The Mechanism By Which PZH Reverses Multiple Drug Resistance Of Colorectal Cancer

Objective:To evaluate the effect of PZH on multi-drug resistance (MDR) of colorectal cancer in vivo and in vitro, and investigate the underlying molecular mechanisms.Methods:1. In vitro study:Cell viability by MTT assay was examined to determine the MDR characteristic of HCT-8/5-FU cells and the reversal effect of PZH. Microscopy and colony formation assay were used to evaluate the cell morphology and survival, respectively. FACS was performed to analyze cell cycle progress. Hoechst staining and activation of Caspase-9and Caspase-3were used to assess cell apoptosis. Transwell assay was performed to investigate cell migration and invasion. Accumulation of Rhodanmine123and5-FU in HCT-8/5-FU cells was determined by FACS and HPLC, respectively. RT-PCR and Western-blot were used to determine the mRNA and protein expression of Bcl-2, Bax, CyclinD1, CDK4and ABCG2.2. In vivo study:Comparison of tumor weight and volume were used to assess the effect of5-FU and PZH on tumor growth. TUNEL assay was used to examine cell apoptosis. Immunohistochemistry (IHC) was performed to detect the expression of proliferative biomarker Ki-67. RT-PCR and Western-blot were used to determine the mRNA and protein expression of Bcl-2, Bax, CyclinD1, CDK4and ABCG2.3. miRNA analysis:Q-PCR was used to determine the expression of miRNAs (miR-200a, miR-200c, miR-582, miR-22).Result:1. In vitro study:The IC50of5-FU and ADM on HCT-8/5-FU cells were significantly higher than that on its parental cells, and the Resistant Inedx (RI) were both higher than1.5(indicating drug resistance), while there was no difference between the IC50of PZH on HCT-8/5-FU and its parental cells (RI<1.5). The combination of PZH (0.25mg/mL) significantly decreased the IC50of5-FU and ADM on HCT-8/5-FU cells, and the Reversal Effect (RE) were both higher than1.5(suggesting the reversal effect on drug resistance). MTT assay showed that PZH treatment significantly decreased the cell viability of HCT-8/5-FU cells in dose-and time-dependent manners. PZH treatment dose-dependently decreased the cell confluence and survival of HCT-8/5-Fu cells. PZH significantly increased the percentage of GO/G1-phase and decreased the percentage of S-phase in HCT-8/5-FU cells. Hoechst staining showed that PZH-treated cells displayed condensed chromatin and fragmented nuclear morphology which are typical apoptotic morphological features. PZH treatment significantly increased the activation of Caspase-9and Caspase-3in HCT-8/5-FU cells. The accumulation of Rhodamine123and5-FU in HCT-8/5-FU cells was significantly increased with the treatment of PZH. Transwell assay showed that PZH treatment significantly decreased the ability of migration and invasion on HCT-8/5-FU cells. RT-PCR and Western-blot analysis showed that PZH treatment significantly down-regulated the expression of anti-apoptotic Bcl-2, cell cycle regulator CyclinD1and CDK4, key transporter ABCG2, but up-regulated the expression of pro-apoptotic Bax.2. In vivo study:(1) PZH treatment significantly inhibited tumor weight and volume in HCT-8/5-FU xenograft mice, whereas5-FU (20mg/kg) did not display therapeutic effect. TUNEL assay indicateed that PZH treatment significantly induced the apoptosis of HCT-8/5-FU cells in tumor tissues. IHC staining indicated that PZH treatment.significantly inhibited the expression of proliferative marker Ki-67. RT-PCR and Western-blot analysis showed that PZH treatment significantly down-regulated the expression of Bcl-2, CyclinD1, CDK4and, ABCG2, but up-regulated that of Bax. Treatment of5-FU did not show above-mention activities.3. miRNA analysis:Q-PCR analysis showed that PZH treatment significantly up-regulated the expression of several MDR-related miRNAs, including miR-200a, miR-200c, miR-582and miR-22.Conclusion:PZH significantly suppresses the expression of several MDR-related miRNAs (miR-200a, miR-200c, miR-582, miR-22) and alters the expression of Bcl-2, CyclinD1, CDK4, Bax and ABCG2both in drug-resistant colorectal cell line HCT-5-FU and xenograft mice. These molecular effects lead to the induction of apoptosis, inhibition of cell proliferation, migration, invasion and function of ABC transporter, eventually resulting in the reversal effect of PZH on MDR of colorectal cancer in vivo and in vitro.