Effects Of SAG On Human Liver Cytochrome P450Enzymes And The Metastasis Of Melanoma

Background: SAG, a benzo[c]phenanthridine alkaloid found in traditional herbalmedicines, has been displayed strong cytotoxic effect on several cancer cell lines invitro and has recognized as an antitumor drug candidate. In view of the toxicity andefficacy are the two prerequisite factors of traditional Chinese medicine monomer in thedrug development, this study aims to investigate the toxicity and antitumor activity ofSAG in depth. Cytochrome P450(CYP) enzymes, a super family of monooxygenaseslocated primarily in hepatocytes, are imperative drug-metabolizing systems inmammals. For most xenobiotics (e.g., food additives, dietary chemicals and drugs),CYP-mediated phase I metabolism is responsible for the key rate-limiting step in wholebiotransformation processes, resulting in unique pharmacological and toxic pattern inthe body. Melanoma is the most serious type of skin cancer in the world. It isresponsible for the major death of cancer metastasis. Clinical trials indicated thatmelanoma shows preferential metastasis to the lung, brain, liver, and skin; meanwhile, itis highly resistant to conventional chemotherapy. Given the rising incidence ofmelanoma and the lack of effective therapies, looking for new chemicals regulating the complex genetic networks involved in melanoma metastasis might be a new avenue forthis devastating disease. Currently in vitro two-dimensional culture model is routinelyadopted for screening anticancer drugs. However, the disadvantages of two-dimensionalculture model are obvious. They include the lack of real tumor tissue microenvironment,the lack of support of the extracellular matrix, the loss of some of the biologicalproperties.Objective:1. To investigate of inhibitory effects of SAG on seven major CYP isoforms1A2,2A6,2E1,2D6,2C8,2C9and3A4.2. To evaluate the anti-metastasis effect of SAG on human melanoma cell451LU bydetecting changes of mRNA and protein expression related to cell-matrigel adhesionand matrigel membrane invasion in vitro. Furthermore, inhibitory effects of SAG onfocal adhesion kinase (FAK) were also determined.3. To establish a three-dimensional culture model of melanoma cell lines451LU，evaluate its morphology and function, and on which basis, the anti-tumor activity ofSAG was evaluated once more.Method:1. To investigate the inhibitory effects of SAG on seven major CYP isoforms by CYPprobe substrate reaction, then, determine of inhibition kinetics parameters throughenzyme-inhibition experiment. At last, give the forecast of the degree of drug-druginteractions of SAG according to the formula.2. Relative toxicity was evaluated by using the sulforhodamine B (SRB) assay. TheFITC-labeled and PI-labeled cells were measured by flow cytometric analyses toinvestigate effects on cell viability of malignant melanoma tumor cells of SAG. Usingtranswell invasion assay tumor cells, wound healing experiments, matrix-cell adhesionassay, endothelial cell proliferation experiments to detect the anti invasion andmetastasis effect of SAG on malignant melanoma cell. Using real time PCR andWestern blot to detect the mRNA and protein level of metastasis related gene andprotein. The effect of SAG on FAK signaling pathway is detected by Western Blot. 3. Culturing the human malignant melanoma cell lines451LU in rat tail collagen, tobuild a three-dimensional culture model. Using histological staining, cell viability assayand immunofluorescence staining to evaluate the morphology of the cell. Using DNAcontent to reflect the proliferation conditions in different culture models. Using realtime PCR to detect the differences expression levels of related genes in the two andthree-dimensional culture model. Evaluate of the impact of four anti-cancer drugs oncell viability of malignant melanoma cells by MTT assay.Results:1. At80μM, SAG did not inhibit the activities of CYP2A6, CYP2D6and CYP2E1. Incontrast, the activities of CYP1A2, CYP2C8, CYP2C9and CYP3A4were inhibited to19.6%,16.6%,22.5%and14.3%of their control activities, respectively. IC50values was4.5,10.2,7.2and3.4μM, respectively. Lineweaver–Burk plots and Dixon plots ofinhibitory kinetic data suggested that the inhibition of CYP1A2, CYP3A4and CYP2C9by SAG was best fit to a competitive way, whereas the inhibition of CYP2C8by SAGwas in a noncompetitive manner. By the secondary plot of the slopes ofLineweaver-Burk plots versus SAG concentrations, Kivalues were estimated to be2.7,8.9,3.8and2.0μM for CYP1A2,2C8,2C9and3A4, respectively. After pre-incubationof SAG with HLM for30min in the presence of NADPH, the percentage of inhibitionof CYP1A2and CYP3A4increased by25.5%and20.3%respectively, compared withthose without NADPH. The study found that SAG catalyzed reaction reflects onCYP1A2, CYP3A4in NADPH-dependent and time-dependent inhibition of behaviorwith KI/kinactvalues of13.3/0.087and5.58/0.029min-1μM-1, respectively.2. We examined the effect of SAG on the proliferation of451LU cell line. SAG caninhibit the proliferation of451LU cell in a dose dependent manner with IC50of3.23±0.53μM. In this study,1,1.5and2μM were chosen for further metastasis relatedassay, because at these concentrations, SAG did not significantly influence the cellgrowth with the cell viability of higher than80%. The results of flow cytometricanalysis also confirmed that SAG did not affect the cell viability of451LU. SAG of1,1.5and2μM all showed significant inhibition effect on the cell adhesion efficiency in dose and time-dependent.1,1.5and2μM SAG all can inhibit the migration of451LU.1,1.5and2μM of SAG can decrease the number of451LU cells on the oppositechamber in transwell invasion assay. In order to clarify molecular mechanism of thisphenomenon, different metastasis related genes were chosen to evaluate the metastaticability of human melanoma cell WM35and metastasis melanoma cell451LU. ICAM-1and MMP-2were significantly increased in451LU cells. The data showed that SAGcan also inhibit the ICAM-1, MMP-2protein expression and reduced expression ofFAK and P-FAK.3. In contrast of451LU cells cultured in three-dimensional culture, the cells intwo-dimensional culture model began to proliferate on the3rd day, began apoptosis onthe5th day and almost apoptosis on the7th day. For the cells cultured in thethree-dimensional culture conditions, the viability of cells was still active on the day14,and the proliferation ability of the three-dimensional culture conditions wassignificantly increased compared with two-dimensional culture samples. Evaluate of theimpact of four anti-cancer drugs (doxorubicin, vinblastine, paclitaxel and SAG) on cellviability of malignant melanoma cells by MTT assay, the IC50values of doxorubicin,vinblastine and SAG on451LU cells were all increased. The mRNA expression ofMMP2, ICAM-1, BRAF-V600E and P-gp were significantly increase in thethree-dimensional culture conditions.Conclusion:1. Invitro–invivo extrapolation from HLM data showed that more than35.9%ofCYP1A2, CYP2C9, CYP2C8and CYP3A4activities invivo could be inhibited bySAG, suggesting that harmful DDIs could occur when SAG orits medical preparationsare co-administered with drugs primarily cleared bythese CYP isoforms. Further invivo studies are needed toevaluate the clinical significance ofthe data presented herein.2. SAG can significantly inhibit the proliferation ability of human malignant melanoma451LU. Meanwhile, SAG can inhibit the angiogenesis, invasion and metastasis abilityof melanoma, which is associated with reducent the MMP2, ICAM1gene and proteinexpression levels. SAG can also reduce the FAK and phosphorylation of FAK protein expression.3. The proliferation ability of the three-dimensional culture conditions was significantlyincreased compared with two-dimensional culture samples. Evaluation of the impact offour anti-cancer drugs on cell viability of malignant melanoma cells by MTT assayshowed that all IC50values were increased. The mRNA expression of MMP2, ICAM-1,BRAF-V600E and P-gp were significantly increased in the three-dimensional cultureconditions.