2014;39(5):711-6.
Abstract
Metastases are known to be responsible for approximately 90% of breast cancer-related deaths. Cyclooxygenase-2 (COX-2) is involved not only in inflammatory processes, but also in the metastasis of cancer cells; it is expressed in 40% of human invasive breast cancers. To comprehensively analyze the effects of cannabidiolic acid (CBDA), a selective COX-2 inhibitor found in the fiber-type cannabis plant (Takeda et al., 2008), on COX-2expression and the genes involved in metastasis, we performed a DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are invasive breast cancer cells that express high levels of COX-2, treated with CBDA for 48 hr at 25 µM. The results obtained revealed that COX-2 and Id-1, a positive regulator of breast cancer metastasis, were down-regulated (0.19-fold and 0.52-fold, respectively), while SHARP1 (or BHLHE41), a suppressor of breast cancer metastasis, was up-regulated (1.72-fold) and CHIP (or STUB1) was unaffected (1.03-fold). These changes were confirmed by real-time RT-PCR analyses. Taken together, the results obtained here demonstrated that i) CBDA had dual inhibitory effects on COX-2through down-regulation and enzyme inhibition, and ii) CBDA may possess the ability to suppress genes that are positively involved in the metastasis of cancer cells in vitro.
- PMID:
- 25242400
- [PubMed – indexed for MEDLINE]
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The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.39, No.5, 711-716, 2014
Toxicomics Report
Down-regulation of cyclooxygenase-2 (COX-2) by cannabidiolic acid in human breast cancer cells
Shuso Takeda1,2, Hiroyuki Okazaki3, Eriko Ikeda1, Satomi Abe1, Yasushi Yoshioka1, Kazuhito Watanabe4 and Hironori Aramaki1,3
1Department of Molecular Biology, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
2Laboratory of Xenobiotic Metabolism and Environmental Toxicology, Faculty of Pharmaceutical Sciences, Hiroshima International University (HIU), 5-1-1 Hiro-koshingai, Kure, Hiroshima 737-0112, Japan 3Drug Innovation Research Center, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
4Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University,
Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan
(Received April 15, 2014; Accepted June 30, 2014)
2Laboratory of Xenobiotic Metabolism and Environmental Toxicology, Faculty of Pharmaceutical Sciences, Hiroshima International University (HIU), 5-1-1 Hiro-koshingai, Kure, Hiroshima 737-0112, Japan 3Drug Innovation Research Center, Daiichi University of Pharmacy, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
4Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University,
Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan
(Received April 15, 2014; Accepted June 30, 2014)
INTRODUCTION:
Cannabidiol (CBD), a major constituent of the fiber-type cannabis plant, exhibits a wide range of biological activ- ities, such as anti-cancer cell proliferation, anti-cerebral infarction, and the inhibition of 15-lipoxygenase (Mishima et al., 2005; Izzo et al., 2009; Takeda et al., 2009; Caffarel et al., 2012; Takeda, 2013). In fresh plant materials, most CBD exists in its acid form, cannabidiolic acid (CBDA, Fig. 1A) (Yamauchi et al., 1967; Turner et al., 1980; Taura et al., 2007). The specific use of the acidic can- nabinoid as an active pharmaceutical ingredient has not yet been achieved because CBDA is recognized as the pharmacologically inactive form (Yamauchi et al., 1967; Razdan, 1986; Burstein, 1999). However, recent studies including ours demonstrated that, in addition to CBD, CBDA by itself exhibits biological actions, such as anti- bacterial effects (Appendino et al., 2008), the inhibition of cyclooxygenase-2 (COX-2) (Takeda et al., 2008), and anti-nausea/emetic effects (Bolognini et al., 2013; Rock et al., 2013).
Cannabidiol (CBD), a major constituent of the fiber-type cannabis plant, exhibits a wide range of biological activ- ities, such as anti-cancer cell proliferation, anti-cerebral infarction, and the inhibition of 15-lipoxygenase (Mishima et al., 2005; Izzo et al., 2009; Takeda et al., 2009; Caffarel et al., 2012; Takeda, 2013). In fresh plant materials, most CBD exists in its acid form, cannabidiolic acid (CBDA, Fig. 1A) (Yamauchi et al., 1967; Turner et al., 1980; Taura et al., 2007). The specific use of the acidic can- nabinoid as an active pharmaceutical ingredient has not yet been achieved because CBDA is recognized as the pharmacologically inactive form (Yamauchi et al., 1967; Razdan, 1986; Burstein, 1999). However, recent studies including ours demonstrated that, in addition to CBD, CBDA by itself exhibits biological actions, such as anti- bacterial effects (Appendino et al., 2008), the inhibition of cyclooxygenase-2 (COX-2) (Takeda et al., 2008), and anti-nausea/emetic effects (Bolognini et al., 2013; Rock et al., 2013).
Approximately 90% of breast cancer-related deaths have been attributed to metastasis to bones and the lungs (Hanahan and Weinberg, 2011). COX-2 is expressed in approximately 40% of human invasive breast cancers (Singh et al., 2007; Holmes et al., 2011), and may analyses.
MATERIALS AND METHODS:
Materials and cell culture
CBDA (purity: 96.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). DuP-697 (puri- ty: > 96%) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All other reagents were of analytical grade, commercially available, and used without further purification. Cell culture (human breast cancer MDA-MB-231 cells) conditions and methods were based on procedures described previously (Takeda et al., 2011, 2013a). Briefly, the MDA-MB-231 cell line (obtained from the American Type Culture Collection, Rockville, MD, USA) was routinely grown in phenol red- containing minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA), supplemented with 10 mM HEPES, 5% fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37°C in a 5% CO2– 95% air-hu- midified incubator. Prior to the 24-hr chemical treatments, the medium was changed to phenol red-free minimum essential medium alpha (Invitrogen) supplemented with 10 mM HEPES, 5% dextran-coated charcoal-treated serum, 100 U/mL of penicillin, and 100 g/mL of strepto- mycin. Cells were then re-seeded into 6-well plates at dif- ferent densities; 30 x 105 cells (31,250 cells/cm2), 40 x 105 cells (41,667 cells/cm2), or 50 x 105 cells (52,083 cells/ cm2)/well. CBDA and DuP-697 were prepared in etha- nol and dimethylsulfoxide (DMSO), respectively. Control incubations contained equivalent additions of ethanol or DMSO…
..RESULTS AND DISCUSSION
CBDA (purity: 96.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). DuP-697 (puri- ty: > 96%) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All other reagents were of analytical grade, commercially available, and used without further purification. Cell culture (human breast cancer MDA-MB-231 cells) conditions and methods were based on procedures described previously (Takeda et al., 2011, 2013a). Briefly, the MDA-MB-231 cell line (obtained from the American Type Culture Collection, Rockville, MD, USA) was routinely grown in phenol red- containing minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA), supplemented with 10 mM HEPES, 5% fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin at 37°C in a 5% CO2– 95% air-hu- midified incubator. Prior to the 24-hr chemical treatments, the medium was changed to phenol red-free minimum essential medium alpha (Invitrogen) supplemented with 10 mM HEPES, 5% dextran-coated charcoal-treated serum, 100 U/mL of penicillin, and 100 g/mL of strepto- mycin. Cells were then re-seeded into 6-well plates at dif- ferent densities; 30 x 105 cells (31,250 cells/cm2), 40 x 105 cells (41,667 cells/cm2), or 50 x 105 cells (52,083 cells/ cm2)/well. CBDA and DuP-697 were prepared in etha- nol and dimethylsulfoxide (DMSO), respectively. Control incubations contained equivalent additions of ethanol or DMSO…
..RESULTS AND DISCUSSION
The anti-proliferative potential of CBDA (Fig. 1A) on MDA-MB-231 cell growth was shown to be marked- ly less than that of CBD (IC50 values of CBD and CBDA = 1~10 M and > 25 M, respectively) (Ligresti et al., 2006; McAllister et al., 2007; Shrivastava et al., 2011). In this study, exposure of MDA-MB-231 cells to CBDA (25 M) for 48 hr did not affect cell viability (data not shown) (Takeda et al., 2012). Under the condition (25 M CBDA for 48 hr), we performed DNA microarray anal- ysis to investigate genes regulated by CBDA in MDA- MB-231 cells (cell density: 30 x 105 cells/well). Among 19,596 genes, CBDA increased the expression of 449 genes (> 2-fold) and decreased the expression 639 genes (< 0.5-fold) in MDA-MB-231 cells. We focused on four genes involved in the metastasis of breast cancer: COX-2, Id-1, CHIP (also known as STUB1), and SHARP1 (also known as BHLHE41). The expression of COX-2 and Id-1 was down-regulated 0.19-fold and 0.52-fold, respective- ly, by CBDA, while SHARP1, a suppressor of breast can- cer metastasis (Montagner et al., 2012) was up-regulated (1.72-fold). However, CHIP, which acts as a suppressor of breast cancer metastasis (Kajiro et al., 2009), was unaf- fected (1.03-fold) by the cannabinoid (Fig. 1B). A previ- ous study reported that the Id-1 gene, a positive regula- tor of breast cancer metastasis/progression (Fong et al., 2003), was down-regulated by CBD (McAllister et al., 2007); therefore, CBDA, the precursor of CBD (see Fig. 1A), may use a similar mechanism(s) to CBD in order to suppress the expression of Id-1.
COX-2 expression was previously suggested to corre- late with tumor cell density (Brueggemeier et al., 1999; Davies et al., 2002). We focused on COX-2 and CHIP, and examined the effects of breast cancer cell density on their expression. As shown in Fig. 2A, COX-2 expres- sion increased slightly as a function of the density of cells ranging from 30 to 50 x 105 cells/well, whereas the addition of CBDA to these cells down-regulated COX-2 expression. In contrast, CHIP expression was not mark- edly modulated by either CBDA or cell density (see also Fig. 2B, 1.12-fold). The down-regulation of COX-2 by CBDA was also demonstrated using real-time RT-PCR analyses (0.45-fold) (Fig. 2B). Although the inability of CBDA to modulate the expression of CHIP in MDA-MB- 231 cells was revealed, CBDA may target gene(s) whose expression can be affected by cancer cell density.
Although we demonstrated for the first time that CBDA was a potent inhibitor of COX-2 (IC50 = 2.2 M) puri- fied from sheep placental cotyledons, which are a general enzyme source for screening (Cayman Chemical Company) (Takeda et al., 2008), a contradictory phenomenon, in which CBDA exhibited a very weak inhibitory potential on COX-2, was recently reported (Ruhaak et al., 2011). Since this research group and we used the same enzyme source, this discrepancy may have been caused by differ- ences in the experimental conditions or purity of CBDA. In the present study, we could not definitively identify the molecular mechanism(s) underlying the CBDA-mediated inhibition of COX-2 expression; however, previous stud- ies reported that sodium salicylate inhibited transcription of the COX-2 gene, which was driven by the transcrip- tion factors, activator protein 1 (AP-1) and nuclear factor- B (NF-B) (Kopp and Ghosh, 1994; Dong et al., 1997; Xu et al., 1999). Since CBDA contains a salicylic acid moiety in its structure (see Fig. 1A, the moiety is high- lighted with a gray inclusion), the possible involvement of this moiety is implicated. Furthermore, we investigat- ed the effect of DuP-697, an established COX-2 inhibi- tor (IC50 = 0.05 M) (Cayman Chemical), on the expres- sion of Id-1, SHARP1, and CHIP genes using real-time RT-PCR analysis. As in the case of CBDA, 25 M DuP- 697 exhibited up-regulation of SHARP1 (1.48-fold) and no remarkable change in CHIP (1.20-fold); however, Id-1 expression, which can be down-regulated by CBDA, was largely unchanged by the inhibitor (0.82-fold) (see Fig. 1B). Thus, it is suggested that stimulation of SHARP1 expression results from COX-2 inhibition, but reduction in expression of Id-1 is caused by CBDA’s property as mentioned above.
Although we demonstrated for the first time that CBDA was a potent inhibitor of COX-2 (IC50 = 2.2 M) puri- fied from sheep placental cotyledons, which are a general enzyme source for screening (Cayman Chemical Company) (Takeda et al., 2008), a contradictory phenomenon, in which CBDA exhibited a very weak inhibitory potential on COX-2, was recently reported (Ruhaak et al., 2011). Since this research group and we used the same enzyme source, this discrepancy may have been caused by differ- ences in the experimental conditions or purity of CBDA. In the present study, we could not definitively identify the molecular mechanism(s) underlying the CBDA-mediated inhibition of COX-2 expression; however, previous stud- ies reported that sodium salicylate inhibited transcription of the COX-2 gene, which was driven by the transcrip- tion factors, activator protein 1 (AP-1) and nuclear factor- B (NF-B) (Kopp and Ghosh, 1994; Dong et al., 1997; Xu et al., 1999). Since CBDA contains a salicylic acid moiety in its structure (see Fig. 1A, the moiety is high- lighted with a gray inclusion), the possible involvement of this moiety is implicated. Furthermore, we investigat- ed the effect of DuP-697, an established COX-2 inhibi- tor (IC50 = 0.05 M) (Cayman Chemical), on the expres- sion of Id-1, SHARP1, and CHIP genes using real-time RT-PCR analysis. As in the case of CBDA, 25 M DuP- 697 exhibited up-regulation of SHARP1 (1.48-fold) and no remarkable change in CHIP (1.20-fold); however, Id-1 expression, which can be down-regulated by CBDA, was largely unchanged by the inhibitor (0.82-fold) (see Fig. 1B). Thus, it is suggested that stimulation of SHARP1 expression results from COX-2 inhibition, but reduction in expression of Id-1 is caused by CBDA’s property as mentioned above.
Taken together, the results of the present study suggest that COX-2 inhibitors that can down-regulate the enzyme may also be useful in inhibiting it, which may abrogate metastasis. Further studies are needed to demonstrate the biological effects of CBDA in vivo.
