Design, Synthesis, and In Vitro Preliminary Cytotoxicity Evaluation of New Anthraquinone-2-Carboxylic Acid Derivatives

Background: Colon cancer is the third leading cause of cancer related deaths in the United States. Epidemiological studies suggest an increase in the intestinal bile-acid load resulting from the consumption of a high-fat diet is a significant risk factor for colon cancer. Bile acids are the endogenous ligands for the farnesoid X receptor (FXR), a ligand-activated transcription factor and member of the nuclear receptor superfamily, and high levels of bile acids can promote colon cancer development. FXR is essential for maintaining bile-acid homeostasis by regulating bile-acid synthesis and transport, preventing the accumulation of intestinal bile acid levels to cancer promoting levels. Previous studies have demonstrated that FXR knockout mice are more susceptible to the development of colon adenocarcinomas, indicating that FXR plays a suppressive role in colon tumor formation. This study investigates the role of FXR in the development of human colon cancer. Methods: Immunohistochemistry was used to label for FXR in normal human colon, colon polyps, and colon adenocarcinomas staged I-IV. SYBR green quantitative PCR and western blot analysis were used to measure expression of FXR and FXR target genes in normal human colon and colon cancers staged I-IV as well as colon cancer cell lines. Reverse phase protein array on colon cancer cell line lysates was used to correlate FXR expression with oncogenic signaling cascades. To test if FXR expression was suppressed by DNA methylation, colon cancer cell lines were treated with a DNA methyltransferase (DNMT) inhibitor and DNMT siRNA and FXR mRNA measured by real-time PCR. Immunoprecipitation with an antibody against 5-methylcytosine (MeDIP) analysis was done in human colon cancer cell lines to determine methylation of NR1H4 (gene encoding FXR) promoter. Results: IHC and qPCR analysis reveals that the expression and function of FXR is markedly reduced early in colon cancer progression, with suppression seen within precancerous lesions. Furthermore, FXR expression in colon cancer cell lines were negatively correlated to oncogenic PI3 kinase signaling cascades and associated with epithelial to mesenchymal transition (EMT). Results suggest DNA methylation as a mechanism of FXR silencing in colon cancer and confirms methylation of the FXR promoter. Conclusion: FXR deficiency in animals indicates FXR serves a tumor suppressive role. Our studies show that FXR is silenced in early in human colon cancer progression possibly by DNA methylation, which could be a cancer promoting event. The overall mechanism of FXR's anti-tumorigenic activity is not fully established but may be due to FXR's role in regulating EMT and bile acid homeostasis. Restoration and enhancement of FXR activity, by blocking DNA methylation or increasing baseline activity of FXR, represents a potential therapeutic option for the treatment of colon cancer. Citation Format: Ann M. Thomas, Le Zhan, Julie Izzo, Dipen Maru, Imad Shureiqi, Veera Baladandayuthapani, Han Liang, Grace L. Guo, Garth Powis. Silencing of farnesoid X receptor in human colon cancer by epigenetic mechanisms is associated with cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3561. doi:10.1158/1538-7445.AM2013-3561

Gastrin is mitogenic for several colon cancers. To assess a possible autocrine role of gastrin in colon cancers, we examined human colon cancer cell lines for expression of gastrin mRNA and various forms of gastrin. Gastrin mRNA was not detected in the majority of colon cancer cell lines by Northern hybridization but was detected in all human colon cancer lines by the sensitive method of reverse transcriptase-polymerase chain reaction (PCR). Gastrin mRNA was quantitated by the competitive PCR method. The majority of cell lines expressed very low levels of gastrin mRNA (< 1-5 copies/cell); only one cell line expressed > 20 copies/cell. The mature carboxyamidated form of gastrin was not detected in any of the cell lines by radioimmunoassay or immunocytochemistry. Results suggested that either gastrin mRNA expressed by colon cancer cells was altered (mutated) or posttranslational processing of progastrin was incomplete. Gastrin cDNA from all the colon cancer cell lines had an identical sequence to the published sequence of human gastrin cDNA. Specific antibodies against precursor forms of gastrin were used, and significant concentrations of nonamidated (glycine-extended) and prepro forms of gastrin were measured in tumor extracts of representative colon cancer cell lines. The presence of precursor forms of gastrin suggested a lack of one or more of the processing enzymes and/or cofactors. Significant concentrations of the processing enzyme (peptidylglycine alpha-amidating monooxygenase) were detected in colon cancer cells by immunocytochemistry. Therefore, lack of other cofactors or enzymes may be contributing to incomplete processing of precursor forms of gastrin, which merits further investigation. Since low levels of gastrin mRNA were expressed by the majority of human colon cancer cell lines and progastrin was incompletely processed, it seems unlikely that gastrin can function as a viable autocrine growth factor for colon cancer cells. High concentrations of glycine-extended gastrin-17 (GG) (> 10(-6) M) were mitogenic for a gastrin-responsive human colon cancer (DLD-1) cell line in vitro. It remains to be seen if GG or other precursor forms of gastrin are similarly mitogenic in vivo, which may then lend credibility to a possible autocrine role of gastrinlike peptides in colon cancers.

Natural products from various plants, microorganisms, fungi etc. have provided considerable value to the pharmaceutical industry over the past half century. But there still remains a vast potential reservoir of possible useful therapeutics, yet to be identifed. Among them, lichens (symbiotic associations between a fungus and an alga and/or a cyanobacterium), which produce more than a 1000 secondary chemicals. The United States is home to a diverse assemblage of lichen species. Lichens play many biological roles; however, their full potential as a source of anticancer drugs has been largely unexplored. To examine the possible anticancer role of several lichen species, we assessed the cytotoxic activity of 17 species collected from various parts of the US. Secondary compounds of these lichens were extracted with acetone and screened against Burkitt's Lymphoma cells (Raji) and the human colon cancer cell line (HT-29). Based on the cytotoxcity of these lichen extracts we selected two species Tuckermannopsis ciliaris and Xanthoparmelia chlorochroa for further testing. IC50 values for these two species showed particular promise - 28.8 and 28.6 μg/ml for Raji cells and 60 and 40 μg/ml for HT-29 cells respectively. The viability and cell growth of both cell lines was significantly reduced by the extracts from these two lichens but these extracts did not affect the viability and growth of normal lymphocytes. We performed morphological detection of apoptosis using Acridine orange and Propidium Iodide. Extracts from both lichens were found to induce apoptosis in both cancer cell lines when treated with the concentrations equivalent to the IC50. Cell proliferation of Raji and HT-29 cells, as measured by 3H-thymidine incorporation, was significantly reduced in treated cells (P &amp;lt;0.05) over 24 hours as compared with controls. To further investigate apoptosis we measured DNA fragmentation using gel electrophoresis. Within 24 hours of treatment, there was partial DNA degradation. Using flow cytometry, it was discovered that extracts from both lichens arrested cell-cycle progression in S phase. Using RT-PCR, it was found that the p53 gene was up-regulated in the treated cells during G1 phase while the DNA repair enzyme Thymidine kinase 1 (TK1) was down regulated. In conclusion, extracts from T. ciliaris and X. chlorochroa were both cytotoxic and induced apoptosis in cancer cells but not in normal cells. Hence extracts from these two lichens could hold potential as a non-toxic anticancer therapy. However, more research is required to further elucidate the molecular mechanisms behind these preliminary findings. Citation Format: Gajendra Shrestha, Atif M. El-Naggar, Sean C. Derenthal, Michael R. Boswell, Evita Weagel, Larry L. St. Clair, Richard Robison, Kim L. O'Neill. Cytototoxicity and mode of action of extract from of two lichens, Tuckermannopsis ciliaria (Ach.) Gyelnik and Xanthoparmelia chlorochroa (Tuck.) Hale against Burkitt's Lymphoma (Raji) cells and the Human colon cancer (HT-29) cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2250. doi:10.1158/1538-7445.AM2013-2250

Tightly coordinated Hedgehog (HH) signaling is critical during normal embryonic development. Aberrant HH signaling is involved in driving cell proliferation in multiple human cancers. Classical HH signaling involves the interaction of the soluble HH ligands with their receptor, Patched (Ptch), thereby releasing a transmembrane protein Smoothened (Smo) from Ptch-mediated inhibition. Smo then activates the Gli family of transcription factors that regulate HH target genes. We have previously reported that HH signaling provides essential survival support to human colon cancer cells and inhibition of this signal induces DNA damage and extensive cell death. However the downstream targets that govern the regulation of cell survival by HH/Gli in cancer cells are not completely defined. This study demonstrates that the Gli proteins transcriptionally regulate the expression of human telomerase reverse transcriptase (hTERT) gene in human cancer cells. hTERT is a known regulator of telomere homeostasis, which determines the replicative potential and hence the life span of cells. Suppression of both Gli1 and Gli2 functions by exogenous expression of a C-terminus truncated Gli3 repressor mutant (Gli3R), or by GANT61, a pharmacological inhibitor of Gli1 and Gli2 activity, reduced hTERT protein expression over a period of 72 hr in human colon, prostate and brain cancer cell lines. Further, exogenously expressed Gli2 significantly increased hTERT protein expression in human colon cancer cell lines. Exposure to GANT61 also inhibited hTERT mRNA expression within 24 hr in human colon cancer cell lines. Insilico analysis of the hTERT promoter revealed 7 putative Gli binding sites suggesting a transcriptional mode of regulation of hTERT expression by Gli2. Chromatin immunoprecipitation with Gli2 antibody precipitated fragments of the hTERT promoter in human colon cancer cell lines, indicating a direct interaction between Gli2 and the hTERT promoter. The binding between Gli2 and hTERT promoter was significantly reduced upon exposure to GANT61. Further, overexpression of the wild type hTERT cDNA in cancer cells prevented the cytotoxic effects of blocking the HH signaling pathway with GANT61. These findings demonstrate hTERT to be a direct transcriptional target and a critical mediator of the HH/Gli signaling pathway, and identify a previously unknown role of the HH/Gli axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/Gli signaling in cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699A. doi:1538-7445.AM2012-4699A

β-Lapachone Induces Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells

In the clinical setting, drug resistance remains a significant obstacle for successful chemotherapy. Bcl-2/adenovirus EIB 19-kDa-interacting protein 3 (BNIP3) is a proapoptotic member of the Bcl-2 family. To address its potential as a therapeutic target for chemosensitisation, this study investigated the effect of BNIP3 expression on chemosensitivity and reversal of oxaliplatin (L-OHP) resistance in human colon cancer cell lines. A plasmid expressing the BNIP3 gene was transfected into human parental colon cancer cell lines (SW620 and colo320) and L-OHP-resistant colon cancer cell lines (SW620/L-OHP and colo320/L-OHP) using Lipofectamine™ 2000, and the transfection efficiency was determined using fluorescence optics. Western blot analysis identified that SW620/L-OHP and colo320/L-OHP cells expressed lower levels of BNIP3 protein compared with the SW620 and colo320 cells. Transfection with the recombinant BNIP3 plasmid revealed an increase in BNIP3 expression in tumour cells. Following transfection with pDsRed-BNIP3, the chemosensitivity of parental and L-OHP-resistant cell lines to L-OHP was increased (P<0.01), as detected by the Cell Counting Kit-8 (CCK8) assay. Hoechst 33342 staining and flow cytometry revealed that the effects on L-OHP-induced apoptosis were enhanced by the overexpression of BNIP3. Chemosensitisation in human colon cancer cells was observed following treatment with the recombinant BNIP3 plasmid in vitro. The results of this study suggest that BNIP3 is a potential therapeutic target for reversing the resistance of L-OHP-resistant colon cancer cells to L-OHP.

The clinical use of anthraquinone antibiotics such as doxorubicin in cancer chemotherapy is limited due to their toxic cardiomyopathy effects. The conjugation of anthraquinones with fatty acids is known to enhance bioactivity by increasing lipophilicity and facilitating cell membrane permeation. Short‐chain saturated fatty acids such as capric, caprylic, caproic acids, and lauric acids are known for their inherent anticancer properties on human colorectal cancer cells. In this study, 2‐hydroxyanthraquinone and saturated fatty acid esters were synthesized and evaluated against human colon cancer cell line HT‐29 and Colo‐205, breast cancer cell lines MDA‐MB‐231 and MCF‐7, and leukemia cell line K‐562 for their in vitro anticancer properties using a sulforhodamine B (SRB) assay. The anthraquinone‐saturated fatty acid conjugates produced excellent activity against human colon and breast cancer cell lines. The octanoic acid and hexanoic acid derivatives of 2‐hydroxy anthraquinone showed excellent anticancer activity with GI 50 values of 0.2 nM and 1.0 nM against human colon cancer cell line Colo‐205 and the human breast cancer cell line MCF‐7, respectively, and emerged as the most active compounds. Furthermore, the in vitro cytotoxicity study against peripheral blood mononuclear cells showed that these derivatives are non‐toxic in nature.

Characterization of insulinlike growth factor I receptors in human colon cancer

Characterization of insulinlike growth factor I receptors in human colon cancer

Liphagal, isolated from the marine sponge Aka coralliphaga, exhibits phosphatidylinositol 3‐kinase alpha (PI3Kα) inhibitory activity and cytotoxic effects in human cancer cells. Siphonodictyal B, the biogenetic precursor of liphagal, also has PI3K inhibitory activity. However, its cytotoxic or antitumor activities have not been evaluated. In this study, we demonstrated that siphonodictyal B inhibits several kinases such as CDK4/6, CDK7, and PIM2 in addition to PI3K in vitro and that siphonodictyal B exhibits more potent cytotoxic effects than liphagal against human colon cancer cell lines. Furthermore, treatment with siphonodictyal B resulted in increased PARP cleavage, a larger sub‐G1 fraction, and a larger annexin V‐positive cell population, all of which are indicative of apoptosis induction. As a mechanism of apoptosis induction, we found that siphonodictyal B activates the p38 MAPK pathway, leading the upregulation of proapoptotic factors. Moreover, siphonodictyal B increased ROS levels, thus promoting p38 MAPK pathway activation. NAC, an ROS scavenger, almost completely reversed both the cytotoxic and p38 MAPK pathway‐activating effects of siphonodictyal B. These results indicate that the p38 MAPK pathway might be involved downstream of ROS signaling as part of the mechanism of siphonodictyal B‐induced apoptosis. Finally, siphonodictyal B displayed antitumor effects in a human colon cancer xenograft mouse model and increased p38 phosphorylation in tumor tissue. These results suggest that siphonodictyal B could serve as the basis of a novel anticancer drug.

To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer functions and its mechanisms in the human esophageal cancer cell line (Eca 109) and human colonic cancer cell line (Ls-174-t). The effect of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After insulin treatment or insulin/5-FU treatment, cell cycle distribution of both cell lines was analyzed by flow cytometry. Western blot assay was used to assess the expression of caspase-3 and thymidylate synthase (TS). Apoptosis was detected by flow cytometry, DNA fragmentation assay, and terminal transferase dUTP nick end labeling assay (TUNEL). Moreover, the changes of 5-FU uptake after insulin pretreatment were detected by HPLC assay and Western blot analysis. We found that insulin enhanced the inhibitory effect of 5- FU on cell proliferation when Eca 109 cells and Ls-174-t cells were pretreated with insulin for the appropriate time. Insulin increased the cell number of the S phase and the uptake of 5-FU. Insulin/5-FU treatment enhanced apoptosis of tumor cells and upregulated the expression of cleaved caspase-3 compared with 5-FU treatment. Moreover, insulin/5-FU treatment induced the changes of free TS and the TS ternary complex level compared with 5-FU treatment in Eca 109 and Ls-174-t cells. These data suggest that insulin enhances anticancer functions of 5- FU when it is treated before 5-FU for the appropriate time in human esophageal and colonic cancer cell lines.

A plasmid carrying DNA to be transcribed into a small interfering RNA against transketolase-like-1 mRNA was constructed and transfected into a human colon cancer cell line. The mRNA expression of transketolase gene family in the human colon cell line was determined by real-time polymerase chain reaction. The effect of anti-transketolase-like-1 small interfering RNA on cell proliferation and cell cycle in the human colon cancer cell line cells was detected by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide. The transketolase-like-1 gene was significantly downregulated in human colon cancer cell line cells transfected with small interfering RNA transketolase-like-1 constructs compared with the cells transfected with control vector and the cells without transfection. In addition, the anti-transketolase-like-1 small interfering RNA construct significantly decreased the level of transketolase in the transfected human colon cancer cell line cells, arrested them in G0/G1 phase and substantially inhibited cell proliferation. No significant difference was found in the other two genes (transketolase and transketolase-like-2 genes) between the transfected human colon cancer cell line cells and the controls (P>0.05). Our data demonstrated that the transketolase-like-1 gene plays an important role in total transketolase activity and in the cell proliferation of human colon cancer. Transketolase-like-1 may serve as a target for novel anticancer therapies.

Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.

Background: Mutations in bacteria frequently occur that display a crucial need for new antimicrobial agents. Metallo-β-lactamases (MBLs) are a growing threat to maintaining the effectiveness of beta-lactam antibiotics. Resistance to beta-lactam antibiotics is one of the most common types of antibiotic resistance, which causes the ineffectiveness of antibiotics. Objectives: This study aimed to identify a novel inhibitor using molecular dynamics simulations to inhibit VIM-2 Metallo-β-lactamases and overcome carbapenem resistance in Pseudomonas aeruginosa strains. Methods: Computational biology tools were employed for this study, including molecular dynamics, binding free energy, virtual screening, and docking. Natural compounds were taken from the ZINC databank and prepared. At the next stage, the prepared compounds were screened based on docking energy in the active site of VIM-2 MBL by Schrödinger (Maestro) software, and better compounds were selected. Captopril was chosen as a positive control inhibitor for VIM-2 MBLs. Ultimately, molecular dynamics simulations were performed using GROMACS software, and outputs were analyzed. Results: Maestro software's screening results showed that ZINC00517765 was the best inhibitor with -12.29 kcal mol-1 docking energy. The ADME investigations revealed that ZINC00517765 had an appropriate range of pharmacokinetics, lipophilicity, and drug-likeness features as an inhibitor of VIM-2 MBL. Molecular dynamics outcomes explicated that VIM-2 MBL in the presence of ZINC00517765 had better stability during simulation. The results of the MM-PBSA study illustrated that ZINC00517765 with -72.29 kJ mol-1 binding free energy was more potent than Captopril with -23.39 kJ mol-1. Conclusions: This study showed that VIM-2 MBL in the presence of ZINC00517765 has suitable stability during simulation. Also, more hydrogen bonds and stronger binding free energy than Captopril confirm that ZINC00517765 is a proper candidate for further studies and laboratory investigation.

&lt;P&gt;Background: Breast cancer and human colon cancer are the most common types of cancer in females and males, respectively. Breast cancer is the most common type of cancer after lung and colon cancers. Natural products are an important source for drug discovery. Boesenbergia rotunda (L.) Mansf. is commonly known as finger root, belonging to the Zingiberaceae family. &lt;/P&gt;&lt;P&gt; Objective: The aim of this study to isolate some natural compounds from the rhizomes of B. rotunda (L.) Mansf., and to investigate their cytotoxicity against the human triple-negative breast cancer cell (MDA-MB-231) and HT-29 colon cancer cell lines. &lt;/P&gt;&lt;P&gt; Methods: The dried rhizomes of B. rotunda were extracted with methanol. The methanolic extract was further used for solvent-solvent extraction. Bioassay-guided extraction and isolation of the rhizomes of the B. rotunda exhibited cytotoxic properties of hexane and dichloromethane fractions. &lt;/P&gt;&lt;P&gt; Results: Six major chemical constituents, pinostrobin (1), pinostrobin chalcone (2), cardamonin (3), 4,5-dihydrokawain (4), pinocembrin (5), and alpinetin (6) were isolated from the rhizomes of the B. rotunda. All the chemical constituents were screened against the human triple-negative breast cancer cell (MDA-MB-231) and HT-29 colon cancer cell lines. The compound cardamonin (3) (IC50 = 5.62&amp;#177;0.61 and 4.44&amp;#177;0.66 &amp;#181;g/mL) and pinostrobin chalcone (2), (IC50 = 20.42&amp;#177;2.23 and 22.51&amp;#177;0.42 μg/mL) were found to be potent natural cytotoxic compounds against MDA-MB-231 and HT-29 colon cancer cell lines, respectively. &lt;/P&gt;&lt;P&gt; Conclusion: Cardamonin (3) and pinostrobin chalcone (2) were found to be the most potential natural compounds against breast cancer cell line MDA-MB-231 and colon cancer HT-29 cell line.&lt;/P&gt;