Sepia ink (sepia esculenta) was purchased from Nanzhen Market (Zhoushan, China), and authenticated by Prof. Sheng-Long Zhao (Zhejiang Ocean University, Zhoushan, China).

Pepsin was purchased from Shanghai Ruji Biological Technology Co., Ltd., China. Sephadex G-25 was purchased from YTHX Biotechnology Co., Ltd. (Beijing, China). The ultrafiltration membrane was purchased from Shanghai Mosu Scientific Equipment (China). Acetonitrile was of LC grade and purchased from Oceanpak Scientific Co., Ltd. (Sweden). CCK-8 assay kit was purchased from Dojindo Laboratories (Japan). Annexin V-FITCC cell apoptosis test kit was purchased from Best Bio (Beijing, China). Rabbit polyclonal antibodies against p53, Caspase-3, Bcl-2, Bax and HRP-conjugated secondary antibodies were purchased from Celling Signaling (Beijing, China). PVDF membrane was purchased from Bio Rad (USA). All other reagents used in the experiment were of the analytica grade.

The sepia ink was separated manually and homogenated by high-speed tissue-masher (Shanghai YouYi limited company, Shanghai, China), as described previously (23). The optimum hydrolysis condition was determined using an orthogonal array design (Table I). An additional orthogonal array design (L16(4⁵)) was applied to optimize the enzymolysis conditions, which consisted of material/distilled water, pH, enzyme dose, enzymolysis temperature, and enzymolysis time. The hydrolysates prepared with Pepsin were inactivated at 95°C for 15 min and centrifuged at 12,000 rpm/min for 10 min (Hitachi Koki Co., Ltd., Japan). Supernatants were lyophilized and stored at −20°C.

SH solutions were fractionated into five groups using four different molecular weight cut-off membranes of 10, 5, 3, 1 kD at 0.2 MPa, 25C. Fractions were collected and named as SH-1, SH-2, SH-3, SH-4, and SH-5 and their MW ranges were: <1, 1–3, 3–5, 5–10 and >10 kDa, respectively. Lyophilized SH-2 (120 mg) was re-suspended in 2 ml distilled water and loaded onto a Sephadex G-25 gel filtration column (2.6×65 cm), which was pr-equilibrated and eluted with distilled water at a flow rate of 1.8 ml/min. The elution solution was collected at 2 min intervals and monitored at 220 nm; three peaks (SH-2-1, SH-2-2, SH-2-3) were pooled and lyophilised. A subfraction of SH-2-2 was further separated by reversed-phase high Performance Liquid Chromatography (RP-HPLC, Agilent 1260 HPLC, Agilent Ltd., USA) on a Xbridge BEH130 C18 column (4.6×250 mm, 5 µm particle size, Waters Scientific, USA) with a linear gradient of acetonitrile at a flow rate of 1.0 ml/min, which was equilibrated by 0.1% trifluoroacetic acid (TFA) in water. The elution (SHP) was monitored at 220 nm, collected, and lyophilized. Finally, the amino acid sequences of the purified peptides was sequenced on a Shimadzu PPSQ-31A automated gas-phase sequencer (Shimadzu, Kyoto, Japan). The purified peptides were dissolved in 10 µl of a 37% CH₃CN (v/v) solution and then applied to TFA-treated glass fiber membranes, precycled with Polybrene (Shimadzu Corporation).

Prostate cancer cells line PC-3 was obtained from the China cell bank of the Institute of Biochemistry and Cell Biology and cultured in F12 medium (Gibco, USA) supplemented with 10% Fetal Calf Serum (FCS), 100 U/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified 5% CO₂ atmosphere at 37°C.

Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) assay, Briefly, exponentially growing cells, at a density of 1×10⁴ cells/100 µl well in 96-well plates, were treated with different concentrations (5, 10, 15, 20 mg/ml) of SHP in complete medium or medium alone. CCK-8 was added to each well containing 200 µl of the culture medium 24, 48, and 72 h later and were then incubated at 37°C in a humidified 5% CO₂ atmosphere for 4–5 h. Absorbance was measured at 450 nm by a microplate reader (Bio-Rad, USA). All experiments were performed in triplicate and repeated three times.

The percentage inhibition of cell proliferation was calculated as follows:

The apoptotic morphologic changes were observed through the AO/EB staining (Acridine orange/Ethidium bromide) method, as described previously (24). PC-3 cells in logarithmic growth phase were plated at a final concentration of 1×10⁵ cells/well and incubated for 24 h. Adherent cells were inoculated with 2 ml/well of varying concentrations (5, 10, and 15 mg/ml) of SHP. F12 medium (10% FCS) was used as a control. The supernatant was aspirated and a AO/EB mixture [20 µl, containing 100 µg/ml AO and 100 µg/ml EB in phosphate buffered saline (PBS)] was added to the cells for 24 h and observed under a fluorescence microscope (Olympus, Japan).

PC-3 cells were seeded in six-well plates, grown for 24 h before, exposed to SHP (0,5, 10, 15 mg/ml) and collected 24 h later, centrifuged, washed twice with cold PBS and stained with annexin V-FITC and PI at room temperature for 15 min in the dark. After incubation, 1X binding buffer was added to each tube and then the cells were immediately analyzed by FACS Calibur flow cytometry (Becton Dickinson, NY, USA).

PC-3cells (2×10⁵ cells/ml) were co-cultured with different concentrations of SHP and harvested after 24 h. The cells lysates were prepared with RIPA buffer (20 mmol/l (PH 7.5), 150 mmol/l NaCl, 1% Triton X-100, sodium pyrophosphate, and EDTA) and collected via centrifugation. Equal amounts of protein were subjected to electrophoresis on 10 or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a PVDF membrane. The membrane were incubated with primary antibody (p53, Bax, Bcl-2, Caspase3, β-actin) in 0.1% BSA at 4°C overnight after blocked 5% dried skilled milk, then rinsed, incubated with secondary antibody at room temperature for 2 h, and rinsed again. Signals were detected by enhanced chemiluminescence (ECL).

One- and two-way analysis of variance (ANOVA) was used to compare the mean of each experiment. Significant differences between the means of parameters were assessed by using Tukey's test (P<0.05). All experimental data was analyzed using SPSS18.0 statistical software.

Orthogonal experimental design is a collection of mathematical and statistical techniques based on the orthogonal Latin square theory and group theory (25); it has been adopted as the primary tool for determining the best processing conditions. As such, we used an orthogonal experimental method (L16(4⁵)) to determine the best condition to hydrolyze sepia ink with pepsin (Table I). The factors affecting the cell proliferation of the hydrolysate were listed in a decreasing order as follows: A>E>B>D>C, where the material/distilled water (A) was found to be the most important factor affecting the cell proliferation inhibitory activity of the hydrolysates. The optimum extraction condition was determined to be A3B2C1D4E4, that is, material/distilled water 1:2, pH was 2.5, enzyme dose 300 U/g, enzymolysis temperature 45°C, and enzymolysis time 8 h. The material/distilled water (A) of sepia ink exhibited a significant effect (P<0.05) on the cell proliferation. Other factors, however, had no significant influence on the cell proliferation inhibitory effects. Purification characterization of anti-cancer peptides.

Our results revealed that co-culture of the sepia ink hydrolysate fractions (10 mg/ml) inhibited the proliferation of PC3 (28.75±2.13, 29.54±0.94, 32.79±1.27, 12.56±1.21%, respectively) (Fig. 1). Based on these results, we utilized the SH-2 fraction for the reemaining experiments as it had the greatest effect on PC3 cells growth (Fig. 2). SH-2 was separated into three subfractions (SH-2-1, SH-2-2, and SH-2-3). Among these subfractions, SH-2-2 (10 mg/ml) exhibited the highest inhibitory activity on cell proliferation (SH-2-1, 33.89±1.53%; SH-2-2, 48.26±1.24%; SH-2-3, 40.99±2.03%). Isolating peptides from SH-2-2 by RP-HPLC.

SH-2-2 purification was initiated using gel chromatography, followed by separation with RP-HPLC, and then samples constituting the peak I were collected (Fig. 3). We found that peak I possessed significant anti-Pca activity, and named it SHP. Its peptide sequence was obtained by N-terminal amino acid sequencing using an Applied Biosystema 494 protein sequencer and determined to be Leu-Lys-Glu-Glu-Asn- Arg-Arg-Arg-Arg-Asp with a molecular mass of 1371.53 Da.

We assessed the inhibitory effect of SHP on the proliferation of PC-3 cells in a CCK-8 assay (Fig. 4). SHP inhibited the growth of PC-3 cells in a time- and concentration-dependent manner and exhibited significant inhibitory effects at concentrations of 5, 10, 15, 20 mg/ml after SHP treatment for 24, 48, and 72 h (P<0.05).

Acridine orange and ethidium bromide (AO/EB) staining was used to identify the morphologic changes in PC-3 cells following co-culture with SHP (5, 10, 15 mg/ml) for 24 h. The results demonstrated that SHP induced clear morphologic changes normally associated with cellular apoptosis. Fig. 5; cells stained green represent viable cells, whereas yellow staining represents early apoptotic cells and reddish or orange staining represents late apoptotic cells. PC-3 cells treated with 5 mg/ml SHP showed clear changes in cellular morphology, including membrane blebbing, chromatin condensation, and fragmented nuclei (26). Similar features were observed in PC-3 cells treated with 10 and 15 mg/ml SHP, but late-stage apoptotic activity-related apoptotic bodies were also observed. Taken together, these results show the concentration-dependency of SHP for cancer cell apoptosis.

To confirm the observed induction of cell apoptosis by SHP, we performed flow cytometry using the Annexin V-FITC/PI assay (Fig. 6, lower right quadrants represent the early apoptotic cells [Annexin V binding and propidium iodide (PI) negative]. As shown in Fig. 6A, the majority of normal cells appeared in the Annexin V-negative section (lower left quadrants) and PC-3 cells treated with SHP (5, 10, and 15 mg/ml) increased the Annexin V-positive cell population from 8.85 to 29% (lower right quadrants).

To understand the mechanisms by which SHP induced apoptosis we examined the expression of proteins typically associated with apoptosis, such as p53, caspase-3, Bcl-2, and Bax (Fig. 7). The results indicated that SHP induced an increase in p53 expression and caspase-3 protein levels (P<0.05). Moreover, Bcl-2 levels were significantly downregulated (P<0.05), whereas Bax expression was enhanced and the ratio of Bcl-2/Bax protein levels was decreased. These findings clearly indicate that SHP induces apoptosis in PC-3 cells via the mitochondrial apoptotic pathway.