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OBJECTIVE@#To investigate the effect of Porphyromonas gingivalis (Pg) infection on IFNGR1 palmitoylation and biological behaviors of esophageal squamous cell carcinoma (ESCC) cells and the clinical implications.@*METHODS@#The expression levels of IFNGR1 protein in ESCC cell lines KYSE30 and KYSE70 were detected using Western blotting at 24 and 48 h after Pg infection, and 2-BP was used to detect IFNGR1 palmitoylation in the cells. KYSE70 cells with wild-type IFNGR1 (IFNGR1-WT cells) and with IFNGR1-C122A palmitoylation site mutation induced by site-specific mutagenesis (IFNGR1-C122A cells) were both infected with Pg, and the changes in palmitoylation of IFNGR1-C122A were analyzed using immunofluorescence and Click-iT assays. The changes in proliferation, migration and invasion ability of the infected cells were evaluated using plate cloning assay, scratch assay and Transwell assay, and IFNGR1 co-localization with lysosomal marker LAMP2 was dected using immunofluorescence assay. Immunohistochemistry was used to detect Pg infection and IFNGR1 protein expression in 50 ESCC tissues, and their correlation with the clinicopathological characteristics and survival outcomes of the patients was analyzed.@*RESULTS@#Pg infection down-regulated the protein expression of IFNGR1 in ESCC and promoted IFNGR1 palmitoylation at site 122. In IFNGR1-WT cells, Pg infection significantly enhanced cell proliferation, migration and invasion (P < 0.05). Similarly, Pg also significantly promoted proliferation, migration and invasion of IFNGR1-C122A cells, but to a lesser extent as compared with the wild-type cells (P < 0.05). Immunofluorescence assay showed that Pg and ZDHHC3 promoted IFNGR1 degradation within the lysosome. Immunohistochemical studies of the ESCC tissue samples showed a negative correlation between IFNGR1 and Pg expression, and a reduced IFNGR1 expression was correlated with a poorer survival outcome of the patient.@*CONCLUSION@#Pg infection enhances IFNGR1 palmitoylation to promote progression of ESCC, and elimination of Pg and inhibiting IFNGR1 palmitoylation may effectively control ESCC progression.
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Humanos , Neoplasias Esofágicas , Porphyromonas gingivalis , Lipoilação , Carcinoma de Células Escamosas do Esôfago , LisossomosRESUMO
Objective:To investigate the effects of Porphyromonas gingivalis ( P. gingivalis) fimbrillin (FimA) on the progression of esophageal squamous cell carcinoma (ESCC). Methods:Wild-type P. gingivalis and fimA gene-deleted P. gingivalis ( fimA-/-P. gingivalis) were used to infect ESCC cells after morphology and PCR identification. Immunofluorescence, CCK-8 and Transwell chamber were used to detect the effects of FimA on the infectivity of P. gingivalis and it influences on cell invasion, proliferation and migration. Western blot was used to detect pSmad2/3 changes. The growth of tumor was detected in a nude mouse model bearing subcutaneous tumor. Results:Deletion of FimA might reduce the interbacterial adhesion of P. gingivalis. Compared with wild-type P. gingivalis, less fimA-/-P. gingivalis could infect NE6-T cells. Moreover, the proliferation, migration and invasion of NE6-T and KYSE30 cells as well as the activation of pSmad2/3 induced by P. gingivalis were inhibited after deletion of FimA. The growth of KYSE30 infected by fimA-/-P. gingivalis in nude mice was significantly slower than that of the wild-type P. gingivalis group. Conclusions:FimA mediated the effects of P. gingivalis on promoting the evolution of ESCC and was a potential target molecule to block the tumor-promoting effect of P. gingivalis.
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Objective:To characterize the histopathological subtypes and their clinicopathological parameters of gender and onset age by common, rare and sparse primary esophageal malignant tumors (PEMT).Methods:A total of 272 437 patients with PEMT were enrolled in this study, and all of the patients were received radical surgery. The clinicopathological information of the patients was obtained from the database established by the State Key Laboratory of Esophageal Cancer Prevention & Treatment from September 1973 to December 2020, which included the clinical treatment, pathological diagnosis and follow-up information of esophagus and gastric cardia cancers. All patients were diagnosed and classified by the criteria of esophageal tumor histopathological diagnosis and classification (2019) of the World Health Organization (WHO). The esophageal tumors, which were not included in the WHO classification, were analyzed separately according to the postoperative pathological diagnosis. The χ 2 test was performed by the SPSS 25.0 software on count data, and the test standard α=0.05. Results:A total of 32 histopathological types were identified in the enrolled PEMT patients, of which 10 subtypes were not included in the WHO classification. According to the frequency, PEMT were divided into common (esophageal squamous cell carcinoma, ESCC, accounting for 97.1%), rare (esophageal adenocarcinoma, EAC, accounting for 2.3%) and sparse (mainly esophageal small cell carcinoma, malignant melanoma, etc., accounting for 0.6%). All the common, rare, and sparse types occurred predominantly in male patients, and the gender difference of rare type was most significant (EAC, male∶ female, 2.67∶1), followed with common type (ESCC, male∶ female, 1.78∶1) and sparse type (male∶ female, 1.71∶1). The common type (ESCC) mainly occurred in the middle thoracic segment (65.2%), while the rare type (EAC) mainly occurred in the lower thoracic segment (56.8%). Among the sparse type, malignant melanoma and malignant fibrous histiocytoma were both predominantly located in the lower thoracic segment (51.7%, 66.7%), and the others were mainly in the middle thoracic segment.Conclusion:ESCC is the most common type among the 32 histopathological types of PEMT, followed by EAC as the rare type, and esophageal small cell carcinoma and malignant melanoma as the major sparse type, and all of which are mainly occur in male patients. The common type of ESCC mainly occur in the middle thoracic segment, while the rare type of EAC mainly in the lower thoracic segment. The mainly sparse type of malignant melanoma and malignant fibrous histiocytoma predominately occur in the lower thoracic segment, and the remaining sparse types mainly occur in the middle thoracic segment.
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Objective To isolate and identify Prevotella nigrescens (P.nigrescens) in gingival cre-vicular fluid of patients with chronic periodontitis and to analyze its tumor-promoting role in esophageal squa-mous cell carcinoma (ESCC). Methods Samples of gingival crevicular fluid were collected from patients with chronic periodontitis and cultured on GAM agar medium under anaerobic conditions. Black colonies on GAM agar plates were picked for subculture, and then the bacteria were isolated and purified. Bacterial identification was conducted using Gram staining and 16S rDNA sequencing analysis. The isolated bacterial species was used to infect ESCC NE6-T cells and the changes in biological characteristics of the infected cells were evaluated. Results Multiple bacterial colonies were observed after anaerobic culturing of gingival cre-vicular fluid samples for 120 h on GAM agar plates. A single bacterial colony with pure black and smooth appearance was obtained from grey black bacterial colonies with streak method. It was a Gram-negative bac-terium with bead-like shape. It showed 99. 78％ 16S rDNA sequence identity with P. nigrescens F0103 and was named P. nigrescens LY01. P. nigrescens LY01 infection promoted the in vitro proliferation, migration and invasion of NE6-T cells and the growth of subcutaneous xenograft in nude mice. In addition, it could al-so induce the upregulation of Ki67 and activation of p-STAT3. Conclusions P.nigrescens inhabiting in gin-gival sulcus might promote the progression of ESCC in human with chronic periodontitis.
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Objective@#To isolate and identify Prevotella nigrescens (P.nigrescens) in gingival crevicular fluid of patients with chronic periodontitis and to analyze its tumor-promoting role in esophageal squamous cell carcinoma (ESCC).@*Methods@#Samples of gingival crevicular fluid were collected from patients with chronic periodontitis and cultured on GAM agar medium under anaerobic conditions. Black colonies on GAM agar plates were picked for subculture, and then the bacteria were isolated and purified. Bacterial identification was conducted using Gram staining and 16S rDNA sequencing analysis. The isolated bacterial species was used to infect ESCC NE6-T cells and the changes in biological characteristics of the infected cells were evaluated.@*Results@#Multiple bacterial colonies were observed after anaerobic culturing of gingival crevicular fluid samples for 120 h on GAM agar plates. A single bacterial colony with pure black and smooth appearance was obtained from grey black bacterial colonies with streak method. It was a Gram-negative bacterium with bead-like shape. It showed 99.78% 16S rDNA sequence identity with P. nigrescens F0103 and was named P. nigrescens LY01. P. nigrescens LY01 infection promoted the in vitro proliferation, migration and invasion of NE6-T cells and the growth of subcutaneous xenograft in nude mice. In addition, it could also induce the upregulation of Ki67 and activation of p-STAT3.@*Conclusions@#P. nigrescens inhabiting in gingival sulcus might promote the progression of ESCC in human with chronic periodontitis.
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Objective To analyze the expression of Kruppel-like factor 4 (KLF4 ) and CD44 in esophageal squamous cell carcinoma (ESCC) tissues and adjacent non-cancerous tissues ,and to investigate their effects on the prognosis .Methods From June 2012 to September 2016 ,in The Second People′s Hospital of Nanyang ,a total of 100 patients with ESCC who receiving operation were selected .The ESCC tissues and the adjacent non-cancerous tissues (control) of the patients were collected .The expression levels of KLF4 and CD44 protein were detected by immunohistochemistry .The expressions of KLF4 and CD44 at mRNA and protein level of 50 paired fresh tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting ,respectively . T-test ,chi-square ,Kaplan-Meier method and Pearson correlation analysis were performed for statistical analysis .Results The positive expression rate of KLF4 protein in ESCC tissues was 55％ (55/100) ,which was lower than that in adjacent non-cancerous tissues (77％ ,77/100) ,and the difference was statistically significant (χ2 =10 .778 ,P=0 .001) .The positive expression rate of CD44 protein in ESCC tissues was 81％ (81/100) ,which was higher than that in adjacent non-cancerous tissues (11％ ,11/100) ,and the difference was statistically significant (χ2=112 .600 ,P<0 .01) .The expression level of KLF4 mRNA in 43 cases was lower than that in adjacent non-cancerous tissues ,the expression level of CD44 mRNA in 46 cases was much higher than that of adjacent non-cancerous tissues ,and the differences were statistically significant (χ2 =51 .837 and 70 .563 ,both P< 0 .01) .There were statistically significant differences in positive expression rates of KLF4 in cancer tissues between different gender , differentiation degree , invasion depth ,TNM stage and lymph node metastasis (all P<0 .05) .Similarly there were statistically significant differences in positive expression rates of CD 44 in cancer tissues between different differentiation degree ,invasion depth ,TNM stage and lymph node metastasis (all P< 0 .05) .The expression of KLF4 was negatively correlated with CD44 expression ,either at protein level or mRNA level (r= -0 .284、-0 .518 ,both P< 0 .01) .The median survival time of patients with positive KLF4 expression in cancer tissues was 33 months ,which was longer than that of patients with negative KLF4 expression (20 months) ,and the difference was statistically significant (χ2 =4 .021 , P= 0 .019) .The median survival time of patients with positive CD44 expression in cancer tissues was 24 months ,which was shorter than that of patients with negative CD44 expression (37 months) , and the difference was statistically significant (χ2 = 4 .379 , P= 0 .016) .The results of univariate analysis showed that TNM stage ,KLF4 expression and CD44 expression were correlated with overall survival time (all P<0 .05) . The results of multivariate analysis indicated that TNM stage ,lower KLF4 expression and higher CD44 expression were the independent risk factors for survival (all P<0 .05) .Conclusion Lower expression of KLF4 and higher expression of CD44 in ESCC may be closely correlated with its occurrence ,development and prognosis .
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Objective To explore the effect of SIRT1 gene silencing on the radiosensitivity of diffuse large B-cell lymphoma (DLBCL) cells.Methods Immunohistochemistry was used to measure the protein expression of SIRT1 in DLBCL tissues.Western blot was used to measure the expression of SIRT1 in DLBCL cell lines (OCI-Ly3,SU-DHL-2,and SU-DHL-4) and the immortalized B cell line HMy2.CIR.After SU-DHL-4 cells were transfected with si-SIRT1 and si-NC using Lipofectamine 2000,the expression of SIRT1 was determined by Western blot.MTT assay and colony-forming assay were used to assess the cell growth and colony formation ability of SU-DHL-4 cells treated with radiation.The group t-test or univariate analysis of variance was used for comparison between groups.Results The expression rate of SIRT1 in DLBCL tissues was 72.6%(103/140),which was significantly higher than that in reactive lymphoid hyperplasia (RLH) tissues (26.5%,8/25)(P=0.001).The SIRT1 expression was significantly higher in DLBCL cells than in HMy2.CIR cells (P=0.020).After SIRT1 gene silencing by si-SIRT1,the expression of SIRT1 was significantly reduced in SU-DHL-4 cells (P=0.008).Besides,SIRT1 gene silencing significantly reduced the growth rate and colony formation ability of SU-DHL-4 cells treated with radiation (P=0.030).Conclusions SIRT1 gene silencing enhances the radiosensitivity of DLBCL cells,providing a novel target for the radiotherapy of DLBCL.
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Objective To investigate the expressions of STAT3 and HIF-1α in esophageal squamous cell carcinoma (ESCC) and its relationships with clinical pathological characteristics and prognosis. Methods The expressions of STAT3 and HIF-1α protein were examined via immunohistochemistry in 50 cases of ESCC and normal esophageal mucosa tissue. Results In the immunohistochemistry of 50 ESCC , the positive expression rates of STAT3 and HIF-1α were 70% and 58%, respectively , significantly higher than those at the adjacent normal mucosa (P < 0.05). The expression of STAT3 in ESCC patients was significantly related to the lymph node metastasis, depth of invasion and TNM stage. The 30-month survival rates were positively correlated with the positive expression of STAT3 (P < 0.05). The expression of HIF-1α in ESCC patients was significantly related to the degree of differentiation and lymph node metastasis. The patients with HIF-1α negative had a higher cumulative survival rate than those with HIF-1α positive (P < 0.05), but the difference was not significant. STAT3 and HIF-1α were positively correlation with statistical significance (r = 0.362,P < 0.05). Conclusion STAT3 and HIF-1α are frequently expressed in ESCC. High expressions of STAT3 and HIF-1αmay contribute to the poor prognosis of human esophageal squamous cell carcinoma.
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Objective To investigate the relationship between expression of DAS-1 in gastric cardia intestinal metaplasia(CIM)and gastric cardia adenocarcinoma (GCA). Methods The cancerous tissues and CIM tissues (2 cm apart from caneer) obtained from 65 patients with GCA were examined for the expression of DAS-1 protein using immunohistoehemistry. The CIM tissues (<2 cm below Z line) obtained from 15 outpatients and inflammatory mucosa from 25 outpatients were also examined for expression of DAS-1 protein. Results The type Ⅲ IM was accounted for 55.4% (36/65) in GCA patients, which was significantly higher than that in outpatients [13.3% (2/15), P<0.01]. The positive rate of DAS-1 expression in cancerous tissues [78.5 % (51/65)] was also significantly higher than that in CIM tissues [38.8 %(30/80), P<0.01]. The expression of DAS-1 protein in IM tissues was gradually increased from type Ⅰ (0%) to type Ⅲ (71.1%) with positive correlation (P<0.01). Conclusions The type Ⅲ IM with over-expression of DAS-1 is closely related to GCA, which might be one of important precancerous lesions for GCA.
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Objective To investigate the sensitivity of tumor cells to photodynamic therapy(PDT), and to select the optimal photo-dose and photo density for PDT to the cultured cells. Methods Cells of SHEE and SHEEC cell lines were divided into 21 groups randomly,after 24 h inoculation, the cells accreted on paries of culture capsule completely, we replaced M199 complete culture solution with 30 μg/rnl Photofrin-Ⅱ solution 100 μl and then replacd with M199 complete culture solution without Photofiin-Ⅱ after 150 min of incubation in Photofrin-Ⅱ. Within 15 min,we dealed the cells with PDT using Photofrin-Diomed 630 under three different power densities:25 raW/cm~2,50 mW/cm~2 and 100 mW/cm~2 for 10 s,20 9,30 s,50 s, 100 s, 150 s and 200 s respectively,and then continue for 24 h culture. We examined the inhibiting effect on cell line SHEE and SHEEC under PDT by the method of CCK-8. Results There was no significant difference in the inhibition ratio with same power density of PDT between cell line SHEE and SHEEC under the concentration of 30 μg/ml Photofrin-Ⅱ. However, the inhibition ratio increased with the raising of photo-dose of PDT,but there was a platform stage after the 2.5～30 J/cm~ 2 photo-dose. Conclu-sion The difference for photodynamic sensitivity between human immortalization esophageal epithelial cell line SHEE and the malignant transformation cell line SHEEC is not significant. The targeting of PDT to malignant tumor cell may not be involved in the photosensitivity for tumor cell.
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Withdrawing medical treatment is quite common among patients with malignant tumors in present clinical practice in China,which is considered as a form of passive euthanasia and serious violation against social ethics and medical purposes.In this paper,the behaviors and reasons for withdrawing treatment of patients with malignant tumors are analyzed,and concerned solutions are explored as well.