Identification Of The Molecular Target Of Acriflavine And Its Relevant Application In Cancer Therapy

Objective:Acriflavine is known as an antibacterial drug initially approved for the clinical treatment. Recently, increasing evidence has shown that acriflavine exhibits potential anti-tumor activity, and has been recognized as an attractive molecule suitable for cancer chemotherapeutics. However, its potential clinical application is greatly undermined by its uncertain molecular targets. Here, a probe-based pull-down approach was applied to comprehensively profile the potential targets of acriflavine. In addition, a target-based drug-in-combination formula that combines acriflavine with other anti-tumor agents was further developed and evaluated both in vitro and in vivo. This study aims to identify the functional target of acriflavine and provide novel insights and opportunities for the development of acriflavine-based antitumor chemotherapy.Methods:(1) Pull-down assay and HPLC-MS/MS were performed to analyze the agarose-bound proteins. (2) Western blotting assay was performed to detect the expression of protein. (3) The anti-proliferation activity of drugs on human cancer cells was determined using CCK-8 kit and SRB method. (4) Propidium iodide staining, Annexin V-PE/7-AAD staining and DAPI staining were performed to determine cell apoptosis. (5) Neutral comet assay and immunofluorescence analysis were used to detect the cellular DSBs level and DNA repair. (6) Nude mouse xenograft models of human cervical cancer cell line Hela or human colon cancer cell line HCT116 were introduced to investigate the in vivo activity of acriflavine and melphalan. (7) TUNEL staining was performed to detect in vivo apoptosis. (8) RNAi technique was used to silence the expression of p21. (9) The gene microarray was used to compare the expression of different genes.Results:Section 1. DNA-PKcs was identified as the direct target of acriflavine and its kinase activity was inhibited by acriflavine both in vitro and in cells. We established and tested a structure-specific pull-down approach based on acriflavine-linked agarose resin (ACF-NHS) to comprehensively profile the direct target of acriflavine. Considering the nuclear location of acriflavine, three nuclear proteins including DNA-PKcs were identified as the potential targets. The results of in silico homology models showed that acriflavine could be docked into the constructed kinase domain of DNA-PKcs, and further experiments clearly demonstrated that acriflavine could inhibit DNA-PKcs activity in vitro and block the autophosphorylation of DNA-PKcs caused by IR and heat stress.Section 2. The combination of acriflavine and melphalan displayed a synergistic cytotoxicity in cancer cells. Melphalan, an alkylating agent clinically used to treat multiple myeloma, ovarian cancer and AL amyloidosis, could induce the activation of DNA-PKcs. The inhibition of DNA-PKcs could enhance its effect on cancer cells. The combination of acriflavine and melphalan exhibited distinct synergistic effect on inhibition of proliferation and induction of caspase-dependent apoptosis. Compared to traditional simultaneous treatment, sequential combination of acriflavine followed by melphalan exerted stronger synergistic efficacy. This effect was accompanied by acriflavine-related dephosphorylation of DNA-PKcs, inhibition of NHEJ repair, increase of DSBs level and sequent induction of apoptosis. In line with the in vitro results, the combined administration of acriflavine and melphalan displayed significant anti-tumor responses in vivo.Section 3. The p53 status dictated the cell fate in response to the combination therapy. The GSEA analysis showed that the combination of acriflavine and melphalan significantly activated p53 signaling pathway. The deficiency of p53 function or expression would impair the synergistic efficacy both in vitro and in vivo. The combination upregulated the transcription of apoptosis-related genes regulated by p53, and increased the mRNA and protein expression of p21, which is involved in cell cycle regulation and hypophosphorylation of CDK2. The silencing of p21 led to decrease in anti-tumor efficacy.Conclusion:We identified acriflavine as a promising DNA-PK inhibitor with greater in vitro inhibitory effect than the existing DNA-PK inhibitor, NU7026. It inhibited DNA-PK-mediated NHEJ repair, increased melphalan-induced DSBs and induced apoptosis. The synergistic efficacy was dependent on the expression of intact p53.