Our studies in FMD have led to

Our studies in FMD have led to the discovery of a reductive damaging mechanism in DNA (Wang et al, 2009; Lu, 2010a; Nguyen et al., 2011). More recently, we further demonstrated the reductive damaging mechanism in human lung and skin normal cells, which causes serious reductive DNA damage and genetic mutations and might be related to the pathology of diseases, especially cancer (Lu et al., 2013). We observed contrast differences in effects of an antioxidant (EGCG) on human normal and abnormal (cancer) cells: It caused DNA damage, mutations and cell death more effectively in normal purchase pppa than cancer cells. Our results provided a compelling explanation for the confirmed observation in clinical trials that increases of some cancers such as lung and skin cancers are associated with antioxidants (The ATBC, 1994; Albanes et al., 1996; Omenn et al., 1996). DeNicola et al. (2011) also recently showed evidence in mice that several oncogenes actively induce transcription of Nrf2 — the transcription factor that mainly regulates physiological antioxidant pathways — that is required for tumor initiation. This Nrf2 antioxidant pathway may therefore lead to a more reduced intracellular environment that enables tumor initiation, though its link to a promotion effect of antioxidant supplementation on human tumorigenesis has been questioned (e.g., Perera and Bardeesy, 2011). Moreover, researchers have also reviewed that the resistance or incurability of (later-stage) metastatic cancers are related to higher-level endogenous antioxidants (e.g., glutathione) in a variety of types of tumor cells/tissues (Calvert et al., 1998; Estrela et al., 2006; Traverso et al., 2013; Watson, 2013). Our finding of a reductive damaging mechanism may lead to effective prevention and therapies. Our mechanistic studies in FMD, described above, have now led to the discovery of a previously unknown class of non-platinum-based compounds, which essentially act as cisplatin analogues. Some general features of these compounds are that they comprise an aromatic ring (rather than a platinum coordinating ion), coupled to two NH2 groups as the electron transfer promoter and one or more halogen atoms (Cl, Br or I), such as 4,5-dichloro/dibromo/diiodo-1,2-diaminobenzene (benzenediamine/phenylenediamine) and 4(3)-chloro/bromo/iodo-1,2-diaminobenzene (benzenediamine/phenylenediamine) (shortened as FMD-nX-DABs or B(NH2)2Xn with X=Cl, Br or I, and n=1, 2). Their structures are shown in Fig. S1 in Supplementary information. We have observed that such FMD compounds are highly effective in DET reaction with weakly-bound electrons (unpublished data; Wang et al.), which may be either intrinsically rich in tumor cells/tissues or exogenously produced by ionizing radiation of biological systems. The resultant radical [B(NH2)2Xn-1] can effectively lead to DNA damage and cell death. An advantage of these FMD agents over XdUs and cisplatin is that FMDs are effective in a DET reaction but are far less toxic due to the absence of the heavy metal (Pt). It has been shown that the cisplatin-induced nephrotoxicity is mainly due to the binding of Pt to proteins in kidneys of cisplatin-treated mice (Townsend and Hanigan, 2002; Zhang et al., 2006). It is the severe toxicity of Pt that led to the call to terminate the attempt to develop new Pt-based anticancer drugs (Reese, 1995). Thus, such non-Pt-based FMD compounds (FMD-nX-DABs) are highly desirable anticancer agents. This study presents in vitro and in vivo results of FMDs as translatable antitumor agents for natural targeted chemotherapy of cervical, breast, ovarian, and lung cancers with minimal toxicity.
Methods Details for cell lines and culture conditions, cell viability assays, DNA DSB assays, apoptosis assays, and cell cycle analysis have been published previously (Luo et al., 2012; Lu et al., 2013). Only brief descriptions and different details are given here. ME-180 and MDA-MB-231 cells were cultured with the ATCC-formulated McCoy\'s 5A medium supplemented with 10% FBS, and L-15 Medium (Leibovitz) with 10% FBS, respectively. All the cells were maintained at 37°C in a humidified atmosphere containing 5% CO2, except for MDA-MB-231 cells maintained at no CO2 (according to ATCC\'s instruction). The cell growth and survival rates with various treatments were measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay (Invitrogen). DNA double-strand breaks (DSBs) were measured by detection of the phosphorylated H2AX foci using the HCS DNA Damage Kit (Invitrogen). The images of cells were acquired with a Nikon Eclipse TS100 fluorescence microscope; quantitative analyses of activated γ-H2AX (DNA DBS yield) in the cells were performed using an Image J software. The CellEvent™ Caspase-3/7 Green Detection Kit (Invitrogen) was used for the detection of activated caspases and apoptotic cells using fluorescence microscope, following the vendor\'s protocol, as described previously, while DNA fragmentation and cell cycle analysis were measured by flow cytometry using a standard APO-BrdU™ TUNEL assay kit (Invitrogen) (Luo et al., 2012). For the latter, the data were analyzed using a FlowJo software for quantitative measurements of apoptotic cells. The DNA histograms were also used for cell cycle analysis. The fits to the DNA histograms of the samples were performed by the Watson model to achieve a lowest RMS (root mean squared) score.