Research On The Effect Of Overcoming Drug Resistance And Its Mechanism Of New Platinum (Ⅳ) Compounds Based On GSTs Inhibitors

Platinum(Ⅱ)drugs,represented by cisplatin,have become the first-line drugs for chemotherapy of various solid tumors.However,serious toxic side effects and drug resistance of platinum drugs are crucial factors for the failure of chemotherapy in the clinc.Platinum(Ⅳ)complexes have kinetic inertness and an octahedral modifiable structure,making them potential candidates for antitumor prodrugs to o vercome the defects of platinum(Ⅱ)drugs,and have attracted widespread attentions.Nitrobenzoxadiazole(NBD)derivatives are a class of compounds containing NBD scaffolds,which can strongly inhibit the activity of glutathione S-transferases(GSTs).Among them,NBDHEX has been widely studied in recent years as a potent GSTs inhib itor.In addition,the platinum(Ⅱ)complexes,namely DN603 and DN604,independently developed by our research group are two kinds of antitumor drugs with high efficiency and low toxicity.The N BDHEX fragments were conjugated with DN603 and DN604 through linking groups,and novel platinum(Ⅳ)complexes DN603-NBD and DN604-NBD were obtained.In vitro cytotoxic activity studies showed that DN603-NBD and DN604-NBD could effectively inhibit the proliferation of non-small cell lung cancer A549 cell line,especially cisplatin-resistant A549/c DDP cell line,and showed superior cytotoxicity to cisplatin.Further studies indicated that the uptake of both two complexes in A549/c DDP cell line was much higher than these of cisplatin.Cell apoptotic assays also revealed that compared with cisplatin,DN603-NBD and DN604-NBD could contribute to a higher rate of apoptosis.Moreover,western blot results confirmed that the two complexes significantly increased the ratio of Bax/Bcl-2,activated caspase 3 and cleaved the DNA repair enzyme PARP,thereby inducing a higher degree of apoptosis in a caspase 3-dependent manner.Since the introduction of NBDHEX,DN603-NBD and DN604-NBD blocked the cell cycles of A549 cell line and A549/c DDP cell line mainly in the S phase,which was the same as the block characteristics of cisplatin.As we know,apoptosis could be induced by the production of excessive ROS,which is also a way for platinum(Ⅱ)drugs to kill tumor cells.Therefore,through the detection of reactive oxygen species(ROS)levels,it was found that the treatments of DN603-NBD and DN604-NBD resulted in higher levels of ROS in both A549 cell line and A549/c DDP cell line.Quantitative determination of ROS fluorescence intensity by flow cytometry also demonstrated that the two complexes could cause the higher fluorescence intensity,indicating that they could induce apoptosis by producing excess ive ROS.Comet assay showed that compared with cisplatin,the DNA damage caused by the two novel platinum(Ⅳ)complexes was greater at the same concentration.Meanwhile,the results of the immunofluorescence assay declared that the green fluorescence intensity of DSBs marker γ-H2 AX was the most significant,indicating that the two complexes could cause strong DN A double-strand break damages.Western blot assay confirmed that the protein expression of GSTP could be at the high level in the cisplatin-resistant A549/c DDP cell line.Immunofluorescence experiments also revealed that two novel platinum(Ⅳ)complexes could target GSTP and inhibit its expression.The results of GSTP enzyme activity assay further indicated that DN603-NBD and DN604-NBD could enhance their inhibitory effects on GSTP and overcome the resistance of platinum drugs to a certain extent.To further explore the relationship between ROS,GSTP and platinum drug-induced resistance,cells were pretreated with NAC and SP600125.NAC is an active oxygen scavenger,and SP600125 is an inhibitor of JNK protein in the JNK signaling pathway closely related to GSTs.In A549 cell line and A549/c DDP cell line,the results of the cell viability assay showed that the number of living cells in DN603-NBD and DN604-NBD groups increased significantly.Western blot analysis of JNK signaling pathway-related proteins also revealed that two novel platinum(Ⅳ)complexes dramatically increased the phosphorylation level of JNK and its downstream target protein c-Jun.In contrast,when NAC and SP600125 were used to pretreat cancer cells respectively,JNK and c-Jun phosphorylation levels were remarkably reduced.These data indicated that DN603-NBD and DN604-NBD could induce the activation of JNK signaling pathway by generating excess ive ROS,reverse the resistance of platinum drugs,and thus induce tumor cel s death.In vivo studies,it was found that DN603-NBD and DN604-NBD could significantly inhibit tumor growth.Among them,the inhibitory effect of DN604-NBD on tumor volume was weaker than that of cisplatin-treated group,while the inhibitory effect of DN603-NBD on solid tumors was comparable to that of cisplatin.The experimental results showed that the tumor inhibition rate of cisplatin at the same dose is 59.83%,and the tumor inhibition rate of DN603-NBD could also reach 48.32%,which had a good inhibitory effect on tumor growth.In addition,compared with cisplatin,the two complexes had no effect on the body weight of nude mice,indicating that they had almost no toxic side effects on model animals in vivo.In general,DN603-NBD and DN604-NBD are two novel potential platinum(Ⅳ)antitumor prodrugs,which deserve further research.