Effects Of CLA On The Growth Of Mammary Cancer Cell Line SKBr3 And Potential Mechanisms

Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids that exist as positional and geometric isomers of linoleic acid (LA; 18:2). CLA possess many physiologic properties including anticarcinogenesis, antiatherosclerosis, antidiabetogenesis, antioxidant, inhibition of body fat accumulation and growth. In addition, CLA modulates immunity and thrombosis as well as fatty acid biochemistry, lipid metabolism and gene expression.Mitogen-activated protein kinases (MAPK) pathway is the most important signal transduction system in cells. Extracellular signal-regulated kinases (ERK) are the clearest member of MAPK family. Its pathway participates in many cell processes including cell growth, development, differentiation and cell division.Cell cycle is the bacis process of cell activities. It contains G1, S, G2 and M phases. The factors that regulate the cell cycle include Cyclin, Cyclin-dependent kinase(CDK), Cyclin-dependent kinase inhibitor(CKI). CyclinDl is the major control switches in the G1 phase. If the expression of CyclinDl is low, the cells can't go through the restriction checkpoint of G1/S and stop in the phase of G1. P27 is a kind of CKIs that binds to Cyclin and CdK blocking the cell move from G1 to S phase.From the beginning of 1970's, the incidence of mammary cancer is rising in Asian countries. In China, especially in developed regions, mammary cancer has become the major malignant tumor that threatens women's health.In our experiments, we adopt mammary cancer cell line SKBr3 to compare the effects of CLA and LA on growth and cell cycle of cancer cells. The characteristics of ERK signal transduction pathway, Cyclin and CKI will provide experimental basement for further understanding the mechanisms of CLA on tumor. Accordingly, the research is also beneficial for us to prevent and cure tumor by using foods rich in CLA or medicine made of effective components of CLA. MethodsIn vitro, SKBr3 cells were incubated with different concentrations (50,100,200 umol/L) of CLA and LA. The growth of the cells was examined by cell counting method and MTT assays; Cells morphological changes were detected under light microscope and transmission electron microscope; The cell cycle was investigated by flow cytometry; ERK 1/2, p-ERKl/2, CyclinDl and P27 were detected by western blotting. ResultsCell counting method and MTT assays showed that CLA inhibited the proliferation of SKBr3 cells significantly in a dose and time-dependent manner. Five days after treatment with CLA at the dose of 200,100 and 50 mol/L, the inhibitory rates were 75.0%, 57.9% and 33.1% respectively. The inhibitory rates of LA groups were 38.4%, 25.4% and 10.1%. Electron microscopy observed typical morphological changes of apoptosis cells in CLA groups. Such changes were not detected in LA groups. Flow cytometry showed that the cell cycle of SKBr3 cells might be inhibited in GI phase by CLA. The G1 phase was 72.2% on the 5th day after treatment with CLA at the dose of 200 mol/L. That of control group was 50.6%. Western blotting indicated that CLA significantly decreased phosphorylation of ERK1/2. The levels of ERK1/2 and p-ERKl/2 had decreased 79.5% and 86.2%(P<0.05). The expression level of CyclinDl treated with 200 mol/L CLA on the 4th day wasreduced 87.4%(n=3, P<0.05), but there was only a moderate effect of CLA on the increase of P27 protein. Compared with that of control group, P27 level of CLA group had increased 1.7 times(n=3, P<0.05). ConclusionsOur research showed that CLA inhibited the proliferation of SKBr3 cells significantly in a dose and time-dependent manner. Induction of apoptosis might be the mechanisms of anticarcinogenesis by CLA. ERK protein, especially phosphorylation of ERK are the critical agents involving in growth and differentiation of cells. CLA decreased the expression of ERK It was hinted that reduction of cell proliferation by CLA might be associated with ERK signal transduction pathway. CLA reduced the expression of CyclinDl greatly. In addition, CLA moderately increased...