Research Article
Curcumin Inhibits the Proliferation and Invasiveness of MHCC97-H Cells via p38 Signaling Pathway
DDR
DRUG DEVELOPMENT RESEARCH 75 : 463–468 (2014)
Kai Zhang,* Xiaojiang Rui, and Xi Yan Department of Gastroenterology, 323 Hospital of People’s Liberation Army, Xi’an, Shaanxi 710054, China
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Curcumin has been reported to be effective as a cancer therapy. However, the antimetastatic effect and molecular mechanism(s) of curcumin in hepatocellular carcinoma (HCC) remain poorly understood. The purpose of this study was to test the effects of curcumin on HCC and its putative mechanism(s). Curcumin inhibited the proliferation of HCC cells and inhibited the migration and invasion of these cells at sub-cytotoxic concentrations. Curcumin also decreased the expression and activity of matrix metalloproteinases (MMP)-2 and MMP-9, and reduced p38 phosphorylation. Combination treatment of HCC cells with curcumin and SB203580 (a p38 signaling pathway inhibitor), generated a synergistic effect on the expression of MMP-2 and MMP-9, suggesting that the anti-metastatic effect of curcumin on HCC may involve a p38 signaling pathway. Drug Dev Res 75 : 463–468, 2014. © 2014 Wiley Periodicals, Inc.
Key words: curcumin; hepatocellular carcinoma; migration and invasion; MMP
INTRODUCTION
MATERIALS AND METHODS
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of death from cancer globally [Villanueva et al., 2010; Zhao et al., 2013]. Metastasis, the major cause of death among cancer patients [Veltri et al., 2010], involves a series of events that are still unclear, but include changes in cell–extracellular matrix (ECM) interactions, disruption of intercellular adhesion, separation of single cells from solid tumor tissue, degradation of the ECM, and transit of tumor cells into this structure [Backus et al., 2005; Mimori et al., 2005]. A fundamental role in tumor metastasis has been attributed to matrix metalloproteinases (MMPs), the expression of which is altered in many cancers [Chen et al., 2013]. Curcumin, a major constituent of the yellow spice turmeric, has been studied as a chemopreventive agent against HCC [Nasr et al., 2014]. In the present study, the anti-metastatic effect of curcumin on HCC was investigated.
Reagents
© 2014 Wiley Periodicals, Inc.
Anti-MMP-2, anti-MMP-9, anti-p38, and anti-pp38 antibodies were from Cell Signaling (Beverly, Massachusetts, USA); anti-β-actin was from Santa Cruz (CA, USA); and curcumin, SB203580, and MTT were from Sigma (St. Louis, MO, USA). This work was supported by grants from science and technology projects in Shaanxi Province (Grant number 2013k1301-09). Conflict of interest: The authors declare that they have no competing interests. *Correspondence to: Kai Zhang, Department of Gastroenterology, 323 Hospital of People’s Liberation, No. 6 Jianshe West Road, Xi’an, Shaanxi 710054, China. E-mail: [email protected] Received 26 May 2014; Accepted 30 June 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21210
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Cell Culture The human HCC cell line MHCC97-H was from the Liver Cancer Institute of Fudan University (Shanghai, China). HepG2 was from ATCC (Rockville, Maryland, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal bovine serum (FBS), 100 U/m penicillin, 100 μg/mL streptomycin, and 2 mmol/l glutamine in a humidified atmosphere of 5% CO2 at 37 C.
Cell Viability Assays Briefly, cells were plated in 96-well culture plates (2 × 103 cells per well). The cells were treated with various concentrations of curcumin. After 24 h, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with 5 mg/mL MTT (Sigma) for 4 h. After incubation, cells were washed with PBS and solubilized with DMSO, and the optical density read in an enzyme-linked immunosorbent assay plate reader.
Migration and Invasion Assays In vitro migration and invasion assays were carried out as described [Wang et al., 2010]. Transwell chambers (Corning Costar, Cambridge, MA, USA) coated with Matrigel or not was used. Cells were trypsinized, suspended at a final concentration of 5 × 105 cells/mL in serum-free medium, and treated with different concentrations of curcumin (0, 10, 20, and 40 μΜ) for 24 h before seeding and then added into each upper transwell chamber. The bottom chamber contained medium with 10% FBS to serve as a chemoattractant. After incubation for 24 h at 37 C under 5% CO2, all noninvaded cells were removed from the upper face of the transwell membrane with a cotton swab. The invaded cells were fixed, stained, and measured.
fluoride (PVDF) membranes that were subsequently blocked in defatted milk (5% in Tris-buffered saline with TWEEN-20 (TBST) buffer) at 37 C for 1 h to prevent nonspecific binding and then incubated overnight with antibodies against p38, p-p38, MMP-2, MMP-9, or β-actin in TBST containing 5% defatted milk at 4 C. The membranes were then incubated with the corresponding second antibody for 1 h at room temperature. The bands were detected with an enhanced chemiluminescence kit (Amersham, ECL Plus, Freiburg, Germany) and exposed by autoradiography. The densitometric analysis was performed using Image J software (GE Healthcare, Bucks, UK), and results were expressed as arbitrary units (a.u.).
Zymography Cells were treated with different concentrations of curcumin or SB203580 at 37 C for 24 h, and then conditioned media were collected and kept. Appropriate volumes of the unboiled samples (adjusted by vital cell number) were separated with 0.1% gelatin-8% SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the gels were washed twice with 2.5% Triton X-100 at room temperature for 30 min and then incubated in reaction buffer (10 mM CaCl2, 40 mM Tris-HCl and 0.01% NaN3, pH 8.0) at 37 C for 12 h. Coomassie brilliant blue R-250 gel stain was then used to stain the gel. The intensities of the gel bands were
Western Blot Analysis First, cells were treated with different concentrations of curcumin, and 1 × 106 cells were suspended in 60 μL of lysis buffer (1 mmol/L Ethylene Diamine Tetraacetic Acid (EDTA), 150 mmol/L KCl, 40mmol/L Tris-HCl, 100 mmol/L NaVO3, 1% Triton X-100, 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF), pH 7.5). Proteins (70 μg) were separated using 8% or 10% Sodium Dodecyl Sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene Drug Dev. Res.
Fig. 1. Cellular viability of MHCC97-H cells treated with curcumin. MHCC97-H cells were treated with various concentrations of curcumin, and after 24 h their viability was measured using an MTT assay. Values represent means ± SD of three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group.
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Fig. 2. Effects of curcumin on the migration and invasion of MHCC97-H cells. (A) For the migration assay, cells treated with different concentrations of curcumin (0, 1, 5, and 10 μM) were plated onto the upper wells of the chamber. FBS (10%) was added to the lower wells for 16 h to induce cell migration. After 24 h, cells on the bottom side of the filter were fixed, stained, and measured. Cell migration spontaneous migration in DMSO was designated as control. (B) The percent migration rate was expressed as a percentage of the control (0 μM). (C) For the invasion assay, the chamber was coated with Matrigel; the other steps are the same with migration assay. (D) The percent invasion rate was expressed as a percentage of the control (0 μM). Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
calculated using an image analysis system (Bio-Rad Laboratories, Richmond, CA, USA).
Statistics Student’s t-test was used to evaluate the significance or P values between groups (*P < 0.05, **P < 0.01). Standard errors of mean values were depicted as error bars in all figures.
RESULTS
Curcumin Inhibits the Proliferation of MHCC97-H Cells The anti-proliferative effects of curcumin (0–60 μM) on MHCC97-H cells are shown in Figure 1.
At 20 μM, curcumin ihibited the proliferation of MHCC97-H cells while concentrations below 20 μM had no effect. Thus, we chose a concentration range of curcumin lower than this for all subsequent experiments.
Curcumin Inhibits the Migration and Invasion of MHCC97-H Cells Curcumin reduced the invasion and migration of MHCC97-H cells in a concentration-dependent manner n MHCC97-H cells treated with 0, 1, 5, and 10 μM curcumin for 16 h (cell migration) and 24 h (cell invasion), respectively (Fig. 2). Similar anti-metastatic effects of curcumin were observed in HepG2 cells (Fig. 3). Drug Dev. Res.
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Fig. 3. Effects of curcumin on the invasion of HepG2 cells. (A) For the invasion assay, the chamber was coated with Matrigel, then cells treated with different concentrations of curcumin were plated onto the upper wells of the chamber. FBS (10%) was added to the lower wells for 16 h to induce cell migration. After 24 h, cells on the bottom side of the filter were fixed, stained, and measured. Cell migration spontaneous migration in DMSO was designated as control. (B) The percent invasion rate was expressed as a percentage of the control. Values represent the means ± SD of three independent experiments performed in triplicate. **P < 0.01 compared with the control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 4. Curcumin suppresses the expression and activity of MMP-2/-9 in MHCC97-H cells. (A) MHCC97-H cells were treated with different concentrations of curcumin (0, 1, 5, and 10 μM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2/-9. (B) Quantification of the protein levels of MMP-2/-9 in MHCC97-H cells. (C) MHCC97-H cells were treated with different concentrations of curcumin (0, 1, 5, and 10 μM) for 24 h and then subjected to gelatin zymography to analyze the activities of MMP-2/-9. (D) Quantification of the activities of MMP-2/-9 in MHCC97-H cells. Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. MMP, metalloproteinase.
Drug Dev. Res.
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Fig. 5. Effect of curcumin on the p38 signaling pathway in MHCC97-H cells. (A) The protein levels of p38 and p-p38. (B) Phosphorylation densities of p38 were digitally scanned. (C) The protein levels of MMP-2/-9 after treating with curcumin and SB203580 (20 μM). (D) Quantification of the protein levels of MMP-2/-9 in MHCC97-H cells. Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. MMP, metalloproteinase.
Curcumin Suppresses the Expression and Activity of MMP-2 and MMP-9 In MHCC97-H cells treated with 1, 5, and 10 μM curcumin for 24 h and subjected to Western blotting, curcumin reduced the protein levels of MMP-2 and MMP-9 in a concentration-dependent manner compared with the control group (Fig. 4A,B). Gelatin zymography also shows that curcumin can reduce the activity of MMP-2 and MMP-9 (Fig. 4C,D). Curcumin also reduces expression of MMP-2/-9 in HepG2 cells (data not shown).
p38 Signaling Is Involved in the Anti-Metastatic Mechanism(s) of Curcumin Given that the p38 signaling pathway plays a critical roles in the invasion of cancer cells via regulation of MMP-2/-9 [Yang et al., 2012], the effect of curcumin on the p38 signaling pathway in MHCC97-H cells was examined. Western blotting showed that curcumin reduces p38 phosphorylation in a concentrationdependent manner (Fig. 5A,B). Treatment with the
p38 inhibitor SB203580 (20 μM) and curcumin reduced MMP-2 and MMP-9 protein expression (Fig. 5C, D). DISCUSSION
Curcumin has anticancer properties [Li et al., 2014; Lim et al., 2014], and in the present curcumin was found to inhibit the proliferation and invasion of HCC cells, an effect that appears to involve a p38 signaling pathway. As curcumin was cytotoxic to MHCC97-H cells at concentrations above 15 μM, lower concentrations were used for subsequent migration and invasion experiments. Using transwell chamber, we found that curcumin could inhibit the migration and invasion of MHCC97-H cells. The metastasis of cancer cells is a complicated process, including the escape from primary site, degration of ECM, invasion into blood and lymph vessel, and reaching new place at last [Chen et al., 2013]. During the process, MMPs play a key role [Merdad et al., 2014; Yang et al., 2014]. Curcumin can Drug Dev. Res.
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exert its anti-metastatic effect via regulating MMPs in colorectal cancer and breast cancer [Abdalla et al., 1997; Kunnumakkara et al., 2009; Mo et al., 2012]. In the study, the results show that curcumin can inhibit the expression and activity of MMP-2/-9, which implies that the anti-metastatic effect of curcumin is closely correlated with the regulation of MMP-2/-9. During the process of metastasis, many signaling pathways play important roles, including p38 signaling pathway [Hsieh et al., 2010; Rao et al., 2014]. Curcumin can exert the anti-tumor effect via regulating p38 signaling pathway [Weir et al., 2007; Chen et al., 2010]. So we analysed the effect of curcumin on p38 signaling pathway in MHCC97-H cells. The results show that curcumin can reduce the phosphorylation level of p38. To further explore the mechanism of the regulation of MMP-2/-9, we detect the effect of SB203580 on the expression of MMP-2/-9. The results show that SB203580 can reduce the expression of MMP-2/-9. In conclusion, our results show that curcumin can inhibit the proliferation and invasion of MHCC97-H cells via regulating the expression and activity of MMP2/-9 through p38 signaling pathway. The finding provides more support for the clinical use of curcumin.
REFERENCES Abdalla SA, Jeziorska M, Schofield P, Woolley DE, Haboubi NY. 1997. Gelatinase B (MMP-9) expression and survival in colorectal cancer patients. Int J Colorectal Dis 12:342–343. Backus J, Laughlin T, Wang Y, Belly R, White R, Baden J, Justus Min C, Mannie A, Tafra L, Atkins D, et al. 2005. Identification and characterization of optimal gene expression markers for detection of breast cancer metastasis. J Mol Diagn 7:327–336. Chen J, Wang G, Wang L, Kang J, Wang J. 2010. Curcumin p38dependently enhances the anticancer activity of valproic acid in human leukemia cells. Eur J Pharm Sci 41:210–218. Chen K, Zhang S, Ji Y, Li J, An P, Ren H, Liang R, Yang J, Li Z. 2013. Baicalein inhibits the invasion and metastatic capabilities of hepatocellular carcinoma cells via down-regulation of the ERK pathway. PLoS ONE 8:e72927. Hsieh MJ, Chen KS, Chiou HL, Hsieh YS. 2010. Carbonic anhydrase XII promotes invasion and migration ability of MDA-MB-231 breast cancer cells through the p38 MAPK signaling pathway. Eur J Cell Biol 89:598–606. Kunnumakkara AB, Diagaradjane P, Anand P, Harikumar KB, Deorukhkar A, Gelovani J, Guha S, Krishnan S, Aggarwal BB. 2009. Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model. Int J Cancer 125:2187–2197.
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Li ZC, Zhang LM, Wang HB, Ma JX, Sun JZ. 2014. Curcumin inhibits lung cancer progression and metastasis through induction of FOXO1. Tumour Biol 35:111–116. Lim TG, Lee SY, Huang Z, Lim do Y, Chen H, Jung SK, Bode AM, Lee KW, Dong Z. 2014. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2. Cancer Prev Res (Phila) 7:466–474. Merdad A, Karim S, Schulten HJ, Dallol A, Buhmeida A, Al-Thubaity F, Gari MA, Chaudhary AG, Abuzenadah AM, Al-Qahtani MH. 2014. Expression of matrix metalloproteinases (MMPs) in primary human breast cancer: MMP-9 as a potential biomarker for cancer invasion and metastasis. Anticancer Res 34:1355–1366. Mimori K, Kataoka A, Yoshinaga K, Ohta M, Sagara Y, Yoshikawa Y, Ohno S, Barnard GF, Mori M. 2005. Identification of molecular markers for metastasis-related genes in primary breast cancer cells. Clin Exp Metastasis 22:59–67. Mo N, Li ZQ, Li J, Cao YD. 2012. Curcumin inhibits TGF-beta1induced MMP-9 and invasion through ERK and Smad signaling in breast cancer MDA- MB-231 cells. Asian Pac J Cancer Prev 13:5709–5714. Nasr M, Selima E, Hamed O, Kazem A. 2014. Targeting different angiogenic pathways with combination of curcumin, leflunomide and perindopril inhibits diethylnitrosamine-induced hepatocellular carcinoma in mice. Eur J Pharmacol 723:267–275. Rao W, Li H, Song F, Zhang R, Yin Q, Wang Y, Xi Y, Ge H. 2014. OVA66 increases cell growth, invasion and survival via regulation of IGF-1R-MAPK signaling in human cancer cells. Carcinogenesis 35:1573–1581. Veltri RW, Isharwal S, Miller MC, Epstein JI, Partin AW. 2010. Nuclear roundness variance predicts prostate cancer progression, metastasis, and death: a prospective evaluation with up to 25 years of follow-up after radical prostatectomy. Prostate 70:1333–1339. Villanueva A, Minguez B, Forner A, Reig M, Llovet JM. 2010. Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy. Annu Rev Med 61:317–328. Wang L, Ling Y, Chen Y, Li CL, Feng F, You QD, Lu N, Guo QL. 2010. Flavonoid baicalein suppresses adhesion, migration and invasion of MDA-MB-231 human breast cancer cells. Cancer Lett 297:42–48. Weir NM, Selvendiran K, Kutala VK, Tong L, Vishwanath S, Rajaram M, Tridandapani S, Anant S, Kuppusamy P. 2007. Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by modulating Akt and p38 MAPK. Cancer Biol Ther 6:178–184. Yang N, Hui L, Wang Y, Yang H, Jiang X. 2014. SOX2 promotes the migration and invasion of laryngeal cancer cells by induction of MMP-2 via the PI3K/Akt/mTOR pathway. Oncol Rep 31:2651– 2659. Yang ZH, Li SN, Liu JX, Guo QX, Sun XW. 2012. MMP-9 polymorphisms are related to serum lipids levels but not associated with colorectal cancer susceptibility in Chinese population. Mol Biol Rep 39:9399–9404. Zhao FT, Jia ZS, Yang Q, Song L, Jiang XJ. 2013. S100A14 promotes the growth and metastasis of hepatocellular carcinoma. Asian Pac J Cancer Prev 14:3831–3836.
Curcumin Inhibits the Proliferation and Invasiveness of MHCC97-H Cells via p38 Signaling Pathway
DDR
DRUG DEVELOPMENT RESEARCH 75 : 463–468 (2014)
Kai Zhang,* Xiaojiang Rui, and Xi Yan Department of Gastroenterology, 323 Hospital of People’s Liberation Army, Xi’an, Shaanxi 710054, China
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT Curcumin has been reported to be effective as a cancer therapy. However, the antimetastatic effect and molecular mechanism(s) of curcumin in hepatocellular carcinoma (HCC) remain poorly understood. The purpose of this study was to test the effects of curcumin on HCC and its putative mechanism(s). Curcumin inhibited the proliferation of HCC cells and inhibited the migration and invasion of these cells at sub-cytotoxic concentrations. Curcumin also decreased the expression and activity of matrix metalloproteinases (MMP)-2 and MMP-9, and reduced p38 phosphorylation. Combination treatment of HCC cells with curcumin and SB203580 (a p38 signaling pathway inhibitor), generated a synergistic effect on the expression of MMP-2 and MMP-9, suggesting that the anti-metastatic effect of curcumin on HCC may involve a p38 signaling pathway. Drug Dev Res 75 : 463–468, 2014. © 2014 Wiley Periodicals, Inc.
Key words: curcumin; hepatocellular carcinoma; migration and invasion; MMP
INTRODUCTION
MATERIALS AND METHODS
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of death from cancer globally [Villanueva et al., 2010; Zhao et al., 2013]. Metastasis, the major cause of death among cancer patients [Veltri et al., 2010], involves a series of events that are still unclear, but include changes in cell–extracellular matrix (ECM) interactions, disruption of intercellular adhesion, separation of single cells from solid tumor tissue, degradation of the ECM, and transit of tumor cells into this structure [Backus et al., 2005; Mimori et al., 2005]. A fundamental role in tumor metastasis has been attributed to matrix metalloproteinases (MMPs), the expression of which is altered in many cancers [Chen et al., 2013]. Curcumin, a major constituent of the yellow spice turmeric, has been studied as a chemopreventive agent against HCC [Nasr et al., 2014]. In the present study, the anti-metastatic effect of curcumin on HCC was investigated.
Reagents
© 2014 Wiley Periodicals, Inc.
Anti-MMP-2, anti-MMP-9, anti-p38, and anti-pp38 antibodies were from Cell Signaling (Beverly, Massachusetts, USA); anti-β-actin was from Santa Cruz (CA, USA); and curcumin, SB203580, and MTT were from Sigma (St. Louis, MO, USA). This work was supported by grants from science and technology projects in Shaanxi Province (Grant number 2013k1301-09). Conflict of interest: The authors declare that they have no competing interests. *Correspondence to: Kai Zhang, Department of Gastroenterology, 323 Hospital of People’s Liberation, No. 6 Jianshe West Road, Xi’an, Shaanxi 710054, China. E-mail: [email protected] Received 26 May 2014; Accepted 30 June 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21210
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Cell Culture The human HCC cell line MHCC97-H was from the Liver Cancer Institute of Fudan University (Shanghai, China). HepG2 was from ATCC (Rockville, Maryland, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal bovine serum (FBS), 100 U/m penicillin, 100 μg/mL streptomycin, and 2 mmol/l glutamine in a humidified atmosphere of 5% CO2 at 37 C.
Cell Viability Assays Briefly, cells were plated in 96-well culture plates (2 × 103 cells per well). The cells were treated with various concentrations of curcumin. After 24 h, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with 5 mg/mL MTT (Sigma) for 4 h. After incubation, cells were washed with PBS and solubilized with DMSO, and the optical density read in an enzyme-linked immunosorbent assay plate reader.
Migration and Invasion Assays In vitro migration and invasion assays were carried out as described [Wang et al., 2010]. Transwell chambers (Corning Costar, Cambridge, MA, USA) coated with Matrigel or not was used. Cells were trypsinized, suspended at a final concentration of 5 × 105 cells/mL in serum-free medium, and treated with different concentrations of curcumin (0, 10, 20, and 40 μΜ) for 24 h before seeding and then added into each upper transwell chamber. The bottom chamber contained medium with 10% FBS to serve as a chemoattractant. After incubation for 24 h at 37 C under 5% CO2, all noninvaded cells were removed from the upper face of the transwell membrane with a cotton swab. The invaded cells were fixed, stained, and measured.
fluoride (PVDF) membranes that were subsequently blocked in defatted milk (5% in Tris-buffered saline with TWEEN-20 (TBST) buffer) at 37 C for 1 h to prevent nonspecific binding and then incubated overnight with antibodies against p38, p-p38, MMP-2, MMP-9, or β-actin in TBST containing 5% defatted milk at 4 C. The membranes were then incubated with the corresponding second antibody for 1 h at room temperature. The bands were detected with an enhanced chemiluminescence kit (Amersham, ECL Plus, Freiburg, Germany) and exposed by autoradiography. The densitometric analysis was performed using Image J software (GE Healthcare, Bucks, UK), and results were expressed as arbitrary units (a.u.).
Zymography Cells were treated with different concentrations of curcumin or SB203580 at 37 C for 24 h, and then conditioned media were collected and kept. Appropriate volumes of the unboiled samples (adjusted by vital cell number) were separated with 0.1% gelatin-8% SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the gels were washed twice with 2.5% Triton X-100 at room temperature for 30 min and then incubated in reaction buffer (10 mM CaCl2, 40 mM Tris-HCl and 0.01% NaN3, pH 8.0) at 37 C for 12 h. Coomassie brilliant blue R-250 gel stain was then used to stain the gel. The intensities of the gel bands were
Western Blot Analysis First, cells were treated with different concentrations of curcumin, and 1 × 106 cells were suspended in 60 μL of lysis buffer (1 mmol/L Ethylene Diamine Tetraacetic Acid (EDTA), 150 mmol/L KCl, 40mmol/L Tris-HCl, 100 mmol/L NaVO3, 1% Triton X-100, 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF), pH 7.5). Proteins (70 μg) were separated using 8% or 10% Sodium Dodecyl Sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene Drug Dev. Res.
Fig. 1. Cellular viability of MHCC97-H cells treated with curcumin. MHCC97-H cells were treated with various concentrations of curcumin, and after 24 h their viability was measured using an MTT assay. Values represent means ± SD of three independent experiments, each performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group.
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Fig. 2. Effects of curcumin on the migration and invasion of MHCC97-H cells. (A) For the migration assay, cells treated with different concentrations of curcumin (0, 1, 5, and 10 μM) were plated onto the upper wells of the chamber. FBS (10%) was added to the lower wells for 16 h to induce cell migration. After 24 h, cells on the bottom side of the filter were fixed, stained, and measured. Cell migration spontaneous migration in DMSO was designated as control. (B) The percent migration rate was expressed as a percentage of the control (0 μM). (C) For the invasion assay, the chamber was coated with Matrigel; the other steps are the same with migration assay. (D) The percent invasion rate was expressed as a percentage of the control (0 μM). Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
calculated using an image analysis system (Bio-Rad Laboratories, Richmond, CA, USA).
Statistics Student’s t-test was used to evaluate the significance or P values between groups (*P < 0.05, **P < 0.01). Standard errors of mean values were depicted as error bars in all figures.
RESULTS
Curcumin Inhibits the Proliferation of MHCC97-H Cells The anti-proliferative effects of curcumin (0–60 μM) on MHCC97-H cells are shown in Figure 1.
At 20 μM, curcumin ihibited the proliferation of MHCC97-H cells while concentrations below 20 μM had no effect. Thus, we chose a concentration range of curcumin lower than this for all subsequent experiments.
Curcumin Inhibits the Migration and Invasion of MHCC97-H Cells Curcumin reduced the invasion and migration of MHCC97-H cells in a concentration-dependent manner n MHCC97-H cells treated with 0, 1, 5, and 10 μM curcumin for 16 h (cell migration) and 24 h (cell invasion), respectively (Fig. 2). Similar anti-metastatic effects of curcumin were observed in HepG2 cells (Fig. 3). Drug Dev. Res.
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Fig. 3. Effects of curcumin on the invasion of HepG2 cells. (A) For the invasion assay, the chamber was coated with Matrigel, then cells treated with different concentrations of curcumin were plated onto the upper wells of the chamber. FBS (10%) was added to the lower wells for 16 h to induce cell migration. After 24 h, cells on the bottom side of the filter were fixed, stained, and measured. Cell migration spontaneous migration in DMSO was designated as control. (B) The percent invasion rate was expressed as a percentage of the control. Values represent the means ± SD of three independent experiments performed in triplicate. **P < 0.01 compared with the control group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 4. Curcumin suppresses the expression and activity of MMP-2/-9 in MHCC97-H cells. (A) MHCC97-H cells were treated with different concentrations of curcumin (0, 1, 5, and 10 μM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-2/-9. (B) Quantification of the protein levels of MMP-2/-9 in MHCC97-H cells. (C) MHCC97-H cells were treated with different concentrations of curcumin (0, 1, 5, and 10 μM) for 24 h and then subjected to gelatin zymography to analyze the activities of MMP-2/-9. (D) Quantification of the activities of MMP-2/-9 in MHCC97-H cells. Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. MMP, metalloproteinase.
Drug Dev. Res.
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Fig. 5. Effect of curcumin on the p38 signaling pathway in MHCC97-H cells. (A) The protein levels of p38 and p-p38. (B) Phosphorylation densities of p38 were digitally scanned. (C) The protein levels of MMP-2/-9 after treating with curcumin and SB203580 (20 μM). (D) Quantification of the protein levels of MMP-2/-9 in MHCC97-H cells. Values represent the means ± SD of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 compared with the control group. MMP, metalloproteinase.
Curcumin Suppresses the Expression and Activity of MMP-2 and MMP-9 In MHCC97-H cells treated with 1, 5, and 10 μM curcumin for 24 h and subjected to Western blotting, curcumin reduced the protein levels of MMP-2 and MMP-9 in a concentration-dependent manner compared with the control group (Fig. 4A,B). Gelatin zymography also shows that curcumin can reduce the activity of MMP-2 and MMP-9 (Fig. 4C,D). Curcumin also reduces expression of MMP-2/-9 in HepG2 cells (data not shown).
p38 Signaling Is Involved in the Anti-Metastatic Mechanism(s) of Curcumin Given that the p38 signaling pathway plays a critical roles in the invasion of cancer cells via regulation of MMP-2/-9 [Yang et al., 2012], the effect of curcumin on the p38 signaling pathway in MHCC97-H cells was examined. Western blotting showed that curcumin reduces p38 phosphorylation in a concentrationdependent manner (Fig. 5A,B). Treatment with the
p38 inhibitor SB203580 (20 μM) and curcumin reduced MMP-2 and MMP-9 protein expression (Fig. 5C, D). DISCUSSION
Curcumin has anticancer properties [Li et al., 2014; Lim et al., 2014], and in the present curcumin was found to inhibit the proliferation and invasion of HCC cells, an effect that appears to involve a p38 signaling pathway. As curcumin was cytotoxic to MHCC97-H cells at concentrations above 15 μM, lower concentrations were used for subsequent migration and invasion experiments. Using transwell chamber, we found that curcumin could inhibit the migration and invasion of MHCC97-H cells. The metastasis of cancer cells is a complicated process, including the escape from primary site, degration of ECM, invasion into blood and lymph vessel, and reaching new place at last [Chen et al., 2013]. During the process, MMPs play a key role [Merdad et al., 2014; Yang et al., 2014]. Curcumin can Drug Dev. Res.
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exert its anti-metastatic effect via regulating MMPs in colorectal cancer and breast cancer [Abdalla et al., 1997; Kunnumakkara et al., 2009; Mo et al., 2012]. In the study, the results show that curcumin can inhibit the expression and activity of MMP-2/-9, which implies that the anti-metastatic effect of curcumin is closely correlated with the regulation of MMP-2/-9. During the process of metastasis, many signaling pathways play important roles, including p38 signaling pathway [Hsieh et al., 2010; Rao et al., 2014]. Curcumin can exert the anti-tumor effect via regulating p38 signaling pathway [Weir et al., 2007; Chen et al., 2010]. So we analysed the effect of curcumin on p38 signaling pathway in MHCC97-H cells. The results show that curcumin can reduce the phosphorylation level of p38. To further explore the mechanism of the regulation of MMP-2/-9, we detect the effect of SB203580 on the expression of MMP-2/-9. The results show that SB203580 can reduce the expression of MMP-2/-9. In conclusion, our results show that curcumin can inhibit the proliferation and invasion of MHCC97-H cells via regulating the expression and activity of MMP2/-9 through p38 signaling pathway. The finding provides more support for the clinical use of curcumin.
REFERENCES Abdalla SA, Jeziorska M, Schofield P, Woolley DE, Haboubi NY. 1997. Gelatinase B (MMP-9) expression and survival in colorectal cancer patients. Int J Colorectal Dis 12:342–343. Backus J, Laughlin T, Wang Y, Belly R, White R, Baden J, Justus Min C, Mannie A, Tafra L, Atkins D, et al. 2005. Identification and characterization of optimal gene expression markers for detection of breast cancer metastasis. J Mol Diagn 7:327–336. Chen J, Wang G, Wang L, Kang J, Wang J. 2010. Curcumin p38dependently enhances the anticancer activity of valproic acid in human leukemia cells. Eur J Pharm Sci 41:210–218. Chen K, Zhang S, Ji Y, Li J, An P, Ren H, Liang R, Yang J, Li Z. 2013. Baicalein inhibits the invasion and metastatic capabilities of hepatocellular carcinoma cells via down-regulation of the ERK pathway. PLoS ONE 8:e72927. Hsieh MJ, Chen KS, Chiou HL, Hsieh YS. 2010. Carbonic anhydrase XII promotes invasion and migration ability of MDA-MB-231 breast cancer cells through the p38 MAPK signaling pathway. Eur J Cell Biol 89:598–606. Kunnumakkara AB, Diagaradjane P, Anand P, Harikumar KB, Deorukhkar A, Gelovani J, Guha S, Krishnan S, Aggarwal BB. 2009. Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model. Int J Cancer 125:2187–2197.
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Li ZC, Zhang LM, Wang HB, Ma JX, Sun JZ. 2014. Curcumin inhibits lung cancer progression and metastasis through induction of FOXO1. Tumour Biol 35:111–116. Lim TG, Lee SY, Huang Z, Lim do Y, Chen H, Jung SK, Bode AM, Lee KW, Dong Z. 2014. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2. Cancer Prev Res (Phila) 7:466–474. Merdad A, Karim S, Schulten HJ, Dallol A, Buhmeida A, Al-Thubaity F, Gari MA, Chaudhary AG, Abuzenadah AM, Al-Qahtani MH. 2014. Expression of matrix metalloproteinases (MMPs) in primary human breast cancer: MMP-9 as a potential biomarker for cancer invasion and metastasis. Anticancer Res 34:1355–1366. Mimori K, Kataoka A, Yoshinaga K, Ohta M, Sagara Y, Yoshikawa Y, Ohno S, Barnard GF, Mori M. 2005. Identification of molecular markers for metastasis-related genes in primary breast cancer cells. Clin Exp Metastasis 22:59–67. Mo N, Li ZQ, Li J, Cao YD. 2012. Curcumin inhibits TGF-beta1induced MMP-9 and invasion through ERK and Smad signaling in breast cancer MDA- MB-231 cells. Asian Pac J Cancer Prev 13:5709–5714. Nasr M, Selima E, Hamed O, Kazem A. 2014. Targeting different angiogenic pathways with combination of curcumin, leflunomide and perindopril inhibits diethylnitrosamine-induced hepatocellular carcinoma in mice. Eur J Pharmacol 723:267–275. Rao W, Li H, Song F, Zhang R, Yin Q, Wang Y, Xi Y, Ge H. 2014. OVA66 increases cell growth, invasion and survival via regulation of IGF-1R-MAPK signaling in human cancer cells. Carcinogenesis 35:1573–1581. Veltri RW, Isharwal S, Miller MC, Epstein JI, Partin AW. 2010. Nuclear roundness variance predicts prostate cancer progression, metastasis, and death: a prospective evaluation with up to 25 years of follow-up after radical prostatectomy. Prostate 70:1333–1339. Villanueva A, Minguez B, Forner A, Reig M, Llovet JM. 2010. Hepatocellular carcinoma: novel molecular approaches for diagnosis, prognosis, and therapy. Annu Rev Med 61:317–328. Wang L, Ling Y, Chen Y, Li CL, Feng F, You QD, Lu N, Guo QL. 2010. Flavonoid baicalein suppresses adhesion, migration and invasion of MDA-MB-231 human breast cancer cells. Cancer Lett 297:42–48. Weir NM, Selvendiran K, Kutala VK, Tong L, Vishwanath S, Rajaram M, Tridandapani S, Anant S, Kuppusamy P. 2007. Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by modulating Akt and p38 MAPK. Cancer Biol Ther 6:178–184. Yang N, Hui L, Wang Y, Yang H, Jiang X. 2014. SOX2 promotes the migration and invasion of laryngeal cancer cells by induction of MMP-2 via the PI3K/Akt/mTOR pathway. Oncol Rep 31:2651– 2659. Yang ZH, Li SN, Liu JX, Guo QX, Sun XW. 2012. MMP-9 polymorphisms are related to serum lipids levels but not associated with colorectal cancer susceptibility in Chinese population. Mol Biol Rep 39:9399–9404. Zhao FT, Jia ZS, Yang Q, Song L, Jiang XJ. 2013. S100A14 promotes the growth and metastasis of hepatocellular carcinoma. Asian Pac J Cancer Prev 14:3831–3836.
