Apigenin Inhibits Growth And Metastasis Of Colorectal Cancer Through Regulating Transgelin

BackgroundColorectal cancer (CRC) continues to be a major healthcare concerns worldwide with more than a million new patients diagnosed annually, accounting for the second leading cause of cancer deaths for both men and women. Its incidence still continues to increase. The precise causes and mechanisms of colorectal cancer development still remain elusive. It is determined by genetic factors and lifestyle-related factors. Surgery is the first choice for early colorectal cancer treatment. Thanks to the introduction of new surgical techniques, the overall survival rate of patients with early colorectal cancer has been increased. However, approximately40%of patients diagnosed with CRC will develop colorectal metastases. Therefore, it is necessary to use adjuvant therapy and palliative care to improving life quality and increasing survival rate of patients with metastases. However, it is lack of effective, low toxicity and low cost chemotherapy drugs. Molecular targeted drugs for colorectal cancer treatment provide new choices, but the application and effect are not very exact, and the price is expensive. As patients becoming resistant to existing chemotherapy drugs, the treatment effect declines obviously.Therefore, it is critical for us to exploit low toxicity, high efficiency and low price natural drugs. Flavonoids are polyphenol chemicals found naturally in fruits, vegetables, tea and other plant origin foods and beverages. Experimental evidence suggests that flavonoids have several potential anti-carcinogenic characteristics due to the different structural features, including anti-oxidant, anti-estrogenic, anti-proliferative and anti-inflammatory properties. In addition, they have been shown to have anti-invasive and/or anti-metastatic activities in a variety of cancer types, including brain, oral cavity, pharynx, thyroid, breast, lung, liver, bile ducts, pancreas, testes, small intestine, colon, kidney, ovarian, cervical, prostate and the epidermal cancers; fibrosarcoma, osteosarcoma, Kaposi’s sarcoma, and melanoma are also inclusive.Apigenin (API) is a natural flavonoid lower in toxicity and higher in efficacy compared with other flavonoids. As a promising molecule for cancer prevention, it receives attention widespread. Apigenin have various biological activities and pharmacological effects. A number of the biological effects of apigenin in numerous mammalian systems in vitro as well as in vivo are related to its antioxidant effects and its role in scavenging free radicals. In addition, it also has anti-mutagenic, anti-inflammatory, antiviral, inhibiting tumor cell proliferation, inducing apoptosis and sensitizing radiotherapy and chemotherapy effects. In recent years, evidence suggests that anti-tumor activity of apigenin is associated with its inhibiting tumor cells proliferation, inducing apoptosis, inhibiting invasion and metastasis of tumor cells and regulating tumor cells signaling pathways. Apigenin can inhibit the occurrence of a variety of cancers such as prostate cancer, ovarian cancer, bladder cancer, colon cancer. Its anti-tumor activities have been demonstrated diversity.However, the molecular mechanism of its anti-tumor activity remains unclear. In this study, we expounded its role playing in colorectal cancer by observing its effect in the biological behavior of human colon cancer cell line SW480including cell proliferations, cell cycle, apoptosis, and cell invasion and metastases. Proteomic techniques allow for identification of the protein changes caused by apigenin in SW480cells. There were12differential spots that simultaneously existed in apigenin exposure compared with the solvent control group. And9protein spots were successfully identified through time-flight mass spectrometry. The protein transgelin (TAGLN, SM22) was choosed to get further study. Transgelin is a22kDa cytoskeletal protein. It is reported that transgelin is closely linked with neoplastic transformation or progression of carcinogenesis. And transgelin was identified as a novel transcriptional regulator of MMP-9expression, transgelin repress MMP-9expression via an amino terminus calponin homology domain by reducing AP-1-dependent trans-activation by way of compromised ERK activation. It is suggested that diminished transgelin expression in cancers may partly account for the elevated MMP-9expression in these tumors. The purpose of this study was to investigate whether the antitumor activation of apigenin was associated with its function in upregulating transgelin and then repressing MMP-9expression and to find a theoretical basis of the molecular mechanism of apigenin antitumor activation.ObjectiveThe purpose of this study was to investigate the influence of apigenin in colorectal cancer, and to explore whether the antitumor activation of apigenin was associated with its function in upregulating transgelin and then repressing MMP-9expression. It may find a theoretical basis of the molecular mechanism of apigenin antitumor activation and provide possible new targets for the treatment of colorectal cancer.MethodsAnimals and Drug AdministrationEstablish the orthotopic visualization animal model of colorectal cancer according to the literature methods. Apigenin was administered by gavage to nude mice at doses of100mg/kg/d for2weeks. It begins to administer when the tumor can be observed in the imaging system. Situ solid tumors were observed in the imaging system after inoculating for42days. Then nude mice with colorectal cancer were randomly allocated to the following groups containing seven animals each. Group Ⅰ:Vehicle controls; Group Ⅱ:100mg/kg/d Apigenin. Nude mice weight was recorded and the tumor in vivo was observed by imaging system before the experiment. During the experiment, nude mice weight was dynamically measured; tumor volume change and tumor metastasis was dynamically observed. After the end of the experiment, solid tumor weight of nude mice was measured; tumor metastasis was observed under the imaging system.Cell Viability AssaySW480cells were seeded in96-well plates (1×104cells/well), cultured in complete grown medium for24h, and then grown in the low serum medium for another24h. Low serum cultured cells were treated with20,40,80,120μM apigenin and vehicle (DMSO) for24and48h. Thereafter the medium was changed and incubated with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for4h. The number of viable cells was directly proportional to the production of fromazan which then dissolved with DMSO, and measured spectrophotometrically at490nm by Emax Microplate reader. Apigenin inhibition of human colon cancer cells according to the following formula:inhibition rate=(1-Experimental group OD value/control group OD value)×100%.Scratch Motility Assay1×106SW480cells were seeded in a6-well plate and grown overnight to confluency in complete media. The cells were low serum cultured for24h. The monolayer was scratched with a pipette tip and then washed with PBS to remove floating cells in low serum media and photographed (0h).40μM apigenin and DMSO groups were applied for24and48h and photographed at the same location as Oh photographed. The migration rate was calculated.Cell Invasion AssayThe SW480cells with green fluorescent fusion protein were treated with40μM apigenin and DMSO (solvent control group) for24or48h, and then the cells was observed via fluorescence microscopy.Cell Cycle DistributionSW480cells were treated with20,40,80,120μM apigenin and DMSO for24or48h, and then harvested, washed with cold PBS and processed for cell cycle analysis. ModFit LT2.0trial cell cycle analysis software was used to determine the percentage of cells in the different phases of the cell cycle.Cell Apoptosis Analysis1) SW480cells were treated with20,40,80,120μM apigenin and DMSO for48h. For apoptosis studies, cells were fixed with methanol and stained with Hoechst33258. Morphological changes of nuclei were observed under a fluorescence microscope.2) SW480cells were treated with40μM apigenin and DMSO for48h and then fixed with4%paraformaldehyde for15min. Annexin V-FITC and propidium iodide were added to the samples for5-15min in the dark. The cells were analyzed under a fluorescence microscope using a dual filter set for FITC+propidium iodide. The cells that showed green staining (early stage) or green with red staining (middle or late stage) were counted as apoptotic cells.Two-dimensional Electrophoresis Screening of Flight Mass Spectrometry Identification of Differentially Expressed ProteinsSW480cells were treated with40μM apigenin and DMSO for48h. The differentially expressed mitochondrial proteins of the two groups were screened by two-dimensional electrophoresis, and then the different protein spots were identified via time flight mass spectrometry.Immunofluorescence StainingSW480cells were treated with40μM apigenin or DMSO for12h or24h. Then differentially expressed mitochondrial proteins were observed by immunofluorescence staining.Real-Time PCRSW480cells were treated with20,40,80,120μM apigenin and DMSO for24h. And then qPCR technology was used to detect mRNA expression.Western Blot Analysis1) SW480cells were treated with40μM apigenin or DMSO for48h. The mitochondrial protein, cytoplasm protein and nucleoprotein were extracted and then bloted with the indicated primary antibodies. Protiens were visualized with the secondary antibodies followed by chemiluminescence detection.2) SW480cells were treated with20,40,80,120μM apigenin group or DMSO for48h. The whole protein was extracted and then bloted with the indicated primary antibodies. Protiens were visualized with the secondary antibodies followed by chemiluminescence detection.Small Interfering RNA TransfectionThe siRNA-transegin were introduced into SW480cells by LipofectamineTM2000according to the manufacturer’s instructions and then the cells were harvested and subjected to western blot with the indicated antibodies or the cell viability was determined by MTT assay.Tissue Microarray and ImmunohistochemistryThe differentially expressed protein of human colorectal adenocarcinoma (n=30), metastatic adenocarcinoma of lymph node (n=25) and liver (n=5) were determined by immunohistochemistry.Statistical analysisAll statistical data were analyzesed using SPSS13.0software. Comparisons between groups were performed using the Student’s t-test or one-way ANOVA followed by Newman-Keuls test if data followed a Gaussian distribution; comparisons of data not following Gaussian distribution were submitted to the Mann-Whitney test. Statistical significance was defined as P<0.05.ResultsApigenin Inhibited Growth and Liver Metastasis of Colorectal Cancer in Nude MiceIn vivo results showed that compared with control group, apigenin decreased relative area and relative fluorescence intensity of tumor, the differences were statistically significant (t values were-10.513and-14.958; P value was less than0.001). The differences of the fluorescence intensity values in intestine of the two groups were statistically significant (t value was-4.630, P value was0.000). Liver metastasis could be observed in only one nude mouse of apigenin group and there was liver metastasis in all6mice of control group. The differences of the fluorescence intensity values in liver of the two groups were statistically significant (t value was-4.917, P value was0.000). The fluorescence semi-quantitative analysis was undertaken by IPP6.0software. The results above showed that apigenin can inhibit colorectal cancer growth and reduce colorectal liver metastasis of nude mice.Apigenin Inhibited Cell Proliferation-Cell Viability AssayThe effects of apigenin on cell proliferation were measured with MTT assay, using SW480cells exposed to different concentrations (20,40,80,120μM) of apigenin for24and48h. Statistical differences in cell proliferation were exhibited as inhibited cell proliferation at all concentrations of apigenin treating for24h or48h (F values were318.645and156.662respectively, P values were less than0.001). At concentrations of20,40and80μM, apigenin significantly decreased cell proliferation in a time and concentration-dependent manner (P values were less than0.05). Cell proliferation decreased up to76.73%when treated with80μM apigenin for48h compared to the control.Apigenin Attenuated Migration of Cells-Wound-Healing AssayWe determined the effects of apigenin on migration of SW480cells by means of wound-healing assay. The determination demonstrated that apigenin attenuated migration of SW480cells at the concentration of40μM for24or48h. There were significant difference between40μM apigenin and DMSO for24or48h (t values were6.469and12.751respectively, P values were less than0.001). Apigenin has markedly decreased the wound closure activity of SW480cells.Apigenin Attenuated Invasion of Cells-Transwell AssayWe determined the effects of apigenin on migration of SW480cells by means of transwell assay. The determination demonstrated that apigenin attenuated invasion of SW480cells at the concentration of40μM for24h and48h. There were significant differences between40μM apigenin and DMSO both24h and48h treating (t values were11.249and-4.859respectively, P values were less than0.05).Apigenin Induced Cell Cycle ArrestCell cycle arrest in SW480cells exposed to apigenin was not observed at all concentrations of apigenin for24h but did occur in cells exposed to apigenin at all concentrations for48h. Compared to vehicle-treated SW480cells,80μM apigenin caused an increase of35.50%in the cell population in the G2/M phase after48h of treatment. The accumulation of the cell population in the G2/M phase was accompanied by a concomitant decrease in the cell population in the G0/G1phase with all concentrations of apigenin.Cell Apoptosis AnalysisBoth Hoechest33258staining and Annexin V-FITC staining assay showed that apigenin did not induce SW480cell apoptosis. There was no significant difference comparing different concentration apigenin groups with DMSO group.Differential Expression Proteins by Apigenin Treating SW480CellsTo determine the differential expression proteins, comparative proteomic analysis of SW480cells treating with apigenin and vehicle was performed. There were12differential spots that simultaneously existed in the two groups.9protein spots were successfully identified by MALDI-TOF MS and by subsequent comparative sequence search in the Mascot database. Transgelin protein was choosed to get further study.Immunofluorescence StainingImmunofluorescence staining results showed that transgelin was expressed in the cytoplasm, nucleus and mitochondria. And its expression in nucleus was the highest. It did not show significant translocation for SW480cells exposed to apigenin for12h or24h, suggesting that the increase of transgelin in mitochondrial for apigenin was not caused by translocation.Fluorescence Quantitative PCRTo confirm and extend the proteomic results, transgelin mRNA expression in SW480cells treating with different concentrations of apigenin and vehicle DMSO was examined by Fluorescence quantitative PCR. The results showed that apigenin increased the expression of transgelin mRNA. The difference was significant (F=498.798, P<0.001.)Western Blot Analysis 1) To confirm and extend the immunofluorescence results, transgelin protein expression in the cytoplasm, nucleus and mitochondria of SW480cells treating with40μM apigenin were examined by western blot analysis. It showed that transgelin was expressed in the cytoplasm, nucleus and mitochondria. And its expression in nucleus was the highest, consistent with immunofluorescence results.2) Transgelin expression in SW480cells treating with different concentrations of apigenin and vehicle DMSO was examined by western blot analysis. Equal protein loading was confirmed by parallel actin immunoblotting, and signal quantification was performed by densitometric scanning. The results showed that apigenin treating for48h up-regulated the expression of transgelin protein in SW480cells, there was significant difference compared with DMSO group (F=13.312, P=0.001). As the concentration of apigenin increasing, the transgelin protein increased more obviously. There was no statistically significant difference between20μM of apigenin group and DMSO group (P>0.05). Comparing with DMSO group,40,80,120μM apigenin groups significantly increased transgelin expression in SW480cells (P=0.042,0.007,0.000). The change pattern of transgelin expression was similar to that observed in the proteomic analysis. Apigenin induced the expression of MMP-9protein downregulation in SW480cells. The difference was statistically significant compared with DMSO group (F=20.396, P<0.001).Transient TransfectionWe examined the effect of transient transfection with transgelin-siRN A on SW480cell viability. Cell viability was increased by transgelin-siRNA. There was significant difference between the transgelin-siRNA groups and the negative control group (F=134.583, P=0.000). The cell growth of siRNA433group was the fastest. It is1.35folds of the NC group. Tissue microarray and immunohistochemistryTissue microarray immunohistochemistry results showed that transgelin expression in human colon adenocarcinoma was higher than its expression in metastatic lymph node and liver tissues. The difference was statistically significant (Z=-2.075, P=0.038).Conclusions1) In vivo, apigenin could inhibit the growth and liver metastasis of colorectal cancer in nude mice. In vitro, apigenin could inhibit the growth and attenuate the migration and invasion of SW480cells.2) Apigenin might be one of the active ingredients of traditional Chinese medicine to combat colorectal cancer. The molecular targeting of transgelin by apigenin might be a useful strategy for chemoprevention and/or chemotherapeutics of colorectal cancers.