Effects Of Oxidized Low-Density Lipoprotein By Thymoquinone In Lipidloaded Mcf-7 Breast Cancer Cells
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Date
2016-07
Authors
Abdul Hamid, Auni Fatin
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Abstract
Breast cancer is the leading cause of death among women worldwide and
recent studies revealed that oxidized low-density lipoprotein (ox-LDL) contributed to
increased risk of breast cancer. To date, thymoquinone (TQ) is widely studied both in
vivo and in vitro in cancer pathogenesis, but there is lack of reports on its lipid
regulation in cancer cells. Thus, the objectives of this study were to elucidate the
effects of ox-LDL on breast cancer cell growth as well as the molecular mechanisms
of lipid metabolism and cell proliferation regulated by TQ. Cytotoxicity of TQ was
analyzed using MTS assay. MCF-7 cells were exposed to 10 μg/ml of ox-LDL,
followed by treatment with 20 μM of TQ for time-dependent study. Localization and
expression of target proteins were studied through immunofluorescence and Western
blot methods. This was followed by qRT-PCR analysis on genomic expression of
VAMP4 and EGLN1. The IC50 value for 24, 48 and 74 hours of TQ treatment was 20
μM, 24 μM and 10 μM respectively. Ox-LDL caused localization of Bcl-2 in the
nucleus, whereas NFκB remained in the cytoplasm of cells after treatment. This
indicated that activation of NFκB was inhibited by ox-LDL. Further analysis showed
that ox-LDL induced MCF-7 cell proliferation through up-regulation of Bcl-2 and
NFκB expression, whereas in FASN, ox-LDL reduced its expression. After treatment
with TQ for 72 hours, LDLR and NFκB showed more reduction in their expression
compared to the other two proteins studied. In addition, FASN was the only protein
that indicated significant inhibition by TQ compared to its expression in native cells
(p-value < 0.05). Further analysis through qRT-PCR experiments revealed that TQ
significantly up-regulated VAMP4 expression at 72 hours TQ treatment (p-value <
0.05) in response to the inhibition of FASN and Bcl-2. EGLN1 level was reduced at
48 hours TQ treatment but the fold change was lower compared to VAMP4. Taken
together, this study provides insight into molecular mechanism of ox-LDL-induced
cell growth as well as regulation of TQ in lipid-loaded cells. The roles of LDLR,
FASN and VAMP4 in lipid-loaded cells could be further studied through their gene
knockdown using siRNA technique and TQ may also be examined in combined
therapy with the existing ER-positive chemotherapeutic agent in cell culture model.
For future studies, it is recommended to use confocal scanning microscope to study
the localization of a protein by scanning many thin sections through a sample to
obtain a clear three-dimensional fluorescent image due to TQ effect.
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Keywords
The effects of ox-LDL on breast cancer cell growth , as well as the molecular mechanisms.