| Prostate diseases are very common in male genitourinary system. Increasing number of men suffer from them due to the aging and changes of lifestyle and environment. However, there are many shortcomings in current treatments, for example, time wasted, critural by-effects and palindromic. More attentions should be paid on the prevention of the disease through the regulation of the diet and lifestyle. We got a stain of Sporidiobolus pararoseus which produces torulene and torularhodin. Torulene and torularhodin share a similar structure with lycopene, which was famous for its protective effect on prostate. Nevertheless, few studies were focused on the physiological activities of them, like antioxidant and tumor suppression effect. In order to clarify the specific function of torulene and torularhodin, torularhodin with a purity above 95% was obtained from the fermentation products of the yeast. Based on it, the protective effect and its mechanism on oxidative injured prostate cells of torulene and torularhodin were investigated. Cell and nude mice models were chosen to evaluate the influence of these two carotenoids on the growth of prostate cancer(PCa) followed. Main works and achievements are revealed below:1. To ensure the functional characteristic research results of torularhodin, a suitable purification apprpach was set. First, the cell wall of the yeast was broken through heating in hydrochloride solution. The crude carotenoids obtained from the step above were then extracted with n-hexane. Followed with it, torularhodin was purified by column chromatography and eluent containing various volume ratios of petroleum ether(30 ~ 60°C boiling range) and acetone(3: 1 ~2: 1 ~ 1: 1). The purification of torularhodin was above 95%, which is capable for the following study. The spectroscopic data of torularhodin were obtained finally by the instruments as follows: HPLC- DAD, LC- ESI- MS, FTIR, and Raman spectroscopy.2. H2O2-induced human prostate stromal immortalized WPMY-1 cells oxidative injury model was established to evaluate the effect and its mechanism of torulene and torularhodin on oxidative injury. The antioxidant indexes, the apoptosis caused by oxidative injury and the mRNA expression level of related genes were measured to access the antioxidant effect of the carotenoids. The results showed that both torulene and torularhodin can reduce the mortality of the oxidative injured WPMY-1 cells through the enhancement of the free radical scavenging ability of the autoxidation system, which was closely to the decreased reactive oxygen species(ROS) intracellular, the increase of antioxidant activities of intracellular antioxidant enzymatic system and the reduction of the production of malondialdehyde(MDA). Besides, the carotenoids can also lower the apoptosis of the oxidative injured cells through the adjustment of the mRNA expression level of Bcl-2 family and Caspase family.3. Androgen-dependent PCa LNCaP and androgen-independent PC-3 cell were chosen to explore the effect of torulene and torularhodin on the growth and proliferation of PCa. Torulene showed a comparable effect with lycopene on PCa cells within the concentration rage from10 μM to 40 μM, while the inhibitory effect of torularhodin was more significant than them. Moreover, with the comparetion of LNCaP cells, PC-3 cells represented a more sensitive reaction to torulene, yet the inhibitory effect of torularhodin was almost the same on both cell lines.4. Hochest33252/PI staining, flow cytometry, western blot and quantitative real-time PCR(Q-PCR) results revealed that the apoptosis mediated via mitochondrial pathway is the principal cause of the inhibitory effect that torulene and torularhodin exerted on the PCa cells. The carotenoids unregulated the expression of p53 at first, which has a modulatory function on the expression of certain members in Bcl-2 family. Followed with the activated expression of the Caspase family, the Cascade reaction was activated, which induced the apoptosis of PCa. In addition, the level of intracellular Ca2+ and the open of the mitochondrial permeability transition pore(MPTP), which led to the change of the mitochondrial membrane potential(MMP), were also influenced by the intervention of torulene and torularhodin. What’s more, torularhodin still influence the growth of androgen-dependent PCa LNCaP cells through AR signaling pathway.5. Human prostate tumor PC-3 cells were injected in BALB/c male nude mice subcutaneously and a xenograft model was established to investigate the effect of torulene and torularhodin on the growth of PCa in vivo. The result indicated that both carotenoids can inhibit the growth of PCa in vivo, the inhibitory effect of torularhodin was similar to lycopene and superior to torulene under the same intragastric dose. HE staining reflected that both carotenoids caused damage in the tumor, and vacuoles and necrosis appeared in group treated with carotenoids at high dose. Immunohistochemistry(IHC) and Q-PCR results showed that torulene and torularhodin induced apoptosis in PCa tumor by mitochondrial pathway, which was similar to the results in vitro. Furthermore, the intake of torulene and torularhodin did not influence the normal growth of nude mice through the result of the living conditions, body weights and viscera indexes.The above results demonstrated that both torulene and torularhodin possess anti-oxidative stress and potential preventive effect on prostate. And torularhodin showed better activity than torulene due to the differences in the structure. What’s more, both carotenoids revealed an inhibitory effect on the growth of PCa in vivo and in vitro, which was related to the apoptosis directed via mitochondrial pathway. The present research provides a novel strategy for the prostate disease prevention, and supply fundamental basis for the comprehensive exploitation and utilization of torulene and torularhodin. |
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