ANTI-MDR Effects And Target For P-GP Mechanisms Study Of Natural Small Molecule Compounds DHA And SM

Exploration of natural products with anti-tumor activity, especially those novel and lead compounds is an important new drug research and development tool. A wide variety of novel small-molecule natural antineoplastic products have recently been reported. These compounds were isolated from marine organism, a variety of animals, plants and microorganisms sources. With the breadth of activity represented in these bioactive small molecules, the future of natural product antineoplastic drug discovery looks bright. Moreover, the structure-activities relationship of compounds and their series derivatives is very important for synthesizing more effective in killing cancer cells and decreasing damage to other healthy tissues. Among the screened small molecule compounds with anti-tumor effects, DHA and SM were two kinds of different structure compounds, however, have the similar activity in anti-MDR cells. Furthermore, the underlying mechanisms were investigated.Multidrug resistance is one of the major causes for clinical chemotherapeutic treatment failure. The failure of the curative treatment of cancer patients often occurs as a result of intrinsic or acquired drug resistance of the tumor to chemotherapeutic agents. The resistance of tumors occurs not only to a single cytotoxic drug used, but also occurs as a cross-resistance to a whole range of drugs with different structures and cellular targets. This phenomenon is called multiple drug resistance (MDR). Once MDR appears, it is ineffective to use high doses of drugs to overcome resistance; oppositely toxic effects will appear and resistance will be further stimulated. Several strategies for overcoming drug resistance have been studied. One approach is to develop a variety of reversal agents such as verapamil, cyclosporin A (CysA) and others. However, the clinical trial results of these drugs are disappointing due to dose-limiting toxicity. The development of novel anticancer drugs insensitive to or bypassing MDR mechanisms is currently a major focus of research. Mechanisms involved in resistance to cancer chemotherapy include decreased drug accumulation (decreased drug uptake and/or increased drug efflux), altered intracellular drug distribution, increased detoxification, diminished drug-target interaction, increased DNA repair, altered cell-cycle regulation, uncoupling of the pathways linking cellular damage with apoptosis, etc. Among these, P-gp mediated multidrug resistance is the main mechanisms of MDR. P-gp, a transmembrane glycoprotein, functions as an adenosine 5’-triphosphate (ATP)-dependent drug transporter which unilaterally transports intracellular drugs out of the cells to acquire drug resistance. Increase in P-gp activity or expression induction leads to both a lower intracellular accumulation and an increased efflux of drugs. Some chemosensitizers directly block P-gp activity by binding to chemotherapeutic drug binding sites or to other modulator binding sites, which result in inhibition of drug binding or transport. The other approach for MDR modulation is down-regulation of P-gp expression.The macrocyclic bisbibenzyls, exclusively isolated from liverworts, have been reported to have diverse biological activities including cytotoxicity, antimicrobial, antifungal effects and reversal effects on multidrug resistance. Dihydroptychantol A (DHA), one of macrocyclic bisbibenzyls which is a class of characteristic natural molecules derived from liverworts was identified to be an antifungal active macrocyclic bisbibenzyl from liverwort Asterella angusta. DHA showed the strongest potency to increase adriamycin cytotoxicity toward K562/A02 cells by MTT assays. Mechanisms of DHA on p-glycoprotein (P-gp)-mediated multidrug resistance (MDR) were further investigated. Based on the flow cytometry, we detected the significant increase of adriamycin and rhodaminel23 accumulation in K562/A02 cells exposed to DHA, meanwhile, notable decrease of rhodamine123 efflux was also observed. Furthermore, P-gp expression was analyzed by flow cytometry and RT-PCR methods. Dose-dependent reduction of P-gp expression was observed in K562/A02 cells pretreated with DHA for 24h. These results demonstrated that DHA reversed effectively MDR by blocking drugs to be pumped out via inhibiting P-gp function and expression.Solamargine (SM), a steroidal glycoalkaloid isolated from Chinese herb Solanum incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. In this study, MTT assay was used to observe the cytotoxicity of SM. DAPI nuclear staining and flow cytometry methods were used to observe the effect of SM on apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR and P-gp protein expression was measured by flow cytometry. The changes in the morphology of actin were examined with immunofluorescence staining. MTT results showed that SM effectively inhibited the proliferation of MDR sublines of K562/A02, KB/VCR and H460/Tax to an equivalent or more sensitive degree comparing with that of their parental K562, KB and H460 cell lines. And SM showed pro-apoptotic effects on MDR K562/A02 cells. Furthermore, SM induced the downregulation of MDR1 mRNA and P-gp expression. In addition, the expression of actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining. These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors.