Antitumor Activity and Mechanism of Action of the Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide

doi:10.21203/rs.3.rs-757762/v1

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Antitumor Activity and Mechanism of Action of the Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide

https://doi.org/10.21203/rs.3.rs-757762/v1

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published 20 Oct, 2021

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Prostate cancer represents the most common cancer in men. For patients with advanced or metastatic form, treatments will be able to slow down the progression but cannot cure it even with the used of new targeted therapies. In this context, the development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2 (DRS-B2), a natural multifunctional antimicrobial peptide isolated from the Amazonian frog skin, has been reported to possess antitumor and antiangiogenic activities. To improve DRS-B2 pharmacological properties and target prostate tumor cells more specifically, we have developed a chimeric molecule, called Hormonotoxin (H-B2) which is composed of a DRS-B2 combined with a hormonal analog, d-Lys⁶-LHRH, to target LHRH-Receptor which is overexpressed in more than 85% of prostate cancers. In vitro H-B2 has a significant antiproliferative effect on the PC3 tumor cell line, with an IC₅₀ value close to that of DRS-B2. The antitumor activity of H-B2 was confirmed in vivo in mouse model xenografted with PC3 tumors and appears to be better tolerated than DRS-B2. Biophysical experiments showed that the addition of the hormonal analog to DRS-B2 did not alter either its secondary structure or its biological activity. Combination of different experimental approaches indicated that H-B2 induces cell death by an apoptotic mechanism whereas DRS-B2 was shown to induce it by necrosis. These results could explain the H-B2 less toxicity compared to DRS-B2. H-B2 represents a promising targeting approach for cancer therapy.

Scientific Communication

General Microbiology

anticancer drug

antimicrobial peptide (AMP)

dermaseptin

frog skin peptides

LHRH

prostate cancer

Phyllomedusa bicolor

therapeutic peptides

Prostate cancer (PCa) is the second most frequent cancer diagnosis made in men and the fifth leading cause of death worldwide in 2018 ¹. For most men with PCa, their disease will follow an indolent course. The 5-year survival rates are encouraging: 98% and 83% in the USA and Europe, respectively ². Localized PCa may be cured with surgery or radionuclide therapy, however, the disease recurs in approximately 20 to 30% of men treated for localized PCa and advanced disease is associated with poor outcomes. Concerning chemotherapy, the current standard treatment for hormone sensitive metastatic PCa is androgen deprivation by luteinizing hormone-releasing hormone (LHRH) agonists or antagonists that induce castration by blocking the gonadotropic axis. Although most men with metastatic PCa initially respond to this Androgen-Deprivation Therapy (ADT), inevitably their cancer progresses on this treatment to a disease state known as castration-resistant prostate cancer (CRPC). Despite the appearance of a panel of new therapies in this indication since 2010, such as second-generation hormone therapy (abiraterone, enzalutamide), taxane-based chemotherapy (docetaxel, cabazitaxel), immunotherapy (sipuleucel-T), targeted therapy (ipilimumab, an anti-CTLA-4 antibody) and metabolic radiotherapy (Ra-223) for men with bone metastasis, the median survival for men with metastatic castration-resistant prostate cancer (mCRPC) is less than 2 years ². In this context, the development of innovative therapies therefore represents a major challenge in the hope of offering to patients a more effective treatment combining low toxicity, reduced side effects and resistance associated with conventional therapies.

Among these new molecules, those from biodiversity could represent a particular interest. A group of interesting peptides from natural sources are antimicrobial peptides (AMPs) ³. Indeed, in recent years, an increasing number of articles show that these AMPs are in fact multi-functional peptides such as anti-cancer agents, immuno-modulators, chemokines, vaccine adjuvants, or regulators of innate defense ^4–6. From this group of AMPs, the cationic antimicrobial peptides (CAPs) offer a concrete development path derived from biodiversity. Characterized for nearly thirty years, they were initially studied for their antimicrobial virtues. Indeed, a growing number of studies have shown that some of these peptides, which are toxic to bacteria but not to normal mammalian cells, have a broad spectrum of cytotoxic activity against cancer cells. Electrostatic interactions between the positive charges carried by CAPs and the anionic components of cell membranes are considered to be the major elements involved in the selective destruction of cancer cells ⁷. Among CAPs, dermaseptins B2 and B3 (DRS-B2 and DRS-B3) are 2 natural antimicrobial peptides isolated from the skin of an Amazonian tree frog of the genus Phyllomedusa bicolor ^8,9. Our team has initially reported significant antitumor activity of DRS-B2 and DRS-B3 on various human cell lines including prostate cancer while they have no effect on normal cells ^5,10. Furthermore, the anti-tumor activity of DRS-B2 was confirmed in vivo in PC3 prostatic tumor cell line xenografted in athymic mice model. Treatment with DRS-B2 at 2.5mg/kg body weight six times a week in peritumoral reduced the tumor growth of 50% after 35 days. Concerning the mechanism of action of DRS-B2, our previous studies suggested a rapid mechanism of cell death, with aggregation at the plasma membrane of cancer cells and penetration into the cytoplasm and the nucleus ^5,11.

These polycationic peptides, that appear to interact and specifically cross the membrane of the tumor cells, and with no effect on normal cells represent an innovative technological platform for the development and design of original molecules that can be used in the targeted treatment of cancers resistant to current therapies. However, its in-vivo use could question the problem of significant toxicity regarding the doses used. To reduce the toxicity of pharmacological molecule, the targeted therapy could represent a promising way. The concept of hormonotoxin (H-B2) was born from this problem, based on tumor targeting by associating DRS-B2 with a hormone (H) whose receptor is over-expressed on the tumor surface. This concept is related to the immunotoxin approach, which combines a toxin with a monoclonal antibody ¹². The specific interaction of the ligand with its receptor would ideally allow targeting tumor cells while optimizing the interaction of the peptide with the membrane. The advantage of peptides is the simplicity of their production by chemical synthesis. Numerous studies have shown that many membrane receptors are over-expressed on the surface of cancer cells and that there are natural or synthetic peptide ligands (agonists or antagonists) that bind to them with very good affinity and selectivity ¹³. This is the case of the luteinizing hormone-releasing hormone receptor (LHRH-R), whose expression rate is greater than 80% in endometrial, ovarian and prostate cancer cells ¹⁴. Available data strongly suggest that about 91% of prostate cancers express LHRH-R high-affinity binding sites ¹⁵. In these cancers, in vitro proliferation may be inhibited by the analogues of LHRH in a dose- and time-dependent manner ^15–17. As a result, LHRH-R appears to be an ideal target for the development of personalized therapeutic treatment of various human cancers. An example is the fusion of LHRH or its analogues with various bacterial and plant toxins that have been used to target and kill cancer cells expressing LHRH receptors ^18–22.

In this paper we reported the design and synthesis of the chimeric peptide H-B2, a molecule composed of dermaseptin-B2 coupled to an LHRH analogue, and studies of its structure and biological activities in vitro and in vivo, as well as the deciphering of its antitumor mechanism of action.

The Hormonotoxin H-B2 induces a significant antiproliferative effect on the hormone-resistant prostate tumor cell line PC3 and has a low hemolytic activity.

Preliminary experiments were performed to investigate the existence of a correlation between LHRH receptor expression and the effect of H-B2. For this purpose, we used four different prostate cell lines (PC3, DU145, 22RV1 and BPH-1) which are presented in Table 1. The PC3, DU145, and 22Rv1 cell lines were used as models for androgen-independent prostate cancer. PC3 and DU145 were derived from bone and brain metastasis, respectively. 22Rv1 cells were originally derived from a primary site of an advanced PCa that was serially transplanted in nude mice and BPH-1 is a prostatic hyperplasic cell line. Western blot analysis indicated that LHRH-R is strongly expressed by metastatic PC3 and DU145 cells, but its expression is very low in 22Rv1 and absent in BPH-1 cells (Fig. 2A). These results are in agreement with studies indicating the overexpression of LHRH-R in human prostatic cancer cells and then could be used to evaluate the anti-proliferative effect of H-B2 ¹⁷. Dose-response of H-B2 and DRS-B2 after 48 h of treatment was evaluated on the different prostate cell lines expressing or not LHRH-R as indicated in Fig. 2A.

Table 1

List of cell lines and their characteristics
Cell line	Characteristics
PC3	Adenocarcinoma derived from bone metastasis
DU145	Adenocarcinoma derived from brain metastasis
22Rv1	Adenocarcinoma derived from a PCa primary tumor that was serially transplanted in nude mice.
BPH-1	Benign Prostatic Hyperplasia Cell Line

Results show that the viability of all cell lines was reduced dose-dependently by DRS-B2 and H-B2 (Fig. 2B). The concentrations of peptides required to inhibit 50% (IC₅₀) of cell proliferation are reported in Fig. 2C. Moreover, comparison of the IC₅₀ shows that H-B2 was slightly more potent than DRS-B2 against cancer cells that express LHRH-R compared to BPH-1 cells that do not express LHRH receptor (Fig. 2B, 2C). Since PC3 cells present a relatively good sensitivity to H-B2 and DRS-B2, we decide to use this cell line for further investigation concerning the in vivo study of H-B2 and its antiproliferative mechanism of action.

To assess the functional role of LHRH-R on the H-B2 antiproliferative activities, LHRH-R was knocked down in PC3 cells. The expression level of LHRH-R was validated by WB analysis (Fig. 3A). The viability test showed that knockdown of LHRH-R in PC3 cells slightly decreased the sensitivity of PC3 cells to H-B2 as compared to si-control or parental PC3 cells who express LHRH-R (Fig. 3B). The calculated IC₅₀ was 3.73 µM for siLHRH-R PC3 compared to 2.95 µM for si-control PC3 and 3.05 µM for PC3 parental cells.

Collectively, these results indicated that the antiproliferative activity of H-B2 is higher in cells which express LHRH receptor, such as prostate cancer cells.

Since CAPs could present hemolytic activity, H-B2 and DRS-B2 were tested on human red blood cells. The hemolytic activity was determined by incubation of DRS-B2 or H-B2 at different doses with human erythrocytes. In parallel, the erythrocytes were incubated in the presence of 0.2% (v/v) Triton representing the positive control (100% hemolysis). Results presented in Fig. 4 show that DRS-B2 and H-B2 have a low hemolytic activity. Indeed, while some cationic antimicrobial peptides show hemolytic activity, DRS-B2 and H-B2 do not cause more than 10% hemolysis at the highest doses tested (50 µM) and could be considered as low cytotoxicity peptides. This finding is encouraging and essential for in vivo testing and for potential therapeutic use.

Hormonotoxin H-B2 significantly inhibits tumor growth in xenografted mice without measurable side effects.

To investigate the antitumor efficacy of the H-B2, we performed in vivo experiments using xenografting PC3 tumor cells in nude mice. Fourteen days after injection of PC3 cells, tumors of around 100 mm³ were developed. The mice were then randomized into four groups (n = 6 mice/group) and treated by IP injection of 2.5 and 5 mg/kg of H-B2 or 2.5 mg/kg of DRS-B2 or vehicle, 3 times per week for five weeks. The results showed that H-B2 has inhibited PC3 tumor growth in a dose-dependent manner (Fig. 5). The inhibition of the tumor growth was about 35% and 54% when mice were treated with H-B2 at 2.5 mg/kg and 5 mg/kg, respectively (Fig. 5A). At the dose of 2.5 mg/kg, H-B2 has a better antitumor effect (35%) than DRS-B2 (26%) (Fig. 5A). Similar results were obtained by analyzing the weights of tumors harvested from tumor-bearing mice (Fig. 5B).

To further define the effect of H-B2 on tumor proliferation in vivo, Ki67 labeling was performed on tumor sections from each group. As shown in Fig. 5C, the proliferation index was significantly inhibited in H-B2-treated mice (35% inhibition at 2.5 mg/kg and 68% at 5 mg/kg) as compared to the control group.

Taken together, these results indicate that HB2 has a better antitumor effect than DRS-B2 and less toxicity in mice (data not shown).

Mechanism of antitumor action of the Hormonotoxin H-B2 in comparison with DRS-B2

The mechanism of antitumor action of these two peptides was addressed by studying, on the one hand, their bioactive structure by three spectroscopic approaches, CD, fluorescence, and 2D-NMR in the presence of micelles mimicking the plasma membranes of the target cells and, on the other hand, by measuring the cell viability after double annexin V-FITC and PI staining, the cytotoxic activity by measuring the cytoplasmic LDH released, and the DNA fragmentation induced by these peptides.

Addition of the hormonal analogue (d-Lys ⁶ -LHRH) to the DRS-B2 did not alter the secondary structure of the hormonotoxin H-B2.

Preliminary indications of the peptides secondary structures obtained by CD measurements in PBS and in zwitterionic detergent (DPC) at different concentrations are shown in Fig. 6. The CD spectrum of DRS-B2 and H-B2 in PBS buffer showed that these peptides have very little ordered structure. However, there is a clear change in the spectra of these two peptides as soon as the critical micellar concentration (CMC) of DPC is reached (Fig. 6A.1 for DRS-B2 and 6B.1 for H-B2) that indicates an enhanced helical content, with minima at 208 and 222 nm. Deconvolution of the spectra allows us to quantify the relative proportions of the secondary structures of our peptides. Thus, in the absence of a micelle in the medium, the observed α-helix ratio is about 11% for DRS-B2 and 10% for HB2 as shown in Fig. 6A.2 and 6B.2, respectively. When the concentration of DPC is increased to 5 mM, the α-helicity rates then reach 25% and 55% for H-B2 and DRS-B2 respectively.

The presence of a W residue in the sequence of DRS-B2 and H-B2 allows us to study the influence of the microenvironment on the structure of these two peptides. Indeed, the fluorescence emission maximum of W is between 320 and 355 nm when excited at 290 nm, and the maximum emission wavelength reflects the exposure of W to the solvent. This fluorescence is measured in aqueous solution (PBS 1x) for observation in a non-structural environment (the peptides does not form an α-helix in water) and in micellar solution to study the effect of a lipid-like microenvironment (Fig. 6A.3 and 6B.3). We observe that beyond 1 mM, which is the CMC of DPC, the fluorescence emission maxima of DRS-B2 and H-B2 shift to shorter wavelengths ("blue shift") and show a strong increase in fluorescence intensity (hyperchromic shift). These spectral changes reflect a change from hydrophilic to hydrophobic environment that can be explained either by the burial of the W residue within the hydrophobic layers of DPC micelles, or by burying the W after folding of the peptide following its conformational changes.

Finally, the structure of H-B2 was investigated using NMR in micellar DPC solution. This technique allows a direct analysis of peptide conformation and flexibility at the residue level. For each residue of the peptide chain, the chemical shift deviation (CSD) of the Hα protons were calculated (Fig. 7A). This corresponds to the difference between the observed chemical shift of the Hα protons and the "random coil" chemical shift (obtained in the absence of structuring and corresponding to values taken from a library of short unstructured peptides). CSD values which deviate from 0 ppm (< − 0.1 or > + 0.1 ppm) are typically used to detect the presence of secondary structures. In the DRS-B2 portion of hormonotoxin H-B2, most residues exhibit significantly negative Hα CSD values (average − 0.23 ppm), indicating the presence of stable helical conformations, as evidenced by CD. H-B2 forms an α-helix along virtually the entire length of the DRS-B2 part (residues 2–31), with greater stability in the first half of the helix. The CSD values are very close to those observed for DRS-B2 alone, proving that the C-terminal attachment of the LHRH segment does not affect the helical structure of the DRS-B2 segment. On the other hand, the LHRH segment appears to be less structured, with possibly the formation of turn structures.

The linewidth of HN protons was also analyzed by measuring the intensity of ¹⁵N-¹H crosspeaks on natural abundance ¹⁵N-¹H 2D HSQC spectrum (Fig. 7B). The proton linewidth is influenced by peptide dynamics and interaction with the larger molecular weight DPC micelle. The LHRH segment shows a different behavior from the DRS-B2 segment in H-B2, with stronger ¹⁵N-¹H crosspeaks intensities. This shows that the LHRH segment is more dynamic and does not appear to strongly interact with the micelle.

The cell death induced by H-B2 and DRS-B2 uses two different mechanisms that are related to apoptosis and necrosis, respectively.

To obtain more information about how H-B2 acts on PC3 tumor cells, the anti-proliferative mechanism of action of this peptide was studied in comparison with that of DRS-B2. Since the known anti-proliferative effect of DRS-B2 on tumor cells resembled more to a membrane killing-like effect, experiments concerning the cytotoxic effect, the induction of apoptosis or necrosis were investigated with H-B2 in comparison with DRS-B2.

A key feature of cells undergoing apoptosis, necrosis, and other forms of cellular damage could be analyzed by measuring the activity of cytoplasmic lactate dehydrogenase (LDH) released by damaged cells. Previously, we have shown that DRS-2 increased this release ¹¹. Thus, PC3 cells were treated with different concentrations (1, 5, 7.5 and 10 µM) of H-B2 or DRS B2 for 24 h and the amount of released cytoplasmic LDH into the medium was measured (Fig. 8). Cells treated with Triton X100 0.9% (v/v) were used as an internal positive control. The results show a low release of cytoplasmic LDH when the PC3 cells are treated with 1 µM of H-B2 or DRS-B2 with 15 and 10% release respectively. The LDH release is maximal upon addition of 5 µM or higher doses of these peptides. However, this maximum release was limited to 65–70%. These data suggested that the cytotoxic effect of H-B2 on PC3 cells is comparable to that of DRS-B2.

To further characterize the effect of H-B2 and DRS-B2 on cell death in PC3 cells, we first examined the viability of cells treated with these peptides by flow cytometry using annexin V-FITC and PI double staining of the cells. As shown in Fig. 9, flow cytometry analysis showed that H-B2 promoted cell apoptosis in a dose-dependent manner. After being treated with H-B2 for 24 h, 4%, 15%, and 63% of double staining A-V⁺/PI⁻ which corresponds to early apoptotic cells were found in PC3 cells treated with 1 µM, 2.5 µM, and 5 µM, respectively. These values were significantly higher than that of untreated cells (1%) and similar (63.5%) to cells treated with H₂O₂ used as a positive control. DRS-B2 presented a lower effect with only 5.5% of double staining A-V+/PI- when PC3 cells were treated with DRS-B2 at 2.5 µM, which agrees with results described by Van Zoggel et al ¹¹.

DNA fragmentation represents the final stage of apoptosis. To examine apoptosis through DNA fragmentation induced by H-B2 or DRS-B2, we used the terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay, in which DNA strand breaks are detected by enzymatic labeling of free 3'-OH ends with modified nucleotides.

The analysis of DNA fragmentation in PC3 cells by the TUNEL technique shows that the percentage of fragmented DNA increases with H-B2 treatments. Treatment of PC3 cells with 5 µM H-B2 induces a DNA fragmentation level of 69% compared to untreated cells (1.5%), whereas it reaches only 9% when treated with the same concentration of DRS-B2. (Fig. 10). PC3 cells treated with 10 nM Taxotere or 1 µM of Staurosporin as positive controls show 96% and 86% DNA fragmentation, respectively (Fig. 10). These data confirm that the mechanism of PC3 cell death induced by H-B2 is different from that induced by DRS-B2, which is necrotic, and could rather correspond to an apoptotic mechanism, in agreement with the flow cytometry results described previously.

With 1.1 million prostate cancers diagnosed worldwide in 2012, of which approximately 6% are metastatic from the start, the development of treatments for mCPRC is a global necessity with a promising market. The development of treatments for the management of castration-resistant metastatic patients is necessary because currently available treatments only allow a few months of survival gain mainly due to problems of drug resistance of cancer cells. In this context, the use of natural or synthetic peptides could offer interesting therapeutic approaches. To limit peripheral toxicity and increase local concentration, numerous cytotoxic molecules conjugated with peptide hormones such as LHRH or somatostatin whose receptors are widely overexpressed on the surface of tumor membranes have been developed ^23–27. Currently, only one cytotoxic peptide is in clinical development (Phase 2 completed), AEZS-108 (zoptarelin doxorubicin) ²⁸.

Starting from our previous studies on the anticancer activity of the CAP dermaseptin-B2 (DRS-B2), a natural peptide issued from the biodiversity, we have developed a synthetic chimeric H-B2 peptide combining DRS-B2 and LHRH peptides ^5,10,11. The use of LHRH was justified by the fact that 86% of prostate cancers express the LHRH receptor. A peptide targeting this receptor therefore should increase the specificity for cancer cells and decrease the in vivo toxicity of DRS-B2.

At the structural level, the coupling of the peptide hormone (H) LHRH to the C-terminus of DRS-B2 does not modify the structure of DRS-B2 nor its conformational behavior in a membrane environment. The characteristics essential to the cytotoxic mode of action of these polycationic peptides and to the targeted molecular interaction are therefore preserved. CAPs are supposed to act on cancer cells in which the outer layer of the plasma membrane is highly negatively charged as for bacteria plasma membranes. In the case of cancer cells, the negatively charged membrane is due to the presence of phosphatidylserine, negatively charged mucin proteins or highly sulfated GAGs (5, 25). After binding dermaseptin peptides accumulate in a carpet like manner on the outside of a lipid bilayer until a threshold concentration is reached, causing them to form pores in which the peptides are inserted with the phospholipid headgroups of the membrane. We have shown that H-B2 was structured as a continuous helix in its "toxin" portion (DRS-B2) and that the "hormone" ligand portion remains free to interact with LHRH-R.

In vitro, H-B2 was slightly more effective than DRS-B2 on the proliferation of the various prostate cancer cell lines tested. Even if the specificity of interaction with LHRH-R could not be demonstrated physically, the knock down expression of the LRHR receptor by RNA interference on PC3 cells slightly decreases the efficiency of H-B2, thus pointing out that addition of the peptide hormone to the DRS-B2 was benefit. We could also mention that cells expressing high level of LHRH receptor like PC3 and DU145 presented a modest but significative gain in IC₅₀ when compared with those of DRS-B2 alone. Targeting cancer cells with an overexpressed receptor like LHRH is a strategy that could permit to improve the efficiency of a peptide like DRS-B2 to gain in its ED₅₀. However, in the case of H-B2, this gain is low. We could imagine that even if the LHRH receptor is overexpressed on PC3 cells, its concentration at the surface of the plasma membrane is too low to guide enough DRS-B2 to membrane to complete its carpet like structure to further form pores in the plasma membrane. Since saturation of the LHRH receptor by H-B2 could not be sufficient to kill cells, therefore, additional part of DRS-B2 in H-B2 structure could be necessary to do it.

In vivo H-B2 inhibited tumor growth by more than 50% and significantly decreased proliferation without any major side effects. Since the addition of the hormone peptide to the DRS-B2 permits a better tolerance when injected in mice, we could conclude that despite significant improvement in IC₅₀ on the cell line, the hormone peptide improved the in vivo activity of DRS-B2 since we could treat mice with 5 mg/kg H-B2 without side effects instead of 2.5 mg/kg with DRS-B2. It is interesting to note that the treatment of prostate tumor cells with H-B2 is globally well tolerated. We did not observe any major abnormalities in the blood workup after H-B2 injection. Importantly, at a dose of 10 mg/kg, DRS-B2 is lethal while H-B2 at the same dose was well tolerated in toxicity tests, with no behavioral changes in the mice or weight loss. The design of the H-B2 chimeric peptide thus made it possible to circumvent the toxicity of DRS-B2 while maintaining its anti-tumor efficacy.

Concerning the mechanism of action, the combination of different experimental approaches used in this study such as cell viability by flow cytometry, cytotoxicity by cytoplasmic LDH release, and DNA fragmentation by TUNEL essay allowed us to show that the mechanism of cell death induced by H-B2 is probably different from that of DRS-B2 and might be similar to apoptosis. Indeed, we showed that both peptides have the same effect on cytoplasmic LDH release but a distinctly different cell labeling when cell viability is analyzed by flow cytometry and DNA fragmentation by TUNEL assays. The differences between the effect of the two peptides observed in the TUNEL assay show that H-B2 induces DNA fragmentation up to 70% in contrast to DRS-B2 which is less than 10%. These results are also in agreement with those obtained in 2012 by Van Zoggel et al who showed a double Annexin V+/PI+ labeling of PC3 cells treated with DRS-B2, suggesting a necrotic cell death mechanism ¹¹.

Apart from DRS-B2 previously studied in the laboratory, two new members of the dermaseptin family have been recently reported to exhibit antitumor activities: Dermaseptin-PP from Phyllomedusa palliata and Dermaseptin-PT9 from Phyllomedusa tarsius ^29,30. Both exhibited antiproliferative activity against various human tumor cells, rapid LDH release activity, and an apoptosis-like cell death mechanism. Among these dermaseptins, the DRS-B2 presented the higher ED₅₀ on various tumor cells.

Finally, the study of the hemolytic effect of the different peptides allowed us to observe that both DRS-B2 and H-B2 have a low hemolytic activity, which is encouraging in the perspective of a therapeutic approach. Further in vivo studies on mice could be necessary to obtain more pharmacokinetic and toxicology information to complete the study.

Peptide synthesis.

The LHRH analogue containing a dK residue (d for D configuration of the Lys residue) in position 6 (peptide A: pEHWSY(dK)LRG-amide) and DRS-B2 (peptide B:

GLWSKIKEVGKEAAKAAAKAAGKAALGACSEAV-acid) were synthesized by X'PROCHEM Company (Lille, France). The chimeric peptide H-B2 (Fig. 1A) composed of the peptide A grafted by its epsilon NH₂ of the dK⁶ residue with the COOH-terminal of the peptide B is determined to be >95% pure by RP-HPLC (Fig. 1B) and its molecular weight determined by ESI-mass spectrometry is [M+4H]/4=1105.2 (Fig. 1C). The peptide is dissolved in sterile water for experimental use.

Circular dichroism (CD) spectroscopy of peptides.

The helical structure of HB2 and DRS-B2 were analyzed by CD spectroscopy using a Jobin Yvon CD6 dichrograph linked to a PC micro-processor as described ³¹. Briefly, measurements were calibrated with (+)-10-camphorsulfonic acid and performed with 10 mM HB2 or DRS-B2 diluted in PBS alone or with increasing concentrations of dodecylphosphocholin (DPC) (10, 30, 100, 1000 and 5000 μM) at 25 ̊C using a quartz cuvette (Hellma) with a path length of 0.1 cm. Spectra, recorded in 1 nm steps, were averaged over five scans, and corrected for the base line. The CD spectra were deconvoluted using CDNN Software ³². Circular dichroism measurements are reported as Δε/n, where Δε is the dichroic increment (M^-1 cm^-1) and n is the number of residues in the peptide. The α-helix content of peptides was obtained using the relation: Pα = -[Δε_222nm x10] (Pα: percentage of α-helix; Δε_222nm: dichroic increment per residue at 222 nm) ³³.

Fluorescence of tryptophan-containing peptides

performed as previously described by Dos Santos et al.⁵ Emission spectra were recorded on a Jobin-Yvon Fluoromax II instrument (HORIBA Jobin-Yvon, France) equipped with an Ozone-free 150 W xenon lamp. The excitation wavelength was 290 nm, and the emission spectra were acquired at 300–360 nm. At least five measurements for each titration point were recorded with an integration time of 1 s. The HB2 and DRS-B2 concentration in PBS was 2 μM, and the DPC concentration varied from 0 to 1000 μM. Tryptophan fluorescence was determined by subtracting spectra without peptide.

Nuclear Magnetic Resonance (NMR) Spectroscopy.

The NMR sample was prepared in 550 μL of H₂O/D₂O (90:10 v/v) in the presence of 85 mM DPC-d₃₈(d₃₈ -dodecylphosphocholin, MAPCHO^â-12-d₃₈, Avanti Polar Lipids) and using a peptide concentration of 1 mM. The pH was set to 4.1 using microliter amounts of NaOH 0.1 M. DSS was added at a concentration of 0.11 mM for chemical shift calibration. The NMR spectra were acquired at 45°C on a 500 MHz (11.7 T) Avance III Bruker^âspectrometer equipped with a TCI cryoprobe. The following experiments were recorded: 2D ¹H-¹H TOCSY (66 ms mixing time), 2D ¹H-¹H NOESY (150 ms mixing time), 2D natural abundance ¹⁵N-¹H and 2D ¹³C-¹H HSQC experiments, as described ³¹.

Cell culture.

Human PCa cell lines PC3, DU145 and 22Rv1 were purchased from ATCC (American Type Culture Collection and were cultured in RPMI supplemented with 10% of fetal bovine serum (FBS). Human hyperplasic cell line BPH1 was purchased from the German Collection of Cell Cultures (DSMZ, Braunschweig, Germany). BPH1 were maintained in RPMI 1640 supplemented by 10% FBS, 20 ng/mL testosterone and 1% insulin-transferrin-selenium. Cell cultures were maintained at 37°C and 5% CO₂in humidified atmosphere. All culture reagents were purchased from Life Technologies (Cergy-Pontoise, France). All experiments were performed as previously described by Dos Santos et al.⁵

Cell-viability assays.

Cells were seeded at a density of 5×10³ cells/well in 96-multiwell plates in complete medium and incubated for 24 h at 37˚C in a controlled humidified 7% CO₂ environment. Cells were then treated with DRS-B2 or HB2 as indicated, for 48 h. Cell viability was measured using the 3-(4,5-dimethylthiazol2-yl)-diphenyltetrazolium bromide (MTT) dye method (Sigma, Saint Quentin Fallavier, France) according to the manufacturer’s instructions. Each experiment was performed in triplicate and at least three independent experiments as previously described by Dos santos et al.⁵. IC₅₀ values were determined by GraphPad Prism 5.0 (GraphPad Software, USA).

Small Interference RNA (siRNA) Assay.

PC3 cell silencing of LHRH-R were performed using Lipofectamine RNaiMax (Invitrogen) according to the manufacturer’s protocol as previously described by Elahouel et al.³⁴. Predesigned siRNAs of either non-targeting siRNA (GGCUACGUCCAGGAG CGCACCTT) as a control or target-specific siRNAs (AAGCAUGGAUUGGAUCAGUAATT) to knock-down LHRH-R were obtained from Eurofins Genomics. Briefly, cells were plated overnight at 50-60% confluence, then transfected with 10 nM of either non-targeting siRNA or LHRH-R target-specific siRNAs, in serum- and antibiotic-free Opti-MEM medium (Invitrogen). After 24 h, transfected cells were treated with HB2 as indicated for 24 h. In addition, transfection efficiency was evaluated by Western blot analysis 24-48 h after transfection.

Western blotting

was performed as previously described by Elahouel et al.³⁴ Cell lysates were prepared with RIPA extraction buffer containing anti-protease inhibitors (Roche). Equal amounts of total proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, then transferred onto polyvinylidene difluoride membrane (Millipore) using standard protocols. Nonspecific binding was prevented by incubating the membranes in 5% (w/v) nonfat milk in PBS containing 0.2% (v/v) Tween-20 (blocking buffer (BB) for 1 h and then incubated with primary antibodies in diluent (5% nonfat milk in TBS-Tween) for 1 h. Membranes were subsequently incubated overnight at 4°C with antibodies, rabbit anti- GnRH-R (Abcam,183079 ; 1 : 2000) or mouse anti- β-actin (SantaCruz Biotechnology; 1 : 2000) and then with HRP-conjugated secondary antibody for 1h at RT (Cell Signaling Technology ; 1:2000). Immunocomplexes were revealed using a chemiluminescence detection system kit “SuperSignal™ West Pico PLUS” (Thermoscientific) and visualized using the GBox system (Syngene).

Hemolysis assessment.

The hemolytic activity of H-B2 and DRS-B2 was determined using fresh human erythrocytes from a healthy donor that was prepared as follows: 4 mL of whole blood was collected and centrifuged at 900 g for 10 min at 4°C to separate plasma from erythrocytes. The pellet was then rinsed with PBS pH 7.4 and centrifuged at 900 g for 10 min at 4°C. After counting and making a red cell solution at 4 × 10⁸ erythrocytes/mL (diluted in PBS pH 7.4), 50 µL of a diluted peptide solution was added in cascade to which 50 µL of the erythrocyte solution was added (made in triplicate). After 1 hour of incubation at 37°C, the tubes were centrifuged at 12 000 rpm for 15 seconds at 4°C. The supernatant was then recovered. The hemoglobin present in the erythrocytes was determined in the supernatants via a plate reader at 450 nm. A parallel incubation in the presence of 0.2% (v/v) Triton was carried out to determine the absorbance associated with 100% hemolysis.

Tumor xenograft Studies

were performed as previously described by Van Zoggel et al.¹¹. PC3 (2 × 10⁶) cells were injected subcutaneously into the right flank of 4-week-old male NMRI nude mice (Janvier, Le Genest-Saint-Isle, France). When the tumor volume reached approximately 100 mm³, the mice were randomly divided into four groups (n=6): control (PBS), DRS-B2 (2.5 mg/kg), H-B2 (2.5 mg/kg), H-B2 (5 mg/kg) by intraperitoneal injection twice a week. Tumor size was measured two times per week with a caliper and tumor volume was calculated with the formula: V= 4/3π*R1²*R2 whereby radius 1 (R1), radius 2 (R2). At the end of the experiment, the mice were sacrificed, and body weight was measured. The tumors were isolated, weighted and then fixed in formalin.

Ethics statement.

All mouse experiments were performed according to ARRIVE guidelines on animal care and approved by Charles Darwin Animal Experimentation Ethics Committee (CEEACD/N°5) and conducted in compliance with European Community.

Immunohistochemical analysis of the tumors

were performed as previously described by Van Zoggel et al.¹¹. The fixed tumors were embedded in paraffin and then 6 µm sections were prepared. The tumors sections were deparaffinized, antigen unmasking was performed, and endogenous peroxidase activity was inactivated with a 2% hydrogen peroxide solution for 10 minutes. Unspecific staining was blocked using Power Block Universal reagent (Biogenex Laboratories/Microm Microtech, Francheville, France) for 10 min at 37°C. Tissues were then incubated 2 hours at room temperature with anti-human Ki67 antibody (Mouse monoclonal, M7240, Dako, 1:50) for 2 hours. Immuno-complexes were revealed using HRP conjugated secondary antibodies and the DAB substrate. Tissues were then counterstained with hematoxylin and cover slipped with Mowiol mounting medium. Quantification of Ki67 positive stained cells was quantified by image J software analysis on the whole tumor section.

Lactate dehydrogenase (LDH) release assay.

The cytoplasm LDH release assay was performed as previously described ¹¹. Briefly, PC3 cells were grown in a 96 well plate (1,500 cells/well/100 μL) in complete medium and treated with various concentrations of H-B2 or DRS-B2. Cell membrane integrity was evaluated by measuring the LDH activity released into the culture media 24 hours after peptide exposure. The CytoTox96 non-radioactive cyto- toxicity assay (Promega; Charbonnières-les-Bains, France) was performed according to the manufacturer’s instructions and quantified by measuring the absorbance at 490 nm. The 100% cytotoxicity corresponds to the LDH released with treatment of the cells with Triton X100 at 0.9% (v/v).

Apoptosis analysis by flow cytometry.

For the apoptosis assay, an FITC-Annexin-V (A-V) and Propidium Iodide (PI) double staining method was used. PC3 cells were grown in a 12-well plate and treated or not with H-B2, DRS-B2 or hydrogen peroxide as described previously ⁵. 24 hours after exposure, cell viability was evaluated by flow cytometry analysis with FITC-Annexin-V (A-V) and propidium iodide staining. Medium and trypsinized cells were collected and washed with PBS. After centrifugation, cells were suspended in PBS to obtain a cell density of 0.5 × 106 cells per mL. One mL of this cell extract was centrifuged, suspended in 200 μL PBS, transferred to a microtiterplate with round bottom and centrifuged again. Resulted cell pellet was resuspended in 200 μL of Binding Buffer 1x (BD PharmingenTM), containing 5 μL of FITC-Annexin-V (BD PharmingenTM) and incubated during 10 minutes in dark at room temperature. The cells were washed with PBS and incubated with 200 μL of Binding Buffer 1x containing PI (final concentration 1 μg/mL) (BD Pharmin-genTM)) for 5 minutes in dark at room temperature. Flow cytometry analysis was performed with LSR Fortessa X20 analyzer (BD Biosciences) and FlowJo V10 software.

Terminal transferase-mediated dUTP Nick End-Labelling (TUNEL) assay.

PC3 cells were cultured in 6-well plates at 150,000 cells per well in complete medium and treated or not with 1 or 2.5 µM of DRS-B2 or H-B2 and with LHRH (5 mM) or Taxotere (10 nM) and Staurosporin (1 mM) as positive control. After 24 hours of treatment, the supernatant was aspirated and the cells trypsinized and plated on a Superfrost PLUS slide and fixed with a 4% formaldehyde solution without methanol. The cells were then permeabilized with a 0.2% Triton X-100 solution in 1X PBS for 5 minutes at room temperature. Terminal transferase-mediated dUTP nick end-labelling (TUNEL) was performed according to the DeadEnd™ Fluorometric TUNEL System kit from Promega®. In addition to the fluorescein labeling, a second labeling with DAPI, intercalating DNA was performed by incubating for 15 minutes at room temperature with a 1 µg/mL solution. The different labelling steps were performed avoiding any exposure to light.

Imaging was performed with an Axiolmager M2 epifluorescence microscope. The images obtained by microscopy were recorded with the Zen 2012 software and analyzed with the ImageJ© software. Each image underwent a thresholding of fluorescence calibrated on the positive control, and which was preserved for all other images. The software then analyzed the number of DAPI and fluorescein fluorescence events to calculate the ratio between the total number of DAPI-labeled cell nuclei and the number of fluorescein-labeled nuclei with fragmented DNA. The experiment was performed in triplicate.

Statistical analysis.

The statistical analyses were performed using the GraphPad PrismTM version 4.00 software from GraphPad Software Inc. (San Diego, CA, USA). The results are expressed as the means ± standard deviation (SD) or standard error mean (SEM) of at least three determinations for each test from three independent experiments. Statistical analyses were carried out using the unpaired t-test. The statistical significance of the differences is given as *p < 0.05; ** p < 0.01; ***p < 0.001; ns: not significant.

ADT: Androgen deprivation therapy

CMC: Critical micellar concentation

CRPC: castration resistant prostate cancer

CSD: Chemical shift deviation

DPC: Dodecylphosphocholin

DRS-B2: Dermaseptin from Phyllomedusa bicolor

dUTP: Deoxyuridine triphosphate

GAG: Glycosaminoglycans

H-B2: Hormonotoxin-dermaseptin B2

IP: Intraperitoneal

LDH: Lactate deshydrogenase

LHRH (GnRH): luteinizing hormone-releasing hormone or Gonadotropin releasing hormone

LHRH-R: luteinizing hormone-releasing hormone-receptor

mCRPC: Metastatic castration resistant prostate cancer

PCa: Prostate cancer

PI: Propidium iodide

Tdt: Terminal deoxynucleotidyl transferase

TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling

ACKNOWLEDGMENTS

We thank Aurelie Guguin and Adeline Henry from the Flow Cytometry facility of IMRB (institute Mondor de recherche biomedicale).

AUTHOR CONTRIBUTIONS

Conceptualization: J.D, Y.H-K, M.A.

Formal analysis: M.C, M.D, M.M-P, Z.L, L.Z, O.L, J.D, Y.H-K, M.A.

Funding acquisition: A.dlT, J.D, Y.H-K, M.A.

Investigation: M.C, M.D, M.M-P, Z.L, P.Z, L.Z, O.L,

Methodology: J.D, Y.H-K, M.A.

Project administration: A.dlT, J.D, Y.H-K, M.A.

Resources: J.D, Y.H-K, M.A.

Supervision: J.D, Y.H-K, M.A.

Validation J.D, Y.H-K, M.A.

Visualization: J.D, Y.H-K, M.A.

Writing original draft: J.D, Y.H-K, M.A.

Writing review & editing: M.C, L.Z, O.L, J.D, Y.H-K, M.A.

FUNDING AND ADDITIONAL INFORMATION

Thanks to the AFU (Association Française d'urologie) and to IMRB which awarded a scholarship to Michaël Miro-Padovani and to Michaël Couty for the realization of a part of this project.

COMPETING INTERESTS:

The authors have declared that no competing interests exist.

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No competing interests reported.

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Antitumor Activity and Mechanism of Action of the Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide

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Abstract

Figures

Introduction

Results

The Hormonotoxin H-B2 induces a significant antiproliferative effect on the hormone-resistant prostate tumor cell line PC3 and has a low hemolytic activity.

Hormonotoxin H-B2 significantly inhibits tumor growth in xenografted mice without measurable side effects.

Mechanism of antitumor action of the Hormonotoxin H-B2 in comparison with DRS-B2

Addition of the hormonal analogue (d-Lys 6 -LHRH) to the DRS-B2 did not alter the secondary structure of the hormonotoxin H-B2.

The cell death induced by H-B2 and DRS-B2 uses two different mechanisms that are related to apoptosis and necrosis, respectively.

Discussion

EXPERIMENTAL PROCEDURES

Peptide synthesis.

Circular dichroism (CD) spectroscopy of peptides.

Fluorescence of tryptophan-containing peptides

Nuclear Magnetic Resonance (NMR) Spectroscopy.

Cell culture.

Cell-viability assays.

Small Interference RNA (siRNA) Assay.

Western blotting

Hemolysis assessment.

Tumor xenograft Studies

Ethics statement.

Immunohistochemical analysis of the tumors

Lactate dehydrogenase (LDH) release assay.

Apoptosis analysis by flow cytometry.

Terminal transferase-mediated dUTP Nick End-Labelling (TUNEL) assay.

Statistical analysis.

Abbreviations

Declarations

ACKNOWLEDGMENTS

AUTHOR CONTRIBUTIONS

FUNDING AND ADDITIONAL INFORMATION

COMPETING INTERESTS:

References

Additional Declarations

Status:

Journal Publication

Version 1

Addition of the hormonal analogue (d-Lys ⁶ -LHRH) to the DRS-B2 did not alter the secondary structure of the hormonotoxin H-B2.