The Mechanism Of The Significantly Enhanced Genotoxic Chemotherapeutics For Incorporation Of HDAC Inhibitory Moiety And DNA Damage Agents

BackgroundCancer is one of the major threats to human health and disease, the therapy forcancer included surgery, chemotherapy, radiation, biotherapy and so on. Chemotherapyincluded a number a families defined by both their chemical structure and mechanism ofaction. Genotoxic drugs represent an important branch of chemotherapy, which kill cancercells by attacking cellular DNA. DNA damage is a crucial threat to cells’ genome integrityand regular metabolism. Among all kinds of damage, DNA double-strand break (DSB) isone of the most threatening damages. DSBs resulting from such attacks are extremely toxic,and one irreparable DSB is sufficient to induce cell death. Despite such potent lethality,cancer cells have developed multiple mechanisms responding to DNA damage. DNAdamage caused by anticancer drugs can be mitigated by cellular DNA repair machinery,thus enabling some cancer cells to survive and ultimately cause treatment failure. Asignificant challenge for treating cancer is to enhance the efficacy of existing DNA damagedrugs. Epigenetics refers to the regulation of gene expression via posttranslationalmodification of protein complexes associated with DNA, without alterations in the DNAsequence. The post-translational modifications of greatest interest in this field areacetylation and deacetylation. These processes are regulated by two groups of enzymeswith opposite activities: histone acetyltransferases (HAT) and histone deacetylases(HDAC). The HDACs involve in DNA repair pathway and regulate genes’ expression.Epigenetic regulation is critical for normal growth and development, and alterations mayresult in a variety of pathological processes including cancer.Considering the drug resistence of DNA damage agents and the the important role ofHDAC inhibition in cancer development. This paper studied the effect of combing HDACinhibition with DNA damage agent in cancer treatment, and further investigated the theunderlying mechanism, the whole paper was divided into three parts:Part1: The underlying effect of incorporation of HDAC inhibitorymoiety and DNA damage agents in cancer cellsObjective: Measure the cell proliferation of Bendamustine and the novel synthesizedNL101by MTT assay and investigate the underlying mechanism.Methods: The KM3and8226cells were treated with NL101and Bendamustine respectively, then measure cell proliferation by MTT assay and the apotosis-relatedγH2AX by Western-Blot. Furthermore, the histone3related acetylated proteins weremeasured in the cells after NL101treated by Western-Blot.Resluts: Compared with bendamustine, NL101significantly enhanced anticancerpotency, and further found that the NL101had similar role with SAHA in HDACinhibition, when cells were treated with NL101, acetylated histone H3was significantaccumulated.Conclusion: NL101has similar cytotoxicity with Bendamustine and also inhibitsHDAC function. It is a novel DNA/HADC dual-targeting drug.Part2: The mRNA expression level of the selected genes in DNArepair pathways after HDAC inhibitor exposure in Hela cellsObjective: Using real-time PCR to measured the mRNA expression of the selectedgenes in DNA repair pathway which repair the DNA damage induced by bendamustine,and further confirm whether these data are consistent with previous reports.Methods: The Hela cells were treated with SAHA at some concentration, after6-8hours, the RNAs were extracted and reversed transcription to cDNAs. Constructed thereal-time PCR oligos of the selected genes. Using real-time PCR to measured the mRNAof the selected genes, the DMSO solvent serves as a negative control.Results: A total of60genes were included, after exposure to SAHA, the mRNAexpressions of CBP、TIP60、MORF、EP300、MSL1and TYMS were downregulateddramatically compared with the control, consistent with previous reports, the data indicatedthat the HDAC inhibitor can influence genes’ mRNA expressions.Conclusion: The HDAC inhibitor influenced genes’ mRNA expressions whichparticipate in DNA damage repair, this suggested that the HDAC inhibitor could suppressDNA repair gene expression and do some roles in drug sensitivity.Part3: Sensitivity effect on knockdown of selected genes whichregulated by SAHA in mouse B lymphoma cell line(Eμ-Mycp19Arf/) on the DNAdamage agents for anti-cancer therapyObjective: Using retro-virus vector as a tool to knocked down the selected genes inEμ-Myc p19Arf/cells, and to investigate the sensitivity effect on DNA damage agents (bendamustine and cisplatin) in cells with selected genes knocked down.Methods: Eμ-Myc p19Arf/cells were stably infected with retroviruses coexpressing6selected genes shRNA respectively (CBP、TIP60、MORF、EP300、MSL1、TYMS) andgreen fluorescent protein (GFP). The stable infected cells were treated with bendamustineand cisplatin. We used GFP-based competition assay by flow cytometry to test sensitivityeffect on DNA damage agents (bendamustine and cisplatin) in selected genes knockeddown mouse B cells, The change in GFP percent can be used to calculate the relative“resistance index”(RI). Cells infected with MLP vector serve as a negative control.Results: Infected of the6selected gene-shRNAs could effectively supressed themRNA expression in Eμ-Myc p19Arf/cells. The GFP percentage of EP300and TYMSreduced rapidly in following days. Knockdown of CBP、TIP60、MORF and MSL1leadEμ-Myc p19Arf/cells more sensitivity to bendamustine and cisplatin than normal cells.Conclusion: The rapidly reduced GFP percentage indicated that EP300and TYMSare essential for cell survival, the sensitivities of bendamustine and cisplatin weresignificant increased after suppression of CBP、TIP60、MORF and MSL1in Eμ-Mycp19Arf/cells.Based on the fact that NL101significantly enhanced anticancer potency. Through afunctional genetic approach, we traced NL101enhanced potency to its newly gainedability to inhibit HDACs. Using this novel DNA damage and HDAC inhibition dualtargeting drug as a tool, we further explored HDAC inhibitor role in DNA repair, histoneacetylase activities are essential for the expression of several DNA repair genes. Thesefindings argue for incorporating HDAC inhibitory moiety into genotoxic drugs, so as toovercome the repair capacity of cancer cells, suggesting that it is pharmacologicallyadvantageous to apply such an approach to enhance traditional genotoxic drugs. Systematicdevelopment of simliar DNA damage and HDAC inhibition dual-targeting drugs mayrepresent a novel opportunity for improving cancer therapy.