The Antitumor Effects And Mechanisms Studies Of Novel Compounds GL3 And SMT-A07

Novel agents are needed to improve the efficacy in treating cancer, the second leading cause of death in the world. GL3, a novel VP16 derivative reformed from the clinical medicine VP16, and SMT-A07, a novel (3-indo-2-yl) indazole derivative designed from novel and reliable docking model were synthesized by pharmacochemistry scientist as potential anti-tumor compounds. Our study was to evaluate the anticancer effect of SMT-A07 and GL3, and further to explore their anticancer mechanisms.PART 1. Study of the Effect and Mechanism of GL3Aims:GL3 was developed as a novel analogue of classical antineoplastic agent VP16 and was specifically designed with bulky nitrous tails on the C-4 side chain. GL3 displayed improved anticancer activity compared to its parent compound VP16. We examined the intrinsic inhibitory profiles of GL3 on TopoⅡand its subsequent events, including apoptosis and G2/M phase arrest, respectively.Methods:MTT was used in proliferation assay to evaluate inhibition effect of GL3 in A2780, PC-3, A549, HepG2, HCT-116, SGC and KB cell lines; TopoⅡactivity inhibited by GL3 was evaluated by measuring TopoⅡ-mediated cleavage-religation complex stabilization; TopoⅠactivity inhibited by GL3 was determined through the measurement of TopoⅠ-mediated cleavage-religation complex stabilization; EB-drug competetive binding assay was introduced to detect whether GL3 could intercalate into DNA chain; Flow cytometry analysis was performed to analyze the apoptosis rates and cell cycle distribution of cells after their being treated with SMT-A07; Western Blotting was used to explore the expression of apoptosis-and cell cycle-related proteins; Comet assay was used to detect the induction of double strand breaks (DSB) caused by GL3.Results: (1) GL3 displayed potent cytotoxicity in a concentration-dependent manner for the cell lines. IC50 values for GL3 against seven cell lines were ranging from 0.82 to 4.31μM measured by cytotoxicity assays. Furthermore. IC50 values indicated that A549 cells were 11-fold more sensitive to GL3 than to VP16.(2) The molecular target of GL3 was TopoⅡ. GL-3 could increase the stability of the TopoⅡ-DNA cleavage complex, and then the activity of TopoⅡwas inhibited. However, GL3 had no effect on TopoⅠ-DNA cleavage complex, and cannot intercalate into DNA chains.(3) GL3 triggered DNA DSB-mediated apoptosis via increasing the stabilization of the TopoⅡ-DNA cleavage complex. There was a dose dependent increase in the apoptotic ratio after cells were exposured to GL3. Up-regulation of the protein levels p53. p-p53, cleaved caspase-3 and cleavage of the 85-KDa fragment of PARP, and down-regulation of expressions of procaspase-8, procaspase-9 and procaspase-3 occured after the concentration of GL3 exceeding 20μM. However, the expressions of proteins underwent no significant changes after treatment with VP16 at 20μM except the up-regulation of p53, p-p53 and bax. yH2AX was up-regulated after incubation with 20μM GL3 for 1 or 2 h, but VP16-treated cells exhibited no obvious phenomenon. Using the neutral single-cell gel electrophoresis, we found that the exposure of A549 cells to GL3 within 2 h induced much more DNA DSBs than that of VP16. However, GL3-induced DNA damage and apoptosis were antagonized by TopoⅡinhibitor Aclarubicin, suggesting that GL3 was a TopoⅡpoison.(4) GL3 induced cell cycle arrest, probably because of the activation of ATM/ATR pathway. GL3 treatment appeared to induce G2/M phase arrest in a concentration-dependent manner. GL3 can increase the expression of p-Chk2, cyclinB1 and Cdc2(T14/T15), as well as decrease the expression of p-Chk 1 and Cdc25C(S216). However, preincubation of ATM/ATR kinase inhibitor caffeine could antagonize GL3-induced G2/M Arrest, suggesting the G2/M arrest caused by GL3 dependented on ATM and/or ATR pathway.Conclusion:With TopoⅡas the molecular target, GL3 displayed potent cytotoxicity by increasing the stability of TopoⅡ-DNA cleavage complex. GL3 triggered DNA DSB-mediated apoptosis much more strongly than its parent compound VP16. Moreover. GL3 probably triggered the activation of ATM/ATR pathway, resulting in G2/M arrest. PART 2. Study of the Effect and Mechanism of SMT-A07Aims:The anticancer effects of SMT-A07, a novel (3-indo-2-yl) indazole derivative, was evaluated in leukemia and solid tumor cells and the mechanisms were investestigated for the purpose of offerring the experimental basis for the (3-indo-2-yl) indazole derivatives.Methods:MTT was used in proliferation assay to evaluate inhibitory effect of SMT-A07 in five leukemia cell lines(CCRF-CEM, HL60, Molt-4, U937, NB4) and ten solid tumor cell lines(HCT-116, SW620, HepG2, Bel-7402, T24,5637, A2780, AGC. BGC, A549); Flow cytometry analysis was performed to analyze the apoptosis rates and cell cycle distribution of cells after their being treated with SMT-A07; DAPI staining was used to observe the morphology of cells; Loss of△Ψm was investigated by JC-1 staining; the expression of apoptosis-related proteins were examined by Western Blotting to explore signalling mechanisms of SMT-A07; Immunofluorescence analysis was used in detection of Cyclin B1 distribution.Results:(1) SMT-A07 was a potential and highly efficient antitumor compound with IC50 values ranging from 0.09 to 1.19μM in five leukemia cell lines and 0.70 to 18.0μM in ten solid tumor cell lines by cytotoxicity assay;(2) SMT-A07 exerted noticeable concentration- and time- dependent cytotoxicity in HL60 and NB4 cells. The apoptosis rates after 24 h treatment with 0.0μM,0.25μM, 0.5μM and 1.0μM SMT-A07 were 6.88±0.64%,14.09±5.39%,31.94±10.81% and 49.72±8.24% in HL60 cells. The apoptosis rates after 0 h,6 h,12 h and 24 h treatment with 1.0μM SMT-A07 were 6.88±0.64%,4.01±1.31%,26.41±1.88% and 49.72±8.24% in HL60 cells. Similarly, SMT-A07 can induce apoptosis in NB4 cells in both concentration- and time- dependent manner. After SMT-A07 incubation, DAPI staining revealed the presence of DNA fragments and perinuclear apoptotic body. SMT-A07 exposure caused down-regulation and cleavage of procaspase-8, procaspase-3, Bid, PARP and up-regulation of cleaved caspase-8, cleaved caspase-3, PARP(Cleaved Factor). Meanwhile, the introduction of broad-range caspase inhibitor BOC-D-FMK prevented cells from caspase-3 and PARP cleavage and subsequent apoptosis. In addition, SMT-A07 also resulted in a loss of△Ψm in both HL60 and NB4 cells.(3) SMT-A07 exerted markable G2/M phase arrest in HCT-116 and SW620 cells. Treatment with 0.0μM 5.0μM.10.0μM and 20.0μM SMT-A07 for 24 h can induce 25.22±4.77%.75.91±2.23%,91.91±0.11% and 96.73±0.34% HCT-116 cells arrest at G2/M phase. Treatment with 20.0μM SMT-A07 for 0 h.6h.12 h and 24 h can arrest 25.22±4.77%,75.91±2.23%.91.91±0.11% and 96.73±0.34% cells at G2/M phase. Treatment of SMT-A07 can also cause G2/M phase arrest in SW620 cells in both concentration- and time- dependent manner.The expressions of cyclinB1 and Cdc2(ser161) in HCT-116 and SW620 cells were increased after the their being treated with SMT A07, while the change of total Cdc2 was not at all evident. Moreover, SMT-A07 treatment maintained cyclinBl in nucleus, indicating that SMT-A07 may induce HCT-116 cells arrest in M phase.Conclusion:SMT-A07 exhibited intense anticancer effect in both leukemia and solid tumor cells. There were two distinct cellular responses to the treatment with SMT-A07. SMT-A07 could induce apoptosis in leukemia cells via caspases cascade, accompanied by a loss of△Ψm in leukemia cells. However, it was not the same in solid tumor cells which did not experience apoptosis with the treatment of SMT A07. But it could induceM phase arrest through suppression of the activity of CyclinBl/Cdc2 complexes in solid tumor cells.