Linxian County in Henan Province, Northern China is known as the region with the highest incidence and mortality rate of esophageal cancer worldwide. Since 1959, the Henan medical team has conducted field work on esophageal cancer prevention and treatment in Linxian. Through three generations of effort exerted by oncologists over 65 years of research on esophageal cancer prevention and treatment in Linxian, the incidence rate of esophageal squamous cell carcinoma in this area has dropped by nearly 50%, and the 5-year survival rate has increased to 40%, reaching the international leading level. This article aims to summarize the history, present opportunities and new challenges, and explore the experience of esophageal cancer prevention and treatment in Linxian (referred to as the “Linxian experience”), providing reference and guidance to cancer prevention and treatment research in China.

Objective To investigate the molecular mechanism underlying Porphyromonas gingivalis(Pg)-induced autophagy in esophageal squamous cell carcinoma(ESCC).Methods After Pg infected KYSE70 cells and KYSE140 cells pretreated with siAtg7 or Chloroquin(CQ),Western blot was used to measure protein levels of Atg7,LC3-Ⅱ/LC3-Ⅰ,and p62;Immunofluorescent confocal imaging analysis was used to detect autophagosome and autolysosome;CCK-8 assay was used to test cell viability;Transwell assay was used for ESCC cell migration and invasion potentials.Likewise,miR-21-5p inhibitor,RASA1 overexpression plasmid,or U0126 were used to block miR-21-5p/RASA1/ERK signaling pathway prior to Pg infection,followed by the aforementioned methods.In addition,immunohistochemistry was used to examine Pg abundance and LC3 protein levels,and RT-PCR was used to evaluate miR-21-5p expression in ESCC and adjacent tissue samples,followed by correlation analyses be-tween Pg and LC3,and Pg and miR-21-5p.Results Pg infected KYSE70 cells and KYSE140 cells showed upreg-ulation Atg7 protein and LC3-Ⅱ/LC3-Ⅰ protein but downregulation of RASA1 protein and p62 protein,enhanced cell proliferation,migration,and invasion as well as immunofluorescent spots of red,green,and yellow in mRFP-GFP-LC3-labeled ESCC cells.Pretreatment with CQ or siAtg7 abolished the above alterations induced by Pg.Con-sistently,pretreatment with miR-21-5p inhibitor,U0126,or RASA1 overexpression plasmid also blocked Pg-stimu-lated autophagy.In ESCC samples,Pg abundance was correlated with upregulation of miR-21-5p and LC3.Con-clusion Pg promotes autophagy in esophageal squamous cell carcinoma via miR-21-5p/RASA1/ERK signaling pathway.

Objective To investigate the mechanism through which RgpB,a virulence factor of Porphyromonas gingivalis(Pg),induces chemoresistance in esophageal squamous carcinoma.Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry.The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation.The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting.Immunofluorescence assay was used to analyze the relationship among Lamp1,Cx43 and TBC1D5.The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique.The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method.Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB,and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5.In Pg-infected esophageal cancer cells,the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells.Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection,which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study.Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy.Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells,thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.

Objective:To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis ( P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods:A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 μg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results:At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1β [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm 2), the thickness of the esophageal wall (median 172.52 μm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1β [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions:P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Objective:To investigate the role of an inflammatory microenvironment induced by Porphyromonasgingivalis ( P. gingivalis) in the occurrence of esophageal squamous cell carcinoma (ESCC) in mice. Methods:A total of 180 C57BL/6 mice were randomly divided into 6 groups, i.e. control group, P. gingivalis group, 4NQO group, 4NQO + P. gingivalis group, 4NQO + P. gingivalis + celecoxib group, and 4NQO + P. gingivalis + antibiotic cocktail (ABC, including metronidazole, neomycin, ampicillin, and vancomycin) group, with 30 mice in each group, using the random number table. All mice were normalized by treatment with ABC in drinking water for 2 weeks. In the following 2 weeks, the mice in the control group and the P. gingivalis group were given drinking water, while the other 4 groups were treated with 30 μg/ml 4NQO in the drinking water. In weeks 11-12, the mice in the P. gingivalis group, the 4NQO + P. gingivalis group, the 4NQO + P. gingivalis + celecoxib group, and the 4NQO + P. gingivalis + ABC group were subjected to ligation of the second molar in oral cavity followed by oral P. gingivalis infection thrice weekly for 24 weeks in weeks 11-34. In weeks 13-34, the mice in 4NQO + P. gingivalis+celecoxib group and 4NQO + P. gingivalis + ABC group were administered with celecoxib and ABC for 22 weeks, respectively. At the end of 34 weeks, gross and microscopic alterations were examined followed by RT-qPCR and immunohistochemistry to examine the expression profiles of inflammatory- and tumor-molecules in esophagi of mice. Results:At 34 weeks, 4NQO treatment alone did not affect the foci of papillary hyperproliferation, diseased area, and the thickness of the esophageal wall, but significantly enhanced the foci of hyperproliferation (median 1.00, P＜0.05) and mild/moderate dysplasia (median 2.00, P＜0.01). In addition, the expression levels of IL-6 [8.35(3.45,8.99)], IL-1β [6.90(2.01,9.72)], TNF-α [12.04(3.31,14.08)], c-myc [2.21(1.80,3.04)], pSTAT3, Ki-67, and pH2AX were higher than those in the control group. The pathological changes of the esophageal mucosa were significantly more overt in the 4NQO + P. gingivalis group in terms of the foci of papillary hyperproliferation (median 2.00), diseased area (median 2.51 mm 2), the thickness of the esophageal wall (median 172.52 μm), the foci of hyperproliferation (median 1.00, P＜0.05), and mild/moderate dysplasia (median 1.00, P＜0.01). In mice of the 4NQO + P. gingivalis group, the expression levels of IL-6 [12.27(5.35,22.08)], IL-1β [13.89(10.04,15.96)], TNF-α [19.56(6.07,20.36)], IFN-γ [11.37(8.23,20.07)], c-myc [2.62(1.51,4.25)], cyclin D1 [4.52(2.68,7.83)], nuclear pSTAT3, COX-2, Ki-67, and pH2AX were significantly increased compared with the mice in the control group. In mice of the 4NQO + P. gingivalis group, the diseased area, invasive malignant foci as well as pSTAT3 and pH2AX expression were significantly blunted by celecoxib. Treatment with ABC markedly reduced the papillary hyperproliferative foci, invasive malignant foci, and pSTAT3 expression but not pH2AX. Conclusions:P. gingivalis promotes the occurrence of esophageal squamous cell carcinoma in mice by inducing an inflammatory microenvironment primed with 4NQO induced DNA damage. Clearance of P. gingivalis with ABC or anti-inflammatory intervention holds promise for prevention of esophageal squamous cell malignant pathogenesis via blockage of IL-6/STAT3 signaling and amelioration of inflammation.

Objective To investigate the mechanism through which RgpB,a virulence factor of Porphyromonas gingivalis(Pg),induces chemoresistance in esophageal squamous carcinoma.Methods The autophagy-regulating factors that interact with RgpB were screened by immunoprecipitation-mass spectrometry.The interaction between RgpB and the autophagy regulator TBC1D5 was investigated using co-immunoprecipitation.The impact of Pg infection on the expression of esophageal cancer cell membrane receptor molecule Cx43 was assessed using Western blotting.Immunofluorescence assay was used to analyze the relationship among Lamp1,Cx43 and TBC1D5.The effect of Pg infection on autophagosome-lysosome fusion was evaluated using autophagy double fluorescence technique.The effects of Pg infection and a Cx43 inhibitor on proliferation of esophageal cancer cells after chemotherapy were examined with plate cloning assay and CCK-8 method.Results Immunoprecipitation-mass spectrometry identified TBC1D5 as an autophagy regulator interacting with RgpB,and co-immunoprecipitation suggested that RgpB could directly bind to TBC1D5.In Pg-infected esophageal cancer cells,the expression of Cx43 on the cell membrane was significantly higher than that in non-infected cells.Immunofluorescence assay showed that the expression of Cx43 on the membrane of esophageal cancer cells increased significantly after Pg infection,which blocked autophagosome-lysosome fusion as shown by stubRFP-sensGFP-LC3 lentivirus study.Plate cloning assay and CCK-8 assay showed that the Cx43 inhibitor significantly attenuated the effect of Pg infection for promoting proliferation of esophageal cancer cells after chemotherapy.Conclusion Pg infection in esophageal cancer blocked autophagosome-lysosome fusion in the tumor cells,thereby preventing Cx43 from lysosomal degradation and leading to chemoresistance of esophageal cancer.

Objective To analyze the effects of Porphyromonas gingivalis(Pg)infection and expression of vitamin D pathway-related proteins on the survival and prognosis of patients with esophageal squamous cell carcinoma(ES-CC).Methods Pg infection and the expression of 24 hydroxylase(CYP24A1),1α hydroxylase(CYP27B1)and vitamin D receptor(VDR)in 173 ESCC tissues were detected by immunohistochemistry.The correlation between each index and the survival time of patients was analyzed.Results The positive rates of Pg,CYP24A1,CYP27B1 and VDR in ESCC were 43.35％,37.57％,20.23％and 21.97％,respectively.The 5-year survival time of ES-CC patients in the Pg+CYP24A1+CYP27B-VDR-high-risk group was shortened(P＜0.05).Conclusion Pg infection and vitamin D pathway-associated proteins can be used as reliable indicators to predict the survival and prognosis of ESCC patients.

Objective: To investigate the effect of Porphyromonas gingivallis ( Pg) infection of esophageal cancer cells on the polarization of tumor associated macrophages (TAMs) and functional changes. Methods: The secretion of tumor⁃related cytokines in the supernatant of Pg infected and uninfected esophageal squamous cell carcinoma (ESCC) cells was detected by ELISA. A co⁃culture model of ESCC cells and macrophages in vitro was established ,and the changes of TAMs surface markers were detected by qPCR , cellular immunofluorescence and flow cytometry. Cytokines secreted by TAMs after co⁃culture were detected by ELISA. ESCC cells were cultured using conditioned medium of co⁃cultured TAMs , and the effects of TAMs on the proliferation , migration and invasion of esophageal cancer cells were evaluated by CCK⁃8 , Wound⁃healing assay and Transwell assay. Results: The expression quantity of IL⁃6 and IL⁃10 of Pg⁃infected ESCC cells increased (P < 0. 01) . The contents of CD163 and CD206 on the surface of TAMs co⁃cultured with Pg⁃infected ESCC cells increased (P < 0. 001) . The cytokines IL⁃6 and IL⁃10 secreted by TAMs co⁃cultured with Pg⁃infected ESCC cells relatively increased (P < 0. 01) . TAMs co⁃cultured with Pginfected ESCC cells were able to enhance ESCC cells proliferation , migration and invasion (all P < 0. 05) . Conclusion Pg infection of ESCC cells can induce the secretion of cytokines , remodel TAMs to polarize toward the M2type immunosuppressive phenotype , thereby promoting the malignant biological behavior of ESCC cells. This study provides data support for the etiology of esophageal cancer and potential target molecules for clinical immunotherapy targeting TAMs.

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.
Humans ; Esophageal Neoplasms ; Porphyromonas gingivalis ; Lipoylation ; Esophageal Squamous Cell Carcinoma ; Lysosomes

Objective : To explore the relationship between Porphyromonas gingivalis(Pg) and mitophagy in esopha- geal cancer cells，and to explore new therapeutic targets for esophageal cancer． Methods : ① Western blot was used to detect the phosphorylation of unc-51-like authophagy activating kinase1 (ULK1) in mitochondria of the Pg infected cells and immunohistochemical method was used to detect the correlation between the expression of Pg and the phosphorylation status of ULK1 in esophageal cancer tissues. ② Western blot，ICC and ELISA were used to de- tect the transfer of nucleotide blinding domain and leucine rich repeat containing family member X1 (NLRX1) from cytoplasm to mitochondria，mitophagy，and the secretion levels of interleukin ( IL) -6 and reactive oxygen species (ROS) under Pg infection. ③ Pg colonization in esophageal tissues of mice in each group was detected by qPCR and Pg colonization in esophageal squamous epithelial cells of mice by RNAscope. Results : Compared with the un- treated group，the phosphorylation level of mitochondrial ULK1 (P＜0．01) ，NLRX1 expression (P＜0. 001) and mitophagy (P＜0. 001) of esophageal cancer cells increased after Pg infection．Compared with the control group， the combined intervention group could inhibit Pg colonization in esophageal tissue and esophageal squamous epithe- lial cells of mice (P＜0. 001) ． Conclusion Pg promotes the translocation of NLRX1 from cytoplasm to mitochon- dria by up-regulating the phosphorylation level of ULK1 in the mitochondria of esophageal cancer cells，and then induces mitophagy，leading to the reduction secretion of IL-6 and ROS，and ultimately maintaining Pg colonization.