2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
Colorectal cancer (CRC) is the second most commonly diagnosed cancer in females and the third in males with over 1.2 million new cancer cases and about 600 000 deaths per year . Although surgical resection followed by radiation and chemotherapy are standard treatments for CRC, it is very difficult to completely resect the metastatic CRC (mCRC), the most diagnosed CRC. Combinations of fluoropyrimidines with oxaliplatin or irinotecan plus a biologic agent (such as bevacizumab) are currently considered to be the standard and best treatment for mCRC [2-3]. However, these current anti-colorectal cancer agents possessed serious side effects, including bone marrow inhibition, nausea and vomiting, and alopecia. Therefore, there is a need to develop novel anti-CRC drugs. Natural products including marine natural products are important sources of anticancer drug leads [4-7].
During the course of our ongoing project for the discovery of novel anti-colorectal cancer and anti-glioma agents from marine organisms [8-15], we identified a new phenolic ester tripolinolate A (TLA, Fig. 1) [12-13] from a halophyte plant Tripolium vulgare Nees, which was collected in a sea beach located at Zhoushan Islands (Zhejiang, China). TLA was found to have significant in vitro activity against the proliferation of colorectal cancer HCT-15 and SW620 cells and glioma U251 and U87-MG cells. A simple and efficient method was also designed to prepare TLA to obtain sufficient amount of TLA to facilitate further investigations of this bioactive compound for its potential in the treatment of CRC. The aim of the present study was to further evaluate the effects of TLA on the proliferation of human normal cells, the apoptosis and cell cycle in the colorectal cancer cells, and the growth of tumors in the tumor-bearing animals.
Male Balb/c mice (weighing 22 ± 2 g) were obtained from Shanghai Slac Laboratory Animal Center (Shanghai, China). All the animals were housed in a standard environment under controlled temperature (24-26 ℃) and light (12 h/12 h light/dark cycle), with free access to feedstuff and tap water. The mice were allowed to acclimate for at least one week before experiment. All procedures involving animal use and care were approved by the Zhejiang University Animal Experimentation Committee. All surgeries on mice were carried out under urethane anesthesia and all efforts to minimize mice's suffering were made.
Human colorectal cancer SW620 cells and mouse colorectal tumor CT-26 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Human normal colorectal CCD-18Co and foreskin fibroblast HFF cells were obtained from Shanghai Bogoo Biotechnology Co., Ltd. Human colorectal cancer SW620 cells were cultured in Leibovitz's L-15 medium, CT-26 cells in HDMEM medium with 10% fetal bovine serum, and CCD-18Co and HFF cells in HDMEM medium with 12.5% fetal bovine serum. SW620 cells were incubated at 37 ℃ in a humidified incubator without CO2, while other cells were incubated at 37 ℃ in a humidified incubator with 5% CO2. Cells after the third passage were used for the experiment.Chemicals and reagents
Tripolinolate A (TLA, 98.6%) was prepared by our group [12-13]. Doxorubicin (DOX, > 98.0%) and urethane were purchased from Sigma-Aldrich Co., LLC. (St. Louis, Missouri, USA), 5-fluorouracil (5-FU, > 98.0%) from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. (Shanghai, China), and Sodium heparin (150 IU·mg-1, Biosharp) from AwardBio Co., Ltd. (Shanghai, China). Annexin V apoptosis detection kits were obtained from Invitrogen (Shanghai, China). De-ionized water was purified by a Milli-Q system (Millipore, Bedford, MA, USA).Instruments
Flow cytometry [Beckman Coulter C500MCL, Beckman Coulter Commercial Enterprise (China) Co., Ltd.] was applied for the quantitation of apoptotic cells. Microplate reader Synergy 2 (BioTek Instruments Inc., Winooski, Vermont, USA) was used for sulforhodamine B (SRB) assay.Sulforhodamine B (SRB) assay
The effects of tripolinolate A (TLA) on the proliferation of human normal colorectal CCD-18Co and foreskin fibroblast HFF cells were evaluated using the SRB assay [10-11]. Briefly, the cells were treated with TLA at various concentrations in a 96-well plate for 72 h. The TLA-treated cells were immobilized with 10% cold trichloroacetic acid solution (50 μL) for 1 h at 4 ℃, rinsed with water five times, dried at room temperature, stained with 0.4% SRB (50 μL) for 10 min, and washed with 1% acetic acid solution five times. The dried dye was dissolved in 10 mmol·L-1 Tris buffer and the OD was measured at 515 nm on a microplate reader.Annexin V-FITC/PI double staining assay
Annexin V-FITC/PI double staining following by flow cytometric analysis [10-11]was used to quantify the apoptosis induced by TLA. Briefly, the cancer cells were treated with TLA for 72 h and the harvested cells (1 × 106 cells) were washed with cold PBS buffer. The washed cells were then re-suspended in 100 μL of 1 × binding buffer mixed with 5 μL of Annexin V-FITC and 1 μL of 100 μg·mL-1 PI working solution. The double stained cells were incubated at room temperature for 15 min and then 400 μL of 1 × binding buffer was added. The fluorescence was determined by flow cytometry at emission wavelengths 530 and 575 nm and excitation wavelength 488 nm.Cell cycle assay
Propidium iodide (PI) DNA staining following by flow cytometric analysis  was applied to determine cell cycle perturbation induced by TLA. Briefly, human colorectal cancer SW620 cells were incubated with TLA at different concentrations for 48 and 72 h, respectively. The cells were harvested, prepared as a single cell suspension in icy PBS, and then fixed with ice-cold 70% ethanol at 4 ℃ overnight. The fixed cells were collected by centrifugation at 1 900 r·min-1 for 7 min, rinsed with PBS twice, re-suspended in PBS with RNase A (50 U·mg-1), incubated for 30 min at 37 ℃, and then stained with PI in dark at 4 ℃ for 30 min. Finally, the cell cycle distributions were determined by flow cytometry.Animal model and drug treatment
One week after the mice acclimated to the environment, mouse tumor CT-26 cells (2 × 106/100 μL) were injected subcutaneously (s.c.) into the armpit of each mouse. After three days of transplantation, the tumor-bearing mice were divided into different groups (n = 6-8/group, including vehicle control group (CON, 10% hydroxypropyl-β-cyclodextrin), different TLA-treated groups (different dosages, each n = 10, dissolved in 10% hydroxypropyl-β-cyclodextrin, each), and positive control group (5-FU, n = 15 mg·kg-1, dissolved in 10% hydroxypropyl-β-cyclodextrin). The animals received TLA by intraperitoneal injection (i.p.) or in situ administration at different dosages with interval of 24 h for 10, 11, or 16 days. Vehicle control group (CON) received the same volume of 10% cyclodextrin at a 24 h-interval. Positive control mice were treated with 5-FU at a dose of 15 mg·kg-1·d-1. All the animals were weighed each day during the treatment period and sacrificed at the 10th, 11th, or 16th day after the transplantation, and then tumors were harvested and weighed. Livers, kidneys, and spleens were also harvested in order to determine the toxicity of TLA. The tumor growth inhibition (TGI) was calculated by the formula: TGI = [(MN)/M] × 100%, where M is the tumor weight of the control group (CON), and N is that of TLA or 5-FU treated group. The liver/kidney/spleen index was calculated by the formula: Liver/Kidney/Spleen Index = W/B, where W is the liver/kidney/spleen weight of every mouse, and B is the whole body weight of every mouse.Statistical analysis
The data are presented as mean ± SD. Differences between groups were analyzed using one way ANOVA, and P < 0.05 were considered statistically significant.Results and Discussion Activity of tripolinolate A (TLA) against the human normal cells
Tripolinolate A (TLA) has been previously shown to significantly inhibit the proliferation of colorectal cancer and glioma cells with IC50 values being 0.99 μmol·L-1 for SW620 cells, 4.58 μmol·L-1 for HCT-15 cells, 0.48 μmol·L-1 for U87-MG cells, and 12.9 μmol·L-1 for U251 cells , respectively The present study evaluated the activity of TLA against human normal colorectal CCD-18Co and foreskin fibroblast HFF cells. The CCD-18Co and HFF cells were incubated with TLA in different concentrations for 72 h, respectively, stained with SRB, and then measured by using microplate reader. The results showed that the IC50 values of TLA against the human normal cells were 76.7 μmol·L-1 for CCD-18Co cells and 63.3 μmol·L-1 for HFF cells (Fig. 2). The ratios of IC50 value for human normal cells to IC50 for colorectal cancer cells were 16.7 to 77.5 for CCD-18Co cells and 13.9 to 63.9 for HHF cells. These data implied that TLA had much less cytotoxicity in the human normal cells than the colorectal cancer cells.
Flow cytometric analysis with annexin V-FITC/PI double staining was applied to quantify the apoptosis induced by TLA in colorectal cancer SW620 cells. The SW620 cells were incubated with TLA (1.0 and 5.0 mmol·L-1) for 72 h. DOX (10.0 μmol·L-1) was used as the positive control. The control group (CON) was treated with vehicle (without TLA or DOX). The results (Table 1 and Fig. 3) demonstrated that the total apoptotic cells including early and late apoptotic cells induced by TLA increased by 57.87% for TLA (1.0 μmol·L-1) and 62.17% for TLA (5.0 μmol·L-1), respectively, compared to the control cells (CON, 3.81%). DOX also induced apoptosis in SW620 cells with a 37.69% increase in total apoptotic cells. Previous study  showed that TLA also induced apoptosis in human glioma U87-MG cells with a 38.5% increase in total apoptotic cells. All the data indicated that TLA remarkably induced apoptosis in the cancer cells.
To test whether an alteration of the cell cycle occurred after the treatment of TLA, the DNA content was measured by flow cytometric analysis. The SW620 cells were initially treated with different concentrations of TLA 5.0, 10.0, 15.0, 20.0, and 25.0 μmol·L-1 for 48 h, but it had no obvious effect on cell cycle. The SW620 cells were further incubated with TLA with the same different concentrations for 72 h. The results (Table 2 and Fig. 4) showed that the proportion of cells in the G2/M phase was increased 5.66%, 8.56%, 11.04%, 13.73%, and 17.33%, respectively, compared with that of the control cells (CON). These data indicated that TLA arrested colorectal cancer SW620 cells at the G2/M phase.
The antitumor activity of TLA was evaluated in the CT-26 tumor-bearing Balb/c mice. To find the effective dose, the antitumor activity of three doses of TLA (15, 30, and 60 mg·kg-1·d-1, n = 5 for each dose) was initially tested. 5-Florouracil (5-FU; 15 mg·kg-1·d-1) was used as the positive control. At the 11th day after the treatment, all the animals were sacrificed and then the parameters were measured. Although all the three doses (i.p.) had some antitumor activity, only 60 mg·kg-1·d-1 showed significant inhibitory effect (56.17%; P < 0.01). Therefore, three additional higher doses (60, 120, and 180 mg·kg-1·d-1, n = 6-8 for each dose) were tested. As shown in Table 3 and Fig. 5, TLA at all the three higher doses significantly inhibited CT-26 tumor growth with inhibition rates being 48.1% (60 mg·kg-1, TLA (60), P < 0.05), 73.9% (120 mg·kg-1, TLA (120), P < 0.01), and 67.2 % (180 mg·kg-1, TLA (180), P < 0.01), respectively, compared to the control group (CON). There was a suggestion of an inverted U-shaped dose response curve with the TLA. 5-FU (15 mg·kg-1, i.p.) also significantly suppressed the growth of CT-26 tumor with an inhibition rate being 81.7% (P < 0.001).
Significant changes (P < 0.05 or P < 0.01) of body weights of the animals treated with 5-FU from the 12th to 16th day were observed, compared to the control group (Table 4). However, there were no significant changes in body weights of the mice treated with TLA at all three doses. The liver/kidney/spleen indices of the animals treated with TLA and 5-FU were also calculated. The results (Fig. 5) showed that both liver index and spleen index for the 5-FU-treated mice were significantly lower (P < 0.05 or P < 0.01), while the liver index for TLA(120)-treated mice was higher (P < 0.05) and the spleen index for TLA(180)-treated mice was lower (P < 0.05), compared to the control group.
The antitumor activity of TLA by in situ administration was also evaluated in the CT-26 tumor-bearing Balb/c mice. In this experiment, the mice received TLA (120 mg·kg-1·d-1, the most effective dosage in above experiment) by in situ administration for 10 days and 5-FU (15 mg·kg-1·d-1) was used as the positive control. The results (Table 3 and Fig. 6) showed that both TLA and 5-FU had significant antitumor effects in the animal model with inhibition rates being 77.65% (P < 0.001) for TLA and 68.33% for 5-FU (P < 0.001), respectively. Significant changes in body weights of the mice treated by 5-FU (P < 0.01 or P < 0.001) and TLA (120) (P < 0.05 or P < 0.01) from the 1th to 8th day were observed (Table 5). However, the body changes from the mice treated by both antitumor agents had no significant value from 9th to 10th. The changes in the liver index of the mice treated by 5-FU and TLA (120) were also significant (P < 0.05, Fig. 6). No changes in the kidney/spleen indices of the mice treated by 5-FU or TLA were found.
Taken together, the data from the present study demonstrated that TLA at dose of 120 mg·kg-1 by both i.p. and in situ administration had significant antitumor effects in the colorectal CT-26 tumor-bearing Balb/c mice and TLA by in situ administration produced more potent antitumor activity than by ip administration. It seemed that both 5-FU and TLA might have some effects on the body weights, the liver/spleen indices of the tumor mice and the effects from 5-FU were more serious than that from TLA.
It has been reported that phenolic ester compounds such as caffeic acid phenethyl ester (CAPE, an antitumor ingredient in propolis) [16-17] and caffeic acid 3, 4-dihydroxyphenethyl ester (CADPE, an antitumor compound isolated from the traditional Chinese medicine Sarcandra glabra) [18-19] are easily hydrolyzed by carboxylesterase in rats and in vitro assay, while CAPE and CADPE are much more stable in the human plasma [16, 19]. This is because the hydrolytic carboxylesterase for CAPE and CADPE only exists in rat blood, but not in human blood [16, 19-20]. TLA is a phenolic ester and is likely to be easily hydrolyzed in rat blood. Our preliminary study also showed that TLA was not detected in both rat plasma and mice microsomes after 30-min incubation, but TLA was stable in both human plasma and human microsomes after 60-min incubation. Therefore, it is possible that TLA might have more potent anticancer effect in humans.Conclusions
In summarily, tripolinolate A (TLA), a new phenolic ester identified from Tripolium vulgare, has been previously found to significantly inhibit the proliferation of different colorectal cancer and glioma cells. The present study further investigated the effects of TLA on the proliferation of human normal cells, the apoptosis and cell cycle in colorectal cancer cells, and the growth of tumors in the colorectal tumor-bearing animals. It was found that: 1) TLA had much less cytotoxicity in the human normal colorectal CCD-18Co and foreskin fibroblast HFF cells than in the colorectal cancer cells; 2) TLA remarkably induced apoptosis in the colorectal cancer SW620 cells and arrested cell cycle at G2/M phase; and 3) TLA (120 mg·kg-1) by both ip and in situ administration had significant antitumor effects in the colorectal CT-26 tumor-bearing Balb/c mice. The antitumor effects of TLA in the other animal models and its antitumor mechanisms are worthwhile to be further investigated.
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