<div>Abstract<p>Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was reported to inhibit proliferation of a variety of cancer cells <i>in vitro</i>. However, the efficacy and <i>in vivo</i> mechanism of action of EF24 in gastrointestinal cancer cells have not been investigated. Here, we assessed the <i>in vivo</i> therapeutic effects of EF24 on colon cancer cells. Using hexosaminidase assay, we determined that EF24 inhibits proliferation of HCT-116 and HT-29 colon and AGS gastric adenocarcinoma cells but not of mouse embryo fibroblasts. Furthermore, the cancer cells showed increased levels of activated caspase-3 and increased Bax to Bcl-2 and Bax to Bcl-xL ratios, suggesting that the cells were undergoing apoptosis. At the same time, cell cycle analysis showed that there was an increased number of cells in the G<sub>2</sub>-M phase. To determine the effects of EF24 <i>in vivo</i>, HCT-116 colon cancer xenografts were established in nude mice and EF24 was given i.p. EF24 significantly suppressed the growth of colon cancer tumor xenografts. Immunostaining for CD31 showed that there was a lower number of microvessels in the EF24-treated animals coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA and protein expression. Western blot analyses also showed decreased AKT and extracellular signal-regulated kinase activation in the tumors. Taken together, these data suggest that the novel curcumin-related compound EF24 is a potent antitumor agent that induces caspase-mediated apoptosis during mitosis and has significant therapeutic potential for gastrointestinal cancers. [Cancer Res 2008;68(6):1962–9]</p></div>
Cancer is one of the leading causes of death in the United States and accounts for approximately 8 million deaths per year worldwide. Although there is an increasing number of therapeutic options available for patients with cancer, their efficacy is time-limited and non-curative. Approximately 50-60% cancer patients in the United States utilize agents derived from different parts of plants or nutrients (complementary and alternative medicine), exclusively or concurrently with traditional therapeutic regime such as chemotherapy and/or radiation therapy. The need for new drugs has prompted studies evaluating possible anti-cancer agents in fruits, vegetables, herbs and spices. Saffron, a spice and a food colorant present in the dry stigmas of the plant Crocus sativus L., has been used as an herbal remedy for various ailments including cancer by the ancient Arabian, Indian and Chinese cultures. Crocetin, an important carotenoid constituent of saffron, has shown significant potential as an anti-tumor agent in animal models and cell culture systems. Crocetin affects the growth of cancer cells by inhibiting nucleic acid synthesis, enhancing anti-oxidative system, inducing apoptosis and hindering growth factor signaling pathways. This review discusses the studies on cancer preventive potential of crocetin and its future use as an anticancer agent.
Abstract We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav‐like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy‐mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5‐year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin‐1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.
Abstract Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors. Citation Format: Scott J. Weir, Partha Ranjarajan, Robyn Wood, Karl Schorno, Prabhu Ramamoorthy, Lian Rajweski, Kathy Heppert, Michael J. McKenna, William McCulloch, Greg A. Reed, Amanda Brinker, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5882.
CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional NH 2 -terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following γ-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3′untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G 2 /M cell cycle arrest, which was overcome by prostaglandin E 2 . Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)1 and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe.
AbstractExpression of anti-apoptotic genes is frequently elevated in tumors, where they increase resistance to chemotherapeutic agents and predict poor patient outcomes. However, key cellular factors regulating anti-apoptotic genes in tumors remain unknown. Increased expression of the regenerating (Reg) genes is commonly observed in gastrointestinal (GI) malignancies including colorectal cancer (CRC). We therefore examined Reg gene expression and associated changes in anti-apoptotic genes in an animal model of GI tumorigenesis. Using real time RT-PCR, we measured expression of Reg genes in human colorectal adenocarcinoma specimens, colon adenocarcinoma cell lines and adenomas from multiple intestinal neoplasia (min) mice heterozygous for a germ-line mutation of the adenomatous polyposis coli (APC) gene. Expression of Reg genes is increased in human colorectal adenocarcinomas and in the intestine of APCmin/+ mice at 4 weeks of age, a time preceding the spontaneous second mutation in the APC gene. Individual Reg genes exhibited regional expression profiles across the GI tract in mice. Adenomas from 14-week old mice had significant increases in at least one member of the Reg gene family, most commonly Reg IV and an associated increase in expression of the anti-apoptotic gene, Bcl-2. Addition of exogenous recombinant human Reg IV to human colon adenocarcinoma cells significantly increased Bcl-2 and Bcl-xL expression and induced resistance to ionizing radiation. These results show that dysregulation of Reg genes occur early in tumorigenesis. Furthermore, increased expression of Reg genes, specifically Reg IV contribute to adenoma formation and lead to increased resistance to apoptotic cell death in CRC.
Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C which coordinates zinc. apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity. His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His61 → Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His61 → Arg abolished RNA binding, while the Glu63 → Gln and Cys96 → Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His61 → Cys, LRR, and Cys93 → Ser mutants increased endogenous editing 2-3-fold, while Glu63 → Gln and His61 → Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme. Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C which coordinates zinc. apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity. His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His61 → Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His61 → Arg abolished RNA binding, while the Glu63 → Gln and Cys96 → Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His61 → Cys, LRR, and Cys93 → Ser mutants increased endogenous editing 2-3-fold, while Glu63 → Gln and His61 → Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.
