Alterations Of NF-κ B And Id-1 In Human Cancerous And Precancerous Lesions, Esophageal Cancer And Immortalized Cell Lines

Esophageal cancer (EC) is one of the six most common malignant diseases in the world with a remarkable geographical distribution. The prognosis of EC is very poor; five-year survival rate is only about 10 percent for the patients at late or advanced stage. Linzhou city (formerly Linxian) and nearby counties in Henan province have been well-recognized as the high incidence area of EC in the world. In the past decades, studies by the scientists from China and abroad have demonstrated that esophageal carcinogenesis was a multistep progressive process involved by multiple genetic changes (accumulation and overlap). The early characteristic for the subjects predisposed to EC is the abnormal proliferation in epithelial cells, morphologically manifested as basal cell hyperplasia (BCH), dysplasia (DVS) and carcinoma in situ (CIS), which could be considered as precancerous lesions of EC. The precancerous lesions are unstable; it can progress to cancer, or to stay for a couple of years without any changes or even return to normal. What is the most important factor to decide theprecancerous lesions developing to different directions, especially in those precancerous lesions with similar morphological changes? What is the key point to drive the mild precancerous lesions to more severe ones? Our hypothesis is that there may be different molecular changes in the precancerous lesions with similar morphology. To further characterize the molecular changes in esophageal carcinogenesis will shed light on elucidating the mechanisms of EC and establishing the biomarkers for early diagnosis and screening high-risk population.Recent results from our laboratory and others suggest that the esophageal carcinogenesis is a multistep progressive process involved by-multiple genetic changes (accumulation and overlap). High frequencies of p53~Rb changes increasing with lesion progression, suggesting that p53-Rb play a fundamental role in molecular mechanisms of esophageal carcinogenesis. However, many biological events in esophageal carcinogenesis could not be explicated by p53-Rb systems. It is important and necessary to identify some molecules associated with p53-Rb system in esophageal carcinogenesis.The nuclear factor kappa B (NF- κ B) superfamily is one of most important transcription factors, and exists in many types of cells, plays an important role in immune response, transcription, apoptosis, cell-cycle control, cell proliferation, and cell transformation. The NF- κ B family of transcription factors has been shown to be constitutively activated in various human cancer progression and invasion and metastasis. Previous studies demonstrated that NF-κ B is a competitor of p53 during apoptosis thus promoting cell survival. The p53/NF- κ B interaction is not confined to the NF-κB subunit and effects on apoptosis. Instead, by influencing the activity of other family members, p53's effects on the cell cycle can in part be mediated by regulating the activity of distinct NF-κ B subunits.The inhibitor of differentiation or DNA binding (Id) helix-loop-helix (HLH) proteins are a group of dominant negative regulators of basic HLH transcriptional factors, which promote cell differentiation. Recent evidence has revealed that Id proteins, especially Id-1, are also able to promote cell proliferation and cell cycle progression through inact i vat ion of tumor suppressor genes and activation of growth promoting pathways in mammalian cells. Upregulation of Id-1 has been found in many types of human cancer and its expression levels are also associated with advanced tumor stage. Id—1 transfected human keratinocytes show neither p53-upregulation nor cell cycle arrest after exposure to a DNA damaging agent, actinomycin D, suggesting that Id— 1 impaired p53-mediated DNA damage response. In addition, the increased Id-1 expression in normal cells is associated with both transcriptional and translational suppression of the tumor suppressors pl6I.\K4a, p21WAFl, p27Kipl. Exogenous Id-1 expression in human primary cells delays senescence through inactivation of the pl6INK4a/Rb and p53 tumor suppressor pathways, resulting in life span extension. These results indicate that the ability of Id-1 in promoting normal cell survival may be through interaction with both the p53 and Rb tumor suppressor pathways.The immortalized cell lines are good models to study relative tumors in vitro, which the human papillomavirus (HPV) whole gene or HPV E6^ E7 gene were used to immortal ize any types cells. Subsequent studies using immunologic and molecular methods have confirmed the presence of HPV in EC, and a pathologic role of HPV has been proposed in the development of EC. Two immortalized esophageal epithelial cell models were set up to imitate the malignant transformation process, the human esophageal epithelial cell primary culture, NECA6, was isolated from surgically resected specimens which had been histologically diagnosed as normal esophageal tissue. HPV type 16 (HPV16) E6 and E7 open reading frames, andhTERT-the catalytic subunit of telomerase-open reading frame were used to infect NECA6. As consequently, two immortalized cell models, NECA6-E6E7 and NECA6-E6E7-hTERT were established. Recent research results by our and others laboratories suggested that there were alteration of some molecules associated with p53-Rb systems in esophageal immortalized cell lines, and these changes were important events in esophageal cell immortalization.There should be a complicated cross-talking between p53-Rb system, NF-k B and Id—1 in cell cycle. It is important and necessary to determine the interaction between p53~Rb system, NF-k B and Id—1 in esophageal carcinogenesis. However, there have been little limited reports on NF- k B^ Id—1 gene changes in esophageal cancer tissue and adjacent tissue with high-risk areas of esophageal cancer. To further elucidate the molecular events involved in the esophageal carcinogenesis, this study was designed to characterize NF-k B^ Id—1 gene expression in EC tissues collected from high-risk area of esophageal cancer and esophageal cancer cells and immortalized cells by immunohistochemical staining, RT-PCR and Western blot, to analyze the effects of pcDNA3/mI k Ba S32A/S36A plasmids, a mutant Ik Ba plasmids of inhibitor of NF-k B, which transfect on the esophageal cancer cell line EC1.1. Materials and Methods1. 1 Surgical and biopsy tissue collection and processingAll the surgically resected primary EC specimens were collected from 65 EC patients in Linzhou, Henan, the high incidence area for EC (35 men and 30 women, 36 to 78 years of age, with a mean +SD of 55 + 7 years). All the patients had not received either chemotherapy or radiotherapy before surgery. Within one hour after excision, the cancer tissue and its adjacent tissue were collected and stored in liquid nitrogen for furtherstudies.1.2 Pathological diagnosisAll the specimens were confirmed by pathology as esophageal squamous cell carcinoma (SCO, and adjacent tissue confirmed by pathology as basal cell hyperplasia (BCH), dysplasia (DYS) and carcinoma in situ (CIS) according to our laboratories established criterion. 1. 3 Cell linesThe esophageal cancer cell lines EC109, EC1, TE1 were provided by Cancer Research laboratory, Medical College of Zhengzhou University.The esophageal epithelial immortalized cell lines \ECA6-E6E7 and NECA6-E6E7-hTERT were established by the collaboration of Cancer Research Laboratory of Medical College of Zhengzhou University and Microbiology Department in Hong Kong University. Two immortalized esophageal epithelial cell models were set up to imitate the malignant transformation process. NECA6. human esophageal epithelial cell primary culture was isolated from surgically resected specimens which had been his to logically diagnosed as normal. HPV type 16 (HPV16) E6 and E7 open reading frames, and hTERT-the catalytic subunit of telomerase-open reading frame were used to infect NECA6. As result, two immortalization cell models, NECA6-E6E7 and NECA6-E6E7-hTERT were established. 1.4 ImmunohistochemistryThe samples were fixed with 8o'-'o ethanol and embedded with paraffin. Each block was sectioned serially for histopathological analysis and immunohistochemistry analysis. Avidin-biotin complex (ABC) method was used for imrnunohistochemi stry analysis. The criteria and procedure ir, detail for immunostaining was the same as reported in our laboratory previously. All the 65 tissues for SCC and the esophageal cancer cell line and immortalized cell line were applied to determine the \F-k B p65, \F-k B p50 and Id— 1 protein expression with immunohistochemistryanalysis. 1.5 RT-PCRSV Total RNA Isolation System Kit (Promega) was used for RNA extraction, ImProm-IITM Reverse Transcription System Kit (Promega) was used for reverse transcription. The cDNA was prepared for PCR amplification. The 20 cases (38 to 76 years of age, with a mean ±SD of 54±6years, 14 males and 6 females) selected from the total 65 cases were applied to detect NF-k B p65, NF-k B p50 and Id-1 mRNA for RT-PCR study. In addition, 10 biopsy samples collected from the symptom-free subjects (33 to 53 years of age with a mean +SD of 41 + 3 years and 6 males and 4 females) were applied as normal control. The esophageal cancer and immortalized cell lines were also applied to detect NF-k B p65, NF- k B p50 and Id-1 mRNA with RT-PCR. 1. 6 Protein extractions and Western blot analysisM-PER Mammalian Protein Extraction Reagent (Pierce) was used for nuclear extracts. The 20 cases (38 to 76 years of age with a mean +SD of 54 ± 6 years and 14 males and 6 females) selected from the total 65 cases were applied for Western blot analysis. In addition, 10 biopsy samples collected from symptom-free subjects (33 to 53 years of age with a mean + SD of 41 ±3 years and 6 males and 4 females) were applied for Western blot analysis as normal control cases. The tissues nuclear extracts were prepared to detect NF- k B p65, NF- k. B p50 and Id-1 proteins for further Western blot analysis. The esophageal cancer and immortalized cell lines were also applied to detect NF- k B p65, NF-k B p50 and Id-1 protein with Western blot analysis.1.7 The pcDNA3/mIk BaS32A/S36A plasmids transfection in esophageal cancer cell line EC1The pcDNA3/mI k B a S32A/S36A was transfected into EC1 by lipofectimine 2000 method. To observe the growth of esophageal cancer cellline EC1 was applied to detect inhibitory effect of pCD.\A3/mI k Ba S32A/S36A plasmids in EC1. Western blot analysis and RT-PCR methods were used to detect the NT- k B p65^ NF- k B p50 protein expression and NF-kB p65 and NF-kB p50 mRNA in EC1. 1.8 StatisticsChi-squared test was performed in SPSS (10. 0 version) to evaluate the paired data. The significant difference was considered when the P value was less than 0.05. 2. Results2. 1 Result of NF-k B p65 2. 1. 1 ImmunohistochemistryThe normal esophageal epithelial were negative for NF-kB p65 staining. However, there were 38. 23%, 53.57%, 53. 33% and 69. 23% positive staining in the BCH, DYS, CIS and SCC respectively, there were statistically significant comparison to the normal esophageal epithelial(P<0.01). Moreover, the expression was significant between BCH and DYS, SCC. P<0. 05 = But there was no significance between the rate of NF-kB p65 positive expression and invasion, differentiation and metastasis in EC. The heterogeneity of NF-k B p65 proteins expression in EC was observed, which there were strong or weak expression and with or without expression and different distribution in different samples and different cancer nests in the same sample.The NF-k B p65 protein was also expressed in esophageal cancer cell lines EC109, ECU TE1 and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 2.1.2 Western blot analysis on NF-kB p65The nuclear extracts were collected from cancer and adjacent tissue and the esophageal cancer and immortalized cells, and then were analyzed for NF-k B p65 protein expression by western blot. A specific expressedprotein bands at Mr 60XIO1 was found as expected by western blot. The rate of NF- k B p65 protein expression in cancer tissue and adjacent tissue were 50% (10/20) vs. 25% (5/20) . The difference was not significant.The NF-k B p65 protein was observed in the esophageal cancer cell lines EC109> ECU TE1 and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 2. 1. 3 RT-PCRThe rate of NF- k B p65 mRNA expression in cancer tissues and adjacent tissues were 65%(13/20)vs. 30%(6/20). The difference was not significant, P>0. 05. There was no NF-kB p65 mRNA expression in normal esophageal epithelial tissue.The NF- k. B p65 mRNA were also observed in the esophageal cancer cell lines EC109^ ECU TE1 and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 2.2 NF-k B p50 2. 2. 1 ImmunohistochemistryThe normal esophageal epithelial were negative for NF-k B p50 staining. However, there were 32. 35%, 46.43%, 46. 67% and 61. 54% positive staining in the BCH, DYS, CIS and SCC respectively, there were statistically significant comparison to the normal esophageal epithelial(P<0.01). Moreover, the expression was significant between BCH and SCC, P<0.05o But there was no significance between the rate of NF- k B p50 positive expression and invasion, differentiation and metastasis in EC. The heterogeneity of .NF- k B p50 proteins expression in EC was observed, which there were strong or weak expression and with or without expression and different distribution in different samples and different cancer nests in the same sample.The NF-k B p50 protein was also expressed in esophageal cancer cell lines EC109> ECK TE1 and esophageal epithelial immortalized cell linesNECA6-E6E7 and NECA6-E6E7-hTERT.2.2.2 Western blotThe nuclear extracts were collected from cancer and adjacent tissue and the esophageal cancerous and immortalized cells and analyzed for NF-k B poO protein expression by western blot. A specific expressed protein bands at Mr 50X10' was seen as expected by western blot. The rate of \F- k B p50 protein expression in cancer tissue and adjacent tissue were 45% (9/20) vs. 20% (4/20) , the difference was not significant.The NF-k B p50 protein was also seen in the esophageal cancer cell lines EC109, ECK TE1 and esophageal epithelial immortalized eel 1 lines NECA6-E6E7 and NECA6-E6E7-hTERT.2.2.3 RT-PCRThe rate of NF- k B p50 mRNA expression in cancer tissues and adjacent tissues were 55%( 11/20) vs. 25%(5-/20). The difference was not significant, P>0.05. There was no NF-k B p50 mRNA expression in normal esophageal epithelial tissue.The NF- k B p50 mRNA were also expressed in the esophageal cancer-cell lines EC109, ECK TE1 and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT.2.2.4 Relationship between NF- k B p65 and NF-k B p50In 65 EC samples, the consistent and different expression rates in NF- k B p50 and NF- k B p65 proteins were 49. 23°o (32/65) vs. 50. 77% (33/65), and there were no significant difference, P>0. 05.2. 3 The pcDNA3/mI k Ba S32A/S36A plasmids transfection in the esophageal cancer cell line EC1The esophageal cancer cell line EC1 trans fee led by the pcDNA3/mI k Ba S32A/S36A plasmids was collected and analyzed by western blot. The molecular weight in target protein presented about Mr 65 X 10! and Mr 50X 10! for NF-k B p65 and NF-k B p50.The two target proteins were detected by western blot in EC1 cells transfected with the pcDNA3. 0 plasmids or without transfection at 0, 24, 48, 96 hours, but not detected the target proteins in the transfected by the pcDNA3/mIk Ba S32A/S36A plasmids.The NF-k B p65 mRNA and NF-k B p50 mRNA were detected by RT-PCR in EC1 cells transfected with by the pcDNA3. 0 plasmids or without, but not detected the target proteins in the transfected by the pcDNA3/mIk B a S32A/S36A plasmids.The tumor cell proliferation analyses demonstrated that the growths of EC1 were inhibited by the pcDNA3/mIk BaS32A/S36A plasmids transfection and without effect in transfected with the pcDNA3. 0 plasmids and without control-transfection by plasmid. Comparisons with EC1 transfected with the pcDNA3. 0 plasmids and without control-transfection, the cell proliferation were significant inhibited in EC1 transfected by the pcDNA3/mIk Ba S32A/S36A plasmids at 0, 24, 48, 96 hours, P<0.05o 2.4 Result of Id-1 2. 4. 1 ImmunohistochemistryThe normal esophageal epithelia were negative for Id—1 staining. However, there were 29. 42%, 46.43%, 60.00% and 63. 07% positive staining in the BCH, DYS, CIS and SCC respectively, there were statistically significant comparison to the normal esophageal epithelial(P<0.01). Moreover, the expression was significant between BCH and DYS and SCC, P<0.05o There was a significant relationship between the rate of Id-1 positive expression and invasion, differentiation and metastasis in EC. The heterogeneity of Id—1 proteins expression in EC was observed, which there were strong or weak expression and with or without expression and different distribution in different samples and different cancer nests in the same sample.The Id-1 protein was also expressed in esophageal cancer cell linesEC109 , EC1 , TEl and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 2.4. 2 Western blotThe nuclear extracts were collected from cancer and adjacent tissue and the esophageal cancer and immortalized cells and analyzed for Id—1 protein expression by western blot. A specific expressed protein band at Mr 15X10' was seen as expected. The rate of Id— 1 protein expression in cancer tissues and adjacent tissues were 55% (11/20) vs. 20% (4/20) , there were no relationship between cancer tissue and adjacent tissue in rate of Icl-1 protein expression, P>0. 05, the difference was not significant.The Id—1 protein was also seen in the esophageal cancer cell lines EC109, EC1, TEl and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 2.4.3 RT-PCRThe rate of Id— L mRNA expression in cancer tissues and adjacent tissues were 75% (15/20) vs. 35% (7/20), the difference was not significant. No Icl-1 mRNA expression was observed in normal esophageal epithelial cells. The Id—1 mRNA was expressed in the esophageal cancer cell lines EC109, EC 1. TEl and esophageal epithelial immortalized cell lines NECA6-E6E7 and NECA6-E6E7-hTERT. 3. Conclusions3. 1 We found that NF- k B p65n NF- k B p50. Id— 1 gene occurred in an early stage for esophageal carcinogenesis, even in BCH. With the lesion progression from mild to severe, the rates of NF-k B p65 and NF- k B p50 expression increased. It indicates that overexpression of NF- k B p65 and NF-k B p50 proteins are frequent molecular events in esophageal precancerous and cancerous lesions, and may play an important role in esophageal carcinogenesis. 3.2 There was a significant relationship between the rate of Id— 1positive expression and development, and invasion, differentiation and metastasis in EC. It suggests that the alterations of Id—1 gene may be one of the key molecular events in esophageal carcinogenesis.3.3 We detected the proteins and mRNA of NF- k B p65^ NF-k B p50> Id—1 gene in esophageal epithelial immortalized cell lines from high-risk areas of esophageal cancer, and these cell lines could be a good model to further understand the potential molecules associated with p53-Rb system.3.4 We found that the growth of the esophageal cancer cell line EC1 was inhibited by the pcDNA3/mI k. B a S32A/S36A plasmids transfection, a mutant I k B a of NF- k B inhibitor, and the expressions of NF- k B p65^ NF- k. B p50 gene were also inhibited by the pcDNA3/mI k B a S32A/S36A plasmids transfection. The present findings indicate that modulation of NF- k B may be a novel target to design the gene therapy for EC, which is of potential in clinical application.