1.1. Colorectal cancer
Cancers are leading cause of mortality in the world. The statistical data demonstrated that the malignancy has been situated the first place of the causes of death in Taiwan [1]. Colorectal cancer is the second leading cause of cancer-related death in the Western world and its incidence is increasing in Asian countries [2]. At least 15% of cases are estimated to have a hereditary background, like familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal carcinoma (HNPCC) [3]. The genetic alterations of adenomatous polyposis coli (APC) were found in the majority of sporadic colorectal neoplasms [4, 5]. Hereditary nonpolyposis colorectal carcinoma also called “Lynch syndrome” that occured at an earlier age than in the general population [6]. Moreover, colon cancer has been found by germline mutations in DNA mismatch repair (MMR) genes, including MLH1, MSH2, and MSH6 [6]. Moreover, high consumption of vegetables and fruits and the low sugar containing foods are likely to reduce risk of colon cancer. Besides, alcohol is another factor to increase risk [7].
1.2. Derivatives of benzimidazoles
The benzimidazole ring system is an important pharmacophore in medicinal
biologically active compounds and exhibit antiviral [8], antihypertension [9], against parasite properties [10] and anticancer activity [11]. The antitumor activity of the benzimidazole derivatives based on interference with the formation of microtubules [12]
and as DNA topoisomerase I inhibitor [13]. For example, FB642 (Methyl-2-benzimidazole carbamate, carbendazim) is a systemic fungicide with antitumor activity both in vivo and in vitro [14-16]. It has been shown that FB642 can induce G2/M arrest and apoptosis [14, 15]. Moreover, FB642 has undergone phase I clinical trials and is under further clinical development for treatment of cancer [16].
ABT-888 (2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide), currently in phase 2 trials [17, 18], is an orally bioavailable poly(ADP-Ribose) polymerase inhibitor with good penetration into the brain that potentiates DNA-damaging agents in preclinical tumor models [12].
1.3. Development of cancer therapy
The traditional therapeutic strategies involve radiotherapy, chemotherapy, immunotherapy, hormonal agent and anti-angiogenesis. There is no effective treatment for late stage and metastatic cancers of colorectal, prostate, pancreatic, breast, glioblastoma and melanoma cancers. Most of the currently available anti-cancer therapeutic strategies rely on the eradication of tumor cells [19]. Novel treatment
modalities are needed for these late stage patients because cytotoxic chemotherapy offers only palliation, usually accompanied with systemic toxicities and poor quality of life [20].
Novel technologies such as genomics and proteomics have increased the number of human genes known to be differentially expressed in normal and malignant tissues [21].
Advances in the understanding of cancer biology and specifically of cell signaling pathways have led to the identification of several potential molecular targets and to development of new agents directed against these targets. Targeted therapies, in the form of monoclonal antibodies and small molecule tyrosine kinase inhibitors have significantly altered the management of many solid tumors and hematologic malignancies [22].
1.4. Apoptosis
Apoptosis plays an important role in the regulation of cell number during development and tissue homeostasis [23]. The term was defined the morphologic features including cytoplasmic blebbing, chromatin condensation, cell shrinkage, nuclear fragmentation and cell rounding (loss of adhesion) [24]. The abnormal regulation of apoptosis resulting in increased or decreased activity is associated with a variety of clinical disorders including cancer, autoimmunity, neurodegenerative diseases,
hematopoietic disorders and infertility [25-27].
Apoptosis can be initiated through extrinsic or death receptor-link and intrinsic or mitochondria-dependent pathways [28]. The extrinsic pathway requires the binding of a ligand to death receptor on cell surface, such as TNF-α, Fas-ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL), all of which can act as extracellular activators of apoptosis upon binding to their respective receptors [29, 30]. The intrinsic pathway is activated in response to intra-cellular stress, such as DNA damage, hypoxia, growth factor deprivation [19] and mediated by the mitochondrial release of cytochrome c [30]. Cytosolic cytochrome c induces the formation of the multisubunit apoptosome composed of apoptotic protease activating factor-1 (Apaf-1), procaspase 9 and either ATP or dATP [31, 32]. Next, caspase 3 activation is detected following the formation of the apoptosome [33]. All caspases possess an active-site cysteine and selectively cleave substrates after aspartic acid residues [33]. Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair, DNA stability, and transcriptional regulation [34], was the first cellular protein to be identified as being specifically cleaved to the signature 89 kDa and 24 kDa fragment during apoptosis [35, 36]. PARP-1 is one of the prime target proteins for caspase 3 [37].
Intracellular reactive oxygen species (ROS) is considered to be a death signal in apoptosis [38, 39]. ROS induces disruption of the mitochondrial membrane potential
(MMP) and release of cytochrome c from mitochondria into the cytosol, where cytochrome c triggers caspase 9 activation and initiates caspases cascade which terminates cell to apoptosis [40, 41].
1.5. Securin and apoptosis
Securin, also known as the pituitary tumor-transforming gene-1 (PTTG1) was isolated from rat pituitary tumor cells in 1997 and identified as a pituitary-derived transforming gene [42]. It consists of a homologous family of proteins expressed in different species that includes Cut2 in fission yeast [43], Pds1 in budding yeast [44], Pim1 in Drosophila [45] and securin in human. PTTG1 is a multifunctional gene located at chromosome 5q33 and encoding 202 amino acid protein [46]. Securin participates in the maintenance of chromosome stability, cell-cycle progression, appropriate cell division [47], DNA repair [48], transactivation activity and apoptosis [49]. PTTG1 overexpression may cause both p53-dependent and p53-independent apoptosis [50]. In our previous study, expression of securin promotes cell apoptosis after radiation in colorectal cancer cell [51]. In contract, inhibition of securin expression increases apoptosis and chromosome instability following arsenite or cytochalasin B [52, 53]. Furthermore, securin depletion sensitizes human colon cancer cells to fisetin-induced apoptosis [54].
1.6. Securin and cell cycle
During most of the cell cycle, securin plays an important role in sister chromatid separation during anaphase [55]. Sister chromatid separation involves the proteolytic cleavage of cohesion proteins, a process that is mediated by separase, a cysteine protease [56]. In normal condition, securin prevents abnormal sister chromatid segregation by binding to the C-terminal domain of separase and inhibits its activity, maintains genomic stability [43, 57]. At the metaphase-anaphase transition, securin degradation ensues as a result of ubiquitination by anaphase-promoting complex (APC) with subsequent release of separase to mediate the separation of sister chromatid by cleavage of the chromosomal cohesion [58, 59].
1.7. Securin and cancer
Securin overexpression has been reported in a variety of endocrine-related tumors, especially pituitary [60], thyroid [61], breast [62], ovarian [63], and uterine tumors [64], as well as nonendocrine-related cancers involving the central nervous system [65], pulmonary system, and gastrointestinal system [66]. Induction of angiogenesis by PTTG was demonstrated the activation of proliferation, migration, and tube formation of human umbilical vein endothelial cells [67]. Overexpression of securin in mouse fibroblast cells results in cellular transformation and promoted tumor formation [42].
Securin may act as a transcription activator and involved in cellular transformation and tumorigenesis through activation of c-myc oncogene [68]. Securin has been identified that it is involved in tumorigenesis [69] and tumor invasiveness [49, 61, 66].
1.8. ATF3 and gamma-H2AX
ATF3 is a stress-inducible gene that encodes a member of the ATF/cyclic AMP
response element-binding (CREB) family of transcription factors that contains a basic region/leucine zipper DNA-binding and binds to the cyclic AMP response element consensus sequence TGACGTCA [70]. In most cases, ATF-3 is induced by external stress signals such as ischemic injuries, mutagens, carcinogens, mitogenic cytokines, or endoplasmic reticulum stresses from abnormal protein processing [71]. ATF3 plays dichotomous roles in the cancer development [72]. It can either promote or suppress the cellular growth depending on the endogenous or exogenous texture of disease conditions. ATF-3 has been reported to affect cell death and cell cycle progression in cancer cells [73]. In tumorigenesis, ATF3 overexpression protected malignant MCF10CA1a human breast cancer cells from apoptosis and promoted their metastatic potential, associated with an up regulation of fibronectin-1, TWIST-1, and Slug transcripts, which are key regulators of cell-cell or cell-extracellular matrix interaction [72]. In contrast several studies have implicated ATF3 as a tumor suppressor, due to its
ability to induce apoptosis and cell cycle arrest. The loss of ATF3 function results in loss of tumor suppression [74].
H2AX is a histone H2A variant that plays essential role in the recruitment and accumulation of DNA repair proteins to sites of double-strand breaks (DSBs) damage [75]. In DSBs generation, the H2AX protein is phosphorylated (termed γ-H2AX) at
serine residue 139 in the unique C-terminal motif SQEY within seconds and forms localized „„foci” at DSBs sites [75]. Moreover, γ-H2AX is formed during apoptosis
initiated by DNA damage. It has been found that γ-H2AX formation is a cellular response to endonuclease-mediated DNA fragmentation downstream from caspase activation during apoptosis [76].
1.9. AKT and survivin
The PI3K/AKT pathway regulates fundamental cellular functions, including cell growth and survival [77]. AKT mediates a variety of biological functions, including glucose uptake, protein synthesis, and inhibition of cell death [78]. AKT regulates cellular survival through phosphorylation of downstream substrates that indirectly or directly control the apoptotic machinery [79]. Survivin, a unique member of the inhibitor of apoptosis protein (IAP) family, plays an important role in regulating both apoptosis and cell division [80]. Survivin has been identified as a negative prognostic
factor in various cancer types and was implicated in resistance to apoptosis induction by anticancer agents [81]. Evidence for the up-regulation of survivin via the PI3K/AKT pathway was first shown in endothelial cells [82]. It has been reported that survivin expression is activated by the PI3K/AKT pathway, conferring cell survival and resistance to apoptosis in various malignant cells, including prostate [83], breast [84], and lung [85]. Several studies have demonstrated resistance of survivin-expressing cells to anticancer drug-induced apoptosis [86].
1.10. The purpose of the study
In this study, the anticancer abilities of ACP-93 on apoptosis and anti-tumorigenesis effects were investigated in the human colon carcinoma cells.
Moreover, we investigated the role of securin in the tumorigenesis. Understanding the anticancer abilities and mechanisms of ACP-93 on apoptosis and anti-tumorigenesis in human colon cancer cells may contribute to potential colon cancer therapy.