| Background:Plant-derived compounds are a major source for cancer therapy all the time.In recent years,triterpenoids have attracted much attention for their effective antitumor biological activities.Asiatic acid(AA;2α,3β,23-trihydroxy-12-ursen-28-oic acid,C3oH48OS),a naturally occurring pentacyclic triterpenoid,existed in a variety of plants,such as centella asiatica,purnella vulgaris,nepeta hindostana,eucalyptus perriniana and psidium guajava.Asiatic acid was primarily used as dermatologic topical agent to prevent UVA-mediated photoaging and wounding healing.Recent research suggested that asiatic acid possessed a wide range of pharmacological properties,including antihepatofibric,neuroprotective,anti-inflammation,anti-diabetics,and antitumor activities.As for antitumor effect,asiatic acid was reported to induce apoptosis death in hepatic and breast cancer cell lines and other tumour cells.Its efficacy was possibly attributed to the inhibition of NF-κB,p38 MAPK and ERK kinases,as well as the change of bcl and caspase family correlative proteins.Despite the above widely described antitumor properties,additional studies are necessarily to investigate the anti-metastatic potential of asiatic acid and to explore the molecular mechanisms of apoptosis induction and metastasis inhibition.Colorectal cancer(CRC)is one of the most common cancer types all over the world.The dietary and life habit change of Chinese people lead that CRC has been the third leading cause of cancer mortality in China.Despite recent advances in the surgical treatment,chemoprevention has emerged as an indispensable option for the control of CRC.The poor prognosis of patients with CRC is considered to primary tumor spread and metastasis.Thus,cancer metastasis is related to be the important causes of death in patients with CRC.As a critical event in the development of carcinoma cells,Epithelial Mesenchymal Transition(EMT)is a procedure that epithelial cells lose cell-cell adhesionand and cell polarity first,and then get migratory and invasive properties to be mesenchymal stem cells.Besides these,E-cadherin,N-cadherin and Vimentin are three important promoters which are responsible for cancer cells migration and invasion through different signaling pathways.PI3K/Akt/mTOR/P70S6K is an important signaling pathway in regulating progression in many tumors.This pathway controls proliferation,growth,translation,migration and survival in cells,and its over-activation is associated with tumor poor prognosis.There are many anti-cancer drugs through the PI3K/Akt/mTOR/p70S6K axis exerting the anticancer effect.For example,targeting insulin-like growth factor 1 receptor(IGF-1R)significantly inhibited growth and metastasis through suppression of the PI3K/Akt/mTOR/p70S6K signaling pathway.In addition,BMP9 inhibited proliferation and migration in breast cancer cells and the PI3K/Akt/mTOR/p70S6K signaling pathway was involved in this process.In another case,activation of the PI3K/Akt/mTOR/p70S6K pathway is involved in S100A4-induced viability and migration in colorectal cancer Cells.Programmed cell death-4(Pdcd4)is a novel tumor suppressor protein that inhibits protein synthesis by suppression of translation.The PI3K/Akt/mTOR pathway constitutively represses Pdcd4 expression in AML,and ATRA induces Pdcd4 through inhibition of this pathway.Besides,Pdcd4 has also been shown to inhibit invasion by activating activator protein-1(AP-1).It is long believed that Pdcd4 and PI3K/Akt/mTOR/p70S6K constitute an important pathway modulating multiple biological processes in carcinoma cells.Particularly,there are rare studies on that the inhibition of PI3K/Akt pathway is always responsible for highly expressed Pdcd4 in cancers including colon carcinoma cells.Objectives:In the present study,we first evaluated the apoptosis and anti-tumour effects of asiatic acid on human colon cancer cells(SW480 and HCT116),and revealed the role of PI3K/Akt signaling pathway as for the underling molecular mechanism.Asiatic acid inhibited proliferation,migration and induced apoptosis by regulating Pdcd4 through PI3K/Akt/mTOR/p70S6K pathway partially.Our study was to testify that asiatic acid can inhibite the PI3K/Akt/mTOR signaling pathway leading to apoptotic cell death,with increasing the expression levels of Pdcd4 and invasion inhibition in cells.Although therapeutics is still in infancy in colon carcinoma,our present study are desirable and asiatic acid may be a novel therapeutic drug for treating colon carcinoma.Methods:1.Asiatic acid inhibited viability of colon carcinoma cells.1.1 Materials.RPMI1640 with 10%heat-inactivated fetal bovine serum(FBS)were purchased from Biological Industries(Biological Industries,Israel).Rapamycin was purchased from Selleckchem(Houston,TX,USA).Antibodies were purchased from Cell Signaling Technology(Danvers,MA,USA).Hoechst33342 dye was purchased from Sigma(Sigma;USA).Fluoresc ein isothiocyanate(FITC)-labeled Annexin V and propidium iodide(PI)were purchased from Keygen Biotech(NanJing,China).1.2 Cell line and asiatic acid samples.The human colon cancer cells(SW480 and HCT116)were obtained from the Cancer Research Institute,Southern Medical University(GuangZhou,China).Cells were incubated in RPMI1640(Biological Industries;Israel)added with either 10%heat-inactivated fetal bovine serum(FBS)(Biological Industries;Israel),at 37 C in a humidified atmosphere containing 5%CO2.Asiatic acid samples.Asiatic acid previously isolated from the urban of Centella asiatica(Umbelliferae)was used.These compounds used for this study were checked by HPLC and were>97%pure.Asiatic acid was dissolved in dimethylsulfoxide(DMSO;Sigma;USA)to make a stock solution at a concentration of 1 mg/ml,which was further diluted to the appropriate concentration with culture medium prior to each experiment.1.3 Cell viability assay.Cell viability was measured by MTT assay.We firstly generated two human colon cancer cell lines.Subsequently,cells(3 × 103)were seeded into 96-well plates(NEST,China)and cultivated to adhere.After the cells were treated with various concentrations of asiatic acid from 24 h to 72 h,15 μl of MTT solution was added to each well for an additional 4 h at 37 ℃,and 150 μl DMSO was also added into each well,follwed by incubation at 37 C for 10 minites with gentle shaking.The absorbance value(OD)of each well was measured at 490 nm using a spectrophotometer(ELx800,Bio Tek Instruments,USA).Experiments were performed in triplicate.1.4 Morphological abnormality assay of cells.The morphological abnormality of cells was observed by optical microscope.Briefly,5 × 105 cells were seeded into per well of six-well plates(NEST,China)and cultivated to adhere.After that,cells were treated with various concentrations of asiatic acid for 24 h.Areas were chosen for ultra-thin sectioning and viewed with an electron microscope(JEM-1011 transmission electron microscope,USA).1.5 Plate colony formation assay.The proliferation of the cells was detected by plate clone formation assay.The cells were seeded into six-well plates(NEST,China)at 2 × 102 cells per well.After incubation for 24 h at 37 C,the medium was replaced by asiatic acid at right concentrations respectively.When there were visible clonings,stop to fix and stain the cells,with counting the number of colony under a microscope.2 Asiatic acid reduced migration of colon carcinoma cells.2.1 Migration assay.For migration assay,1 × 102 cells in 200 μl RPMI1640 were seeded on the upper chamber of transwell with 8.0-μm-pore polycarbonate membrane insert.RPMI1640(600 μl)with 10%fetal bovine serum was added into the lower chamber as a chemoattractant.After the cells were incubated for 8 h,the insert was washed with PBS,and cells on the top surface of the insert were removed gently by a cotton swab.Cells adhering to the lower surface were fixed by methanol,stained by 0.1%crystal violet,and counted under microscope in 6-8 predetermined fields.For each experiment,three independent filters were analyzed.2.2 Western blotting assays.The cells were harvested and lysed with protein lysate.Same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS PAGE,10%)and transferred to polyvinylidene fluoride(PVDF)membranes.The membranes were incubated with antibodies(Anti-E-cadherin,anti-N-cadherin,anti-vimentin,anti-β-antin,anti-GAPDH)(Cell Signaling Technology,USA)overnight at 4 C,until they were blocked with 5%nonfat dried milk for 1 h.Then,the immunoreactive bands were visualized by enhanced chemiluminescence using HRP conjugated IgG secondary antibodies.The bands were visualized by eECL Western Blot Kit(CWBIO Technology).The images were captured with ChemiDocTM CRS+Molecular Imager(Bio-Rad).3 Analysis of cell apoptosis3.1 Analysis of cell apoptosis with Hoechst33342.Cells were distributed into control and treatment groups with asiatic acid at certain concentrations for 24 h.After washing with PBS for 1-2 times,cells were added at the concentration of 5 μg/ml of Hoechst33342 dye and were incubated in the dark for 5 minutes.Then,the cells were observed with fluorescence microscope(Nikon TE2000,Beijing,China)and were photographed in 350 nm excitation wavelength and 460 nm emission wavelength.3.2 For the analysis of the apoptosis rate using flow cytometry.Each cell line was maintained at a density of 2 × 105 cells in six-Well plates.After treatment with asiatic acid,the cells were harvested,rinsed with ice-cold PBS,and were treated for 30 minites with fluorescein isothiocyanate(FITC)-labeled Annexin V and propidium iodide(PI)(Keygen Biotech;NanJing,China)for 10 minites at room temperature(RT)in the dark,and binding buffer(400 μl)was added to each tube according to the supplier’s protocols,and then were analyzed with a flow cytometer within 1 h.3.3 Analysis of cell apoptosis for DNA contentCells were distributed into six-well plates,and each cell group had three wells.After incubation for 12 hours at 37 ℃,cells were treated with asiatic acid from 0 to 25 μg/ml respectively.To evaluate the DNA content,the nuclei were fixed in methanol,stained with 15 μg/ml of 4,60-diamidino-2-phenylindole(PI)and 100μg/ml DNase free RNase A for 30 minites at 37 C,rinsed twice with PBS.The DNA content of labeled cells was acquired using FACS cytometry assay(BD Biosciences company).All experiments were carried out in triplicate.3.4 Western blotting assays.The cells were harvested and lysed with protein lysate.Same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS PAGE,10%)and transferred to polyvinyhdene fluoride(PVDF)membranes.The membranes were incubated with antibodies(anti-phosphorylated Akt(Ser473),anti-Akt,anti-phosphorylated mTOR,anti-mTOR,anti-phosphorylated p70S6K,anti-p70S6K,anti-Pdcd4,anti-p-antin,anti-GAPDH)(Cell Signaling Technology,USA)overnight at 4 C,until they were blocked with 5%nonfat dried milk for 1 h.Then,the immunoreactive bands were visualized by enhanced chemiluminescence using HRP conjugated IgG secondary antibodies.The bands were visualized by eECL Western Blot Kit(CWBIO Technology).The images were captured with ChemiDocTM CRS+Molecular Imager(Bio-Rad).4 Statistical analysis.All data are expressed as the mean value ± Standard error of the mean(SEM)from at least three independent experiments.The comparisons of different groups were performed using Student’s t-test and one-way of variance(ANOVA).Statistical analyses were performed with the SPSS 13.0 statistical software package(SPSS Inc.Chicago,IL,USA).All statistical tests were two-sided,and P<0.05 was considered to be statistically significant.Results1 Asiatic acid inhibited viability of colon carcinoma cells.MTT assay was used to determine the effects of asiatic acid on the viability of SW480 and HCT116 cells.Cells were treated with various concentrations of asiatic acid(0-50 μg/ml)for 24,48,and 72 h.Asiatic acid significantly inhibited the growth of SW480 and HCT116 cells in a dose-and time-dependent manner.Thus,we select some indicated concentrations and tretment-tine of asiatic acid for the subsequent studies(p<0.05,p<0.01,P<0.001).Cells were treated with asiatic acid at the indicated concentrations(15 and 25 μg/ml)for 24 h.The control groups grew significantly,while the morphology of drug-treated groups became small and pyknotic,and kept their cytoplasm condensed.Cell organelles were damaged with lumps of different sizes,which confirmed asiatic acid had cytotoxic activity in human colon carcinoma cells.Colony formation of SW480 and HCT116 cells was inhibited by asiatic acid.As expected,these cell lines treated with asiatic acid presented an obviously reduced cell growth and colony formation relative to the control cell lines.With the single clone area reduction,the number of clones treated with asiatic acid in groups significantly was reduced,which indicated that the activity of single cell proliferation was also decreased.2 Asiatic acid reduced migration of colon carcinoma cells.To gain an insight into the role of asiatic acid in the cell migration and tumorigenesis of SW480 and HCT116 cells,we implement the transwell migration assay.It was showed that there were lesser cells penetrating the membranes in drug-treated group than in control group.All in all,the result certified that asiatic acid could reduced the migration of SW480 and HCTI16 cells.Besides,the number of control group of SW480 was more than HCT116,which indicated the ability of migration between SW480 and HCT116 was different on the other hand.As the initiator of the metastatic cascade in the tumour cells.EMT-interrelated factors such as Vimentin,N-cadherin and E-cadherin were evaluated.Consistent with the result of transwell migration assay,we found the expression level of E-cadherin was increased while Vimentin and N-cadherin were reduced.These appearances showed that the inhibitory ability of asiatic acid on the migration in colon carcinoma cells related to EMT.As a critical event in the development of carcinoma cells,Epithelial Mesenchymal Transition(EMT)is a procedure that epithelial cells lose cell-cell adhesionand and cell polarity first,and then get migratory and invasive properties to be mesenchymal stem cells.Therefore,we discussed the expression of EMT relevant factors.As expected,asiatic acid significantly increased the level of E-cadherin and decreased the level of N-cadherin,and vimentin in SW480 and HCT116,indicating that EMT participated in the inhibitory of asiatic acid on migration of partial colon carcinoma cells.3 Asiatic acid induced apoptosis of colon carcinoma cells.we investigated that asiatic acid could induce apoptosis in colon carcinoma cells again.We first performed Hoechst33342 nucleus staining.The formation of cytoplasmic agglutination,which are suggestive of active apoptosis,was observed in SW480 and HCT116 cells treated with 15 and 25 μg/ml asiatic acid for 24 h,whereas none were observed in the control groups.Flow cytometric evaluation displayed that apoptotic cells appeared more and more in the drug-treated group,with the increasing concentration of asiatic acid.Following the observation,we next found that apoptotic rates of the drug groups were higher relative to the control cells group.More interestingly,an increase in apoptosis was observed in the sub-GI population of SW480 and HCT116 cells treated with asiatic acid.Meanwhile,the cell cycle also showed notable change with the decreased percentage of GI phase and increased percentage of G2+M with S phase cells(P<0.05).The PI3K/Akt/mTOR/p70S6K signaling pathway is one of the significant pathways regulating proliferation,apoptosis and migration of cancerous cells.Then,we decided to investigate whether asiatic acid affected this pathway in colon carcinoma cells.As we can see,asiatic acid obviously decreased the phosphorylation of PI3K,Akt(Ser473),mTOR,and p70S6K in a treatment-concentration-dependent manner.Besides,protein expression of the programmed cell death factor Pdcd4,the downstream factor of p70S6K,was increased.Then we used the mTOR inhibitor rapamycin to explore whether this pathway got involved in the proliferation,migration and apoptosis in colon cancer cells.Pretreatment of asiatic acid and rapamycin further decreased the expression of mTOR and p70S6K and increased the expression of Pdcd4,testifying that asiatic acid exerted anticancer effect by regulating Pdcd4 via PI3K/Akt/mTOR/p70S6K pathway in colon cancer cells partially.Conclusion:1 MTT assay and colony formation assay indicated asiatic acid inhibited viability of colon carcinoma cells.Treated colon carcinoma cells revealed the antiproliferative effects of asiatic acid;2 Transwell migration assay showed that asiatic acid could reduced the migration of SW480 and HCT116 cells.Asiatic acid moulated epithelial mesenchymal transition marker protein in colon carcinoma cells.Asiatic acid significantly increased the level of E-cadherin and decreased the level of N-cadherin,and vimentin in SW480 and HCT116,indicating that EMT participated in the inhibitory of asiatic acid on migration of partial colon carcinoma cells.3 Asiatic acid induced apoptosis and cell cycle arrest of colon carcinoma cells.Our study found asiatic acid inhibited the PI3K/Akt/mTOR signaling pathway leading to apoptotic cell death,with increasing the expression levels of Pdcd4 and invasion inhibition in cells. |
PDF Full Text Request