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Potentiation of drug-induced cytotoxicity by conjugated linoleic acids (CLA) in in vitro models of drug-resistant cancer
Abstract
Preclinical studies indicating that conjugated isomers of linoleic acid suppress mitosis, restore apoptosis, induce differentiation and suppress angiogenesis in animal models of cancer formed the rationale for an in vitro investigation of their potential benefits in targeting drug-resistant cancer cells. Additionally, several lines of evidence implicate a role for sphingolipids in multiple drug resistance. The objective of this study was to evaluate the effects of conjugated linoleic acids (CLA) and various anticancer drugs on growth of MCF-7 and MCF-7/cis cell lines and to evaluate the effects of co-treatments
of CLA and doxorubicin (Dox) on cell growth, intracellular drug accumulation, sphingolipid content and cellular lipid composition in a drug-resistant melanoma model.
Initially an investigation of the potential of co-treatment with a commercial mixture of CLA isomers (CLA-mix) and two single isomers c9,t11-CLA and t10,c12-CLA to enhance the cytotoxic effects of Doxorubicin (Dox) on a drug naive MDA-MB435-S-F (MDA) cancer cell line was carried out. It was found that each preparation of CLA enhanced cytotoxicity of Dox in a dose dependant manner with an increase of over 3-fold with 6 μg/ml CLA-mix. Similarly, both c9,t11- and t10,c12-CLA purified isomers yielded an increase in doxorubicin cytotoxicity of up to 3 fold. Toxicity assays carried out on a Dox-pulsed MDA cell line, MDA-MB435-S-F/Adr10p10p (A10p10p), demonstrated a similar pattern of enhanced toxicity with up to 5, 7 and 12-fold increase when co-treated with CLA-mix, c9,t11-CLA and t10,c12-CLA respectively. Dox retention as determined by liquid chromatography tandem mass spectrometry (LCMS)
was significantly enhanced by the CLA-mix in A10p10p cells and by c9,t11-CLA in MDA and A10p10p cells. Analysis of sphingomyelin metabolites by High Performance Liquid Chromatography (HPLC) revealed that CLA treatments in combination with Dox increased ceramide levels in both cell lines compared with Dox alone. Treatment of both MDA and A10p10p cells with CLA-mix resulted in down-regulation of Her2/neu coded p185 c-erbB2/c-neu oncoprotein expression. Eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) resulted in similar down-regulation of this protein in the
A10p10p cell line. Fatty acid composition of MDA and A10p10p cells was analysed by gas chromatography (GC). There was a marked increase in the unsaturation index of
both cell lines following all fatty acid treatments, suggesting inhibition of fatty acid synthase (FAS) activity, a key enzyme in the de novo synthesis of fatty acids. With the exception of the CLA-mix in the resistant line, all fatty acids in both cell lines caused a
decrease in the ratio of C18:1/18:0 suggesting the D 9 desaturase enzyme stearoyl-CoA desaturase (SCD) as a potential target for inhibition by fatty acid treatments. The data suggest that CLA-induced changes in ceramide status, intracellular drug accumulation and lipid profiles may underlie the enhanced chemosensitivity of the resistant cell line and represent a new avenue to develop chemotherapy regimes for patients with drugresistant
tumours leading to improved response and survival rates in these patients.
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Purpose: In preclinical studies, sequential exposure to irinotecan (CPT-11) then fluorouracil (5-FU) is superior to concurrent exposure or the reverse sequence; a 24-hour infusion of CPT-11 may be better tolerated than shorter infusions.
Experimental Design: CPT-11 was first given at four levels (70-140 mg/m²/24 hours), followed by leucovorin 500 mg/m²/0.5 hours and 5-FU 2,000 mg/m²/48 hours on days 1 and 15 of a 4-week cycle. 5-FU was then increased in three cohorts up to 3,900 mg/m²/48 hours.
Results: Two patients had dose-limiting toxicity during cycle 1 at 140/3,900 of CPT-11/5-FU (2-week delay for neutrophil recovery; grade 3 nausea despite antiemetics); one of six patients at 140/3,120 had dose-limiting toxicity (grade 3 diarrhea, grade 4 neutropenia). Four of 22 patients with colorectal cancer had partial responses, two of which had prior bolus CPT-11/5-FU. The mean 5-FU plasma concentration was 5.1 μmol/L at 3,900 mg/m²/48 hours. The end of infusion CPT-11 plasma concentration averaged 519 nmol/L at 140 mg/m²/24 hours. Patients with UDP-glucuronosyltransferase (UGT1A1; TA)6/6 promoter genotype had a lower ratio of free to glucuronide form of SN-38 than in patients with ≥1 (TA)7 allele. Thymidylate synthase genotypes for the 28-base promoter repeat were 2/2 (13%), 2/3 (74%), 3/3 (13%); all four responders had a 2/3 genotype.
Conclusions: Doses (mg/m²) of CPT-11 140/24 hours, leucovorin 500/0.5 hours and 5-FU 3,120/48 hours were well tolerated.