Anti-tumor Effects And Mechanisms Research Of ADS, An Andrographolide Derivatives In Vitro

Andrographis paniculate(Burm.f.)Nees was found in India and planted in east, middlesouth and southwest China. It is used extensively as an antipyretic, anti-inflammtory and antivirus medicine for a long time. Recently, it’s newly pharmacological antitumor activity was found. ADS, an andrographolide derivatives, was synthesized by semi-synthetic method, and its had strong antitumor activity on4T-1and LLC cancer in vivo.The purpose of this paper was to investigate the anti-proliferation effects of ADS and its potential anti-tumor mechanisms in4T-1and LLC cells.The anti-proliferation effects of ADS were assessed by MTT assay in4T-1and LLC cells. Using PI staining and flow cytometry-based cell cycle analysis to evaluate its effects on cell cycle progression. Apoptotic morphological changes of4T-1and LLC cells after ADS treatment were observed by light microscope and high content screening (HCS) after AO/EB double staining, PI/Hoecsht33258double staining. Changes in the mitochondrial membrane potential (△Ψm) were assessed with Rh123/Hoechst33258double staining by high content screening (HCS). The expression of caspase-3, caspase-9, Bcl-2, Bax and cytochrome c (Cyto c) were measured using western bloting.MTT assay demonstrated that ADS exerted potent anti-tumor effect in a significant concentration-dependent manner and time-dependent manner with the IC50values of (5.5±1.36) uM and (4.4±0.93) μM (4T-1),(4.12±1.27) μM and (5.15±1.01) μM (LLC) after24h and48h treatment respectively. And the anti-tumor effects were more powerful than andrographolide both in4T-1and LLC cells.Cell cycle analysis results showed that3.2μM ADS treatment induced4T-1cell cycle arrest in G2/M phase with the cell percentage increasing from (7.95±0.1)%to (22.44±0.7)%, and exerted significant difference compared with the blank control. However,1.8μM ADS treatment induced LLC cell cycle arrest in G0/G1phase with the cell percentage increasing from (35.52±3.9)%to (55.44±2.7)%,2.5μM and3.0μM ADS treatment induced LLC cell cycle arrest in G2/M phase with the cell percentage increasing from (6.41±0.6)%to (14.4±0.8)%,(16.13±1.6)%respectively, and both exerted significant difference compared with the blank control.Changes in cell morphology under light microscope were observed both in4T-1and LLC cells. The results showed that cells revealed characteristics apoptotic displayed shrunken morphology like cell shrinkage, cell turnned round and the formation of apoptotic bodies after2.0μM ADS treatment for24h.AO/EB double staining and PI/Hoecsht33258double staining showed that chromoplasm-pyknosis and fragmented nuclei were observed in ADS treatment cells, and its degraded in a concentration-dependent manner and time-dependent manner. But the morphous of cell nucleus were intact.The mitochondrial membrane potential was measured with Rh123/Hoechst33258double staining by HCS. The results indicated that ADS could induce the△Ψm loss and destroy membrane integrity of4T-1and LLC cells in a concentration-dependent and time-dependent manner.Western bloting results showed that ADS could cause activation of the caspase families, increased expression of caspase-3, caspase-9and cytochrome c, Bcl-2families also involved in the process of apoptosis, Bcl-2expression lowered, Bax expression increased.ADS exerted potent growth inhibitory effect in4T-1and LLC cells, induced cell cycle arrest and further induced apoptosis via mitochondrial pathway.