| In recent years, the morbility and mortality of prostate cancer is increasing year after year. Many prostate cancer patients were usually diagnosed at an advanced stage because of lack of clinical symptoms at early stage. Clinically, androgen ablation is the standard treatment for advanced prostate cancer. But prostate cancer usually progresses from androgen-dependent to androgen-independent stage, making antiandrogen therapy ineffective leading to an increase in metastatic potential and incurable malignancy. Novel treatment modalities are therefore needed to delay or prevent progression of advanced prostate cancer. Searching for new naturally occurring botanicals and dietary substances are gaining increasing attention as cancer chemopreventive agents. In this regard, for prostate cancer chemoprevention at the present time, there is considerable emphasis in identifying novel botanicals that selectively induces apoptosis and growth arrest of prostate cancer cells without producing cytotoxic effects on normal cells.Inula Britannica L. belongs to the Compositae, having the effects of dissolving phlegm, line water, and preventing vomiting. Acetylbritannilactone (ABL), a new active extract isolated from a traditional Chinese medicinal herb Inula Britannica L, is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. However, the effects of inhibiting growth and inducing apoptosis of tumor cells were limited. We further synthesized the compound 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methyl- -ene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen -2-yl)propanoate (ABL-N), which in preliminary studies showed that had exceptional anti-proliferative activity against several human cancer cell types with good security. In the present study, we demonstrated the antiproliferative and proapoptotic effects of ABL-N in human Prostate cancer cells and delineate the mechanism of the effects.The present study included three sections as following:Section 1: Effects of ABL-N on inhibition of prostate cancer cell proliferation and promotion apoptosis in vitro.Objective: To investigate the effects of ABL-N on the growth and apoptosis of prostate cancer cells.Methods: In this part of experiment, we selected androgen dependence cells (LNCap), androgen independence cells (DU145, PC3) and normal human prostate epithelial PrEc cells as the objects of study. Through methods, such as MTT assay, staining for TUNEL/DAPI, annexinV/PI staining with the flow cytometer (FCM), DNA fragmentation using a Cell Death Detection ELISAPlus kit, we detected the changing of proliferation and apoptosis of prostate cancer cells with ABL-N. And the effects on the migration of ABL-N were evaluated by the wound migration assays.Results:1 ABL-N inhibits the cell viability of PC3, DU145 and LNcap cells.To evaluate the effects of ABL-N on cell viability of human and normal prostate epithelial cell, MTT assay was done. We observed that ABL-N of different concentration(0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40μmol/L) treatment to human prostate PC3, LNCaP and DU145 cells resulted in a dose-dependent inhibition of cell growth after 24h,without any substantial effect on normal human prostate epithelial PrEc cells. We found that about (73.34±4.41)% of PrEC cells were viable following a 24 h exposure to 40μmol/LABL-N, whereas only (11.92±2.31)% of PC3, (12.55±1.94)% of LNcap, (13.28±2.26)% of DU145 cells survived under similar conditions of ABL-N treatment. Half effectively suppressing concentration (IC50) of the three kinds of cells was closed, respectively as (13.65±1.45)μmol/L, (16.92±1.91)μmol/L, (17.01±1.73)μmol/L.According to the experimental results of MTT, we chose the final concentration of ABL-N, which respectively were 0, 5, 10, 20, 40μmol/L, as the investigative objects using for the further experimental study. Taking into accunt the more aggressive and highly metastatic nature of prostate cancer, PC3 cells were selected then as a model system to conduct mechanistic studies in the further experiments.2 The effect of ABL-N on the apoptosis of PC3 cellsTo test whether ABL-N-mediated decrease cell viability is due to induction of apoptosis, we conducted flow cytometric analysis and TUNEL assay in ABL-N-treated cells. After culturing in the ABL-N (0, 5, 10, 40μmol/L) for 24h, PC3 cells were stained for TUNEL/DAPI to assess the effects of ABL-N on apoptosis. The TUNEL staining clearly showed that the appearance of apoptosis in the cells exposed to ABL-N in a concentration-dependent increase. Also DAPI staining showed that the condensed and fragmented nuclei increased with the ABL-N treatment in a dose-dependent manner. Nucleosome fragmentation further determined by Cell Death Detection ELISA PLUS confirmed that DNA fragments increased obviously with the increasing of ABL-N concentration.Finally, evidence for ongoing apoptosis is supported by annexinV/PI staining with the flow cytometer (FCM), which is a characteristic marker for apoptosis. After the effect of ABL-N, PC3 cells were distributed in four quadrants (Q1: apoptosis cells, Q2: middle-late apoptosis cells, Q3: normal cells, Q4: early apoptosis cells). With the concentration of ABL-N increasing, cells of quadrants Q3 decreased obviously, and gradually shift to quadrants Q4, Q2, and Q1. The results showed that the percents of early apoptosis cells were (0.5±0.12) % in control group and (25±1.16) % in 20μmol/L ABL-N group. And the percents of late apoptosis cells were (28±1.64)% in 20μmol/L group and (50±1.98)% in 40μmol/L ABL-N group. Meaning that the number of apoptosis cells increased. Data showed a significant induction of apoptosis by ABL-N in doses-dependent manner, which was evident from the significant enhancement in AnnexinV/PI staining.3 The effect of ABL-N on cell migrationIn the wound-healing assay, PC3 cells were grown to confluence before wounding with a sterile pipette tip and monitored after 24 h for migration into the wound area. The experimental results of wound healing appeared that the migration of PC3 cells obviously decreased with the ABL-N concentration increasing. The results showed that the migration activity of PC3 cells with ABL-N (20μmol/L) were obviously inhibited about 90% compared with control group. The wound migration assays revealed that ABL-N significantly inhibited migration of prostate cancer cells.Conclusion: ABL-N inhibited proliferation and promoted apoptosis of prostate cancer cells in a dose-dependent and significantly inhibited migration of prostate cancer cells.Section 2: Mechanisms of ABL-N inhibiting proliferation and inducing apoptosis in prostate cancer cellsObjective: In order to discussing the mechanism of ABL-N on inducing the apoptosis of prostate cancer cell, providing experimental basis on the clinical practice of extractive from Inula, which was used as an antineoplastic agent.Methods: Taking into account the more aggressive and highly metastatic nature of prostate cancer, PC3 cells were selected as a model system to conduct mechanistic studies in the further experiments.To further explore mechanism of ABL-N inhibiting proliferation and promoting apoptosis of prostate cancer cells, We observed the expression level of relative proteins of apoptosis Bax and Bcl-2, and related proteins on tumorous growth and aggressiveness, such as Klf5, Stat5b, ICAM-1. To test whether caspases are involved in apoptosis induction by ABL-N, we first evaluated the active caspases in ABL-N-treated cells. In order to discussing the mechanism of ABL-N on inducing the apoptosis of prostate cancer cell.Results:1 The effect of ABL-N on prostate cancer related proteinsTo evaluate the underlying mechanism by which ABL-N-induced apoptosis, we carried out western blotting to investigate the induction of cancer-related protein Stat5b and Klf5 in PC3 cells. ABL-N considerably decreased Stat5b and Klf5 protein in a dose-dependent manner (P<0.05). The results showed that the expressional quantity of protein Klf5 in 20μmol/L ABL-N group (0.87) decreased (51.54±4.53) % comparing with the control group (1.79). Also the expressional quantity of protein Stat5b in 20μmol/L ABL-N group (0.57) decreased (53.12±4.31) % comparing with the control group (1.23).We also investigated the expression of the proangiogenic factor ICAM-1 in PC3 cells, which correlated with increased metastatic potential of prostate cancer cells, and found that ABL-N resulted in a dose-dependent inhibition of ICAM-1 expression in PC3 cells. The results showed that the expressional quantity of protein ICAM-1 in 20μmol/L ABL-N group (0.63) decreased (67.75±4.88) % comparing with the control group (1.95).2 The effect of ABL-N on the expression of Bcl-2 family membersExposure of human PC3 cells to ABL-N resulted in a marked increase of Bax protein expression in a dose-dependent manner (P<0.05). The results showed that the expressional quantity of protein Bax in 20μmol/L ABL-N group increased (91.49±6.38) % comparing with the control group. However, the levels of Bcl-2 proteins were not affected obviously after ABL-N treatment. This resulted in a substantial increase in Bax/Bcl-2 ratio (P<0.05), which favors apoptosis, may collectively form a molecular basis for the apoptotic action of ABL-N. The results showed that the ratio of protein Bax/Bcl-2 in 20μmol/L ABL-N group (1.18) was about 2 times of that in the control group (0.57).3 The effect of ABL-N on the activation of CaspasesTo test whether caspases are involved in apoptosis induction by ABL-N, caspase-2, -3, -6, -8 and caspase-9 were assayed colorimetrically to determine their role in ABL-N induced apoptosis. As shown in result, caspase-3 activation in PC3 cells was dose-dependent enhancement after ABL-N treatment (P<0.05). The caspase-3 activity with ABL-N 20μmol/L (0.95) is significantly being about 4.2-fold of the control cells (0.24) after 24 hours (P<0.05). However, under similar conditions, ABL-N also stimulated caspase-9, caspase-2, caspase-6 and caspase-8 activities in a lesser extent.4 The influence of caspase inhibitor effect on the apoptotic action of ABL-NTo define the role of caspase activation in ABL-N-induced apoptosis, we treated prostate cancer PC3 cells with pan-caspase inhibitor z-VAD-fmk (50μmol/L) and caspase-3-specific inhibitor z-DEVD-fmk (50μmol/L) before challenge with ABL-N (20μmol/L).Z-VAD-fmk pretreatment for 4h abrogated ABL-N-induced apoptosis as measured by the nucleosome fragmentation. The caspase-3-specific inhibitor z-DEVD-fmk also reduced ABL-N-induced apoptosis in PC3 cells. The results indicated that, with z-VAD-fmk (50μmol/L) pretreatment, DNA fragments distinctly decreased about 66.04% (P<0.05) with ABL-N (20μmol/L) for 24h. And also indicated that, with z-DEVD-fmk (50μmol/L) pretreatment, DNA fragments distinctly decreased about 54.38% (P<0.05) with ABL-N (20μmol/L) for 24h.To define the role of caspase-3 activation in ABL-N-induced apoptosis, we treated prostate cancer PC3 cells with caspase-3-specific inhibitor Z-DEVD-fmk (50μmol/L) pretreatment for 4h before challenge with ABL-N (20μmol/L) for 24h. As shown in result, caspase-3 activation in PC3 cells was decreased about 65.77% comparing with that ABL-N treatment (P<0.05).These results suggested that activation of caspase cascade was essential for ABL-N-induced apoptosis in prostate cancer cells.Conclusion: ABL-N plays an important role in anti-tumors by inhibiting the expression of tumor related factors Klf5, Stat5b, ICAM-1, increasing the ratio of Bax/Bcl-2, and through mitochondrial way to induce apoptosis of prostate cancer cell.Section 3: Effects of ABL-N on the tumor growth and the expression of tumor-related proteins in xenograft modelObjective: To investigate the effect of ABL-N on inhibiting prostate cancer in vivo, and its influence of the expression of tumor related protein in the general specimens.Methods: Because ABL-N was observed to be effective in inhibiting the growth of PCa cells in vitro, we next determined whether these results could be translated into an in vivo xenograft mouse model. We succeeded in building the model of prostate cancer xenograft. And further explore the effect ABL-N on inhibiting the growth of prostate cancer in vivo through measuring the volume of xenograft and observing the state of nude mice. The expression of tumor related protein Klf5,Stat5,ICAM-1,Bax,Bcl-2 in the general specimens were also detected by Immunohistochemistry.Results:1 ABL-N inhibiting the growth of prostate cancer xenograftTo investigate the ability of ABL-N to inhibit tumor growth in vivo, we established a xenograft model of PC3 cells in nude mice. The treatment with ABL-N (15mg/kg) significantly suppressed the growth of PC3 tumors when after about 14days. Next, we evaluated the extent of tumor growth inhibition in ABL-N-treated animals and found that the volume of nude mice tumor decreased about (25.65±2.14) % (P<0.01) comparing with the vehicle control group after 32 days. The observed differences for tumor development in delphinidin-treated mice compared with control mice were statistically significant (P <0.01).2 The influence of ABL-N on the weight and general condition of tumor-burdened nude miceABL-N treatment did not cause any loss in the body weight and food intake, indicating that the dosages used were not toxic to the animals. From these data, we conclude that delphinidin is an effective anticancer agent that has the potential to inhibit or slow the tumorigenicity of prostate cancer PC3 cells in in vivo system. This initial in vivo experiment suggested that ABL-N might be an effective anticancer agent at dosages that induced negligible toxic effects.Meanwhile, it was demonstrated that the hepatorenal organization of nude mice treated by ABL-N didn't have pathological changes compared with control nude mice using immunohistochemistry.3 ABL-N inhibits Stat5b, ICAM-1 and Klf5 expression in PC3 originated tumors in athymic nude mice.Because ABL-N treatment was observed to modulate the expression levels of Bax, Bcl-2 and the ratio of Bax/Bcl-2 under in vitro conditions, we determined the effect of ABL-N administration on the expression levels of Bax and Bcl-2 in tumors excised from both groups of animals. It was evident from the immunohistochemical analysis of tumors that animals receiving ABL-N exhibited significant increase the expression level of Bax protein. The expression level of Bcl-2 in tumor tissues of animals treated with ABL-N was not influenced significantly compared with vehicle-treated animals.4 ABL-N inhibits Stat5b, ICAM-1 and Klf5 expression in PC3 originated tumors in athymic nude mice.As ABL-N treatment was observed to inhibit or decrease tumorigenic potential of PC3 cells in vivo with the suppression of the cancer-related protein Stat5b, ICAM-1 and Klf5, we next determined the effect of ABL-N on the protein levels of Stat5b, ICAM-1 and Klf5. It was evident from the immunohistochemical analysis of tumors that animals receiving ABL-N exhibited significant decrease of Stat5b, ICAM-1 and Klf5 compared with vehicle-treated animals (P<0.05), suggesting the anticancer efficacy of ABL-N under in in vivo conditions through suppression of the cancer-related protein Stat5b, ICAM-1 and Klf5.Conclusion: We demonstrated that ABL-N could inhibit the growth of tumor through enhancing the expression of protein Bax, increasing the ratio of Bax/Bcl-2, inhibiting the expression of cancer-related protein ICAM-1, Stat5 and Klf5 in vivo model. These results suggested that the anti-tumor efficacy of ABL-N under in in vivo conditions. |
PDF Full Text Request