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α-Lipoic acid reduces matrix metalloproteinase activity in MDA-MB-231 human breast cancer cells
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Nutrition Research 30 (2010) 403 – 409 www.nrjournal.com
α-Lipoic acid reduces matrix metalloproteinase activity in MDA-MB-231 human breast cancer cells Hyun Sook Lee a , Mi Hee Na b , Woo Kyoung Kim b,⁎ a
Department of Foods and Nutrition, Kookmin University, Sungbuk-Gu, Seoul 136-702, South Korea Department of Food Science and Nutrition, Dankook University, Yongin-si, Gyeonggi-do 448-701, South Korea Received 3 February 2010; revised 7 June 2010; accepted 11 June 2010
b
Abstract α-Lipoic acid (LA), a naturally occurring molecule in animal and plant cells, is a potent antioxidant that reportedly exerts beneficial effects on cell proliferation and apoptosis in various cancer cell lines. However, the molecular mechanisms behind the antimetastatic property of LA are not well understood. The present study investigates the effect of LA on metastasis in a cell system. Our hypothesis is that LA inhibits metastasis via inhibition of matrix metalloproteinase (MMP) in vitro. MDA-MB-231 cells, a human breast cancer cell line, were treated with various concentrations of LA (0, 250, 500, or 1000 μmol/L) to measure metastasis, MMP activity, and mRNA expression. The viability of cells was examined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The effect of LA on metastasis was evaluated using the motility, migration, and invasion assay in vitro. The activity and mRNA expression of MMP-2 and MMP-9 were measured. After LA treatment, cell motility and cell migration were significantly decreased (P b .05). α-Lipoic acid also reduced cell invasion through a Matrigel-coated chamber (P b .05). Activities of MMP-2 and MMP-9 were decreased by LA treatment in a dose-dependent manner. RT-PCR analysis confirmed the reduction in mRNA expression level of MMP-2 and MMP-9 by LA treatment. We conclude that in this cell culture model, LA treatment inhibits cancer metastasis, and this inhibition is likely due to the decrease in the activity and mRNA expression levels of MMP-2 and MMP-9 caused by LA. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
α-Lipoic acid; Metastasis; Breast cancer cells; MMP-2; MMP-9 ECM, extracellular matrix; FBS, fetal bovine serum; LA, α-Lipoic acid; MMP, matrix metalloproteinase; SFM, serum-free media.
1. Introduction Breast cancer is the leading cause of cancer-related mortality in Korea, as well as in the Western world; thus, effective chemoprevention could significantly impact this rising trend of cancer-related mortality. However, successful use of dietary antioxidants for tumor chemoprevention in humans remains an unmet goal [1]. ⁎ Corresponding author. Tel.: +82 31 8005 3172; fax: +82 31 8005 3170. E-mail address: [email protected] (W.K. Kim). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2010.06.009
It has been proposed that antioxidants play a protective role in breast cancer [2]. α-Lipoic acid is a potent antioxidant that is naturally present in animal and plant cells [3,4] and is involved in many important biological and biochemical cellular processes [5]. α-Lipoic acid recycles vitamin C and E, and it increases intracellular glutathione concentrations [3,4], which reduces oxidative stress in vivo. α-Lipoic acid induces cell cycle arrest and apoptosis in transformed cells, while protecting their normal cell counterpart [6]. In experimental cancer therapy and cancer chemotherapy in humans, LA can decrease the toxicity of anticancer drugs
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(eg, doxorubicin), which are known to cause high rates of free radical formation. α-Lipoic acid, with its free radical scavenging capacity, has the potential to become a very useful substance for interfering with processes occurring in malignant cells. Moreover, by following this long-term administration of LA in both animals and humans, a low incidence of side effects (even at high concentrations of 400500 mg/kg of LA) supports the overall feasibility of using LA as a dietary supplement [3]. Hence, LA has been proposed to be an effective agent in cancer prevention. The ability of LA to prevent cancer, however, is dependent on the proper selection of an appropriate dose, which varies in different organs. Berkson et al [7] reported that at the low concentration of 1 μmol/L, LA acted as a growth factor, although it functioned as an antiproliferative agent at the concentration of 100 μmol/L. Organ-dependent effects have also been reported for colorectal [8,9], lung, breast, and prostate [10] cancer. α-Lipoic acid may have opposite effects on colon cancer vs breast cancer, as it was shown to inhibit the former but stimulate the latter [11]. There is limited knowledge regarding the effects of LA on cancer in terms of metastasis and matrix metalloproteinases (MMPs), through which it may be exerting its antitumor effects. The most well-known extracellular matrix (ECM)– degrading enzymes are the MMPs. MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the ECM. MMPs are structurally and functionally homologous proteins that (by structure and substrate specificity) can be divided into 4 families: collagenases, gelatinases, stromelysins, and membraneassociated MMPs. Among the human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are key enzymes that degrade type IV collagen [12]. These 2 MMPs share structural and catalytic similarities, but their transcription is independently regulated because of distinct arrays of regulatory elements in their gene promoters. In several studies, MMP-2 and MMP-9 were shown to be expressed in breast carcinoma tumor cells [13,14]. Therefore, the inhibition of MMP activity by dietary factors holds great promise for the prevention or inhibition of metastasis. We hypothesize that LA inhibits metastasis via inhibition of MMP activity. The purpose of this study is twofold: first, document whether LA inhibits metastasis in the MDA-MB231 human breast cancer cell lines; and second, to clarify whether the antimetastatic effect of LA occurs via reduction of MMP-2 and MMP-9 activities.
2. Methods and materials 2.1. Materials and reagents α-Lipoic acid was purchased from Sigma (St. Louis, MO, USA), dissolved in ethyl alcohol, and diluted in cell culture media. MDA-MB-231 cells were purchased from the American Type Culture Collection (Rockville, Md). The following reagents and chemicals were obtained from
the respective suppliers: Dulbecco modified Eagle medium/ Nutrient Mixture Ham's F12 (DMEM/F12), streptomycin, and penicillin were obtained from Gibco/BRL (Grand Island, NY, USA); and RIA-grade bovine serum albumin, transferrin, and other reagents were purchased from Sigma. 2.2. Cell culture The MDA-MB-231 human breast cancer cell line was maintained in DMEM/F12 containing 100 mL/L fetal bovine serum (FBS) with 100 000 U/L penicillin and 100 mg/L streptomycin. For our experiments, the cells were plated with and allowed to attach in DMEM/F12 containing 10% FBS. The cell monolayer was rinsed and starved of serum for 24 hours using DMEM/F12 supplemented with 5 mg/L transferrin, 1 g/L bovine serum albumin, and 5 μg/L selenium before being treated with LA. The plates were replaced with fresh serum-free media (SFM) with or without the indicated concentrations of LA. Viable cell numbers were estimated 12 and 24 hours after the cells were exposed to LA using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, as described previously [15]. 2.3. Boyden chamber motility assay PVDF filters (8-μm-diameter pore size) were coated with a 0.01% gelatin solution for 16 hours at room temperature. The MDA-MB-231 cells (2 × 106 cells/mL) were resuspended in media containing 0, 250, 500, or 1000 μmol/L LA and carefully transferred into the upper chambers. The lower chambers were filled with 10% FBS medium as a chemoattractant. The Boyden chamber was incubated at 37°C with 5% CO2 for 12 hours. After gently removing the filter from the chamber, the cells on the upper side of the chamber's filter were removed by wiping with a paper. The filter was stained with Diff-Quick stain solution (Dade Behring, Network, NJ), and the cells on the lower surface of the filter, which had successfully penetrated through the pores of the gelatin-coated filter, were fixed onto a glass slide. The cells in the lower slide were counted [16]. Three separate motility assays were performed. 2.4. Wound healing migration assay Wound-healing migration assays are based on the repopulation of wounded cultures as described previously [17]. The cells were seeded into 12-well culture plates at 5 × 105 cell/mL and cultured in medium containing 10% FBS till confluent. The confluent cell monolayers were incubated for 1 hour with 1 μg/mL of mitomycin C to stop cell proliferation. The monolayers were carefully wounded using a yellow pipette tip, and any cellular debris that was present was removed by washing with phosphate buffered saline (PBS). The wounded monolayers were then incubated for 24 hours in SFM containing 0, 250, 500, and 1000 μmol/ L LA. Photographs of the wounded areas were taken at time 0 and again after 12 and 24 hours.
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2.5. Matrigel invasion assay For invasion studies, wells of a Matrigel chamber (BD Bioscience, San Jose, CA, USA) were filled with SFM and brought to room temperature. The MDA-MB-231 cells (1 × 106 cell/mL) were resuspended in media containing 0, 250, 500 or 1000 μmol/L LA, and they were carefully transferred into the upper chambers. The lower chambers were filled with 10% FBS medium as a chemoattractant. The Matrigel chambers were incubated for 12 hours at 37°C with 5% CO2. Then, the cells on the upper surface of the filters were removed by wiping with a paper. The filters were stained with Diff-Quick stain solution (Dade Behring) and the cells on the lower surface of the filter were fixed onto a glass slide. The cells in 5 randomly selected microscopic fields (×400) of the lower surface of the filter were counted. Three separate experiments were performed. 2.6. Matrix metalloproteinase activity (gelatin zymography) The cells were seeded into a 6-well plate at 1 × 103 cells/ mL and incubated in a medium containing 10% FBS to form a confluent cell monolayer. Cells were treated with various concentrations of the LA. After 24 hours incubation, the media were collected and concentrated using Centricon devices, and the MMP activities were investigated using gelatin zymography. Zymography was performed using gels with 10% polyacrylamide and 1% gelatin [18]. Matrix metalloproteinase activity was visualized by staining with Coomassie blue.
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among the group means were tested at α = .05 using Duncan multiple range test. For each variable, at least 3 independent experiments were carried out. Data are given as the means ± SD. 3. Results 3.1. Effect of LA on MDA-MB-231 cell proliferation To examine the effect of LA on MDA-MB-231 breast cancer cell growth, cells in a monolayer culture were treated with LA (0-1000 μmol/L) for 0, 12, or 24 hours in SFM, and viable cell numbers were estimated. The number of viable MDA-MB-231 cells did not differ by LA treatment within 24 hours (Fig. 1). However, after 24-hour incubation with LA concentrations more than 250 μmol/L, the viable cell number significantly decreased (data not shown). To show that the antimetastatic effect of LA was independent of reduction in cell proliferation, LA treatment time was never more than 24 hours in this study. 3.2. Effects of LA on motility and invasion Both tumor metastasis and angiogenesis entail cell migration, first by the spreading of tumor cells outside of their primary site and, second, by cancer endothelial cells crossing the basement membrane and moving toward an angiogenic stimulus [21]. We examined whether LA is able to prevent the motility of MDA-MB-231 breast cancer cells
2.7. Reverse transcriptase–polymerase chain reaction Reverse transcriptase–polymerase chain reaction was performed as previously described [19]. Total RNA was isolated using Tri-reagent (Sigma), and cDNA was synthesized using 2 μg of total RNA with SuperScript reverse transcriptase (Invitrogen). For cDNA amplification, primers for MMP-2 (upstream primer, 5′-CAGGCTCTTCTCCTTTCSCAAC-3′; downstream primer, 5′-AAGCCACGGCTTGGTTTTCCTC-3′) and MMP-9 (upstream primer, 5′TGGGCTACGTGACCTATGACCAT-3′; downstream primer, 5′-GCCCAGCCCACCTCCACTCCTC-3′) were used. The primers were allowed to anneal at 55°C for 1 minute through 35 cycles. The expression of human β-actin transcripts was examined as an internal control, as described previously [20]. The PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The bands corresponding to each specific PCR product were quantified by densitometric scanning of the exposed film using the Bio-profile Bio-IL application (Vilber-Lourmat, Eberhardzell, Germany). 2.8. Statistical analyses Statistical analyses were performed using the Statistical Analysis System software (SAS Institute, Cary, NC). Data analysis between groups was performed by a 1-way analysis of variance, and significance of differences
Fig. 1. Effects of LA on cell proliferation in MDA-MB-231 cells. MDAMB-231 human breast cancer cells were plated at a density of 2.5 × 104 cells/ mL in 24-well plates with DMEM/F12 supplemented with 10% FBS for 24 hours. The monolayers were serum-starved with DMEM/F12 supplemented with 5 μg/mL transferrin, 5 ng/mL selenium, and 1 mg/mL bovine serum albumin for 24 hours. After serum starvation, the monolayers were incubated in serum-free medium with 0, 250, 500, and 1000 μmol/L LA. Viable cell numbers were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide assay. Values are the means ± SD from 3 separate experiments. Bars having different letters are significantly different (P b .05).
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using a Boyden chamber assay. Treatment of the MDA-MB231 cells with increasing concentrations of LA led to dosedependent decreases in cell motility (Fig. 2) and wound healing cell migration (Fig. 3). α-Lipoic acid also showed a dose-dependent inhibitory effect on cell invasion through a Matrigel chamber (Fig. 4). 3.3. α-Lipoic acid inhibits MMP activities and mRNA expressions
Fig. 2. Effects of LA on cell motility in MDA-MB-231 cells. The cells were cultured in the presence of various concentrations of LA for 12 hours with a Boyden chamber. Microphotograph of filters (A) and quantitative analysis of the Boyden chamber assay (B) are shown. Motility is expressed as a percentage of the control (0 μmol/L). Each bar represents the means ± SD calculated from 3 independent experiments. Bars having different letters are significantly different (P b .05).
To determine whether LA affects MMP-2 and MMP-9, which degrade the ECM to allow metastasis of malignant tumors, the activity and mRNA expression of these MMPs were tested. Fig. 5 shows that MMP-2 and MMP-9 activities were decreased in MDA-MB-231 cells by LA treatment in a dose-dependent manner. To determine whether the inhibition of MMP enzyme expression by LA is due to decreased levels of transcription, we performed RT-PCR. In the RT-PCR analysis, treatment of the MDA-MB-231 cells with LA decreased levels of MMP-2 and MMP-9 mRNA expression (Fig. 6). 4. Discussion α-Lipoic acid is a naturally occurring compound that is synthesized by animals and humans. It functions as a cofactor in several mitochondrial multienzyme complexes
Fig. 3. Effects of LA on migration in MDA-MB-231 cells. The cells were plated in 12-well plates at a density of 0.5 × 106 cells/well with DMEM/F12 supplemented with 10% FBS. Confluent monolayers were wounded and then incubated in serum-free medium in the presence of 0, 250, 500, and 1000 μmol/L LA. At 0, 12, and 24 hours after wounding, the cells were photographed under a phase contrast microscope.
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Fig. 4. Effects of LA on invasion in MDA-MB-231 cells. The cells were cultured in the presence of various concentrations of LA for 12 hours within a Matrigel invasion chamber. Microphotograph of filters (A) and quantitative analysis of the Matrigel chamber invasion assay (B) are shown. Invasion is expressed as a percentage of the control (0 μmol/L). Results are the means ± SD from 3 independent experiments. Bars having different letters are significantly different (P b .05).
by catalyzing the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids [22,23]. α-Lipoic acid is consumed in a typical daily diet, absorbed through the blood-brain barrier, and taken up and transformed by cells and tissues into dihydrolipoic acid [24]. α-Lipoic acid, an antioxidant, may protect DNA from free radicals, reduce the chance of oncogene mutations, and inhibit cancer cell proliferation. Many studies indicate that LA is able to suppress tumor growth. α-Lipoic acid was found to induce apoptosis in cancer cells such as leukemia and colon cancer cells but not in normal cells [9,25]. α-Lipoic acid acted as a growth factor at a low dose of 1 μmol/L, but it functioned as an antiproliferative agent at a higher concentration of 100 μmol/L [7]. In this study, LA had no effect on cell proliferation until 24 hours after high concentrations of LA (250-1000 μmol/L) treatment. In this study, 24 hours was selected for the rest of study because viability was not affected at this time point. This investigation attempted to address the role of LA in metastasis because LA may be exerting its antitumor effects through this process. Metastasis is a complex multistep process involving cell motility and invasion. Tumor cell invasion and metastasis involve the proteolytic degradation of basement membranes and the ECM, the alteration of cell to cell adhesion, and the physical movement of tumor cells [26]. Degradation of the ECM is initiated by proteinases secreted by different cell types participating in the tumor cell invasion, and increased expression or activity of every
Fig. 5. α-Lipoic acid decrease MMP-2 and MMP-9 activity in MDA-MB231 cells. MDA-MB-231 cells were plated in 6-well plates at a density of 1 × 106 cell/well with DMEM/F12 supplemented with 10% FBS for 48 hours; the monolayers were incubated in serum-free medium in the absence or presence of 0, 250, 500, and 1000 μmol/L LA for 24 h. The media were collected and the activities of MMP-2 (A) and MMP-9 (B) were measured by zymography. (A) Photograph of the MMP bands. (B) Quantitative analysis of the bands. Results are the means ± SD from 3 independent experiments. Bars having different letters are significantly different (P b .05).
known class of proteinases has been linked to malignancy and tumor cell invasion [27]. MMPs play crucial roles in tumor invasion [28]. The regulation of MMPs occurs at 3 levels: gene expression, proenzyme processing, and inhibition of enzyme activity [29,30]. Among the MMPs, MMP-2 and MMP-9 are key enzymes that degrade type IV collagen [12]. Several studies have already shown increased expression of MMP-2 and MMP-9 in malignant breast cancer [13,31]; hence, the interruption of one or more of these regulatory steps is one possible approach for antimetastatic therapy. To date, no direct evidence suggests that LA exerts such an effect on the development of cancer metastasis. Therefore, we first examined whether LA could affect metastasis in vitro using MDA-MB-231 breast cancer cell lines.
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Fig. 6. Effects of LA on MMP-2 and MMP-9 mRNA expression in MDAMB-231 cells. For RT-PCR, the MDA-MB-231 cells were treated with LA. Total RNA was isolated and RT-PCR was performed to investigate the mRNA expressions of MMP-2 (A) and MMP-9 (B). (A) Photographs of ethidium bromide-stained gels, which were representative of 3 independent experiments, are shown. (B) Quantitative analysis of RT-PCR. The relative abundance of each band was estimated by Image J Launcher (provided by NCBI). Results are means ± SD calculated from 3 independent experiments. Bars having different letters are significantly different (P b .05).
Motility is another property of cancer cells that is necessary for their migration from the primary tumor site to a secondary organ. The present study showed that treatment with doses more than 250 μmol/L of LA reduced the motility of MDA-MB-231 cells. α-Lipoic acid inhibited the migration and invasion of cells in a dose-dependent manner. Others have reported that LA inhibits migration in Jurkat cells [32] and vascular smooth muscle cells [33]. The observed alterations in metastatic cell phenotypes confirm our hypothesis that LA inhibits cancer metastasis in vitro. This study also investigated breast cancer cell invasiveness using a transwell chamber system. To successfully penetrate the filter, the cells must successfully degrade and
traverse the Matrigel-coated insert. The process of tumor cell invasion and metastasis requires the degradation of connective tissue associated with vascular basement membranes and interstitial connective tissue [34]. The basement membrane is the thick barrier between a free malignant cell and the bloodstream, and it must be traversed before malignant cells can enter circulating blood [35]. Thus, invasion through the basement membrane is a critical step in metastasis [36]. The present study shows that LA inhibits the invasion of MDA-MB-231 breast cancer cells in a dose-dependent manner. Cell invasion was decreased in the 24-hour treatment with 250 to 1000 μmol/L LA, although cell proliferation was not affected in this time frame. Therefore, these data suggest that the inhibitory effect of LA on cell invasion occurs independent of its effect on cell proliferation. In this study, we show that the activity and mRNA level of MMP-2 and MMP-9 are decreased in MDA-MB231 cells treated with LA. Others have also reported that LA inhibits MMP-9 activity and expression in a dosedependent manner [33,37]. Kim et al found that LA inhibits MMP-9 expression and nuclear factor-κB activity in high-glucose–induced, tumor necrosis factor-α–stimulated vascular smooth muscle cell (VSMC). They demonstrated that LA can inhibit MMP-9 promoter activity and expression; these effects are mediated by the suppression of the nuclear factor-κB pathway [33]. Marracci et al reported that LA chelates metallic ions and may sequester Zn2+ away from the active site of MMP-9 [37]. These results suggest that the decrease in metastasis observed with LA treatment may be due to its inhibitory effect on MMP-2 and MMP-9 activities. Therefore, we accept our hypothesis that LA reduces MMP-2 and MMP-9 activities. In conclusion, we have shown that LA inhibits the metastasis of MDA-MB-231 breast cancer cells. Our data suggest that the anticancer effects of LA may contribute to inhibition of metastasis through a decrease in the activity and mRNA expression of MMP-2 and MMP-9. The major limitations of our study are that there is no in vivo study to demonstrate the antimetastatic effect of LA and that high doses of LA were used. Thus, the mechanism behind the anticancer properties of LA needs to be investigated in vivo by others and additional research on more physiological levels of LA is needed. Acknowledgment This research was supported by the research fund of Dankook University in 2009. References [1] Hung HC, Joshipura KJ, Jiang R, Hu FB, Hunter D, Smith-Warner SA, et al. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst 2004;96:1577-84. [2] Touillaud MS, Thiebaut AC, Fournier A, Niravong M, Boutron-Ruault MC, Clavel-Chapelon F. Dietary lignan intake and postmenopausal
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Nutrition Research 30 (2010) 403 – 409 www.nrjournal.com
α-Lipoic acid reduces matrix metalloproteinase activity in MDA-MB-231 human breast cancer cells Hyun Sook Lee a , Mi Hee Na b , Woo Kyoung Kim b,⁎ a
Department of Foods and Nutrition, Kookmin University, Sungbuk-Gu, Seoul 136-702, South Korea Department of Food Science and Nutrition, Dankook University, Yongin-si, Gyeonggi-do 448-701, South Korea Received 3 February 2010; revised 7 June 2010; accepted 11 June 2010
b
Abstract α-Lipoic acid (LA), a naturally occurring molecule in animal and plant cells, is a potent antioxidant that reportedly exerts beneficial effects on cell proliferation and apoptosis in various cancer cell lines. However, the molecular mechanisms behind the antimetastatic property of LA are not well understood. The present study investigates the effect of LA on metastasis in a cell system. Our hypothesis is that LA inhibits metastasis via inhibition of matrix metalloproteinase (MMP) in vitro. MDA-MB-231 cells, a human breast cancer cell line, were treated with various concentrations of LA (0, 250, 500, or 1000 μmol/L) to measure metastasis, MMP activity, and mRNA expression. The viability of cells was examined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The effect of LA on metastasis was evaluated using the motility, migration, and invasion assay in vitro. The activity and mRNA expression of MMP-2 and MMP-9 were measured. After LA treatment, cell motility and cell migration were significantly decreased (P b .05). α-Lipoic acid also reduced cell invasion through a Matrigel-coated chamber (P b .05). Activities of MMP-2 and MMP-9 were decreased by LA treatment in a dose-dependent manner. RT-PCR analysis confirmed the reduction in mRNA expression level of MMP-2 and MMP-9 by LA treatment. We conclude that in this cell culture model, LA treatment inhibits cancer metastasis, and this inhibition is likely due to the decrease in the activity and mRNA expression levels of MMP-2 and MMP-9 caused by LA. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:
α-Lipoic acid; Metastasis; Breast cancer cells; MMP-2; MMP-9 ECM, extracellular matrix; FBS, fetal bovine serum; LA, α-Lipoic acid; MMP, matrix metalloproteinase; SFM, serum-free media.
1. Introduction Breast cancer is the leading cause of cancer-related mortality in Korea, as well as in the Western world; thus, effective chemoprevention could significantly impact this rising trend of cancer-related mortality. However, successful use of dietary antioxidants for tumor chemoprevention in humans remains an unmet goal [1]. ⁎ Corresponding author. Tel.: +82 31 8005 3172; fax: +82 31 8005 3170. E-mail address: [email protected] (W.K. Kim). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2010.06.009
It has been proposed that antioxidants play a protective role in breast cancer [2]. α-Lipoic acid is a potent antioxidant that is naturally present in animal and plant cells [3,4] and is involved in many important biological and biochemical cellular processes [5]. α-Lipoic acid recycles vitamin C and E, and it increases intracellular glutathione concentrations [3,4], which reduces oxidative stress in vivo. α-Lipoic acid induces cell cycle arrest and apoptosis in transformed cells, while protecting their normal cell counterpart [6]. In experimental cancer therapy and cancer chemotherapy in humans, LA can decrease the toxicity of anticancer drugs
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(eg, doxorubicin), which are known to cause high rates of free radical formation. α-Lipoic acid, with its free radical scavenging capacity, has the potential to become a very useful substance for interfering with processes occurring in malignant cells. Moreover, by following this long-term administration of LA in both animals and humans, a low incidence of side effects (even at high concentrations of 400500 mg/kg of LA) supports the overall feasibility of using LA as a dietary supplement [3]. Hence, LA has been proposed to be an effective agent in cancer prevention. The ability of LA to prevent cancer, however, is dependent on the proper selection of an appropriate dose, which varies in different organs. Berkson et al [7] reported that at the low concentration of 1 μmol/L, LA acted as a growth factor, although it functioned as an antiproliferative agent at the concentration of 100 μmol/L. Organ-dependent effects have also been reported for colorectal [8,9], lung, breast, and prostate [10] cancer. α-Lipoic acid may have opposite effects on colon cancer vs breast cancer, as it was shown to inhibit the former but stimulate the latter [11]. There is limited knowledge regarding the effects of LA on cancer in terms of metastasis and matrix metalloproteinases (MMPs), through which it may be exerting its antitumor effects. The most well-known extracellular matrix (ECM)– degrading enzymes are the MMPs. MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the ECM. MMPs are structurally and functionally homologous proteins that (by structure and substrate specificity) can be divided into 4 families: collagenases, gelatinases, stromelysins, and membraneassociated MMPs. Among the human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are key enzymes that degrade type IV collagen [12]. These 2 MMPs share structural and catalytic similarities, but their transcription is independently regulated because of distinct arrays of regulatory elements in their gene promoters. In several studies, MMP-2 and MMP-9 were shown to be expressed in breast carcinoma tumor cells [13,14]. Therefore, the inhibition of MMP activity by dietary factors holds great promise for the prevention or inhibition of metastasis. We hypothesize that LA inhibits metastasis via inhibition of MMP activity. The purpose of this study is twofold: first, document whether LA inhibits metastasis in the MDA-MB231 human breast cancer cell lines; and second, to clarify whether the antimetastatic effect of LA occurs via reduction of MMP-2 and MMP-9 activities.
2. Methods and materials 2.1. Materials and reagents α-Lipoic acid was purchased from Sigma (St. Louis, MO, USA), dissolved in ethyl alcohol, and diluted in cell culture media. MDA-MB-231 cells were purchased from the American Type Culture Collection (Rockville, Md). The following reagents and chemicals were obtained from
the respective suppliers: Dulbecco modified Eagle medium/ Nutrient Mixture Ham's F12 (DMEM/F12), streptomycin, and penicillin were obtained from Gibco/BRL (Grand Island, NY, USA); and RIA-grade bovine serum albumin, transferrin, and other reagents were purchased from Sigma. 2.2. Cell culture The MDA-MB-231 human breast cancer cell line was maintained in DMEM/F12 containing 100 mL/L fetal bovine serum (FBS) with 100 000 U/L penicillin and 100 mg/L streptomycin. For our experiments, the cells were plated with and allowed to attach in DMEM/F12 containing 10% FBS. The cell monolayer was rinsed and starved of serum for 24 hours using DMEM/F12 supplemented with 5 mg/L transferrin, 1 g/L bovine serum albumin, and 5 μg/L selenium before being treated with LA. The plates were replaced with fresh serum-free media (SFM) with or without the indicated concentrations of LA. Viable cell numbers were estimated 12 and 24 hours after the cells were exposed to LA using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, as described previously [15]. 2.3. Boyden chamber motility assay PVDF filters (8-μm-diameter pore size) were coated with a 0.01% gelatin solution for 16 hours at room temperature. The MDA-MB-231 cells (2 × 106 cells/mL) were resuspended in media containing 0, 250, 500, or 1000 μmol/L LA and carefully transferred into the upper chambers. The lower chambers were filled with 10% FBS medium as a chemoattractant. The Boyden chamber was incubated at 37°C with 5% CO2 for 12 hours. After gently removing the filter from the chamber, the cells on the upper side of the chamber's filter were removed by wiping with a paper. The filter was stained with Diff-Quick stain solution (Dade Behring, Network, NJ), and the cells on the lower surface of the filter, which had successfully penetrated through the pores of the gelatin-coated filter, were fixed onto a glass slide. The cells in the lower slide were counted [16]. Three separate motility assays were performed. 2.4. Wound healing migration assay Wound-healing migration assays are based on the repopulation of wounded cultures as described previously [17]. The cells were seeded into 12-well culture plates at 5 × 105 cell/mL and cultured in medium containing 10% FBS till confluent. The confluent cell monolayers were incubated for 1 hour with 1 μg/mL of mitomycin C to stop cell proliferation. The monolayers were carefully wounded using a yellow pipette tip, and any cellular debris that was present was removed by washing with phosphate buffered saline (PBS). The wounded monolayers were then incubated for 24 hours in SFM containing 0, 250, 500, and 1000 μmol/ L LA. Photographs of the wounded areas were taken at time 0 and again after 12 and 24 hours.
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2.5. Matrigel invasion assay For invasion studies, wells of a Matrigel chamber (BD Bioscience, San Jose, CA, USA) were filled with SFM and brought to room temperature. The MDA-MB-231 cells (1 × 106 cell/mL) were resuspended in media containing 0, 250, 500 or 1000 μmol/L LA, and they were carefully transferred into the upper chambers. The lower chambers were filled with 10% FBS medium as a chemoattractant. The Matrigel chambers were incubated for 12 hours at 37°C with 5% CO2. Then, the cells on the upper surface of the filters were removed by wiping with a paper. The filters were stained with Diff-Quick stain solution (Dade Behring) and the cells on the lower surface of the filter were fixed onto a glass slide. The cells in 5 randomly selected microscopic fields (×400) of the lower surface of the filter were counted. Three separate experiments were performed. 2.6. Matrix metalloproteinase activity (gelatin zymography) The cells were seeded into a 6-well plate at 1 × 103 cells/ mL and incubated in a medium containing 10% FBS to form a confluent cell monolayer. Cells were treated with various concentrations of the LA. After 24 hours incubation, the media were collected and concentrated using Centricon devices, and the MMP activities were investigated using gelatin zymography. Zymography was performed using gels with 10% polyacrylamide and 1% gelatin [18]. Matrix metalloproteinase activity was visualized by staining with Coomassie blue.
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among the group means were tested at α = .05 using Duncan multiple range test. For each variable, at least 3 independent experiments were carried out. Data are given as the means ± SD. 3. Results 3.1. Effect of LA on MDA-MB-231 cell proliferation To examine the effect of LA on MDA-MB-231 breast cancer cell growth, cells in a monolayer culture were treated with LA (0-1000 μmol/L) for 0, 12, or 24 hours in SFM, and viable cell numbers were estimated. The number of viable MDA-MB-231 cells did not differ by LA treatment within 24 hours (Fig. 1). However, after 24-hour incubation with LA concentrations more than 250 μmol/L, the viable cell number significantly decreased (data not shown). To show that the antimetastatic effect of LA was independent of reduction in cell proliferation, LA treatment time was never more than 24 hours in this study. 3.2. Effects of LA on motility and invasion Both tumor metastasis and angiogenesis entail cell migration, first by the spreading of tumor cells outside of their primary site and, second, by cancer endothelial cells crossing the basement membrane and moving toward an angiogenic stimulus [21]. We examined whether LA is able to prevent the motility of MDA-MB-231 breast cancer cells
2.7. Reverse transcriptase–polymerase chain reaction Reverse transcriptase–polymerase chain reaction was performed as previously described [19]. Total RNA was isolated using Tri-reagent (Sigma), and cDNA was synthesized using 2 μg of total RNA with SuperScript reverse transcriptase (Invitrogen). For cDNA amplification, primers for MMP-2 (upstream primer, 5′-CAGGCTCTTCTCCTTTCSCAAC-3′; downstream primer, 5′-AAGCCACGGCTTGGTTTTCCTC-3′) and MMP-9 (upstream primer, 5′TGGGCTACGTGACCTATGACCAT-3′; downstream primer, 5′-GCCCAGCCCACCTCCACTCCTC-3′) were used. The primers were allowed to anneal at 55°C for 1 minute through 35 cycles. The expression of human β-actin transcripts was examined as an internal control, as described previously [20]. The PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The bands corresponding to each specific PCR product were quantified by densitometric scanning of the exposed film using the Bio-profile Bio-IL application (Vilber-Lourmat, Eberhardzell, Germany). 2.8. Statistical analyses Statistical analyses were performed using the Statistical Analysis System software (SAS Institute, Cary, NC). Data analysis between groups was performed by a 1-way analysis of variance, and significance of differences
Fig. 1. Effects of LA on cell proliferation in MDA-MB-231 cells. MDAMB-231 human breast cancer cells were plated at a density of 2.5 × 104 cells/ mL in 24-well plates with DMEM/F12 supplemented with 10% FBS for 24 hours. The monolayers were serum-starved with DMEM/F12 supplemented with 5 μg/mL transferrin, 5 ng/mL selenium, and 1 mg/mL bovine serum albumin for 24 hours. After serum starvation, the monolayers were incubated in serum-free medium with 0, 250, 500, and 1000 μmol/L LA. Viable cell numbers were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide assay. Values are the means ± SD from 3 separate experiments. Bars having different letters are significantly different (P b .05).
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using a Boyden chamber assay. Treatment of the MDA-MB231 cells with increasing concentrations of LA led to dosedependent decreases in cell motility (Fig. 2) and wound healing cell migration (Fig. 3). α-Lipoic acid also showed a dose-dependent inhibitory effect on cell invasion through a Matrigel chamber (Fig. 4). 3.3. α-Lipoic acid inhibits MMP activities and mRNA expressions
Fig. 2. Effects of LA on cell motility in MDA-MB-231 cells. The cells were cultured in the presence of various concentrations of LA for 12 hours with a Boyden chamber. Microphotograph of filters (A) and quantitative analysis of the Boyden chamber assay (B) are shown. Motility is expressed as a percentage of the control (0 μmol/L). Each bar represents the means ± SD calculated from 3 independent experiments. Bars having different letters are significantly different (P b .05).
To determine whether LA affects MMP-2 and MMP-9, which degrade the ECM to allow metastasis of malignant tumors, the activity and mRNA expression of these MMPs were tested. Fig. 5 shows that MMP-2 and MMP-9 activities were decreased in MDA-MB-231 cells by LA treatment in a dose-dependent manner. To determine whether the inhibition of MMP enzyme expression by LA is due to decreased levels of transcription, we performed RT-PCR. In the RT-PCR analysis, treatment of the MDA-MB-231 cells with LA decreased levels of MMP-2 and MMP-9 mRNA expression (Fig. 6). 4. Discussion α-Lipoic acid is a naturally occurring compound that is synthesized by animals and humans. It functions as a cofactor in several mitochondrial multienzyme complexes
Fig. 3. Effects of LA on migration in MDA-MB-231 cells. The cells were plated in 12-well plates at a density of 0.5 × 106 cells/well with DMEM/F12 supplemented with 10% FBS. Confluent monolayers were wounded and then incubated in serum-free medium in the presence of 0, 250, 500, and 1000 μmol/L LA. At 0, 12, and 24 hours after wounding, the cells were photographed under a phase contrast microscope.
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Fig. 4. Effects of LA on invasion in MDA-MB-231 cells. The cells were cultured in the presence of various concentrations of LA for 12 hours within a Matrigel invasion chamber. Microphotograph of filters (A) and quantitative analysis of the Matrigel chamber invasion assay (B) are shown. Invasion is expressed as a percentage of the control (0 μmol/L). Results are the means ± SD from 3 independent experiments. Bars having different letters are significantly different (P b .05).
by catalyzing the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids [22,23]. α-Lipoic acid is consumed in a typical daily diet, absorbed through the blood-brain barrier, and taken up and transformed by cells and tissues into dihydrolipoic acid [24]. α-Lipoic acid, an antioxidant, may protect DNA from free radicals, reduce the chance of oncogene mutations, and inhibit cancer cell proliferation. Many studies indicate that LA is able to suppress tumor growth. α-Lipoic acid was found to induce apoptosis in cancer cells such as leukemia and colon cancer cells but not in normal cells [9,25]. α-Lipoic acid acted as a growth factor at a low dose of 1 μmol/L, but it functioned as an antiproliferative agent at a higher concentration of 100 μmol/L [7]. In this study, LA had no effect on cell proliferation until 24 hours after high concentrations of LA (250-1000 μmol/L) treatment. In this study, 24 hours was selected for the rest of study because viability was not affected at this time point. This investigation attempted to address the role of LA in metastasis because LA may be exerting its antitumor effects through this process. Metastasis is a complex multistep process involving cell motility and invasion. Tumor cell invasion and metastasis involve the proteolytic degradation of basement membranes and the ECM, the alteration of cell to cell adhesion, and the physical movement of tumor cells [26]. Degradation of the ECM is initiated by proteinases secreted by different cell types participating in the tumor cell invasion, and increased expression or activity of every
Fig. 5. α-Lipoic acid decrease MMP-2 and MMP-9 activity in MDA-MB231 cells. MDA-MB-231 cells were plated in 6-well plates at a density of 1 × 106 cell/well with DMEM/F12 supplemented with 10% FBS for 48 hours; the monolayers were incubated in serum-free medium in the absence or presence of 0, 250, 500, and 1000 μmol/L LA for 24 h. The media were collected and the activities of MMP-2 (A) and MMP-9 (B) were measured by zymography. (A) Photograph of the MMP bands. (B) Quantitative analysis of the bands. Results are the means ± SD from 3 independent experiments. Bars having different letters are significantly different (P b .05).
known class of proteinases has been linked to malignancy and tumor cell invasion [27]. MMPs play crucial roles in tumor invasion [28]. The regulation of MMPs occurs at 3 levels: gene expression, proenzyme processing, and inhibition of enzyme activity [29,30]. Among the MMPs, MMP-2 and MMP-9 are key enzymes that degrade type IV collagen [12]. Several studies have already shown increased expression of MMP-2 and MMP-9 in malignant breast cancer [13,31]; hence, the interruption of one or more of these regulatory steps is one possible approach for antimetastatic therapy. To date, no direct evidence suggests that LA exerts such an effect on the development of cancer metastasis. Therefore, we first examined whether LA could affect metastasis in vitro using MDA-MB-231 breast cancer cell lines.
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Fig. 6. Effects of LA on MMP-2 and MMP-9 mRNA expression in MDAMB-231 cells. For RT-PCR, the MDA-MB-231 cells were treated with LA. Total RNA was isolated and RT-PCR was performed to investigate the mRNA expressions of MMP-2 (A) and MMP-9 (B). (A) Photographs of ethidium bromide-stained gels, which were representative of 3 independent experiments, are shown. (B) Quantitative analysis of RT-PCR. The relative abundance of each band was estimated by Image J Launcher (provided by NCBI). Results are means ± SD calculated from 3 independent experiments. Bars having different letters are significantly different (P b .05).
Motility is another property of cancer cells that is necessary for their migration from the primary tumor site to a secondary organ. The present study showed that treatment with doses more than 250 μmol/L of LA reduced the motility of MDA-MB-231 cells. α-Lipoic acid inhibited the migration and invasion of cells in a dose-dependent manner. Others have reported that LA inhibits migration in Jurkat cells [32] and vascular smooth muscle cells [33]. The observed alterations in metastatic cell phenotypes confirm our hypothesis that LA inhibits cancer metastasis in vitro. This study also investigated breast cancer cell invasiveness using a transwell chamber system. To successfully penetrate the filter, the cells must successfully degrade and
traverse the Matrigel-coated insert. The process of tumor cell invasion and metastasis requires the degradation of connective tissue associated with vascular basement membranes and interstitial connective tissue [34]. The basement membrane is the thick barrier between a free malignant cell and the bloodstream, and it must be traversed before malignant cells can enter circulating blood [35]. Thus, invasion through the basement membrane is a critical step in metastasis [36]. The present study shows that LA inhibits the invasion of MDA-MB-231 breast cancer cells in a dose-dependent manner. Cell invasion was decreased in the 24-hour treatment with 250 to 1000 μmol/L LA, although cell proliferation was not affected in this time frame. Therefore, these data suggest that the inhibitory effect of LA on cell invasion occurs independent of its effect on cell proliferation. In this study, we show that the activity and mRNA level of MMP-2 and MMP-9 are decreased in MDA-MB231 cells treated with LA. Others have also reported that LA inhibits MMP-9 activity and expression in a dosedependent manner [33,37]. Kim et al found that LA inhibits MMP-9 expression and nuclear factor-κB activity in high-glucose–induced, tumor necrosis factor-α–stimulated vascular smooth muscle cell (VSMC). They demonstrated that LA can inhibit MMP-9 promoter activity and expression; these effects are mediated by the suppression of the nuclear factor-κB pathway [33]. Marracci et al reported that LA chelates metallic ions and may sequester Zn2+ away from the active site of MMP-9 [37]. These results suggest that the decrease in metastasis observed with LA treatment may be due to its inhibitory effect on MMP-2 and MMP-9 activities. Therefore, we accept our hypothesis that LA reduces MMP-2 and MMP-9 activities. In conclusion, we have shown that LA inhibits the metastasis of MDA-MB-231 breast cancer cells. Our data suggest that the anticancer effects of LA may contribute to inhibition of metastasis through a decrease in the activity and mRNA expression of MMP-2 and MMP-9. The major limitations of our study are that there is no in vivo study to demonstrate the antimetastatic effect of LA and that high doses of LA were used. Thus, the mechanism behind the anticancer properties of LA needs to be investigated in vivo by others and additional research on more physiological levels of LA is needed. Acknowledgment This research was supported by the research fund of Dankook University in 2009. References [1] Hung HC, Joshipura KJ, Jiang R, Hu FB, Hunter D, Smith-Warner SA, et al. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst 2004;96:1577-84. [2] Touillaud MS, Thiebaut AC, Fournier A, Niravong M, Boutron-Ruault MC, Clavel-Chapelon F. Dietary lignan intake and postmenopausal
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