The Effect And Mechanism Of Hedyotis Diffusa Willd Reverse Multi-drug Resistance In Colorectal Cancer

Colorectal cancer (CRC) is one of the most common gastrointestinal cancer. More than 1.2 million new CRC cases and 0.6 million deaths occurred worldwide every year. In recent years, the incidence and mortality of CRC is increasing in China. And CRC had a tendency toward young, which threatens patient’s life and health seriously. Surgical resection is the preferred way for the treatment of CRC. However, chemotherapy is the major therapy for patients who have postoperative recurrence, metastasis, or are unsuitable for surgery. But drug resistance limits the effectiveness of current CRC chemotherapy, leading to recurrence and metastasis. Traditional chemical reversal, gene reversal and immune reversal agent are limited in clinical application, due to the single mechanism, serious side effect or inconvenient in use. Therefore, it’s necessary to find out a safe and effective drug resistance reversal agent to enhance the efficacy of chemotherapy and overcome the resistance of current chemotherapeutic agents, which has great influence for improvement of the clinical effect and living quality of patients.Multidrug resistance (MDR) refers to the cellular resistance to numerous drugs differing in mechanisms of action and/or chemical structures. Formation of multidrug resistance is a complicated process, which depends on drug concentration in the tumor cells, drug targets, and so on. Different mechanisms contribute to MDR, mainly including changes in drugs transportation, DNA repair, apoptosis, related signal pathways and microRNA epigenetic regulation. Thus, these studies can provide a new sight into the effect and molecular mechanism of the MDR reversal agent in CRC.Previously, we reported that Hedyotis Diffusa Willd (HDW) induces the apoptosis and reduces the proliferation of CRC cells, inhibits the tumor angiogenesis and reverses MDR. However, the mechanism of HDW in MDR reversal of CRC is not clarified. Therefore, we study the effects and mechanisms of HDW to provide theory basis for clinical treatment.Part Ⅰ Preparation of HDW extractionObjective:Preparation of stable and reliable HDW extraction.Methods:HDW was extracted by ethanol. Then the contents of flavonoids and phenolic compounds were detected with rutin and gallic acid as standard, respectively. HPLC analysis was used to obtain the fingerprint spectrum and quality control of EEHDW. pH value was determined by means of acidimeter.Results:The total flavonoids and the total phenols were extracted effectively from EEHDW by ethanol aqueous solution. The total flavonoids were 71.534, and the total phenols were 111.272. The HPLC fingerprint can be used as the quality control. The pH of EEHDW is 7.0, and is good in precision, stability and replication.Conclusion:We obtained standardized EEHDW with stable quality, which provide guarantee support for further study in vivo and in vitro.Part Ⅱ The study on the resistance reversion effect and mechanism of EEHDW in CRC cells.Objective:To study the reversal effects of EEHDW on multi-drug resistance (MDR), and the role in intracellular accumulation, apoptosis, proliferation, activation of related signal pathways and expression of microRNA in HCT-8/5-FU cells.Methods:The viability of HCT-8/5-FU cells was determined by MTT assay to confirm the prosperity of MDR model in vitro and the reversal effects of EEHDW on MDR. Intracellular 5-FU, Adriamycin and rhodamine 123 were determined by HPLC, inverted fluorescence microscope and flow cytometry (FCM) were used to confirm the drug efflux function in HCT-8/5-FU cells. Moreover, MTT assays, morphology observation, cell colony formation and cell cycles analysis by FCM were used to confirmed the effects of EEHDW on HCT-8/5-FU cells proliferation. DAPI staining and Annexin V/PI staining by FCM were used to confirm the effect of EEHDW on HCT-8/5-FU cells apoptosis. Transwell and cell adhesion assay were used to confirm the effect of EEHDW on HCT-8/5-FU cell migration, invasion and adhesion ability. RT-PCR and Western blotting were performed to confirm the effect of EEHDW on the mRNA and protein expression of drug efflux factors (ABCC1/MRP1, ABCB1/P-gp and ABCG2/BCRP), cell proliferation regulatory factors (Cyclin D1, CDK4 and p21) and cell apoptosis regulatory factors (Bcl-2 and Bax) in HCT-8/5-FU cells. Additionally, Western blotting was used to confirm the effect of EEHDW on the activation of ERK, JNK, p38 and PI3K/AKT pathways in HCT-8/5-FU cells. Finally, miRNA microarrays and QPCR were used to confirm the effect of EEHDW on the expression of miRNAs in HCT-8/5-FU cells.Results:HCT-8/5-FU cells showed more resistant to different chemotherapeutics compared to HCT-8, thus could be used as a model for further research. EEHDW could increase the sensitivity of HCT-8/5-FU cells to 5-FU, increase accumulation of 5-FU, ADM and Rh123 and inhibit the drug efflux in HCT-8/5-FU cells. EEHDW could induce cell apoptosis and inhibit cell proliferation, migration, invasion and adhesion. EEHDW could markedly downregulate the mRNA and protein expression of ABCC1/MRP1, ABCB1/P-gp, ABCG2/BCRP, Cyclin D1, CDK4 and Bcl-2, and upregulate p21 and Bax in a dose-dependent manner. EEHDW could reduce the activation of ERK1/2, JNK, p38 and PI3K/AKT pathways in HCT-8/5-FU cells. Besides, EEHDW had a wide regulatory role in the expression of miRNAs in HCT-8/5-FU cells, such as upregulating the expression of miR-92b, miR-1247, miR-4800-5p, miR-1224, miR-1260, miR-4669 and miR-4298, and downregulating the expression of miR-582-5p, miR-4454, miR-222, miR-483-5p and miR-4443.Conclusion:HCT-8/5-FU cells process a characteristic of MDR. EEHDW can reverse the MDR of CRC cells and its reversal effect is mainly through the regulation in the expression of MDR-related, apoptosis-and proliferation-related factors, and the effect on miRNAs and the activation of multiple signal pathways.Part Ⅲ The study on the inhibitory effect and mechanism of EEHDW on the growth of multidrug resistant CRC xenograft in nude miceObjective:To investigate the reversal effects of EEHDW on growth of CRC xenografts in nude mice and its molecular mechanism in vivo.Methods:Male BALB/c athymic nude mice were subcutaneously injected with HCT-8/5-FU cells and HCT-8 cells in the right flank area to establish an MDR model of CRC.5-FU treatment was used to reflect the response of MDR model. Then, mice with HCT-8/5-FU xenografts were randomized into 4 groups and treated with saline, EEHDW,5-FU, and EEHDW-5-FU combination for 6 weeks. The effect of EEHDW on growth of transplantation tumors was determined by measurement of volume and weight of tumors. PCNA and TUNEL assays were used to confirm the effect of EEHDW on proliferation and apoptosis of xenograft tissues. RT-PCR and IHC were used to confirm the effect of EEHDW on the mRNA and protein expression of drug efflux factors (ABCC1/MRP1, ABCB1/P-gp and ABCG2/BCRP), cell proliferation regulatory factors (Cyclin D1, CDK4 and p21) and cell apoptosis regulatory factors (Bcl-2 and Bax) in xenograft tumor tissues. Western blotting was used to confirm the effect of EEHDW on the activation of ERK, JNK, p38 and PI3K/AKT pathways in xenograft tumor tissues. QPCR was used to study the effect of EEHDW on the expression of miRNAs in xenograft tumor tissues.Results:HCT-8/5-FU xenograft tumors showed less response to 5-FU than HCT-8 xenograft tumors, thus could be used as an MDR model for further research. EEHDW or combination treatment could inhibit growth of xenograft tumors, and combination treatment showed best therapy effect. EEHDW or combination treatment could induce cell apoptosis and inhibit cell proliferation in xenograft tumors. EEHDW or combination treatment could markedly downregulate the mRNA and protein expression of ABCC1/MRP1, ABCB1/P-gp, ABCG2/BCRP, Cyclin D1, CDK4 and Bcl-2, and upregulate the mRNA and protein expression of p21 and Bax in a dose-dependent manner in in xenograft tumors. EEHDW or combination treatment could reduce the activation of ERK1/2, JNK, p38, PI3K/AKT pathways in xenograft tumor tissues and played a wide regulatory role in the expression of miRNA, such as upregulating the expression of miR-92b, miR-1247, miR-4800-5p, miR-1224, miR-1260, miR-4669 and miR-4298, and downregulating the expression of miR-582-5p, miR-4454, miR-222, miR-483-5p and miR-4443.Conclusion:We successfully established the MDR model of CRC in nude mice. EEHDW can inhibit the growth of xenograft tumors and increase the sensitivity of tumor cells to 5-FU. EEHDW plays a significant role in the mediation of MDR-related, proliferation and apoptosis-related factors, and remarkably affects the activation of related signaling pathways and the expression of miRNAs.