Effects of curcumin on the gene expression profile of L-02 cells
- Authors:
- Published online on: May 4, 2015 https://doi.org/10.3892/br.2015.460
- Pages: 519-526
Abstract
Introduction
Curcumin [1,7-bis (4-hydroxy-3-methoxypheny1)-1, 6-heptadiene-3,5-dione, C21H20O6] is a hydroalcoholic extracted compound of the traditional Chinese medicine, Rhizoma Curcumae Longae, which has several essential biological functions, including anti-inflammatory, antioxidant, anticarcinogenic, hypercholesterolemia and cardioprotective activities (1–3). Animal studies have suggested that curcumin has a wide range of potential therapeutic effects. Aggarwal et al (3) reported that curcumin, which is responsible for the majority of the biological activity of turmeric, has been used historically as a component of Indian Ayurvedic medicine to treat a wide variety of ailments. Curcumin has been used extensively to treat cardiovascular diseases (4) and cancer (2). According to recent studies, curcumin is an active lipid-mediating compound that can significantly decrease the level of serum lipid peroxides, increase high-density lipoprotein cholesterol (HDL-C) and decrease the total serum cholesterol (5,6).
With the development of microarray technology, it is possible to simultaneously detect the expression of millions of target genes (7). In a previous study, microarray technology was used to detect the differential gene expression of animals treated with curcumin (8). However, the effects of curcumin treatment on the profiles of total gene expression in vitro are seldom reported.
Further studies have exhibited more pharmacological roles of curcumin. There is extensive experimental evidence that curcumin is an important factor involved in the initiation and development of numerous diseases by regulating the expression of genes or activating signaling pathways, but only a few of the functional genes regulated by curcumin are assessed and reported. Jain et al (9) reported that the liver was considered as the major organ responsible for the metabolism of curcumin. Accordingly, it is desirable to compare the gene expression profile on hepatocyte cells L-02 treated with and without curcumin in order to identify the molecular mechanism and action sites of curcumin at the cellular level, subsequently gain highly specific and sensitive drug action marker genes of curcumin. The aim of the present study was to provide the gene expression and possible gene signaling pathways affected in L-02 cells by curcumin treatment.
Materials and methods
Reagents and chemicals
Curcumin was obtained from the Delta Information Centre for Natural Organic Compounds (Xuacheng, Anhui, China). The L-02 cell line was provided by Shanghai Cell Bank, China Academy of Science. Cell culture medium was from Gibco Invitrogen Life Technologies (Carlsbad, CA, USA). TRIzol was purchased from Invitrogen Life Technologies. The two-step reverse transcription-polymerase chain reaction (RT-PCR) kit obtained from Hangzhou Bioer Technology Co., Ltd. (Hangzhou, China); and reagents regarding DNA microarray and standard protocols were provided by KangChen Bio-tech Inc. (Shanghai, China).
Culture of L-02 and total RNA extraction
i) L-02 hepatocyte cells were seeded in two 25-cm2 flasks and cultured in RPMI-1640 medium with 10% bovine calf serum in a 5% CO2 atmosphere at 37°C. The medium was refreshed every 24 h. When the cell density reached ~80% confluence, test samples were treated with the medium containing 20 µM curcumin and the control samples were treated with normal medium. ii) After 24 h, total RNA was extracted using the TRIzol reagent according to the manufacturer's instructions. TRIzol reagent (1 ml) was added to each flask and homogenized with a homogenizer. iii) Following extraction with chloroform, RNA was precipitated with isopropanol. The resultant pellet was finally re-suspended in RNAase-free water. iv) Following a DNase digestion step, RNA quantification and quality assurance were assessed by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) at 260/280 nm and RNA integrity and genome DNA contamination were tested by denaturing agarose gel electrophoresis.
Microarray analysis
Microarray chips, associated protocols and equipment for the processing of these chips were from KangChen Bio-tech Inc. Probe synthesis from the total RNA samples, hybridization, detection and scanning were performed according to standard protocols.
i) First and second strand cDNA were synthesized from total RNA using the two-step RT-PCR kit according to the manufacturer's instructions. Following this, aRNA was synthesized according to recommended instructions by KangChen Bio-tech Inc. ii) Following purification of aRNA, the solution was transferred to NucAway Spin Columns and the aRNA was labeled using CyDye according to the manufacturer's instructions. iii) The efficiency of aRNA labeling was assessed by NanoDrop ND-1000. The efficiency of aRNA labeling = concentration of aRNA labeling/total aRNA. iv) The labeled aRNA was hybridized with a highly sensitive Homo sapiens array according to the standard protocol. Following hybridization, the arrays were washed in a GeneChip Fluidics Station 450. v) After washing and drying, hybridization signals derived from CyDye were scanned with a GenePix 4000B and processed with Agilent array. vi) Subsequent further analysis, including data preprocessing and normalization, differential, statistical and cluster analysis, were performed by software carried in GenePix 4000B.
Results
The total RNA with A260/A280 value >1.5 was accepted for subsequent use in the DNA chip analysis. The OD260/OD280 of the test sample was 1.87 while that of the control sample was 1.56, reaching the standard required.
The aRNA labeling with an A260:A280 value between 1.8 and 2.1 and an efficiency of aRNA labeling of >0.9 was accepted for subsequent use in the DNA chip analysis. The efficiency of aRNA labeling treated with curcumin was 0.96 and the control sample was 0.93, while the quantification and quality of aRNA reached the accepted standard of GeneChip analysis.
The test and control samples were examined by GeneChip and each sample was tested in triplicate to reduce technical errors. Altogether, 6 GeneChips were used to examine the gene expression profile of L-02 cells. Genes with 5-fold higher expression compared to the control were regarded as upregulated genes, while genes with 0.33-fold lower expression were regarded as downregulated genes.
All gene expression is included in Table I. A total of 80 genes was found to be significantly differentially expressed, of which 47 (58.8%) were upregulated and 33 (41.3%) downregulated; 21 (26.3%) were associated with cancer, cell cycle and cell differentiation and 4 (5%) were associated with lipid metabolism and immunoreactions. These genes were classified based upon their functions and primary biological processes by referring to the National Center for Biotechnology Information.
Discussion
Using DNA microarray, the differences in gene expression profiling of human L-02 cells treated with curcumin compared to the control sample were identified. Future studies should clone and analyze these functional differential genes. After comparing the differential expression of the two samples, the data of the present study indicates the same conclusions as certain other research studies regarding the effects of curcumin, such as the study by Kakar and Roy (10) on the antitumor effect of myc and extensive cardioprotective effects against diabetic cardiovascular complications reported by Hong et al (8). Other novel potential marker genes are also revealed by the present study, such as leption and oxysterol-binding protein-like 7.
Curcumin is capable of developing physiological reactions and functions by regulating the expression and affecting the signal transduction pathway.
The KIAA0937 protein is a regulator of the notch signaling pathway involved in cell-cell communications that regulates a broad spectrum of cell-fate determinations (11). Following treatment with curcumin, the expression of the KIAA0937 protein was only 29.66% of the control sample, which indicates that curcumin reduced the communication between cells, which may be involved in suppressing metastasis.
Binding of platelet-activating factor (PAF) to the PAF receptor stimulates numerous signal transduction pathways, including phospholipase C, D, A2, mitogen-activated protein kinases and the phosphatidylinositol-calcium second messenger system (12). From the data of the DNA chip, curcumin upregulated the expression of platelet-activating factor receptor (PTAFR). With the increased level of PTAFR, PAF participates in these physiological processes more effectively. Following PAF stimulation, PTAFR becomes phosphorylated, internalized and downregulated, and subsequently the downstream pathway is affected, such as platelet aggregation and degranulation, inflammation and anaphylaxis (13).
RAB1A is a member of the Ras superfamily of guanosine triphosphatases (GTPases) and functions through binding GTP and guanosine diphosphate and possessing intrinsic GTPase activity (14). Phosphorylation and downregulation of RAB1A by curcumin are involved in numerous biological activities. Once these signaling pathways are activated, various physiological phenomena are improved and diseases may be cured.
Curcumin also regulated a few of the genes functioning as transcriptional activators or transcriptional repressors. Zinc fingers and homeoboxes 1 is a member of the zinc fingers and homeoboxes gene family, which belongs to nuclear homodimeric transcriptional repressors. By regulating these transcriptional factors, diseases are cured following different expression levels of proteins associated with vital physical activities.
In addition to the differentially expressed genes involved in the signaling pathways presented above, there are certain potential marker genes associated with diseases. In conclusion, these genes were classified and concluded according to the effects of curcumin on diseases.
Extensive research has indicated that the antitumor mechanism of curcumin may include the induction of apoptosis of tumor cells, inhibiting various tumor cell growth and proliferation, preventing tumor invasion and metastasis. As a potential anticancer drug, curcumin regulates 21 genes associated with cell cycle and its regulation, differentiation, proliferation and apoptosis. These genes provide further insights into cancer and the likely effects of modifying the encoded targets therapeutically.
Tumor protein p63 (TP63) encodes a member of the p53 family of transcription factors, which functions as a tumor suppressor that is involved in preventing cancer. TP63 may combine with TP73/p73 for initiation of p53/TP53-dependent apoptosis in response to genotoxic insults and the presence of activated oncogenes (15). TP63 may be involved in the notch signaling pathway by possibly inducing Jagged1 (JAG1) and JAG2, and may play a role in the regulation of epithelial morphogenesis (16). The higher expression of TP63 indicates curcumin induced apoptosis of tumor cells and regulated the epithelial tissue and cells by the notch signaling pathway.
Chen and Tan (17) reported that curcumin plays a role in the inhibition of cell growth through inhibiting the c-Jun N-terminal kinase (JNK) pathway and the data of the DNA chip analysis also shows that the mitogen-activated protein kinase binding protein 1 is upregulated by curcumin, which is involved in the JNK signaling pathway to induce apoptosis.
MYC-associated factor X is a member of the basic helix-loop-helix leucine zipper family of transcription factor (18). It is able to form the MYC-MAX complex. Myc is an oncoprotein involved in cell proliferation, differentiation and apoptosis. According to the Kakar and Roy (10) study, the expression of mRNA of myc is lowered only 60% following incubating with 10 nmol curcumin. In the present study, the expression of the myc gene in the sample with curcumin treatment is only 21.95% compared to that of the control sample, which proves the same conclusion that curcumin may inhibit the growth of tumor cell by downregulating the myc gene.
There is extensive evidence indicating that the caspase family is essential in cell apoptosis or programmed cell death. The DNA chip analysis reveals that curcumin downregulated the expression of WD repeat domain 35, which functions in the caspase pathway. The same conclusion is reported by Khar et al (19) that the antitumor activity of curcumin functions through the caspase pathway mediated by the induction of apoptosis.
Protein kinase is considered as an extremely important target in the study of antitumor drug. According to Reddy and Aggarwal (20), curcumin may inhibit the activity of a variety of protein kinases involved in tumor cell growth. The DNA chip reveals that numerous curcumin-sensitive genes are associated with protein kinases, such as myotubularin-related protein 4, which functions in phosphorylated on Ser, Thr and Tyr residues and lowered molecular weight phosphatase substrate para-nitrophenylphosphate (21) and GTPase-activating protein binding protein 1, which may be a phosphorylation-dependent sequence-specific endoribonuclease and involved in the Ras-GTPase pathway by binding specifically to the Ras-GTPase-activating protein (22). The microarray results provide evidence for the Reddy and Aggarwal (20) study that curcumin is an inhibitor of phosphorylase kinase.
The data of the DNA chip showed that curcumin may upregulate heat-shock cognate protein, a member of the heat-shock protein 70 gene family. These proteins play a role in cell proliferation, cell protection, apoptosis and antitumor immune therapy stress response and maintenance of the mitochondria (23). A recent study has proved that heat shock 70 kDa protein 9 (HSPA9) has been shown to interact with fibroblast growth factor 1 (24) and p53 (25), which indicated that HSPA9 and its pathway are promising targets for tumor therapy.
In experiments on tumors, curcumin inhibits the growth of new cancers directly and irreversibly, and it also suppresses the onset of tumors, as well as their growth and metastasis. The present data suggest that curcumin may make important contributions to prevent cancer and decrease cancer mortality through normalizing the expression of tumor-relevant genes.
Curcumin also showed cardioprotective effects by decreasing the level of blood lipid. There are 4 primary genes associated with cardiovascular and cerebrovascular disease that are regulated by curcumin.
A previous study indicated the key contribution of apolipoproteinB (apoB) to atherogenesis as the main inductor of its earlier steps. The apoB receptor is one of the most important elements regarding cholesterol metabolism, which is also a curcumin-sensitive element. The apoB receptor is involved in biological processes, such as cholesterol, lipid and steroid metabolism, and lipid transport, while apoB receptor could bind lipoprotein containing apoB48 and apoB100 (26). The present study shows that the level of apoB receptor in L-02 cells treated with curcumin was upregulated 26.07 times compared to the control, which supports considerable evidence for the previous experimental results that curcumin has enough capability to lower the level of low-density lipoprotein (LDL) cholesterol, as well as triglycerides and inhibit LDL oxidation (27) and it would indeed be the novel potential drug in hypercholesterolemia or atherosclerosis therapy. Another study shows that a daily oral administration of the curcumin extract significantly decreases LDL and apoB and increases HDL and apoA of healthy subjects (28).
In conclusion, the present DNA chip data and previous finding of a lowering effect of curcumin on blood lipid and lipoprotein peroxide justifies further study to investigate the molecular mechanism and highly sensitive target of curcumin. Curcumin may be useful as an anti-atherogenic agent.
Inflammation and other immune reactions effect almost all disease progression. Atherosclerosis is an inflammatory disease and the inflammatory cell is an indispensable participant in the neoplastic process, fostering proliferation and survival. According to the data of the DNA chip and research regarding curcumin, curcumin plays an important role in various immune reactions and inflammatory processes.
The data show that curcumin may increase the expression of peptidogly recognition protein 1 (PGLYRP-1), one of the pattern receptors, which is part of the innate immune system and binds to peptidoglycan, a component of bacterial cell walls that has also been found in human atherosclerotic lesions (29). Rohatgi et al (30) found that chronic exposure to bacterial antigens may cause or exacerbate the inflammatory response to lipid deposition within arterial walls, so PGLYRP-1 levels were not only involved in innate immunity, but were also independently associated with atherosclerosis phenotypes. Therefore, it was also suggested that an association may exist between innate immunity and atherosclerosis, and we could draw a conclusion that the upregulation of PGLYRP-1 by curcumin means that curcumin not only prevents bacterial invasion and strong inherent immunity, but also slows down the development of atherosclerosis.
Leptin protein may be involved in the interaction of cytokine receptors, which function as a regulator of immune and inflammatory responses, hematopoiesis, angiogenesis and wound healing (31). As db/db mice have defects of the leptin receptor gene, the leptin protein may be involved in obese type II diabetes with high blood sugar, high cholesterol, insulin resistance and other characteristics. Therefore, the lower expression of leptin protein in the curcumin-treated sample indicates that curcumin may function in reducing cholesterol and blood sugar, reversing insulin resistance.
In the present study, a microarray approach was employed to identify the potential effects of curcumin. The results of the gene chip suggested that curcumin possesses a broad spectrum of biological activities, which includes inhibiting tumor cell growth, antioxidant, anti-inflammatory agent and antithrombotic agent. The biological functions of curcumin are the same as the majority of previous research findings, and additionally, other novel and potential targets spots and pathways have been revealed. All these data construct a genes database regarding curcumin. With further research regarding curcumin, the genes database will be completed and available. From the present study, we can accurately position the target genes and pathway, which supports an overall background of the function of curcumin and make our further research more advisable and appropriate.
The data of the study not only indicated the same conclusion as certain other studies regarding the effects of curcumin, but also revealed novel potential marker genes.
Acknowledgements
The present study was supported by the Foundation of Zhejiang Provincial Natural Science (grant nos. Y2090891 and LY14H290007), Science Foundation of TCM Administration Bureau of Zhejiang Province (grant no. 2010ZB027) and the Zhejiang Provincial Key Laboratory project (grant no. 2012E10002). Thanks to Mr. Mike Genko Berger (USA) for improving the article language.
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