| With the development of society, the improvement in the quality of people’s life, the increase in health awareness, as well as the increasing incident of cancer due to the imbalance of nutrition proportion and structure, the requirement for food has been changed from the quantity to the quality. Reasonable arrangement of diet structure and the scientific intake of nutritional ingredient have been prevalent to improve their physical quality, prevent and reduce the occurrence of cancer. Natural active ingredients from dietary plants are known as“the eighth major nutrients” due to its effects on scavenging free radicals leading to antioxidation and antitumor.Curcuminoids(curcumin, demethoxycurcumin and bisdemethoxycurcumin) are a common natural color agent and a flavouring agent in food industry, which have been proved to possess wide anti-cancer activity. However, its low bioavailability in vivo has restricted the antioxidative and antitumor activity after absorbance, which limits its application scope and application value. In this study, natural borneol(NB), a common food flavour, has been used as a absorption promoters, and the inhibitory effect of NB potentiating curcuminoids on cancer cell growth and the molecular mechanisms of their combined inhibitory effects on cancer cells have been investigated and elucidated. The study is expected to reveal the molecular mechanism underlying NB-potentiated the anticancer activity of curcuminoids and to provide theoretical foundation for the efficient and accurate utilization of NB,curcuminoids and others natural nutrition factors and also for dietary prevention intervention of cancer and others chronic diseases, which may aid in the application of NB and curcuminoids as two common food additives. In this study, Hep G2 cells were selected as a research object to investigate the inhibitory effect of the combination of NB and curcuminoids on Hep G2 cells growth and clarify their relevant molecular mechanism. In detail, the research contents are as follows:1. Optimization of the proportion of the combination of NB and curcuminoids and their treatment timesFirstly, MTT assay was used to investigate the effect of NB and curcuminoids alone withdifferent concentrations and times on the cell viability of Hep G2 cells. The results showed that the cell viability of Hep G2 cells gradually decreased with the increasing of curcumin(Cur) concentration and treatment time, indicating that Cur inhibited Hep G2 cells growth in a dose-dependent manner and a time-dependent manner. However, demethoxycurcumin(DCur)and bisdemethoxycurcumin(BDCur) inhibited Hep G2 cells growth only in a dose-dependent manner. Moreover, the anticancer activity of curcuminoids is Cur>DCur>BDCur, indicating that with the decrease in the mount of methoxyl base, the anticancer activity gradually decreased. NB alone with different concentration did not show inhibitory effect on Hep G2 cells. Different concentration of NB and curcuminoids in combination were then selected to investigate their combined inhibitory effect on Hep G2 cells. According to the results, NB could enhance the anticancer activities of curcuminoids. Moreover, 20 μg/ml NB and 20 μM Cur, 20 μg/ml NB and 40 μM DCur, and 20 μg/ml NB and 40 μM BDCur in combination co-treatment for 24 h showed the strongest cytotoxicity on Hep G2 cells. Therefore, we selected 20 μg/ml NB, 20 μM Cur, 40 μM DCur, and 40 μM BDCur for single or combiantion treatments for 24 h in all subsequent experiments.2. Investigation on the pathway of curcuminoids entering into cells and cellular localization of curcuminoidsFluorescence spectrometry and fluorescence microscope were used to investigate the pathway of curcuminoids entered into cells and cellular localization of curcuminoids to elucidate the distribution and uptake mechanism in Hep G2 cells. The results showed that NB could significantly increase the curcuminoids content in Hep G2 cells to enhance the anticancer activity of curcuminoids. In addition, curcuminoids have been shown to enter into Hep G2 cells by the pathway of transferrin receptors(Tf R) and activated downstream signals in the cytoplasm.3. Investigation on the enhancing effect mechanism of NB enhancement of anticancer activity of curcuminoidsWestern blot assay and membrane permeability assay were used to investigate the enhancing effect mechanism of NB enhancement of anticancer activity of curcuminoids. Theresults showed NB could improve the permeability of cell membrane to make more curcuminoides into the cells. Moreover, NB could down-regulate the expression of ABCB1,ABCC1 and ABCG2 transport proteins to reduce the efflux of intracellular curcuminoids,which resulted in an increase in the curcuminoids content in Hep G2 cells and increased the anticancer activity of curcuminoids. Both of the two explations contributed to the enhancing effect of NB.4. Investigation on the molecular mechanism of NB and curcuminoids in combination inhibition on Hep G2 cells growthThe molecular mechanism of NB and curcuminoids in combination inhibition on Hep G2 cells growth were systematic studied by flow cytometry assay, Western blot assay,DHE Fluorescence spectrometry and proteomics assay. As shown by the results, compared to curcuminoids, the combination of NB and curcuminoids significantly decrease the population of G2/M phase, indicating that NB enhances curcuminoids-induced G2/M cell arrest in Hep G2 cells. Further molecular mechanism studies showed the combination of NB and curcuminoids significantly decrease the expression of cyclin B1 and cdc2 and increase the expression of p-p53, p-ATM and p-MDM2, indicating that the combination of NB and curcuminoids activated p53 signaling pathway. Moreover, the down-regulation of p-Akt and p-ERK expression and the up-regulation of p-JNK and p-p38 MAPK expression played important roles in activating p53 pathway. Meanwhile, proteomics assay showed PSMA5,NPM and hn RNPC1/C2 also played important roles in p53 pathway. Further studies showed that reactive oxygen species(ROS) is upstream of NB and curcuminoids in combination-induced G2/M cell cycle arrest signaling pathway. Therefore, NB popentiates curcuminoids-inhibited cells growth by mediating ROS overproduction-activated p53 pathway to induce G2/M phase arrest in Hep G2 cells. |
PDF Full Text Request