For the acute toxicity testing, a total dosage of 1000 mg/kg of P2 was administered to the mice by intraperitoneal injection (i.p.) or intravenous injection (i.v.). The general behavior, body weight and fatality of treated mice were monitored daily for two weeks. As a result, the general behavior of P2 treated mice did not exhibit much variation for 14 days, except for lethargy on the first day after administration compared with the control mice. No fatality was observed during the experiment and no macroscopic lesions were seen on the organs of treated mice when being dissected after 14 days’ observation. The body weights of the treated mice had a bit of an alteration, but recovered five days after administration in both the i.p. group and i.v. group (Table 1). The results of histopathologic analysis revealed no gross morphologic difference between P2-treated mice and control mice (Figure 5).

Mice were treated with 1000 mg/kg of P2 by i.p. or i.v. After 14 days’ observation, the mice were sacrificed, and five kinds of organs, including the heart, liver, spleen, lung and kidney, were excised. The organs were then fixed by paraformaldehyde and stained by H & E to make histopathologic analysis; magnification (200×, Nikon, Tokyo, Japan).

To understand the inhibitory effect of P2 on the growth of tumor cells in vivo, mice hepatoma cells H-22 and sarcoma cells S-180 were selected to evaluate the in vivo antitumor effects of P2. The effects of P2 on mice transplanted with S-180 or H-22 are presented in Table 2 and Figure 6. The results revealed that P2 significantly decreased the tumor weights of S-180 tumor-bearing mice and H-22 tumor-bearing mice. The inhibitory rates were 34.0%, 45.8% and 60.1% for S-180 tumor-bearing mice and 26.4%, 41.4% and 46.4% for H-22 tumor-bearing mice at the dosages of 7, 21, 63 mg/kg/day, respectively. Furthermore, P2 could raise the spleen index, but had no obvious influence on the thymus index of the tumor-bearing mice (Table 2).

Cyclophosphamide (CTX) was purchased from Shanxi Pude pharmaceutical Co., Ltd. China. Dimethyl sulfoxide (DMSO), 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), Penicillin G and streptomycin were obtained from Sigma Chemical (St. Louis, MO, USA). RPMI-1640 and Fetal Bovine serum (FBS) were purchased from GIBCO Invitrogen Corporation (San Diego, CA, USA). All other chemicals used were of analytical grade, available locally.

A. subcrenata was purchased from Huangsha seafood market (Guangzhou, China). A. subcrenata was shelled and washed by distilled water three times. To obtain the active constituents, phosphate-buffered saline solution was added to A. subcrenata at 3:1 volume to weight ratio for homogenation and centrifugation to collect the supernatant. Ammonium sulphate powder was slowly added to the supernatant to precipitate the protein components. The precipitate was collected for further purification by ion-exchange chromatography eluted with sodium chloride solution of different concentrations. The effluent fractions were detected by a protein detector at 280 nm. The effluent fraction of P2 was concentrated and dialyzed. A dry powder of P2 was obtained by vacuum freeze-drying.

Cell lines used for evaluation of the in vitro cytotoxicity in this study included seven tumor cell lines and one normal cell line, namely HeLa (cervical cancer cell line), HT-29 (colon cancer cell line), HepG2 (hepatocellular carcinoma cell line), K562 (leukemia cell line), DU145 (prostate cancer cell line), SPC-A-1 (lung adenocarcinoma cell line), B16-F10 (murine melanoma cell line) and L-02 (human normal liver cell line). All of cell lines were provided by the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and the cells were cultured in RPMI-1640 medium, which was supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin and cultured in an atmosphere of 5% CO₂ at 37 °C. Cells were collected for the experiments in the logarithmic growth phase.

The cell lines used for evaluation of the in vivo antitumor activity in this study included mice sarcoma S-180 and hepatoma cells H-22, which were provided by Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. S-180 ascite and H-22 ascite were maintained in vivo in mice by transplantation of 0.2 mL of ascites (1 × 10⁷ cell) from the infected mice to the non-infected mice.

Kunming mice (KM mice, 20 ± 2 g) were purchased from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Mice were housed in a temperature (23 °C) and humidity (55%)-controlled room on a 12 h light/dark cycle and given free access to water and food.

For the antitumor screening tests, P2 was dissolved and diluted to 1.56, 6.25, 25, 100, 400 and 1600 μg/mL by RPMI-1640 medium. For the tests of time-dependent and dose-dependent effects, the concentrations of P2 were 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 μg/mL. The HeLa and HT-29 cells were treated with different concentrations of P2 for 24, 48 and 72 h. For the cytotoxicity on normal cells, human liver L-02 cells were treated with P2 for 48 h. All tests were performed by MTT assay according to the paper [31], with some necessary variation. Briefly, cells were seeded in the well of 96-well microtiter plates and incubated with different concentrations of P2 for a setting time. After that, 20 μL of MTT (5 mg/mL) was added to each well, and then, the plates were incubated for another 4 h at 37 °C. The supernatant was aspirated and MTT-formazan crystals were dissolved in 200 μL of DMSO. Absorbance was measured spectrophotometrically at 570 nm. Cell growth inhibition was evaluated by comparing the absorbance of treated and untreated cells. The percentage of cell growth inhibition was calculated as the following formula: (1)

Eighty mice were randomly divided into four groups of either of the sexes. Two groups of mice received a single dose of P2 at 1000 mg/kg. The sample was administered via the caudal vein and abdominal cavity (0.2 mL/10 g). The control groups were given normal saline solution (0.9% NaCl). Mouse survival and body weight variations were monitored daily for 14 days. After 14 days’ observation, mice were sacrificed, and five kinds of organs, including the heart, liver, spleen, lung and kidney, were excised. The organs were fixed by paraformaldehyde and stained by hematoxylin and eosin (H & E) to make a histopathologic analysis. The MTD was defined as the highest dose that induced no more than 15% weight loss comparing with control, caused no toxic death and no visible organ lesions [32].

Animal care and treatment were performed at Jinan University’s experimental animal center. A total of 70 mice (male, body weight 20 ± 2 g) were injected with 6 × 10⁶ cells/mouse of S-180 (sarcoma-180) or H-22 (mice hepatoma cells) subcutaneously into the right front armpit. 24 h after implantation of tumor cells, the mice were randomly divided into five test groups with 14 mice per cohort. The mice were daily treated by P2 (7, 21, 63 mg/kg/day), CTX (25 mg/kg/day) or normal saline (NS, 10 mL/kg) for nine days. The mice were sacrificed, and the tumors were excised and weighed for evaluating the tumor growth inhibition at 24 h after the end of treatment. The spleen and thymus were also segregated and weighed to calculate the spleen index and thymus index for assessing the effects of P2 on the immune system of the tumor-bearing mice. The tumor inhibitory rate and organ index were calculated by the following formulae: (2) (3) W_control and W_treated were the average tumor weights of the control and treated mice, respectively. W_{spleen/thymus} and W_body stand for the average weights of spleen/thymus and body of the mice.

Data are shown as mean ± SD. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test. All comparisons were made relative to untreated controls and significant difference is indicated as * p < 0.05 and ** p < 0.01.