Structural Feature Of Schiff Base

Published Date: 02 Nov 2017

The main structural feature of Schiff base is the azomethine group with a general formula RHC=N-R1, where R and R1 are alkyl, aryl, cyclo alkyl or heterocyclic groups which may be variously substituted. The presence of lone pair electrons in a sp2 hybridized orbital of nitrogen atom of the azomethine group is of considerable chemical and biological importance. Intramolecular hydrogen bonding between OH hydrogen and C=N nitrogen atoms of Schiff bases determines the properties of various molecular systems and plays a significant role in many biochemical mechanisms (Szady-Chelmieniecka, Grech et al. 2001).

In our aims to search for small molecular entities with conventional anticancer property and anti-angiogenesis activity for cancer treatment, we investigated Salicylidene-aniline and Salicylidene-pyridine Schiff base derivatives. The advantages of small molecule drugs are the ability to diffuse into cells and can act on targets inside the cell. Most Schiff bases derivatives up to date are metal complexes studying against cancer cell proliferation (Y. Xiao, C. Bi et al. 2008; Yan, Sun et al. 2010) and angiogenesis(Yan, Sun et al. 2010). However, to our knowledge no detail studies have been done on angiogenesis using Schiff base ligand without the metal complexes. Herein, we provide evidences that colon cancer cell proliferation and angiogenesis are potently inhibited by BPMP-S5 and PEBB-S6.

Cytotoxic and Apoptotic Properties of Salicylidene-aniline and Salicylidene-pyridine Schiff base Derivatives.

In this study, we evaluated anti-proliferative and antiangiogenic properties of Salicylidene-aniline and Salicylidene-pyridine Schiff base derivatives. These compounds were measured for cytotoxicity effects on four cancerous human cell lines (HCT 116, MCF-7, MDA-MB-231, and K562) and normal human cell lines (HUVECs and CCD-18Co), using colormetric method of MTT (Mosmann 1983).

While both BPMP-S5 and PEBB-S6 were discovered to specifically inhibit HCT 116 and HUVEC cells proliferation, the other Schiff bases, MPMP-S1 and BMMP-S2 were not toxic towards all the tested cell lines. The cytotoxicity activities of these compounds can be connected to the structure of each Schiff bases derivatives. BMMP-S2 showed better cytotoxicity than MPMP-S1 because the 4-bromoaniline moiety enhanced the lipophilicity properties of BMMP-S2 as the log P of 4-bromoaniline is higher than 2-aminopyridine of MPMP-S1. Among Salicylidene-aniline Schiff bases derivatives, BMMP-S2 is found to be less potent then BPMP-S5, due to the existence of methoxy moiety at the ortho position of BMMP-S2, thus reducing its lipophilicity. Whereas for PEBB-S6 and MPMP-S1 of Salicylidene-pyridine Schiff bases derivatives, the addition of bromine to salicylaldehyde core improved the lipophilicity of PEBB-S6, hence making it more potent in inhibiting HCT 116 and HUVEC cell proliferation. Further studies on BPMP-S5 and PEBB-S6 induced cell death in HCT116 cells revealed that the DNA content of HCT116-treated with both compounds declined after 48 hours of incubation which supports the detrimental effect of BPMP-S5 and PEBB-S6 on HCT116 cells.

The incorporation of halogen atoms into Schiff bases increased the lipophilic property and so less water soluble. Consequently, halogen atoms are used to improve the penetration of lipid membranes. Halogenation also enhances the blood-brain barrier ( BBB) permeability and this is a pre-requisite for the drugs that need to reach the CNS(Gentry, Egleton et al. 1999) . In this work, the introduction of bromine atoms to the Schiff bases derivatives also could lead to volumetric and conformational changes because its bulky groups that tend to occupy all the active site of molecular targets, including the deeper pockets (Hernandes, Cavalcanti et al. 2010). The incorporation of bromine atoms including other halogen atoms also increase the membrane permeability and therefore improve the oral absorption(Gerebtzoff, Li-Blatter et al. 2004).

In order to estimate the drug effects on each colony,

The number of cells within each colony depends on the number of cell doubling and can be used as an estimate of the effects, if any, of the drug on the cell doubling time. A clonogenic assay can thus discriminate between cytotoxic (cell kill) and cytostatic (decreased growth rate) effects. Since a cytotstatic effect may be lost upon removal of the drug, a cytotoxicity assay based on colony formation is also described since this allows continuos drug exposure. Both BPMP-S5 and PEBB-S6 showed to be cytotoxic agents as they inhibited the colony formation of HCT116 after 48 hours of treatment at different concentrations (Fig. 2A-2B).

Apoptosis plays important role in the development and homeostasis of multicellular organisms (Daniel and Abrahamson 2000). Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive structural pathology (Bursch, Kleine et al. 1990) . In order to determine whether BPMP-S5 and PEBB-S6 induced apoptosis in HCT 116 colon cancer cells, we have assessed the morphological changes and quantified DNA fragmentation. In control cell which is treated with 1 % DMSO (vehicle), the morphology of most HCT 116 cells was still intact and there were a lot of mitotic cells. Only a small percentage of apoptosis cells existed. Hoechst 33258 staining of treated HCT 116 cells with BPMP-S5 displayed chromatin condensation, DNA fragmentation, and apoptotic bodies. A time-dependent pattern can be seen for HCT 116 cells that were treated with 90 µM of BPMP-S5 for 6 hours to 48 hours (Fig. 4). For HCT 116 cells treated with 25 µM to 500 µM BPMP-S5 for 48 hours, the apoptotic index also increased dose-dependently until 250 µM. However, at the highest concentrations, 500 µM of BPMP-S5 treatment caused some of the HCT 116 cells morphology to be damaged. Hence, the reduction of apoptotic index at this concentration (Fig. 5).

Hoechst 33258 staining of treated HCT 116 cells with PEBB-S6 also displayed chromatin condensation, DNA fragmentation, and apoptotic bodies. A time-dependent pattern can be seen for HCT 116 cells that were treated with 70 µM of PEBB-S6 for 6 hours to 48 hours (Fig. 3A-3B). The apoptotic index increased as the time of treatment increased. For HCT 116 cells treated with 25 µM -500 µM PEBB-S6 for 48 hours, the apoptotic index also increased as the concentration increased until treatment with 250 µM PEBB-S6. However, at the highest concentrations, 500 µM of PEBB-S6 treatment caused some of the HCT 116 cells morphology to be damaged. Hence, the reduction of apoptotic index at this concentration (Fig. 3C-3D).

Diphenylamine was used to assess DNA fragmentation in BPMP-S5 and PEBB-S6 treated HCT 116 cells. Diphenylamine reagent reacts with DNA to form a blue product and the amount of blue colour is linearly related to the amount of DNA present. HCT 116 cells were treated with 50, 100, and 250 μM of BPMP-S5 and PEBB-S6 for 48 hours produced a dose- dependent manner of DNA fragmentation (Fig. 6).

ROS production is often observed in apoptotic processes triggered by various stimuli (Dröge 2002). ROS also can trigger a transient increase of mitochondrial ROS production through the activation of the mitochondrial permeability transition pore by oxidation of thiol groups in the adenine nucleotide translocase (Halestrap and Brenner 2003). High ROS concentrations induce apoptotic cell death in various cell lines, suggesting that ROS contributes to cell death whenever they are generated in apoptotic process (Manda, Nechifor et al. 2009). The level of ROS concentrations in HCT 116 cells treated with BPMP-S5 is significantly increased in a time-dependent manner. For HCT 116 cells treated with 90 µM of BPMP-S5, ROS production can be seen started at 6 hours of treatment and time-dependently increased to 24 hours of treatment. However as the treatment reached 48 hours, the ROS level is significantly decreased. This is because some cells already undergone apoptosis process (Fig. 7A). HCT 116 cells that were treated with 25 µM to 500 µM of BPMP-S5 for 48 hours showed that a decrease in ROS production as the concentration increased as the morphology of the cells changed from normal to apoptotic cells (Fig. 7B).

The level of ROS concentrations in HCT 116 cells treated with PEBB-S6 is also significantly increased in a time-dependent manner. ROS production can be seen started at 6 hours of treatment and time-dependently increased to 24 hours of treatment for HCT 116 cells treated with 70 µM of PEBB-S6. However as the treatment reach 48 hours, the ROS level of was significantly decreased (Fig. 5A). This is because some cells already undergone apoptosis process. HCT 116 cells that were being treated with 25 µM -500 µM of PEBB-S6 for 48 hours showed that the ROS production decreased as the concentration increased as the morphology of the cells changed from normal to apoptotic cells (Fig. 5B).

ROS also triggers a transient increase of mitochondrial ROS production through the activation of the mitochondrial permeability transition pore which leads in turn to collapse of mitochondrial membrane potential and simultaneous increased ROS generation through the mitochondrial electron transport chain (Manda, Nechifor et al. 2009). The collapse of MMP is also considered as a feature in the early stages of apoptosis. Thus, we investigated the effect of BPMP-S5 and PEBB-S6 on mitochondria membrane potential using Rhodamine 123, a cationic fluorescent dye which distributes according to the negative membrane potential across the mitochondrial inner membrane(Chazotte 2011). Loss of potential will result in the loss of the dye and therefore the green fluorescence intensity. As shown in this study, both BPMP-S5 and PEBB-S6 disrupted mitochondrial membrane potential in a time- and dose-dependent manner (Fig. 8 and 9). Thus, the cell structural changes correlate with a decrease in MMP.

Caspases have proteolytic activity and able to cleave proteins at aspartic acid residues. Once activated, caspases cleave, and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade (Shi 2004). The caspase family can be grouped into two classes, depending on their point of entry into the apoptotic cascade. There are the initiator (or upstream) caspases (such as caspase-2, -8, -9 and -10) and the effector (or downstream) caspases (like caspase-3, -6, -7) (Elmore 2007). In this study, the results show that BPMP-S5 and PEBB-S6 caused induction of caspase-9 which consequently triggered the caspase-3/7 leading to HCT 116 cell death. Caspase-3 is one of the important "executioner" caspase enzymes, triggering the cleavage of numerous proteins that lead to the ordered breakdown of the cell. However, caspase-8 is found to be inactivated in HCT 116 cells treated with BPMP-S5 as well as PEBB-S6. Therefore, both BPMP-S5 and PEBB-S6 have shown to induce apoptosis through the intrinsic pathway.

The Anti-angiogenic Properties of Salicylidene-aniline and Salicylidene-pyridine Schiff base derivatives

Since Folkman introduced his concept, angiogenesis has been recognized to play a critical role in the pathogenesis of many diseases. Angiogenesis consists of a series of cascades involving activation of the endothelial cells, degradation of basement membrane, endothelial sprouting from the parent vessel, invasion of the extracellular matrix, endothelial proliferation, vessel elongation, branching, anastomosis, increases in vessel diameter, basement membrane formation, pericyte acquisition, and remodelling (West, Burbridge et al. 2009). Tumor-induced angiogenesis is important in supporting tumor growth and and progression, not only by providing the necessary blood supply but also by allowing metastatic cells to enter circulation. Numerous approaches to tumor angiogenesis have been utilized for the treatment of human cancer beyond traditional chemotherapy. Besides, inhibition of angiogenesis provides additional benefits, including broad applicability to many tumor types, independence of tumor cell resistance and lower dosage of chemotherapy.

The use of agents that that specifically inhibit the growth of the endothelial cells is one of successful approach to modulate angiogenesis. One of the first compounds identified to exhibit inhibitory effects on cell growth with specificity for endothelial cells was O-chloroacetylcarbamoyl fumagillol or AGM-1470/TNP-470, an analog of the fungus-derived antibiotic fumagillin (Ingber, Fujita et al. 1990; Kusaka, Sudo et al. 1991) mechanism of action of this compound was found to be prevention of endothelial cells to enter G1 phase of the cell cycle, resulting in a decrease in proliferation (Castronovo and Belotti 1996). As both BPMP-S5 and PEBB-S6 selectively inhibited HUVEC cell lines proliferation, we deepened out studies to determine the antiangiogenic properties of both compounds.

Nicosia and Ottineti introduced the rat aortic ring assay in 1990 to assess angiogenesis in an ex vivo model. The rat aortic ring assay bridged the gap between in vitro and in vivo systems (Nicosia and Ottinetti 1990). In the present study, all four Schiff base compounds and Suramin as positive control were first screened for their anti-angiogenic properties using the rat aortic ring assay (Brown, Maynes et al. 1996). The results showed that both BPMP-S5 and PEBB-S6 Schiff bases potently inhibited the outgrowth of new blood vessels in a dose-dependent manner. By matching the results of anti-proliferative effect on HUVECs and rat aortic ring assay together, both BPMP-S5 and PEBB-S6 demonstrate potential anti-angiogenic effect which can be elaborated through cell migration, formation of capillary network, and VEGF expression in the endothelial cells.

Endothelial cell migration is a motile process which is regulated by chemotaxis, the directional migration toward a gradient of soluble chemoattractants; haptotaxis, the directional migration toward a gradient of immobilized ligands; and mechanotaxis, the directional migration generated by mechanical forces (Li, Huang et al. 2005). This process also involves degradation of the extracellular matrix to enable progression of the migrating cells. It requires the activation of several signaling pathways that converge on cytoskeletal remodeling. Then, it follows a series of events in which the endothelial cells extend, contract, and throw their rear toward the front and progress forward (Lamalice, Le Boeuf et al. 2007). In this study, the findings illustrated effective reduction in the migratory capacity of BPMP-S5 and PEBB-S6 treated HUVEC cells are compared to control in which cells treated dose-dependently with PEBB-S6 exhibited greater reduction in inhibiting cell migration.

The in vitro tube formation on Matrigel demonstrated the steps required during angiogenesis including proliferation, migration, and differentiation leading to the formation of capillaries in vivo (Madri, Pratt et al. 1988). The inhibition of formation of three-dimensional branching and tube-like structures of endothelial cells in Matrigel basement membrane elucidates a useful sign of antiangiogenic potency of the agent on microvascular growth(Murray, Martin et al. 2009). Our results showed a concentration-dependent manner in inhibiting the formation of endothelial tubes.

Vascular endothelial growth factor (VEGF) is the key signal used by oxygen-hungry cells to promote blood vessels growth. It binds to tyrosine kinase receptors that cause endothelial cell proliferation, migration, and new vessel formation (Hoeben, Landuyt et al. 2004). Evidence from preclinical and clinical studies indicates VEGF is the predominant angiogenic factor in human colon cancer and is associated with formation of metastases and poor prognosis (Ellis, Takahashi et al. 2000). VEGF expression is upregulated in numerous solid malignancies(Berse, Brown et al. 1992; Ferrara, Gerber et al. 1999), including primary and metastatic carcinomas of the gastrointestinal tract (Brown, Berse et al. 1993). Several studies have implicated VEGF in human colon cancer angiogenesis (Takahashi, Kitadai et al. 1995). VEGF function can also be inhibited by small molecules that block or prevent activation of VEGF-A receptor tyrosine kinases for example; SU5416 and SU6668 inhibited metastases, microvessel formation, and cell proliferation while increasing tumor cell and endothelial cell apoptosis. These results showed that targeting the VEGF receptor/ligand system is a rational approach to inhibiting tumor growth and prolonging survival (Ishigami, Arii et al. 1998).

As application of Schiff base as VEGF inhibitor is a new attempt in drug design , recent studies have shown that Schiff base derivatives of nimesulide, a well-known cyclooxygenase-2 (COX-2) inhibitor and its copper conjugate exhibited modulating effects on VEGF in BxPC3 pancreatic cancer cells where the inhibition of VEGF levels by copper conjugate is less than its ligand (Ambike, Adsule et al. 2007). In addition, docking studies of 3-(naphthalen-1-ylimino) indolin-2-one and 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one, an isatin Schiff base derivatives exhibited higher docking binding energies with VEGF receptor (Azizian, Mohammadi et al. 2012). In the present study, both BPMP-S5 and PEBB-S6 demonstrated dose-dependently reduced the VEGF expression in HUVEC cells.

The chorioallantoic membrane system (CAM) is a simple inexpensive method that can be considered as an interface between in vitro and in vivo models. The chick embryo model has long been used for the investigation of angiogenesis and oncogenesis (Brooks, Silletti et al. 1998; Zijlstra, Mellor et al. 2002). This model provides a naturally immunodeficient host that accepts transplantation from various tissues and species and extra-embryonic membranes that are connected to the embryo through a continuous circulatory system and that are readily accessible for experimental manipulation and observations (Isachenko, Mallmann et al. 2012). In this study, treatments with BPMP-S5 and PEBB-S6 onto the CAM have caused changes in the vascularisation pattern, which can be regarded as angiogenic response.

Studies on the interaction of Salicylidene-aniline and Salicylidene-pyridine Schiff base derivatives with DNA and their relationship with antiangiogenic activity.

DNA binding studies involving Schiff bases complexes have been studied extensively over the past years. Most of these Schiff bases can cause DNA strand breaks due to the presence of metals such as copper, which consequently trigger apoptosis in various cancer cell lines (Filomeni, Cerchiaro et al. 2007; Qiao, Ma et al. 2011). However, the study of metal-free Schiff bases interaction with the nucleic acid is still lacking and research on their anti-tumour potential is not well established.

Molecules that target the DNA minor groove a relatively sequence specific and they can be excellent carrier structure for chemotherapeutic compounds which can help to minimize side effects (Khan, Shah et al. 2012). Recently, our group have discovered two isomeric diaminobenzene Schiff bases derivatives, N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene that possessed strong binding affinity to the AT rich region of DNA minor groove with mild cytotoxicity towards colon cancer cells but neither were found toxic towards human breast or liver cancer cell lines (Helal, Al-Mudaris et al. 2012).

In our present study, all four Schiff bases compounds, MPMP-S1, BMMP-S2, BPMP-S5, and PEBB-S6 bind to the minor groove of DNA at the GC rich region. Even though from the docking energy of salicylidene-pyridine Schiff bases, the binding affinity of MPMP-S1 is better than PEBB-S6, it possesses more potent activity in inhibiting HCT 116 and HUVECs cells proliferation as well as the outgrowth of blood vessels in the rat aortic ring assay. This probably because PEBB-S6 is more lipophilic than MPMP-S1 that helps it penetrates into the cell membranes and causing apoptosis in HCT 116 cells and suppressing the outgrowth of blood vessels in the rat aortic ring. Same condition also applied between salicylidene-aniline Schiff bases, where BPMP-S5 having lesser binding affinity than BMMP-S2.

Most proteins make sequence-specific interactions with DNA in the major groove due to a larger number of functional groups of the DNA bases are accessible at the region. However, some proteins and many small molecules interact with DNA in the minor groove. The DNA-protein interactions that are essential to cell processes occur when proteins bind at the DNA grooves through hydrogen bonds and nonspecific binding (Cai, Gray et al. 2009).

Transcriptional regulation is exerted by the combinatorial action of proteins binding to distinct promoter and enhancer elements. Usually a limited number of cis-acting DNA elements are recognized not only by a single transcription factor but by a set of different proteins which are often structurally related. The most important and widely distributed promoter elements are G-rich elements such as the GC-box (GGGGCGGGG) and the related GT/CACCC-box (GGTGTGGGG). These elements are required for the appropriate expression of many ubiquitous, tissue-specific and viral genes. In addition, they occur frequently in the regulatory region of genes which are under a specific mode of control such as hormonal activation, cell cycle regulation, and developmental patterning (Suske 1999). One of these transcription factor is Sp1 that has been considered as a ubiquitous transcription factor, increasing evidence suggests that it plays a major role in regulating expression of cell differentiation, cell cycle, and apoptosis-related genes affecting cellular growth (Black, Black et al. 2001)

Previous studies have clearly shown that GC-selective DNA binding drugs can block the interactions between Sp1 and DNA. The binding site of transcription factor Sp1 ((5’-GGG GGC GGG GGC-3’) is a GC-rich element which is the target of the minor groove binder such as mithramycin and chromomycin that bind selectively to GC-rich DNA sequences is capable to block preferentially binding Sp1 transcription factors to GC-rich elements in gene promoters (Blume, Snyder et al. 1991; Previdi, Malek et al. 2010). Therefore, inhibit gene transcription (Snyder, Ray et al. 1991; Albertini, Jain et al. 2006; Kwak, Park et al. 2006), which in turn alter the regulation of cell proliferation and differentiation (Remsing, Bahadori et al. 2003; Koutsodontis and Kardassis 2004; Albertini, Jain et al. 2006; Kwak, Park et al. 2006). Thus, the abilities of BPMP-S5 and PEBB-S6 to inhibit angiogenesis can be associated with their competency to bind to the GC-rich DNA regions of DNA minor groove, hence halting the binding of transcription factor Sp1 to its gene promoter, hence suppressing the VEGF production.