International Journal of
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
Coniferyl Aldehyde Reduces Radiation Damage Through Increased Protein Stability of Heat Shock Transcriptional Factor 1 by Phosphorylation Seo-Young Kim, MS,* Hae-June Lee, DVM, PhD,y Joo-Won Nam, PhD,* Eun-Kyoung Seo, PhD,* and Yun-Sil Lee, PhD* *Graduate School of Pharmaceutical Sciences, Ewha Women’s University, Seoul, Korea; and yDivision of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul, Korea Received Apr 17, 2014, and in revised form Nov 10, 2014. Accepted for publication Nov 19, 2014.
Summary Development of effective protectors against radiation is of great importance in view of their potential application during both radiation therapy and accidental exposure after nuclear accidents. Coniferyl aldehyde (CA) induced stability in HSF1 protein by phosphorylation, which was accompanied by increased expression of its transcriptional targets HSP27 and HSP70. HSF1 induction by CA exhibited cytoprotective functions against radiation and chemotherapeutic agents in normal cells. Taken together, CA may be a good candidate as a cytoprotective agent targeting HSF1.
Purpose: We previously screened natural compounds and found that coniferyl aldehyde (CA) was identified as an inducer of HSF1. In this study, we further examined the protective effects of CA against ionizing radiation (IR) in normal cell system. Methods and Materials: Western blotting and reverse transcription-polymerase chain reaction tests were performed to evaluate expression of HSF1, HSP27, and HSP70 in response to CA. Cell death and cleavage of PARP and caspase-3 were analyzed to determine the protective effects of CA in the presence of IR or taxol. The protective effects of CA were also evaluated using animal models. Results: CA increased stability of the HSF1 protein by phosphorylation at Ser326, which was accompanied by increased expression of HSP27 and HSP70. HSF1 phosphorylation at Ser326 by CA was mediated by EKR1/2 activation. Cotreatment of CA with IR or taxol in normal cells induced protective effects with phosphorylation- dependent patterns at Ser326 of HSF1. The decrease in bone marrow (BM) cellularity and increase of terminal deoxynucleotidyl transferase dUTP nick end labelingepositive BM cells by IR were also significantly inhibited by CA in mice (30.6% and 56.0%, respectively). A549 lung orthotopic lung tumor model indicated that CA did not affect the IRmediated reduction of lung tumor nodules, whereas CA protected normal lung tissues from the therapeutic irradiation. Conclusions: These results suggest that CA may be useful for inducing HSF1 to protect against normal cell damage after IR or chemotherapeutic agents. Ó 2015 Elsevier Inc.
Reprint requests to: Yun-Sil Lee, PhD, Graduate School of Pharmaceutical Sciences, Ewha Women’s University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, Korea. Tel: 82-2-3277-3022; E-mail: [email protected] This work was supported by Nuclear Research and Development Program grants NRF2011-0031696 and NRF2013M2A2A704043384 from Int J Radiation Oncol Biol Phys, Vol. 91, No. 4, pp. 807e816, 2015 0360-3016/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.11.031
the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning. Conflict of interest: none. S-Y Kim and H-J Lee contributed equally to this work. Supplementary material for this article can be found at www.redjournal.org.
808
Kim et al.
Introduction Heat shock factor 1 (HSF1), a master regulator of the heat shock response, facilitates cell survival and proliferation in eukaryotes (1, 2). HSF1 is maintained in an inactive state in the cytoplasm, but upon exposure to thermal or other proteotoxic stressors, heat shock proteins (HSP) are titrated away from the interaction with HSF1 (3, 4). HSF1 is also involved in protection of normal cells. In this way, HSF1 may be a notable pharmacologic target for cytoprotection (5-7). Nearly 40% of cancer patients require radiation therapy during management of their disease (8). Although radiation therapy improves the survival of a significant number of cancer patients, it is also associated with significant toxicity and injury to normal tissues (9). Furthermore, management of victims of radiation injury following the Chernobyl (1986) and Fukushima (2011) disasters has awakened the need to develop new methods to protect individuals against the effects of radiation. Recently, the effects of clinical radiation have been targeted toward decreasing normal cell damage (10, 11). Along these same lines, there have been numerous attempts to develop new radiation protectors. Amifostine, a radiation protector clinically approved by the U.S. Food and Drug Administration (FDA) (12, 13), induces serious side effects. Some drug candidates based on synthetic compounds such as superoxide dismutase, nitroxides, and bis-benzimidazole are also relatively toxic. Therefore, nontoxic phytochemicals have taken center stage in the effort to develop new radiation protectors (14, 15). The need to protect normal tissues against radiation injury during management of radiation therapy or exposure to radiation hazards remains a critical issue in the field of radiation biology (16). The present study investigated whether coniferyl aldehyde (CA), which was isolated from natural products (17), activates HSF1 to protect normal cells against radiation and chemotherapeutic agents.
International Journal of Radiation Oncology Biology Physics
phosphorylation mutant constructs HSF1 (S230A and S326A) were constructed using overlapped extension primers.
Survival day detection and bone marrow damage experiments Female ICR mice (5-6 weeks old) were purchased from Central Lab Animal, Inc. For survival day detection, mice received 7 Gy of whole-body radiation using a gamma irradiator (3.81 Gy/min). CA (5 or 10 mg/kg, 1 pretreatment of CA before ionizing radiation [IR] and 3 post-treatments of CA after IR) was administered by intraperitoneal (IP) injection. Each group contained 10 mice. For bone marrow (BM) damage experiments, female ICR mice (5-6 weeks old) received 4.5 Gy of whole-body gamma irradiation (2.41 Gy/min) (19). CA was IP injected using the same protocol as the survival day experiment. After 9 days of IR, mice were euthanized (see Fig. 5B). Each group contained 5 mice. All protocols involving mice were approved by the Institutional Animal Care and Use Committee of (Ewha Womans University, South Korea) Laboratory Animal Genomic Center.
Orthotropic lung tumor models Human non-small-cell lung carcinoma A549 cells (American Type Culture Collection), at 2 106 cells in 100 ml of Hanks’ balanced salt solution, were injected into the tail veins of 6-week-old female BALB/c nude mice (Orient Bio). At 4 weeks after cell injection, mice were treated with CA (5 doses of 10 mg/kg) and/or 10 Gy of thoracic IR. The lung was harvested and analyzed at 7 days after IR (see Fig. 6A). Protocols involved in this study were approved by the Institutional Animal Care and Use Committee of Korea Institute of Radiological and Medical Sciences.
Results Methods and Materials Compounds Coniferaldehyde glucoside was isolated from the bark of Eucommia ulmoides (Fig. E1; available online at www. redjournal.com) (17). Compounds were dissolved in dimethyl sulfoxide and diluted in cell culture medium. CA was purchased from Sigma-Aldrich, Inc. (catalog 382051). Paclitaxel was purchased from Selleck (catalog no. NSC125973). Celastrol was isolated and purified from Tripterygium refelii (18). U0126 (a MEK1/2 inhibitor) was purchased from Cell Signaling Technology (catalog no. 9903).
Plasmids Wild-type (WT) human HSF1 was cloned into p3XFLAGMyc-CMV containing an N-terminal Flag tag. The
CA increased expression of HSF1 protein, accompanied by transcriptional activation of HSPs We previously screened several natural compounds (Fig. E1; available online at www.redjournal.com) isolated from bark of Eucommia ulmoides to identify induction of HSPs and HSF1, using L132 normal lung cells. CA glucoside induced the highest expression of HSPs and HSF1 expression among the compounds tested (17). The glucoside-free form of CA exhibited similar effects on the patterns of expression of HSF1 and HSPs in the glucoside form, and thus, the glucoside-free form of CA was used during our experiments (Fig. 1A; Fig. E2; available online at www.redjournal.com). Expression levels of HSP27 and HSP70 mRNA were increased by CA, but levels of HSF1 mRNA were not altered by CA (Fig. 1B). The protein expression levels of HSF1 and HSPs were up-regulated by
Volume 91 Number 4 2015
Coniferyl aldehyde reduces IR damage CA (hr)
B 0
O
6
Relative mRNA (fold)
A
12 Celastrol
H HO OCH3 Coniferyl aldehyde (CA)
1.8 1.6 1.4 1.2 1.0 0.8 0
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6
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1.0 0.8
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2
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3
Fig. 1. Coniferyl aldehyde induces HSF1. (A) Structure of coniferyl aldehyde (CA). (B) RT-PCR after treatment of 3 mM CA in L132 cells was performed. Western blotting was performed at 12 hours after CA treatment (C) or 3 mM CA at the indicated times (D). Band intensities were measured and normalized to that of the respective loading control band (gapdh or b-actin). Values are expressed as the fold change relative to the negative control (mean SD of 3 experiments). Celastrol (1 mM, 12 hours) was used as a positive control. CA in a time- and dose-dependent manner, with maximal induction occurring at 3 mM and 12 hours of treatment (Fig. 1C and 1D). Celastrol was used as a positive control for induction of HSP (20).
CA protects cells against taxol or IR in an HSF1dependent manner Candidate HSF1-inducing agents should not be toxic to normal cells. Therefore, the L132 cells were treated with various concentrations of CA. L132 cells exhibited a significant decrease in cell viability upon treatment with taxol or celastrol. However, cells treated with CA showed no significant decrease in cell viability at 20 mM (Fig. E3; available online at www.redjournal.com). To examine whether HSF1 induction by CA affected cellular damage induced by cytotoxic agents, cells were pretreated with CA, followed by incubation with or without taxol or IR. Treatment with taxol or IR resulted in increased PARP and caspase-3 cleavage, whereas pretreatment with CA significantly reduced these effects (Fig. 2A). Specifically, taxol
and IR increased cytotoxicity in the absence of CA, whereas pretreatment of CA markedly inhibited taxol- or IR-mediated cytotoxicity (Fig. 2B). To confirm whether CA-mediated cytoprotective effects were dependent on HSF1, we tested its effects in HSF1/ and HSF1þ/þ cells. Pretreatment of CA reduced taxol- or IR-induced cytotoxicity and apoptosis in HSF1þ/þ cells. However, in the case of HSF1/cells, CA did not inhibit taxol- or IR-induced cytotoxicity and apoptosis (Fig. 2C and 2D).
CA-induced cytoprotection is mediated by phosphorylation of HSF1 at Ser326 CA did not alter the mRNA expression of HSF1, and thus, we next analyzed the effects of CA on HSF1 protein stability. Control cells exhibited a rapid degradation of HSF1 protein, whereas CA-treated cells exhibited increased HSF1 stability (Fig. 3A). Protein stability is commonly regulated by phosphorylation status (21), and thus, we next tested various phosphorylated HSF1 sites by
International Journal of Radiation Oncology Biology Physics
Kim et al.
B Con –
IR +
–
Taxol +
–
CA
+
Cleaved PARP Cleaved caspase3 β-Actin
IR CA
– –
– +
+ –
80
**
60
***
40 20 0 IR CA
+ +
– –
– +
+ –
– –
– +
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+ +
++ –
++ +
HSF1+/+ IR CA
+ +
*
100
100
HSF1–/–
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C
120
120
Cell viability (% of control)
A
Cell viability (% of control)
810
– +
– –
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– +
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– –
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HSF1 Cleaved PARP Cleaved caspase3 β-Actin HSF1 +/+ HSF1 –/–
100 **
80 60 40 20 0 IR CA
– –
– +
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+ +
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Cell viability (% of control)
120
Cell viability (% of control)
D
HSF1 +/+ HSF1 –/–
100 *
80 60 40 20 0 Taxol – – CA
– +
+ –
+ +
Fig. 2. Cytoprotective effects of coniferyl aldehyde (CA) against taxol or IR. (A) Pretreatment of L132 cells with CA (3 mM) was performed 1 hour prior to taxol (0.3 or 0.5 mM) or IR (5 or 10 Gy). After 24 hours of taxol treatment or 48 hours of IR, Western blotting (A) or MTT assay (B) was performed. Values are expressed as percentages relative to negative control. (C) HSF1þ/þ and HSF1/ MEF cells were treated with taxol (0.3 or 0.5 mM) or IR (5 or 10 Gy) with or without a 1-hour pretreatment with CA (3 mM). After 24 hours of taxol treatment or 48 hours of IR exposure, Western blotting or MTT assay (D) was performed. Data represent means SD of 3 independent experiments.*P
Radiation Oncology biology
physics
www.redjournal.org
Biology Contribution
Coniferyl Aldehyde Reduces Radiation Damage Through Increased Protein Stability of Heat Shock Transcriptional Factor 1 by Phosphorylation Seo-Young Kim, MS,* Hae-June Lee, DVM, PhD,y Joo-Won Nam, PhD,* Eun-Kyoung Seo, PhD,* and Yun-Sil Lee, PhD* *Graduate School of Pharmaceutical Sciences, Ewha Women’s University, Seoul, Korea; and yDivision of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul, Korea Received Apr 17, 2014, and in revised form Nov 10, 2014. Accepted for publication Nov 19, 2014.
Summary Development of effective protectors against radiation is of great importance in view of their potential application during both radiation therapy and accidental exposure after nuclear accidents. Coniferyl aldehyde (CA) induced stability in HSF1 protein by phosphorylation, which was accompanied by increased expression of its transcriptional targets HSP27 and HSP70. HSF1 induction by CA exhibited cytoprotective functions against radiation and chemotherapeutic agents in normal cells. Taken together, CA may be a good candidate as a cytoprotective agent targeting HSF1.
Purpose: We previously screened natural compounds and found that coniferyl aldehyde (CA) was identified as an inducer of HSF1. In this study, we further examined the protective effects of CA against ionizing radiation (IR) in normal cell system. Methods and Materials: Western blotting and reverse transcription-polymerase chain reaction tests were performed to evaluate expression of HSF1, HSP27, and HSP70 in response to CA. Cell death and cleavage of PARP and caspase-3 were analyzed to determine the protective effects of CA in the presence of IR or taxol. The protective effects of CA were also evaluated using animal models. Results: CA increased stability of the HSF1 protein by phosphorylation at Ser326, which was accompanied by increased expression of HSP27 and HSP70. HSF1 phosphorylation at Ser326 by CA was mediated by EKR1/2 activation. Cotreatment of CA with IR or taxol in normal cells induced protective effects with phosphorylation- dependent patterns at Ser326 of HSF1. The decrease in bone marrow (BM) cellularity and increase of terminal deoxynucleotidyl transferase dUTP nick end labelingepositive BM cells by IR were also significantly inhibited by CA in mice (30.6% and 56.0%, respectively). A549 lung orthotopic lung tumor model indicated that CA did not affect the IRmediated reduction of lung tumor nodules, whereas CA protected normal lung tissues from the therapeutic irradiation. Conclusions: These results suggest that CA may be useful for inducing HSF1 to protect against normal cell damage after IR or chemotherapeutic agents. Ó 2015 Elsevier Inc.
Reprint requests to: Yun-Sil Lee, PhD, Graduate School of Pharmaceutical Sciences, Ewha Women’s University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, Korea. Tel: 82-2-3277-3022; E-mail: [email protected] This work was supported by Nuclear Research and Development Program grants NRF2011-0031696 and NRF2013M2A2A704043384 from Int J Radiation Oncol Biol Phys, Vol. 91, No. 4, pp. 807e816, 2015 0360-3016/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.11.031
the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning. Conflict of interest: none. S-Y Kim and H-J Lee contributed equally to this work. Supplementary material for this article can be found at www.redjournal.org.
808
Kim et al.
Introduction Heat shock factor 1 (HSF1), a master regulator of the heat shock response, facilitates cell survival and proliferation in eukaryotes (1, 2). HSF1 is maintained in an inactive state in the cytoplasm, but upon exposure to thermal or other proteotoxic stressors, heat shock proteins (HSP) are titrated away from the interaction with HSF1 (3, 4). HSF1 is also involved in protection of normal cells. In this way, HSF1 may be a notable pharmacologic target for cytoprotection (5-7). Nearly 40% of cancer patients require radiation therapy during management of their disease (8). Although radiation therapy improves the survival of a significant number of cancer patients, it is also associated with significant toxicity and injury to normal tissues (9). Furthermore, management of victims of radiation injury following the Chernobyl (1986) and Fukushima (2011) disasters has awakened the need to develop new methods to protect individuals against the effects of radiation. Recently, the effects of clinical radiation have been targeted toward decreasing normal cell damage (10, 11). Along these same lines, there have been numerous attempts to develop new radiation protectors. Amifostine, a radiation protector clinically approved by the U.S. Food and Drug Administration (FDA) (12, 13), induces serious side effects. Some drug candidates based on synthetic compounds such as superoxide dismutase, nitroxides, and bis-benzimidazole are also relatively toxic. Therefore, nontoxic phytochemicals have taken center stage in the effort to develop new radiation protectors (14, 15). The need to protect normal tissues against radiation injury during management of radiation therapy or exposure to radiation hazards remains a critical issue in the field of radiation biology (16). The present study investigated whether coniferyl aldehyde (CA), which was isolated from natural products (17), activates HSF1 to protect normal cells against radiation and chemotherapeutic agents.
International Journal of Radiation Oncology Biology Physics
phosphorylation mutant constructs HSF1 (S230A and S326A) were constructed using overlapped extension primers.
Survival day detection and bone marrow damage experiments Female ICR mice (5-6 weeks old) were purchased from Central Lab Animal, Inc. For survival day detection, mice received 7 Gy of whole-body radiation using a gamma irradiator (3.81 Gy/min). CA (5 or 10 mg/kg, 1 pretreatment of CA before ionizing radiation [IR] and 3 post-treatments of CA after IR) was administered by intraperitoneal (IP) injection. Each group contained 10 mice. For bone marrow (BM) damage experiments, female ICR mice (5-6 weeks old) received 4.5 Gy of whole-body gamma irradiation (2.41 Gy/min) (19). CA was IP injected using the same protocol as the survival day experiment. After 9 days of IR, mice were euthanized (see Fig. 5B). Each group contained 5 mice. All protocols involving mice were approved by the Institutional Animal Care and Use Committee of (Ewha Womans University, South Korea) Laboratory Animal Genomic Center.
Orthotropic lung tumor models Human non-small-cell lung carcinoma A549 cells (American Type Culture Collection), at 2 106 cells in 100 ml of Hanks’ balanced salt solution, were injected into the tail veins of 6-week-old female BALB/c nude mice (Orient Bio). At 4 weeks after cell injection, mice were treated with CA (5 doses of 10 mg/kg) and/or 10 Gy of thoracic IR. The lung was harvested and analyzed at 7 days after IR (see Fig. 6A). Protocols involved in this study were approved by the Institutional Animal Care and Use Committee of Korea Institute of Radiological and Medical Sciences.
Results Methods and Materials Compounds Coniferaldehyde glucoside was isolated from the bark of Eucommia ulmoides (Fig. E1; available online at www. redjournal.com) (17). Compounds were dissolved in dimethyl sulfoxide and diluted in cell culture medium. CA was purchased from Sigma-Aldrich, Inc. (catalog 382051). Paclitaxel was purchased from Selleck (catalog no. NSC125973). Celastrol was isolated and purified from Tripterygium refelii (18). U0126 (a MEK1/2 inhibitor) was purchased from Cell Signaling Technology (catalog no. 9903).
Plasmids Wild-type (WT) human HSF1 was cloned into p3XFLAGMyc-CMV containing an N-terminal Flag tag. The
CA increased expression of HSF1 protein, accompanied by transcriptional activation of HSPs We previously screened several natural compounds (Fig. E1; available online at www.redjournal.com) isolated from bark of Eucommia ulmoides to identify induction of HSPs and HSF1, using L132 normal lung cells. CA glucoside induced the highest expression of HSPs and HSF1 expression among the compounds tested (17). The glucoside-free form of CA exhibited similar effects on the patterns of expression of HSF1 and HSPs in the glucoside form, and thus, the glucoside-free form of CA was used during our experiments (Fig. 1A; Fig. E2; available online at www.redjournal.com). Expression levels of HSP27 and HSP70 mRNA were increased by CA, but levels of HSF1 mRNA were not altered by CA (Fig. 1B). The protein expression levels of HSF1 and HSPs were up-regulated by
Volume 91 Number 4 2015
Coniferyl aldehyde reduces IR damage CA (hr)
B 0
O
6
Relative mRNA (fold)
A
12 Celastrol
H HO OCH3 Coniferyl aldehyde (CA)
1.8 1.6 1.4 1.2 1.0 0.8 0
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D
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Fig. 1. Coniferyl aldehyde induces HSF1. (A) Structure of coniferyl aldehyde (CA). (B) RT-PCR after treatment of 3 mM CA in L132 cells was performed. Western blotting was performed at 12 hours after CA treatment (C) or 3 mM CA at the indicated times (D). Band intensities were measured and normalized to that of the respective loading control band (gapdh or b-actin). Values are expressed as the fold change relative to the negative control (mean SD of 3 experiments). Celastrol (1 mM, 12 hours) was used as a positive control. CA in a time- and dose-dependent manner, with maximal induction occurring at 3 mM and 12 hours of treatment (Fig. 1C and 1D). Celastrol was used as a positive control for induction of HSP (20).
CA protects cells against taxol or IR in an HSF1dependent manner Candidate HSF1-inducing agents should not be toxic to normal cells. Therefore, the L132 cells were treated with various concentrations of CA. L132 cells exhibited a significant decrease in cell viability upon treatment with taxol or celastrol. However, cells treated with CA showed no significant decrease in cell viability at 20 mM (Fig. E3; available online at www.redjournal.com). To examine whether HSF1 induction by CA affected cellular damage induced by cytotoxic agents, cells were pretreated with CA, followed by incubation with or without taxol or IR. Treatment with taxol or IR resulted in increased PARP and caspase-3 cleavage, whereas pretreatment with CA significantly reduced these effects (Fig. 2A). Specifically, taxol
and IR increased cytotoxicity in the absence of CA, whereas pretreatment of CA markedly inhibited taxol- or IR-mediated cytotoxicity (Fig. 2B). To confirm whether CA-mediated cytoprotective effects were dependent on HSF1, we tested its effects in HSF1/ and HSF1þ/þ cells. Pretreatment of CA reduced taxol- or IR-induced cytotoxicity and apoptosis in HSF1þ/þ cells. However, in the case of HSF1/cells, CA did not inhibit taxol- or IR-induced cytotoxicity and apoptosis (Fig. 2C and 2D).
CA-induced cytoprotection is mediated by phosphorylation of HSF1 at Ser326 CA did not alter the mRNA expression of HSF1, and thus, we next analyzed the effects of CA on HSF1 protein stability. Control cells exhibited a rapid degradation of HSF1 protein, whereas CA-treated cells exhibited increased HSF1 stability (Fig. 3A). Protein stability is commonly regulated by phosphorylation status (21), and thus, we next tested various phosphorylated HSF1 sites by
International Journal of Radiation Oncology Biology Physics
Kim et al.
B Con –
IR +
–
Taxol +
–
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+
Cleaved PARP Cleaved caspase3 β-Actin
IR CA
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810
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HSF1 +/+ HSF1 –/–
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Fig. 2. Cytoprotective effects of coniferyl aldehyde (CA) against taxol or IR. (A) Pretreatment of L132 cells with CA (3 mM) was performed 1 hour prior to taxol (0.3 or 0.5 mM) or IR (5 or 10 Gy). After 24 hours of taxol treatment or 48 hours of IR, Western blotting (A) or MTT assay (B) was performed. Values are expressed as percentages relative to negative control. (C) HSF1þ/þ and HSF1/ MEF cells were treated with taxol (0.3 or 0.5 mM) or IR (5 or 10 Gy) with or without a 1-hour pretreatment with CA (3 mM). After 24 hours of taxol treatment or 48 hours of IR exposure, Western blotting or MTT assay (D) was performed. Data represent means SD of 3 independent experiments.*P
