Archives of Biochemistry and Biophysics 571 (2015) 40–49
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-a-induced ovary cancer cell migration Mariko Suzuki a,b, Rie Matsushima-Nishiwaki b, Gen Kuroyanagi b, Noriko Suzuki a, Reika Takamatsu c, Tatsuro Furui a, Naoki Yoshimi c, Osamu Kozawa b,⇑, Ken-ichirou Morishige a a b c
Department of Obstetrics and Gynecology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan Department of Pathology and Oncology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan
a r t i c l e
i n f o
Article history: Received 31 October 2014 and in revised form 20 February 2015 Available online 27 February 2015 Keywords: HSP22 Ovary cancer TGF-a Migration
a b s t r a c t Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-a-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-a-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-a-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24 – 109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-a-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Heat shock proteins (HSPs)1 are induced when the cells are exposed to biological stresses such as heat and chemical stress [1]. The HSP family has recently been classified into seven groups, including HSPA (HSP70), HSPB (small HSPs), HSPC (HSP90) and HSPH (HSP110) [2,3]. High-molecular-weight HSPs, such as HSPA (HSP70), HSPC (HSP90) and HSPH (HSP110), have been well characterized and are firmly established to function as molecular chaperones which prevent the aggregation of unfolded proteins, ⇑ Corresponding author. E-mail address: [email protected] (O. Kozawa). Abbreviations used: HSPs, heat shock proteins; TGF, transforming growth factor; EGFR, epidermal growth factor receptor; MYPT-1, myosin phosphatase targeting subunit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DAB, 3,30 -diamino-benzidine tetrahydrochloride; IHC, immunohistochemical; PBS, phosphate-buffered saline; DAPI, 40 ,6diamidino-2-phenylin-dole; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; FIGO, International Federation of Gynecology and Obstetrics; PFS, progression free survival. 1
http://dx.doi.org/10.1016/j.abb.2015.02.030 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
giving them a cytoprotective function [1,2]. On the other hand, small HSPs (HSPB) with monomeric molecular masses from 15 to 30 kDa has significant similarities in amino acid sequences, the a-crystallin domain [3,4]. Among the HSPB group, HSP27 (HSPB1), aB-crystallin (HSPB5), HSP20 (HSPB6) and HSP22 (HSPB8) are recognized to be ubiquitously expressed in cells and tissues. Although HSPBs are well known to function as molecular chaperones as well as high-molecular-weight HSPs, each HSPB has unique properties in various physiological and pathological processes such as regulation of smooth muscle contraction or relaxation [3]. In addition, accumulating evidence suggests that HSPBs are implicated in the proliferation, invasion and metastasis of a wide range of human cancers [5]. HSP22 (HSPB8) that belongs to the HSPB group was initially identified as a H11 protein kinase in human melanoma cells. The sequence of HSP22 has the protein kinase domain, which is similar to the large subunit of herpes simplex virus type 2 ribonucleotide reductase [3]. It has been shown that HSP22 is overexpressed in various types of cancer cells [3]. In addition, HSP22 reportedly possesses anti-apoptotic activities in glioblastoma, melanoma and breast cancer cells [6–8]. However, the exact roles of HSP22 in cancer cells remain to be clarified.
M. Suzuki et al. / Archives of Biochemistry and Biophysics 571 (2015) 40–49
Ovarian cancer is the one of the most common gynecologic cancers. Epithelial ovarian cancer comprises 90% of all ovarian cancers [9]. At the time of diagnosis most patients of ovarian cancer are in advanced stage, which is associated with a substantially poorer prognosis than its early-stage counterparts. Consequently, ovarian cancer is the most lethal gynecological malignant disease. Ovarian cancer spreads initially by direct extension into adjacent organs, such as fallopian tube, uterus, and contralateral adnexa, and occasionally invades to the rectum, bladder, pelvic sidewall [10]. It is generally recognized that the metastatic pattern of ovarian cancer is different from that of most other epithelial malignant diseases [11]. After direct extension, epithelial ovarian cancer most frequently disseminates via the transcoelomic route [11]. Transcoelomic metastasis contributes substantially to the morbidity associated with this cancer [9]. Therefore, therapeutic advantages could be gained from understanding the process of transcoelomic metastasis. Metastasis is a multistep process that involves cellular migration and invasion. It is well recognized that cancer cell migration is a key process for metastasis. However, the details of migration have not yet been elucidated. Transforming growth factor (TGF)-a is a ligand for epidermal growth factor receptor (EGFR) which activates signaling pathways of cell proliferation, differentiation, and is also involved in cancer development. It has been shown that EGFR is frequently overexpressed in the ovarian cancer and is strongly associated with the mortality of the ovarian cancer patients [12]. In addition, high expression of HER-2, one of the EGFR family is reportedly associated with decreased survival rate from ovarian cancer [13]. Accumulating evidence suggests that TGF-a as an autocrine or paracrine factor via EGFR activation regulates ovarian cancer cells [14]. TGF-a has been shown to play carcinogenic actions in growth, migration and progression of epithelial ovarian cancer [15–18]. In this study, we evaluated the expression levels of HSP22 (HSPB8) in 20 patients with ovarian serous carcinoma and investigated the involvement of HSP22 in TGF-a-induced migration of ovarian cancer cells using a serous ovarian cancer cell line, SKOV3.ip1 with highly expression of HER-2.
Materials and methods Antibodies and chemicals Recombinant human TGF-a was obtained from R&D systems Inc. (Minneapolis, MN). SB203580, SP600125, PD98059, AKT inhibitor, rapamycin and Y27632 were purchased from CalbiochemNovabiochem Co. (La Jolla, CA). HSP22 antibodies, phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phosphospecific SAPK/JNK antibodies, SAPK/JNK antibodies, phosphospecific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific AKT (Thr-308) antibodies, AKT antibodies, phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies and phospho-specific myosin phosphatase targeting subunit (MYPT-1) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HSP22 antibodies for immunohistochemistry and the BCA protein assay kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Human HSP22 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Cusabio Biotech Co., Ltd. (Wuhan, Hubei, China). An ECL Western blotting detection system was purchased from GE Healthcare (Waukesha, WI). Control small interfering RNA (siRNA) (SliencerÒ Negative Control No. 1 siRNA, Neg) and HSP22-siRNAs (SliencerÒ Validated siRNA, 935 and 933) were obtained from Ambion (Austin, TX). siLentFect was purchased from
41
Bio-Rad (Hercules, CA). Other materials and chemicals were obtained from commercial sources. SB203580, SP600125, PD98059, rapamycin, AKT inhibitor and Y27632 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the cell migration assay or Western blot analysis. Cell culture Human ovarian serous carcinoma SKOV3.ip1 cells were kindly gifted from Dr. Kenjiro Sawada (Department of Gynecology, Osaka University, Osaka, Japan). The SKOV3.ip1 cells, which were established from ascites of nude mouse which was intraperitoneally injected with human ovarian cancer derived SKOV3 cells, demonstrates a high metastatic potential and higher expressions of HER-2 [19]. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2/95% air. The cultured cells proceeded by the sodium alginate method for cell block technique to examine the expression of HSP22 immunohistochemically. In brief, after centrifuging the cells in 10 ml DMEM for 10 min, the supernatant was decanted. The sediment was added with 0.5 ml of 1% sodium alginate-formalin solutions, centrifuged for 10 min, and then added with 1 or 2 drops of 1 M calcium chloride solution. They were left for 5–10 min, fixed in 20% formalin solutions, and then processed to make paraffinblocks. Tissue specimens Twenty patients undergoing surgical resection from April, 2009 to July, 2012, at the Department of Gynecology, Gifu University Hospital were diagnosed with ovarian serous carcinoma. The diagnoses were pathologically established by using the WHO classification [20] and no patients had been treated before surgical removal of the tumors. The resected tissues of ovarian cancer were obtained according to protocol approved by the committee for conduct of human research at Gifu University Graduate School of Medicine. Written informed consent was obtained from each patient. Each excised tissue was divided to two parts, and one part was then fixed with 20%-buffered formalin overnight, and another part was snap-frozen in liquid nitrogen and stored at 80 °C until used for Western blot analysis and ELISA. The 20% formalin-fixed tissues were embedded in a paraffin wax and then cut with 3 lm thickness to examine histopathologically and immunohistochemically. Immunohistochemical examination The specimens in above formalin-fixed and paraffin-embedded blocks were immunohistochemically examined by Dako EnVision™ FLEX detection system (Dako, Glostrup, Denmark) to investigate the expression and localization of HSP22 in the tissues and cells. Briefly, the deparaffinized sections were treated with 3% H2O2 in methanol for 10 min to inhibit endogenous peroxidase activity. Sections were pretreated with Target Retrieval solution (High pH) (Dako) at 97 °C for 20 min, and then incubated with HSP22 antibodies for 20 min at room temperature. Each section was treated sequentially with HRP-conjugated EnVision™ FLEX at room temperature for 20 min. The section was then treated with 3,30 -diamino-benzidine tetrahydrochloride (DAB) as a chromogen for 10 min, following as a counter staining with hematoxylin for 5 min. For analyzing the HSP22 immunoreactivity in each tissue, the immunohistochemical (IHC) score (0–6) was calculated by multiplying the proportion score, which means the distribution of the stained area and is ranged from 0 to 3 (0 = 0% of positively
42
M. Suzuki et al. / Archives of Biochemistry and Biophysics 571 (2015) 40–49
Fig. 1. Expression of HSP22 in SKOV3.ip1 cells. Immnohistochemical analysis of HSP22 in SKOV3.ip1 cells (HSP22) and the corresponding hematoxylin and eosin staining (HE).
stained cells; 1 = 1–10% of positively stained cells; 2 = 11–50% of positively stained cells; 3 = 51–100% of positively stained cells), and the intensity score, which was classified from 0 to 2 (0 = negative; 1 = weak or scant in each cytoplasm; 2 = strong or diffuse in each cytoplasm). Then the HSP22 IHC scores of the patients were divided into high expression group (>2) and low expression group (
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-a-induced ovary cancer cell migration Mariko Suzuki a,b, Rie Matsushima-Nishiwaki b, Gen Kuroyanagi b, Noriko Suzuki a, Reika Takamatsu c, Tatsuro Furui a, Naoki Yoshimi c, Osamu Kozawa b,⇑, Ken-ichirou Morishige a a b c
Department of Obstetrics and Gynecology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan Department of Pathology and Oncology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan
a r t i c l e
i n f o
Article history: Received 31 October 2014 and in revised form 20 February 2015 Available online 27 February 2015 Keywords: HSP22 Ovary cancer TGF-a Migration
a b s t r a c t Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-a-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-a-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-a-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24 – 109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-a-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Heat shock proteins (HSPs)1 are induced when the cells are exposed to biological stresses such as heat and chemical stress [1]. The HSP family has recently been classified into seven groups, including HSPA (HSP70), HSPB (small HSPs), HSPC (HSP90) and HSPH (HSP110) [2,3]. High-molecular-weight HSPs, such as HSPA (HSP70), HSPC (HSP90) and HSPH (HSP110), have been well characterized and are firmly established to function as molecular chaperones which prevent the aggregation of unfolded proteins, ⇑ Corresponding author. E-mail address: [email protected] (O. Kozawa). Abbreviations used: HSPs, heat shock proteins; TGF, transforming growth factor; EGFR, epidermal growth factor receptor; MYPT-1, myosin phosphatase targeting subunit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DAB, 3,30 -diamino-benzidine tetrahydrochloride; IHC, immunohistochemical; PBS, phosphate-buffered saline; DAPI, 40 ,6diamidino-2-phenylin-dole; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; FIGO, International Federation of Gynecology and Obstetrics; PFS, progression free survival. 1
http://dx.doi.org/10.1016/j.abb.2015.02.030 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
giving them a cytoprotective function [1,2]. On the other hand, small HSPs (HSPB) with monomeric molecular masses from 15 to 30 kDa has significant similarities in amino acid sequences, the a-crystallin domain [3,4]. Among the HSPB group, HSP27 (HSPB1), aB-crystallin (HSPB5), HSP20 (HSPB6) and HSP22 (HSPB8) are recognized to be ubiquitously expressed in cells and tissues. Although HSPBs are well known to function as molecular chaperones as well as high-molecular-weight HSPs, each HSPB has unique properties in various physiological and pathological processes such as regulation of smooth muscle contraction or relaxation [3]. In addition, accumulating evidence suggests that HSPBs are implicated in the proliferation, invasion and metastasis of a wide range of human cancers [5]. HSP22 (HSPB8) that belongs to the HSPB group was initially identified as a H11 protein kinase in human melanoma cells. The sequence of HSP22 has the protein kinase domain, which is similar to the large subunit of herpes simplex virus type 2 ribonucleotide reductase [3]. It has been shown that HSP22 is overexpressed in various types of cancer cells [3]. In addition, HSP22 reportedly possesses anti-apoptotic activities in glioblastoma, melanoma and breast cancer cells [6–8]. However, the exact roles of HSP22 in cancer cells remain to be clarified.
M. Suzuki et al. / Archives of Biochemistry and Biophysics 571 (2015) 40–49
Ovarian cancer is the one of the most common gynecologic cancers. Epithelial ovarian cancer comprises 90% of all ovarian cancers [9]. At the time of diagnosis most patients of ovarian cancer are in advanced stage, which is associated with a substantially poorer prognosis than its early-stage counterparts. Consequently, ovarian cancer is the most lethal gynecological malignant disease. Ovarian cancer spreads initially by direct extension into adjacent organs, such as fallopian tube, uterus, and contralateral adnexa, and occasionally invades to the rectum, bladder, pelvic sidewall [10]. It is generally recognized that the metastatic pattern of ovarian cancer is different from that of most other epithelial malignant diseases [11]. After direct extension, epithelial ovarian cancer most frequently disseminates via the transcoelomic route [11]. Transcoelomic metastasis contributes substantially to the morbidity associated with this cancer [9]. Therefore, therapeutic advantages could be gained from understanding the process of transcoelomic metastasis. Metastasis is a multistep process that involves cellular migration and invasion. It is well recognized that cancer cell migration is a key process for metastasis. However, the details of migration have not yet been elucidated. Transforming growth factor (TGF)-a is a ligand for epidermal growth factor receptor (EGFR) which activates signaling pathways of cell proliferation, differentiation, and is also involved in cancer development. It has been shown that EGFR is frequently overexpressed in the ovarian cancer and is strongly associated with the mortality of the ovarian cancer patients [12]. In addition, high expression of HER-2, one of the EGFR family is reportedly associated with decreased survival rate from ovarian cancer [13]. Accumulating evidence suggests that TGF-a as an autocrine or paracrine factor via EGFR activation regulates ovarian cancer cells [14]. TGF-a has been shown to play carcinogenic actions in growth, migration and progression of epithelial ovarian cancer [15–18]. In this study, we evaluated the expression levels of HSP22 (HSPB8) in 20 patients with ovarian serous carcinoma and investigated the involvement of HSP22 in TGF-a-induced migration of ovarian cancer cells using a serous ovarian cancer cell line, SKOV3.ip1 with highly expression of HER-2.
Materials and methods Antibodies and chemicals Recombinant human TGF-a was obtained from R&D systems Inc. (Minneapolis, MN). SB203580, SP600125, PD98059, AKT inhibitor, rapamycin and Y27632 were purchased from CalbiochemNovabiochem Co. (La Jolla, CA). HSP22 antibodies, phospho-specific p38 MAP kinase antibodies, p38 MAP kinase antibodies, phosphospecific SAPK/JNK antibodies, SAPK/JNK antibodies, phosphospecific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific AKT (Thr-308) antibodies, AKT antibodies, phospho-specific p70 S6 kinase antibodies, p70 S6 kinase antibodies and phospho-specific myosin phosphatase targeting subunit (MYPT-1) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). HSP22 antibodies for immunohistochemistry and the BCA protein assay kit were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Human HSP22 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Cusabio Biotech Co., Ltd. (Wuhan, Hubei, China). An ECL Western blotting detection system was purchased from GE Healthcare (Waukesha, WI). Control small interfering RNA (siRNA) (SliencerÒ Negative Control No. 1 siRNA, Neg) and HSP22-siRNAs (SliencerÒ Validated siRNA, 935 and 933) were obtained from Ambion (Austin, TX). siLentFect was purchased from
41
Bio-Rad (Hercules, CA). Other materials and chemicals were obtained from commercial sources. SB203580, SP600125, PD98059, rapamycin, AKT inhibitor and Y27632 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the cell migration assay or Western blot analysis. Cell culture Human ovarian serous carcinoma SKOV3.ip1 cells were kindly gifted from Dr. Kenjiro Sawada (Department of Gynecology, Osaka University, Osaka, Japan). The SKOV3.ip1 cells, which were established from ascites of nude mouse which was intraperitoneally injected with human ovarian cancer derived SKOV3 cells, demonstrates a high metastatic potential and higher expressions of HER-2 [19]. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2/95% air. The cultured cells proceeded by the sodium alginate method for cell block technique to examine the expression of HSP22 immunohistochemically. In brief, after centrifuging the cells in 10 ml DMEM for 10 min, the supernatant was decanted. The sediment was added with 0.5 ml of 1% sodium alginate-formalin solutions, centrifuged for 10 min, and then added with 1 or 2 drops of 1 M calcium chloride solution. They were left for 5–10 min, fixed in 20% formalin solutions, and then processed to make paraffinblocks. Tissue specimens Twenty patients undergoing surgical resection from April, 2009 to July, 2012, at the Department of Gynecology, Gifu University Hospital were diagnosed with ovarian serous carcinoma. The diagnoses were pathologically established by using the WHO classification [20] and no patients had been treated before surgical removal of the tumors. The resected tissues of ovarian cancer were obtained according to protocol approved by the committee for conduct of human research at Gifu University Graduate School of Medicine. Written informed consent was obtained from each patient. Each excised tissue was divided to two parts, and one part was then fixed with 20%-buffered formalin overnight, and another part was snap-frozen in liquid nitrogen and stored at 80 °C until used for Western blot analysis and ELISA. The 20% formalin-fixed tissues were embedded in a paraffin wax and then cut with 3 lm thickness to examine histopathologically and immunohistochemically. Immunohistochemical examination The specimens in above formalin-fixed and paraffin-embedded blocks were immunohistochemically examined by Dako EnVision™ FLEX detection system (Dako, Glostrup, Denmark) to investigate the expression and localization of HSP22 in the tissues and cells. Briefly, the deparaffinized sections were treated with 3% H2O2 in methanol for 10 min to inhibit endogenous peroxidase activity. Sections were pretreated with Target Retrieval solution (High pH) (Dako) at 97 °C for 20 min, and then incubated with HSP22 antibodies for 20 min at room temperature. Each section was treated sequentially with HRP-conjugated EnVision™ FLEX at room temperature for 20 min. The section was then treated with 3,30 -diamino-benzidine tetrahydrochloride (DAB) as a chromogen for 10 min, following as a counter staining with hematoxylin for 5 min. For analyzing the HSP22 immunoreactivity in each tissue, the immunohistochemical (IHC) score (0–6) was calculated by multiplying the proportion score, which means the distribution of the stained area and is ranged from 0 to 3 (0 = 0% of positively
42
M. Suzuki et al. / Archives of Biochemistry and Biophysics 571 (2015) 40–49
Fig. 1. Expression of HSP22 in SKOV3.ip1 cells. Immnohistochemical analysis of HSP22 in SKOV3.ip1 cells (HSP22) and the corresponding hematoxylin and eosin staining (HE).
stained cells; 1 = 1–10% of positively stained cells; 2 = 11–50% of positively stained cells; 3 = 51–100% of positively stained cells), and the intensity score, which was classified from 0 to 2 (0 = negative; 1 = weak or scant in each cytoplasm; 2 = strong or diffuse in each cytoplasm). Then the HSP22 IHC scores of the patients were divided into high expression group (>2) and low expression group (
