Renata Brum Martucci

Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines

BackgroundLycopene, a major carotenoid component of tomato, has a potential anticancer activity in many types of cancer. Epidemiological and clinical trials rarely provide evidence for mechanisms of the compound’s action, and studies on its effect on cancer of different cell origins are now being done. The aim of the present study was to determine the effect of lycopene on cell cycle and cell viability in eight human cancer cell lines.MethodsHuman cell lines were treated with lycopene (1–5 μM) for 48 and 96 h. Cell viability was monitored using the method of MTT. The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) and by DAPI.ResultsOur data showed a significant decrease in the number of viable cells in three cancer cells lines (HT-29, T84 and MCF-7) after 48 h treatment with lycopene, and changes in the fraction of cells retained in different cell cycle phases. Lycopene promoted also cell cycle arrest followed by decreased cell viability in majority of cell lines after 96 h, as compared to controls. Furthermore, an increase in apoptosis was observed in four cell lines (T-84, HT-29, MCF-7 and DU145) when cells were treated with lycopene.ConclusionsOur findings show the capacity of lycopene to inhibit cell proliferation, arrest cell cycle in different phases and increase apoptosis, mainly in breast, colon and prostate lines after 96 h. These observations suggest that lycopene may alter cell cycle regulatory proteins depending on the type of cancer and the dose of lycopene administration. Taken together, these data indicated that the antiproliferative effect of lycopene was cellular type, time and dose-dependent.

β‐Carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat‐storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of β‐carotene and retinoic acid (RA), although the direct biogenesis of RA from β‐carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of β‐carotene in HSCs. GRX cells were plated in 25 cm2 tissue culture flasks, treated during 10 days with 3 μmol/L β‐carotene and subsequently transferred into the standard culture medium. β‐Carotene induced a full cell conversion into the fat‐storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high‐performance liquid chromatography for β‐carotene and retinoids. Cells accumulated 27.48 ± 6.5 pmol/L β‐carotene/106 cells, but could not convert it to ROH nor produced retinyl esters (RE). β‐Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 ± 1.23 pmol/L/106 cells), promoting the phenotype conversion. After 24‐h chase, cells contained 20.15 ± 1.12 pmol/L β‐carotene/106 cells and steadily released β‐carotene into the medium (6.69 ± 1.75 pmol/ml). We conclude that HSC are the site of the liver β‐carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin‐A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat‐storing lipocyte phenotype. Circulating retinol is associated with plasma retinol‐binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [3H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high‐affinity membrane receptor for RBP–retinol complex (KD = 4.93 nM), and both cell types expressed a low‐affinity one (KD = 234 nM). The RBP–retinol complex, but not the BSA‐delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein–protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane‐bound retinol. RBP‐delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin‐delivered retinol was mainly associated with membranes. Disturbing the clathrin‐mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo‐RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.

Susceptibility of a continuous murine cell line (GRX) to viral infection

The ability of a murine cell line (GRX) to support viral replication was evaluated. GRX cell cultures were infected with different DNA or RNA viruses. It was observed that the GRX cell line is susceptible to the replication of Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Mayaro virus (MAY), Sindbis virus (SIN), and West equine encephalitis virus (WEE), and can be used as substrate for viral replication studies. Viral replication induced cytopathic effect (CPE) 24-48 h post-infection. The 2.4 5.4 GRX cells yielded infectious virus titers between 10 TCID (Tissue Culture Infectious Dose ) /25 mL and 10 TCID /25 mL 50 50 50 in the first viral passage. These results demonstrate that GRX cells efficiently sustain viral replication and therefore can be used as a valuable tool in the virology laboratory.The ability of a murine cell line (GRX) to support viral replication was evaluated. GRX cell cultures were infected with different DNA or RNA viruses. It was observed that the GRX cell line is susceptible to the replication of Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Mayaro virus (MAY), Sindbis virus (SIN), and West equine encephalitis virus (WEE), and can be used as substrate for viral replication studies. Viral replication induced cytopathic effect (CPE) 24-48 h post-infection. The 2.4 5.4 GRX cells yielded infectious virus titers between 10 TCID (Tissue Culture Infectious Dose ) /25 mL and 10 TCID /25 mL 50 50 50 in the first viral passage. These results demonstrate that GRX cells efficiently sustain viral replication and therefore can be used as a valuable tool in the virology laboratory.

Susceptibilidad de un linaje celular murino continuo (GRX) a la infección viral

The ability of a murine cell line (GRX) to support viral replication was evaluated. GRX cell cultures were infected with different DNA or RNA viruses. It was observed that the GRX cell line is susceptible to the replication of Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Mayaro virus (MAY), Sindbis virus (SIN), and West equine encephalitis virus (WEE), and can be used as substrate for viral replication studies. Viral replication induced cytopathic effect (CPE) 24-48 h post-infection. The 2.4 5.4 GRX cells yielded infectious virus titers between 10 TCID (Tissue Culture Infectious Dose ) /25 mL and 10 TCID /25 mL 50 50 50 in the first viral passage. These results demonstrate that GRX cells efficiently sustain viral replication and therefore can be used as a valuable tool in the virology laboratory.The ability of a murine cell line (GRX) to support viral replication was evaluated. GRX cell cultures were infected with different DNA or RNA viruses. It was observed that the GRX cell line is susceptible to the replication of Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), Mayaro virus (MAY), Sindbis virus (SIN), and West equine encephalitis virus (WEE), and can be used as substrate for viral replication studies. Viral replication induced cytopathic effect (CPE) 24-48 h post-infection. The 2.4 5.4 GRX cells yielded infectious virus titers between 10 TCID (Tissue Culture Infectious Dose ) /25 mL and 10 TCID /25 mL 50 50 50 in the first viral passage. These results demonstrate that GRX cells efficiently sustain viral replication and therefore can be used as a valuable tool in the virology laboratory.