| Background:Gallbladder cancer, a very rare, highly-lethal disease, is the most common malignant tumor of the extrahepatic biliary tract and the seventh common gastrointestinal carcinoma. Gallbladder cancer is highly invasive and aggressive and carries a dismal prognosis. The 5-year survival rate for all stages of gallbladder cancer is approximately 5%. Most patients will die by one year following the surgery. Although gallbladder cancer is an uncommon disease, there is a very high incidence of gallbladder cancer in some regions, such as Chile, where gallbladder cancer has the highest mortality rate. The incidence of gall bladder cancer is increasing in China as well as north central India. Thus, it is important to study gallbladder cancer in relation to human health. In the absence of a cure, the search for new effective chemopreventive and/or chemotherapeutic agents against gallbladder cancer becomes exceedingly vital.Natural products are one of the main sources for discovery of new drug compounds. Due to their diverse biological effects, cyclic triterpenoids such as ursolic acid and tubeimoside have attracted much attention in the field of cancer research. Pachymic acid (PA) is a lanostrane-type triterpenoid from P. cocos, which is also called Fuling in Chinese medicine. In addition to P. cocos, pachymic acid has also been reported to be isolated from the European fungus Fomitopsispinicola. Meanwhile, PA also possesses anti-emetic, anti-inflammatory, and anti-cancer properties, PA is able to inhibit PMA-induced mouse skin tumor formation in mouse following initiation with 7,12 dimethylbenz [a] anthracene (DMBA), and Epstein-Barr virus early antigen activation in Raji cells. These findings suggest that PA serves as a potential anti-cancer agent.Recently, many studies showed that PA is able to inhibit cancer growth, invasion or metastasis in different cancers, including lung, breast and pancreatic cancer. However, there hasn’t been any research on effect of PA on tumorigenesis in gallbladder cancer. Therefore, we pay more attention to the effects of PA on gallbladder carcinogenesis.Objectives:The objectives of this study were to assess the effects of PA on cell growth, migration, adhesion and invasion using human gallbladder carcinoma cells, to assess the effect of PA inhibiting subcutaneous tumor growth in nude mice. Our data suggest that PA is able to effectively inhibit gallbladder cancer development. These results provide compelling evidence that PA may be useful as anti-cancer agents for treatment and/or prevention of gallbladder cancer.Materials and methods:Cell culture and treatmentThe human gallbladder cell line, GBC-SD, was propagated at 37℃ in a 5% CO2 humidified incubator in DMEM medium containing 10% fetal bovine serum,10 mM HEPES, and antibiotics. PA was reconstituted in DMSO at a stock concentration of 20 mM and subsequently diluted to the working concentrations for experimental procedures. Control treatments contained an equivalent concentration of DMSO (0.1%) for all experiments.Cell viability assayThe anti proliferative effect of PA on GBC-SD cells was evaluated using a cell counting kit-8 following manufacturer’s protocol. Briefly, after indicated treatment, 10μl of CCK-8 solution was added into each well, and following one hour incubation, the absorbance was measured at 450 nm using a microplate reader.Cell cycle assayAfter treatment, cells were trypsinized, harvested, and fixed in 1 ml 80% cold ethanol in test tubes and incubated at 4℃ for 15 min. After incubation, cells were centrifuged at 1,500 rpm for 5 min and the cell pellets were resuspended in 500μl propidium iodine (10μg/ml) contain -ing 300μg/ml RNase. Then cells were incubated on ice for 30 min and filtered with 53μm nylon mesh. Cell cycle distribution was calculated from 10,000 cells with ModFit LT TM software using FACS caliber.Western blot analysisProteins were prepared with RIP A buffer. Each sample (30μg) was subjected to SDS-polyacrylamide (10%) gel electrophoresis and electrotransferred onto a PVDF membrane. After blocked, the membranes were incubated with appropriate primary antibodies in blocking buffer overnight at 4℃. Then the membranes were incubated with secondary antibody. At last, the membranes were visualized with ECL Plus reagent (GE Healthcare) and developed onto X-ray film (Kodak). The antibodies used as follow:total Akt (1:1000), pAkt (1:1000), total ERK1/2 (1:1000), pERK1/2 (1:1000) and GAPDH (1:1500) are from Cell Signaling Technology (USA), PCNA (1:10000), ICAM-1 (1:200) and RhoA (1:1000).Migration and invasion assayEqual numbers of cells (1×105) in 0.5 ml DMEM complemented with 1% FBS were added to the upper compartment of the chamber and kept at 37℃ for 16 hours. As a chemoattractant, the lower compartment contained DMEM supplemented with 10% FBS. At the end of the incubation period, cells from the upper surface of the filter were wiped off with a cotton swab. The lower surface of the filter was stained with crystalviolet. The number of cells having migrat ed to the bottom of the chamber was counted in the light microscope on ten randomly select ed fields. The mean number of cells was calculated per field. Three sets of experiments were carried out, each in triplicate.Cell adhesion assay12-well plates were coated with fibronectin overnight at 4℃. Wells were rinsed with 1X PBS the following day, preheated to 37℃, for surface neutralization. Remaining binding sites were blocked with 0.1% bovine serum albumin (BSA) in PBS for a period of 1 hr. Cells were plated with GBC-SD cells After incubation for 60 minutes, cells were treated with percoll flotation medium and per coll fixative for 15 minutes, washed with PBS and treated with 0.5% crystal violet staining, washed again and allowed to dry. Then cells were counted.Animal experimentBALB/c (nu/nu) male nude mice were selected and received subcutaneous injection of 1 X 10’GBC-SD cells in logarithmic growth phase on abdominal region. After tumors grew to diameters of about 1 cm, the mice were randomly divided into two groups:experimental and control group each with 15 mice.2 mL saline with 2 mg PA was given to mice through gastric tube every day for 10 days, while mice in control group received normal saline. Every day we measured the diameter of tumors and drew tumor growth curve. Three days after termination of administration, all mice were sacrificed and tumors were dissected. We measured the diameter and weight of all tumors and calculated the tumor control rate of PA. The diameter of tumors in 2 groups was compared.Results:PA suppresses growth of human gallbladder carcinoma cellsCell growth was inhibited by 10μg/ml PA 12 h after treatment, and a concentration of 50μg/ml further reduced cell growth. And we also found that the growth of cells was suppressed in a time- and dose-dependent manner. After 48 h treatment, about 25%,40% and 70% of the cell growth were inhibited by PA at concentration of 10μg/ml,20μg/ml and 50μg/ml, respectively. Our results suggest that PA also inhibits the growth of gallbladder carcinoma cells in a time-dependent and dose-dependent manner. To confirm these observations by an alternative method, we did cell cycle analysis using flow cytometry analysis. Our data showed that there was a dose-dependent increase of the cells in G1 phase and dose-dependent decrease of cells in S phase, which support that PA inhibit the growth of gallbladder carcinoma cells by inducing cell cycle arrest at G1 phase. However, the cells in G2/M phase were not affected by PA treatment. Our data strongly support that PA treatment inhibits the growth of gallbladder carcinoma cells.PA reduces gallbladder carcinoma cell migration and invasionThe migration ability of cells was inhibited in a dose-dependent manner by PA all the groups (10μg/ml,20μg/ml and 50μg/ml). Our qualification data demonstrated significantly reduces migrated cells in PA treated groups. To further confirm our finding, we did transwell assay with Matrigel in the small chamber, which detect the invasion ability of the cells. The invasion ability of cells was also dramatically suppressed by PA treatment. Our data suggested that PA treatment can effective inhibit the migration and invasion ability of gall bladder carcinoma cells.PA reduces gallbladder carcinoma cell adhesion.As adhesion molecules play a pivotal role in the development of recurrent, invasive, and distant metastasis, we further investigated whether cell adhesion ability was affected by PA treatment in gallbladder carcinoma cells. As shown in Figure 4, PA treatment at concentration of 10μg/ml reduced the adhesion cells (P=0.059). Relatively high concentration of PA treatment, such as 20μg/ml and 50μg/ml significantly inhibit cell adhesion ability. Thus, our data indicate the PA treatment can inhibit the adhesion of gallbladder carcinoma cells.PA inhibits many signaling pathways involved in cancerWe detected many important proteins expression in our cells by western blot. We found that PA treatment can dose-dependently downregulated PCNA, ICAM-1, RhoA, which are important proteins that affect cancer cell growth, adhe -sion and migration. Importantly, we also observed a dose-dependent inhibition of phosphorylated-Akt and phosphorylated-ERK1/2 without affect the total expression of Akt and ERK1/2. Our data suggest that PA treat ment significantly inhibits Rho A, Akt and ERK1/2 pathway in gallbladder carcinoma cells.PA inhibits subcutaneous tumor growth in nude miceAll the tumors were entirely dissected from mice and measured, the tumor control rate of PA to tumors was 25.6%. We measured the diameter of each mouth for 13 days and drew the tumor growth curve using its mean value. Through t-test, we found the difference between experimental and control groups was statistically significant(P<0.05)Conclusion:PA can significantly inhibit cell growth, migration, invasion and adhesion in gallbladder cancer cells. We also demonstrate the PA inhibits cell growth through inducing cell cycle arrest, PA inhibits subcutaneous tumor growth in nude mice. Moreover, we find the PA is able to function through down-regulation of Akt and ERK1/2 pathways. Therefore, we speculate that PA may be used in treatment of gallbladder cancer. Together, these results encourage further studies of PA as a promising candidate for gallbladder cancer therapy. |
PDF Full Text Request