K092A and K092B, Two Peptides Isolated from the Dogfish (Scyliorhinus canicula L.), with Potential Antineoplastic Activity Against Human Prostate and Breast Cancer Cells

Cancer therapy is currently a major challenge within the research community, especially in reducing the side effects of treatments and to develop new specific strategies against cancers that still have a poor prognosis. In this context, alternative strategies using biotechnologies, such as marine peptides, have been developed based on their promise of effectivity associated with a low toxicity for healthy cells. The purpose of the present paper is to investigate the active mechanism of two peptides that were isolated from the epigonal tissue of the lesser spotted dogfish Scyliorhinus canicula L., identified NFDTDEQALEDVFSKYG (K092A) and EAPPEAAEEDEW (K092B) on the in vitro growth inhibition of ZR-75-1 mammary carcinoma cells and MDA-Pca-2b prostate cancer cells. The effects of the peptides on cell proliferation and cell death mechanisms were studied by the flow cytometry and immunofluorescence microscopy approaches. The results have shown the onset of both K092A- and K092B-induced early cytoskeleton changes, and then cell cycle perturbations followed by non-apoptotic cell death. Moreover, impedance perturbation and plasma membrane perforation in ZR-75-1 K092A-treated cell cultures and autophagy inhibition in MDA-Pca-2b K092B-treated cells have been observed. In conclusion, these two bioactive peptides from dogfish exhibit antineoplastic activity on the human prostate and breast cancer cells in vitro.


Introduction
Bioactive peptides are promising for therapeutic use due to several advantages: they are of small size and often constitute the functional part of proteins and, therefore, they are highly functionally efficient, easily synthesized, and modified to enhance their properties; they often specifically target cancer cells without damaging normal cells; they do not accumulate in specific organs; and, their degradation generates amino acids, which minimizes their toxicity and side effects and they are not usually genotoxic [1][2][3]. However, their rapid clearance is a disadvantage for long-term activity [4].
The mitochondrial activity of the cell culture was measured while using the WST-1 test at 6 h, 12 h, 24 h, 48 h, 72 h, and 96 hours post-treatment (hpt) on ZR-75-1 ( Figure 1) and MDA-Pca-2b ( Figure 2) cells grown with: (i) culture media, (ii) culture media and 0.01 M ammonium bicarbonate, and (iii) culture media and K092A ( Figure 1A) or K092B ( Figure 2B) dissolved in 0.01 M ammonium bicarbonate at the final concentration that corresponded to the IC50. This assay showed a gradual increase of the mitochondrial activity in both controls and for the two types of cells, reflecting their proliferation over time with one exception for the ZR-75-1 cells at 96 hpt. A significant decrease of the mitochondrial activity for K092A-treated ZR-75-1 cells when compared to the ammonium bicarbonate control was observed at 6 hpt (0.245 ± 0.017 for treated vs. 0.312 ± 0.029 for control), from 48 hpt (0.394 ± 0.050 for treated vs. 0.597 ± 0.145 for control) and until 96 hpt (0.183 ± 0.021 for treated vs. 0.321 ± 0.047 for control) ( Figure 1). Significantly, a decrease by half of the mitochondrial activity for K092B-treated MDA-Pca-2b cells when compared to the ammonium bicarbonate control was observed each time, from 6 hpt (0.088 ± 0.005 for treated vs. 0.137 ± 0.014 for control) and until 96 hpt (0.316 ± 0.088 for treated vs. 0.708 ± 0.067 for control) (Figure 2A). Furthermore, microscopic observations at 6 h, 48 h, 72 h, and 96 hpt revealed that the K092A-treated ZR-75-1 cells exhibited a decrease in the number of cells as well as the presence of more round suspended cells and abnormal cells, as illustrated at 72 hpt ( Figure 1B). Microscopic observations also showed that the K092B-treated MDA-Pca-2b cells presented a decrease of the cell number as well as the presence of more round suspended cells, as illustrated at 24 hpt ( Figure 2B). perturbation, and non-apoptotic cell death. Interestingly, the action mode of both peptides starts with the induction of cytoskeleton disruption. This event seems to drive the growth inhibition for ZR-75-1 and MDA-Pca-2b cells through different ways. Finally, this work confirms that marine organisms are a good source of bioactive peptides and emphasizes the fact that dogfish is a potent source of antineoplastic peptides.

Decrease in Mitochondrial Activity and Cell Number Was Reported in K092A-Treated Human Mammary Carcinoma and K092B-Treated Human Prostate Cancer Cells
The mitochondrial activity of the cell culture was measured while using the WST-1 test at 6 h, 12 h, 24 h, 48 h, 72 h, and 96 hours post-treatment (hpt) on ZR-75-1 ( Figure 1) and MDA-Pca-2b ( Figure  2) cells grown with: (i) culture media, (ii) culture media and 0.01 M ammonium bicarbonate, and (iii) culture media and K092A ( Figure 1A) or K092B ( Figure 2B) dissolved in 0.01 M ammonium bicarbonate at the final concentration that corresponded to the IC50. This assay showed a gradual increase of the mitochondrial activity in both controls and for the two types of cells, reflecting their proliferation over time with one exception for the ZR-75-1 cells at 96 hpt. A significant decrease of the mitochondrial activity for K092A-treated ZR-75-1 cells when compared to the ammonium bicarbonate control was observed at 6 hpt (0.245 ± 0.017 for treated vs. 0.312 ± 0.029 for control), from 48 hpt (0.394 ± 0.050 for treated vs. 0.597 ± 0.145 for control) and until 96 hpt (0.183 ± 0.021 for treated vs. 0.321 ± 0.047 for control) ( Figure 1). Significantly, a decrease by half of the mitochondrial activity for K092B-treated MDA-Pca-2b cells when compared to the ammonium bicarbonate control was observed each time, from 6 hpt (0.088 ± 0.005 for treated vs. 0.137 ± 0.014 for control) and until 96 hpt (0.316 ± 0.088 for treated vs. 0.708 ± 0.067 for control) (Figure 2A). Furthermore, microscopic observations at 6 h, 48 h, 72 h, and 96 hpt revealed that the K092A-treated ZR-75-1 cells exhibited a decrease in the number of cells as well as the presence of more round suspended cells and abnormal cells, as illustrated at 72 hpt ( Figure 1B). Microscopic observations also showed that the K092B-treated MDA-Pca-2b cells presented a decrease of the cell number as well as the presence of more round suspended cells, as illustrated at 24 hpt ( Figure 2B).  These results illustrate the potential in vitro antineoplastic activity of K092A and K092B on the ZR-75-1 cells and MDA-Pca-2b cells, respectively.

K092A-and K092B-Induced Perturbation of Electric Impedance in Culture Plates of Treated Cells
Real-time electric impedance sensing measurements of culture plates were investigated from the time of cell seeding until 72 hpt while using the XCelligence system for K092A-treated ZR-75-1 cells ( Figure 3A) or for K092B-treated MDA-Pca-2b cells ( Figure 3B). All culture conditions presented the same impedance until the treatment time and both of the controls exhibited a similar increase of impedance, even if the ammonium bicarbonate control reached an amplitude that was slightly lower than the one observed for the culture media control from 45 h until 72 hpt ( Figure 3A). When compared with the controls, K092A-treated cells presented a fast increase of impedance from treatment time until 6 hpt, followed by a decrease until 48 hpt. Subsequently, impedance was stabilized at the corresponding initial level until the end of the culture time ( Figure 3A). For K092Btreated MDA-Pca-2b cells culture, the profile of impedance was similar to the ones that were observed for controls, but of lower amplitude ( Figure 3B).
These data show that K092A and K092B have induced a perturbation of the electrical impedance in the ZR-75-1 and MDA-Pca-2b cell cultures, respectively. The different profiles observed suggested that the two peptides drive the perturbation of cell adhesion through different ways. These results illustrate the potential in vitro antineoplastic activity of K092A and K092B on the ZR-75-1 cells and MDA-Pca-2b cells, respectively.

K092A-and K092B-Induced Perturbation of Electric Impedance in Culture Plates of Treated Cells
Real-time electric impedance sensing measurements of culture plates were investigated from the time of cell seeding until 72 hpt while using the XCelligence system for K092A-treated ZR-75-1 cells ( Figure 3A) or for K092B-treated MDA-Pca-2b cells ( Figure 3B). All culture conditions presented the same impedance until the treatment time and both of the controls exhibited a similar increase of impedance, even if the ammonium bicarbonate control reached an amplitude that was slightly lower than the one observed for the culture media control from 45 h until 72 hpt ( Figure 3A). When compared with the controls, K092A-treated cells presented a fast increase of impedance from treatment time until 6 hpt, followed by a decrease until 48 hpt. Subsequently, impedance was stabilized at the corresponding initial level until the end of the culture time ( Figure 3A). For K092B-treated MDA-Pca-2b cells culture, the profile of impedance was similar to the ones that were observed for controls, but of lower amplitude ( Figure 3B).
These data show that K092A and K092B have induced a perturbation of the electrical impedance in the ZR-75-1 and MDA-Pca-2b cell cultures, respectively. The different profiles observed suggested that the two peptides drive the perturbation of cell adhesion through different ways. Cells were seeded (black arrow) 24 h before the treatment (red arrow) and results were represented by mean ± SD of 4 replicates.

K092A Induced Cytoskeleton Perturbation Followed by Membrane Destabilization and Necrosis in ZR-75-1 Cells
Cell death mechanisms (Table 3, Figures 6 and 7) and cytoskeleton integrity ( Figure 8) were investigated at 24 hpt, 48 hpt and 72 hpt and at 6 hpt and 48 hpt, respectively, to further explore the mechanism of K092A involved in impedance perturbation and antiproliferative effect.
These results show that K092A and K092B have promoted an antiproliferative effect, starting at 24 hpt in ZR-75-1 cells and at 8 hpt in MDA-PCa-2b cells, respectively.

K092A Induced Cytoskeleton Perturbation Followed by Membrane Destabilization and Necrosis in ZR-75-1 Cells
Cell death mechanisms (Table 3, Figures 6 and 7) and cytoskeleton integrity ( Figure 8) were investigated at 24 hpt, 48 hpt and 72 hpt and at 6 hpt and 48 hpt, respectively, to further explore the mechanism of K092A involved in impedance perturbation and antiproliferative effect. Table 3. Apoptosis, necrosis, destabilized membranes, and cell fragments in ZR-75-1 control and K092A-treated cells at 24-72 h post-treatment (%).

Cell Death
Sample   Representative flow cytometry data set at 72 hpt for ZR-75-1 control and K092A-treated cells, annexin V/propidium iodide staining for apoptosis and necrosis, SYBR Green I/propidium iodide staining for membrane integrity study. Significant results were highlighted for apoptosis (light red box), necrosis (light orange box), and destabilized membranes (light green box). When the cytoskeleton of cells was examined, the population that was treated with K092A presented more cells with the aggregation of actin and fewer adherent cells at 6 hpt ( Figure 8A  These results show that K092A has driven a drastic cytoskeleton perturbation, followed by membrane destabilization and necrosis.

K092B Induced Prolonged Necrosis and Membrane Destabilization in MDA-Pca-2b Cells
Apoptosis, necrosis, and membrane destabilization were investigated at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 hpt in order to correlate the antiproliferative effect and the cell death mechanisms induced by K092B on MDA-Pca-2b cells (Table 4, Figure 9).
When the cytoskeleton of cells was examined, the population that was treated with K092A presented more cells with the aggregation of actin and fewer adherent cells at 6 hpt ( Figure 8A,B) and at 48 hpt ( Figure 8C,D). Similar observations were made for tubulin at 6 hpt ( Figure 8E,F) and at 48 hpt ( Figure 8G,H).
These results show that K092A has driven a drastic cytoskeleton perturbation, followed by membrane destabilization and necrosis.  Figure 9).   These results show that K092B has induced cell death by membrane destabilization and necrosis.
These results show that K092B has induced cell death by membrane destabilization and necrosis.

K092B Induced Early Autophagy Inhibition, Increase of Neutral Red Retention and Cytoskeleton Perturbation in MDA-Pca-2b Cells
The neutral red retention capacity of lysosomes was studied at 6 h, 12 h, 24 h, 48 h, and 72 hpt ( Figure 10A), autophagy at 4 h, 6 h, and 12 hpt ( Figure 10B,C), as well as cytoskeleton integrity at 6 h and 48 hpt in order to further explore the mechanism involved in early antiproliferative and cell death effect observed in K092B-treated cells ( Figure 11).
In the control cells, the increase of neutral red retention capacity that was observed between 24 hpt and 72 hpt seems to correlate with the increase of cell number during the same period. K092B-treated cells presented a two-fold increase in the lysosome retention capacity at 6 hpt (1.273 ± 0.181 for treated vs. 0.707 ± 0.123 for control), 12 hpt (1.084 ± 0.179 for treated vs. 0.599 ± 0.106 for control), and at 24 hpt (1.069 ± 0.230 for treated vs. 0.560 ± 0.153 for control) when compared to the control cells ( Figure 10A). Autophagy study also showed that K092B-treated cells presented fewer autophagosomes than control cells at 6 hpt (60% ± 15% for treated vs. 100% for control) and 12 hpt (83% ± 3% for treated vs. 100% for control) ( Figure 10B,C).  Figure 10A). Autophagy study also showed that K092B-treated cells presented fewer autophagosomes than control cells at 6 hpt (60% ± 15% for treated vs. 100% for control) and 12 hpt (83% ± 3% for treated vs. 100% for control) ( Figure 10B,C). When the cytoskeleton of cells was examined, the population that was treated with K092B presented more cells with aggregated actin cytoskeleton and fewer adherent cells at 6 hpt ( Figure  11A,B) and at 48 hpt ( Figure 11C,D). Similar observations were made for tubulin cytoskeleton at 6 hpt ( Figure 11E,F) and 48 hpt (Figure 11G,H). Moreover, the presence of multinucleated cells was also detected in K092B-treated cells at 6 hpt ( Figure 11B).

cells (white arrows) from cells with agglutinated actin (black arrows) and multinucleated cells (blue arrows). (E-H) Tubulin was stained using anti-tubulin antibody and nuclei using DAPI at 6 hpt (E,F) and 48 hpt (G,H) for the control cells (E,G) and K092B-treated cells (F,H). Inserts (d-f) were focused in order to distinguish spread and adherent normal cells (white arrows) from cells with agglutinated tubulin (black arrows). The white scale bar is 40 µm and yellow bar 10 µm.
These results show that K092B induced early cytoskeleton perturbation and autophagy inhibition.

Discussion
This study reported that the first effects that were observed for K092A treatment in ZR-75-1 cells were a durable perturbation in electric impedance of cell culture and a perturbation of the cytoskeleton. These events are quickly followed by a slowing-down of the cell cycle, an important cell membrane destabilization, and finally, cell death by necrosis. The X-Celligence system has been previously used to study the kinetics and effects of peptides or drugs on cells, since electric impedance was correlated with various cell properties, such as cell size, cell proliferation rate, cell adherence intensity, and cell number [34][35][36][37][38]. In the present study, the rapid and transient increase of cell impedance during the first 24 hours of treatment of ZR-75-1 cells suggested quick cytoskeletal changes, as observed by phenethyl isothiocyanate [38] or nocodazole in lung cancer cells [36]. Moreover, the late stability of impedance suggested a durable antiproliferative effect. These results appear to be correlated with cytoskeleton perturbation, as observed for both actin and tubulin cytoskeletons at 6 hpt and 48 hpt, and allow for us to exclude an increase of cell junction formation, which could also be responsible for the transient increase of cell impedance, because of the decrease of cell size and cell interactions observed. Such antiproliferative effects resulting on cytoskeleton disruption have been described after microtubule polymerization disruption induced by cucurbitacin B in human breast cancer cells [39] or after actin filaments reorganization induced by ophiobolin A in human glioblastoma cells [40]. In the present study, an antiproliferative effect has also been reported by WST-1 assay and flow cytometry analyses, which indicate that cell proliferation strongly decreased in K092A-treated ZR-75-1 cells as soon as 48 hpt. When considering the close relationship between cell cycle completion and cytoskeleton dynamics and the timing of observed antiproliferative effects and cytoskeleton perturbation, we could hypothesize that K092A affects the cell cycle progression of ZR-75-1 cells through the alteration of their cytoskeleton network. Indeed, direct perturbation of the dynamics of microtubule induced cell cycle arrest before metaphase often associated with mitotic spindle aberrations, as reported in many studies on various cancer cell lines When the cytoskeleton of cells was examined, the population that was treated with K092B presented more cells with aggregated actin cytoskeleton and fewer adherent cells at 6 hpt ( Figure 11A,B) and at 48 hpt ( Figure 11C,D). Similar observations were made for tubulin cytoskeleton at 6 hpt ( Figure 11E,F) and 48 hpt ( Figure 11G,H). Moreover, the presence of multinucleated cells was also detected in K092B-treated cells at 6 hpt ( Figure 11B).
These results show that K092B induced early cytoskeleton perturbation and autophagy inhibition.

Discussion
This study reported that the first effects that were observed for K092A treatment in ZR-75-1 cells were a durable perturbation in electric impedance of cell culture and a perturbation of the cytoskeleton. These events are quickly followed by a slowing-down of the cell cycle, an important cell membrane destabilization, and finally, cell death by necrosis. The X-Celligence system has been previously used to study the kinetics and effects of peptides or drugs on cells, since electric impedance was correlated with various cell properties, such as cell size, cell proliferation rate, cell adherence intensity, and cell number [34][35][36][37][38]. In the present study, the rapid and transient increase of cell impedance during the first 24 h of treatment of ZR-75-1 cells suggested quick cytoskeletal changes, as observed by phenethyl isothiocyanate [38] or nocodazole in lung cancer cells [36]. Moreover, the late stability of impedance suggested a durable antiproliferative effect. These results appear to be correlated with cytoskeleton perturbation, as observed for both actin and tubulin cytoskeletons at 6 hpt and 48 hpt, and allow for us to exclude an increase of cell junction formation, which could also be responsible for the transient increase of cell impedance, because of the decrease of cell size and cell interactions observed. Such antiproliferative effects resulting on cytoskeleton disruption have been described after microtubule polymerization disruption induced by cucurbitacin B in human breast cancer cells [39] or after actin filaments reorganization induced by ophiobolin A in human glioblastoma cells [40]. In the present study, an antiproliferative effect has also been reported by WST-1 assay and flow cytometry analyses, which indicate that cell proliferation strongly decreased in K092A-treated ZR-75-1 cells as soon as 48 hpt. When considering the close relationship between cell cycle completion and cytoskeleton dynamics and the timing of observed antiproliferative effects and cytoskeleton perturbation, we could hypothesize that K092A affects the cell cycle progression of ZR-75-1 cells through the alteration of their cytoskeleton network. Indeed, direct perturbation of the dynamics of microtubule induced cell cycle arrest before metaphase often associated with mitotic spindle aberrations, as reported in many studies on various cancer cell lines [39,41,42]. Finally, the membrane perforation that was observed in K092A-treated cells seems to trigger necrotic death due to the interactions between cytoskeleton and plasma membrane [43] and could result from cytoskeleton disruption.
This study also reported that the first noticeable effects of K092B on MDA-Pca-2b cells, as observed at 6 hpt, consisted of: a cytostatic effect that was observed by real-time electric impedance sensing measurements, autophagy inhibition, and cytoskeleton aggregation. They were followed by necrosis, membrane destabilization, and the inhibition of cell proliferation. Autophagy is a complex mechanism that allows for the turnover of organelles and of proteins, therefore protecting cells from various stress conditions [44]. In cancer cells, according to its intensity, autophagy can promote either cell survival allowing tumor survival and development, or cell death by apoptosis or necrosis [44][45][46][47][48][49]. The inhibition of autophagy was correlated with an increase of caspase-dependent apoptosis in various cells, such as HeLa or lymphoma cells [48] and necrosis in human glioblastomes [49]. Consistent with these cell behaviors, our results have shown that necrosis features at 8 h and 12 hpt followed the inhibition of autophagy, as observed at 6 hpt, which suggests that K092B induces early MDA-Pca-2b cell death by autophagy inhibition. In light of the various interactions described between cytoskeleton and autophagy [50][51][52], in particular for lysosomes and endosomes trafficking [53], we could hypothesize that the autophagy inhibition that was observed on K092B-treated cells was induced by the cytoskeleton perturbations that were observed at the same time. Such correlation between microtubule remodeling and autophagy inhibition has been previously reported on mouse neural cells [54]. Moreover, the early increase of lysosomal retention capacity that was detected in K092B-treated cells could be correlated to autophagy inhibition and similarly with the cytoskeleton perturbation. Indeed, the fusion of lysosomes with others organelles is needed to form autophagosomes and autolysosomes [46], which suggests that autophagy inhibition might involve an increase in the number of lysosomes due to a lack of fusion.
Such a mechanism of action, causing cytoskeleton destabilization, cell cycle perturbation, and finally, cell death, has been previously described. For example, the 17-amino acids peptide C36L1 inhibits in vitro the migration, the invasion, and proliferation of melanoma cells by causing direct depolymerization of microtubules [55]. In this study, Figueiredo et al. were able to identify that C36L1 directly binds to the tubulin protein, causing depolymerization [55]. This direct interaction between bioactive peptide and tubulin can also lead to polymerization inhibition, cell cycle arrest, and cell death, as reported for the peptide MMAE [10]. These kinds of peptide are very promising for improved therapy, since, for example, the peptide MMAE has been approved by the U.S. Food and Drugs Administration in 2011 for the treatment of Hodgkin and systemic anaplastic large cell lymphoma [10].

Cell Culture and Treatments Conditions
The ZR-75-1 cell line was established from the metastatic site of breast glands of a 63 years old caucasian woman with a ductal breast carcinoma (cell line obtained from the global bioresource center ATCC; www.atcc.org/). This cell line was grown in vitro and in vivo and it expressed wild type and a variant of estrogen receptor as well as the progesterone receptor. MDA-PCa 2b-cell line was established from the bone metastasis of a 63 years old black man with androgen-independent adenocarcinoma of the prostate (bioresource center ATCC; www.atcc.org/). The ZR-75-1 cells were maintained in RPMI 1640 media (supplemented with 10% SVF, 2 mM L-Glu, 10 mM beta estradiol, 1 mM Na pyruvate) at 27 • C in a 5% CO 2 air incubator. MDA-PCa-2b cells were maintained in Ham's F12 media (supplemented with 20% SVF, 5 mM L-Glu, 10 ng/mL EGF, hydrocortisone 100 pg/mL) at 37 • C in a 5% CO 2 air incubator. Prior to the experiment, the cells were grown to approximately 80% confluence three times and then exposed to K092A (1.22 µg/µL) or K092B (1.3 µg/µL). K092A (94% purity) and K092B (98% purity) was purchased from the Bionexus company (Strasbourg, France), stored at −20 • C, and then dissolved in bicarbonate ammonium (0.1 M) when needed. The results were compared with those that were obtained for both controls, cells grown in culture media, and in culture media with bicarbonate ammonium (0.1 M).

Mitochondrial Activity Assay
Mitochondrial activity was measured by the WST-1 colorimetric assay (Roche; Ref.: 11 644 807 001) that was based on the cellular capacity to metabolize the WST-1 by mitochondrial enzymatic complex. The cells were seeded into 96-well plates (ZR-75-1: 7500 cells/well; MDA-Pca-2b: 5000 cells/well) and treated in replicates for 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. WST-1 was added, as recommended by suppliers, and the plates were incubated for 2 h before being analyzed in a spectrophotometer (BioRad; iMark; Marnes-la-Coquette, France). Optical density (OD) at 620 nm that corresponded to background level was subtracted from OD at 450 nm, corresponding to WST-1 degradation. For each study, statistical analysis was performed while using the Mann and Whitney test (p < 0.05) on three independent experiments (N = 3; n = 9).

Lactate Dehydrogenase Activity Assay
Lactate dehydrogenase (LDH) activity was measured by the LDH cytotoxic colorimetric assay (Roche; Ref.: 11 644 793 001) based on the reduction of NAD by the cytoplasmic LDH that was released in extracellular medium after membranes destabilization. The resulting NADH is utilized in the stoichiometric conversion of a tetrazolium dye into red formazan salt by a catalyst complex that was included in the LDH assay kit. The cells were seeded into 96-well plates (ZR-75-1: 7500 cells/well) and treated in replicates for 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. The catalyst complex solution was added, as recommended by the suppliers, and the plates were incubated for 30 min. before being analyzed in a spectrophotometer (BioRad; iMark; Marnes-la-Coquette, France). Optical density (OD) at 620 nm, which corresponded to background level, was subtracted from OD at 500 nm, which corresponded to formazan salt detection. Statistical analysis was performed while using the Mann and Whitney test (p < 0.01) on three independent experiments (N = 3; n = 9).

Neutral Red Retention Assay
Neutral red retention capacity was based on the cellular capacity to sequestrate the neutral red inside lysosomes. The cells were seeded into 96-well plates (MDA-Pca-2b: 5000 cells/well) and treated in replicates for 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. Neutral red was added (0.033%) and incubated with cells for two hours at 37 • C. The cells were washed, fixed (0.5% formaldehyde in PBS), and the neutral red was released from lysosomes while using solubilization solution (1% acetic acid, 50% ethanol in PBS), which was analyzed in a spectrophotometer (BioRad; iMark; Marnes-la-Coquette, France. Optical density (OD) at 650 nm, corresponding to background level, was subtracted from OD at 540 nm corresponding to neutral red. Statistical analysis was performed while using the Mann and Whitney test (p < 0.01) on three independent experiments (N = 3; n = 9).

Apoptosis/Necrosis and Membranes Integrity Analysis
Both apoptosis/necrosis and membrane integrity were quantified by flow cytometry using two double stainings: propidium iodide (PI) and annexin V (AV), or PI and SYBR Green I (SGI) respectively. PI and SGI were both used to stain DNA, but only the SGI could pass through the cellular membranes without perforation. Annexin V allowed for apoptosis/necrosis detection by its high affinity to bind phosphatidylserines that were exposed at the extracellular side of apoptotic and necrotic cells. The cells were seeded into 24 well-plates (ZR-75-1: 44,531 cells/well; MDA-Pca-2b: 29,688 cells/well) and treated in replicates for 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h. The cells were then collected (170 U trypsin/mL), washed, and then stained in two ways: (i) apoptosis/necrosis analysis using PI and AV (both 1:500, as described by suppliers); (ii) membrane integrity analysis while using SGI at 1:1 × 10 6 and PI at 1:500 with an incubation time of 30 min. in dark conditions. The samples were then analyzed by flow cytometry (Gallios cytometer, Beckman Coulter Life Sciences, Villepinte, France). For each sample, 20,000 events were counted and statistical analyses were performed on three independent experiments while using the Mann and Whitney test (p < 0.05) (N = 3; n = 6).

Cell Cycle Analysis
Cell cycle repartition was investigated at 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h by flow cytometry with PI staining. On ethanol fixed cells, PI staining allowed for DNA quantification corresponding to cell populations in G0/G1 (2C); S (between 2C and 4C) or G2/M (4C). The cells were seeded in replicates into 24-well plates (ZR-75-1: 44,531 cells/well; MDA-Pca-2b: 29,688 cells/well) and treated. Afterwards, they were collected (170 U trypsin/mL), washed, fixed, and stored in 70% ethanol at −20 • C. Before flow cytometry analysis, the cells were washed at 37 • C and stained for 30 min in dark conditions with PI, as described by suppliers. The samples were then analyzed by flow cytometry (Gallios cytometer with Kaluza licence, Beckman Coulter Life Sciences, Villepinte, France). For each sample, 20,000 events were counted and statistical analysis was performed on three independent experiments while using the Mann and Whitney test (p < 0.05) (N = 3; n = 6).

Electric Impedance Measurement
Real-time electric impedance was analyzed over the whole cell culture duration by the XCelligence system (Roche, Basel, Switzerland) under a 5% CO 2 atmosphere at 37 • C. The cells were seeded on a 96-wells-E-plate (Roche) with the bottoms surface area covered by interdigitated gold microelectrodes. The general principle is based on the presence of cells in the plate that affect the electric transmission between microelectrodes, and so leads to an increase of current resistance. This current resistance was called "cell index" or "impedance", and it was directly dependent on cell line, culture media, and cell properties, such as cell number, cell proliferation, cell adherence intensity, or cell morphologic changes [34]. After cell seeding into replicates (ZR-75-1: 7500 cells/well), the E-plate was incubated in the XCelligence system that was set up in a CO 2 incubator at 37 • C. The cells were allowed to grow for 24 h before treatment with K092A (1.22 µg/µL). Impedance was automatically analyzed every five minutes until 72 h and every hour until the end of the culture during the first 48 h of cell culture (including 24 h without treatment).

Autophagy Measurement
Autophagy, which was directly correlated with the quantity of autophagosomes, was investigated at 4 h, 6 h, and 12 h post-treatment while using acridine orange assay and flow cytometry analysis. Acridine orange was used to stain autophagosome organelles (red fluorescence) and nucleic acids in cytoplasm and nucleus (green fluorescence). The percentage of autophagosomes (R; R = 100% for controls) was determined by normalization of the red fluorescence by the green fluorescence: R = FL3 INT/FL1 INT. The cells were seeded in replicates into 24-well plates (29,688 cells/well) and then treated. Cells were then stained with AO (5 µg/mL) for 10 min at 37 • C, collected (170 U trypsin/mL), washed, and analyzed by flow cytometry. For each sample, 20,000 events were counted and statistical analysis was performed on two independent experiments while using the Mann and Whitney test (p < 0.05) (N = 2; n = 4).

Immunocytochemistry Analysis
Tubulin and actin cytoskeletons were observed at 6 h and 48 h post-treatment while using a mouse anti-tyrosine tubulin antibody (Sigma: T9028; L'Isle d'Abeau Chesnes, France) and phalloidin staining (5 µg/mL), respectively. The cells were seeded in replicates on microscope cover glass slides into 24-well plates (ZR-75-1: 44,531 cells/well; MDA-Pca-2b: 29,688 cells/well) and treated. Cells were then fixed in 4% PFA for 10 min, washed in 1% BSA-PBS solution, and then incubated 1 h with rhodamine-phalloidin (1:200) or anti-tubulin (1:200). In the second case, the cells were then washed, incubated with secondary antibody (1:500; goat anti-mouse IgG Alexa fluor 488; Invitrogen: A11001; Thermo Fisher Scientific, Paris, France) for 1h in dark conditions, and then washed, while, after rhodamine-phalloidin incubation, the cells were only washed. Cover glass slides were finally mounted on microscope slides with a mounting solution containing DAPI (Prolong gold antifade with DAPI; Invitrogen P36935). Observations, pictures, and merge were taken with an Eclipse 80i microscope coupled to a DXM1200-C camera (Nikon, Champigny sur Marne, France).

Conclusions
K092A and K092B isolated from male dogfish genital tract are potential antineoplastic marine peptides that are able to strongly inhibit the in vitro growth of ZR-75-1 and MDA Pca2b cells, respectively. The main as successive steps of K092A action in the ZR-75-1 cee-line are: (i) cytoskeleton disruption at 6 hpt; (ii) decrease of WST1, arrest in G0/G1, and decrease in the rate of S phase progression towards G2/M phases, increase in damaged membranes, and cell fragments at 48 hpt; and, (iii) increase of necrosis at 72 hpt. The main as successive steps of K092B action in MDA Pca2b cee-line are: (i) decrease of WST1, increase of necrosis and lysosomes/autophagosomes and of multinucleate cells at 6 hpt; (ii) arrest in G0/G1 and decrease in the rate of S phase progression towards G2/M phases; (iii) increase in damaged membranes and cell fragments at 48 hpt; and, (iv) prolonged necrosis at 72 hpt.
Both of the peptides appear to target a cytoskeleton element, quickly disrupt, and induce an early cytostatic fate, leading to a durable cell cycle perturbation and cell-number stabilization. Subsequently, the maintenance of cytoskeleton perturbation seems to be responsible for a later cytotoxic effect triggering non-apoptotic cell death mechanisms, such as necrosis and/or membrane disruption. Further investigations should be done in order to confirm this hypothesis. In any case, this study evidences that the dogfish could be a promising source of bioactive peptides against cancer cell growth.
MTV of 1.69 ± 0.91 mm 3 (N = 5) compared to 2.23 ± 0.25 mm 3 for control (N = 5) and a T/C of 107.03%. For the 130 mg/kg treated group, the results have also shown the complete regression of the tumor for one mouse, a MTV of 1.52 ± 0.61 mm 3 (N = 5) compared to 2.23 ± 0.25 mm 3 for control (N = 5) and a T/C of 72.90%. Furthermore, frozen sections of tumors were analyzed by immunohistochemistry by using a rat IgG2a anti-mouse CD31 (PECAM-1: platelet endothelial cell adhesion molecule 1) (1:250; Cedarlane CL8930AP, clone 390) in order to follow angiogenesis of tumors. Results have shown an immunostaining at the center and at the periphery of tumors in the control group (N = 5) ( Figure A1A) and, comparatively, a very low staining ay the center of tumors in the peptide-treated groups (N = 5 for each dose) ( Figure A1B). At higher magnification, no cuboidal cells were stained in treated group ( Figure A1D,E) compared to control group ( Figure A1C) even if some isolated cells or trabecular tubules remained positive. These results suggested and antiangiogenesis activity of the K092B peptide.  Figure A1(A)) and, comparatively, a very low staining ay the center of tumors in the peptide-treated groups (N = 5 for each dose) ( Figure A1(B)). At higher magnification, no cuboidal cells were stained in treated group ( Figure A1 (D-E)) compared to control group ( Figure  A1(C)) even if some isolated cells or trabecular tubules remained positive. These results suggested and antiangiogenesis activity of the K092B peptide.