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Experimental Biology and Medicine 230:421-428 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Targeted Ablation of Prostate Carcinoma Cells Through LH Receptor Using Hecate-CGß Conjugate: Functional Characteristic and Molecular Mechanism of Cell Death Pathway

Gabriel Bodek*,1, Anna Kowalczyk*, Agnieszka Waclawik*, Ilpo Huhtaniemi{dagger},{ddagger} and Adam J. Ziecik*

* Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-714 Olsztyn, Poland; {dagger} Department of Physiology, University of Turku, Finland; and {ddagger} Institute of Reproductive and Developmental Biology, Imperial College London, London W12 ONN, United Kingdom

1To whom requests for reprints should be addressed at Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-714 Olsztyn, Poland. E-mail: gbodek{at}pan.olsztyn.pl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A Hecate-CGß conjugate (lytic peptide and ß-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.

Key Words: lytic peptide • prostate cancer • Hecate-CGß conjugate


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Carcinoma of the prostate is one of the most frequent solid tumors in human males (1). The mainstay of therapy for advanced disease is androgen deprivation. Thirty percent of cancers do not initially respond to androgen deprivation, and up to 70% of cases with initial good response will relapse after 2–3 years (2). However, all hormonal therapies based on androgen deprivation provide a remission of only limited duration, and most patients eventually relapse because of androgen-independent prostatic cancer. The prognosis of patients with androgen refractory prostate cancer is very poor, and no effective therapy exists at present (3). Selective drug delivery to cancer tissue is one of the major problems in classical chemotherapy, and targeted chemotherapy, which represents a modern oncological strategy designed to improve the effectiveness of cytotoxic drugs, can differentiate between carcinoma cells and normal ones.

Widespread throughout the animal and plant kingdoms, antibacterial lytic peptides, which are fundamental weapons in multicellular organisms, can become a new class of selective anticancer drugs (4, 5). Despite ancient lineage, animals and plants use lytic peptides as effective defensive weapons against microbes (5). Many lytic peptides, such as maganins or tachyplesins, preferentially bind to membranes possessing a higher content of anionic phospholipids in the outer leaflet (6). After contact, lytic peptides insert into negatively charged membranes, creating physical holes that cause leakage of cellular contents, leading to cell death (7). Disintegration mechanisms of gram-negative cytoplasmatic membrane involve displacement of the divalent cations necessary to maintain its integrity and lead to total lysis of cells in a short time (8, 9). Membranes of prokaryotic cells and most tumor cells possess a negatively charged outer leaflet, making them slightly different from normal cells; thus, many lytic peptides, such as tachyloplesins (10) or maganins (11), selectively cause plasma membrane perturbation in transformed cells. However, the molecular mechanism by which some lytic peptides bind preferentially to tumor cell membranes compared with nontransformed cells is still poorly understood.

To increase selectivity of the membrane lytic peptide Hecate (12) toward negatively charged prostate cancer cell membranes, we connected Hecate with a fragment of human chorionic gonadotropin (CG) ß-chain, which possesses high affinity toward luteinizing hormone receptors (LHR; Ref. 13) and which, in addition to gonads (14), are also localized in various female and male nongonadal tissues (15–17). The presence of LHR also results in tumor prostate cells (18) serving as targets for peptide ligands linked to the lytic peptide Hecate. The ability of the selective destruction of the Hecate-CGß conjugate was tested on various tumor cells (19–21) in vitro and in vivo.

In the present study, for the first time we characterized the dynamics of the lysis process by the Hecate-CGß conjugate toward prostate cancer cells expressing LHR. We also investigated the possibility of saturation of the LHR and kept control of the lysis process by physiological doses of CG, which is a natural competitor of Hecate-CGß conjugate. We were particularly interested in the molecular mechanism of the cell death pathway, which is poorly understood and exists in the action of the Hecate-CGß conjugate on various cancer cells. A high concentration of melittin induced cell death by necrosis (22). Whether the Hecate analog of melittin, conjugated with part of the CG could activate necrosis or apoptosis was unknown. Selective fragmentation of cellular DNA is an important step in cells undergoing apoptosis (23). To explain the potential molecular mechanisms of the cell death pathways after plasma membrane perturbation by the Hecate-CGß conjugate, we focused on internucleosomal DNA fragmentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures.
Human prostate cancer cell line PC-3 (24) was maintained in vitro in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin (Sigma), and 0.5 g/l streptomycin (Sigma). The murine Leydig cell line BLT-1 (25) was cultured in complete DMEM/F12 (Sigma) with 10% FCS and antibiotics (0.1 g/l gentamicin; Sigma). The murine Leydig cell line MLTC-1 (26) was routinely cultured in Waymouth’s medium (Sigma) with 9% horse serum (HS) (Sigma), 4.5% FCS, and antibiotics (0.1 g/l gentamicin). All cell lines were cultured and maintained in 75-cm2 flasks (Costar, Cambridge, MA) at 37°C in a humidified atmosphere of 5% CO2, 95% air.

Assessment of LHR mRNA by RT-PCR.
Total RNA was extracted from human prostate PC-3 cancer cells with the total RNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland) according to the manufacturer’s suggested procedure. To examine LHR expression in human prostate cancer cells, the sense primer corresponding to nucleotides 199–225 (5'-CAA GCT TTC AGA GGA CTT AAT GAG GTC-3') and the antisense primer (5'-AAA GCA CAG CAG TGG CTG GGG TA-3') corresponding to nucleotides 845–823 of the human LHR cDNA were used. Fifty microliters of RT-PCR mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 0.001% gelatin, 0.5 µM of each primer, 0.2 mM dNTP, 20 IU of RNase inhibitor RNasin (Sigma), 20 IU of AMV RT (Sigma), and 2 IU Taq polymerase (Sigma). Two micrograms of total RNA were reverse transcribed at 50°C for 50 mins. Then, after 3 mins of primary denaturation at 97°C, the reverse-transcribed cDNA was amplified at 35 cycles, including denaturation at 96°C for 1 min, primer annealing at 57°C for 1 min, extension at 72°C for 2 mins, and a final extension for 10 mins. For negative controls, the reactions were also run on RNA isolated from human colon carcinoma cell line HT-29, on blank-only buffer samples, and in the absence of the reverse transcriptase enzyme.

Preparation of Drugs.
Hecate and Hecate-CGß conjugate were synthesized and purified in the Peptide and Protein Laboratory, Department of Virology, Hartman Institute, University of Helsinki, as described earlier (20).

Cytotoxic Studies of the Lytic Peptide Hecate and Hecate-CGß Conjugate: Determination of Cell Viability.
The PC-3 cells were plated 48 hrs before treatment on 24-well culture dishes at a density of 105 cells per well in 1 ml of complete culture medium. The cells were washed once with phosphate buffered saline (PBS), and 500 µl of incubation medium (without FCS) were added with different concentrations (0.1, 0.5, 1, 5, 10 µM) of Hecate-CGß conjugate or Hecate alone. Incubation was stopped after 2 hrs, and released lactate dehydrogenase (LDH) in the medium was measured immediately to estimate cell viability by monitoring the decrease in absorbance due to oxidation of NADH at 339 nm.

Time-Dependent Toxicity of Hecate Alone and Hecate-CGß Conjugate.
The PC-3 cells were prepared as described previously, and the Hecate alone and the Hecate-CGß conjugate were added in concentrations of 0.1, 0.5, 1, 5, and 10 µM. Incubation was stopped after 15, 30, 45, and 60 mins, and LDH released was measured immediately to estimate cell viability. Each experiment was repeated a minimum of three times and was assessed in quadruplicate.

Competition Studies.
The PC-3 cells were plated 48 hrs before treatment on 24-well plates at a density of 105 cells per well in 1 ml of complete culture medium. The cells were washed once with PBS, and 1 ml of incubation medium was added with different concentrations, 10 and 100 ng/ml, of CG (CR-127) for 1, 3, 6, and 24 hrs. After incubation (with CG), the Hecate-CGß conjugate was added in concentrations of 0.1, 0.5, 1, 5, and 10 µM, and incubation was prolonged an additional 2 hrs. The LDH released was measured immediately following the experiment to estimate cell viability.

Assessment of Apoptosis and Analysis of DNA Fragmentation by Agarose Gel Electrophoresis.
Cells were plated onto six-well plates in complete medium and incubated at 37°C in an atmosphere of 95% air and 5% CO2 for 24 hrs. Cells were washed once with PBS and then incubated for 4 hrs with the Hecate alone or Hecate-CGß conjugate in concentrations of 0.5 and 1 µM. For positive control, 0.1% of hydrogen peroxide was added to the culture medium. Both adherent and floating cells were collected for each sample. Cellular DNA was isolated from cells and analyzed by 1.5% agarose gel electrophoresis as described (Ref. 27, with modification). The gels were stained with ethidium bromide, and DNA ladders were visualized under UV light.

Apoptotic morphology was assessed by observation of cells without fixation under normal illumination using an inverted microscope.

Statistical Analysis.
A PC version 3.0 of the GraphPad Instat program (GraphPad Software Inc., San Diego, CA) was used for one-factor analysis of variance, followed by Dunnett’s multiple comparison test. All data are presented as the mean ± SEM, and differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LHRs in Prostate PC-3 Cancer Cell Line.
Application of RT-PCR confirmed the presence of LHR expression in human prostate cancer cell line (PC-3). A product formed as a result of this reaction had about 647 base pairs, and its size was equivalent to the fragment of LHR (Fig. 1Go; line PC-3). In contrast to this result, no positive reaction was obtained when RNA of cancer HT-29 cells were used as a template or the template was omitted (Fig. 1Go; line HT-29 and line 0).



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  		Figure 1. LH/CG receptor gene expression in PC-3 cell line detected by reverse transcription-polymerase chain reaction. Negative controls: HT-29 (colon carcinoma); 0, reaction on blank, without template; M, DNA marker.

 
Cytotoxic Effect of Hecate Alone and Hecate-CGß Conjugate.
Each treatment of human prostate cell line PC-3 with Hecate alone (Fig. 2AGo) or Hecate-CGß conjuate (Fig. 2BGo) showed an increase of cytotoxic activity, depending on the dosage. Sensitivity of the PC-3 cells measured as percent of LDH release significantly increased at a concentration of 1 µM Hecate-CGß conjugate (P < 0.001; 1 vs. 0 µM; Fig. 2BGo). At this concentration of the conjugate, LDH activity was 2.6-fold higher compared to LDH activity found earlier following incubation of PC-3 cells with the same concentration of Hecate alone (Fig. 2AGo). The dramatic increase of LDH activity was observed at concentrations of 5 and 10 µM of the Hecate-CGß conjugate and was 8.5 and 10.5 times higher than control (0 µM), respectively.



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  		Figure 2. Cytotoxicity determined by measuring lactate dehydrogenase release (mean ± SEM) after Hecate alone (A) or Hecate-CGß conjugate (B) treatment in prostate cancer cell line PC-3; *** P < 0.001.

 
Sensitivity of the PC-3 cells to Hecate alone significantly increased at a concentration of 5 µM of preparate (P < 0.001; 5 vs. 0 µM; Fig. 2AGo), and LDH activity was about 6.8-fold higher than control without Hecate. The LDH release at a concentration of 10 µM was similar to that of a concentration of 5 µM of the Hecate-CGß conjugate.

The Hecate-CGß conjugate compared with Hecate alone caused a higher release of LDH at all effective doses: 1, 5 and 10 µM (for 1 µM, P < 0.05; for 5 and 10 µM, P < 0.001).

Time-Dependent Toxicity of Hecate-CGß Conjugate and Hecate Alone.
The Hecate-CGß conjugate caused a rapid increase of LDH release from the prostate cancer cells during the first 15 mins of incubation at a concentration of 1 µM (P < 0.001; 1 vs. 0 µM; Fig. 3AGo). Prolonging the time of incubation up to 30, 45, and 60 mins maintained the significant (P < 0.001) LDH leak from prostate cells when compared to control (0 µM). The higher doses of Hecate-CGß conjugate caused a time-dependent increase of the LDH release until 45 mins. At this point, LDH activity reached the highest level (4.9–5.3-fold higher than control, respectively). A decreasing tendency of LDH activity after 45 mins of incubation with a Hecate-CGß conjugate concentration of 5–10 µM (Fig. 3BGo) was observed.



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  		Figure 3. Time-dependent effect of Hecate-CGß conjugate and Hecate alone on lactate dehydrogenase (LDH) release of PC-3 cells. Effect of treatment by Hecate-CGß conjugate (A) and Hecate (C) after the first 15 mins. Pattern of LDH leakage after prolonging time incubation up to 30, 45, and 60 mins with Hecate-CGß conjugate (B) and Hecate (D). Data, reported as % of control, represent the mean ± SEM of three independent experiments. Solid lines, statistically significant compared to 0 µM; dashed lines, statistically insignificant compared to 0 µM; *** P < 0.001.

 
The Hecate alone did not increase LDH activity after treatment at low concentrations (0.1–1 µM) in first 15 mins and until 1 hr (Fig. 3C, DGo). However, at concentrations of 5–10 µM, the LDH release was significantly higher after 15 mins of incubation (Fig. 3CGo; P < 0.01). A significant, time-dependent increase in LDH release was observed until 60 mins of incubation (Fig. 3DGo; P < 0.01).

Effect of Preincubation of the PC-3 Cells with CG on LDH Activity.
To determine whether the Hecate-CGß conjugate toxic activity against prostate cancer cells depends on their specific LH/CG binding sites, the PC-3 cells were preincubated with CG to saturate LHR before the conjugate treatment. Preincubation of prostate cells with 10 ng/ml CG for 1 hr did not cause a decrease of the Hecate-CGß conjugate toxicity (Fig. 4AGo). However, a dose of 100 ng/ml of CG added at the same time significantly decreased LDH release at 1-, 5-, and 10-µM concentrations of the conjugate (Fig. 4BGo). A similar effect was observed when the preincubation with CG (100 ng/ml) was extended to 3 and 24 hrs (data not shown).



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  		Figure 4. Effect of PC-3 cells preincubation with 10 ng/ml CG for 1 hr (A) or with 100 ng/ml CG for 1 hr (B). Data, reported as % of control, represent mean ± SEM of three independent experiments; *** P < 0.001.

 
The highest dose of CG (100 ng/ml) significantly reduced LDH release at higher concentrations of the Hecate-CGß conjugate (5 and 10 µM; Fig. 5Go). Preincubation of PC-3 cells during 1–3 hrs caused a 4-fold decrease of LDH activity when compared to control cells where CG preincubation was omitted (Fig. 5Go; D–G vs. C, P < 0.001). After the longest period of PC-3 cell preincubation with CG (24 hrs), the LDH release still remained inhibited (Fig. 5, H, IGo). However, the reduction of LDH activity was 1.8-fold weaker at a concentration of 10 µM (Fig. 5, IGo) of the conjugate compared to prostate cancer cells with CG added for 1–3 hrs (Fig. 5, E, GGo; P < 0.001).



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  		Figure 5. Comprehensive diagram showing the effects of time on preincubation of PC-3 cells with 100 ng/ml CG and high doses (5 µM, D, F, H; 10 µM, E, G, I) of Hecate-CGß on lactate dehydrogenase release. A, B, and C cells were not preincubated with CG. Means with no superscripts in common are different (a < b, c, d, e, P < 0.01; b < c, P < 0.01; d < e P < 0.05).

 
Analysis of DNA Fragmentation and Assessment of Morphological Changes.
Cancer cells were exposed to the Hecate-CGß conjugate or Hecate alone for 4 hrs, and then DNA was extracted and analyzed by agarose gel electrophoresis. The apoptotic cell death in cells (positive control) was induced by using 0.1% H2O2. Whereas H2O2 induced marked internucleosomal DNA fragmentation and cell death via apoptosis (Fig. 6A–CGo; line H2O2), the Hecate and the Hecate-CGß conjugate stimulation did not show such an effect (Fig. 6A–CGo; respective lines).



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  		Figure 6. Lack of DNA fragmentation in human prostate cancer PC-3 cells (A), murine Leydig tumor BLT-1 (B), and MLTC-1 cells (C) after treatment with the Hecate-CGß conjugate or the Hecate alone. Line C, untreated control cells; line H2O2, induced DNA laddering by 0.1% of hydrogen peroxide; line 0.5, 1, cells treated with different concentrations (0.5–1 µM) of the Hecate-CGß conjugate or Hecate alone. Samples were harvested and DNA was separated using 1.5% agarose gel electrophoresis for 4 hrs (gels A and B) and 8 hrs (gel C). DNA ladders were visualized under UV light and documented by photography.

 
Light microscopy revealed morphological changes, including the loss of cell volume and misplacement of adhesion to the surrounding cells after treating PC-3 cells with 0.1% H2O2 (Fig. 6BGo). Most of the cells treated for 4 hrs were detached and floating compared to controlled untreated cells (Fig. 6AGo). In contrast, cells stimulated by the Hecate-CGß conjugate did not lose volume or adhesion to the surface (Fig. 7CGo). Cells were swelled rather than contracted, numerous cells surfaces had broken off, and leakage of cellular components was observed.



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  		Figure 7. Morphology of prostate cancer PC-3 cells after 4 hrs of exposure to 0.1% hydrogen peroxide (B) and 1 µM Hecate-CGß conjugate (C). Photo A illustrates the normal prostate cancer PC-3 cells in a culture. Black arrowheads (B) indicate membrane-bound apoptotic bodies. Black arrows (C) indicate surface break-off and leakage of cellular components. All pointed cells (black arrows) were stained by Trypan blue. Magnification: x400.

 
The effect of the lytic peptide Hecate alone or Hecate-CGß conjugate on cell membranes did not induce DNA fragmentation in prostate PC-3 cells (Fig. 6AGo). Also, we did not observe DNA fragmentation in murine Leydig BLT-1 (Fig. 6BGo) or MLTC-1 (Fig. 6CGo) tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The selective affinity of lytic peptides toward negatively charged cancer cells is known (28). The cancer cells die because of cell membrane disruption when they come in contact with lytic peptides. We used Hecate-CGß conjugate to target in vitro anticancer activity of the lytic peptide Hecate (12) in prostate tumor cells possessing LHR (18). Prostate cancer PC-3 cells were more susceptible to lytic action by the Hecate-CGß conjugate than the Hecate alone. The presence of LHR in PC-3 cells has been described earlier (18) and confirmed in this study. The previous findings showing that the conjugate preferentially destroys various cancer cells expressing LHR (13, 19, 20, 21) are also in accord with our results. The lysis of the prostate cancer cell membranes by the conjugate is relatively fast. After 15 mins of incubation, cellular membrane parturition induced by the Hecate-CGß conjugate leads to uncontrolled leaks of cellular components and consequently death of the cell. A low concentration of the conjugate (1 µM) during 15 mins of incubation induced ionic imbalance in cells, probably by membrane pore formation (6). Higher concentrations of the conjugate (5–10 µM) caused a steep increase in ion imbalance or membrane disruption that finally led to decreased cell viability. At these higher concentrations of the Hecate-CGß conjugate, the lytic process increases dramatically and seems irreversible.

The interesting finding of this study is that the lytic activity of the Hecate-CGß conjugate can be limited by CG. We suggest that short-term preincubation (1–3 hrs) with 100 ng/ml gonadotropin leads to saturation of LHR and protects cells against the toxicity of the conjugate at a concentration of 1 µM and reduces LDH release at higher (5–10-µM) concentrations of the conjugate. These results confirmed our earlier report, where we showed the competition between the Hecate-CGß conjugate and CG for LH/CG receptors in luteal cell membrane fraction (20). Nevertheless, we cannot exclude the mechanism that decreased cytotoxicity of the conjugate can depend partially on the involved signal pathway induced by CG after binding to LHR and thus protects cells from lysis. Toxicity of the Hecate-CGß conjugate after long-term preincubation (24 hrs) of cells with CG slightly increased as measured by LDH release. We suggest that down-regulation of LHR could additionally reduce the integrity of cell membranes and that tumor cells possessing the negatively charged outer leaflet of membranes become vulnerable to the conjugate.

Our observations demonstrated that cells treated by different concentrations of the Hecate-CGß conjugate did not exhibit an internucleosomal DNA fragmentation pattern. DNA fragmentation was not seen in low, not effective (0.5 µM) and effective (1 µM) doses of the conjugate. However, 0.1% of hydrogen peroxide was able to induce substantial DNA fragmentation in PC-3 cells, but the Hecate-CGß conjugate and Hecate alone did not induce DNA fragmentation. Moreover, we have recently carried out flourescent-activated cell sorter analysis and caspase-3 activation that clearly proves a lack of the apoptosis process after the Hecate-CGß conjugate treatment. Further, under a light microscope, morphological changes characterized as apoptotic bodies were observed after treatment of PC-3 cells by an apoptic switcher, hydrogen peroxide. These changes did not arise following treatment of the cells with the conjugate. Instead, in the apoptotic bodies, we observed numerous irregular surface break-offs and loss of cellular components in cell cultures treated by the conjugate.

In summary, we characterized the action of Hecate-CGß conjugate on prostate PC-3 cells in vitro. We have shown that cytotoxic activity of the conjugate induced plasma membrane disruption in a short period of time. The most interesting finding of this study is that lytic activity of the conjugate can be controlled and inhibited by physiological doses of CG. An additional effort was made to explain the killing molecular mechanisms of the conjugate. A lack of DNA fragmentation after the Hecate-CGß conjugate treatment (independently of doses and time of incubation) showed that cells died by necrosis rather than apoptosis.


   Acknowledgments
 
I would like to thank Dr. Nafis A. Rahman and Dr. Adolfo Rivero-Muller for their valuable contributions to this study. We acknowledge Dr. A.F. Parlow, Harbor–UCLA Medical Center, and NIDDKD’s NHPP for the gift of human CG (CR-127). This project was supported by the Committee of Scientific Research in Poland, grant 5PO6K00917, partially supported by the Stefan Batory Foundation (to G.B.) and the Gordon D. Cain Foundation.

Received for publication August 6, 2004. Accepted for publication March 15, 2005.


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 Materials and Methods
 Results
 Discussion
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C. Leuschner and W. Hansel
Targeting Breast and Prostate Cancers Through Their Hormone Receptors
Biol Reprod, November 1, 2005; 73(5): 860 - 865.
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