Oncology Research, Vol. 21, pp. 165–171 Printed in the USA. All rights reserved. Copyright Ó 2014 Cognizant Comm. Corp.
0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504014X13887748696662 E-ISSN 1555-3906 www.cognizantcommunication.com
Leptin Promotes Metastasis by Inducing an Epithelial–Mesenchymal Transition in A549 Lung Cancer Cells Helin Feng,*1 Qingyi Liu,†1 Ning Zhang,‡ Lihua Zheng,§ Meixiang Sang,¶ Jiangang Feng,* Jinming Zhang,* Xiangyun Wu,# and Baoen Shan¶ *Department of Orthopedics, The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, China †Department of Thoracic Surgery, The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, China ‡Department of Cardiology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China §Master of Hebei Medical University, Shijiazhuang, China ¶Research Center, Fourth Hospital of Hebei Medical University and Hebei Cancer Institute, Shijiazhuang, China #Department of Laboratory, The Third Affiliated Hospital of Hebei Medical University, Shijiazhuang, China
Leptin, an adipocyte-derived cytokine associated with obesity, has been reported to participate in carcinogenesis. Epithelial–mesenchymal transition (EMT) is also considered as a key event in tumor metastasis. The aim of this study is to investigate the mechanism of leptin in the promotion of EMT leading to metastasis in A549 lung cancer cells. We investigated the effect of leptin on migration of A549 cells using wound healing and transwell assays. The incidence of EMT in A549 cells was examined by real-time PCR and immunofluorescence staining. The expression of TGF-b in A549 cells was detected by real-time PCR, and blocking of TGF-b in A549 cells was achieved by siRNA techniques. Additional work was performed using 100 patient samples, which included samples from 50 patients diagnosed with lung cancer and an additional 50 patients diagnosed with lung cancer with metastatic bone lesions. Leptin expression was measured using immunohistochemistry techniques. We demonstrated that leptin can effectively enhance the metastasis of human lung cancer A549 cell line using both wound healing and transwell assays. We also found the incidence of EMT in A549 cells after leptin exposure. Furthermore, we detected the expression of TGF-b in A549 cells, which had been reported to play an important role in inducing EMT. We showed that leptin can significantly upregulate TGF-b at both the mRNA and protein levels in A549 cells. Using siRNA to block the expression of TGF-b in A549 cells, we confirmed the role of TGF-b in the promotion of metastasis and induction of EMT. Furthermore, we found that in patient samples leptin was present at higher levels in samples associated with diagnosis of lung cancer bone metastases tissue than lung cancer tissue. Our results indicated that leptin promoted the metastasis of A549 human lung cancer cell lines by inducing EMT in a TGF-b-dependent manner. Key words: Leptin; Lung cancer; Metastasis; Epithelial–mesenchymal transition (EMT)
INTRODUCTION Lung cancer is a leading cause of tumor-associated mortality and the 5-year survival rate is around 10–15% (1,2). Lung cancer cells often escape from primary sites and spread to other places such as pleural space, brain, skeleton, and liver. The metastasis can be detected in approximately 30% of non-small cell lung carcinoma (NSCLC) patients at stage I disease even if the tumor was resected completely (3). Importantly, complications associated with the metastasis of lung cancer often result in patient death. Leptin, which is secreted mainly by adipose tissue, is usually involved in the regulation of energy balance and food intake (4). It is the product of the ob gene that controls body
weight homeostasis by affecting food intake and energy expenditure through a negative feedback on hypothalamic nuclei (5). Several studies showed that leptin may be produced by other tissues, such as pituitary gland (5), stomach, and fetal cartilage (6–8). Leptin can modulate insulinassociated signal pathways, which affect gene transcription and regulate the growth of cells (9,10). Additionally, leptin demonstrates physiological functions in lipid metabolism, hematopoiesis, and angiogenesis (11–14). Recently, emerging evidence has shown that leptin plays an important role in tumorigenesis, angiogenesis, and metastasis (15–18). Leptin receptors (OBR) are also found in many tissues in several forms (19,20). Yang et al. (17) demonstrated that leptin enhanced the migration of
These authors provided equal contribution to this work and should be considered co-first authors. Address correspondence to Baoen Shan, Research Center, Fourth Hospital of Hebei Medical University and Hebei Cancer Institute, Shijiazhuang, 050011, China. Tel: +86 311 86095283; Fax: +86 311 86992004; E-mail: [email protected]
1
Delivered by Ingenta to: 165 New York University IP: 91.200.80.120 On: Mon, 13 Mar 2017 03:50:45 Article(s) and/or figure(s) cannot be used for resale. Please use proper citation format when citing this article including the DOI,
166 feng ET AL.
chondrosarcoma cells by increasing avb3 integrin expression through the OBR1/IRS-1/PI3K/Akt/NF-kB signal transduction pathway. Leptin may increase bone mass by stimulating osteoblast proliferation through activation of the PI(3)-K and MAPK signaling pathways (18). In order to better understand the role of leptin in lung cancer metastasis, we examined the effect of leptin on the metastasis of lung cancer and the potential mechanism. Our results indicated that leptin enhanced the metastasis of A549 human lung cancer cell lines by inducing epithelial–mesenchymal transition (EMT) in a tumor growth factor- b (TGF-b)-dependent manner. MATERIALS AND METHODS Cell Lines The human lung cancer A549 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 50 units of penicillin, and 50 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Wound Healing and Transwell Assays The methods for wound healing and the Transwell assays have been described previously (17–21). In wound healing assay, cells were seeded on a six-well dish and incubated for 24 h; the monolayer was then disrupted with a cell scraper (1.2 mm width). The speed of wound closure was monitored after 24 and 48 h by measuring the ratio of the distance of the wound at 0 h. Cell migration assay was performed using Transwell (pore size, 8 μm; Costar, Corning Life Sciences, Acton, MA) in a 24-well plate. About 1 × 104 A549 cells in 200 μl of serum-free medium containing leptin was placed in the upper chamber, and 300 μl of the same medium was placed in the lower chamber. The plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. At the end of the experiment, the cells that passed through the filters were stained with crystal violet solution and counted under a microscope. These experiments were performed in triplicate. Immunofluorescence A459 cells (1 × 104) were seeded in a 48-well dish. After 24 h, the cells were washed in PBS twice and fixed in 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and blocked with PBS containing 3% bovine serum albumin (BSA). After blocking, cells were simultaneously incubated with primary antibodies for 1 h at room temperature. After washing with PBS, cells were incubated with secondary antibodies for 1 h at room temperature. Cell nuclei were stained with DAPI.
All matched samples were photographed with an immunofluorescence microscope. Real-Time Polymerase Chain Reaction (PCR) The A549 cells were extracted the total mRNA with RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. One microgram of total RNA was used to generate the first-strand cDNA using random primers and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed in triplicate using the SYBR PrimeScript RT-PCR Kit (Takara, Dalian). The primer sets used for PCR-based amplification were as follows: TGF-b, 5¢-GCCGAGCCCTGGACACCAAC-3¢ (forward) and 5¢-GCGCCCGGGTTATGCTGGTT-3¢ (reverse). The expres sion of GAPDH was measured as an internal control. Thermocycler conditions included an initial hold at 50°C for 2 min and then 95°C for 10 min, followed by a two-step PCR program of 95°C for 15 s and 60°C for 60 s, repeated for 40 cycles on an Mx4000 system (Stratagene, La Jolla, CA), on which data were collected and quantitatively analyzed. The expression level of mRNA was reported as fold change relative to the control group. Western Blot Cell lysis, SDS-polyacrylamide gel electrophoresis, and immunoblotting were carried out as described previously (22) by using the primary antibodies: polyclonal anti-TGF-b (Abcam), polyclonal anti-actin (Sigma), and goat anti-rabbit secondary antibody (Invitrogen). Short Interfering RNA (siRNA)-Mediated Knockdown To knock down the expression of TGF-b, A549 cells were transfected with the chemically synthesized siRNA targeting TGF-b or with the control siRNA (Dharmacon, Chicago, IN) using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Total RNA and cell lysates were prepared 48 h after transfection. Immunohistochemistry Lung tumor samples were obtained from 50 patients (36 males and 14 females, with a mean age of 56 years) diagnosed with lung cancer. Furthermore, we also collected bone metastasis of lung cancer tissue from 50 patients (34 males and 16 females, mean age 58). The patients’ age and gender from the two specimens were not statistically different. Specimens from the fresh tumor mass were fixed in 10% formalin and then paraffin embedded. Paraffin samples were cut into 4-μm-thick slices, dewaxed conventionally, and underwent heat-induced antigen retrieval utilizing citrate buffer. After blocking endogenous peroxidase activity, the samples were incubated with monoclonal anti-human leptin antibody at room temperature for 1 h. Detection of
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EFFECT OF LEPTIN ON A549 LUNG CANCER METASTASIS the primary antibody was performed by rabbit anti-mouse antibody and streptavidin–biotin–horseradish complex (SABC/HRP, DAKO A/S, Denmark). Statistical Analysis Each experiment was carried out at least three times with consistent results. The representative gel or blot for each experiment is presented in this study. The statistical significance was analyzed using Student’s t test. A value of p
0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504014X13887748696662 E-ISSN 1555-3906 www.cognizantcommunication.com
Leptin Promotes Metastasis by Inducing an Epithelial–Mesenchymal Transition in A549 Lung Cancer Cells Helin Feng,*1 Qingyi Liu,†1 Ning Zhang,‡ Lihua Zheng,§ Meixiang Sang,¶ Jiangang Feng,* Jinming Zhang,* Xiangyun Wu,# and Baoen Shan¶ *Department of Orthopedics, The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, China †Department of Thoracic Surgery, The Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, China ‡Department of Cardiology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China §Master of Hebei Medical University, Shijiazhuang, China ¶Research Center, Fourth Hospital of Hebei Medical University and Hebei Cancer Institute, Shijiazhuang, China #Department of Laboratory, The Third Affiliated Hospital of Hebei Medical University, Shijiazhuang, China
Leptin, an adipocyte-derived cytokine associated with obesity, has been reported to participate in carcinogenesis. Epithelial–mesenchymal transition (EMT) is also considered as a key event in tumor metastasis. The aim of this study is to investigate the mechanism of leptin in the promotion of EMT leading to metastasis in A549 lung cancer cells. We investigated the effect of leptin on migration of A549 cells using wound healing and transwell assays. The incidence of EMT in A549 cells was examined by real-time PCR and immunofluorescence staining. The expression of TGF-b in A549 cells was detected by real-time PCR, and blocking of TGF-b in A549 cells was achieved by siRNA techniques. Additional work was performed using 100 patient samples, which included samples from 50 patients diagnosed with lung cancer and an additional 50 patients diagnosed with lung cancer with metastatic bone lesions. Leptin expression was measured using immunohistochemistry techniques. We demonstrated that leptin can effectively enhance the metastasis of human lung cancer A549 cell line using both wound healing and transwell assays. We also found the incidence of EMT in A549 cells after leptin exposure. Furthermore, we detected the expression of TGF-b in A549 cells, which had been reported to play an important role in inducing EMT. We showed that leptin can significantly upregulate TGF-b at both the mRNA and protein levels in A549 cells. Using siRNA to block the expression of TGF-b in A549 cells, we confirmed the role of TGF-b in the promotion of metastasis and induction of EMT. Furthermore, we found that in patient samples leptin was present at higher levels in samples associated with diagnosis of lung cancer bone metastases tissue than lung cancer tissue. Our results indicated that leptin promoted the metastasis of A549 human lung cancer cell lines by inducing EMT in a TGF-b-dependent manner. Key words: Leptin; Lung cancer; Metastasis; Epithelial–mesenchymal transition (EMT)
INTRODUCTION Lung cancer is a leading cause of tumor-associated mortality and the 5-year survival rate is around 10–15% (1,2). Lung cancer cells often escape from primary sites and spread to other places such as pleural space, brain, skeleton, and liver. The metastasis can be detected in approximately 30% of non-small cell lung carcinoma (NSCLC) patients at stage I disease even if the tumor was resected completely (3). Importantly, complications associated with the metastasis of lung cancer often result in patient death. Leptin, which is secreted mainly by adipose tissue, is usually involved in the regulation of energy balance and food intake (4). It is the product of the ob gene that controls body
weight homeostasis by affecting food intake and energy expenditure through a negative feedback on hypothalamic nuclei (5). Several studies showed that leptin may be produced by other tissues, such as pituitary gland (5), stomach, and fetal cartilage (6–8). Leptin can modulate insulinassociated signal pathways, which affect gene transcription and regulate the growth of cells (9,10). Additionally, leptin demonstrates physiological functions in lipid metabolism, hematopoiesis, and angiogenesis (11–14). Recently, emerging evidence has shown that leptin plays an important role in tumorigenesis, angiogenesis, and metastasis (15–18). Leptin receptors (OBR) are also found in many tissues in several forms (19,20). Yang et al. (17) demonstrated that leptin enhanced the migration of
These authors provided equal contribution to this work and should be considered co-first authors. Address correspondence to Baoen Shan, Research Center, Fourth Hospital of Hebei Medical University and Hebei Cancer Institute, Shijiazhuang, 050011, China. Tel: +86 311 86095283; Fax: +86 311 86992004; E-mail: [email protected]
1
Delivered by Ingenta to: 165 New York University IP: 91.200.80.120 On: Mon, 13 Mar 2017 03:50:45 Article(s) and/or figure(s) cannot be used for resale. Please use proper citation format when citing this article including the DOI,
166 feng ET AL.
chondrosarcoma cells by increasing avb3 integrin expression through the OBR1/IRS-1/PI3K/Akt/NF-kB signal transduction pathway. Leptin may increase bone mass by stimulating osteoblast proliferation through activation of the PI(3)-K and MAPK signaling pathways (18). In order to better understand the role of leptin in lung cancer metastasis, we examined the effect of leptin on the metastasis of lung cancer and the potential mechanism. Our results indicated that leptin enhanced the metastasis of A549 human lung cancer cell lines by inducing epithelial–mesenchymal transition (EMT) in a tumor growth factor- b (TGF-b)-dependent manner. MATERIALS AND METHODS Cell Lines The human lung cancer A549 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 50 units of penicillin, and 50 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Wound Healing and Transwell Assays The methods for wound healing and the Transwell assays have been described previously (17–21). In wound healing assay, cells were seeded on a six-well dish and incubated for 24 h; the monolayer was then disrupted with a cell scraper (1.2 mm width). The speed of wound closure was monitored after 24 and 48 h by measuring the ratio of the distance of the wound at 0 h. Cell migration assay was performed using Transwell (pore size, 8 μm; Costar, Corning Life Sciences, Acton, MA) in a 24-well plate. About 1 × 104 A549 cells in 200 μl of serum-free medium containing leptin was placed in the upper chamber, and 300 μl of the same medium was placed in the lower chamber. The plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. At the end of the experiment, the cells that passed through the filters were stained with crystal violet solution and counted under a microscope. These experiments were performed in triplicate. Immunofluorescence A459 cells (1 × 104) were seeded in a 48-well dish. After 24 h, the cells were washed in PBS twice and fixed in 3.7% formaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and blocked with PBS containing 3% bovine serum albumin (BSA). After blocking, cells were simultaneously incubated with primary antibodies for 1 h at room temperature. After washing with PBS, cells were incubated with secondary antibodies for 1 h at room temperature. Cell nuclei were stained with DAPI.
All matched samples were photographed with an immunofluorescence microscope. Real-Time Polymerase Chain Reaction (PCR) The A549 cells were extracted the total mRNA with RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. One microgram of total RNA was used to generate the first-strand cDNA using random primers and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed in triplicate using the SYBR PrimeScript RT-PCR Kit (Takara, Dalian). The primer sets used for PCR-based amplification were as follows: TGF-b, 5¢-GCCGAGCCCTGGACACCAAC-3¢ (forward) and 5¢-GCGCCCGGGTTATGCTGGTT-3¢ (reverse). The expres sion of GAPDH was measured as an internal control. Thermocycler conditions included an initial hold at 50°C for 2 min and then 95°C for 10 min, followed by a two-step PCR program of 95°C for 15 s and 60°C for 60 s, repeated for 40 cycles on an Mx4000 system (Stratagene, La Jolla, CA), on which data were collected and quantitatively analyzed. The expression level of mRNA was reported as fold change relative to the control group. Western Blot Cell lysis, SDS-polyacrylamide gel electrophoresis, and immunoblotting were carried out as described previously (22) by using the primary antibodies: polyclonal anti-TGF-b (Abcam), polyclonal anti-actin (Sigma), and goat anti-rabbit secondary antibody (Invitrogen). Short Interfering RNA (siRNA)-Mediated Knockdown To knock down the expression of TGF-b, A549 cells were transfected with the chemically synthesized siRNA targeting TGF-b or with the control siRNA (Dharmacon, Chicago, IN) using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Total RNA and cell lysates were prepared 48 h after transfection. Immunohistochemistry Lung tumor samples were obtained from 50 patients (36 males and 14 females, with a mean age of 56 years) diagnosed with lung cancer. Furthermore, we also collected bone metastasis of lung cancer tissue from 50 patients (34 males and 16 females, mean age 58). The patients’ age and gender from the two specimens were not statistically different. Specimens from the fresh tumor mass were fixed in 10% formalin and then paraffin embedded. Paraffin samples were cut into 4-μm-thick slices, dewaxed conventionally, and underwent heat-induced antigen retrieval utilizing citrate buffer. After blocking endogenous peroxidase activity, the samples were incubated with monoclonal anti-human leptin antibody at room temperature for 1 h. Detection of
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EFFECT OF LEPTIN ON A549 LUNG CANCER METASTASIS the primary antibody was performed by rabbit anti-mouse antibody and streptavidin–biotin–horseradish complex (SABC/HRP, DAKO A/S, Denmark). Statistical Analysis Each experiment was carried out at least three times with consistent results. The representative gel or blot for each experiment is presented in this study. The statistical significance was analyzed using Student’s t test. A value of p
