Tumor Biol. DOI 10.1007/s13277-014-1714-6
RESEARCH ARTICLE
Biochemical/metabolic changes associated with hepatocellular carcinoma development in mice Nahla E. El-Ashmawy & Hoda A. El-Bahrawy & Maha M. Shamloula & Ola A. El-Feky
Received: 15 December 2013 / Accepted: 29 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality after lung and stomach cancers. This work was undertaken to investigate some of the biochemical mediators/pathways associated with or implicated in the pathogenesis of HCC. Male albino mice were classified into two groups: normal control group and HCC group. Early stage HCC was induced by injection of diethylnitrosamine (DEN) i.p. 200 mg/kg as a single dose, and after 2 weeks, the mice were given i.p. injection of thioacetamide (TAA) 100 mg/kg twice per week for 4 weeks. Mice were left for further 2 weeks without any treatment, after which, mice were sacrificed; blood and liver samples were collected. Serum was used for determination of activities of glucose-6-phosphate dehydrogenase (G6PDH) and aldolase as well as levels of insulin-like growth factor-1 (IGF-1) and epithelial cadherin (E-cadherin). One portion of the liver was used for histopathological examination and immunohistochemical staining of the tumor suppressor p53 protein. Another portion of the liver was used for determination of citrate synthase activity. Induction of HCC in mice resulted in significant increase in G6PDH and aldolase activities, and Ecadherin level, but significant decrease in IGF-1. HCC mice group showed moderate expression of p53 protein. These results suggest that the molecular pathogenesis of HCC in mice involves reduction of serum level of IGF-1 and increased serum level of E-cadherin accompanied by dysregulation of
N. E. El-Ashmawy : H. A. El-Bahrawy : O. A. El-Feky (*) Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt e-mail: [email protected] M. M. Shamloula Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
p53 protein expression. HCC was also associated with reprogrammed metabolic profile shifted toward increased glycolysis and lipogenesis. Keywords Hepatocellular carcinoma . IGF-1 . E-cadherin . p53 . G6PDH . Aldolase
Introduction Hepatocellular carcinoma (HCC) is the most common cause of primary liver neoplasms and the fourth most frequent type of cancer worldwide following lung, breast, and bowel cancers with an increasing incidence, causing one million deaths per year [10]. A study conducted by Cairo Liver Center in 2010 revealed that HCC has nearly doubled over the last decade, and there is a growing incidence of HCC in Egypt (10–120 cases/ 100,000), which represents the leading cause of death among all other cancer sites [11]. Several factors contribute to the pathogenesis of HCC; hepatitis B and C account for more than 70 % of HCC worldwide. Additional etiological factors include toxins and drugs (e.g., alcohol, aflatoxins, anabolic steroids, and vinyl chloride), metabolic liver diseases (e.g., hereditary hemochromatosis, α1-antitrypsin deficiency), steatosis, nonalcoholic fatty liver disease, and diabetes [7]. Different genes have been implicated in hepatocarcinogenesis including genes involved in growth inhibition and apoptosis (e.g., tumor suppressor gene; p53), and genes responsible for cell-cell interaction and signal transduction. Mutations in the p53 tumor suppressor gene are among the most common alterations which play an important role in either initiation or progression of HCC [7]. Hepatocellular carcinoma also showed resistance to apoptosis mediated by death receptors such as Fas receptor. The
Tumor Biol.
majority of the HCCs show one or more alterations in the Fas pathway molecules, which inhibit Fas-mediated apoptosis. Loss of response to Fas in HCC cells may be produced either by downregulation of Fas expression, concomitant with decreased expression of downstream molecules, such as Fasassociated protein with death domain (FADD), or by upregulation or overactivation of molecules that counteract its proapoptotic effect, including nuclear factor kappa B (NF-κB), Bcl-2–associated X protein (Bcl-2), or B-cell lymphoma-extra large (Bcl-xl) [33]. The development of HCC is a multistage process. In the molecular aspect, dysregulation of pleiotropic growth factors (e.g., IGF-1, transforming growth factor-alpha, hepatocyte growth factor, and EGF), adhesive molecules (e.g. Ecadherin), and metabolic pathways represents a central protumorigenic principle in human hepatocarcinogenesis [9, 45]. Some autocrine signal activators, such as epidermal growth factor receptor (EGFR) ligands, might protect liver tumor cells from apoptosis induced by stress, physiological factors, or pro-apoptotic drugs. Dysregulation of growth factor signaling, including EGF and IGF-1 pathways, has been well established in human HCCs [2]. The transforming growth factor-beta (TGF-β) family of cytokines plays a physiological role during embryonic development, and its misregulation can result in tumorigenesis. TGF-β1 is an important regulatory suppressor factor in hepatocytes, inhibiting proliferation and inducing cell death. Paradoxically, TGF-β may also modulate other pro-tumorigenic processes, such as cell invasion, immune regulation, or microenvironment modification. The escape from the antiproliferative and pro-apoptotic actions of TGF-β might be a prerequisite for hepatocarcinoma progression [25]. This study was conducted to identify some of the pathogenetic mediators or molecular mechanisms implicated in HCC chemically induced in mice.
Materials and methods Experimental design Sixty male albino mice were utilized in this study, 15–30 g each. Mice were purchased from the animal house of Giza Institute of Ophthalmology, Cairo, Egypt. Mice were weighed and housed in wire cages for 2 weeks under identical environmental conditions for adaptation and allowed free access to balanced laboratory diet and water ad libitum. The diet contains about 54 % carbohydrates, 37.2 % protein, and 6 % fat [15]. After acclimatization period, mice were weighed and randomly divided into two groups: group 1: normal control group, 10 mice received the vehicle; group 2: HCC group, 50 mice. For induction of HCC, 200 mg/kg of diethylnitrosamine (DEN) (Sigma-Aldrich Inc. USA) was injected i.p. as a single
dose [22]. After 14 days, the mice were subjected to i.p. injection of thioacetamide (TAA) (Sigma-Aldrich Inc. USA) 100 mg/kg twice per week for 4 weeks [32]. Then the mice were left for further 2 weeks without any treatment. At the end of the experiment (8 weeks), mice were weighed then anesthetized by ether and blood was collected by cardiac puncture. The survival rate within each group was calculated as number of live animals after 8 weeks/number of animals at the start of the experiment×100 [8]. Blood samples were centrifuged for 12 min at 3,000 rpm, at 4 °C using cooling centrifuge (Sigma 3K15, Germany). The obtained serum was subdivided into four portions; one portion (250 μL) was used for immediate determination of activity of glucose-6phosphate dehydrogenase enzyme (G6PDH), and other portions were stored at −20 °C until used for biochemical analysis of aldolase enzyme activity (250 μL serum), IGF-1 (10 μL serum), and E-cadherin (50 μL serum). Mice were sacrificed and their livers were dissected. Fresh liver was washed twice with ice-cold saline, dried on clean paper towels, and weighed. Relative liver weight was calculated as liver weight (g)/final body weight (g)×100. The liver was minced quickly and divided into two portions. One portion of the liver was kept in 10 % formalin for histopathological examination and immunohistochemical staining of the tumor suppressor gene p53 product. The second portion was kept frozen in liquid nitrogen at −80 °C till determination of the activity of citrate synthase enzyme. Serum analysis IGF-1 and E-cadherin were determined by mouse IGF-1 and mouse E-cadherin ELISA kits, respectively, according to manufacturer instructions. The ELISA kits were purchased from Boster Biological Technology, Ltd. (China). The concentrations of IGF-1 and E-cadherin were determined by ELISA kit according to the manufacturer procedures and expressed as nanogram per milliliter. The enzyme activities of G6PDH [43] and aldolase [35] were determined by measurement of the rate of absorbance change at 340 nm using kits obtained from Randox Laboratories Ltd. Company (England). Determination of liver citrate synthase activity Liver extract was prepared according to Morgunov and Srere [30]. For 50-mg liver tissue samples, Cellytic MT reagent (Sigma-Aldrich Inc. USA) was added in the ratio of 1:20 w/v and protease inhibitor cocktail (Sigma-Aldrich Inc. USA) was added in the ratio of 20:1w/v. The mixture was homogenized under cooling, centrifuged at 15,000×g for 10 min at 4 °C, and the protein-containing supernatant was separated and used for the determination of citrate synthase activity. The reaction mixture contains the
Tumor Biol.
following: 100 μL of 1.01 mM dithionitro benzoic acid (DTNB), 25 μL of 10 % triton X-100, 50 μL of 10 mM oxalacetate, 25 μL of 12.2 mM acetyl-CoA, and 790 μL of redistilled water. Supernatant (20 μL) was added, mixed carefully, and incubated for 10 min at 30 °C. The yellow product 5-thio-2-nitrobenzoic acid (TNB) was measured spectrophotometrically at 412 nm [44]. The protein content was determined according to Fleury and Eberhard [12] using kits obtained from Biodiagnostics Co. Ltd. (Egypt), and citrate synthase activity was expressed as micromole per minute per milligram of protein.
Histopathology The liver sections were prepared (3–5 μm thick) and stained with hematoxylin and eosin (H and E). Then, sections were investigated under light microscope (Leica, Switzerland) using image analysis system under magnification ×400. The liver sections were investigated by a pathologist. Hepatocytes were seen with blue nuclei and pink to red cytoplasm.
Immunohistochemical detection of p53 protein p53 protein was detected by immunostaining of the liver sections prepared from formalin-fixed, paraffin-embedded liver, using an Invitrogen kit (HistostainTM-SP kit). The kit utilizes the labeled streptavidin-biotin (LAB-SA) staining methodology. The primary antibody was mouse monoclonal antibody specific for p53 (PAb 240, Invitrogen Corporation, Camarillo, CA, USA). The slides were investigated with light microscopy (Leica, Switzerland) by a pathologist for number of positive cells and color intensity. Strongly p53-stained cells (+++) are those showing nuclei/cytoplasm with dark brown color and highest number of apoptotic bodies or figures. Moderately p53-stained cells (++) are those showing intermediate golden brown color and modest number of apoptotic bodies or figures. Weakly p53-stained cells (+) are those showing light brown color and least number of apoptotic bodies or figures [19].
Results Survival rate, body weight, and relative liver weight The survival rate was 20 % in the HCC group compared to 80 % in the normal control group (Fig. 1). HCC group showed a significant body weight loss (P0.01, ↑24.69 %) compared with normal control group (Table 2). G6PDH, aldolase, and citrate synthase activities HCC group showed a significant increase in serum G6PDH activity (P
RESEARCH ARTICLE
Biochemical/metabolic changes associated with hepatocellular carcinoma development in mice Nahla E. El-Ashmawy & Hoda A. El-Bahrawy & Maha M. Shamloula & Ola A. El-Feky
Received: 15 December 2013 / Accepted: 29 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality after lung and stomach cancers. This work was undertaken to investigate some of the biochemical mediators/pathways associated with or implicated in the pathogenesis of HCC. Male albino mice were classified into two groups: normal control group and HCC group. Early stage HCC was induced by injection of diethylnitrosamine (DEN) i.p. 200 mg/kg as a single dose, and after 2 weeks, the mice were given i.p. injection of thioacetamide (TAA) 100 mg/kg twice per week for 4 weeks. Mice were left for further 2 weeks without any treatment, after which, mice were sacrificed; blood and liver samples were collected. Serum was used for determination of activities of glucose-6-phosphate dehydrogenase (G6PDH) and aldolase as well as levels of insulin-like growth factor-1 (IGF-1) and epithelial cadherin (E-cadherin). One portion of the liver was used for histopathological examination and immunohistochemical staining of the tumor suppressor p53 protein. Another portion of the liver was used for determination of citrate synthase activity. Induction of HCC in mice resulted in significant increase in G6PDH and aldolase activities, and Ecadherin level, but significant decrease in IGF-1. HCC mice group showed moderate expression of p53 protein. These results suggest that the molecular pathogenesis of HCC in mice involves reduction of serum level of IGF-1 and increased serum level of E-cadherin accompanied by dysregulation of
N. E. El-Ashmawy : H. A. El-Bahrawy : O. A. El-Feky (*) Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt e-mail: [email protected] M. M. Shamloula Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
p53 protein expression. HCC was also associated with reprogrammed metabolic profile shifted toward increased glycolysis and lipogenesis. Keywords Hepatocellular carcinoma . IGF-1 . E-cadherin . p53 . G6PDH . Aldolase
Introduction Hepatocellular carcinoma (HCC) is the most common cause of primary liver neoplasms and the fourth most frequent type of cancer worldwide following lung, breast, and bowel cancers with an increasing incidence, causing one million deaths per year [10]. A study conducted by Cairo Liver Center in 2010 revealed that HCC has nearly doubled over the last decade, and there is a growing incidence of HCC in Egypt (10–120 cases/ 100,000), which represents the leading cause of death among all other cancer sites [11]. Several factors contribute to the pathogenesis of HCC; hepatitis B and C account for more than 70 % of HCC worldwide. Additional etiological factors include toxins and drugs (e.g., alcohol, aflatoxins, anabolic steroids, and vinyl chloride), metabolic liver diseases (e.g., hereditary hemochromatosis, α1-antitrypsin deficiency), steatosis, nonalcoholic fatty liver disease, and diabetes [7]. Different genes have been implicated in hepatocarcinogenesis including genes involved in growth inhibition and apoptosis (e.g., tumor suppressor gene; p53), and genes responsible for cell-cell interaction and signal transduction. Mutations in the p53 tumor suppressor gene are among the most common alterations which play an important role in either initiation or progression of HCC [7]. Hepatocellular carcinoma also showed resistance to apoptosis mediated by death receptors such as Fas receptor. The
Tumor Biol.
majority of the HCCs show one or more alterations in the Fas pathway molecules, which inhibit Fas-mediated apoptosis. Loss of response to Fas in HCC cells may be produced either by downregulation of Fas expression, concomitant with decreased expression of downstream molecules, such as Fasassociated protein with death domain (FADD), or by upregulation or overactivation of molecules that counteract its proapoptotic effect, including nuclear factor kappa B (NF-κB), Bcl-2–associated X protein (Bcl-2), or B-cell lymphoma-extra large (Bcl-xl) [33]. The development of HCC is a multistage process. In the molecular aspect, dysregulation of pleiotropic growth factors (e.g., IGF-1, transforming growth factor-alpha, hepatocyte growth factor, and EGF), adhesive molecules (e.g. Ecadherin), and metabolic pathways represents a central protumorigenic principle in human hepatocarcinogenesis [9, 45]. Some autocrine signal activators, such as epidermal growth factor receptor (EGFR) ligands, might protect liver tumor cells from apoptosis induced by stress, physiological factors, or pro-apoptotic drugs. Dysregulation of growth factor signaling, including EGF and IGF-1 pathways, has been well established in human HCCs [2]. The transforming growth factor-beta (TGF-β) family of cytokines plays a physiological role during embryonic development, and its misregulation can result in tumorigenesis. TGF-β1 is an important regulatory suppressor factor in hepatocytes, inhibiting proliferation and inducing cell death. Paradoxically, TGF-β may also modulate other pro-tumorigenic processes, such as cell invasion, immune regulation, or microenvironment modification. The escape from the antiproliferative and pro-apoptotic actions of TGF-β might be a prerequisite for hepatocarcinoma progression [25]. This study was conducted to identify some of the pathogenetic mediators or molecular mechanisms implicated in HCC chemically induced in mice.
Materials and methods Experimental design Sixty male albino mice were utilized in this study, 15–30 g each. Mice were purchased from the animal house of Giza Institute of Ophthalmology, Cairo, Egypt. Mice were weighed and housed in wire cages for 2 weeks under identical environmental conditions for adaptation and allowed free access to balanced laboratory diet and water ad libitum. The diet contains about 54 % carbohydrates, 37.2 % protein, and 6 % fat [15]. After acclimatization period, mice were weighed and randomly divided into two groups: group 1: normal control group, 10 mice received the vehicle; group 2: HCC group, 50 mice. For induction of HCC, 200 mg/kg of diethylnitrosamine (DEN) (Sigma-Aldrich Inc. USA) was injected i.p. as a single
dose [22]. After 14 days, the mice were subjected to i.p. injection of thioacetamide (TAA) (Sigma-Aldrich Inc. USA) 100 mg/kg twice per week for 4 weeks [32]. Then the mice were left for further 2 weeks without any treatment. At the end of the experiment (8 weeks), mice were weighed then anesthetized by ether and blood was collected by cardiac puncture. The survival rate within each group was calculated as number of live animals after 8 weeks/number of animals at the start of the experiment×100 [8]. Blood samples were centrifuged for 12 min at 3,000 rpm, at 4 °C using cooling centrifuge (Sigma 3K15, Germany). The obtained serum was subdivided into four portions; one portion (250 μL) was used for immediate determination of activity of glucose-6phosphate dehydrogenase enzyme (G6PDH), and other portions were stored at −20 °C until used for biochemical analysis of aldolase enzyme activity (250 μL serum), IGF-1 (10 μL serum), and E-cadherin (50 μL serum). Mice were sacrificed and their livers were dissected. Fresh liver was washed twice with ice-cold saline, dried on clean paper towels, and weighed. Relative liver weight was calculated as liver weight (g)/final body weight (g)×100. The liver was minced quickly and divided into two portions. One portion of the liver was kept in 10 % formalin for histopathological examination and immunohistochemical staining of the tumor suppressor gene p53 product. The second portion was kept frozen in liquid nitrogen at −80 °C till determination of the activity of citrate synthase enzyme. Serum analysis IGF-1 and E-cadherin were determined by mouse IGF-1 and mouse E-cadherin ELISA kits, respectively, according to manufacturer instructions. The ELISA kits were purchased from Boster Biological Technology, Ltd. (China). The concentrations of IGF-1 and E-cadherin were determined by ELISA kit according to the manufacturer procedures and expressed as nanogram per milliliter. The enzyme activities of G6PDH [43] and aldolase [35] were determined by measurement of the rate of absorbance change at 340 nm using kits obtained from Randox Laboratories Ltd. Company (England). Determination of liver citrate synthase activity Liver extract was prepared according to Morgunov and Srere [30]. For 50-mg liver tissue samples, Cellytic MT reagent (Sigma-Aldrich Inc. USA) was added in the ratio of 1:20 w/v and protease inhibitor cocktail (Sigma-Aldrich Inc. USA) was added in the ratio of 20:1w/v. The mixture was homogenized under cooling, centrifuged at 15,000×g for 10 min at 4 °C, and the protein-containing supernatant was separated and used for the determination of citrate synthase activity. The reaction mixture contains the
Tumor Biol.
following: 100 μL of 1.01 mM dithionitro benzoic acid (DTNB), 25 μL of 10 % triton X-100, 50 μL of 10 mM oxalacetate, 25 μL of 12.2 mM acetyl-CoA, and 790 μL of redistilled water. Supernatant (20 μL) was added, mixed carefully, and incubated for 10 min at 30 °C. The yellow product 5-thio-2-nitrobenzoic acid (TNB) was measured spectrophotometrically at 412 nm [44]. The protein content was determined according to Fleury and Eberhard [12] using kits obtained from Biodiagnostics Co. Ltd. (Egypt), and citrate synthase activity was expressed as micromole per minute per milligram of protein.
Histopathology The liver sections were prepared (3–5 μm thick) and stained with hematoxylin and eosin (H and E). Then, sections were investigated under light microscope (Leica, Switzerland) using image analysis system under magnification ×400. The liver sections were investigated by a pathologist. Hepatocytes were seen with blue nuclei and pink to red cytoplasm.
Immunohistochemical detection of p53 protein p53 protein was detected by immunostaining of the liver sections prepared from formalin-fixed, paraffin-embedded liver, using an Invitrogen kit (HistostainTM-SP kit). The kit utilizes the labeled streptavidin-biotin (LAB-SA) staining methodology. The primary antibody was mouse monoclonal antibody specific for p53 (PAb 240, Invitrogen Corporation, Camarillo, CA, USA). The slides were investigated with light microscopy (Leica, Switzerland) by a pathologist for number of positive cells and color intensity. Strongly p53-stained cells (+++) are those showing nuclei/cytoplasm with dark brown color and highest number of apoptotic bodies or figures. Moderately p53-stained cells (++) are those showing intermediate golden brown color and modest number of apoptotic bodies or figures. Weakly p53-stained cells (+) are those showing light brown color and least number of apoptotic bodies or figures [19].
Results Survival rate, body weight, and relative liver weight The survival rate was 20 % in the HCC group compared to 80 % in the normal control group (Fig. 1). HCC group showed a significant body weight loss (P0.01, ↑24.69 %) compared with normal control group (Table 2). G6PDH, aldolase, and citrate synthase activities HCC group showed a significant increase in serum G6PDH activity (P
