Polychlorinated biphenyls (PCBs) are a family of structurally related chlorinated aromatic hydrocarbons. Numerous studies have documented a wide spectrum of biological effects of PCBs on human health, such as immunotoxicity, neurotoxocity, estrogenic or antiestrogenic activity, and carcinogensis. The role of PCBs as etiologic agents for breast cancer has been intensively explored in a variety of in vivo, animal and epidemiologic studies. A number of investigations indicated that higher levels of PCBs in mammary tissues or sera correlated to breast cancer risk, and PCBs might be implicated in advancing breast cancer progression.
In the current study, we for the first time report that PCBs greatly promote the ROCK activity and therefore increase cell motility for both non-metastatic and metastatic human breast cancer cells in vitro. In the in vivo study, PCBs significantly advance disease progression, leading to enhanced capability of metastatic breast cancer cells to metastasize to bone, lung and liver. Additionally, PCBs robustly induce the production of intracellular reactive oxygen species (ROS) in breast cancer cells; ROS mechanistically elevate ROCK activity.
Citation: Liu S, Li S, Du Y (2010) Polychlorinated Biphenyls (PCBs) Enhance Metastatic Properties of Breast Cancer Cells by Activating Rho-Associated Kinase (ROCK). PLoS ONE 5(6): e11272. doi:10.1371/journal.pone.0011272
Editor: Syed A. Aziz, Health Canada, Canada
Received: May 8, 2010; Accepted: May 28, 2010; Published: June 23, 2010
Copyright: © 2010 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the “Hundreds Talents” program of the Chinese Academy of Sciences (http://english.cas.cn/), and National Natural Science Foundation of China (NSFC) (http://www.nsfc.gov.cn/Portal0/default106.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Polychlorinated biphenyls (PCBs) are chlorinated aromatic hydrocarbons with 209 congeners. They are highly stable and lipophilic chemicals widely distributed in the environment. Although the production and massive use of PCBs have been banned in the 1970 s, they persist in the environment, biomagnify through food chains and consequently accumulate in fat tissues in humans , , . For many years, PCBs are concerned as etiologic agents for breast cancer and other cancers. Initial studies indicated higher levels of PCBs in mammary tissues or sera corresponded to increased risk of breast cancer , ; however, later studies showed no positive association between PCB exposure and breast cancer development, with possible positive correlations to some specific PCB congeners or among particular populations , , . Some recent investigations suggested that PCBs were implicated in promoting breast cancer progression, and some particular PCB congeners might contribute to high-grade tumors and overall poor prognosis in breast cancer patients , , .
Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among women. The real threat to lives of breast cancer patients is not the primary tumor in the breast, but secondary tumors in other organs (e.g. bone and lung) which accounts for 90% of deaths of breast cancer patients. Once breast cancer cells metastasize and form tumors in distant organs, the disease is incurable with available therapeutics . Until now, the underlying mechanisms responsible for breast cancer metastasis remain to be elucidated, and there is by far no metastasis-specific therapy in clinical practice.
The Rho-associated kinases, ROCK 1 and 2 (here referred to as ROCK), are major mediators of Rho activity . ROCK is implicated in the regulation of in vitro invasion and motility and in vivo metastasis of cancers . Clinical studies show that the expression of ROCK is significantly increased in human breast cancer tumors with metastases than those without metastases , . And increased expression of ROCK is associated with higher pathological grades and later stages, and its expression level is strongly correlated to the overall survival in breast cancer patients . Overexpression of ROCK can significantly enhance in vitro cell invasion/migration in cancer cells , , ; however, the expression of the dominant-negative ROCK and the ROCK inhibitor, Y27632, can massively suppress in vitro cancer cell invasion/migration and in vivo motility and dissemination , , , .
In this study, to delineate the associations between the PCB exposure and breast cancer risk and progression, we assess the effects of PCBs on the tumorigenic and metastatic features of breast cancer cells. Overall, we describe a novel role of PCBs in enhancing metastatic properties of breast cancer cells by activating the ROCK signaling.
Results and Discussion
The concerns with respect to likely carcinogenic influence on mammary tissues with exposure to PCBs derived from the estrogenic activity of these compounds, which have the ability to mimic or interfere with the action of endogenous hormones , , . Because of their lipophilic nature, PCBs tend to accumulate in adipose tissues, and therefore could be detectable in human mammary tissues and even in milk. Epidemiological studies suggested a number of congeners are simultaneously detected in serum, breast milk and adipose tissue in humans , , , . Due to their estrogenic activity, PCBs have been documented to disrupt the endocrine systems of animals and humans , , and the combination of different PCBs could behave synergistically in presenting their estrogenic potency . To mimic co-exposure to PCB congeners, in this study we used the commercially available PCB mix (equal amount of No. 28, 52, 101, 138, 153, 180 and 209) of frequently detected PCBs in human subjects. We employed estrogen receptor-positive (ER+) MCF-7 cells and estrogen receptor-negative (ER-) MDA-MB-231 cells in order to discriminate the ER-dependent and -independent effects. There was a rather mild effect of the PCB mix on cell growth and survival at low concentrations (<60 nM) for both ER+ MCF-7 and ER- MDA-MB-231 cells (data not shown); however, PCBs exerted significant cytotoxicity on these cells and induce cell death at high concentrations (>60 nM). As shown in Fig. 1A, distinct apoptotic morphology was observed in MDA-MB-231 cells treated with 60 nM PCBs for 24 hrs compared to the vehicle control, as the PCB-treated cells became round and tended to detach from the plate. The apoptosis induced by PCBs was confirmed by an analysis of flow cytometry using FITC-Annexin V and propidium iodide (PI) stains. PCBs dramatically induced cell death as indicated by increased early apoptotic cells (37.05% VS 2.93%), necrotic cells (13.70% VS 3.04%) and late apoptotic/necrotic cells (8.04% VS 1.31%) (P<0.001, n = 3), compared to the control (Fig. 1B).
(A) Phase-contrast images of cell morphology from MDA-MB-231 cells treated with/without the PCB mix at 60 nM for 24 hrs. Original maginification, ×200. (B) FACS analysis of apoptosis induced by PCBs at 60 nM for 24 hrs in MDA-MB-231 cells using FITC-Annexin V and PI stains. Early apoptotic cells (lower right), necrotic cells (upper left) and late apoptotic/necrotic cells (upper right) are shown as arrows indicate.
The previous studies indicated that PCBs were correlated with more aggressive breast cancers, and might promote tumor progression , , . Thus, the cell motility influenced by the PCB mix was assessed in a transwell migration assay as previously described . Compared to the non-metastatic cells, MCF-7, metastatic cells, MDA-MB-231, displayed robust migration ability, as the number of transmigrated cells was 8 times greater than that of MCF-7 cells (P<0.001, n = 6) (Fig. 2A&B). Upon the stimulation of PCBs at 30 nM for 24 hrs, the cell motility was tremendously increased (∼10 times) for both ER+ MCF-7 and ER- MDA-MN-231 (Fig. 2A&B, P<0.001), suggesting the presence of ER is not an indispensable for PCB-facilitated cell migration. At higher concentrations of PCBs (e.g. 60 nM), the cell motility was greatly reduced (Fig. 2A&B, P<0.001) presumably due to their cytotoxicity as discussed above (Fig. 1). In addition, all PCB congeners (namely 28, 52, 101, 138, 153, 180 and 209) used in this study are not structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has the ability to bind to and activate the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). These non-dioxin-like PCBs therefore have no ability to bind to the AhR and activate its downstream signaling . Thus, the PCB-promoted effect on cell motility is independent of the ER signaling and the AhR signaling as well.
Cell motility was examined from a transwell migration assay in MCF-7 and MDA-MB-231 cells treated with the PCB mix at 30 or 60 nM for 24 hrs. After the DAPI staining (blue), two images were randomly taken from three individual replicates under a microscope, and transmigrated cells in the chamber filters in each image were counted. Representative images for MCF-7 (A) and MDA-MB-231 (B) cells are shown, and the numbers of transmigrated cells were quantified (n = 6). *, P<0.001, compared with the vehicle control and the 60 nM group.
To gain insight into the role of PCBs in regulating cell migration/invasion, we looked into the changes of metastatic capability of MDA-MB-231 cells upon exposure of PCBs in vivo using a mouse model of breast cancer metastasis (Fig. 3A). As shown in Fig. 3B, the growth of primary tumors was little affected after 4 weeks of PCB exposure, consistent with the in vitro observation on cell proliferation (data not shown). However, PCBs greatly enhanced the capability of MDA-MB-231 cells to metastasize to lung, liver and bone. 2 out of 4 mice developed lung metastasis, and 1 out of 4 mice developed liver metastasis among the PCB-treated mice. In contrast, no mice in the vehicle control group developed either lung or liver metastasis (Fig. 4A&C). Although 50% of mice for both groups developed bone metastasis in bilateral hind limbs, the mass of metastatic tumors in the PCB-treated mice was significantly greater than that in the control mice (Fig. 4B&C, n = 4, P<0.05). This observation is consistent with a previous epidemiological finding that high concentrations of PCBs in mammary adipose tissues were associated with high-grade tumors and overall poor prognosis in breast cancer patients .
(A) A schematic of the mouse model used in this study. The details about the model are described in the Methods. (B) The average weight of primary tumors in the PCB-treated mice and the vehicle control mice (n = 8).
(A) The occurrence of metastases in all organs tested in the PCB-treated mice and vehicle control mice. Metastases were examined using the Xenogen 2000 and the IVIS software as previously described . (B) The quantified data of metastatic tumors (reflected by photon flux, photons/sec ) in mouse skeleton (n = 4). (C) Representative images of metastases in mouse liver, lung and skeleton from the bioluminescent imaging.
To shed light on the mechanism by which PCBs promote in vitro cell motility and in vivo metastasis of breast cancer cells, we investigated the potential downstream targets of PCBs. Illuminated by the increased cell motility, we assessed the effect of PCBs on the ROCK activity. PCBs significantly activated ROCK activity by 30% or so, and this effect could be attenuated by a specific ROCK inhibitor, Y27632(Fig. 5A, n = 3, P<0.05). Corresponding to the changes of ROCK activity, the substrate of ROCK, myosin light chain (MLC), was more phosphorylated upon the treatment of PCBs than the vehicle control; the increase of phosphorylated MLC (P-MLC) was weakened by Y27632 (Fig. 5A). PCBs largely induced the production of intracellular reactive oxygen species (ROS) in breast cancer cells by 40% (Fig. 5B), along with the increase of ROCK activity and the level of phosphorylated MLC (Fig. 5C). Importantly, we demonstrated that the increase of ROS mechanistically led to elevated ROCK activity induced by PCBs, as the increase for both the ROCK activity and the level of phosphorylated MLC could be undermined by the pre-treatment of beta-mercaptoethanol (β-ME) (Fig. 5C, n = 3, P<0.05).
(A) The relative ROCK activity and Western blot analysis of P-MLC in MDA-MB-231 cells treated with the PCB mix (30 nM) with or without the ROCK inhibitor, Y27632 (10 µM) for 24 hrs (n = 3). (B) ROS production in MDA-MB-231 cells upon PCB treatment. DCF fluorescence in cells were measured by FACS analysis after 6-hr PCB treatment with or without β-ME (14.3 µM) (n = 3). (C) The relative ROCK activity and Western blot analysis of P-MLC in cells upon PCB treatment for 6 hrs with or without β-ME (n = 3). *, P<0.05, compared with the vehicle control and the PCBs+β-ME group. The intensities of autoradiogram in Western blots were quantified with Image J (rsbweb.nih.gov/ij). The quantified data for P-MLC were normalized to those of GAPDH.
Although the ability of PCBs in provoking tumor initiation and development has been observed, the mechanism remains to be elucidated. Numerous possible mechanisms have been explored, such as the estrogenic activity, effects on vitamin A metabolism and intercellular communication, and induction of oxidative stress (ROS) , .The potential interaction between ROCK- and ROS-signal transduction pathways has been suggested in previous studies , , : ROCK is activated by arsenic trioxide (As2O3), a strong ROS-inducer . Further, hypoxia-induced ROS activates the RhoA/ROCK pathway .
PCBs are widespread and persistent environmental pollutants that impose potential hazards on human health. The possible correlation between environmental PCB exposure and breast cancer risk exists biologically plausible, and mounting evidence indicates that PCB exposure contributes to the aggressiveness and metastases of breast cancer. The findings from the current study suggest that PCBs potently augment the ROCK signaling to advance breast cancer metastases, and the activation of ROCK by PCBs is dependent on ROS, but independent of the ER signaling and the AhR signaling (Fig. 6). Inhibition of ROCK, therefore, appears to represent a novel therapeutic approach for metastases in breast cancer upon PCB exposure.
At low concentrations, PCBs activate ROCK kinase activity to regulate the actin-myosin-dependent contraction by phosphorylating motor proteins, such as the regulatory MLC. The resulting effect of ROCK activation leads to increased cell motility and potentially metastasis. At high concentrations, PCBs cause cell death via apoptosis, which may be dependent on ROS or not.
Materials and Methods
The PCB congener mix (equal amount of No. 28, 52, 101, 138, 153, 180 and 209) was purchased from Sigma. Stock solutions of these compounds were diluted in 100% ethanol and added to the culture medium immediately before use. Equal volume of PBS corresponding to different concentrations of the PCB mix was diluted in 100% ethanol as the vehicle control.
Human Breast cancer cell lines and cell culture
All mouse care and experimentation were approved by the Committee of Animal Care at the RCEES, Chinese Academy of Sciences. Immunodeficient (NOD/SCID) mice were maintained under aseptic sterile conditions. Surgeries were performed under sterile conditions and mice received antibiotics (trimethoprim sulfa) in the drinking water for 2 weeks following all surgical procedures. Experimental set-up was similar to the methods described in our previous publication . Briefly, bilateral fourth mammary fat pads (MFPs) were injected with 1 million MDA-MB-231 breast cancer cells after a midline incision was made and the fourth MFP was visualized on each side of the mouse. Cells were injected in 1∶3 diluted 20 µl matrigel (BD Biosciences):sterile PBS using a Hamilton syringe (Hamilton Co.). 10 wks later when the tumor size reached about 200 mm3, the mouse was sacrificed. The primary tumors were removed and cut into small pieces in a cube shape with a scissor. 0.02 g of tumor tissues were implanted underneath MFPs of 8 wk old mice. 2 weeks later when primary tumors reached ∼75 mm3, one group of mice received one dose of intraperitoneal (i.p.) injection of the PCB mix diluted in ethanol (3 nM/kg, in 100 µl).Control mice received ethanol-diluted PBS.
Bioluminescent imaging was performed 4 wks post the injection, with both primary tumors and metastases examined using the Xenogen 2000 and the IVIS software. Mice were administrated i.p. injections of luciferin (100 nl of 1 mg/mL; Molecular Probes), and after 10 minutes, anesthetized (isoflurane inhalation) for imaging of primary tumors. Following euthanization using CO2, mouse hind limbs, lungs, livers, kidneys and other tissues were removed for separate imaging, and detection of metastases.
Western blot analysis
Protein extracts were prepared from cells, and 20 µg of each was used for separation by 4–12% SDS-PAGE and processed for Western blot analyses as described previously . Antibodies used were the anti-GAPDH (1∶1,000, Santa Cruz Biotechnology), and anti–phosphorylated MLC (1∶1,000, Sigma).
Apoptosis analysis was analyzed using FITC-conjugated Annexin V and PI as described previously . The level of cellular ROS was examined by FACS analysis using cells treated with 5 µM of DCF (Molecular Probes; Invitrogen) at 37°C as previously described . Briefly, cells were incubated with DCF in DMEM medium for 30 minutes and then treated with PCBs (60 nM) with or without β-ME (14.3 µM) for 6 hrs. Cells were then collected for analyses.
ROCK kinase activity assay
Breast cancer cells were treated with PCBs in medium with 1% FBS for 24 hrs at 37°C and 5% CO2. After two cold PBS washes, 1×106 cells were lysed with M-PER Mammalian Protein Extraction Reagent (Pierce). ROCK kinase activity was determined by the Rho-kinase Assay kit (Cyclex Co, Japan) according to the manufacturer's protocol. The kinase activity in the vehicle control was defined as 1.
Transwell migration assay
After breast cancer cells were serum-starved for 24 hrs, cell motility was assessed by the transwell (Corning) migration assay following the standard protocol. Cells were plated on the upper chamber (1×104/insert) in 1% serum medium with or without PCBs and allowed to migrate for 24 hrs with 10% serum medium in the bottom as chemoattractant. Cells on the filter-side of the upper chamber were cleaned with cotton swabs and the filters were stained with 1×DAPI solution after cold methanol fix. Cell nuclei on the filters were visualized under a fluorescent microscope.
Conceived and designed the experiments: SL YD. Performed the experiments: SL SL. Analyzed the data: SL SL. Contributed reagents/materials/analysis tools: SL YD. Wrote the paper: SL SL YD.
- 1. DeCaprio AP, Johnson GW, Tarbell AM, Carpenter DO, Chiarenzelli JR, et al. (2005) Polychlorinated biphenyl (PCB) exposure assessment by multivariate statistical analysis of serum congener profiles in an adult Native American population. Environ Res 98: 284–302.
- 2. Park JS, Linderholm L, Charles MJ, Athanasiadou M, Petrik J, et al. (2007) Polychlorinated biphenyls their hydroxylated metabolites (OH-PCBS) in pregnant women from eastern Slovakia. Environ Health Perspect 115: 20–27.
- 3. Humphrey HE, Gardiner JC, Pandya JR, Sweeney AM, Gasior DM, et al. (2000) PCB congener profile in the serum of humans consuming Great Lakes fish. Environ Health Perspect 108: 167–172.
- 4. Salehi F, Turner MC, Phillips KP, Wigle DT, Krewski D, et al. (2008) Review of the etiology of breast cancer with special attention to organochlorines as potential endocrine disruptors. J Toxicol Environ Health B Crit Rev 11: 276–300.
- 5. Calle EE, Frumkin H, Henley SJ, Savitz DA, Thun MJ (2002) Organochlorines breast cancer risk. CA Cancer J Clin 52: 301–309.
- 6. Golden R, Kimbrough R (2009) Weight of evidence evaluation of potential human cancer risks from exposure to polychlorinated biphenyls: an update based on studies published since 2003. Crit Rev Toxicol 39: 299–331.
- 7. Millikan R, DeVoto E, Duell EJ, Tse CK, Savitz DA, et al. (2000) Dichlorodiphenyldichloroethene, polychlorinated biphenyls, and breast cancer among African-American and white women in North Carolina. Cancer Epidemiol Biomarkers Prev 9: 1233–1240.
- 8. Holford TR, Zheng T, Mayne ST, Zahm SH, Tessari JD, et al. (2000) Joint effects of nine polychlorinated biphenyl (PCB) congeners on breast cancer risk. Int J Epidemiol 29: 975–982.
- 9. Demers A, Ayotte P, Brisson J, Dodin S, Robert J, et al. (2000) Risk and aggressiveness of breast cancer in relation to plasma organochlorine concentrations. Cancer Epidemiol Biomarkers Prev 9: 161–166.
- 10. Snedeker SM (2001) Pesticides and breast cancer risk: a review of DDT, DDE, and dieldrin. Environ Health Perspect 109: Suppl 135–47.
- 11. Woolcott CG, Aronson KJ, Hanna WM, SenGupta SK, McCready DR, et al. (2001) Organochlorines and breast cancer risk by receptor status, tumor size, and grade (Canada). Cancer Causes Control 12: 395–404.
- 12. Suva LJ, Griffin RJ, Makhoul I (2009) Mechanisms of bone metastases of breast cancer. Endocr Relat Cancer 16: 703–713.
- 13. Tang Y, Olufemi L, Wang MT, Nie D (2008) Role of Rho GTPases in breast cancer. Front Biosci 13: 759–776.
- 14. Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4: 446–456.
- 15. Lane J, Martin TA, Watkins G, Mansel RE, Jiang WG (2008) The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer. Int J Oncol 33: 585–593.
- 16. Liu S, Goldstein RH, Scepansky EM, Rosenblatt M (2009) Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone. Cancer Res 69: 8742–8751.
- 17. Bourguignon LY, Zhu H, Shao L, Zhu D, Chen YW (1999) Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. Cell Motil Cytoskeleton 43: 269–287.
- 18. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, et al. (1999) An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med 5: 221–225.
- 19. Li B, Zhao WD, Tan ZM, Fang WG, Zhu L, et al. (2006) Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells. FEBS Lett 580: 4252–4260.
- 20. Imamura F, Mukai M, Ayaki M, Akedo H (2000) Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res 91: 811–816.
- 21. Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E (2006) ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol 16: 1515–1523.
- 22. Yoshioka K, Foletta V, Bernard O, Itoh K (2003) A role for LIM kinase in cancer invasion. Proc Natl Acad Sci U S A 100: 7247–7252.
- 23. Shiraishi F, Okumura T, Nomachi M, Serizawa S, Nishikawa J, et al. (2003) Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls. Chemosphere 52: 33–42.
- 24. Negri E, Bosetti C, Fattore E, La Vecchia C (2003) Environmental exposure to polychlorinated biphenyls (PCBs) and breast cancer: a systematic review of the epidemiological evidence. Eur J Cancer Prev 12: 509–516.
- 25. Bonefeld-Jorgensen EC, Andersen HR, Rasmussen TH, Vinggaard AM (2001) Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity. Toxicology 158: 141–153.
- 26. Murphy LE, Gollenberg AL, Louis GM, Kostyniak PJ, Sundaram RMaternal serum preconception polychlorinated biphenyl concentrations and infant birth weight. Environ Health Perspect 118: 297–302.
- 27. Zhao XR, Qin ZF, Yang ZZ, Zhao Q, Zhao YX, et al. Dual body burdens of polychlorinated biphenyls and polybrominated diphenyl ethers among local residents in an e-waste recycling region in Southeast China. Chemosphere 78: 659–666.
- 28. Fossi MC, Massi A, Lari L, Marsili L, Focardi S, et al. (1995) Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organochlorine accumulation. Environ Pollut 90: 15–24.
- 29. Safe S (2004) Endocrine disruptors and human health: is there a problem. Toxicology 205: 3–10.
- 30. Arnold SF, Klotz DM, Collins BM, Vonier PM, Guillette LJ Jr, et al. (1996) Synergistic activation of estrogen receptor with combinations of environmental chemicals. Science 272: 1489–1492.
- 31. Carpenter DO (2006) Polychlorinated biphenyls (PCBs): routes of exposure and effects on human health. Rev Environ Health 21: 1–23.
- 32. Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, et al. (1995) The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect 103: Suppl 7113–122.
- 33. Korach KS, Sarver P, Chae K, McLachlan JA, McKinney JD (1988) Estrogen receptor-binding activity of polychlorinated hydroxybiphenyls: conformationally restricted structural probes. Mol Pharmacol 33: 120–126.
- 34. Yamaguchi T, Oishi K, Uchida M, Echizen H (2003) Edaravone, a radical scavenger, may enhance or produce antiproliferative effects of fluvastatin, amlodipine, ozagrel, GF109203X and Y27632 on cultured basilar artery smooth muscle cells. Biol Pharm Bull 26: 1706–1710.
- 35. Potin S, Bertoglio J, Breard J (2007) Involvement of a Rho-ROCK-JNK pathway in arsenic trioxide-induced apoptosis in chronic myelogenous leukemia cells. FEBS Lett 581: 118–124.
- 36. Dada LA, Novoa E, Lecuona E, Sun H, Sznajder JI (2007) Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells. J Cell Sci 120: 2214–2222.
- 37. Kuperwasser C, Dessain S, Bierbaum BE, Garnet D, Sperandio K, et al. (2005) A mouse model of human breast cancer metastasis to human bone. Cancer Res 65: 6130–6138.
- 38. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, et al. (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3: 537–549.
- 39. Liu S, Suragani RN, Wang F, Han A, Zhao W, et al. (2007) The function of heme-regulated eIF2alpha kinase in murine iron homeostasis and macrophage maturation. J Clin Invest 117: 3296–3305.
- 40. Liu S, Bhattacharya S, Han A, Suragani RN, Zhao W, et al. (2008) Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency. Br J Haematol 143: 129–137.