A novel targeted therapy of Leydig and granulosa cell tumors through the luteinizing hormone receptor using a hecate-chorionic gonadotropin beta conjugate in transgenic mice

Abstract

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Figure 1

Total gonadal volumes of male (a) and female (c) wild-type (WT) control littermate mice and inhα/Tag transgenic (TG) mice, before and after 3 weeks of Hecate-CGβ conjugate (Panels A), Hecate (Panels B), or sham treatment (Panel C). (b and d) Tumor burden volumes (tumor volume/g body weight). The values are mean ± SEM of both gonads. *P < .05, Hecate, Hecate-CGβ conjugate, or sham-treated gonadal volume after treatment versus before treatment. WT, wildtype control littermate mice; TG, transgenic mice expressing the inhibinα Simian Virus 40 T-antigen (inhα/Tag).

Figure 1

Total gonadal volumes of male (a) and female (c) wild-type (WT) control littermate mice and inhα/Tag transgenic (TG) mice, before and after 3 weeks of Hecate-CGβ conjugate (Panels A), Hecate (Panels B), or sham treatment (Panel C). (b and d) Tumor burden volumes (tumor volume/g body weight). The values are mean ± SEM of both gonads. *P < .05, Hecate, Hecate-CGβ conjugate, or sham-treated gonadal volume after treatment versus before treatment. WT, wildtype control littermate mice; TG, transgenic mice expressing the inhibinα Simian Virus 40 T-antigen (inhα/Tag).

Figure 1

Total gonadal volumes of male (a) and female (c) wild-type (WT) control littermate mice and inhα/Tag transgenic (TG) mice, before and after 3 weeks of Hecate-CGβ conjugate (Panels A), Hecate (Panels B), or sham treatment (Panel C). (b and d) Tumor burden volumes (tumor volume/g body weight). The values are mean ± SEM of both gonads. *P < .05, Hecate, Hecate-CGβ conjugate, or sham-treated gonadal volume after treatment versus before treatment. WT, wildtype control littermate mice; TG, transgenic mice expressing the inhibinα Simian Virus 40 T-antigen (inhα/Tag).

Figure 1

Total gonadal volumes of male (a) and female (c) wild-type (WT) control littermate mice and inhα/Tag transgenic (TG) mice, before and after 3 weeks of Hecate-CGβ conjugate (Panels A), Hecate (Panels B), or sham treatment (Panel C). (b and d) Tumor burden volumes (tumor volume/g body weight). The values are mean ± SEM of both gonads. *P < .05, Hecate, Hecate-CGβ conjugate, or sham-treated gonadal volume after treatment versus before treatment. WT, wildtype control littermate mice; TG, transgenic mice expressing the inhibinα Simian Virus 40 T-antigen (inhα/Tag).

Figure 2

Northern hybridization analysis of LHR mRNA expression in Hecate and Hecate-CGβ conjugate-treated or sham-treated testicular and ovarian tissues of inhα/Tag transgenic (TG) males and females. Each lane contains 20 mg of total RNA. The migration of the 28S and 18S rRNA are shown on the left side of the LHR panel. The sizes of the different LHR mRNA splice variants (in kb) are presented on the right. Two lanes for each type of sample are depicted. One lane of WT male and female gonad mRNA expression is shown on the right as a positive control for LHR mRNA. The upper panel shows on the top the ethidium bromide (EtBr) staining of the 28S rRNA for RNA loading control. The middle panel shows Northern hybridization for LHR mRNA. The lower panel shows the densitometric quantification of the longest (7.0 kb) LHR mRNA splice variant (open bar for Hecate and filled bars for sham-treated and Hecate-CGβ-treated) in arbitrary densitometric units (mean of TG sham-treated testis and ovaries regarded as 100%) corrected for intensity of the 28S rRNA band. Each bar represents the mean ± SEM of three independent experiments in duplicates. **P < .01, *P < .05 (P < .01 and P < .05; Hecate-CGβ-treated versus Hecate-treated and/or sham-treated TG control group). TG, transgenic mice expressing the inhibinα Simian Virus 40 T-antigen (inhα/Tag); TG-C, shamtreated TG mice.

Figure 3

Serum progesterone (A) and LH (B) concentrations in inhα/Tag transgenic female and male mice after the 3-week treatment with either Hecate, Hecate-CGβ conjugate, or sham (TG-C). **P < .01, *P < .05 (P < .01 and P < .05; Hecate-CGβ-treated versus Hecate-treated and/or shamtreated TG-C control group). TG, transgenic mice expressing the inhibin; Simian Virus 40 T-antigen (inhα/Tag); TG-C, sham-treated TG mice.

Figure 4

Histologic pictures of hematoxylin/eosin-stained ovarian granulosa (A and B) and testicular Leydig cell (C and D) tumors of inhα/Tag mice treated with Hecate (A and C) or Hecate-CGβ conjugate for three consecutive weeks. Arrows indicate that the interstitial tissue (B and D) has dramatically shrunken after Hecate-CGβ conjugate treatment (the bar in panel A is 40 µm; panels B-D have the same magnification as panel A).

Figure 5

Fluorescent and light microscopic analysis of cocultured Leydig tumor mLTC-1 (LHR-positive, bigger cells) and colon carcinoma HT-29 (LHR-negative, smaller cells) (marked with arrows). (a) Pretreated with 0.5 µM Hecate-CGβ conjugate for 15 minutes in the presence of nonpermeable PI in the media. The left panels (A and B) showed the light microscopic pictures of two different cell mixtures. The upper and lower middle darker field panels (C and D) showed PI fluorescent microscopy. The cell membranes of the mLTC-1 cells were preferentially perturbed by the Hecate-CGβ conjugate. Panels E and F are merged pictures of light and PI fluorescent microscopy (the bar in panel A is 40 µm; panels B–F have the same magnification as panel A). PI, propidium iodide. (b) Pretreated for 30 minutes with an increasing dose of Hecate: (A and D) 0.5 µM, (B and E) 1 µM, and (C and F) 5 µM Hecate in the presence of nonpermeable PI in the media. The left panels (A–C) show the light microscopy pictures. The right darker panels (D–F) show PI fluorescent microscopy. Hecate killed both mLTC-1 cells and HT-29 cells with the highest (5 µM) concentration used not depending on their LHR content. A concentration of 1 µM Hecate killed only HT-29 cells (the bar in panel A is 40 µm; panels B–F have the same magnification as panel A). PI, propidium iodide. (c) Light microscopic picture of a single mLTC-1 Leydig tumor cell incubated in 0.5 µM Hecate-CGβ conjugate for 0, 15, and 30 minutes. Cell swelling could be observed at 30 minutes (C), which strongly suggests necrosis as the mode of cell death (the bar in the panel A is 40 µm; panels B–C have the same magnification as panel A).

Figure 6

Flow cytometric analysis of mLTC-1 cells treated for 4 hours with concentrations of 0.1, 0.5, and 1 µM Hecate-CGβ conjugate or Hecate. Cells treated with 0.1% H₂O₂ for 4 hours were used as positive apoptotic controls. After incubation, cells were lysed in a hypotonic solution containing nonpermeable PI and analyzed by FACS (FACSCalibur flow cytometer; Becton Dickinson). Neither Hecate-CGβ conjugate nor Hecate treatment induced nuclear fragmentation in mLTC-1 Leydig cells. The numbers on the top left quadrant of the panels represent the percentage of apoptotic cells. The data are representative of three separate experiments with similar results.

Figure 7

Western blot analysis of caspase-3. mLTC-1 cells were treated with 0.5, 1, and 5 µM of either Hecate or Hecate-CGβ conjugate for 60 minutes. Adherent and detached cells were assayed for cleaved caspase-3. No detectable active caspase-3 activity in Hecate- and Hecate-CGβ-treated Leydig tumor mLTC-1 cells could be observed. “C”—mLTC-1 cells treated with 0.1% H₂O₂ showing cleaved caspase-3, positive control for apoptosis; on the left side, the molecular marker sizes are shown in kilodaltons.