The Role Of Autophagy In The Reversal Of Multidrug Resistence By Macrocyclic Bisbibenzyls And In Paclitaxel Chemotherapy

Tumour is one of the most common and frequently encounted diseases. In particular, malignant tumor is the leading cause of death worldwide. Chemotherapy plays a very important role in the treatment of malignant tumours. However, the effective treatment of cancer by chemotherapeutic agents is frequently impaired as a result of either de novo or acquired multidrug resistance (MDR) of cancer cells. MDR in cancer is a phenomenon that occurs when cancer cells become simultaneously resistant to structurally unrelated chemotherapeutic agents, and leading to the failure of cancer treatment. Many researches were carried out to elucidate the mechanisms of MDR and to find compounds which re-sensitize MDR cells to chemotherapeutic drugs.Although several cellular mechanisms are associated with the development of MDR, P-glycoprotein (P-gp), an ATP- binding cassette (ABC) transporter, is the most extensively characterised mechanism of MDR. P-gp protein is capable of extruding various drugs from cells in energy-dependent manner, leading to the decrease of drug concentrations within the cell and reducing efficacy of drugs, and subsequent promotion of MDR. Numerous efforts have been made in developing compounds capable of modulating the activity of P-gp as a means of reversing the MDR phenotype. A number of noncytotoxic drugs which sensitize MDR cells to chemotherapeutic drugs have been identified, including calcium channel blockers, immunosuppressive drugs and natural occurring polyphenolic compounds. Although these drugs can sensitize MDR cells to chemotherapeutic drugs in vitro, they failed to be applied in clinic because of their severe side effects and poor pharmaco kinetics in vivo. Therefore, the development or discovery of safe and effective MDR reversal agents is much more desired to reversing MDR in the clinic.Macrocyclic bisbibenzyls are a series of phenolic natural products that are found exclusively in bryophytes. These natural products exert a variety of biological activities, including antifung, antimicrobial, antioxidation, muscle-relaxing, and cytotoxicity due to the diversity of their structures. To date, more than 100 types of bisbibenzyls with excellent biological activities have been discovered from the liverworts and some of them could be the prospective leading compounds for the drug discovery. After screening of over 100 bisbibenzyl compounds, MarchantinC (MC) and dihydroptychantol A (DHA) were gained attention because of their cytotoxicity and reversal of MDR in cancer cells. In attempt to elucidate the mechanisms underlying their reversal effect on MDR, MC and their analogues (MC1,MC2, MC3), and derivatives of DHA (DHA1,DHA2, DHA3) were chemically synthesized, respectively. Their reversal activities and possible mechanisms were investigated in our present study. Interestingly, autophagy induced by DHA2 was involved in its MDR reversal effect. In addition to our testing compounds, autophagy was also examined in the treatment of chemotherapeutic agents, such as paclitaxel. We found that autophagy induced by DHA2 or paclitaxel all represented a defense mechanism in DHA2-mediated reversal of drug-resistance or paclitaxel-induced apoptosis.Part 1 The effect of marchantin C and its analogues on reversing multidrug resistence in drug-resistant cancer cellsObjective:To examine the effect of marchantin C and its analogues on reversing multidrug resistence.Methods:1. The cytotoxicity and multidrug resistance reversal activity were analyzed by MTT assay.2. The effect of MC2 on the expression of P-gp was determined by western-blot analysis.3. The effect of MC2 on the transport function of P-gp was determined by Rh123 accumulation and efflux assay4. Nucleic morphological changes of apoptosis were determined by staining cell with DAPI. The expression level of Bcl-2, Bax and PARP was determined by western-blot.Results:1. Marchantin C and its derivatives exerted their cytotoxicity on KB/VCR cell line, overexpression of P-gp protein. Of four derivatives, MC2 had low cytotoxicity and exhibited more potent effect on MDR reversal in drug-resistant cells.2. MC2 increased the accumulation of ADR and Rh123,and reduced the leakage of Rh123 from the cells. However, MC2 didn't affect the expression of P-gp under the same condition, indicating that MC2 inhibited the transport activity of P-gp, but not the protein abundance.3. The combination treatment of MC2 and vincristine(VCR) significantly increased vincristine-induced apoptosis in drug-resistant KB/VCR cells.Conclusion:Marchantin C and its synthetic deviatives could reverse P-gp-mediated MDR in cancer cells. Of four compounds, MC2 exhibited most potent effect on the reversal of MDR by directly inhibiting the drug efflux function of P-gp. The low cytotoxicity, non-interference on the expression of P-gp, and reversible blocking action of P-gp suggest that MC2 may be a promising candidate for further investigation.Part 2 Targeting autophagy augments the activity of DHA2 to overcome p-gp mediated multidrug resistanceObjective:To study the effect of the analogues of DHA on the reversing multidrug resistence, and evaluate the role of autophagy in DHA2-mediated reversal activity of MDR in drug resistant cellsMethods:1. The cytotoxicity and multidrug resistance reversal activity of the analogues of DHA was analyzed by MTT assay.2. The effect of DHA2 on the expression of P-gp was determined by western-blot analysis.3. The effect of MC2 on the transport function of P-gp was evaluated by Rh123 and adriamycin accumulation assay.4. The expression and the processing of LC3 was examined by western-blot. Fluorescence microscopic examinations were done to detect the redistribution of LC3 during autophagosome formations. Transmission electron microscopy was used to detect the formation of vacuoles.5. ATP levels were measured by the luciferin-luciferase method using an ATP Bioluminescence Assay Kit.6. To clarify the interconnection of DHA2-induced autophagy and the reversal activity of DHA2, we manipulated autophagic activity using autophagy inhibitor 3-MA or siRNA target beclinl.Results:1. The analogues(DHA1, DHA2, DHA3) of DHA potentiates the cytotoxicity of anticancer drugs in the P-gp overexpressing cells. Of them, DHA2 was the most effective chemical on reversal of MDR.2. DHA2 didn't alter the expression of P-gp. But the transport of Rh123 and ADR by P-gp is significantly inhibited by DHA2. These results suggested that DHA2 reversed P-gp-mediated MDR in cancer cells by directly inhibiting the drug efflux function of P-gp.3. The treatment of DHA2 lead to the upregulation of LC3 and the modification of LC3-I to LC3-II, the appearance of characteristic punctate fluorescent patterns and the increase of the vacuoles in DHA2-treated cells. These observations indicated that DHA2 treatment caused autophagy in MDR cancer cells.4. The reversal activity of DHA2 was dramatically enhanced after 3-MA inhibited autophagy. The reversal effect of DHA2 was also significantly increased by beclin 1 siRNA but only slightly affected by random siRNA. These results indicated that autophagy induced by DHA2 represents a defense mechanism in MDR cancer cells.5. The ATP levels was further declined in the cells treated with DHA2 in the presence of 3-MA. SiRNA-mediated suppression of beclin 1 also significantly dcreased the ATP level after the treatment of DHA2.Conclusion:DHA2 significantly reversed P-gp-mediated MDR in cancer cells by directly inhibiting the drug-transport activity of P-gp. DHA2 could induce autophagy in MDR cancer cells. The reversal activity of DHA2 was significantly exacerbated after inhibition of autophagy. These results suggested that autophagy induced by DHA2 represents a defense mechanism in MDR cancer cells.Part 3 Autophagy inhibition promotes paclitaxel-induced apoptosis in cancer cellsObjective:To determine if paclitaxel could induce autophagy and investigate the role of autophagy in paclitaxel-induced apoptosis.Methods:1. Morphological changes of apoptosis were determined by DAPI staining. The effect of paclitaxel on the expression of Bcl-2, Bax, PARP was determined by immunoblotting assay.2. The change in the expression level of the LC3, Beclinl and Atg5 was investigated by immunoblot analysis. Paclitaxel-induced autophagic flux was also determined after the cells was transiently transfeced with an expression plasmid containing GFP-LC3.3. We confirmed the autophagic response to paclitaxel by analysis of acidic vesicular organelles (AVOs) formation by staining the cells with acridine orange (AO).4. The interconnection between paclitaxel-induced autophagy and apoptosis was investigated using autophagy inhibitor 3-MA. The apoptosis of cancer cell was detected by AnnexinV/PI staining.5. The role of autophagy in the paclitaxel-mediated cytotoxicity was further studied by knocking down the beclin 1 expression using siRNA.6. Animal experiments was designed to test whether treatment with paclitaxel would cause autophagy in vivo and that inhibition of autophagy would potentiate the effects of paclitaxel. A549 cells were injected in the right flank of nude mice. When the tumour size reached a predetermined size, tumor-bearing animals were treated with PBS,3-MA, paclitaxel, or a combination of 3-MA and paclitaxel.7. Paclitaxel-induced autophagic vacuoles was detected in GFP-LC3 stably transfected U87 cells and in GFP-LC3 transiently transfeced PC3 and HT-29 cells by fluorescence microscopy. Alternations in the expression levels of LC3, Beclin1 and Atg5 proteins was determined by western-blot analysis.3-MA was used to inhibit autophagy and then evaluated the role of autopgy in the paclitaxel-induced apoptosis in U87, PC3 and HT-29 cells.Results:1. Treatment with paclitaxel induced apoptosis in A549 cells.2. Paclitaxel treatment led to the up-regulation of Atg5, Beclin 1 andLC3, the increase of punctate fluorescent signals in A549 cells, and the formation of acidic vesicular organelles (AVOs). These data indicate that paclitaxel was able to induce autophagy in A549 cells.3. Inhibition of autophagy by 3-MA potentiates paclitaxel-mediated apoptotic cell death. Inhibition of autophagy by beclin 1 siRNA also increases the cytotoxic effect of paclitaxel.4. Paclitaxel induce autophagy in vivo and suppression of autophagy enhanced the cytotoxicity of paclitaxel.5. The autophagy-activation effect of paclitaxel was comfirmed in other cancer cells including human glioma cells U87, human prostate cancer PC-3 and colon cancer HT-29 cells.Conclusion:Our data indicate that autophagy was induced in cancer cells along with paclitaxel-induced apoptosis, and inhibition of autophagy could result in potentiation of the proapoptotic effect of paclitaxel. Our findings suggest that appropriate modulation of autophagy is necessary for sensitizing tumor cells to anticancer therapy.