Modulation of thymidilate synthase and p53 expression by HDAC inhibitor vorinostat resulted in synergistic antitumor effect in combination with 5FU or raltitrexed.

Despite the introduction of several novel anticancer agents almost 50% of colorectal cancer (CRC) patients die for cancer suggesting the necessity of new therapeutical approaches. In this study we demonstrated that the HDAC inhibitor vorinostat exerted potent antiproliferative effect in a panel of mut- and wt-p53 human CRC cell lines. Moreover, in combination with 5-fluorouracil modulated by folinic acid (5FU-FA) or with Raltitrexed (RTX), both commonly used in the treatment of this disease, it showed a clear schedule-dependent synergistic antiproliferative interaction as demonstrated by calculating combination indexes. Only simultaneous, or 24 h pretreatment with vorinostat followed by either agent, produced synergistic effect paralleled by evident cell cycle perturbations with major S-phase arrest. Moreover, we provided for the first time evidences that vorinostat can overcome resistance to both 5FU and RTX. Downmodulation of Thymidilate synthase (TS) protein induced by vorinostat within 24 h, represented a key factor in enhancing the effects of both drugs in sensitive as well as resistant tumor cells. Furthermore, p53, whose wild-type expression is critical for sensitivity to 5FU and RTX, was upregulated by vorinostat in wt- and downregulated in mut-p53 cells, suggesting an additional mechanism of the antiproliferative synergistic interactions observed. Overall these data add new insights in the mechanism of vorinostat antitumor effect and suggested that the association of vorinostat plus 5FU-FA and/or RTX should be clinically explored

Arsenic trioxide/ascorbic acid therapy in patients with refractory metastatic colorectal carcinoma: A clinical experience

Arsenic trioxide (As2O3) has demonstrated effectiveness in treating acute promyelocytic leukemia (APL). Therefore the FDA has approved it to treat APL. In patients with refractory metastatic colorectal carcinoma (CRC), we assessed the efficacy and toxicity of As2O3/AA (ascorbic acid) as the outcome of this trial. Five patients with refractory metastatic CRC who failed all previous standard chemotherapy were enrolled in this study. They were treated with 0.25 mg/kg body weight/day As2O3 and 1000 mg/day of ascorbic acid for 5 days a week for 5 weeks. Each treatment cycle extended for 7 weeks with 5 weeks of treatment and 2 weeks of rest. All the patients developed moderate to severe toxic side effects to arsenic trioxide/AA therapy and therefore the study was discontinued. No CR (complete remission) or PR (partial remission) was observed. CT scans demonstrated stable or progressive disease. Three of the five patients died within 2 to 5 months after cessation of the therapy. None of the deaths could be related to this clinical trial. Two years of follow-up study showed that two patients were alive with stable disease. Under the current treatment regimen all patients developed moderate to severe side effects with no clinically measurable activity. As an alternate, efforts may be made to reduce the dose and arsenic trioxide may be combined with other standard regimen in reversing the chemo resistance

Hypoxia signature of splice forms of tryptophanyl-tRNA synthetase marks pancreatic cancer cells with distinct metastatic abilities

Pancreatic cancer is one of most deadly because of its aggressive growth and high metastatic ability that correlates with intratumoral hypoxia. Earlier diagnosis and prognosis marker of pancreatic cancer is not yet available. In colorectal cancer, protein biosynthesis enzyme, tryptophanyl-tRNA synthetase (TrpRS), is up-regulated in good-prognosis tumors and down-regulated in metastatic poor-prognosis tumors. Tryptophanyl-tRNA synthetase status in pancreatic cancer is unknown. To correlate metastatic ability with hypoxia and TrpRS as a possible prognostic marker, we examined mRNA and protein expression in 2 human pancreatic cancer cell lines with different metastatic abilities and TrpRS levels using our site-specific monoclonal antibodies directed to conformation-dependent epitopes on pancreatic TrpRS. Pancreatic MIAPaCa-2, Panc-1, cervical HeLa, and prostate cancer PC-3 cells were cultivated under normoxia or in hypoxic chamber. Expression of full-length TrpRS, antiangiogenic TrpRS, cyclin B1, hypoxia-inducible factor 1α, and Glut-1 was determined with reverse transcriptase-polymerase chain reaction, immunoblotting, and immunocytochemistry. We demonstrate that hypoxia regulates differentially TrpRS splice forms. Pronounced down-regulation of full-length TrpRS by hypoxia is concomitant with higher metastatic ability. Tryptophanyl-tRNA synthetase down-regulation by hypoxia may be a factor responsible for low TrpRS in tumors with high metastatic ability. Tryptophanyl-tRNA synthetase recognizability is important for pancreatic cancer prognosis and as a new target for metastasis treatment

Chronic exposure of colorectal cancer cells in culture to fluoropyrimidine analogs induces thymidylate synthase and suppresses p53. A molecular explanation for the mechanism of 5-FU resistance

Resistance to chemotherapy is a major issue in treating malignant diseases. 5-Fluorouracil (5-FU) is the drug of choice in managing colorectal cancer (CRC) patients. However, 5-FU resistance leads to eventual treatment failure. Therefore, delaying or reversing the onset of 5-FU resistance will benefit these terminally ill patient populations. A metabolite of 5-FU irreversibly binds thymidylate synthase (TS) thus inhibiting its activity. Many studies demonstrated that these resistant patients had an increased intratumoral TS level. We used TS-siRNA to reduce TS and resensitize HT29FU CRC cells back to this uracil analogue. We exposed the CRC cell line HT29 to an increasing concentration of 5-FU or 5-fluorouridine (FUR) and established a derivative cell line (HT29FU and HT29FUR). Using real-time polymerase chain reaction (PCR) and Western immunodetection assays, we analyzed the expression of TS and p53 mRNA and protein in control and experimental groups. Cytotoxicity to 5-FU was determined by reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT assay) or trypan blue dye exclusion assay. The HT29FU and HT29FUR cells have a distinct morphology: they are generally asteroid shaped. The half maximal inhibitory concentration (IC(50)) values for the resistant cell line for 5-FU is over 148 microM compared to 5 microM for the sensitive parental cell line. The resistant cell lines expressed more of TS and less of p53. TS-siRNA suppressed TS only. Other pathways were not significantly altered. It also marginally (20%) re-sensitized resistant cells to 5-FU. Restoration of partial sensitivity to 5-FU by TS-siRNA reiterates the primacy of the DNA synthesis pathway in 5-FU mode of action. We speculate that the short half-life of the transiently transfected siRNA may contribute to the marginal restoration of sensitivity. By integrating TS-siRNA expression vector into the genome and regulating its expression, we may be able to reverse 5-FU resistance and make the cells as sensitive as the parental cell line

In vitro global gene expression analyses support the ethnopharmacological use of Achyranthes aspera

Achyranthes aspera (family Amaranthaceae) is known for its anticancer properties. We have systematically validated the in vitro and in vivo anticancer properties of this plant. However, we do not know its mode of action. Global gene expression analyses may help decipher its mode of action. In the absence of identified active molecules, we believe this is the best approach to discover the mode of action of natural products with known medicinal properties. We exposed human pancreatic cancer cell line MiaPaCa-2 (CRL-1420) to 34 μg/mL of LE for 24, 48, and 72 hours. Gene expression analyses were performed using whole human genome microarrays (Agilent Technologies, USA). In our analyses, 82 (54/28) genes passed the quality control parameter, set at FDR ≤ 0.01 and FC of ≥±2. LE predominantly affected pathways of immune response, metabolism, development, gene expression regulation, cell adhesion, cystic fibrosis transmembrane conductance regulation (CFTR), and chemotaxis (MetaCore tool (Thomson Reuters, NY)). Disease biomarker enrichment analysis identified LE regulated genes involved in Vasculitis—inflammation of blood vessels. Arthritis and pancreatitis are two of many etiologies for vasculitis. The outcome of disease network analysis supports the medicinal use of A. aspera, viz, to stop bleeding, as a cure for pancreatic cancer, as an antiarthritic medication, and so forth

Abstract 4019: Regulatory role of Gli motifs in thymidylate synthase expression

Abstract Fluoropyrimidine analogue 5-Flurorouracil (5-FU) is the mainstay of CRC treatment. It was designed as a competitive inhibitor of the enzyme thymidylate synthase [TS; TYMS]. All the new agents (Irinotecan, CPT-11, Avastin etc) are effective only in combination with 5-FU. The enzyme thymidylate synthase is pivotal for DNA replication. It controls cell survival and proliferation. Increases in TS level that follows 5-FU administration renders the treatment ineffective. Considerable efforts have been made to reverse 5-FU resistance by down-regulating TS expression. In this context, we demonstrated that arsenic trioxide (ATO) sensitized 5-FU resistant colorectal cancer cells by inhibiting TS mRNA expression both in vitro and in clinic. However, the precise mechanism of TS reduction by arsenic trioxide is not known. Many investigators, including ourselves, have shown a direct correlation between Glioma associated oncogene homolog (Gli) and TS expression in cultured cells as well in patient samples, who received arsenic trioxide and 5-FU combination therapy. However, the link between ATO, Gli and TS is not known. Gli is an important molecule in hedgehog (hh) signaling. It plays a vital role in development and cancer. As a transcription factor, Gli binds the sequence 5’ GACCACCCA 3’ a conserved 9 bp DNA motif. We hypothesized that TS promoter may harbor one or more of the 9 bp Gli motifs. We queried for Gli motifs in the TS gene regulatory region. Position specific base frequencies for the TF consensus sequence for Gli1-3, Ci, cECF, TGM were prepared using the data from Hallikas et al. (2006). We used FIMO (Find Individual Motif Occurrences) tool in the Meme Suite (http://meme.nbcr.net) to search for Gli motifs in the query sequence. Anti-Gli antibodies and non-specific IgG were used to precipitate chromatin immuno complex. The immuno precipitates were probed with TS specific primer. This search yielded 35 possible Gli motifs in TS promoter. From among the 35 motifs with significant homology (p≤0.001), only top eight matches had passed FDR threshold (q≤0.05). However, these eight matches represented three distinct binding sites on TS promoter. Thus we computationally determined that TS promoter has three unique Gli motifs viz BS1, BS2 and BS3. BS-2 in TS promoter is identical to BS-3 on the Jun and BS1 is identical to the BS 1 of Fgf15 which had been experimentally confirmed to be regulated by Gli1. The binding site was subsequently confirmed by ChIP assays. It appears that the inhibition of Gli1 by ATO down regulates TS expression. This cascade of reactions initiated by ATO reverts refractory tumors, once again sensitive to 5-FU. Taken together the results strongly indicate a direct role of Gli in TS transcription. Citation Format: Subbarayan R. Pochi, Nikesh Doshi, Xiaoqing Han, Zhiqiang Wang, Anthony Capobianco, David Robbins, Bach Ardalan. Regulatory role of Gli motifs in thymidylate synthase expression . [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 4019. doi:10.1158/1538-7445.AM2013-4019</jats:p