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Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
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ABSTRACT |
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gonadotropin-releasing hormone, gonadotropin-releasing hormone receptor, human breast cancer, human chorionic gonadotropin, human prostate cancer, LH/CG receptors, luteinizing hormone, lytic peptic conjugates, mammary glands, metastases
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INTRODUCTION |
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By 1997, it was evident that luteinizing hormone/chorionic gonadotropin (LH/CG) receptors are expressed in prostate, breast, ovarian, testicular, and endometrial cancer cells [8–12]. This led us to test the hypothesis that these cancer cells could be targeted and destroyed by membrane-disrupting lytic peptides joined to LH/CG ligands. LH/CG receptors in prostate and breast cancer cells can be up-regulated both in vivo and in vitro by treatment with other hormones, most notably through their estrogen and FSH receptors [13, 14].
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MEMBRANE DISRUPTING LYTIC PEPTIDES AND THEIR LH LIGANDS |
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Based on naturally occurring lytic peptides and their similarities in destroying bacterial and tumor cells, a number of synthetic membrane-disrupting peptides have been synthesized and linked through peptide bonds to LH/CG. These include Hecate, a 23-amino acid amphipathic lytic peptide; Phor14, a 14-amino acid peptide; and Phor21, a 21-amino acid peptide joined with a 15-amino acid segment (amino acids 81–95) of the beta subunit of hCG [13]. Phor21 and Phor14 have also been linked to the same 15-amino acid fragment of ßCG in which the cysteines have been replaced by alanines. This 15-amino acid segment of ßCG was shown to possess nearly 70% of the binding activity of the entire molecule [25]; it competes with LH or CG for binding sites on bovine luteal cells and PC-3 prostate cancer cells [13].
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PROOF OF CONCEPT |
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PROSTATE CANCER CELLS |
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Two separate studies [13, 14] determined the abilities of Hecate and Phor14, alone and conjugated with ßCG, to kill androgen-dependent and androgen-independent prostate cancer cells. The toxicities of each compound were related to the expression and binding capacities of LH/CG receptors in each cell line. Viability of the treated cells was measured by trypan blue-exclusion, by release of LDH into the medium, and the methylthiazoletetrazolium assay, a quantitative colorimetric assay for live cells.
The results, shown in Figure 1 for Hecate-ßCG, indicate that the abilities of the lytic peptide-ßCG conjugates to kill prostate cancer cells are closely related to their LH/CG receptor capacities (R2 = 0.709). However, both Hecate and Phor14 alone were capable of killing androgen-sensitive and androgen-insensitive cell lines in vitro at low molar concentrations. When steroids were removed from the culture media by charcoal extraction, the sensitivity of BRF41T, PC-3, and LNCaP cells to both Hecate-ßCG and Phor14-ßCG was lowered by about 50% (P < 0.02). The LH/CG binding capacity was also significantly reduced (P < 0.005) by steroid removal. Addition of estradiol to the charcoal-extracted medium restored the sensitivity of both PC-3 and LNCaP cells to Phor14-ßCG, whereas the addition of tamoxifen, which competes with estradiol for receptors, blocked the effect of added estradiol. Addition of FSH to the culture medium also restored the sensitivity of the PC-3 cells to Phor14-ßCG. In competition experiments, the ßCG fragment competed with the lytic peptide-ßCG conjugate for binding sites on the LH receptor in LNCaP and PC-3 prostate cells [13, 14].
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In Vivo Studies with Hecate-ßCG and Phor14-ßCG
The targeted destruction of xenografts of human prostate cancer cells by lytic peptide-CG conjugates was reported in 2001 [13, 14]. Athymic Balb/c male nude mice implanted s.c. at 6 wk of age with 1 x 106 PC-3 cells suspended in PBS and Matrigel were treated by tail vein injections of Hecate-ßCG or Phor14-ßCG once a week for 3 wk, and killed 7 days after the last treatment. The first injection of Phor14-ßCG, at either 12 or 24 mg/kg, stopped tumor growth. Administration of 12 mg/kg reduced tumor burden from 60.2 mg/g of body weight (BW) to 27.2 mg/g BW (P < 0.0001) and administration of 24 mg/kg reduced tumor burden to 11.8 mg/g BW (P < 0.0001). Pretreatment of tumor-bearing mice with estradiol or FSH before administration of 12 mg/kg Phor14-ßCG resulted in a reduction of tumor burden equal to that produced by 24 mg/kg alone [13].
Histologically, the tumors of mice treated with both Hecate-ßCG and Phor14-ßCG consisted primarily of necrotic cells and fluid. Necrosis was most severe in tumors of mice pretreated with FSH or estradiol. All tumors of treated mice were poorly vascularized, although some intact vessels were present. All of the control animals had large solid tumors, and their tumor burdens were 100 ± 13 mg/g BW. In contrast, 73% of the treated animals had no visible tumor tissue. The remaining animals had only small residual tumors, and the tumor burden was 22 mg/g BW. No histological abnormalities were noted in livers, spleens, hearts, kidneys, adrenals, pancreas, lungs, and pituitaries of the treated mice. However, primary and secondary spermatozoa and spermatids were virtually absent from the testicular tubules and epididymides. The interstitial cells were shrunken and pyknotic.
Up to 80% of human prostate cancers also express GnRH receptors and GnRH-Hecate has also been shown to reduce tumor burden in male nude mice bearing PC-3 xenografts [27].
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BREAST CANCER CELLS |
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A number of human breast cancer cell lines are known to express LH/CG and GnRH receptors [12, 31]. In 2003, we established that Hecate-ßCG has the ability to destroy human breast cancer cells in a dose-dependent manner. In in vitro experiments identical to those described above for prostate cancer cell lines, Hecate and Hecate-ßCG were added to cultures of MCF-7 (estrogen-dependent) and MDA-MB-435S (estrogen-independent) breast cancer cell lines. The LH/CG binding capacity for the MDA-MB-435S cells was higher than for the MCF-7 cells (133.1 ± 8 vs. 63.2 ± 11 fmol/106 cells; P < 0.03) and the EC50 was lower (9.51 ± 1.04 vs. 18.01 ± 1.6; P < 0.02). As was the case for prostate cancer cells, removal of steroids from the culture medium decreased sensitivity of the MCF-7 cells to both conjugates, and this sensitivity was restored by adding estradiol to the medium. To test the hypothesis that toxicity of the Hecate-ßCG conjugate depended on the availability of binding sites for LH/CG, an experiment was conducted in which CG was used to compete with Hecate-ßCG for binding sites on the LH/CG receptors in MDA-MB-435S cells. In the presence of CG, the sensitivity of these cells to Hecate-ßCG was significantly decreased (P < 0.02). These in vitro results were confirmed in a subsequent report by Bodek et al. [29].
In Vivo Experiments
Nude mice at 6 wk of age (22–27 g BW) were injected s.c. with 1 x 106 MDA-MB-435S cells suspended in 0.1 ml of PBS and 0.3 ml of Matrigel [26]. When the tumor volume reached 30–50 mm3 the mice were treated with Hecate-ßCG (10 mg/kg) by tail vein injections once a week for 3 wk, and were killed 7 days after the last treatment. Control mice received saline injections. At the time of death, complete necropsies were performed and tissues were fixed for subsequent histological study.
As was the case with prostate cancer cells, the first injection of Hecate-ßCG stopped growth of the tumors (P < 0.003 compared to saline-injected controls). Tumor burden was decreased from 47 ± 7 mg/g BW in control animals to 7 ± 5 mg/g BW in treated animals (P < 0.001). Body weights, liver weights, and weights of uteri and oviducts were not affected by treatment. Histologically, the tumor tissue of treated animals consisted primarily of necrotic cells, whereas the tumors of untreated mice contained sheets of cells with large vesicular hyperchromatic nuclei, prominent nucleoli, and many mitotic figures. Metastatic tumor cell aggregates were detected in lymph nodes of untreated animals, but not in treated animals. Although ovarian weights were unaffected, cellular degeneration and necrosis of granulosa cells were prominent in follicles, and recently formed corpora lutea were absent, indicating failure of ovulation.
In a recent report [30], Zaleska et al. demonstrated that Hecate-ßCG given once a week for 3 wk inhibits mammary gland tumor growth induced in rats by prenatal exposure to diethylstilbestrol and postnatal exposure to dimethylbenz(a) anthracene. The percentage of tumor volume increase in the animals treated with Hecate-ßCG was 45.3 ± 27.6 mm3 compared to 324.8 ± 78.1 mm3 for the animals treated with Hecate alone, and 309 ± 51.2 for the saline-treated control group (P < 0.001). Tumor burden for Hecate-ßCG-treated animals was 9.5 ± 2.1 mg/g BW, compared to 17.6 ± 1.6 in animals treated with Hecate alone, and 21.6 ± 2.9 mg/ g BW in controls (P < 0.01 vs. controls). Although the reduction of tumor burden due to treatment with Hecate alone was statistically significant (P < 0.05), it was small and due primarily to a slowing of tumor growth shortly after the first injection. Only Hecate-ßCG caused an important decrease of tumor growth after the third injection. Furthermore, mitotic figures were common in tumors of control and Hecate-treated animals, but not in animals treated with Hecate-ßCG (19.2 ± 2.5 mitoses per 1000 nuclei for controls, 16.3 ± 2.1 for rats treated with Hecate, and 8.1 ± 1.2 for rats treated with Hecate-ßCG; P < 0.05 and P < 0.01 for Hecate and controls vs. Hecate-ßCG). Hecate treatment-alone results did not differ from those of controls. In agreement with previous studies, the number of recently formed corpora lutea in the ovaries of animals treated with Hecate-ßCG was reduced compared to saline-treated control rats.
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OVARIAN CANCER CELLS |
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Tumors of ovarian surface epithelium constitute more than 80% of all ovarian carcinomas, and 40% of epithelial ovarian carcinomas express LH/CG receptors [31–33]. Hecate and Hecate-ßCG were added to OVCAR-3 cells in complete and charcoal-treated media. Both Hecate and Hecate-ßCG destroyed OVCAR-3 cells in a dose-dependent manner [28]. Removal of steroids from the culture medium significantly (P < 0.01) reduced the sensitivity of the OVCAR-3 cell line to Hecate-ßCG, but not to Hecate. Addition of estradiol to the charcoal-treated medium restored the sensitivity of the cells to Hecate-ßCG (P < 0.01), and the estrogen effect was blocked by the addition of tamoxifen. However, the addition of FSH had no effect on the toxicity of Hecate-ßCG.
Hecate-ßCG was recently reported to induce rapid cell-specific membrane destruction in LH receptor-expressing granulosa cell tumors in transgenic mice [34].
In Vivo Studies
Tumor volumes 9 wk after inoculation of nude mice with OVCAR-3 cells were highly variable (768 ± 200 to 1231 ± 271 mm3) after treatment once a week for 3 wk [28]. The percentage increase in tumor volume was lower (P < 0.05) in groups treated with Hecate, Hecate-ßCG, and estradiol plus Hecate-ßCG compared to saline-injected controls. Histological examination revealed multifocal areas of necrotic cells in the tumors of Hecate-ßCG-treated mice, but the massive necrosis previously observed in the prostate and breast cancer tumors from animals treated with Hecate-ßCG and Phor14-ßCG was not evident. All animals treated with Hecate-ßCG showed abnormal ovarian morphology, including follicular atresia and absent or poorly organized corpora lutea. The presence of LH/CG receptors in OVCAR-3 ovarian cancer cells was demonstrated by an immunocytochemical method, but receptor numbers were not measured. Thus, the effect of Hecate-ßCG on OVCAR-3 xenografts was not as pronounced as on prostate or breast cancer xenografts, possibly because of a difference in numbers of LH/CG receptors in the OVCAR-3 xenografts. Furthermore, the primary tumors consisted of a more heterogeneous cell population.
In contrast, Hecate-ßCG significantly (P < 0.05) decreased the tumor burden in transgenic mice bearing spontaneous granulosa cell tumors [34].
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TESTICULAR CANCER CELLS |
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Zaleska et al. [35] determined that Hecate-ßCG destroys Leydig cancer cells (BLT-1) in vitro. The toxicity of the conjugate was closely correlated with the number of LH/ CG receptors per cell; cells lacking the LH receptor (spleen cells and KK-1 granulosa cells) were sensitive to Hecate-ßCG only at very high concentrations (i.e., 5 mM and higher). Bodek et al. [34] recently reported that Hecate-ßCG is highly effective in reducing tumor volume and tumor burden in LH receptor-expressing spontaneous testicular tumors in transgenic mice.
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LYTIC PEPTIDE-ßCG CONJUGATES DESTROY METASTATIC CELLS |
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PC-3·luc cells (1 x 106) were injected, along with Matrigel, into the interscapular region of male nude mice. In two separate experiments, the efficacy of Phor14-ßCG(ala) at concentrations of 0.02, 0.2, 2.5, and 10 mg/kg in regressing primary tumors and killing metastatic cells was tested in the presence and absence of the primary tumor. Single (1x/wk) and multiple (3x/wk) injections for 3 wk beginning on Day 35 after tumor propagation were compared. In the absence of the tumor, lymph node metastases, as measured by luciferase-positive cells [36, 37] (Fig. 2) were nearly eliminated by injections of 0.02 mg/kg or higher doses of Phor14-ßCG(ala). Multiple injections were more effective than single injections at all concentrations (P < 0.05). Metastatic cells were markedly reduced in bones, kidneys, and lungs. Metastatic cells were also destroyed by all concentrations of Phor14-ßCG(ala) in the presence of the primary tumor. As in previous experiments, the primary tumors were regressed as a result of treatment in both experiments. In untreated animals, resection of the primary tumor increased the number of metastatic cells in the axillary and interscapular lymph nodes (210 ± 64 vs. 929 ± 396 luciferase positive cells/node; P < 0.05). Similar studies conducted with MDA-MB-435S breast cancer xenografts treated with Hecate-ßCG [24] have given similar results.
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PHOR21-ßCG(ala) |
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Phor21-ßCG(ala) was accepted into the National Cancer Institute Rapid Access to Intervention Development program, in which further pharmacokinetic and cytotoxicity studies have been carried out. These studies agree with our own studies, indicating that plasma concentrations decline to unmeasurable levels within 4 h after injection. In contrast, the lytic peptide-ßCG compounds are quite stable in vitro. These results suggest that Phor21-ßCG(ala) may be used to target disseminated prostate cancer cells resulting from metastasis, without damaging normal tissues that do not express LH/CG receptors.
Phor21-ßCG(ala) proved to be effective in destroying human breast cancer xenografts in nude mice at much lower dose levels than either Hecate-ßCG or Phor14-ßCG. Doses as low as 0.2 mg/kg BW reduced metastatic cells in bones and lymph nodes to nearly undetectable levels. Phor 21 alone had no effect on tumor volume. Histopathological studies of reproductive organs of mice treated with Phor21-ßCG(ala) indicated that, like Hecate-ßCG and Phor14-ßCG, Phor21-ßCG(ala) adversely affected ovarian follicle growth and corpus luteum formation, but did not damage other organs.
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SUMMARY AND CONCLUSIONS |
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In in vivo experiments, Hecate-ßCG, Phor14-ßCG, and Phor21-ßCG(ala) each caused remarkable reductions in tumor volume and tumor burden in nude mice bearing prostate or breast cancer xenografts and in rats bearing diethylstilbestrol/dimethylbenz(a) anthracene-induced mammary tumors. In addition to regressing the primary tumors, the lytic peptide conjugates were highly effective in targeting and destroying prostate and breast cancer metastatic cells in lymph nodes, bones, kidneys, lungs, and other organs.
Although Hecate, Phor14, and Phor21 alone at micromolar concentrations kill prostate and breast cancer cells in vitro [13, 14, 28], they have no effect in vivo in male mice bearing human prostate or breast cancer xenografts [27], and only a small effect in rats bearing diethylstilbestrol/ dimethylbenz(a) anthracene-induced rat mammary gland tumors [30].
Histological examinations of the tumors of control mice revealed viable tumor cells and invasion of adjacent tissues. Lytic peptide conjugate-treated mice had no tumors, or tumors composed largely of necrotic cells. Cells die by necrosis, not apoptosis, after lytic peptide-ßCG treatment [38]. The treatments did not affect vital organs or tissues, including heart, lung, liver, spleen, intestines, pancreas, brain, or kidney. Only gonadal tissue showed impaired function due to treatment with Hecate-ßCG or Phor21-ßCG(ala). Sperm production was inhibited and interstitial cells were damaged. Blood vessels within the tumors and in adjacent nonneoplastic tissues were not affected by any treatment. Treatment with lytic peptide conjugates did not alter counts of leukocytes, erythrocytes, or platelets. Liver function was not altered. These membrane-disrupting peptide conjugates are not hemolytic, they have low antigenicity, and are rapidly metabolized in vivo.
These findings suggest that lytic peptide-LH/CG or GnRH conjugates may be effective in treating hormone-independent breast and prostate cancers that express LH or GnRH receptors, their metastases, and disseminated cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Carola Leuschner, Reproductive Biology, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808-4124. FAX: 225 763 2525; Leuschc{at}pbrc.edu
Received: 4 May 2005.
First decision: 2 June 2005.
Accepted: 15 July 2005.
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REFERENCES |
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ABSTRACT
INTRODUCTION
