The Anti-tumor Effect And Mechanism Of Blocking Fatty Acid Synthesis And Uptake On Breast Cancer Cell

Breast cancer is one of the most common neoplasms and the leading cause of cancer-related deaths in women worldwide.Since the 1990 s,the incidence of breast cancer in China has increased more than twice as many around the world.Nowadays,the prevention and treatment of breast cancer is of importance.As other malignant tumors,the formation,proliferation and metastasis of breast cancer are the result of the interaction of multiple genes and multiple factors.In addition to genetic factors such as heredity,age,menstrual history,marriage history and other known deterministic risk factors,epidemiological studies have shown that obesity and high fat diet are closely related to the development of breast cancer.Obesity is not only a risk factor for breast cancer,but also associated with poor prognosis of breast cancer patients.Indicating that the occurrence and development of breast cancer and body fat metabolism is closely related.This suggests that the occurrence and development of breast cancer is closely related to lipid metabolism.Moreover,as is known,Cancer cells exhibit metabolic alterations characterized by increased glycolysis and lipogenesis,which meet the need of macromolecules and energy to support rapid growth and proliferation of cancer cells.The most important and fundamental role of FAs is as building blocks for newly-synthesized membrane phospholipids.Besides,FAs can also be used to produce protumorigenic signalling lipids and be used for mitchondrial ?-oxidation to produce ATP.De novo synthesis is the primary way for tumor cells to obtain FA.Thus,the FA synthesis pathway has been an attractive cancer target for quite some time,and attentions are primarily focused on FA synthase with the production of long-chain SFAs.However,MUFAs play a more important role in dynamic or rapidly dividing cancer cells and are a major precursor to form membranelipids including phospholipids,cholesterol esters,and glycerides and can also function as signaling molecules.SCD1 is the critical enzyme in synthesis of MUFAs,oleic and palmitoleic acid,from SFA,stearic and palmitic acid.SCD1 may represent a final rate-limiting point in FA synthesis pathway.Evidences suggest that the expression of SCD1 increased abnormally in many metabolic disorders including fatty liver,diabetes,obesity and some types of tumors.SCD1 may be closely related to lipid metabolic disorders even the key point of these disease.Furthermore,in anatomy of mammary gland,adipocytes represent one of the most prominent cell types,thus,cancerous breast glands are embedded in the mammary fat pad.Mammary adipocytes store and secrete FAs,adipokines,and have a potential to influence neighboring cells by paracrine and endocrine mechanisms.Mammary adipocytes appear capable of translocating stored lipids to breast cancer cells as another key source of FAs.Adipocytes can produce large amounts of long chain FA(LCFA)through fat mobilization.Evidence shows that FAs especially LCFAs are actively transported across cell membrane by specialized proteins instead of passive diffusion.The protein-mediated import of LCFAs is of greatest significance when the metabolic requirements for LCFAs are high or when the level of FFAs is low.Although,several proteins have been implicated in facilitating FA uptake,CD36 is the best characterized as a FA translocase which enhance LCFA uptake by overexpression or translocation from intracellular stores to the plasma membrane.Accordingly,we hypothesize that besides de novo lipogenesis,breast cancer cells can also uptake exogenous FAs via transmembrane channel FA translocase—CD36.The therapeutic efforts aimed to starve cancer cells to death should suppress both FA synthesis and uptake pathways.SCD1 not only regulates the cell lipid synthesis,but also plays an important role in the maintenance of malignant phenotypes of tumor cells.However,the complex mechanism of SCD1 regulating lipid metabolism and biological characteristics of tumor cells has not been fully elucidated.Cellgrowth and proliferation depend on the regulation of anabolism and catabolism.In mammalian cells,two major signaling proteins Akt and AMP-dependent protein kinases(AMPK)regulate a number of anabolism and catabolism.The process of lipid synthesis is also regulated by Akt and AMPK pathways.Therefore,we further explore the mechanism of Akt and AMPK pathways in SCD1 induced proliferation in breast cancer cells,and provide a new direction targeting lipid metabolism for anti-tumor treatment.Part 1 The anti-proliferation effect of SCD1 inhibitor on breast cancer MCF-7 cells and the rescue effect of oleic acidObjective: To study the anti-proliferation effect of SCD1 inhibitor MF-438 on MCF-7 cells,and to find a new target for breast cancer treatment.Methods: MCF-7 cells were treated with MF-438 from 100 nmol/L to100 ?mol/L in 10% or 2% FBS conditions or in the absence or presence of oleic acid(OA)or palmitic acid(PA),cell viability was measured after 48 h using MTS assay and the IC50 values of MF-438 under different culture conditions were calculated.Cells were treated with 5?M MF-438 or DMSO in the absence or presence of 100 ?M OA for 48 h.Apoptosis morphology was observed by fluorescence microscope after Hoechst 33342 staining and cell apoptosis and death rate was determined by PI staining using flow cytometry.Cell cycle distribution was determined by PI staining using a flow cytometer.Cell migration ability was measured by scratch closure rate.Results:1 MF-438 showed a significant dose dependent proliferation inhibition in MCF-7 cells in 10% or 2% FBS conditions.Under 10% FBS condition,the IC50 value was determined to be 16.7 ?mol/L,whereas the IC50 in 2% FBS was 3.7 ?mol/L.Supplementation with OA showed a dose dependent rescue of cell viability to MF-438 treatment,whereas addition of PA produced a modest viability reduction.2 The typical apoptotic morphology was observed in most MCF-7 cells treated with MF-438 including cell shrinkage,chromatin condensation,marginalization and fragmentation as well as the apoptotic bodies.Furthermore,the result of PI staining indicated that the rate of late apoptosis and death cells was significantly higher in MF-438 treated group than DMSO control(P<0.05).The apoptotic cells were significantly decreased and cell death rate was reduced after OA addition.3 MF-438 treatment caused a significant reduction in the population of cells in S phase and G2/M phase,and a concomitant increase in the percentage of cells in G0/G1 phase compared to DMSO control(P<0.05).However,the populations of cells in S phase,G2/M phase and G0/G1 phase were all reversed by OA supplement in MF-438 treated group compared to DMSO control.4 The scratches of the cells treated with MF-438 were wider than DMSO control at the same time point(P<0.05).However,when OA was added in cells treated with MF-438,the scratches became significantly narrower compared to MF-438 group.Conclusion:1 Blockade in SCD1 activity can not enable MCF-7 cells to get through the early stages of the cell cycle,lead cells to apoptosis and made cells migrated more slowly.2 Exogenous OA markedly reversed the effect caused by SCD1 inhibition.Part 2 CD36 depletion prevents oleic acid from reversing the antitumor effect of SCD1 inhibitionObjective: To study the effect of SCD1 inhibitor on the proliferation of breast cancer MCF-7 cells after CD36 knockdown.To explore ways in which breast cancer cells obtain fatty acid,so as to block the source of fatty acid acquisition and achieve a stable and lasting anti-tumor effect.Methods: Construct tissue microarray and then the expression of SCD1 and CD36 protein were detected by immunohistochemistry techniques in human breast cancer tissues and normal breast tissues.MCF-7 cells were transfected with siRNA targeting human CD36 gene.MCF-7 cells or cells transfected with CD36 siRNA were cultured with 100 ?M OA for 48 h,thenstained with Oil Red O for intracellular lipid deposits detection.CD36 depleted MCF-7 cells was treated with MF-438 from 100 nmol/L to 100?mol/L in the absence or presence of 100 ?M OA,cell viability was measured after 48 h using MTS assay.CD36 depleted MCF-7 cells were incubated with5 ?M MF-438 or DMSO for 48 h in the absence or presence of 100 ?M OA.Apoptosis morphology was observed by fluorescence microscope after Hoechst 33342 staining and cell apoptosis and death rate was determined by PI staining using flow cytometry.Cell cycle distribution was determined by PI staining using a flow cytometer.Cell migration ability was measured by scratch closure rate.Results:1 SCD1 exhibited diffuse cytoplasmic staining and highly expressed in nearly all breast cancer samples,whereas it was low or no expression in adjacent normal breast tissues.Similarly,the majority of breast cancer samples also stained for CD36 mainly at the plasma membrane,part of the breast cancer showed cytoplasmic staining and a small part of breast cancer was low expression,whereas normal breast cancer tissue is not expressed.2 The content of red lipid droplets in MCF-7 cells was decreased in 2%FBS condition,and more cytoplasmic lipid droplets accumulated in MCF-7cells but not in CD36 silenced MCF-7 cells after OA supplementation.3 MF-438 treatment also exhibited dose-dependent inhibition of cell proliferation.CD36 depletion treatment slightly decreased cell proliferation compare with normal MCF-7 cells.However,supplementation with oleic acid did not show an obvious rescue effect of cell viability to MF-438 in CD36 depleted MCF-7 cells.4 The typical apoptotic morphology was found in most CD36 depleted MCF-7 cells treated with MF-438.PI staining showed that the rate of late apoptosis and death cells was significantly higher when CD36 silenced MCF-7 cells treated with MF-438(P<0.05)whether oleic acid was added or not.5 A significant reduction in the population of CD36 depleted cells in Sphase and G2/M phase with MF-438 treatment compared to siRNA-CD36control(P < 0.05),and a concomitant increase in the percentage of G0/G1phase(P<0.05).OA supplement did not alter the cell cycle profile in Oleic acid combined with MF-438 treatment compared to MF-438 treatment alone(P>0.05).6 The scratches of the CD36 silenced MCF-7 cells treated with MF-438 were wider than the DMSO control(P < 0.05).When oleic acid was supplemented,There were no obvious changes in the width of the scratches compared to MF-438 treated group(P>0.05).Conclusion:1 Breast cancer MCF-7 cells can uptake FA via CD36,as an alternative source of FA acquisition,to meet the needs of proliferation.2 When the CD36 gene is silenced,the anti-tumor effect of SCD1 inhibitor can not be reversed by exogenous OA in inhibiting cell growth and proliferation,inducing apoptosis,blocking cell cycle and inhibiting cell migration.3 SCD1 inhibition combined with CD36 silencing has a synergistic anti-tumor effect on breast cancer MCF-7 cells.Part 3 MF-438 inhibits the proliferation of breast cancer cells through Akt and AMPK pathwaysObjective: To investigate the role of Akt pathway and AMPK pathway in the anti-tumor effect of SCD1 inhibitor on breast cancer MCF-7 cells and to explore the mechanism of SCD1 in breast cancer.Methods: MCF-7 cells were treated with 5?M SCD1 inhibitor MF-438 for 48h,then western blot was used to detect the expression of total Akt,pAkt,SREBP-1,pAMPK and pACC.Results:1 The expression of pAkt and SREBP-1 was significantly decreased compared with control group(P <0.05),and the ratio of p Akt to total Akt decreased by about 59% and SREBP-1 decreased by 38%(P<0.05).The expression of pAkt increased with the addition of OA in the medium(P <0.05),whereas p Akt was further decreased after adding PA without statistically significant(P>0.05).2 Compared with control group,the expression of pAMPK increased about 65% and p ACC increased about 74% after MF-438 intervention,the difference was statistically significant(P<0.05).After OA supplement,the expression of pAMPK and pACC were significantly reduced compared to MF-438 alone group(P <0.05).While exogenous PA did not significantly alter the expression of pAMPK and pACC.Conclusion:1 SCD1 inhibitor down-regulates Akt pathway by reducing Akt phosphorylation,then decreases the expression of SREBP-1,thereby down-regulating FA synthesis.Exogenous OA can reverse the reduction of Akt phosphorylation caused by SCD1 inhibitor,then activate Akt pathway and promote cell proliferation,but PA has the opposite effect.2 SCD1 inhibitors up-regulates AMPK pathway by increasing AMPK phosphorylation,increase the inactivation form of ACC phosphorylation,thereby down-regulating FA synthesis.Exogenous OA reduces phosphorylation of AMPK,induced ACC dephosphorylation,then restored FA synthesis,thereby partially reverse SCD1 inhibitor induced FA synthesis reduction.While PA further reduced FA synthesis,but did not significantly alter the expression of pACC and pAMPK.