Antimultidrug-resistant Effect And Mechanism Of A Serial Of β-O-demethyl-epipodophyllotoxin Analogues

Aims:To evaluate the anticancer effect and anti-multidrug resistant effect of a series of novel etoposide 4-bata-amino podophyllotoxin analogues,in vivo and in vitro,and explore the anticancer mechanisms with the chosen compound.Methods:The structure of TopoIIA bound to G-segment DNA(PDB ID:2RGR)28 was chosen for the molecular docking of TOPO-IIA-DNA Complex and Compounds Vp16 and 5a-5n;MTT method was used in proliferation assay to evaluate the inhibition effect of 5a-5n on human cancer cells(K562,Ha549,U251),IC₅₀ values were calculated; Topoâ…¡-mediated cleavage- religation complex stabilization assay was used to evaluate Topoâ…¡activity;The MDR1 expression and p-gp expression leaver were analyzed by Real-Time RT-PCR and westrn blot;MTT method was used in the anti-multidrug resistant activity evaluate of 5k-5n on HL60,HL60R,KB and KBR cells;in vivo activity of 5k was assayed through mice beating murine carcinoma S180 model,the weight of tumor were measured and the inhibitory rate were calculated;The in vivo antiproliferation and antimultidrug-resistant activity of 5k was evaluated using human tumor models xenografted KB and KB/VCR in nude mice;Activity of P-gp ATPase in response to VP-16 and 5k was measured by Pgp-Glo assay system;Propidium Iodide (PI) Staining for Flow Cytometry was used to evaluate cell cycle distribution; Immunofluorescence analyse was used in the detection of Cyclin B1 distribution; Annexin V-FITC/Propidium iodide(PI) apoptosis detection kit was used to analyse apoptosis;All the proteins expression and activation were measured by western blotting; Carboxy-DCFDA stain and flow cytometry were used to test the content of ROS;JC1 stain and flow cytometry were used to test the change of mitochondrial membrane potential(△Ψm);LC-MS/MS was used in the in vivo pharmacokinetic studies in xenograft nu/nu Mice Model.Results: 1.5k-5n showed highly anticancer proliferation on K562,A549 and U251 cells,with the IC50 values(0.3-14.5μM),all far below?? the responding values of Vp-16 (5.9-84.9μM);2.5k-5n inhibited Topâ…¡by stabilizing the Topoâ…¡-DNA cleavage-religation complex and showed a higher activity than VP-16;3.The cytotoxicity of 5k-5n and Vp-16 were examined in two drug-sensitive cell lines and their MDR-positive sublines(HL60/HL60R and KB/KBR),VP-16 showed much higher IC₅₀(158.8μM) against KB/VCR cells than against KB(14.0μM).In contrast, all two cells exhibited dose-dependent sensitivity to 5k-5n with the IC₅₀＜3μM in KB and IC₅₀＜8μM in KB/VCR;4.To characterize the resistant cell lines(HL60R and KBR),we used Real-Time RT-PCR and western blotting to identify mRNAs and p-gp that were differentially expressed between the parental and daughter cell lines;5.Pgp-Glo Assay system was used to detect whether 5k was the substrate for human recombination P-gp in vitro,the results came out that 5k might not the substrate of human recombination P-gp;6.S180 sarcoma beating mice were used to evaluate the effect of 5k on tumor growth in mice.5k produced dose dependent effect in mice S-180 sarcoma models;7.The in vivo antiproliferation and antimultidrug-resistant activity assay showed that both 5k(2.5 mg/kg) and VP-16(5 mg/kg) exhibited moderate-to-good therapeutic activity on KB xenograft model,with the T/C%were 53%and 59%,while in drug-resistant KB/VCR xenograft model,5k(2.5 mg/kg) showed significantly therapeutic activity on KB/VCR xenograft model with a T/C%value of 45%.However, as a MDR tumor model,KB/VCR xenograft appeared resistant to VP-16,with a T/C% value of 72%.5k(2.5 mg/kg) showed 2.4-fold higher growth inhibition rate against multidrug-resistant cancer KB/VCR than VP-16(5 mg/kg);8.γ-H2AX was upregulated in KB cells incubated with 20μM 5k for 2h,which was significantly higher than levels in that treated with VP-16;9.5k treatment induced a strong G₂/M phase arrest in a dose and time dependent manner.Importantly,5k showed better active than VP-16.The G₂/M distribution rate reached 91.7±5.1%at 24 h after treatment with 5k(80.0 nM) and it was 26.1%,46.1% and 80.1%at 24h after treatment with 5k;10.5k(80 nM) treatment induced the upregulation and accumulation of Cyclin B1 at cytoplasm which indicated that 5k may induce KB cells arrest in G₂ phase;11.Western blotting results showed Cyclin B1 levels increased significantly in a dose dependent manner upon 5k treatment,Cdc2 protein levels were not changed much while phospho-cdc2(Thr161) levels were decreased by the treatment of 5k.Meanwhile, 5k was found to induce the phosphorylation of Cdc25C(Ser218),Chk1(Ser317) and Chk2(Thr68),but Chk1 was observed decreased slightly while Chk 2 was not changed after the treatment.Further more,exposure of cells to 20.0-80.0 nM 5k for 24 h enhanced the lever of P53 and phosphorylation of P53 on Ser20 slightly,and the downstream protein of P53,P21 were not upregulated obviously after the treatment;12.Preincubation with 2 mM caffeine for 30 min markedly abrogated 5k-induced G₂ arrest,indicating the ATM/ATR axis was involved in 5k-induced DNA damage response.Consistent with cell cycle distribution,caffeine pre-treatment of cells reversed the 5k-induced decrease of the Thr161 phosphorylation of Cdc2,increase the Ser317 phosphorylation of Chk1 and Thr68 phosphorylation of Chk2,blocked the accumulation of Cyclin B1;13.5k triggers apoptosis at high doses.The percentages of apoptosis came up to 34.56%,48.00%,59.80%at 1.25μM,2.50μM,5.00μM 5k treatments in KB cells respectively much higher than 19.25%at 5μM VP-16 treatment.14.5k obviously decreases the protein levels of procaspase-3,procaspase-9 and XIAP, and induces the cleavage of PARP,caspase-3 and caspase-9 in a dose dependent manner. Whereas,it was not that obviously apoptosis in VP-16(5μM) treated KB cells. 15.Compared to the corresponding control,5k caused an obvious decrease of△Ψm in KB cells,71.6%cells presented low△Ψm at 48 h incubation while 15.5%in control group;16.Cells were exposed to 5.0μM 5k for 1-5 h prior to staining with carboxy-DCFDA and flow cytometric analysis.A peak of mean green fluorescence(3.5-fold over control) was detected at 3-h treatment with 5k.Afterward,ROS production showed a decreased trend at 5-6 h after the addition of 5k.However,no protective effect was observed with NAC at the percentage of apoptotic cells indicating that pre-treatment with NAC failed to abrogate apoptosis of 5k in KB cells;17.Exposure to 5k(48 h;1.25-5.00μM) resulted in slight decrease of Bcl-2 and no obvious increase of Bax,with a slight increase in Bax/Bcl-2 ratio,we then examined the protein expression of Bax from the mitochondrial fraction.The data suggested that there was an increased amount of Bax in mitochondrial fraction;18.After a single 2.5 mg/kg i.v.dose of 5k,quantification analysis was measured in KB xenografted nude mice plasma by LC-MS/MS.The result indicated that serum concentration of 5k reached 1.67±0.19μM after 10min treatment but declined lower than 0.034μM after 24 h.Conclusion:5k exhibits high anticancer and anti-multidrug resistant effect both in vivo and in vitro.Targeting Topoâ…¡,5k could drive high lever of DNA damage.There were two distinct cellular responses to DNA damage with the treatment of 5k,the induction of a cell cycle distribution at the G2/M phase at low doses and the activation of apoptosis at high doses.5k-induced G₂ phase arrest caused through DNA damage were sensed by ATM and/or ATR,while 5k-induced apoptosis signaling was demonstrated to mediate through mitochondrial death pathways.