The expression of phosphorylated Akt in oral carcinogenesis and The expression of phosphorylated Akt in oral carcinogenesis and its its induction by nicotine and alkaline stimulation induction by nicotine and alkaline stimulation Chieng-Ming Ma 1 ,Ho-Tai Wu 2 , Tsung-Yun Liu 3 , Shou-Yen Kao 2, 3 Introduction Introduction In Taiwan, the incidence and the mortality of oral squamous cell In Taiwan, the incidence and the mortality of oral squamous cell carcinoma (OSCC) were ranked 4th in males carcinoma (OSCC) were ranked 4th in males among all malignancies. In male OSCC cases, 90% were areca nut c among all malignancies. In male OSCC cases, 90% were areca nut chewers and more than 70% were cigarette hewers and more than 70% were cigarette smokers. Nicotine, an important component of cigarette smoke, ha smokers. Nicotine, an important component of cigarette smoke, has previously been identified as a compound s previously been identified as a compound affecting signal transduction pathways. Previous studies suggest affecting signal transduction pathways. Previous studies suggest that nicotine that nicotine-induced tumourigenesis signaling induced tumourigenesis signaling pathways include activation of the phosphatidylinositol pathways include activation of the phosphatidylinositol-3-kinase kinaseȞPI3 PI3-Kȟ/Akt pathway, resulting in cell survival and /Akt pathway, resulting in cell survival and an anti an anti-apoptosis response. Previous studies reported that nicotine per apoptosis response. Previous studies reported that nicotine permeates into buccal mucosa was correlated meates into buccal mucosa was correlated to increasing pH values. The alkaline oral environment in areca to increasing pH values. The alkaline oral environment in areca nut chewers might potentially alter the pH and affect nut chewers might potentially alter the pH and affect oral carcinogens. In this study, we primarily explore the potent oral carcinogens. In this study, we primarily explore the potent ial significance of Akt expression in the process of oral ial significance of Akt expression in the process of oral carcinogenesis by examining human tissues in vivo, and further, carcinogenesis by examining human tissues in vivo, and further, to examine the effect of Akt phosphorylation under to examine the effect of Akt phosphorylation under different time exposure or concentrations of nicotine and diffe different time exposure or concentrations of nicotine and diffe rent pH environments in vitro. rent pH environments in vitro. Materials and Methods Materials and Methods 1. 1. Patients and tissue samples Patients and tissue samples Tissue specimens of were obtained from the Oral Tissue specimens of were obtained from the Oral-Maxillofacial Surgery Division of the Taipei VGH. The tissue Maxillofacial Surgery Division of the Taipei VGH. The tissue groups were comprised of 30 patients diagnosed with OSCC, 30 pat groups were comprised of 30 patients diagnosed with OSCC, 30 pat ients with precancerous lesions and the ients with precancerous lesions and the control group was from 10 patients receiving pre control group was from 10 patients receiving pre-prosthetic surgery. The tissues were stained with hematoxylin prosthetic surgery. The tissues were stained with hematoxylin and eosin, and examined after sectioned in 0.5 and eosin, and examined after sectioned in 0.5 μm thick tissue paraffin block. m thick tissue paraffin block. 2. 2. Immunohistochemistry (IHC) Immunohistochemistry (IHC) Immunohistochemistry staining was performed using the Immunohistochemistry staining was performed using the AutoProbe AutoProbe II ABC universal staining kit according to II ABC universal staining kit according to the manufacturer the manufacturer’s instructions. Then sections were incubated at 4 s instructions. Then sections were incubated at 4∘C overnight with anti C overnight with anti-p-Akt (Ser473) in 1:50 Akt (Ser473) in 1:50 dilution. The immunoreactivity of phosphorylated Akt dilution. The immunoreactivity of phosphorylated Akt Ȟp-Akt Aktȟ in oral epithelium was randomly collected from in oral epithelium was randomly collected from five fields in each section and observed under 200X magnificatio five fields in each section and observed under 200X magnification. Scoring was divided into three grades: grade n. Scoring was divided into three grades: grade 0), which exhibited less than 20% cellularity in oral epithelium 0), which exhibited less than 20% cellularity in oral epithelium having positive staining of anti having positive staining of anti-p-Akt; grade 1 Akt; grade 1 (moderate expression), which had 20% (moderate expression), which had 20%-50% cellularity having positive staining of anti 50% cellularity having positive staining of anti-p-Akt; grade 2 (strong Akt; grade 2 (strong expression), which had more than 50% cellularity having positive expression), which had more than 50% cellularity having positive staining of anti staining of anti-p-Akt. Akt. 3. 3. Cell culture and pharmacologic treatments Cell culture and pharmacologic treatments Normal human bronchial epithelial cells (NHBE), oral human epith Normal human bronchial epithelial cells (NHBE), oral human epithelial carcinoma M1 cell line elial carcinoma M1 cell line ȞOEC OEC-M1 M1ȟ, and , and Immortalized human epithelial cells ( Immortalized human epithelial cells (HaCaT HaCaT) were maintained under F12 ) were maintained under F12-K medium, RPMI 1640 medium, K medium, RPMI 1640 medium, DMEM medium containing 10% Fetal Bovine Serum ( DMEM medium containing 10% Fetal Bovine Serum (FBS),individually FBS),individually. For time . For time-dependent induction of Akt dependent induction of Akt phosphorylation, 100 phosphorylation, 100 μM nicotine was used in 5, 15, 30, 45, and 60 minutes. Dose nicotine was used in 5, 15, 30, 45, and 60 minutes. Dose-dependent induction of Akt dependent induction of Akt phosphorylation for OEC phosphorylation for OEC-M1 and M1 and HaCaT HaCaT was generated at 30 and 60 minutes following 0.01 was generated at 30 and 60 minutes following 0.01 μM, 0.1 , 0.1 μM, 1 , 1 μM, 10 , 10 μM, and 100 , and 100 μM nicotine stimulation. Two different pH conditions were prepared nicotine stimulation. Two different pH conditions were prepared, at 7.4 and 8.0 for , at 7.4 and 8.0 for t h e c o n s e c u t i v e e x p e r i m e n t s o f t h e c o n s e c u t i v e e x p e r i m e n t s o f dose/time dose/time-dependent effects for OEC dependent effects for OEC-M1. M1. 4. Western blotting 4. Western blotting After treatments, cell palates were prepared with lysis buffer . After treatments, cell palates were prepared with lysis buffer . Protein was extracted after 10,000g centrifugation. Protein was extracted after 10,000g centrifugation. Equivalent protein amounts were loaded, and the lysates were sep Equivalent protein amounts were loaded, and the lysates were separated by 10% SDS arated by 10% SDS-PAGE and transferred to PAGE and transferred to a nitrocellulose sheet. The sheets were incubated with the follo a nitrocellulose sheet. The sheets were incubated with the following antibodies: monoclonal anti wing antibodies: monoclonal anti-Akt, monoclonal Akt, monoclonal anti anti-p-Akt (Thr308)), monoclonal anti Akt (Thr308)), monoclonal anti-p-Akt (Ser473) and monoclonal anti Akt (Ser473) and monoclonal anti-β-actin actin under 1:1000 dilution. The under 1:1000 dilution. The bound antibodies were visualized using the enhanced chemilumines bound antibodies were visualized using the enhanced chemiluminescence detection (ECL) system and cence detection (ECL) system and quantitated quantitated by by ImageQuant ImageQuant and normalized by Akt and and normalized by Akt and β-Actin Actin. . 5.Statistical Analysis 5.Statistical Analysis Un Un-paired t test and the Fisher exact test were used to analyze the paired t test and the Fisher exact test were used to analyze the p-Akt immunoreactivity. Differences were Akt immunoreactivity. Differences were considered significant when the P value < 0.05. considered significant when the P value < 0.05. Results Results A higher expression in the cancer than in the normal mucosa (P = A higher expression in the cancer than in the normal mucosa (P = 0.0002) and precancerous (P = 0.0049) groups 0.0002) and precancerous (P = 0.0049) groups was observed (showed in Fig 1). Time and dose dependent increase was observed (showed in Fig 1). Time and dose dependent increase of p of p-Akt in the NHBE and Akt in the NHBE and HaCaT HaCaT cell lines cell lines were observed (showed in Fig 2) The time and dose dependent incr were observed (showed in Fig 2) The time and dose dependent increase of p ease of p-Akt by nicotine treatment in OEC Akt by nicotine treatment in OEC- M1 cells was obvious, with maximal effect at 1 M1 cells was obvious, with maximal effect at 1 μM (showed in Fig 3). A higher p (showed in Fig 3). A higher p-Akt expression in a more Akt expression in a more alkaline environment of pH 8.0 than pH 7.4 was observed in OEC alkaline environment of pH 8.0 than pH 7.4 was observed in OEC- M1 cells (showed in Fig 4). M1 cells (showed in Fig 4). Discussions Discussions During the carcinogenesis of the cells, the abnormal cells are m During the carcinogenesis of the cells, the abnormal cells are modulated by Akt so that increased proliferation potential odulated by Akt so that increased proliferation potential exists which may allow them to escape from apoptosis . Furthermo exists which may allow them to escape from apoptosis . Furthermore, the more advanced cancers (stages III, IV) re, the more advanced cancers (stages III, IV) present a higher degree of Akt expression than the early stages present a higher degree of Akt expression than the early stages (stages I, II) of cancers, which implies a potential (stages I, II) of cancers, which implies a potential association of Akt function with tumor invasiveness and malignan association of Akt function with tumor invasiveness and malignancy. Our data showed a significant increase in Akt cy. Our data showed a significant increase in Akt expression when normal, precancerous lesions developed into canc expression when normal, precancerous lesions developed into cancer, implying the possibility of mutated oral epithelial er, implying the possibility of mutated oral epithelial cells escaping from apoptosis during oral carcinogenesis. These cells escaping from apoptosis during oral carcinogenesis. These precancerous cells transformed into early stage cancer precancerous cells transformed into early stage cancer cells, which further gained a higher grade of invasiveness when cells, which further gained a higher grade of invasiveness when they progressed to advanced stage cancer cells. The they progressed to advanced stage cancer cells. The overexpression of p overexpression of p-Akt in tumor cells is capable of inducing proliferation; resisti Akt in tumor cells is capable of inducing proliferation; resisti ng environmental stress and inhibiting ng environmental stress and inhibiting apoptosis just coincides with the theory of clonally expansion a apoptosis just coincides with the theory of clonally expansion and metastasis of tumor cells. In this study, the specimens nd metastasis of tumor cells. In this study, the specimens were all harvested from patients who were followed for an averag were all harvested from patients who were followed for an average period of 2 years or less. Thus, in order to determine e period of 2 years or less. Thus, in order to determine the significance of Akt expression in their prognosis, a longe the significance of Akt expression in their prognosis, a longer period of clinical follow r period of clinical follow-up and even more samples of up and even more samples of precancerous lesions or cancer are required from our study group precancerous lesions or cancer are required from our study group. . 1 Taipei Municipal Jianguo High School 2 Oral & Maxillofacial Surgery, Veterans General Hospital, Taipei 3 School of Dentistry, National Yang-Ming University, Taipei Our study used a similar Our study used a similar NHBEs NHBEs model proposed by West model proposed by West et al ( et al (J Clin Invest 2003).which verified the effect of p which verified the effect of p-Akt Akt expression that was obtained by that study, and upheld the reli expression that was obtained by that study, and upheld the reli ability of our experimental model ability of our experimental model ΰFig. 2 Fig. 2α. Both dose . Both dose- dependent experiments in OEC dependent experiments in OEC-M1 and M1 and HaCaT HaCaT cell lines were unanimous in illustrating that p cell lines were unanimous in illustrating that p-Akt activation could occur Akt activation could occur when the nicotine concentration was as low as 1 when the nicotine concentration was as low as 1 μM. The experiment revealed that earlier and higher p . The experiment revealed that earlier and higher p-Akt expression Akt expression may implicate nicotine stimulation enhancement, leading to AKT p may implicate nicotine stimulation enhancement, leading to AKT phosphorylation, which increases as pH increases. The hosphorylation, which increases as pH increases. The activation downstream of the PI3 activation downstream of the PI3-K/Akt pathway could enhance cell survival and anti K/Akt pathway could enhance cell survival and anti-apoptosis effects, leading to apoptosis effects, leading to damaged cells escaping from programmed cell death and a conseque damaged cells escaping from programmed cell death and a consequent increase in carcinogenesis. This was confirmed nt increase in carcinogenesis. This was confirmed indirectly by a Taiwanese epidemiological study that showed the indirectly by a Taiwanese epidemiological study that showed the cancer risk in those populations simultaneously having cancer risk in those populations simultaneously having the habits of smoking and areca nut chewing was a lot higher tha the habits of smoking and areca nut chewing was a lot higher than those with only one habit, or the normal population. n those with only one habit, or the normal population. In summary, our experiments demonstrate the important findings o In summary, our experiments demonstrate the important findings of an increase in p f an increase in p-Akt expression during the malignant Akt expression during the malignant transformation process of oral epithelia. The importance of nico transformation process of oral epithelia. The importance of nicotine tine-induced p induced p-Akt expression seems closely related to Akt expression seems closely related to oral carcinogenesis. The enhanced nicotine absorption in oral ke oral carcinogenesis. The enhanced nicotine absorption in oral keratinocytes at a higher pH value reveals the clinical ratinocytes at a higher pH value reveals the clinical importance and novelty of our findings, and strongly suggests th importance and novelty of our findings, and strongly suggests that a higher risk of oral malignancy exists for those who at a higher risk of oral malignancy exists for those who smoke cigarettes and chew the areca nut . To find out whether t smoke cigarettes and chew the areca nut . To find out whether the p he p-Akt could be a biomarker in predicting the prognosis Akt could be a biomarker in predicting the prognosis or progression in OSCC, we will require a longer period of clini or progression in OSCC, we will require a longer period of clinical follow cal follow-up of our study cohort. up of our study cohort. Acknowledge: The study was grant supported by grants from NSC-95-2314-B-075-075, VGH-95C1-090, Cl-95-16. Normal Precancer Cancer 0.0 0.5 1.0 1.5 2.0 *** P =0.0002 P =0.06 ** P =0.0049 Grades Figure 1. Increases in Positive IHC Staining in Different Oral Figure 1. Increases in Positive IHC Staining in Different Oral Tissue. A gradual increase in positive IHC staining in p Tissue. A gradual increase in positive IHC staining in p-Akt from normal oral mucosa (A Akt from normal oral mucosa (A-C), precancerous C), precancerous lesions (D lesions (D-F), and cancer (G F), and cancer (G-I) was observed. I) was observed. A B C D E F G H I Timeΰminα pAkt (S473) pAkt (T308) Akt β- Actin 0 5 15 30 45 60 1 1.6 1.8 1.8 1.7 1.3 1 1.6 1.5 1.4 1.2 1.4 OEC-M1 10 0.01 0.1 1 0 100 Conc. (μM) pAkt (S473) pAkt (T308) Akt β- Actin 1 1 2.9 1.6 3.5 1.7 6.7 3.3 7.2 5.4 2.9 2.2 OEC-M1 Figure 2. Western Blot Analysis Figure 2. Western Blot Analysis – NHBE Cells & NHBE Cells & HaCaT HaCaT cells Treated cells Treated with Nicotine with Nicotine Figure 3. Western Blot Analysis Figure 3. Western Blot Analysis – OEC OEC-M1 Cells Treated with Nicotine M1 Cells Treated with Nicotine Dose (μM) pAkt (ser) Akt β- Actin 0.01 0.1 1 0 0.01 0.1 1 pH 7.4 pH 8.0 pAkt (ser) Akt β- Actin 5 15 30 0 5 15 30 pH 7.4 pH 8.0 Time (Mins) Figure 4. The effect of Akt phosphorylation in OEC Figure 4. The effect of Akt phosphorylation in OEC-M1 cells treated with nicotine at pH 7.4 and 8.0 in time M1 cells treated with nicotine at pH 7.4 and 8.0 in time-dependent and dose dependent and dose-dependent manner. dependent manner.