| Part 1 Effect and mechanism of apigenin on human bladder cancer T24 cellsBackgroundIn our country, the incidence of bladder cancer ranks first in the urinary tract tumors. In recent years, the incidence of bladder cancer is still in an increasing trend. Main treatments of bladder cancer currently are surgery and chemotherapy, etc. Apigenin is a kind of natural flavonoid compounds, which widely distributes in our daily food like fruits and vegetables. Apigenin is able to suppress a variety of tumors, but its effect on bladder cancer has not been verified.PurposeIn this study we explore the influence and the mechanism of apigenin on T24 bladder cancer cell line in vitro, and confirm the experimental results in nude mice in VIVO.MethodsT24 cells were treated with different concentrations (10-80 μM) of apigenin and in different time (24-72 hours). MTT assay was used to detect the change of cell proliferation and viability. Flow cytometry was used to detect the change of cell cycle and apoptosis. Transwell experiment was taken to test the change of cell movement ability. The expression of the corresponding proteins was tested by Western Blot experiments. Bisulfite Sequencing PCR was used to test the methylation level of PTEN. Then we made a nude mouse tumor model to verify the effect of apigenin on T24 cells in vivo.Results1. Apigenin inhibited the proliferation of T24 bladder cancer cell in a time-dose dependent way. With the concentration higher and the treating time longer, the inhibition ability got stronger.2. The inhibition of proliferation in T24 cell was not obvious with apigenin concentration of 10 and 20μM. While the concentration increased to 40 and 80μM, the proliferation was significantly inhibited. The inhibitory concentration 50% values for apigenin treatment were estimated to be 82.5,52.9, and 43.8 μM for 24,48, and 72 hours, respectively.3. After T24 cells were treated with 10 and 20μM apigenin for 24hours, Transwell test showed that cell migration and invasion ability decreased significantly.4. After T24 cells were treated with 20,40 and 80μM apigenin for 24hours, by the flow cytometry (PI and annexin V double staining), there is an obvious apoptosis of the cells, mainly with late apoptosis, and with the increase of concentration, the apoptosis rate increased accordingly.5. After T24 cells were treated with 40,80,120 and 160μM apigenin for 24hours, by the flow cytometry detection, there appeared a significant G2/M phase arrest, and with the increase of concentration, the extent of G2/M phase arrest became more obvious.6. After T24 cells were treated with 20,40 and 80μM apigenin for 24hours, with the increase of concentration, the expression of pro caspase-3 gradually declined, the expression of active caspase-3 gradually rose and the expression of PARP fragmentation gradually increased.7. After T24 cells were treated with 20,40 and 80μM apigenin for 24hours, with the increase of concentration, the expression of p-Akt, PDK, and PI3K gradually declined.8. After T24 cells were treated with 20,40 and 80 μM apigenin for 24hours, with the increase of concentration, the expression of pro-apoptotic proteins Bax and Bad gradually rose, and the expression of anti-apoptotic proteins Bcl-xl and Bcl-2 gradually declined, while the expression of Akt had no obvious change.9. T24 cells was treated with 20 μM PI3 kinase inhibitor LY294002 for 30 min and then with 40 μM apigenin for another 24 h. PI3K inhibitor, LY294002 decreased the protein levels of cleaved PARP and active casepase-3 which were activated by apigenin.10. After T24 cells were treated with 20,40 and 80μM apigenin for 24hours, with the increase of concentration, the expression of PTEN gradually increased. Apigenin decresed the methylation level of PTEN in T24, UM-UC-3 and 5637 baldder cancer cells.11. In nude mice experiment, with the increase of concentration of apigenin locally injected, tumor volumes gradually shrinked.ConclusionApigenin inhibits cell proliferation, induces apoptosis and G2/M phase cell cycle arrest and inhibits migration ability of T24 cells in a dose-time dependent way. The inhibition of apigenin on T24 cell was induced by the activation of PI3K/PDK/Akt pathway and the regulation of the Bcl-2 protein family.Part 2 Apigenin enhances the anti-tumor effects of cisplatin by down-regulating ERCC-1 DNA repair proteinBackgroundThe combination chemotherapies with methotrexate plus vinblastine, doxorubicin and cisplatin (MVAC or CMV regimens) or gemcitabine plus cisplatin represent the standard as first-line therapy for patients with metastatic bladder cancer. Platinum is the most effective chemotherapy drug for bladder cancer. However, the median progression free survival (PFS) of cisplatin-based first-line therapy ranges from 7 to 9 months. The nucleotide excision repair (NER) system is a crucial mechanism in cisplatin resistance and excision repair cross complementing group 1 (ERCC-1) is an important part of NER.PurposeThe aim of this study was to investigate the effect and mechanism of apigenin in combination with cisplatin on T24 cells.MethodsCisplatin treated T24 cells with different concentrations (5-30 μM) for 24 hours and MTT assay was used to detect the cell proliferation. Cisplatin (15μM) treated T24 cells alone or in combination with apigenin (40μM). MTT assay and clone formation assay were used to detect cell proliferation. The expression of ERCC mRNA and protein were measured by RT-PCR and Western Blotting experiment, respectively.Results1. Cisplatin inhibited the proliferation of T24 bladder cancer cell in a dose-dependent way. With the concentration higher, the inhibitory effect became more obvious. The IC50 of Cisplatin on T24 cells for 24 hours was about 14.8μM.2. Apigenin enhanced the inhibitory effect of cisplatin on T24 cell proliferation.3. The clone formation assay showed that apigenin enhanced the inhibitory effect of cisplatin on T24 cell.4. Transwell test showed that Apigenin enhanced the inhibitory effect of cisplatin on migration in T24 cell.5. RT-PCR and western blotting experiments showed that cisplatin increased the expression of ERRC-1 mRNA and protein, and apigenin significantly inhibited the up-regulation of ERRC-1 mRNA and protein expression caused by cisplatin.ConclusionCisplatin inhibited the proliferation of T24 bladder cancer cell in a dose-dependent way. Apigenin can obviously enhance inhibitory effect of cisplatin on T24 cell, and significantly inhibited the up-regulation of ERRC-1 mRNA and protein expression caused by cisplatin.Part 3 Effect and mechanism of apigenin on the ability of migration and invasion in human prostate cancer DU145 cellsBackgroundIn Europe and the United States, prostate cancer is of the highest incidence of malignant tumors in male, and is the second leading cause of cancer-associated mortality. In our country, the incidence of prostate cancer is rising year by year. Compared with the European and American countries, the lower incidence of prostate cancer in Asian men may due to dietary factors. Studies have shown that apigenin has inhibitory effect in prostate cancer, but it was unknown whether apigenin is able to inhibit the metastasis of prostate cancer.PurposeThe aim of this study was to investigate the effect and mechanism of apigenin on the movement of prostate cancer DU145 cells.MethodsDU145 cells are treated with different concentrations (10-80μM) of apigenin and in different time (24-48 hours). MTT assay is used to detect the change of cell proliferation and viability. Flow cytometry is used to detect the change of cell cycle. Wound healing assay and Transwell experiment are taken to test the change of cell movement ability. The expression of the corresponding proteins is tested by Western Blot experiments.Results1. Apigenin inhibited the proliferation of DU145 prostate cancer cell in a time dose dependent way. With the concentration higher and the treating time longer, the inhibition ability got stronger.2. While the concentration increased to 40 and 80μM, the proliferation of DU145 cells was significantly inhibited. The inhibitory concentration 50% values for apigenin treatment were estimated to be 80.8, and 49.3μM for 24 and 48 hours, respectively.3. After DU145 cells were treated with 10 and 20μM apigenin for 24hours, wound healing assays showed that cell movement ability decreased significantly.4. After DU145 cells were treated with 10 and 20μM apigenin for 24hours, wound healing assays showed that cell migration and invasion ability decreased significantly.5. After DU145 cells were treated with 20,40, and 80 μM apigenin for 24hours, by the flow cytometry detection, there appeared a significant G2/M phase arrest, and with the increase of concentration, the extent of G2/M phase arrest became more obvious.6. After DU145 cells were treated with 5,10 and 20μM apigenin for 24hours, with the increase of concentration, the expression of E-cadherin gradually rose and the expression of Snail and Vimentin gradually declined.7. After DU145 cells were treated with 5,10 and 20μM apigenin for 24hours, with the increase of concentration, the expression of MMP-2 and MMP-9 mRNA and protein declined.ConclusionApigenin inhibits cell proliferation, induces G2/M phase cell cycle arrest and inhibits movement ability of DU145 cells in a dose-time dependent way. The inhibition of apigenin on DU145 cells was induced by the suppression of epithelial mesenchymal transition (EMT) and MMPs. |
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