Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population

Future of 5-fluorouracil in cancer therapeutics, current pharmacokinetics issues and a way forward

Background: In addition to exhibiting antitumor potential, antitumor drugs exhibit toxicity due to a poor pharmacokinetic profile. An enormous amount of research has been carried out and is still ongoing to obtain more targeted, potent, and safe drugs to treat cancer, and pharmacokinetic evaluations of anticancer drugs are needed. Objectives: The present review examined different delivery systems and methodologies designed in recent years to investigate the pharmacokinetics of the anticancer drug, 5-fluorouracil (5-FU). These methodologies highlight how the issues of bioavailability, absorption, half-life, targeted neoplastic cell potential, and high therapeutic index of 5-FU are resolved. Results: A number of naturally occurring macromolecules such as modified starch, porphyran, peptides, and folic acids have been found to be successful in vitro to improve the permeability and retention effect of 5-FU against solid tumors. A promising approach for targeted 5-FU delivery to oncoproteins has resulted in a number of potentially sound anticancer nanocomposites. Chitosan nanoparticles loaded with 5-FU have been shown to exhibit cytotoxicity equivalent to 5-FU injections against gastric carcinoma. At the level of inter- and intra-molecular interactions, the co-crystal approach has been found to be successful against colorectal cancer proteins. Because of the 5-FU ligand-like nature and its metal-binding potential, researchers have shifted attention toward the synergistic co-administration of gold complexes with this drug. Conclusions: This study highlighted the techniques used to improve the pharmacokinetics of 5-FU and that “nanocarriers” are a promising approach in this field. The conclusion is supported by solid evidence

Randomized trial of calcipotriol combined with 5-fluorouracil for skin cancer precursor immunotherapy

BACKGROUND. Actinic keratosis is a precursor to cutaneous squamous cell carcinoma. Long treatment durations and severe side effects have limited the efficacy of current actinic keratosis treatments. Thymic stromal lymphopoietin (TSLP) is an epithelium-derived cytokine that induces a robust antitumor immunity in barrier-defective skin. Here, we investigated the efficacy of calcipotriol, a topical TSLP inducer, in combination with 5-fluorouracil (5-FU) as an immunotherapy for actinic keratosis. METHODS. The mechanism of calcipotriol action against skin carcinogenesis was examined in genetically engineered mouse models. The efficacy and safety of 0.005% calcipotriol ointment combined with 5% 5-FU cream were compared with Vaseline plus 5-FU for the field treatment of actinic keratosis in a randomized, double-blind clinical trial involving 131 participants. The assigned treatment was self-applied to the entirety of the qualified anatomical sites (face, scalp, and upper extremities) twice daily for 4 consecutive days. The percentage of reduction in the number of actinic keratoses (primary outcome), local skin reactions, and immune activation parameters were assessed. RESULTS. Calcipotriol suppressed skin cancer development in mice in a TSLP-dependent manner. Four-day application of calcipotriol plus 5-FU versus Vaseline plus 5-FU led to an 87.8% versus 26.3% mean reduction in the number of actinic keratoses in participants (P < 0.0001). Importantly, calcipotriol plus 5-FU treatment induced TSLP, HLA class II, and natural killer cell group 2D (NKG2D) ligand expression in the lesional keratinocytes associated with a marked CD4(+) T cell infiltration, which peaked on days 10–11 after treatment, without pain, crusting, or ulceration. CONCLUSION. Our findings demonstrate the synergistic effects of calcipotriol and 5-FU treatment in optimally activating a CD4(+) T cell–mediated immunity against actinic keratoses and, potentially, cancers of the skin and other organs. TRIAL REGISTRATION. ClinicalTrials.gov NCT02019355. FUNDING. Not applicable (investigator-initiated clinical trial)

High incidence of Angina pectoris in patients treated with 5-fluorouracil - A planned surveillance study with 102 patients

Objective: Angina pectoris, arrhythmic sudden death and myocardial infarction, all these cardiac events have occasionally been reported during 5-fluorouracil (5-FU) chemotherapy. Underlying mechanisms leading to these events are unknown; damage to the myocytes or vasospasms have been discussed. Methods: 102 consecutive and unselected patients were monitored with 12-lead ECG, echocardiography and radionuclide ventriculography prior to the first cycle of 5-FU chemotherapy and 3 months from baseline. Results: 19% of the patients developed reversible symptoms of angina pectoris during treatment which lasted up to 12 h after cessation of the infusion. Most of the 19 patients showed corresponding ECG changes. 6 out of the 19 patients with severe angina pectoris had subsequent coronary angiography. In none of these patients the coronary angiography showed coronary artery disease, but it showed low ventricular function (ejection fraction <50%) in 2 patients. The ejection fraction did not increase overtime. Arrhythmias were screened for with Holter monitoring during 5-FU chemotherapy. The frequency of bradycardia and ventricular extrasystoles increased significantly (p < 0.05) during treatment compared to arrhythmias in Holter monitoring 3 months later. Furthermore the Qtc time in the ECG 3 months later was significantly prolonged (p < 0.05) compared to baseline values. Conclusions:The incidence of angina pectoris in patients during 5-FU treatment seems higher than previously suspected. As myocardial ischemia can be fatal, attentiveness to these symptoms and immediate treatment are crucial. Copyright (C) 2003 S. Karger AG, Basel

Early growth response protein-1 promoter-mediated synergistic antitumor effect of hTERTC27 gene therapy and 5-flurorouracil on nasopharyngeal carcinoma

hTERTC27 is a newly constructed polypeptide that can induce telomere dysfunction. To study the synergistic antitumor effects of the hTERTC27 polypeptide driven by the early growth response protein-1 (Egr-1) promoter and chemotherapeutic 5-flurorouracil (5-FU) on nasopharyngeal carcinoma, a series of in vitro and in vivo experiments were performed. The results showed that hTERTC27 expression was significantly increased up to 7.21-folds by the 5-FU-activated Egr-1 promoter in C666-1 cells. Overexpressed hTERTC27 made the cells more sensitive to 5-FU, and additionally, inhibited cell proliferation about 20.41%. Combinational therapy of overexpressed hTERTC27 driven by the 5-FU-activated Egr-1 promoter and 5-FU synergistically inhibited cell proliferation and promoted apoptosis of C666-1 cells for about 4.75-fold and 1.76-fold in comparison with a sole therapy of hTERTC27 or 5-FU in vitro. In vivo experiments showed that overexpressed hTERTC27 driven by 5-FU-activated Egr-1 promoter and 5-FU synergistically reduced tumor volume, tumor weight, and local infiltration, which may be relative to tumor cell apoptosis. These results suggest that combinational therapy of overexpressed hTERTC27, which is driven by the 5-FU-activated Egr-1 promoter, and 5-FU may provide a novel approach to treat nasopharyngeal cancer. © 2012 Mary Ann Liebert, Inc.published_or_final_versio

Green synthesis and biological evaluation of novel 5-fluorouracil derivatives as potent anticancer agents

This study reports the formation of 5-FU co-crystals with four different pharmacologically safe co-formers; Urea, Thiourea, Acetanilide and Aspirin using methanol as a solvent. Two fabrication schemes were followed i.e., solid-state grinding protocol, in which API and co-formers were mixed through vigorous grinding while in the other method separate solutions of both the components were made and mixed together. The adopted approaches offer easy fabrication protocols, no temperature maintenance requirements, no need of expensive solvents, hardly available apparatus, isolation and purification of the desired products. In addition, there is no byproducts formation, In fact, a phenomenon embracing the requirements of green synthesis. Through FTIR analysis; for API the Nsingle bondH absorption frequency was recorded at 3409.02 cm−1 and that of single bondCdouble bondO was observed at 1647.77 cm−1. These characteristics peaks of 5-FU were significantly shifted and recorded at 3499.40 cm−1 and 1649.62 cm−1 for 5-FU-Ac (3B) and 3496.39 cm−1 and 1659.30 cm−1 for 5-FU-As (4B) co-crystals for Nsingle bondH and single bondCdouble bondO groups respectively. The structural differences between API and co-crystals were further confirmed through PXRD analysis. The characteristic peak of 5-FU at 2θ = 28.79918o was significantly shifted in the graphs of co-crystals not only in position but also with respect to intensity and FWHM values. In addition, new peaks were also recorded in all the spectra of co-formers confirming the structural differences between API and co-formers. In addition, percent growth inhibition was also observed by all the co-crystals through MTT assay against HCT 116 colorectal cell lines in vitro. At four different concentrations; 25, 50, 100 and 200 µg/mL, slightly different trends of the effectiveness of API and co-crystals were observed. However; among all the co-crystal forms, 5-FU-thiourea co-crystals obtained through solution method (2B) proved to be the most effective growth inhibitor at all the four above mentioned concentrations

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Degradation rate of 5-fluorouracil in metastatic colorectal cancer. A new predictive outcome biomarker?

BACKGROUND: 5-FU based chemotherapy is the most common first line regimen used for metastatic colorectal cancer (mCRC). Identification of predictive markers of response to chemotherapy is a challenging approach for drug selection. The present study analyzes the predictive role of 5-FU degradation rate (5-FUDR) and genetic polymorphisms (MTHFR, TSER, DPYD) on survival. MATERIALS AND METHODS: Genetic polymorphisms of MTHFR, TSER and DPYD, and the 5-FUDR of homogenous patients with mCRC were retrospectively studied. Genetic markers and the 5-FUDR were correlated with clinical outcome. RESULTS: 133 patients affected by mCRC, treated with fluoropyrimidine-based chemotherapy from 2009 to 2014, were evaluated. Patients were classified into three metabolic classes, according to normal distribution of 5-FUDR in more than 1000 patients, as previously published: poor-metabolizer (PM) with 5-FU-DR ≤ 0,85 ng/ml/106 cells/min (8 pts); normal metabolizer with 0,85 < 5-FU-DR < 2,2 ng/ml/106 cells/min (119 pts); ultra-rapid metabolizer (UM) with 5-FU-DR ≥ 2,2 ng/ml/106 cells/min (6 pts). PM and UM groups showed a longer PFS respect to normal metabolizer group (14.5 and 11 months respectively vs 8 months; p = 0.029). A higher G3-4 toxicity rate was observed in PM and UM, respect to normal metabolizer (50% in both PM and UM vs 18%; p = 0.019). No significant associations between genes polymorphisms and outcomes or toxicities were observed. CONCLUSION: 5-FUDR seems to be significantly involved in predicting survival of patients who underwent 5-FU based CHT for mCRC. Although our findings require confirmation in large prospective studies, they reinforce the concept that individual genetic variation may allow personalized selection of chemotherapy to optimize clinical outcomes

Gemcitabine: Progress in the treatment of pancreatic cancer

Unresectable pancreatic cancer has a dismal prognosis with a median survival of 3-5 months in untreated disease. Since the introduction of gemcitabine, pancreatic cancer may no longer be regarded a chemotherapy-resistant tumor. Treatment with single-agent gemcitabine achieved clinical benefit and symptoms improvement in 20-30% of patients. While 1-year survival was observed in 2% of 5-fluorouracil (5-FU)-treated patients, it was raised to 18% by single-agent gemcitabine. Good treatment tolerability and low incidence of side effects are clear advantages of single-agent gemcitabine. Improvement of efficacy is, however, expected from combination treatment. Gemcitabine and cisplatin given as first-line treatment in three studies achieved a median survival of 7.4-8.3 months. One-year survival was raised to 28% as reported in one study. Comparable activity was obtained by a combination of gemcitabine with 5-FU. Nine studies using gemcitabine in combination with standard-dose or high-dose 5-FU reported a median survival ranging from 5.5 to 13 months. Notwithstanding these promising results, recommendations regarding palliative chemotherapy of pancreatic cancer remain tentative and still need confirmation by presently ongoing phase III trials. Inclusion of pancreatic cancer patients into clinical trials should be a major goal. Outside clinical trials, patients should present with an adequate PS (Karnofsky-performance index greater than or equal to 70) to qualify for chemotherapy. Copyright (C) 2001 S. Karger AG, Basel

Efficacy of Combined 5-Fluorouracil and Photodynamic Therapy in Glioma Spheroids

Standard treatment regimens consisting of surgery, radiation and chemotherapy have proven ineffective for the treatment of high-grade gliomas such as glioblastoma multiforme (GBM). An effective cure requires elimination of nests of tumor cells that have migrated from the resection margin and infiltrated normal brain. A number of localized therapies, including light-based approaches such as photodynamic therapy (PDT) and photochemical internalization (PCI) are currently under investigation for the management of GBM patients. Several studies have demonstrated a high degree of synergy between PDT and bleomycin, via the PCI mechanism, in a variety of in vitro and in vivo models, including glioma cell lines. The purpose of this study was to examine the efficacy of combined treatments consisting of PDT and the chemotherapeutic agent, 5-fluorouracil (5-FU) in a 3-dimensional spheroid model consisting of F98 rat glioma cells. Spheroids were incubated with a photosensitizer (aluminum phthalocyanine disulfonate; AlPcS2a) and irradiated with 670 nm laser light. Three different wash protocols (0, 4 and 24 h) were employed to determine whether any observed interactions between PDT and 5-FU could be attributed to the PCI mechanism, or were simply due to different cytotoxic pathways of the two treatment modalities. Although the combined PDT + 5-FU treatments resulted in greater suppression of spheroid growth compared to either treatment alone, no statistically significant differences in growth effects were observed between 0 and 4 h wash protocols suggesting that the combined treatment effects were due to different mechanisms of cytotoxicity, rather than a PCI effect