Radiat Environ Biophys DOI 10.1007/s00411-014-0540-y
ORIGINAL PAPER
Complementary and alternative medicine in reducing radiation-induced skin toxicity Jennifer J. Hu • Tengjiao Cui • Jorge L. Rodriguez-Gil Glenn O. Allen • Jie Li • Cristiane Takita • Brian E. Lally
•
Received: 9 January 2014 / Accepted: 6 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Radiation therapy-induced acute and late effects, particularly skin toxicities, have significant impact on cancer patients’ quality of life and long-term survival. To date, no effective topical agents have been routinely used in the clinical setting to prevent skin toxicity. Using SKH-hr1 hairless mice, we investigated two complementary and alternative medicine in their effects on inflammation and ionizing radiation (IR)-induced skin toxicity: Calendula officinalis (CO) and Ching Wan Hung (CWH). They were applied immediately following each IR dosing of 10 Gy/day for 4 days. Skin toxicity and inflammatory factors were evaluated at multiple time points up to 15 days post-radiation. Serum interleukin (IL)-1a, monocyte chemotactic protein-1 (MCP1), keratinocyte-derived chemokine (KC), and granulocyte colony-stimulating factor (G-CSF) were significantly induced by radiation. Both CO and CWH significantly inhibited IR-induced MCP1 (p \ 0.01), KC (p \ 0.05), and G-CSF (p \ 0.001). IRinduced erythema and blood vessel dilation were J. J. Hu (&) J. L. Rodriguez-Gil G. O. Allen Department of Public Health Sciences, University of Miami School of Medicine, 1120 NW 14th St., CRB 1511, Miami, FL 33136, USA e-mail: [email protected] J. J. Hu J. L. Rodriguez-Gil G. O. Allen J. Li C. Takita B. E. Lally Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA T. Cui J. Li Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA C. Takita B. E. Lally Department of Radiation Oncology, University of Miami Miller School of Medicine, Miami, FL, USA
significantly reduced by CWH (p \ 0.001) but not by CO at day 10 post-IR. Both agents inhibited IR-induced IL-1a (p \ 0.01), MCP1 (p \ 0.05), and vascular endothelial growth factor (p \ 0.05). There were continuous inhibitory effects of CWH on IR-induced skin toxicities and inflammation. In contrast, CO treatment resulted in skin reactions compared to IR alone. Our results suggest that both CO and CWH reduce IR-induced inflammation and CWH reduced IR-induced erythema. In summary, CWH showed promising effects in reducing IR-related inflammation and skin toxicities, and future proof-of-principal testing in humans will be critical in evaluating its potential application in preventing IR-induced skin toxicities. Keywords Radiation protection Complementary and alternative medicine Skin toxicity Inflammation
Introduction Adjuvant radiotherapy (RT) is recommended for the majority of patients receiving breast conservation surgery (BCS) (Dragun et al. 2011; Sjovall et al. 2010). RT has been shown to reduce the relative incidence of recurrent tumor in ipsilateral breast by approximately 2/3 when compared to women with BCS alone (Fisher et al. 2002). In the Early Breast Cancer Trialists’ Group meta-analysis, the 5-year absolute risk of local recurrence was reduced by 15.7 % with the use of RT while 15-year mortality was reduced by 3.8 % (Darby et al. 2011), demonstrating a clear effect of RT on local control and long-term survival in breast cancer. However, the most commonly reported side effect was fatigue followed by skin reactions and pain (Sjovall et al. 2010). In light of the recommendation of RT after BCS in breast cancer, there are efforts to reduce RT-
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related side effects which can influence the quality of life and be a limiting factor to effective adjuvant RT. Adjuvant RT is a vital component for local control of tumor and long-term survival, following BCS. However, patients undergoing RT may develop erythema (reddening) within hours to weeks following treatment depending on the dose of radiation delivered (Stone et al. 2004). Erythema can be categorized in two stages: an early phase where reddening is seen within a few hours and is transient with symptoms subsiding by 24–48 h and a later phase which begins 2–3 weeks following irradiation and may be associated with inflammatory processes and blood vessel dilation. Methods to adequately manage adverse reactions are actively being investigated. A clear consensus on the effectiveness of specific topical agents is still lacking. During the course of RT, cells of the basal layer are destroyed resulting in disruption of the normal tissue turnover. With continued radiation insult, further tissue destruction and inflammation occur (Ryan 2012). Ionizing radiation (IR) induces several molecules such as interleukin (IL)-6, tumor necrosis factor (TNF)-a, IL-1a, IL1b, intercellular adhesion molecule (ICAM)-1, transforming growth factor (TGF)-b, and the chemokines IL-8 and eotaxin in skin cells (Zhao and Robbins 2009; Muller and Meineke 2007) which contribute to the skin damage. Approximately 90 % of patients may develop radiationinduced dermatitis. This reaction can be categorized as dermatitis and/or dry desquamation where erythema, hyper-pigmentation, scaling, and pruritus of the irradiated skin occur, or moist desquamation where the epidermis sloughs off and exposes the dermal layer (Harper et al. 2004). While individual genetic factors may impact the severity of adverse skin reactions (Barnett et al. 2009; Mayer et al. 2011), application of selected topical agents including mometasone furoate, topical agents derived from plant sources, and corticosteroids has been shown to reduce the effect of RT-induced skin reactions in several small studies (Rizza et al. 2010; Miller et al. 2011). Unfortunately, a number of trials with two of the most commonly used agents, aloe vera gel and Biafine, showed no effect on the onset or severity of RT-induced dermatitis (McQuestion 2011). The efficacy of corticosteroids in preventing dermatitis has been inconclusive. Though studies showed some benefit, the use of corticosteroids was not recommended. The use of hyaluronic acid cream demonstrated some beneficial effect, but a recommendation for use could not be made (McQuestion 2011). Studies evaluating the use of Calendula officinalis (CO), an extract from marigold, showed some promising result (Pommier et al. 2004). A reduction in skin reactions and pain was observed with use of this topical treatment. In addition, the results from a recent study suggest that topical application of
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amitriptyline, ketamine, and lidocaine can alleviate neuropathic pain from radiation dermatitis (Uzaraga et al. 2011). Ching Wan Hung (CWH) is a Chinese herbal ointment which is sold as an over-counter soothing lotion for burns. Some of the active ingredients include lobelia, myrrh, tangkuei, borneol, sanguisorba, chaenomeles, frankincense, carthamus, and pistacia (http://www.itmonline.org/jintu/ chingwan.htm). There is no scientific literature related to this product. However, the results from two previous studies suggest the anti-inflammatory and analgesic activities of myrrh (Dolara et al. 1996; Su et al. 2012). Furthermore, the ethanol extract of Sanguisorba officinalis has a potent anti-inflammatory activity mediated by nuclear factor kappaB (NF-jB), and activator protein (AP)-1 inhibitory properties linked to the suppression of sarcoma (Src) and mitogen-activated protein (MAP) kinase activation (Yu et al. 2011). Based on the potential anti-inflammatory and analgesic properties of CWH, we hypothesize that CWH may be efficacious in reducing RT-induced dermatitis and skin lesions. Using a mouse model system, the current study evaluated whether CWH has similar effects as CO on preventing IR-induced skin toxicity.
Methods Radiation treatment and serum sample collection Eight-week-old female SKH-hr1 hairless mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a pathogen-free environment under a 12-h light–dark cycle. CO extract, available with the trade name Pommade au Calendula Par Digestion, was obtained from Boiron Ltd (Levallois-Perret, France). Ching Wan Hung was manufactured by the Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd. (Tianjin, China) and distributed by the Solstice Medicine Company (Los Angeles, CA) in the USA. Experiments were performed in compliance with the University of Miami Institutional Animal Care and Use Committee (IACUC). Mice were divided into four groups (ten animals in each group): (1) control; (2) IR: 10 Gy/day of for 4 days; (3) IR ? CO; and (4) IR ? CWH. IR was applied once a day on 4 consecutive days. IR was generated using a GC-40 irradiator which features dual Cs sources at both top and bottom of the irradiation chamber. Irradiation from the bottom source was blocked using a 1-in. lead plate so that animals only received irradiation from the top source. During irradiation, animal body, except for the limbs, was protected by a customized body shield made of 5-mm lead. Additional four 1/8-in. lead plates were placed on top of the body shield to provide extra shielding to vital organs.
Radiat Environ Biophys
Under these settings, 46 cGy/min was delivered to the hind limbs for 21 min and 45 s for a cumulative dose of 10 Gy. CO and CWH (*0.6 mg) were applied topically using a cotton swab immediately following each IR dosing on both hind limbs. Immediately after animals were sacrificed at different time points, blood was collected from the heart and used for serum preparation. Blood samples were allowed to clot for 30 min at room temperature before centrifuging at 2,0009g for 15 min at 4 °C. After centrifugation, serum was stored at -80 °C until assay. Assessment of skin toxicity Mice were evaluated every 2 days following IR with and without topical treatment of CO and CWH. IR-induced skin reactions, erythema, blood vessel dilation, and crust/ scaling, were noted. The physical changes of the mouse skin at the irradiated region were photographed every 2 days using a SONY Cybershot camera coupled to a DermLite II Pro Dermatoscope (3Gen LLC, San Juan Capistrano, CA). Gross assessment of early radiation dermatitis, erythema, edema, dyspigmentation, desquamation, exudation, and ulceration, was conducted using the dermatoscope, which allows visualization of superficial structures to deeper pigmentation. Skin toxicity was evaluated and scored as either 0 (no visible lesion), 1 (moderate lesion), or 2 (severe lesion), for both erythema and blood vessel dilation. ELISA Serum levels of inflammatory factors were evaluated using ELISA kits for vascular endothelial growth factor (VEGF), IL-1a, monocyte chemotactic protein (MCP)-1, KC, soluble intercellular adhesion molecule (sICAM)-1, and granulocyte colony-stimulating factor (G-CSF) (R&D Systems, Minneapolis, MN). Whole or diluted serum from days 5 and 15 was analyzed according to the manufacture’s procedures. Optical density at 450 nm was determined using the BioTek Synergy HT microplate reader (Winooski, VT). Duplicate samples from each mouse were evaluated. Statistical analysis To compare the proportions of erythema and blood vessel dilation in the four groups of mice, the Fisher’s exact test was performed using the SPSS software (version 15.0; SPSS, Inc., Chicago, Illinois). The effects of CO or CWH on IR-induced cytokines and angiogenic factors were examined using Student’s t test for pair-wise comparisons. The means and standard deviations (SD) were presented.
Results Gross assessment of dermatitis The assessment of IR-induced erythema and blood vessel dilation is presented in Table 1. Gross assessment revealed no obvious IR-induced dermatitis until day 8 among all groups (data not shown). In the group of mice irradiated and then treated with CO, nine of ten mice had detectable erythema. Similar to the IR-only group, no mice irradiated and treated with CWH had detectable erythema at day 8 post-IR. Erythema was evident in IR-only mice at day 10 post-IR (Table 1). All 10 mice within the IR-only group exhibited moderate erythema (score of 1). No mice irradiated and then treated with CWH had detectable erythema (score of 0; p \ 0.001). In contrast, all 10 mice irradiated and then treated with CO displayed erythema. Moreover, four and six mice showed moderate and severe erythema, respectively. At day 12, erythema began to wane in the IRonly group with only six of ten mice showing moderate erythema. All the mice treated with CO and CWH had no detectable erythema. At day 14 post-IR, 3 mice in the IRonly group still showed moderate erythema. The COtreated group had two mice with moderate erythema, while the CWH-treated group had no mice with erythema. Blood vessel dilation was obvious in 100 % of the IRonly group at day 10 post-IR (Table 1), with two mice being moderate (score of 1) and eight mice being severe (score of 2). Mice in the IR ? CWH group showed no vessel dilation (score of 0, p \ 0.001). Conversely, mice irradiated and treated with CO all displayed severe vessel dilation (score of 2). At day 12, vessel dilation was similar with the IR-only group where all mice exhibited dilated vessels. However, three were moderate, while seven were severe. All mice irradiated and treated with CWH displayed moderate vessel dilation (p \ 0.001). In contrast, mice irradiated and treated with CO all showed severe vessel dilation. At day 14, all mice receiving IR only showed moderate vessel dilation, while mice irradiated and treated with either CO or CWH all showed severe vessel dilation. Radiation-induced inflammatory and angiogenic factors We first evaluated the effects of IR and subsequent treatment with CWH on the expression of 40 inflammatory and angiogenic factors using two proteomic profiler arrays (data not shown). We observed that several factors, including G-CSF, MCP1, and KC, were induced by IR and subsequently reduced with CWH treatment. Previous reports suggest both IL-1a and ICAM-1 were induced by IR and may contribute to skin inflammation and damage (Muller and Meineke 2007; Gallet et al. 2011), and we also
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Radiat Environ Biophys Table 1 Skin toxicity in response to IR and modulation by Calendula Officinalis and Ching Wan Hung
Time
Toxicity group
0 Day 8
Day 12
a
Fisher’s exact test
IR ionizing radiation, CO Calendula officinalis, CWH Ching Wan Hung
0
1
2
10
0
0
10
0
0
10
0
0
10
0
0
1
9
0
IR ? CWH post
10
0
0
Control
\0.001
10
0
0
10
0
0 0
10
0
0
10
0
IR
0
10
0
0
2
8
IR ? CO post
0
4
6
0
0
10
\0.001
IR ? CWH post
10
0
0
10
0
0
Control
10
0
0
10
0
0
4
6
0
0
3
7
IR ? CO post IR ? CWH post
10 10
0 0
0 0
0 0
0 10
10 0
Control
10
0
0
10
0
0
IR
7
3
0
0
10
0
IR ? CO post
8
2
0
0
0
10
10
0
0
0
0
10
IR ? CWH post
observed IR-induced increase of IL-1a, soluble ICAM-1, or VEGF using the proteomic array. We then assessed the effect of irradiation and topical treatment on the levels of the three IR-induced factors using specific ELISAs: MCP1, KC, and G-CSF in addition to VEGF, sICAM-1, and IL-1a. At day 5, IR significantly induced IL-1a (p = 0.02), MCP1 (p \ 0.01), KC (p \ 0.01), and G-CSF (p \ 0.001) (Table 2). There was no detectable IR-induced effect on VEGF or sICAM1 at this time point. Treatment with CO or CWH reduced the effect of IR on MCP1 (p \ 0.01), KC (p \ 0.02), and G-CSF (p \ 0.001). However, IL-1a was marginally reduced with CO while CWH augmented IL-1a (p \ 0.01) levels. At day 15, IR significantly induced VEGF (p = 0.02) IL-1a (p \ 0.02), KC (p \ 0.01), and G-CSF (p \ 0.001) (Table 2). Similar to the results from day 5, the levels of sICAM1 were not altered by IR. A marginal induction of MCP1 was observed (p = 0.051). Application of CO to the irradiated skin reduced the level of VEGF (p = 0.01), IL-1a (p \ 0.01), MCP1 (p = 0.002), and KC (p \ 0.04). Similar effects were observed with CWH reduction of IR-induced VEGF (p \ 0.01), IL-1a (p = 0.002), and MCP1 (p = 0.014). However, while KC was reduced with CWH, the effect was not significant (p = 0.14). In addition, CWH augmented the effect of IR on G-CSF (p \ 0.02).
Discussion Topical agents derived from plant sources have been tested in the management of adverse skin reactions and found to
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2
Control
IR
Day 14
1
p valuea
Blood vessel dilation
IR IR ? CO post Day 10
p valuea
Erythema
\0.001
0.033
N/A
\0.001
\0.001
\0.001
be effective at reducing erythema (Rizza et al. 2010). In this study, we compared the effects of CO, a formulation of plant extracts, which has been used in human clinical trials, and the CWH herbal balm. Treatment with CWH inhibited IR-induced erythema at different time points was assessed. In contrast, CO showed elevated skin reaction in the absence of IR, suggesting that in this animal model, CO alone caused adverse reactions not seen with CWH. Associated with a reduction in early vessel dilation and erythema, the IR-induced inflammatory factors, VEGF, IL1a, MCP1, KC, and G-CSF, were differentially reduced with the CWH topical treatment. Interestingly, CO reduced these inflammatory factors while still inducing erythema and vessel dilation. In light of published reports indicating that pro-inflammatory cytokines induced by irradiation (Zhao and Robbins 2009; Muller and Meineke 2007) may contribute to skin damages and inhibition of such inflammation may aid in reduction in skin toxicity (Ryan 2012), our findings suggest that CWH may have clinical application. Similar to a previous report (Benderitter et al. 2007), we observed an IR-induced elevation in MCP1. However, we did not detect elevated IL-6, TNF-a, and TGF-b in our model system. It is not clear whether the difference is due to the type of samples used. In our study, serum was used for the cytokine analysis while Benderitter and colleagues evaluated cytokine expression in the skin tissue (Benderitter et al. 2007). In addition, ICAM1 was elevated in irradiated skin tissue (Muller et al. 2006) but not in serum as suggested in our study. The elevation of IL-1a, MCP1, KC, and G-CSF at day 5 of treatment would imply increasing recruitment of
Radiat Environ Biophys Table 2 Changes of inflammatory factors in response to IR and modulation by Calendula Officinalis and Ching Wan Hung Inflammatory factors
Day 5 pair-wise comparison a
Group VEGF (pg/mL)
IL-1 a (pg/mL)
MCP 1 (pg/mL)
KC (pg/mL)
sICAM1 (ng/mL)
G-CSF (pg/mL)
Day 15 pair-wise comparison
Mean (SD)
p value
b
Groupa
p valueb
Mean (SD)
1
96.6 (1.5)
1
93.0 (5.6)
2
103.0 (16.6)
1 versus 2
0.64
2
123.3 (3.0)
1 versus 2
0.02*
3
99.8 (3.0)
2 versus 3
0.82
3
107.3 (1.5)
2 versus 3
0.01**
4
90.2 (4.5)
2 versus 4
0.40
2 versus 4
\0.01**
1
46.4 (3.2)
2
68.3 (3.0)
1 versus 2
3 4
45.0 (20.7) 114.4 (5.8)
2 versus 3 2 versus 4
1
4
75.2 (4.5)
1
110.3 (14.5)
0.02*
2
186.4 (2.8)
1 versus 2
0.02*
0.26 \0.01**
3 4
103.1 (10.2) 32.6 (9.9)
2 versus 3 2 versus 4
\0.01** 0.002**
93.0 (11.6)
1
68.1 (14.9)
2
228.6 (8.3)
1 versus 2
\0.01*
2
119.6 (8.6)
1 versus 2
0.051
3
113.4 (11.5)
2 versus 3
\0.01**
3
73.4 (1.5)
2 versus 3
\0.02**
4
90.0 (1.5)
2 versus 4
\0.01**
2 versus 4
1
235.43 (0)
2
324.6 (10.0)
1 versus 2
3
233.8 (15.1)
2 versus 3
4
210.5 (22.2)
2 versus 4
1
299.0 (63.4)
2
280.7 (28.1)
1 versus 2
0.47
2
295.0 (54.1)
1 versus 2
0.02*
3
328.6 (35.1)
2 versus 3
\0.02**
3
400.8 (20.3)
2 versus 3
\0.001**
4
244.0 (31.2)
2 versus 4
\0.04**
4
425.5 (75.6)
2 versus 4
0.002**
1
82.8 (32.9)
1
34.3 (14.2)
2 3
660.2 (48.3) 242.1 (126.9)
1 versus 2 2 versus 3
\0.001* \0.001**
2 3
447.4 (162.5) 233.2 (61.98)
1 versus 2 2 versus 3
\0.001* 0.01**
4
143.8 (85.1)
2 versus 4
\0.001**
4
688.3 (54.2)
2 versus 4
\0.02**
4
69.2 (1.5)
1
104.5 (5.4)
0.014**
\0.01*
2
314.6 (24.1)
1 versus 2
\0.01*
\0.02**
3
229.3 (4.3)
2 versus 3
\0.04**
0.022
4
245.8 (32.0)
2 versus 4
0.14
1
364.8 (32.9)
a Group 1 = no irradiation; Group 2 = irradiation; Group 3 = irradiation followed by CO treatment; Group 4 = irradiation followed by CWH treatment b
p value, ANOVA tests
* Significant p value for IR induction ** Significant p value for topical treatment compared to IR induction
immune cells to the site of irradiation and correlates with subsequent erythema in irradiated mice. At day 15 when the effect of IR was waning, VEGF, MCP1, and IL-1a were significantly inhibited by CWH. However, at this time point, increase in vessel dilation was observed in the CWHtreated group. While the majority of the tested factors were inhibited by CWH at day 15, G-CSF was elevated in the CWH-treated group. G-CSF is known to increase production and recruitment of neutrophils from the bone marrow (Cornish et al. 2009). In contrast to the previous clinical findings (Pommier et al. 2004), our mouse model system did not support the beneficial effects of CO in preventing IR-induced erythema and blood vessel dilation. Although CO did not inhibit IRinduced dermatitis, a reduction in IR-induced MCP1, KC, G-CSF, and IL-1a was observed. This finding implies that CO may have beneficial effects on cytokine reduction but
not on overall skin toxicity. The failure of CO to reduce skin toxicity in our model system was surprising since CO has been shown to be effective in preventing RT-associated skin toxicity in a phase III trial (Pommier et al. 2004). However, no further validation study has been performed, and this agent has not been incorporated into routine clinical practice. Neither CO nor CWH has been tested in animal models. This initial test of both agents in an animal model has provided preliminary data which may have implications in humans. Nevertheless, animal-to-human extrapolation may have limitations, and different radiation sources with various dosage intensities may result in altered responses. To the best of our knowledge, this is the first study evaluating the effects of CWH in a mouse model system of radiation dermatitis. In addition to the gross assessment of skin reaction, we showed a diminution of inflammatory and
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angiogenic factors that contribute to inflammation in the skin. Future studies with higher doses or specific radiation sources may be helpful to elucidate the molecular mechanisms of CWH-mediated effects on inflammation and IRinduced skin toxicity. Our data suggest the potential use of CWH in reducing RT-induced adverse skin reactions. Therefore, future clinical trials comparing CWH and CO are warranted to test their efficacy in preventing radiationinduced skin toxicities. Acknowledgments We dedicate this paper to the memory of senior research associate Venetta Thomas, who has provided technical support. We are thankful to Ahmed Mansoor and Xiadong Wu for technical support. This study was supported by a Department of Defense Concept Award (BC076685) and to J. J. Hu. Conflict of interest disclose.
The authors have no conflicts of interest to
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Mayer C, Popanda O, Greve B, Fritz E, Illig T, Eckardt-Schupp F, Gomolka M, Benner A, Schmezer P (2011) A radiation-induced gene expression signature as a tool to predict acute radiotherapyinduced adverse side effects. Cancer Lett 302:20–28. doi:10. 1016/j.canlet.2010.12.006 McQuestion M (2011) Evidence-based skin care management in radiation therapy: clinical update. Semin Oncol Nurs 27:e1–17. doi:10.1016/j.soncn.2011.02.009 Miller RC, Schwartz DJ, Sloan JA, Griffin PC, Deming RL, Anders JC, Stoffel TJ, Haselow RE, Schaefer PL, Bearden JD 3rd, Atherton PJ, Loprinzi CL, Martenson JA (2011) Mometasone furoate effect on acute skin toxicity in breast cancer patients receiving radiotherapy: a phase III double-blind, randomized trial from the North Central Cancer Treatment Group N06C4. Int J Radiat Oncol Biol Phys 79:1460–1466. doi:10.1016/j.ijrobp. 2010.01.031 Muller K, Meineke V (2007) Radiation-induced alterations in cytokine production by skin cells. Exp Hematol 35:96–104. doi:10.1016/j.exphem.2007.01.017 Muller K, Kohn FM, Port M, Abend M, Molls M, Ring J, Meineke V (2006) Intercellular adhesion molecule-1: a consistent inflammatory marker of the cutaneous radiation reaction both in vitro and in vivo. Br J Dermatol 155:670–679. doi:10.1111/j.13652133.2006.07407.x Pommier P, Gomez F, Sunyach MP, D’Hombres A, Carrie C, Montbarbon X (2004) Phase III randomized trial of Calendula officinalis compared with trolamine for the prevention of acute dermatitis during irradiation for breast cancer. J Clin Oncol 22:1447–1453. doi:10.1200/JCO.2004.07.063 Rizza L, D’Agostino A, Girlando A, Puglia C (2010) Evaluation of the effect of topical agents on radiation-induced skin disease by reflectance spectrophotometry. J Pharm Pharmacol 62:779–785. doi:10.1211/jpp.62.06.0015 Ryan JL (2012) Ionizing radiation: the good, the bad, and the ugly. J Invest Dermatol 132:985–993. doi:10.1038/jid.2011.411 Sjovall K, Strombeck G, Lofgren A, Bendahl PO, Gunnars B (2010) Adjuvant radiotherapy of women with breast cancer: information, support and side-effects. Eur J Oncol Nurs 14:147–153. doi:10.1016/j.ejon.2009.09.002 Stone HB, Moulder JE, Coleman CN, Ang KK, Anscher MS, Barcellos-Hoff MH, Dynan WS, Fike JR, Grdina DJ, Greenberger JS, Hauer-Jensen M, Hill RP, Kolesnick RN, Macvittie TJ, Marks C, McBride WH, Metting N, Pellmar T, Purucker M, Robbins ME, Schiestl RH, Seed TM, Tomaszewski JE, Travis EL, Wallner PE, Wolpert M, Zaharevitz D (2004) Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3–4, 2003. Radiat Res 162:711–728 Su S, Hua Y, Wang Y, Gu W, Zhou W, Duan JA, Jiang H, Chen T, Tang Y (2012) Evaluation of the anti-inflammatory and analgesic properties of individual and combined extracts from Commiphora myrrha, and Boswellia carterii. J Ethnopharmacol 139:649–656. doi:10.1016/j.jep.2011.12.013 Uzaraga I, Gerbis B, Holwerda E, Gillis D, Wai E (2011) Topical amitriptyline, ketamine, and lidocaine in neuropathic pain caused by radiation skin reaction: a pilot study. Support Care Cancer. doi:10.1007/s00520-011-1240-7 Yu T, Lee YJ, Yang HM, Han S, Kim JH, Lee Y, Kim C, Han MH, Kim MY, Lee J, Cho JY (2011) Inhibitory effect of Sanguisorba officinalis ethanol extract on NO and PGE production is mediated by suppression of NF-kappaB and AP-1 activation signaling cascade. J Ethnopharmacol 134:11–17. doi:10.1016/j. jep.2010.08.060 Zhao W, Robbins ME (2009) Inflammation and chronic oxidative stress in radiation-induced late normal tissue injury: therapeutic implications. Curr Med Chem 16:130–143
ORIGINAL PAPER
Complementary and alternative medicine in reducing radiation-induced skin toxicity Jennifer J. Hu • Tengjiao Cui • Jorge L. Rodriguez-Gil Glenn O. Allen • Jie Li • Cristiane Takita • Brian E. Lally
•
Received: 9 January 2014 / Accepted: 6 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Radiation therapy-induced acute and late effects, particularly skin toxicities, have significant impact on cancer patients’ quality of life and long-term survival. To date, no effective topical agents have been routinely used in the clinical setting to prevent skin toxicity. Using SKH-hr1 hairless mice, we investigated two complementary and alternative medicine in their effects on inflammation and ionizing radiation (IR)-induced skin toxicity: Calendula officinalis (CO) and Ching Wan Hung (CWH). They were applied immediately following each IR dosing of 10 Gy/day for 4 days. Skin toxicity and inflammatory factors were evaluated at multiple time points up to 15 days post-radiation. Serum interleukin (IL)-1a, monocyte chemotactic protein-1 (MCP1), keratinocyte-derived chemokine (KC), and granulocyte colony-stimulating factor (G-CSF) were significantly induced by radiation. Both CO and CWH significantly inhibited IR-induced MCP1 (p \ 0.01), KC (p \ 0.05), and G-CSF (p \ 0.001). IRinduced erythema and blood vessel dilation were J. J. Hu (&) J. L. Rodriguez-Gil G. O. Allen Department of Public Health Sciences, University of Miami School of Medicine, 1120 NW 14th St., CRB 1511, Miami, FL 33136, USA e-mail: [email protected] J. J. Hu J. L. Rodriguez-Gil G. O. Allen J. Li C. Takita B. E. Lally Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA T. Cui J. Li Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, FL, USA C. Takita B. E. Lally Department of Radiation Oncology, University of Miami Miller School of Medicine, Miami, FL, USA
significantly reduced by CWH (p \ 0.001) but not by CO at day 10 post-IR. Both agents inhibited IR-induced IL-1a (p \ 0.01), MCP1 (p \ 0.05), and vascular endothelial growth factor (p \ 0.05). There were continuous inhibitory effects of CWH on IR-induced skin toxicities and inflammation. In contrast, CO treatment resulted in skin reactions compared to IR alone. Our results suggest that both CO and CWH reduce IR-induced inflammation and CWH reduced IR-induced erythema. In summary, CWH showed promising effects in reducing IR-related inflammation and skin toxicities, and future proof-of-principal testing in humans will be critical in evaluating its potential application in preventing IR-induced skin toxicities. Keywords Radiation protection Complementary and alternative medicine Skin toxicity Inflammation
Introduction Adjuvant radiotherapy (RT) is recommended for the majority of patients receiving breast conservation surgery (BCS) (Dragun et al. 2011; Sjovall et al. 2010). RT has been shown to reduce the relative incidence of recurrent tumor in ipsilateral breast by approximately 2/3 when compared to women with BCS alone (Fisher et al. 2002). In the Early Breast Cancer Trialists’ Group meta-analysis, the 5-year absolute risk of local recurrence was reduced by 15.7 % with the use of RT while 15-year mortality was reduced by 3.8 % (Darby et al. 2011), demonstrating a clear effect of RT on local control and long-term survival in breast cancer. However, the most commonly reported side effect was fatigue followed by skin reactions and pain (Sjovall et al. 2010). In light of the recommendation of RT after BCS in breast cancer, there are efforts to reduce RT-
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related side effects which can influence the quality of life and be a limiting factor to effective adjuvant RT. Adjuvant RT is a vital component for local control of tumor and long-term survival, following BCS. However, patients undergoing RT may develop erythema (reddening) within hours to weeks following treatment depending on the dose of radiation delivered (Stone et al. 2004). Erythema can be categorized in two stages: an early phase where reddening is seen within a few hours and is transient with symptoms subsiding by 24–48 h and a later phase which begins 2–3 weeks following irradiation and may be associated with inflammatory processes and blood vessel dilation. Methods to adequately manage adverse reactions are actively being investigated. A clear consensus on the effectiveness of specific topical agents is still lacking. During the course of RT, cells of the basal layer are destroyed resulting in disruption of the normal tissue turnover. With continued radiation insult, further tissue destruction and inflammation occur (Ryan 2012). Ionizing radiation (IR) induces several molecules such as interleukin (IL)-6, tumor necrosis factor (TNF)-a, IL-1a, IL1b, intercellular adhesion molecule (ICAM)-1, transforming growth factor (TGF)-b, and the chemokines IL-8 and eotaxin in skin cells (Zhao and Robbins 2009; Muller and Meineke 2007) which contribute to the skin damage. Approximately 90 % of patients may develop radiationinduced dermatitis. This reaction can be categorized as dermatitis and/or dry desquamation where erythema, hyper-pigmentation, scaling, and pruritus of the irradiated skin occur, or moist desquamation where the epidermis sloughs off and exposes the dermal layer (Harper et al. 2004). While individual genetic factors may impact the severity of adverse skin reactions (Barnett et al. 2009; Mayer et al. 2011), application of selected topical agents including mometasone furoate, topical agents derived from plant sources, and corticosteroids has been shown to reduce the effect of RT-induced skin reactions in several small studies (Rizza et al. 2010; Miller et al. 2011). Unfortunately, a number of trials with two of the most commonly used agents, aloe vera gel and Biafine, showed no effect on the onset or severity of RT-induced dermatitis (McQuestion 2011). The efficacy of corticosteroids in preventing dermatitis has been inconclusive. Though studies showed some benefit, the use of corticosteroids was not recommended. The use of hyaluronic acid cream demonstrated some beneficial effect, but a recommendation for use could not be made (McQuestion 2011). Studies evaluating the use of Calendula officinalis (CO), an extract from marigold, showed some promising result (Pommier et al. 2004). A reduction in skin reactions and pain was observed with use of this topical treatment. In addition, the results from a recent study suggest that topical application of
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amitriptyline, ketamine, and lidocaine can alleviate neuropathic pain from radiation dermatitis (Uzaraga et al. 2011). Ching Wan Hung (CWH) is a Chinese herbal ointment which is sold as an over-counter soothing lotion for burns. Some of the active ingredients include lobelia, myrrh, tangkuei, borneol, sanguisorba, chaenomeles, frankincense, carthamus, and pistacia (http://www.itmonline.org/jintu/ chingwan.htm). There is no scientific literature related to this product. However, the results from two previous studies suggest the anti-inflammatory and analgesic activities of myrrh (Dolara et al. 1996; Su et al. 2012). Furthermore, the ethanol extract of Sanguisorba officinalis has a potent anti-inflammatory activity mediated by nuclear factor kappaB (NF-jB), and activator protein (AP)-1 inhibitory properties linked to the suppression of sarcoma (Src) and mitogen-activated protein (MAP) kinase activation (Yu et al. 2011). Based on the potential anti-inflammatory and analgesic properties of CWH, we hypothesize that CWH may be efficacious in reducing RT-induced dermatitis and skin lesions. Using a mouse model system, the current study evaluated whether CWH has similar effects as CO on preventing IR-induced skin toxicity.
Methods Radiation treatment and serum sample collection Eight-week-old female SKH-hr1 hairless mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained in a pathogen-free environment under a 12-h light–dark cycle. CO extract, available with the trade name Pommade au Calendula Par Digestion, was obtained from Boiron Ltd (Levallois-Perret, France). Ching Wan Hung was manufactured by the Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd. (Tianjin, China) and distributed by the Solstice Medicine Company (Los Angeles, CA) in the USA. Experiments were performed in compliance with the University of Miami Institutional Animal Care and Use Committee (IACUC). Mice were divided into four groups (ten animals in each group): (1) control; (2) IR: 10 Gy/day of for 4 days; (3) IR ? CO; and (4) IR ? CWH. IR was applied once a day on 4 consecutive days. IR was generated using a GC-40 irradiator which features dual Cs sources at both top and bottom of the irradiation chamber. Irradiation from the bottom source was blocked using a 1-in. lead plate so that animals only received irradiation from the top source. During irradiation, animal body, except for the limbs, was protected by a customized body shield made of 5-mm lead. Additional four 1/8-in. lead plates were placed on top of the body shield to provide extra shielding to vital organs.
Radiat Environ Biophys
Under these settings, 46 cGy/min was delivered to the hind limbs for 21 min and 45 s for a cumulative dose of 10 Gy. CO and CWH (*0.6 mg) were applied topically using a cotton swab immediately following each IR dosing on both hind limbs. Immediately after animals were sacrificed at different time points, blood was collected from the heart and used for serum preparation. Blood samples were allowed to clot for 30 min at room temperature before centrifuging at 2,0009g for 15 min at 4 °C. After centrifugation, serum was stored at -80 °C until assay. Assessment of skin toxicity Mice were evaluated every 2 days following IR with and without topical treatment of CO and CWH. IR-induced skin reactions, erythema, blood vessel dilation, and crust/ scaling, were noted. The physical changes of the mouse skin at the irradiated region were photographed every 2 days using a SONY Cybershot camera coupled to a DermLite II Pro Dermatoscope (3Gen LLC, San Juan Capistrano, CA). Gross assessment of early radiation dermatitis, erythema, edema, dyspigmentation, desquamation, exudation, and ulceration, was conducted using the dermatoscope, which allows visualization of superficial structures to deeper pigmentation. Skin toxicity was evaluated and scored as either 0 (no visible lesion), 1 (moderate lesion), or 2 (severe lesion), for both erythema and blood vessel dilation. ELISA Serum levels of inflammatory factors were evaluated using ELISA kits for vascular endothelial growth factor (VEGF), IL-1a, monocyte chemotactic protein (MCP)-1, KC, soluble intercellular adhesion molecule (sICAM)-1, and granulocyte colony-stimulating factor (G-CSF) (R&D Systems, Minneapolis, MN). Whole or diluted serum from days 5 and 15 was analyzed according to the manufacture’s procedures. Optical density at 450 nm was determined using the BioTek Synergy HT microplate reader (Winooski, VT). Duplicate samples from each mouse were evaluated. Statistical analysis To compare the proportions of erythema and blood vessel dilation in the four groups of mice, the Fisher’s exact test was performed using the SPSS software (version 15.0; SPSS, Inc., Chicago, Illinois). The effects of CO or CWH on IR-induced cytokines and angiogenic factors were examined using Student’s t test for pair-wise comparisons. The means and standard deviations (SD) were presented.
Results Gross assessment of dermatitis The assessment of IR-induced erythema and blood vessel dilation is presented in Table 1. Gross assessment revealed no obvious IR-induced dermatitis until day 8 among all groups (data not shown). In the group of mice irradiated and then treated with CO, nine of ten mice had detectable erythema. Similar to the IR-only group, no mice irradiated and treated with CWH had detectable erythema at day 8 post-IR. Erythema was evident in IR-only mice at day 10 post-IR (Table 1). All 10 mice within the IR-only group exhibited moderate erythema (score of 1). No mice irradiated and then treated with CWH had detectable erythema (score of 0; p \ 0.001). In contrast, all 10 mice irradiated and then treated with CO displayed erythema. Moreover, four and six mice showed moderate and severe erythema, respectively. At day 12, erythema began to wane in the IRonly group with only six of ten mice showing moderate erythema. All the mice treated with CO and CWH had no detectable erythema. At day 14 post-IR, 3 mice in the IRonly group still showed moderate erythema. The COtreated group had two mice with moderate erythema, while the CWH-treated group had no mice with erythema. Blood vessel dilation was obvious in 100 % of the IRonly group at day 10 post-IR (Table 1), with two mice being moderate (score of 1) and eight mice being severe (score of 2). Mice in the IR ? CWH group showed no vessel dilation (score of 0, p \ 0.001). Conversely, mice irradiated and treated with CO all displayed severe vessel dilation (score of 2). At day 12, vessel dilation was similar with the IR-only group where all mice exhibited dilated vessels. However, three were moderate, while seven were severe. All mice irradiated and treated with CWH displayed moderate vessel dilation (p \ 0.001). In contrast, mice irradiated and treated with CO all showed severe vessel dilation. At day 14, all mice receiving IR only showed moderate vessel dilation, while mice irradiated and treated with either CO or CWH all showed severe vessel dilation. Radiation-induced inflammatory and angiogenic factors We first evaluated the effects of IR and subsequent treatment with CWH on the expression of 40 inflammatory and angiogenic factors using two proteomic profiler arrays (data not shown). We observed that several factors, including G-CSF, MCP1, and KC, were induced by IR and subsequently reduced with CWH treatment. Previous reports suggest both IL-1a and ICAM-1 were induced by IR and may contribute to skin inflammation and damage (Muller and Meineke 2007; Gallet et al. 2011), and we also
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Radiat Environ Biophys Table 1 Skin toxicity in response to IR and modulation by Calendula Officinalis and Ching Wan Hung
Time
Toxicity group
0 Day 8
Day 12
a
Fisher’s exact test
IR ionizing radiation, CO Calendula officinalis, CWH Ching Wan Hung
0
1
2
10
0
0
10
0
0
10
0
0
10
0
0
1
9
0
IR ? CWH post
10
0
0
Control
\0.001
10
0
0
10
0
0 0
10
0
0
10
0
IR
0
10
0
0
2
8
IR ? CO post
0
4
6
0
0
10
\0.001
IR ? CWH post
10
0
0
10
0
0
Control
10
0
0
10
0
0
4
6
0
0
3
7
IR ? CO post IR ? CWH post
10 10
0 0
0 0
0 0
0 10
10 0
Control
10
0
0
10
0
0
IR
7
3
0
0
10
0
IR ? CO post
8
2
0
0
0
10
10
0
0
0
0
10
IR ? CWH post
observed IR-induced increase of IL-1a, soluble ICAM-1, or VEGF using the proteomic array. We then assessed the effect of irradiation and topical treatment on the levels of the three IR-induced factors using specific ELISAs: MCP1, KC, and G-CSF in addition to VEGF, sICAM-1, and IL-1a. At day 5, IR significantly induced IL-1a (p = 0.02), MCP1 (p \ 0.01), KC (p \ 0.01), and G-CSF (p \ 0.001) (Table 2). There was no detectable IR-induced effect on VEGF or sICAM1 at this time point. Treatment with CO or CWH reduced the effect of IR on MCP1 (p \ 0.01), KC (p \ 0.02), and G-CSF (p \ 0.001). However, IL-1a was marginally reduced with CO while CWH augmented IL-1a (p \ 0.01) levels. At day 15, IR significantly induced VEGF (p = 0.02) IL-1a (p \ 0.02), KC (p \ 0.01), and G-CSF (p \ 0.001) (Table 2). Similar to the results from day 5, the levels of sICAM1 were not altered by IR. A marginal induction of MCP1 was observed (p = 0.051). Application of CO to the irradiated skin reduced the level of VEGF (p = 0.01), IL-1a (p \ 0.01), MCP1 (p = 0.002), and KC (p \ 0.04). Similar effects were observed with CWH reduction of IR-induced VEGF (p \ 0.01), IL-1a (p = 0.002), and MCP1 (p = 0.014). However, while KC was reduced with CWH, the effect was not significant (p = 0.14). In addition, CWH augmented the effect of IR on G-CSF (p \ 0.02).
Discussion Topical agents derived from plant sources have been tested in the management of adverse skin reactions and found to
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2
Control
IR
Day 14
1
p valuea
Blood vessel dilation
IR IR ? CO post Day 10
p valuea
Erythema
\0.001
0.033
N/A
\0.001
\0.001
\0.001
be effective at reducing erythema (Rizza et al. 2010). In this study, we compared the effects of CO, a formulation of plant extracts, which has been used in human clinical trials, and the CWH herbal balm. Treatment with CWH inhibited IR-induced erythema at different time points was assessed. In contrast, CO showed elevated skin reaction in the absence of IR, suggesting that in this animal model, CO alone caused adverse reactions not seen with CWH. Associated with a reduction in early vessel dilation and erythema, the IR-induced inflammatory factors, VEGF, IL1a, MCP1, KC, and G-CSF, were differentially reduced with the CWH topical treatment. Interestingly, CO reduced these inflammatory factors while still inducing erythema and vessel dilation. In light of published reports indicating that pro-inflammatory cytokines induced by irradiation (Zhao and Robbins 2009; Muller and Meineke 2007) may contribute to skin damages and inhibition of such inflammation may aid in reduction in skin toxicity (Ryan 2012), our findings suggest that CWH may have clinical application. Similar to a previous report (Benderitter et al. 2007), we observed an IR-induced elevation in MCP1. However, we did not detect elevated IL-6, TNF-a, and TGF-b in our model system. It is not clear whether the difference is due to the type of samples used. In our study, serum was used for the cytokine analysis while Benderitter and colleagues evaluated cytokine expression in the skin tissue (Benderitter et al. 2007). In addition, ICAM1 was elevated in irradiated skin tissue (Muller et al. 2006) but not in serum as suggested in our study. The elevation of IL-1a, MCP1, KC, and G-CSF at day 5 of treatment would imply increasing recruitment of
Radiat Environ Biophys Table 2 Changes of inflammatory factors in response to IR and modulation by Calendula Officinalis and Ching Wan Hung Inflammatory factors
Day 5 pair-wise comparison a
Group VEGF (pg/mL)
IL-1 a (pg/mL)
MCP 1 (pg/mL)
KC (pg/mL)
sICAM1 (ng/mL)
G-CSF (pg/mL)
Day 15 pair-wise comparison
Mean (SD)
p value
b
Groupa
p valueb
Mean (SD)
1
96.6 (1.5)
1
93.0 (5.6)
2
103.0 (16.6)
1 versus 2
0.64
2
123.3 (3.0)
1 versus 2
0.02*
3
99.8 (3.0)
2 versus 3
0.82
3
107.3 (1.5)
2 versus 3
0.01**
4
90.2 (4.5)
2 versus 4
0.40
2 versus 4
\0.01**
1
46.4 (3.2)
2
68.3 (3.0)
1 versus 2
3 4
45.0 (20.7) 114.4 (5.8)
2 versus 3 2 versus 4
1
4
75.2 (4.5)
1
110.3 (14.5)
0.02*
2
186.4 (2.8)
1 versus 2
0.02*
0.26 \0.01**
3 4
103.1 (10.2) 32.6 (9.9)
2 versus 3 2 versus 4
\0.01** 0.002**
93.0 (11.6)
1
68.1 (14.9)
2
228.6 (8.3)
1 versus 2
\0.01*
2
119.6 (8.6)
1 versus 2
0.051
3
113.4 (11.5)
2 versus 3
\0.01**
3
73.4 (1.5)
2 versus 3
\0.02**
4
90.0 (1.5)
2 versus 4
\0.01**
2 versus 4
1
235.43 (0)
2
324.6 (10.0)
1 versus 2
3
233.8 (15.1)
2 versus 3
4
210.5 (22.2)
2 versus 4
1
299.0 (63.4)
2
280.7 (28.1)
1 versus 2
0.47
2
295.0 (54.1)
1 versus 2
0.02*
3
328.6 (35.1)
2 versus 3
\0.02**
3
400.8 (20.3)
2 versus 3
\0.001**
4
244.0 (31.2)
2 versus 4
\0.04**
4
425.5 (75.6)
2 versus 4
0.002**
1
82.8 (32.9)
1
34.3 (14.2)
2 3
660.2 (48.3) 242.1 (126.9)
1 versus 2 2 versus 3
\0.001* \0.001**
2 3
447.4 (162.5) 233.2 (61.98)
1 versus 2 2 versus 3
\0.001* 0.01**
4
143.8 (85.1)
2 versus 4
\0.001**
4
688.3 (54.2)
2 versus 4
\0.02**
4
69.2 (1.5)
1
104.5 (5.4)
0.014**
\0.01*
2
314.6 (24.1)
1 versus 2
\0.01*
\0.02**
3
229.3 (4.3)
2 versus 3
\0.04**
0.022
4
245.8 (32.0)
2 versus 4
0.14
1
364.8 (32.9)
a Group 1 = no irradiation; Group 2 = irradiation; Group 3 = irradiation followed by CO treatment; Group 4 = irradiation followed by CWH treatment b
p value, ANOVA tests
* Significant p value for IR induction ** Significant p value for topical treatment compared to IR induction
immune cells to the site of irradiation and correlates with subsequent erythema in irradiated mice. At day 15 when the effect of IR was waning, VEGF, MCP1, and IL-1a were significantly inhibited by CWH. However, at this time point, increase in vessel dilation was observed in the CWHtreated group. While the majority of the tested factors were inhibited by CWH at day 15, G-CSF was elevated in the CWH-treated group. G-CSF is known to increase production and recruitment of neutrophils from the bone marrow (Cornish et al. 2009). In contrast to the previous clinical findings (Pommier et al. 2004), our mouse model system did not support the beneficial effects of CO in preventing IR-induced erythema and blood vessel dilation. Although CO did not inhibit IRinduced dermatitis, a reduction in IR-induced MCP1, KC, G-CSF, and IL-1a was observed. This finding implies that CO may have beneficial effects on cytokine reduction but
not on overall skin toxicity. The failure of CO to reduce skin toxicity in our model system was surprising since CO has been shown to be effective in preventing RT-associated skin toxicity in a phase III trial (Pommier et al. 2004). However, no further validation study has been performed, and this agent has not been incorporated into routine clinical practice. Neither CO nor CWH has been tested in animal models. This initial test of both agents in an animal model has provided preliminary data which may have implications in humans. Nevertheless, animal-to-human extrapolation may have limitations, and different radiation sources with various dosage intensities may result in altered responses. To the best of our knowledge, this is the first study evaluating the effects of CWH in a mouse model system of radiation dermatitis. In addition to the gross assessment of skin reaction, we showed a diminution of inflammatory and
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angiogenic factors that contribute to inflammation in the skin. Future studies with higher doses or specific radiation sources may be helpful to elucidate the molecular mechanisms of CWH-mediated effects on inflammation and IRinduced skin toxicity. Our data suggest the potential use of CWH in reducing RT-induced adverse skin reactions. Therefore, future clinical trials comparing CWH and CO are warranted to test their efficacy in preventing radiationinduced skin toxicities. Acknowledgments We dedicate this paper to the memory of senior research associate Venetta Thomas, who has provided technical support. We are thankful to Ahmed Mansoor and Xiadong Wu for technical support. This study was supported by a Department of Defense Concept Award (BC076685) and to J. J. Hu. Conflict of interest disclose.
The authors have no conflicts of interest to
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