Chinese Journal of Cancer Research 17(3):203-206, 2005
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GROWTH-INHIBITORY EFFECTS OF CURCUMIN ON Raji CELLS AND ITS MECHANISMS :1 * CHEN Yan (~,~,~,),
WU Qing ( ~ ) ,
LI Xin-gang ( ~ [ ~ l J )
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 C L C number: R730.5
Document code: A
Article ID: 1000-9604(2005)03-0203-04
ABSTRACT
Objective: To study the growth-inhibitory effects of curcumin on B-NHL cell line Raji ceils in vitro and its molecular mechanisms. Methods: The growth inhibition rates of Raji cells, after being treated with 6.25 ixmol/L - 50 ltmol/L curcumin for 12 h - 48 h, were examined by MTT assay. The apoptosis rate was detected by flow cytometry (FCM), the protein expression levels of bcl-2 and p53 in Raji cells were examined by SP immunohistochemistry. The expression of p53 in Raji cell were checked by RT-PCR. Results: After being treated by various concentrations of curcumin, the growth of Raji cells was inhibited significantly. The rates of apoptosis were 11.8% -79.7% (P<0.01), the down regulation of p53 expression was observed within 24 h after the treatment of curcumin by RT-PCR. The expression of bcl-2 and p53 was decreased, which depended on the action time. Conclusion: Curcumin could significantly inhibit the growth of Raji cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms. Key words: Curcumin; Raji cell; Gene expression; Apoptosis
Since anti-carcinogenic agents can also induce apoptosis in normal ceils in recent years, effort has been directed at looking for new effective anti-cancer drugs with low toxicity. Curcumin is the major active component of curcumin, which is a Traditional Chinese Medicine and has been used as a dietary ingredient and as a natural condiment. Recent studies showed I1] that this agent could decrease the proliferative potential and selectively induce apoptosis in cancer cells. It is reported that curcumin can inhibit the proliferation of tumor cells by repressing the activation of PTK and induce apoptosis by blocking signal transduction pathways [2]. However, its exact molecular mechanism has not been understood TM.
Received date: Apr. 21, 2005; Accepted date: May 26, 2005 Foundation item: This work was supported by the National Natural Science Foundation of China (No. 30271672). *Author to whom correspondence should be addressed. Phone: (0086-27)-85726387; Fax: (0086-27)-85485531; E-mail: yanchen @public.wh.hb.cn Biography: CHEN Yah (1952-), male, doctor of medicine, professor, Tongji Medical College, worked at Essen University Medical College, Germany in 1992-1994, and FMI Institute, Basel, Switzerland in 1999-2000, as a visiting scholar, majors in hematology.
In this study, we explored the growth-inhibitory effects of curcumin on B-NHL cell line Raji cell, an observed its influence on cellular gene expression of bcl-2 and p53.
M A T E R I A L S AND M E T H O D S
Reagents Curcumin, purity > 99%, purchased from Sigma Chemical Co. (USA), was dissolved in the dimethylsulfoxide (DMSO). Lymphoma Raji cell line was kindly provided by Cell Culture Center of Shanghai Academy of Sciences. Antibodies against bcl-2 and p53 were purchased from Jing Mei Company.
Cell Culture B-cell Non-Hodgkin's lymphoma cell line Raji cells were cultured in RPIM-1640 medium supplemented with 10% heat inactivated Newborn calf serum and 100 U/ml penicillin and 100 U/ml streptomycin in a humidified 5% incubator at 37°C. Ceils were passaged twice weekly and routinely examined for mycoplasma contamination.
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MTT Assay MTT assay was used to examine proliferation inhibition of Raji cells treated with 6.25 p.mol/L, 12.5 gmol/L, 25 gmol/L and 50 gmol/L curcumin for 0 h, 12 h, 24 h and 48 h. The cells without treatment of curcumin served as control. Cellular proliferation inhibition rates (CPIR) = (1-Average a value of experimental group/ average a value of control group) x 100%.
Flow Cytometry (FCM) Detection In order to analyze the content of DNA, cells treated with PBS, and resuspended in PBS containing 20 mg/L PI and 1 g/L ribonuclease A. 1x 1 0 6 of fixed cells were examined per experimental condition by flow cytometry, and percentage of degraded DNA was determined by the number of cells displaying subdiploid (sub-G1) DNA divided by total number of cell examined.
RT-PCR According to the manufacturer's instructions, total cellular RNA was extracted from the cells, using Trizol reagent (Sigma). Concentration and purity of the total cellular RNA were determined by the fluorescent pectrophotometric analysis (A260/A280>l:8). Two sets of primers used for PCR were designed by ourselves and synthesized by Shanghai Biological Project Company. The p53 primers' sequences were Pu 5'-TCTGTGACTTGCACGTACTC-3' and Pd 5'-CACGGATCTGAAGGGTGAAA-3' with amplified product of 349 bp. 13-actin primers were Pu 5'-ACCACCATGTACCCAGGCAT-3' and Pd 5'-CTCTCTTTGCACTCCCTGGGGG-3' with amplified product of 150 bp. PCR was performed as follows: 95°C, 5 rain, 72°C, 1 min, 94°C, 1 rain, 5°C, 1 min, 72°C, 7 min by 36 cycles. PCR products were resolved on 1% agarose gel and 10 I.tl was loaded into every slot. At the same time, the marker of 100 bpm was loaded into a slot. The gel was run at 40 V of voltage. The band density was analyzed by Gel Documentation and Analysis System (Kodak EDAS 290). The result was showed as the ratio of density of the object band to the 13-action band.
Gene Expression Assay of bcl-2 and p53 The percentage of bcl-2 and p53 protein expression was determined by using the strept peroxidase (SP) immunohistochemical method. After treatment with 6.25~50 lamol/L curcumin for 24 h, Raji cells were rinsed with PBS and fixed with acetone (4°C) for 15 rain. Non-specific binding sites were blocked with 10% normal goat serum for 30 rain. When the serum was removed, the cells were incubated with monoclonal
Chinese Journal of Cancer Research 17(3).'203-206, 2005
mouse anti-human bcl-2 and p53 antibodies (4°C, 24 h) and goat anti-mouse IgG (25°C, 30 rain). Then the cells were treated with avidin DH-biotinylated horse radish peroxidase H complex (ABC) and counterstained with haematoxylin. For scoring of positivity, 500 cells were counted under light microscope to analyze the positive intensity and calculate the positive rate.
Statistic Analysis All data were statistically analyzed by statistic software SPSS 11.5 and t-test.
RESULTS
Cellular Proliferation Different concentrations of curcumin (6.25~50 gmol/L) effectively inhibited the proliferation of Raji cells in vitro after 12~48 h treatment, in a time-and dose-dependent manner (Figure t). Compared with control group, inhibitory rate of Raji cell proliferation was 36.05%~80.36%, after treatment with various concentrations of curcumin for 48 h. The differences among various concentrations groups and those among the various time groups were very significant (P<0.01). 100
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Fig. 1. The growth inhibitory effects of curcumin at various concentrations on Raji cells
Cell Apoptotic Rates Curcumin at 12.5 gmol/L could induce apoptosis when cultured with Raji cells for 36~48 h, the apoptotic rate was over 50% (Figure 2).
Effect of Curcumin on p53 Expressions The ratios of absorbance A of p53 mRNA to absorbance A of 13-actin mRNA were calculated for every group. 6.25~12.5 (gmol/L) groups and control group had no significant change between every two groups (P>0.05). However, the ratios of other two groups:
Chinese Journal o f Cancer Research 17(3).203-206, 2005
25, 50 (Iamol/L) groups had statistically significant difference between every two groups (P<0.05) (Figure 3).
The Effect of Curcumin on bcl-2 and p53 Protein Expression
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After treatment with curcumin for 24 h, the expressions of bcl-2 and p53 proteins in Raji cells decreased in a dose-dependent manner. The differences among various concentration groups and control group were very significant (P<0.01, Table 1).
Table 1. The effects o f curcumin at various concentrations on cellular bcl-2 and p53 expression [ ( x _ + s ) %] determined by SP immunohistochemistry
Group Time of tests/n Control group 5 6.25 lamol/L Cur 5 12.5 p.mol/L Cur 5 25 ~tmol/L Cur 5 50 ~tmol/L Cur 5 *P<0.0I, as compared with control group
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Fig. 2. Cell apoptotic rates concentration of curcumin (~tmol/L)
48
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p53 13-actin 1
2
3
4
5
Fig. 3. Agarose gel analysis of RT-PCR of p53 mRNA in Raji cells 1-0 ~mol/L, 2-6.25 p.mol/L, 3-12 ~tmol/L, 4-25 p.mol/L, 5-501amol/L
1.0 "~ 0.8 1"411 1.2 ¢~ 0.6 0.4 0.2 0
i
l
i i
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1
p53 67.46 + 3.58 59.61 + 4.02 40.70 + 1.75 26.61 + 3.76* 15.06 + 1.42°
DISCUSSION
6.25
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bcl-2 90.71 + 2.53 83.14 + 1.85 63.54 _+2.33 49.21 + 1.50" 35.79 + 3.69"
i
2
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3
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4
i
5
Fig. 4. Effects of curcumin on expression of p53 mRNA in Raji cell ( x + s, n=3) 1-control, 2-6.25 gmol/L Cur, 3-12.5 gmol/L Cur, 4-25 gmol/L Cur, 5-50 ~.mol/L Cur. The density of the scanned. •P<0.01 vs control.
Curcumin is widely used as a spice and coloring agent for foods, such as curry, mustard, bean cake, French fries and potato chips, as well as in cosmetics and drugs. At the same time, it is also a Traditional Chinese Medicine, which is used to treat inflammations. Curcumin (diferulomethane, C12H3605, FW364.5) is a major active component of curcuma, which has been demonstrated to possess a variety of pharmacological effects. Recently, curcumin has been considered by FDA to be a very promising anti-tumor agent but its exact molecular mechanisms of action has not been fully understood [a]. It is proposed that curcumin may suppress tumor progression by blocking signal transduction pathways in target cells. In vitro studies showed that curcumin inhibits the proliferation of cancer cells and induces apoptosis by means of many pathways and by adjusting a large array of tumor surface makers. The target sites of its action may be at levels of DNA, mRNA and protein tS' 6]. In this study, we explored whether some apoptotic signal transduction genes, such as bcl-2 and p5, play roles in the induction of apoptosis by curcumin. Up to now, the apoptosis-inducing activity of curcumin has been demonstrated in many tumor cell lines. However, there have been few reports about the effects of curcumin on B-NHL cell line Raji cells. Our results of MTT assay indicated that curcumin could inhibit the in vitro growth and cause apoptosis of Raji cells, the apoptosis rates varied with the concentrations of the drugs. Bcl-2 protein is distributed on the cytoplasm surface of the outer membrane of mitochondria, endoplasmic membrane, and maintains the trans-membrane potential by working on proteins related to permeability transition (PT) pore. There are quite a few death-promoting factors (DPF) in the inter membrane of mitochondria, such as
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Chinese Journal of Cancer Research 17(3):203-206, 2005
cytochrome C and caspase. The action of bcl-2 protein takes place upstream of the activation of caspase-3 and inhibits the activation of caspase-3. After treatment with curcumin of various concentrations, the level of bcl-2 protein expression was decreased in a dose-dependent manner, which makes it easier for the treated cells to proceed to so-called programmed cell death I7'81. The mutation of the p53 gene, located in the center of the cell apoptotic regulation, contributes to the development of malignant tumors. One of the most important biological function of MDM2 is to combine with p53 protein, thereby directly speeding up the degradation of p53. Mutated p53 could inhibit the biochemical function of p53 such as transcription, but also suppress the biological function of p53 such as Gl arrest, GE-M arrest and the cell apoptosis dependent on p53 signal pathway, p53 was found to be activated by acetylation modification, to bind DNA in a sequence-specific manner, to transactivated down-stream target gene transcription, such as GADD45, mdm2 an p21, to arrest cell cycle process and to induce apoptosis/91. Our results show that after being treated by curcumin at various concentrations, the level of p53 protein expression was decreased in a dose-dependent manner and our data also indicated that curcumin down-regulated the p53 mRNA expression in a dose-dependent manner. Similar findings were reported in recent literatures El°l. To sum up, curcumin could significantly inhibit the growth of B-NHL cell line Raji cells in vitro in a time-dose-dependent manner. Down-regulating the expression of bcl-2 and p53 genes to induce cell apoptosis is one of the molecular mechanisms.
REFERENCES
[1]
Xu JH, Chen YZ, Huang XW, et al. Curcumin inducing apoptosis and inhibiting expressing of P210ber/ablprotein in human chronic myelogenous leukemia K562 cells [J]. FASEB J 2000; 14:A1516-9. [2] Shi Wen Long, Liu Yan. Effect of curcumin on anticancer [J]. Chin Pharm J 2004; 3:164-7. [3] Jiang MC, Yang HF, Yen JJ, et al. Curcumin induces apoptosis immortalized NIH 3T3 and malignant cancer cells lines [J]. Nutr Cancer 1996;26:111-20. [4] Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies [J]. Anticancer Res 2003; 23:363-8. [5] Li JK, Lin Shia SY. Mechanisms of cancer chemoprevention by curcumin [J]. Proc Natl Sci Counc Repub China B 2001; 25:59-66. [6] Mukhopadhyay A, Bueso Ramos C, Chatterjee D, et al. Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines [J]. Oncogene 2001; 20:7597-609. [7] Ramachandran C, You W. Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin [J]. Breast Cancer Res Treat 1999; 54:269-78. [8] Anto ILl, Mukhopadhyay A, Denning K, et al. Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome C release: its suppression by ectopic expressing of Bcl-2 and Bcl-xl [J]. Carcunogenesis 2002; 23:143-50. [9] Gu W, Roeder RG. Activation of p53 sequence-specific DNA bind by acetylation of the p53 C-terminal domain [J]. Cell 1997;90:595-606. [10] Choudhuri T, Pal S, Agwarwal ML, et al. Curcumin induces apoptosis in human breast cancer cells through p53 dependent Bax induction [J]. FEBS Lett 2002; 512:334-40.