals but not in treated animals. All other organs of treated animals appeared normal, except for the ovaries, which contained many atretic follicles and were devoid of recently formed corpora lutea, indicating a failure of ovulation.
All three lytic peptide-βCG conjugates (Hecate-βCG, Phor14-βCG and Phor21-βCG) were also effective in targeting and destroying human prostate (PC-3) tumor cells in nude mice. Figure 8 illustrates the remarkable ability of the LHRH-Hecate conjugate to reduce the prostate tumor (PC-3) volume and weight as compared to saline treated control animals and animals treated with unconjugated LHRH and Hecate.
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