Riccardin D Inhibited The Growth Of Human Tumor By Targeting DNA Topoisomerase â…¡

BackgroundsTopoisomerase is a kind of ribozyme which is essential for the survival of eukaryote and can mediate the single-stranded or double-stranded DNA instantaneous breaks. Topoisomerase plays an important role in DNA replication, transcription, recombination and chromosome segregation. On the basis of different ways of DNA breaks, topoisomerase can be divided into topoisomerasel and topoisomerasell. To interfer with DNA replication by targeting topoisomerase have a predictive value in the adjuvant therapy of malignant tumor. Topoisomerases are the targets of many clinically useful anticancer drugs. But still some problems were shown in the application process such as the generation of multi-drug resistance, the emergence of secondary acute myeloid leukemia due to the long-term use of Topo agents, cardiotoxicity. Therefore, it is of great significance to develope safe, efficient, low toxicity drugs targeting topoisomerase which can reverse multidrug resistance.RiccardinD, a novel macrocyclic bisbibenzyl compound, was extracted from the Chinese liverwort plant Dumortiera hirsutafrom. Previous studies have shown that riccardinD can inhibit the formation of the biofilms of Candida albicans, and its analogues Plagiochin E can reverse fungi multidrug resistance and tumor multidrug resistance, and the other analogues Marchantin C can lead to tumor cell apoptosis by microtubule depolymerization. Marchantin C also can arrest tumor cells in G2/M phase.The preliminary studies suggested that RiccardinD may have anti-tumor effect and reversal of multidrug resistance. The mechanism studies are underway. Methods1. The effects of riccardinD on leukemia cell apopotosisHuman chronic myelogenous leukemia cell line K562 and human acute myelogenous leukemia cell line HL60 were obtained for the detection of apoptosis using Hoechst33258, FITC-Annexin V staining and DNA Ladder detection. Caspase-3, Caspase-9, cleaved PARP expression were determined by Western blotting. RT-PCR was employed to estimate the ratio between anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax-a. Finally, the two key moleculars cytochromec and caspase8 in the mitochondrial and death receptor-mediated apoptosis pathways were detected by western blotting.2. The effects of riccardinD on topoisomerasesⅡactivityThe effects of riccardinD on topoisomerase I and topoisomeraseⅡactivity were tested with pBR322 DNA as substrate and etoposide as a positive control. The ativity of topoisomerasell which was extracted from K562 cells after treatment with riccardinD were detected. Real-time quantitative PCR was employed to detect the topoisomerasella gene levels. SPR analysis was used to evaluate the binding affinity between riccardinD and topoisomeraseⅡα. TopoisomeraseⅡα-deficient cell lines HL60/MX2 and topoisomerasella-overexpression cell lines were used to detect the effects of riccardinD on cell proliferation.3. The effects of riccardinD on the growth of human non-small cell lung cancer in vitro and in vivoTo test the effects of riccardinD on non-small cell lung cancer cell apoptosis in vitro using MTT, Hoechst33258 and FITC-Annexin V double staining. In addition, the scratch assay, transwell chamber assay and gelatin zymography activity assay were employed to investigate the effects of riccardinD on the anti-invasion and metastasis ability of non-small cell lung cancer cell. Nude mice inoculated with H460 cells were administrated riccardinD when the tumor grew to 100mm3-300mm3. Administration was performed every 3 days for three consecutive weeks. The rates of tumor growth inhibition were defined as a ratio to the vehicle tumor weight. The specimens were removed for TUNEL staining and Western blot analysis for the apoptosis protein Caspase-3, Caspase-9 and cleaved PARP and invasion and metastasis associated protein MMP-2, MMP-9, VEGF, and Erkl/2.4. The effects of riccardinD on the multi-drug resistanceTo test the effects of riccardinD alone, doxorubicin alone and paclitaxel alone on the cell proliferation of K562/A02 named doxorubicin resistant strain and H460/RT named paclitaxel resistant strain using MTT assay. The inhibitory effects of the combination of doxorubicin, paclitaxel and riccardinD were evaluated respectively and the reversal fold was calculated. The nude mice inoculated with H460 cells were administrated to the more effective combination of paclitaxel and riccardinD when the tumor grew to 100mm3-300mm3. The rates of tumor growth inhibition for the combination group and alone were defined as a ratio to the vehicle tumor weight. The specimens were removed for Western blot analysis for the apoptosis protein and multi-drug resistance protein. The effects of riccardinD on multi-drug resistance protein and gene expression were detectd in K562/A02 cells.Results1. RiccardinD induced apoptosis of human leukemia cellsMTT results showed that riccardin D (10μM-60μM) significantly inhibited the proliferation of K562 and HL60 cells after 24h,48h and 72h treatment. The maximum inhibition rate was 90.6%. Hoechst33258 staining results showed that 10μM,20μM, 30μM RiccardinD induced the nuclear chromatin, condensation, or gathering around to the nuclear membrane, or in blocks fragmentation change role in leukemia cells after 48h treatment. FITC-AnnexinⅤdouble staining showed that apoptosis rates were 25.55% and 27.56% after treatment with 10μM riccardin D in K562 and HL60 cells, respectively. DNA Ladder results showed the formation of DNA ladder fragments after treatment with different concentrations of riccardin D with the dose-dependence. Western blot results showed that 10μM-40μM Riccardin D can significantly increase the expression of Caspase-3, Caspase-9, cleaved PARP protein in K562 and HL60 cells. RT-PCR showed the Bcl-2/Bax-αratio was significantly decreased. In addition,10μM-40μM Riccardin D can significantly increase the release of K562 cells cytochrome c with no significant effect on the expression of procaspase8. The activity form was not detected.2. RiccardinD induced apoptosis by targeting DNA topoisomeraseⅡTopoisomerase activity testing results show that riccardin D had no effect on topoisomeraseⅠactivity.200uM-800μM riccardin D inhibited the topoisomeraseⅡactivity significantly in a dose dependent manner. And the inhibitory effects of riccardin D on topoisomeraseⅡactivity were significantly stronger than the positive control etoposide. The topoisomeraseⅡactivity in cells treated with riccardin D was also obviously reduced. Real-time quantitative PCR showed ricca rdin D can significantly reduce the DNA levels of topoisomeraseⅡα. SPR analysis showed that riccardin D revealed a high binding affinity for topoisomeraseⅡwith the equilibrium dissociation constant (KD) of 2.68E-06. Riccardin D did not inhibit the proliferation of topoisomeraseⅡαdeficient cell lines HL60/MX2 with the highest rate 30%. Apoptosis induction was not obvious, either. Compared with the normal K562 cells, riccardin inhibited more significantly for topoisomeraseⅡ-overexpression cell lines.3. Riccardin D inhibited the growth of human non-small cell lung cancer in vitro and in vivoMTT, Hoechst33258, FITC-Annexin V staining and Western blotting results showed that, riccardin D inhibited the cell proliferation of non-small cell lung cancer cells and induced apoptosis and increased the expression of apoptosis protein in vitro. Scratch assay results showed that the 5μM-10μM riccardin D inhibited A549 cells to the migration of cell-free scratch area. Transwell chamber assay showed that at the range of 2.5μM,5μM and 10μM of riccardin D, the number of cells penetrating the Matrigel-coated polycarbonate filters were inhibited by 40.7%,56.5% and 65.4%, respectively. Gelatin zymography results showed the MMP-2, MMP-9 activity was markedly inhibited in the supernatants of A549 and H460 cells exposed to riccardin D. 20 mg/kg of riccardin D delayed the growth of H460 xenografts by 44.5%. Etoposide (20 mg/kg), the positive control drug, inhibited the growth of H460 xenografts by 59.7%. Riccardin D treatment was generally well tolerated by mice with no significant loss of body weight. While, a markedly decrease of body weight was measured in etoposide treated-animals TUNEL staining of tumor tissue showed the cells in riccardinD-treated group were positive staining with blue nuclei. The apoptosis rate was 36.9%.4. Riccardin D reversed the multi-drug resistanceThe combined treatment with 10μM Riccardin D and doxorubicin in K562/A02 reversed the doxorubicin resistance. The reversal fold was 2.31. The combined treatment with 10μM Riccardin D and paclitaxel in H460/RT reversed the paclitaxel resistance. The reversal fold was 8.42. Riccardin D alone, paclitaxel alone and the combination treatment in nude mice inoculated H460/RT cells were evaluated. The inhibition rates were 46.8%,48.1% for alone group. The tumor inhibition rate was 57.3% in the combined group with paclitaxel. Electrophoresis showed that the apoptosis protein in tumor tissue of combination group was significantly increased. Riccardin D significantly reduced P-gp protein and the gene expression Mdrl in K562/A02 cells.ConclusionRiccardin D inhibited the tumor growth in nude mice inoculated H460 cells significanty, and also had the ability of anti-invasion and metastasis and reversed the multidrug resistance.The mechanisms were involved in targeting topoisomerasell. Riccardin D revealed a high binding affinity with topoisomerasell and obviously inhibited the topoisomerasell activity. Finally, riccardinD induced apoptosis of leukemia cells through the mitochondrial mediated apoptosis pathway.