Skip to main content
Download PDF

Antitumor effects of cecropin B-LHRH’ on drug-resistant ovarian and endometrial cancer cells

BMC Cancer volume 16, Article number: 251 (2016) Cite this article

Abstract

Background

Luteinizing hormone-releasing hormone receptor (LHRHr) represents a promising therapeutic target for treating sex hormone-dependent tumors. We coupled cecropin B, an antimicrobial peptide, to LHRH’, a form of LHRH modified at carboxyl-terminal residues 4–10, which binds to LHRHr without interfering with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. This study aimed to assess the antitumor effects of cecropin B-LHRH’ (CB-LHRH’) in drug-resistant ovarian and endometrial cancers.

Methods

To evaluate the antitumor effects of CB-LHRH’, three drug resistant ovarian cancer cell lines (SKOV-3, ES-2, NIH:OVCAR-3) and an endometrial cancer cell line (HEC-1A) were treated with CB-LHRH’. Cell morphology changes were assessed using inverted and electron microscopes. In addition, cell growth and cell cytotoxicity were measured by MTT assay and LDH release, respectively. In addition, hemolysis was measured. Furthermore, radioligand receptor binding, hypersensitization and minimal inhibitory concentrations (against Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii) were determined. Finally, the impact on tumor growth in BALB/c-nu mice was assessed in an ES-2 xenograft model.

Results

CB-LHRH’ bound LHRHr with high-affinity (dissociation constant, Kd = 0.252 ± 0.061nM). Interestingly, CB-LHRH’ significantly inhibited the cell viability of SKOV-3, ES-2, NIH:OVCAR-3 and HEC-1A, but not that of normal eukaryotic cells. CB-LHRH’ was active against bacteria at micromolar concentrations, and caused no hypersensitivity in guinea pigs. Furthermore, CB-LHRH’ inhibited tumor growth with a 23.8 and 20.4 % reduction in tumor weight at 50 and 25 mg/kg.d, respectively.

Conclusions

CB-LHRH’ is a candidate for targeted chemotherapy against ovarian and endometrial cancers.

Peer Review reports

Background

Sex hormone-dependent tumors, including ovarian, endometrial, breast and prostate carcinomas, are the most common reproductive system tumors. Ovarian cancer is often detected at a late stage [1–3]; despite cytoreductive surgery and paclitaxel/platinum-based chemotherapy it frequently recurs, resulting in poor prognosis [4–6]. Although endometrial carcinoma presents at an early stage and responds well to surgery [7, 8], frequent recurrence also results in poor prognosis [8, 9]. Chemotherapy of sex hormone-dependent cancers is limited by the intrinsic or acquired drug resistance of tumor cells as well as the toxicity to normal cells of chemotherapeutic agents [5, 10, 11], whose side effect profiles limit the chemotherapeutic dosing [12, 13].

Targeted cytotoxic agents enable selective treatment of primary tumors and their metastases, reducing side effects and improving efficacy [2, 14–17]. Luteinizing hormone-releasing hormone receptor (LHRHr) may represent a useful target in sex hormone-dependent tumors as it is expressed in human breast (52 %), ovarian (80 %), endometrial (80 %), and prostate (86 %) carcinomas [18]. Potential drawbacks of targeted chemotherapy using natural LHRH as a binding partner include side effects resulting from interference with pituitary secretion of LH and FSH [19]. Previous LHRHr targeted cytotoxic therapies include cytotoxins, such as doxorubicin, which have non-specific cytotoxicity. Other LHRHr targeted therapies used chemical approaches linking the cytotoxin to LHRH; however, such conjugates are readily hydrolyzed in the blood stream, releasing cytotoxic radicals before reaching their therapeutic targets, therefore inducing non-specific cytotoxicity [19]. Refined and improved strategies for LHRHr chemotherapy are necessary if these approaches are to be useful.

LHRH is a decapeptide that binds to receptors on pituitary gonadotropes, stimulating biosynthesis and secretion of FSH and LH, which regulate gonadal steroidogenesis and gametogenesis in both sexes [20]. The carboxyl-terminal residues 4–10 of LHRH are involved in receptor binding, while amino-terminal residues 1–3 activate the receptors [21]. As targeted chemotherapy using natural LHRH as a carrier may interfere with LH and FSH secretion, we constructed a modified peptide, LHRH’, in which amino-terminal residues 1–3 are not included. Thus, LHRH’ is expected to target LHRHr positive carcinoma cells, while not interfering with LH and FSH secretion.

Cecropin B is an antimicrobial peptide (AMP) first characterized in 1980 [22, 23]; since then, thousands of similar molecules have been isolated from a wide range of organisms [24, 25]. AMPs are short peptides possessing net cationic charges, selective toxicity, rapid cytotoxic effects, broad antimicrobial spectra, and no documented resistance [24–26]. Some AMPs, including cecropins, are highly potent against cancer cells but not normal mammalian cells [27–31], making them attractive for the treatment of some cancers.

In this study, we developed a novel LHRHr cytotoxin by linking Cecropin B to the modified LHRH’ receptor ligand, Cecropin B-LHRH’ (CB-LHRH’) [32]. We hypothesized that the novel CB-LHRH’ may bind to LHRH receptors and deliver an effective broad-spectrum toxin specifically to tumor cells, without affecting healthy cells or readily inducing resistance. Therefore, we aimed in this study to assess the antitumor effects of CB-LHRH’ in the treatment of drug-resistant ovarian and endometrial cancers, using BALB/c-nu mice (a common model for cancer studies) harboring ES-2 xenografts.

Methods

The study was approved by the ethical committee of Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Informed consent was obtained from all individual participants included in the study.

Peptides and cytotoxic agents

CB-LHRH’ recombinant polypeptide (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALSYGLRPG) (Shanghai Sangon Biological Engineering Technology & Services Corporation, Shanghai, China) was synthesized by solid phase synthesis to a purity of 96 % as evaluated by high-performance liquid chromatography. It was identified by mass spectrographic analysis (MW 4,566.57D), dissolved in phosphate-buffered saline (PBS), and diluted to the desired concentration before use.

Drug-resistant tumor cell lines

Four drug-resistant cancer cell lines, including three ovarian cancer cell lines (SKOV-3, ES-2, NIH:OVCAR-3) and one endometrial cancer cell line (HEC-1A) (Cell Bank of Shanghai Biological Institute, Shanghai, China), were cultured in RPMI 1640 with 10 % fetal bovine serum, 1,000 IU/mL penicillin, and 100 mg/mL streptomycin at 37 °C and 5 % CO2. Cell cultures and subculture procedures were performed according to the recommendations of the ATCC Global Bioresource Center. ES-2, HEC-1A, and NIH:OVCAR-3 cells express LHRHr, while the SKOV-3 does not express LHRHr [33–35].

Clonogenic assay

A clonogenic assay was carried out as previously described [36]. 300 cells were seeded into 6-well dishes in 2 mL of medium. After overnight incubation, CB-LHRH’ was added at a final concentration of 12.5 μM and cells were further cultured for 10 d. Cells were then stained with crystal violet (0.5 % w/v). Colonies containing a minimum of 50 cells were counted using a dissection microscope. Each treatment was performed in triplicate and each experiment repeated three times.

Cytotoxicity assay

Lactate dehydrogenase (LDH) release was quantified using LDH Cytotoxicity Detection Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, 100 μL of exponentially growing tumor cells (6 × 104 cells/mL) were seeded into 96-well plates, cultured overnight, and washed with RPMI 1640. Then, several CB-LHRH’ amounts (6.25, 12.5, 25, 50, and 100 μM) were completed to 100 μL with RPMI and added to cells, followed by 4 h incubation. LDH release was assessed by measuring absorbance at 490 and 630 nm, respectively, on a microplate reader (Bio-Tek Instruments Inc., Vermont, USA). Cytotoxicity was determined by the following formula: Cytotoxicity (%) = (exp. value - low control)/(high control - low control) x 100. All conditions were carried out in triplicate, and the experiments repeated three times.

Proliferation assay

The anti-proliferative activity of CB-LHRH’ was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [37]. Briefly, 100 μL of exponentially growing tumor cells (6 × 104 cells/mL) were seeded in 96-well plates and cultured overnight. B-LHRH’ was then added to each well at final concentrations of 6.25, 12.5, 25, 50, and 100 μM. After another 24 or 48 h, 10 μL of fresh MTT (Sigma–Aldrich, St. Louis, USA) at 5 mg/mL in Hank’s salt solution was added for 4 h. After careful removal of the medium, 100 μL dimethylsulfoxide (Sigma–Aldrich, St. Louis, USA) was added to wells followed by absorbance reading on a microplate reader (Bio-Tek Instruments Inc, Vermont, USA) at 490 and 630 nm, respectively. Cell viability was determined as follows: Cell viability (%) = (absorbance of treated wells - absorbance of blank control)/(absorbance of negative control - absorbance of blank control) x 100. The inhibition rate was derived as 1-Cell viability. Each experiment was performed in triplicate and repeated three times.

Cell morphology was monitored using inverted and electron microscopes after 1 h exposure to CB-LHRH’ as previously described [38].

Electron microscopy

For transmission electron microscopy ES-2 cells grown on sterile slides were fixed with 2.5 % glutaraldehyde in pH 7.4 phosphate buffer for 1 h at 4 °C, followed by post-fixation in 1 % osmium tetroxide 1 h at 4 °C and dehydrated in an alcohol gradient (50 to 100 %). The attached cells were then dried by Lyophilization and coated with Au before examination with transmission electron microscope (S-4800 SEM, Hitachi, Ltd. Tokyo, Japan).

For scanning electron microscopy ES-2 cells were grown and fixed under similar conditions, without slides. After post-fixation cells were infiltrated with Epon resin and sectioned with Leica UC6 ultramicrotome (Leica Microsystems Inc., LKB-II, Wetzlar, Germany). Sections of about 100 nm were post-stained with lead citrate and uranyl acetate. Grids were examined with scanning electron microscope (JEM-200CX TEM, JEOL, Ltd. Tokyo, Japan).

Cytotoxicity of CB-LHRH’ in normal eukaryotic cells

The ability of CB-LHRH’ to induce cytotoxicity in normal eukaryotic cells was assessed as previously described [39]. With informed consent and institutional review board approval, peripheral blood samples were obtained from healthy human donors. Red blood cells were separated by centrifugation at 1,000 g for 5 min followed by three washes with PBS. Washed red blood cells were resuspended in sterile Alsever’s solution (2.05 % dextrose, 0.8 % sodium citrate, 0.055 % citric acid, and 0.42 % sodium chloride, pH 6.1, hematocrit 5 %) in a plate and treated with CB-LHRH’ at 100, 200, 300, 400, or 500 μM for either 30 min or 4 h at 37 °C with shaking. After centrifugation for 10 min at 600 g, the supernatant was collected for hemolysis measurement at 490 nm (hemoglobin absorbance). Adding equal volumes of water to red blood cells provided 100 % hemolysis (positive control); cell-free Alsever’s solution was used as negative control. Each experiment was performed in triplicate and repeated three times.

Radioligand receptor binding assays

Under approved animal research protocol standards, binding capacity to LHRHr was assessed in rat pituitary membranes by radioligand-binding assays as previously described [40]. The experiment was approved by the animal care and use committee of Fudan University.

Preparation of membrane fractions for primary pituicytes

To obtain primary pituicytes, SD male and female rats (180–240 g) were sacrificed and the pituitary glands dissected and washed twice with 10 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM PMSF, 1.2 mM MgCl2, 0.01 mM EDTA-Na2. Tissue aliquots were homogenized using an automatic glass Potter homogenizer at 2000 rpm for 3 × 10 s. After nucleus and debris removal by centrifugation at 2000 g and 4 °C for 5 min, membrane preparation aliquots were collected by centrifugation at 2000 g and 4 °C for 20 min. Protein concentrations were determined by the Lowry method using bovine serum albumin (BSA) as a standard, diluted with 10 mM Tris-HCl buffer to 2 mg/mL stock at −70 °C.

Radioligand receptor binding assay

CB-LHRH’ peptide was radioactively labeled with Iodine-125 (Perkin Elmer Life and Analytical Sciences, Waltham, USA) using Chloramine-T (Sigma–Aldrich, St. Louis, USA) and purified on Sephadex G15 chromatography (Sigma–Aldrich) using 0.1 mM aqueous acetic acid, containing 2.5 g/L BSA as eluent. The specific activity of 125I-CB-LHRH’ was about 0.2 ~ 0.24 mCi/mmol with a purity greater than 95 %. In the binding assay, polypropylene tubes were pretreated overnight at 4 °C with 10 mM Tris-HCl, pH 7.4 containing 1 % BSA. Membrane fraction aliquots (100 μg protein in 50 μL per tube) were incubated with different concentrations of 125I-CB-LHRH’ at 37 °C for 1 h in a total volume of 150 μL. To examine the binding specificity, 2 μg gonadorelin (Tash Biotechnology, Shanghai, China) was used as competitor. Reactions were terminated by adding 1 mL of 10 mM Tris-HCl buffer (pH 7.4, 4 °C). The solutions were subsequently filtered onto Whatman filters, washed with 1 mL of 5 % trichloroacetic acid and transferred to a counting tube. The radioactivity was measured by a gamma counter (GC-300, AnHui ustc ZonKia Scientific Instruments co. LTD, Hefei, China). Specific binding was determined by subtracting nonspecific from total binding. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were derived by the Scatchard method. Kd represents the concentration of drug inducing the half (50 %) biggest effect. Bmax indicates the maximum binding volume combined per mg receptor proteins. Each experiment was performed in triplicate and repeated three times.

Hypersensitization test

Under approved animal research protocol standards, the hypersensitive reaction against CB-LHRH’ (2 mg/mL, equal to 438 μM) was assessed in guinea pigs. According to the Chinese Pharmacopoeia, six animals with an average weight of 250 g (XiFengYang Special Economic Animal Farm, Huzhou, China) were administered an intraperitoneal injection of 0.5 mL of CB-LHRH’ every other day three times, while three additional animals received PBS physiological saline injections. Of the six animals administered CB-LHRH’, three received an intravenous injection of 1 mL CB-LHRH’ at day 15, and the remaining three at day 22. Allergic reactions, such as piloerection, sneezing, lacrimation, dyspnoea, and hyperspasmia, were then monitored for the next 30 min.

Antibacterial assay

The antibacterial activity of CB-LHRH’ was evaluated by the agar dilution method [41]. Muller Hinton’s agar medium plates containing serial dilutions of CB-LHRH’ were inoculated with 2 μL of tenfold-diluted 0.5 McF bacteria and incubated for 22 h at 35 °C. The lowest concentration of CB-LHRH’ inhibiting bacterial growth was considered the minimal inhibitory concentration (MIC). Nine clinical strains of the following species were evaluated: Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

Xenograft studies

Five- to six-week-old female (15–20 g) athymic nude mice (BALB/c-nu) were obtained from Shanghai SLAC Laboratory Animal Corporation, China. The animals were housed in sterile cages under laminar flow hoods in a temperature controlled room with a 12-hour light/12-hour dark cycle, and fed autoclaved chow and water ad libitum. All experiments were carried out in accordance with the guidelines for the welfare ethics of experimental animals [42]. All the animal-related procedures were approved by the Animal Ethical Committee of Zhejiang University.

Experimental protocol

Cultured ES-2 ovarian cancer cells were resuspended at 1 × 107/ml and inoculated subcutaneously into the nude mouse armpit. When tumors reached 60 mm3, they were extracted, cut into pieces of about 2 mm3, and inoculated subcutaneously into the nude mouse armpit. When the tumors reached an average volume of about 100 mm3, the mice were randomly divided into five groups (n = 10). Groups 1–3 received intra-tumor injection of CB-LHRH’ twice daily at 12.5, 25 or 50 mg/kg.d, respectively. Group 4 received 2 mg/kg cisplatin, once every 3 days [43], and group 5 the same volume of PBS, administered twice daily.

Tumor volumes were assessed every 3 d as 0.5 × length × width2 (where length is the longest diameter across the tumor and width the corresponding perpendicular diameter as measured by calipers). On day 13, 4 h after the last dose, mice were sacrificed, and tumors excised and weighed; tumor growth inhibition rate was derived as (1-tumor weight treated/tumor weight control) × 100 %.

Response criteria

Anti-tumor activity was evaluated as relative growth of tumor volume (T/C, value), the mean relative tumor volume (RTV) for the treatment group divided by the mean RTV for the control group, as follows: T/C (%) = TRTV/CRTV X 100 %. RTV = Vt/V0, where Vt is the volume on any given day, and V0 the volume at treatment start. Agents producing a T/C of >60 % were considered to be inactive, those with a mean T/C of ≤60 % considered to be active.

General toxicity was evaluated based on body weight, measured weekly.

Statistical analysis

Data are mean ± standard deviation (SD). Normality and homogeneity of variance assumptions were assessed. One way analysis of variance (ANOVA) was used for multiple group comparison, as required. SPSS 16.0 (SPSS, USA) was used for the statistical analysis. p < 0.05 was considered statistically significant.

Results

CB-LHRH’ inhibited proliferation of cancer cell lines

Three drug-resistant ovarian cancer cell lines (SKOV-3, ES-2, NIH:OVCAR-3) and one drug-resistant endometrial cancer cell line (HEC-1A) were incubated with 12.5 μM CB-LHRH’. After 10 d there were almost no colonies after CB-LHRH’ treatment, while control wells contained approximately 30–50 colonies, indicating that 12.5 μM CB-LHRH’ significantly inhibited the growth of drug-resistant cancer cells.

Damage to all four cell lines was observed within 24 h of exposure to 12.5 μM CB-LHRH’ by light microscopy (data not shown). And the effect of CB-LHRH’ on ES-2 cell morphology was shown in Fig. 1. Typical cell damage included aggregation, detachment from the plate, inflation, cytoplasm leak, disintegration into granules, and decreased cell number (Fig. 1).

Fig. 1
figure 1

Cecropin B-LHRH’ induces changes in ES-2 morphology. Untreated cells (a–c, 10, 20, and 40×, respectively), display normal morphology, while cells treated with 25 μM cecropin B-LHRH’ for 24 h (d–f, 10, 20, and 40×, respectively) showed reduced number, and aggregated, detached from the plate, inflated, released cytoplasm, and disintegrated into granules. Morphological changes observed in ES-2 cell line

Full size image

Electron microscopy revealed cell membrane blebbing, the disappearance or reduction of microvilli, cell shrinkage, increased cellular granularity, the formation and separation of apoptotic bodies, and cytoplasm leak in response to 25 μM CB-LHRH’ in ES-2 cell (Fig. 2).

Fig. 2
figure 2

Cecropin B-LHRH’ induces ultrastructural changes in ES-2 cells. Transmission electron microscope images (a–e), and scanning electron microscope images (f) of a normal ES-2 cell (a); ES-2 cells incubated with 25 μM cecropin B-LHRH’ for 1 h (b–f). Figures b–d illustrate cell membrane blebbing, the disappearance or reduction of microvilli, cell shrinkage, increased cellular granularity, and the formation and separation of apoptotic bodies. e and f show cytoplasm leak. Morphological changes observed in ES-2 cell line

Full size image

All four cell lines incubated for 4 h with CB-LHRH’ exhibited concentration-dependent cytotoxicity (P < 0.001) as shown in Fig. 3. And there were no differences among the four cell lines (Fig. 3). In contrast, CB-LHRH’ caused no hemolysis of red blood cells from healthy human donors (data not shown).

Fig. 3
figure 3

Cytotoxicity of cecropin B-LHRH’ against the four cancer cell lines. Cytotoxicity was detected in SKOV-3, ES-2, NIH:OVCAR-3, and HEC-1A cells incubated overnight with CB-LHRH’ by LDH Cytotoxicity Detection Kit. Concentration-dependent cytotoxicity was observed in all four cell lines

Full size image

Proliferation of all four drug-resistant cancer cell lines was inhibited by CB-LHRH’ in a concentration-dependent manner (P < 0.001) (Fig. 4a–d). And the greatest inhibition was obtained for ES-2 cells, and CB-LHRH’ inhibited cell proliferation in ES-2 cell in a time-dependent manner (P < 0.05) (Fig. 4c). Inhibition of SKOV-3 and HEC-1A cell proliferation was weaker at the dose of lower than 25 μM and 50 μM, respectively (Fig. 4a and 4b). And inhibition of NIH:OVCAR-3 cell proliferation was up to 40 % even at the high dose of 100 μM (Fig. 4d).

Fig. 4
figure 4

Cecropin B-LHRH’ inhibition of cancer cell proliferation. Proliferation inhibition was detected in (a) SKOV-3, (b) HEC-1A, (c) ES-2 and (d) NIH:OVCAR-3 cells incubated with CB-LHRH’ at the dose of 6.25 μM, 12.5 μM,25 μM,50 μM, and 100 μM for 24 h or 48 h by the MTT assay. Proliferation was inhibited in a time- and concentration-dependent manner in ES-2 cell line (p = 0.026), and in a concentration-dependent manner in other three cell lines (p < 0.001 vs 6.25 μM). Data are presented as mean ± SD, n = 3

Full size image

CB-LHRH’ is a high-affinity ligand for LHRHr

The binding affinity of CB-LHRH’ to the LHRH receptor was assessed in rat pituitary membranes by radioligand-binding assays. Kd and Bmax values were 0.2526 ± 0.061 nM and 110.4 ± 17.68 fmol/mg protein, respectively (Fig. 5), indicating that 125I-CB-LHRH’ is a high-affinity ligand for LHRHr.

Fig. 5
figure 5

Saturation curve and Scatchard plot of LHRHr-specific 125I-cecropin B-LHRH’ binding. The binding affinity of CB-LHRH’ to the LHRH receptor was assessed in rat pituitary membranes by radioligand-binding assays. TB, total binding; SB, specific binding; NSB, non-specific binding. Each experiment was performed in triplicate and repeated three times

Full size image

CB-LHRH’ does not induce hypersensitivity

The potential of CB-LHRH’ to induce a hypersensitive reaction in mammals was assessed in the guinea pig model. Guinea pigs administered 0.5 mL 2 mg/ml CB-LHRH’ intraperitoneally every other day three times (n = 6) survived the 15 days of treatment and their weights did not differ significantly from animals administered PBS (n = 3). Afterwards, three animals were administrated an additional 1 mL of 2 mg/ml CB-LHRH’. No anaphylaxis occurred in any of the six guinea pigs treated with CB-LHRH’ (data not shown).

CB-LHRH’ inhibits Gram-negative bacteria

The antibacterial activity of CB-LHRH’ was evaluated by the agar dilution method. CB-LHRH’ killed or inhibited the growth of all tested gram-negative bacteria at micromolar concentrations. The minimal inhibitory concentrations (MIC) were 1.93–7.72 μM, 3.86–7.72 μM, 3.86–7.72 μM, 3.86–7.72 μM, ≥15.44 μM, and >15.44 μM for Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus, respectively (Table 1).

Table 1 The MIC of the Cecropin B-LHRH’ against the six kinds of clinically isolated bacteria
Full size table

CB-LHRH’ inhibits ES-2 Xenograft growth in vivo

Nude mice were implanted with ES-2 and administered intra-tumoral injection of CB-LHRH’ twice daily at 0, 12.5, 25 or 50 mg/kg.d, or 2 mg/kg cisplatin, once every 3 days (n = 10); thirteen days later 9, 8, 9, 7 and 8 surviving mice were obtained, respectively (Tables 2 and 3).

Table 2 Therapeutic effect of Cecropin B-LHRH’ on the volume of ES-2 tumors xenografted into nude mice (Mean ± SD)
Full size table
Table 3 Therapeutic effects of Cecropin B-LHRH’ on the weight of ES-2 tumors xenografted into nude mice (Mean ± SD)
Full size table

However, administration of CB-LHRH’ at 50 and 25 mg/kg significantly inhibited the growth of ES-2 Xenografts by day 13, resulting in tumor sizes of 50 % and 59 % the control values (P < 0.01), and 23.8 % and 20.4 % decrease in tumor weights (P < 0.05), respectively; no differences were found in animal weights.

Discussion

In this study, we developed a novel LHRHr cytotoxin strategy by linking Cecropin B to the modified LHRH’ receptor ligand in order to deliver an effective broad-spectrum toxin specifically to tumor cell targets, without affecting gonadotrophin secretion in healthy cells or inducing resistance.

CB-LHRH’ binds to LHRHr with a low dissociation constant of 0.252 ± 0.061 nM, indicating the high-affinity of the modified peptide, which should have broad-spectrum toxicity against cancer cells expressing LHRHr. Indeed, CB-LHRH’ was cytotoxic to ES-2 and NIH:OVCAR-3 (ovarian cancer cells) and HEC-1A (endometrial cancer cells) which express LHRHr, while displaying the weakest cytotoxicity in the LHRHr negative ovarian cancer cell line SKOV-3. Of note, the four cancer cell lines sensitive to CB-LHRH’ are known to be resistant to several cytotoxic drugs, including diphtheria toxin, doxorubicin, cisplatin, and adriamycin, among others, according to the ATCC Global Bioresource Center (USA). For instance, ES-2 cells were derived from an ovarian clear cell carcinoma characterized by unique clinical features such as high incidence in stage I of the disease, relatively strong resistance to conventional platinum or taxane-based chemotherapies, and poor prognoses [44–46]. The overt cytotoxicity demonstrated for CB-LHRH’ against several multidrug-resistant cancer cell-lines, particularly ES-2 cells, indicates that the new peptide might help in the treatment of some cancers resistant to currently available chemotherapeutics; indeed, CB-LHRH’ is a promising candidate for the treatment of LHRHr dependent cancers.

The mechanisms underlying the antimicrobial and anticancer effects of cecropin include plasma membrane disruption via micellization, or pore formation by peptide-lipid interactions [47, 48]. Cationic peptides target characteristic, cell-surface, anionic lipids by electrostatic attraction that are ubiquitous and exclusive to micro-organisms, followed by membrane permeation and disruption. This simple electrostatic discrimination provides selective toxicity, as well as a broad spectrum of antimicrobial activity. Unspecific molecular recognition makes the development of resistance difficult. Eukaryotic cell membranes have important amounts of sterols, and the membrane-stabilizing cholesterol protects normal eukaryotic cells from attacks by therapeutic peptides [47, 48]. Changes in lipid compositions of cancer cell membranes may be a reason why these peptides show specific toxicity to cancer cells [30]. In this study, loss of cytoplasm and disintegration of cancer cells, with no hemolytic activity in normal red blood cells, supports similar anticancer mechanisms of plasma membrane disruption.

CB-LHRH’ also induced no hypersensitive reactions in guinea pigs, suggesting that CB-LHRH’ may not cause severe allergy, a common problem of peptide drugs. This benign profile distinguishes the newly developed polypeptide from other chemotherapeutics, as the safety of chemotherapeutics is one of the most important features of contemporary drug treatment. However, these results need to be replicated in human subjects in order to rule out potential relevant inter-species differences. In addition, the anticancer effects of CB-LHRH’ was assessed after intra-tumoral administration, which does not fully recapitulate the targeting of tumors in the peritoneal space.

CB-LHRH’ also inhibited the growth of clinically isolated bacteria at micromolar concentrations, indicating a potent antibacterial effect. The antibacterial and anticancer functions of CB-LHRH’ suggest that the carboxyl-terminal extension of CB doesn’t alter its antibacterial properties. In addition to inhibiting growth of cancer cells, CB-LHRH’ may also benefit cancer patients with its antibacterial function, since bacterial infections are a major cause of morbidity and mortality in neutropenic patients following chemotherapy for malignancy [49].

Conclusion

LHRH’ may represent a more promising therapeutic than LHRH, and cecropin B appears to be a potentially valuable component of targeted therapies. The peptide drug CB-LHRH’ described here is a potent candidate for cancer therapy, with broad anti-cancer spectrum and minimal toxicity to normal eukaryotic cells.

Ethics approval and consent to participate

The study was approved by the ethical committee of Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article.

Abbreviations

AMP:

antimicrobial peptide

Bmax:

maximum binding capacity

BSA:

bovine serum albumin

FSH:

follicle-stimulating hormone

Kd:

equilibrium dissociation constant

LDH:

lactate dehydrogenase

LH:

luteinizing hormone

LHRH:

luteinizing hormone-releasing hormone

LHRH’:

modified LHRH

LHRHr:

luteinizing hormone-releasing hormone receptor

MIC:

minimal inhibitory concentration

MTT:

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

PBS:

phosphate-buffered salt solution

RTV:

relative tumor volume

T/C value:

RTV of the treatment group/RTV of the control group

References

  1. McLemore MR, Miaskowski C, Aouizerat BE, Chen LM, Dodd MJ. Epidemiological and genetic factors associated with ovarian cancer. Cancer Nurs. 2009;32:281–8. quiz 9–90.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Banerjee S, Gore M. The future of targeted therapies in ovarian cancer. Oncologist. 2009;14:706–16.

    Article  CAS  PubMed  Google Scholar 

  3. Collinson F, Jayson G. New therapeutic agents in ovarian cancer. Curr Opin Obstet Gynecol. 2009;21:44–53.

    Article  PubMed  Google Scholar 

  4. del Carmen MG, Birrer M, Schorge JO. Carcinosarcoma of the ovary: a review of the literature. Gynecol Oncol. 2012;125:271–7.

    Article  PubMed  Google Scholar 

  5. Martin LP, Schilder RJ. Management of recurrent ovarian carcinoma: current status and future directions. Semin Oncol. 2009;36:112–25.

    Article  PubMed  Google Scholar 

  6. Metzger-Filho O, Moulin C, D'Hondt V. First-line systemic treatment of ovarian cancer: a critical review of available evidence and expectations for future directions. Curr Opin Oncol. 2010;22:513–20.

    Article  CAS  PubMed  Google Scholar 

  7. Engelsen IB, Akslen LA, Salvesen HB. Biologic markers in endometrial cancer treatment. APMIS. 2009;117:693–707.

    Article  CAS  PubMed  Google Scholar 

  8. Hill EK, Dizon DS. Medical therapy of endometrial cancer: current status and promising novel treatments. Drugs. 2012;72:705–13.

    Article  CAS  PubMed  Google Scholar 

  9. Dellinger TH, Monk BJ. Systemic therapy for recurrent endometrial cancer: a review of North American trials. Expert Rev Anticancer Ther. 2009;9:905–16.

    Article  PubMed  Google Scholar 

  10. Eckhardt S. Recent progress in the development of anticancer agents. Curr Med Chem Anticancer Agents. 2002;2:419–39.

    Article  CAS  PubMed  Google Scholar 

  11. Lee C, Raffaghello L, Longo VD. Starvation, detoxification, and multidrug resistance in cancer therapy. Drug Resist Updat. 2012;15:114–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Colombo P, Gunnarsson K, Iatropoulos M, Brughera M. Toxicological testing of cytotoxic drugs (review). Int J Oncol. 2001;19:1021–8.

    CAS  PubMed  Google Scholar 

  13. Wolf S, Barton D, Kottschade L, Grothey A, Loprinzi C. Chemotherapy-induced peripheral neuropathy: prevention and treatment strategies. Eur J Cancer. 2008;44:1507–15.

    Article  CAS  PubMed  Google Scholar 

  14. Li J, Chen F, Cona MM, Feng Y, Himmelreich U, Oyen R, et al. A review on various targeted anticancer therapies. Target Oncol. 2012;7:69–85.

    Article  PubMed  Google Scholar 

  15. Liu SV, Liu S, Pinski J. Luteinizing hormone-releasing hormone receptor targeted agents for prostate cancer. Expert Opin Investig Drugs. 2011;20:769–78.

    Article  CAS  PubMed  Google Scholar 

  16. Tagawa T, Morgan R, Yen Y, Mortimer J. Ovarian cancer: opportunity for targeted therapy. J Oncol. 2012;2012:682480.

    Article  PubMed  Google Scholar 

  17. Ledermann JA, Raja FA. Targeted trials in ovarian cancer. Gynecol Oncol. 2010;119:151–6.

    Article  CAS  PubMed  Google Scholar 

  18. Nagy A, Schally AV. Targeting of cytotoxic luteinizing hormone-releasing hormone analogs to breast, ovarian, endometrial, and prostate cancers. Biol Reprod. 2005;73:851–9.

    Article  CAS  PubMed  Google Scholar 

  19. Nagy A, Plonowski A, Schally AV. Stability of cytotoxic luteinizing hormone-releasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: implications for the design of preclinical studies. Proc Natl Acad Sci U S A. 2000;97:829–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR. Gonadotropin-releasing hormone receptors. Endocr Rev. 2004;25:235–75.

    Article  CAS  PubMed  Google Scholar 

  21. Cheng CK, Leung PC. Molecular biology of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and their receptors in humans. Endocr Rev. 2005;26:283–306.

    Article  CAS  PubMed  Google Scholar 

  22. Hultmark D, Steiner H, Rasmuson T, Boman HG. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 1980;106:7–16.

    Article  CAS  PubMed  Google Scholar 

  23. Steiner H, Hultmark D, Engstrom A, Bennich H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981;292:246–8.

    Article  CAS  PubMed  Google Scholar 

  24. Piotto SP, Sessa L, Concilio S, Iannelli P. YADAMP: yet another database of antimicrobial peptides. Int J Antimicrob Agents. 2012;39:346–51.

    Article  CAS  PubMed  Google Scholar 

  25. Seshadri Sundararajan V, Gabere MN, Pretorius A, Adam S, Christoffels A, Lehvaslaiho M, et al. DAMPD: a manually curated antimicrobial peptide database. Nucleic Acids Res. 2012;40:D1108–12.

    Article  PubMed  Google Scholar 

  26. Smolarczyk R, Cichon T, Kamysz W, Glowala-Kosinska M, Szydlo A, Szultka L, et al. Anticancer effects of CAMEL peptide. Lab Invest. 2010;90:940–52.

    Article  CAS  PubMed  Google Scholar 

  27. Hoskin DW, Ramamoorthy A. Studies on anticancer activities of antimicrobial peptides. Biochim Biophys Acta. 2008;1778:357–75.

    Article  CAS  PubMed  Google Scholar 

  28. Dennison SR, Whittaker M, Harris F, Phoenix DA. Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Curr Protein Pept Sci. 2006;7:487–99.

    Article  CAS  PubMed  Google Scholar 

  29. Papo N, Shai Y. Host defense peptides as new weapons in cancer treatment. Cell Mol Life Sci. 2005;62:784–90.

    Article  CAS  PubMed  Google Scholar 

  30. Mader JS, Hoskin DW. Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment. Expert Opin Investig Drugs. 2006;15:933–46.

    Article  CAS  PubMed  Google Scholar 

  31. Hilchie AL, Doucette CD, Pinto DM, Patrzykat A, Douglas S, Hoskin DW. Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts. Breast Cancer Res. 2011;13:R102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li XY, Li HL, Zheng GY. Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function. Zhonghua Fu Chan Ke Za Zhi. 2007;42:477–81.

    CAS  PubMed  Google Scholar 

  33. Arencibia JM, Bajo AM, Schally AV, Krupa M, Chatzistamou I, Nagy A. Effective treatment of experimental ES-2 human ovarian cancers with a cytotoxic analog of luteinizing hormone-releasing hormone AN-207. Anticancer Drugs. 2002;13:949–56.

    Article  CAS  PubMed  Google Scholar 

  34. Westphalen S, Kotulla G, Kaiser F, Krauss W, Werning G, Elsasser HP, et al. Receptor mediated antiproliferative effects of the cytotoxic LHRH agonist AN-152 in human ovarian and endometrial cancer cell lines. Int J Oncol. 2000;17:1063–9.

    CAS  PubMed  Google Scholar 

  35. Gunthert AR, Grundker C, Bottcher B, Emons G. Luteinizing hormone-releasing hormone (LHRH) inhibits apoptosis induced by cytotoxic agent and UV-light but not apoptosis mediated through CD95 in human ovarian and endometrial cancer cells. Anticancer Res. 2004;24:1727–32.

    PubMed  Google Scholar 

  36. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.

    Article  CAS  PubMed  Google Scholar 

  37. MTT RS. Methods in molecular biology. vitro toxicity testing protocols. Totowa: Humana Press Inc.; 1995.

  38. Ito E, Nei H, Noda M, Saito T, Koizumi M, Kudo R. Electron microscopic examination of cytologic samples. Acta Cytol. 1998;42:1095–103.

    Article  CAS  PubMed  Google Scholar 

  39. Li Q, Dong C, Deng A, Katsumata M, Nakadai A, Kawada T, et al. Hemolysis of erythrocytes by granulysin-derived peptides but not by granulysin. Antimicrob Agents Chemother. 2005;49:388–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bylund DB, Deupree JD, Toews ML. Radioligand-binding methods for membrane preparations and intact cells. Methods in molecular biology. Totowa: Humana Press Inc; 2004.

    Google Scholar 

  41. Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008;3:163–75.

    Article  CAS  PubMed  Google Scholar 

  42. Ritchie AA, Langdon SP. Estrogen-responsive ovarian cancer xenografts. Methods in molecular medicine. Totowa: Humana Press Inc; 2001.

    Google Scholar 

  43. Ming-fanga Y, Si-sunb L, Jiab H. Effect of Combination of Selenium Dioxide and Cisplatin on Human Ovarian Cancer Xenograft in Nude Mice [J]. Acta Academiae Medicinae Jiangxi. 2009;5:014.

    Google Scholar 

  44. Kajiyama H, Shibata K, Mizuno M, Umezu T, Suzuki S, Yamamoto E, et al. Long-term clinical outcome of patients with recurrent epithelial ovarian carcinoma: is it the same for each histological type? Int J Gynecol Cancer. 2012;22:394–9.

    Article  PubMed  Google Scholar 

  45. Lee YY, Kim TJ, Kim MJ, Kim HJ, Song T, Kim MK, et al. Prognosis of ovarian clear cell carcinoma compared to other histological subtypes: a meta-analysis. Gynecol Oncol. 2011;122:541–7.

    Article  PubMed  Google Scholar 

  46. del Carmen MG, Birrer M, Schorge JO. Clear cell carcinoma of the ovary: a review of the literature. Gynecol Oncol. 2012;126:481–90.

    Article  PubMed  Google Scholar 

  47. Matsuzaki K. Why and how are peptide-lipid interactions utilized for self defence? Biochem Soc Trans. 2001;29:598–601.

    Article  CAS  PubMed  Google Scholar 

  48. Henriques ST, Craik DJ. Importance of the cell membrane on the mechanism of action of cyclotides. ACS Chem Biol. 2012;7:626–36.

    Article  CAS  PubMed  Google Scholar 

  49. Gafter-Gvili A, Fraser A, Paul M, Vidal L, Lawrie TA, van de Wetering MD, et al. Antibiotic prophylaxis for bacterial infections in afebrile neutropenic patients following chemotherapy. Cochrane Database Syst Rev. 2012;1:CD004386.

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This study was supported by grant from the National Natural Science Foundation of China (No. 30700896 and No. 81270672).

Author information

Authors and Affiliations

  1. Department of Gynecology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Xiaoyong Li & Xinmei Zhang

  2. Institute of Radiation Medicine, Fudan University, Shanghai, China

    Bo Shen

  3. Central Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Qi Chen

  4. Department of Women Health Care, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Xiaohui Zhang

  5. Pharmacy Division, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Yiqing Ye & Fengmei Wang

Authors
  1. Xiaoyong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bo Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaohui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yiqing Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fengmei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinmei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xinmei Zhang.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

XYL conceived and designed the experiments and write most of the manuscript. BS carried out Radioligand Receptor Binding Assays and analyzed the data. QC performed experiments and analyzed the data. XHZ participated in study design and performed the statistical analysis. YQY, FMW carried out Antibacterial Assays. XMZ conceived of the study, and participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, X., Shen, B., Chen, Q. et al. Antitumor effects of cecropin B-LHRH’ on drug-resistant ovarian and endometrial cancer cells. BMC Cancer 16, 251 (2016). https://doi.org/10.1186/s12885-016-2287-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12885-016-2287-0

Keywords

  • Luteinizing hormone releasing-hormone receptor
  • Cecropin B peptide
  • Ovarian cancer
  • Endometrial cancer

BMC Cancer

ISSN: 1471-2407

Contact us