1.Comparison Of Three Colorimetric Assays:CCK-8, MTT And SRB For Antitumor Drug Screening In Vitro 2.the Study Of 37 Ent-kaurananoids On Antitumor Activities, Mechanisms And Structure-activity Relationship

OBJECTIVE To compare the correlation, repeatability, stability and sensitivity between CCK-8, SRB and MTT assay. To analyse their advantages and disadvantages in the application for antitumor drug screening in vitro.METHODS Ten different cell densities of K562 and HepG2 cell lines were seeded in 96 well-plate. Their optical density (OD) were measured by CCK-8, MTT and SRB assay. The correlation coefficients between living cells and OD values for three assays were calculated respectively. Triplicate tests were performed for each assay and the repeatability of three assays were compared. At an optimal seeding density, the OD values of three assays were analysed at different time point and the stability of three assays were measured. The IC₅₀ values of 4 natural products and 2 antitumor drugs were evaluated by the three assays respectively, and the sensitivity of three assays were analysed.RESULTS For CCK-8, MTT and SRB assay, the correlation coefficient between living cells and OD values was over 0. 888(P<0.05); the OD values measured by triplicate tests were well correlated, the correlation coefficient was over 0. 956 (P<0.05). The OD values measured by CCK-8 and MTT assay varied in a time dependent manner (P<0.05), especially with CCK-8 assay, while the OD values measured by SRB assay were stable. No obvious difference was found between IC₅₀ values of six samples determined by MTT and SRB assay, while the IC₅₀ values determined by CCK-8 assay were lower than that of MTT and SRB assay.CONCLUSION The OD values measured by CCK-8,MTT and SRB assay are well relevanced with the number of living cells. The repeatability of CCK-8,MTT and SRB assay is nearly good. The SRB assay has better stability than that of MTT and CCK-8 assay. All the three assays can be used in antitumor drug screening for both cells growing in suspension and in adherence, and have equal effectivity for antitumor activity. The CCK-8 assay is more sensitive and more simple than MTT and SRB assay, but is also more expensive. The CCK-8 assay will be more suitable for the screen of samples with micro-quantity. OBJECTIVE To study the antitumor activities of 37 ent-kaurananoids from Isodon xerophilus in vitro and their mechanisms, and preliminary analysis of their structure-activity relationship, then to establish a basis for developing new antitumor drugs from this kind of ent-kaurananoids.METHODS The effects of 37 diterpenoids on the proliferation of 3 human tumor cell lines and murine spleen T and B lymphocytes were measured by CCK-8 assay, and their structure-activity relationship were studied. Two compounds with high cytotoxicity were selected for further mechamism study. Their activity on apoptosis induction for HepG2 cell was detected with morphology and DNA agarose gel of electrophoresis. Their effects on cell cycle was analysed by Flow Cytometry(FCM).RESULTS Among the 37 compounds, 13 of them showed cytotoxicity withα,β-unsatered ketone functional group, but another 13 compounds had no effect on three human tumor cells. Majority of these compounds could affect the proliferation of murine T,B lymphocytes.It seemed that theα,β-unsatered ketone was not necessary for their cytotoxity on murine T,B lymphocytes. Compound SXER32A and SXER32B could induce the apoptosis of HepG2 cell. Compound SXER32A could block the cell cycle into G2/M phase at low concentration, and it blocked that into S phase at high concentration. Compound SXER32B could block the cell cycle of few cells into S phase at low concentration. At high concentration, it could both block the cell cycle into S and G0/G1 phases.CONCLUSION Compounds SW11 exhibit strong cytotoxicity for 3 human tumor cell lines and low immunotoxicity for murine T and B lymphocytes. It has the potential to be a candidate for antiumor drug. SXER43, SXER55, SXER57 and SXER68 have no any activity against tumor cells, but have effects on the inhibition of proliferation for murine T and B lymphocytes. They have the potential to be a candidate as immunodepressants. SXER48 could promote T and B lymphocyte proliferation, and had no effect on hunman tumor cells proliferation. It could be a candidate of immunopotentiator. The structure-activity relationship analysis of 37 ent-kaurananoids shows thatα,β-unsatered ketone functional group is an essential active centre for the antitumor activity, while the functional group is not depend on lymphocyte cytotoxicity. The antitumor activity of SXER32A and SXER32B result from that they block the cell cycle and induce cell to apoptosis in HepG2 cells.