Study On Multidrug Resistance Mechanism And Anti-tumor Activity Of Compound RY10-4

Flavonoids are one kind of widespread compounds in plants, which are included in people's diet. They are good for people's healthy because of their physiological, pharmacological and healthy care activities. Flavonoids have been playing a unmeasurable role from regulating routine healthy condition to preventing of tumor, cardiovascular disease and so on. Recently, more and more studies have focused on some Flavonoids due to their low toxicity, high efficiency and natural properties in treating diseases.Macrothelypteris torresiana(Gaud.)Ching, which belongs to genus Macrothelypteris in family Thelypteridaceae, is widespread in south of the Yangtze. A Chinese common folk view says that this plant can clean heat and toxin, resolve hard lump, so it is often used to stanch bleeding and reduce swelling in traditional Chinese medical science. In recent studies, it has been found that protoapigenone, a special kind of flavonoids in Macrothelypteris torresiana(Gaud.)Ching, shows good antitumor activities in vivo and in vitro experiments. More exciting, protoapigenone has a wide antitumor spectrum, which shows obvious cytotoxicity to tumor cells of different origins. By further research, a series of flavonoids,whose B ring are not aromatic nucleus, as exemplified by protaopigenone, all show markable antitumor activities in vivo and vitro screening experiments. However, the traditional flavonoids, which is similar to protoapigenone in structure, show poor antitumor activities. Besides, some other studies find that protoapigenone can regular the expressing of MDR1 and other relative genes, contributing to its treating and reversing multidrug resistance(MDR) phenomenon.On the basis of previous studies and using protaopigenone as a lead compound, our group has chemically synthesized a new compound 2-(1-hydroxy-4-oxo-2,5-cyclohexadien-1-yl)-4H-Pyran-4-one by structure-activity relationship(SAP),containing 1-hydroxycyclohexa-2,5-dien-4-one group, named as RY10-4, which has lower cytotoxicity and higher efficacy. In this dissertation, we studied the characteristics of reversing MDR of breast cancer cells and anti-lung cancer cells activities of RY10-4, which has a specific antitumor effect. The main content was divided into two parts. In the first part, we studied the effect and related signal pathways of RY10-4's reversing MDR of breast cancer cells,MCF-7/ADR; In the second part, we studied the effects of RY10-4 against the cell proliferation and invasion of human lung cancer cells A549 and induced their apoptosis, and explored some related mechanism of its anti-lung cancer activities.PART I:Study on the effect of RY10-4 reversed MDR in breast cancer MCF-7/ADR cell line in vitro and in vivoObjective:In consideration of the fact that protoapigenone can reverse MDR of tumor cells, and in earlier study, we found that protoapigenone analog RY10-4 could reverse phosphorylated AKT(p-AKT) activated by adriamycin (ADR), indicating that it might play a major role in antitumor effect of ADR resistance tumor cells in clinic potentially, here we discuss whether RY10-4 can reverse MDR of tumor cells and the potential mechanism in this process. First, we studied the effect of RY10-4 on the cell growth of MCF-7/ADR cell line of breast cancer. Second, we studied the effect of RY10-4 on the susceptibility of ADR in treating MCF-7/ADR cells and the process of drug molecules transport. Further, we explored the related signaling pathways participating in RY10-4 mediated reversing MDR of MCF-7/ADR cells.Methods:We used the human breast cancer cell line MCF-7 and its MDR counterpart MCF-7/ADR known as over-expression of P-glycoprotein (P-gp) to study the antitumor and MDR reversing effect of RY10-4. (1) Employing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay to detect the effect of RY10-4 on the cell growth of MCF-7 and MCF-7/ADR cells according to the growth curve; Employing flow cytometry assay with Annexin V/PI staining to detect the effect of RY10-4 on the apoptosis station MCF-7 and MCF-7/ADR cells. (2) Employing MTT assay to detect the effect of RY10-4 on the drug susceptibility of MCF-7 and MCF-7/ADR cells in vitro. (3)Employing rhodamine excretion assay to detect the effect of RY10-4 on the drug molecules transport of MCF-7 and MCF-7/ADR cell member; Employing Western Blot assay to detect the effect of RY10-4 on P-gp expression of MCF-7/ADR cells; Employing Reverse-transcriptase (RT) PCR and Real-time PCR assay to detect the effect of RY10-4 on the expression of MDR gene MDR1 in MCF-7/ADR cells. (4) Employing intracellular ATP level assay to detect the effect of RY10-4 on the over-expression P-gp in MCF-7/ADR cells. (5) Employing Western Blot and RT-PCR assay to detect the effect of RY10-4 on the expression of the intracellular protein AKT, p-AKT, Nuclear factor Kappa B (NF-Kb) in MCF-7/ADR cells, and further in combined with AKT inhibitor LY294002 and NF-?B inhibitor PDTC to decect the effect on the expression of MDRlgene and P-gp protein. (6) the human multidrug-resistant breast tumor xenograft model established with MCF-7/ADR cell line was used to investigate the antitumor activity of RY 10-4 in vivoResult:RY10-4 has strong reversal effect of multidrug resistance in MCF-7/ADR. (1) Through the growth curve, we find RY10-4 shows an inhibitory effect on the growth of both MCF-7 and MCF-7/ADR cells. Compared to control group(ADR), RY10-4 is more powerful to inhibit the growth of MCF-7/ADR cells; Through flow cytometry assay with Annexin V/PI staining, we find that RY10-4 can remarkably induce the apoptosis of MCF-7/ADR cells. (2) Through MTT assay, we find that MCF-7/ADR cells is sensitive to RY10-4, and RY10-4 can enhance its susceptibility to chemotherapy drugs. (3) Through rhodamine excretion assay, we find that RY10-4 can promote rhodamine to accumulate in MCF-7/ADR cells, indicating that RY10-4 can reduce drug transport activities of MCF-7/ADR cytomembrane; Through Western Blot assay, we find that RY10-4 can promote the up-regulation of P-gp expression in MCF-7/ADR cells; Through RT-PCR and Real-time PCR assay, we find that RY10-4 can up-regulate the transcription level of MDR1 in MCF-7/ADR cells. (4) Through intracellular ATP level assay, we find that RY10-4 can inhibit the the activity of ATP enzyme and reduce the ATP level, as reversing the MDR phenomenon. (5) Through Western Blot and RT-PCR assay, we find that RY10-4 can regulate MDR of drug resistance breast cancer cells-MCF-7/ADR mediated by MDR/P-gp through inhibiting PI3K/AKT/NF-?B signaling pathway. (6) RY10-4 was effect to reverse multidrug-resistant breast cancer in vivo.Conclusion:As a protoapigenone analog, RY10-4 works in two ways. On one hand it can inhibit the growth of drug resistance breast cancer cells, induce their apoptosis, down-regulate the ATP level and inhibit the excretion of P-gp. On the other, it can down-regulate the expression of MDR1/P-gp, reversing MDR of MCF-7/ADR cells through inhibiting PI3K/AKT/NF-?B signaling pathway. Therefore, RY10-4 has both antitumor and MDR reversing effects. Although the anti breast cancer tumor activities of RY10-4 was studied deeply before, our research is the first time to report and prove that RY10-4 is able to reverse MDR of ADR-selected human breast tumor cells. Further more, its potent MDR reversing function ensure its safety in application, which is better than traditional MDR reversal agent-Verapamil. Meanwhile, our research show that AKT and NF-?B can work as a potential target of MDR reversal agents. Besides, this research gives the theoretical basis for the possible clinical applications of RY10-4 alone or in combination with other chemotherapeutic drugs in the treatment of MDR tumors.PART II:Study on the antitumor activity of RY10-4 against lung cancer in vitroObjective:To study the cell proliferation inhibition of RY10-4 on human lung cancer cells A549 in vitro, and to determine the biological activity and the underlying mechanisms and signaling pathways of RY10-4 on lung cancer cells by using in vitro experimental models.Methods:We used the human lung cancer cell line A549 to study the antitumor effect of RY10-4 in vitro. (1) Employing MTT and Trypan blue exclusion assay to detect the inhibition effect of RY10-4 on A549 cells in vitro. (2) Employing fluorescent staining with Hoechst 33258 and flow cytometry assay with Annexin V/PI staining assay to detect the effect of RY10-4 on the induction of apoptosis. (3) Employing flow cytometry assay with propidium iodide (PI) staining to detect the effect of RY10-4 on cell cycle distribution. (4) Employing Transwell assay to detect the effect of RY10-4 on A549 cells invasion ability in vitro. (5)Employing Western Blot assay to detect the effect of RY10-4 on invasion and apoptosis related protein and signaling pathways related protein expression level. (6) Employing flow cytometry assay to detect the effect of RY10-4 on the level of ROS.Result:RY10-4 has strong effect on human lung cancer cells. (1) The proliferation viability of A549 cells in vitro significantly decreased after the treatment of RY10-4. (2) RY10-4 significantly induced the apoptosis of A549 cells, the ratio of apoptotic cells significantly increased in groups treated with RY10-4, and the typical morphological characteristics of apoptotic cell was observed. Besides, pretreatment with Signal transducer and activator of transcription 3 (STAT3) inhibitor AG490 could significantly promote the apoptosis induced by RY10-4. (3) The percentage of cells in the G1 phase significantly increased in groups treated with RY10-4. (4) After treatment with RY10-4 of high concentration, the numbers of cells passing through the polycarbonate membrane decreased notably, indicating that RY10-4 had an inhibitory effect on invasion ability of A549 cells. (5) After treatment with RY10-4, we found in A549 cells that cytochrome c was released from mitochondria to cytoplasm, Procaspase-3,-9 was activated, the ratio of Bcl-2/Bax decreased, the ERK and P38 MAPK signaling pathways was activated by phosphorylation. After pretreatment with inhibitor, we found that ERK-selected inhibitor U0126 and STAT3-selected inhibitor AG490 could significantly promote apoptosis induced by RY10-4, whereas the apoptosis was blocked by P38-selected inhibitor SB203580. (6) RY10-4 could increase the level of ROS.After pretreatment with inhibitors, we found that U0126 and AG490 could promote the level of ROS induced by RY10-4,whereas SB203580 could decrease the level of ROS induced by RY10-4.Conclusion:RY10-4 exhibited high antitumor activity toward A549 cells in vitro by inhibiting proliferation, promoting apoptosis, blocking the cell cycle process and inhibiting invasion. STAT3 signaling pathway mediated by ERK and P38 MAPK was an important mechanism in inducing apoptosis and inhibiting invasion of RY10-4.