Associations of Porphyromonas gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters and Survival of Esophageal Squamous Cell Carcinoma Patients.

Purpose: The present study focused on exploring the associations of Porphyromonas gingivalis (P. gingivalis) infection and low Beclin1 expression with clinicopathological parameters and survival of esophageal squamous cell carcinoma (ESCC) patients, so as to illustrate its clinical significance and prognostic value.
Methods: Immunohistochemistry (IHC) was used to detect P. gingivalis infection status and Beclin1 expression in 370 ESCC patients. The chi-square test was adopted to illustrate the relationship between categorical variables, and Cohen's kappa coefficient was used for correlation analysis. Kaplan-Meier survival curves with the log-rank test were used to analyse the correlation of P. gingivalis infection and low Beclin1 expression with survival time. The effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptotic abilities of ESCC cells in vitro were detected by Cell Counting Kit-8, wound healing and flow cytometry assays. For P. gingivalis infection of ESCC cells, cell culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70-80%, cells were inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10. Result: P. gingivalis infection was negatively correlated with Beclin1 expression in ESCC tissues, and P. gingivalis infection and low Beclin1 expression were associated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis in ESCC patients. In vitro experiments confirmed that P. gingivalis infection and Beclin1 downregulation potentiate the proliferation, migration and antiapoptotic abilities of ESCC cells (KYSE150 and KYSE30). Our results provide evidence that P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC.
Conclusion: Long-term smoking and alcohol consumption causes poor oral and esophageal microenvironments and ESCC patients with these features were more susceptible to P. gingivalis infection and persistent colonization, and exhibited lower Beclin1 expression, worse prognosis and more advanced clinicopathological features. Our findings indicate that effectively eliminating P. gingivalis colonization and restoring Beclin1 expression in ESCC patients may contribute to preventation and targeted treatment, and yield new insights into the aetiological research on ESCC.

Introduction

Esophageal cancer is one of the most common malignancies worldwide, and more than 95% of esophageal cancer cases in China are cases of esophageal squamous cell carcinoma (ESCC) [1, 2]. ESCC generally manifests as general symptoms of digestive system diseases in the early phase, which are easy to ignore and are generally diagnosed at an advanced stage [3]. Therefore, the 5-year survival rate of ESCC is less than 20% [4]. The causes of ESCC are diverse and include smoking, alcohol consumption [5], diet [6], and chronic infections [7]. Our previous study confirmed that Porphyromonas gingivalis (P. gingivalis) is distributed more in the upper digestive tract and less in the lower part near the cardia and associated with a worse prognosis for ESCC patients, and it colonized ESCC cells for a long time to promote its occurrence and resistance to neoadjuvant chemotherapy [8-10]. However, the specific carcinogenic mechanism is not completely clear.

Recent studies have revealed that chronic infection and long-term colonization by a variety of pathogenic microorganisms interfere with the autophagy process via the regulation of autophagy-related proteins in host cells, which provides favourable conditions for the long-term colonization of host cells [11]. Autophagy is an important pathway for the degradation of pathogenic microorganisms in eukaryotic cells [12]. Autophagosomes transport damaged organelles or mutated cell components and pathogenic microorganisms to lysosomes for degradation and clearance [13]. The internal blood supply of tumor cells gradually becomes insufficient with malignant proliferation and induces a state of nutritional deficiency, at which point tumor cells may gain energy from autophagosomes and degrade their internal components to support tumor proliferation and metabolism [14]. Beclin1 is the key regulatory factor of autophagy, and it is encoded by the Becn1 gene [15]. Beclin1 plays an important role in tumorigenesis [16], neurodegeneration [17] and autophagy [13], and it is negatively correlated with the occurrence and development of a variety of tumors [16, 18]. A recent study showed that continuous infection with human papillomavirus and Helicobacter pylori inhibited the autophagy protein Beclin1, which resulted in abnormal autophagy and the malignant proliferation of cervical cancer and gastric cancer cells [19, 20]. Beclin1 deletion is closely associated with the degree of differentiation, lymph node metastasis, chemotherapy resistance, and a poor prognosis in multiple malignancies [21, 22].

The present study detected P. gingivalis infection and Beclin1 expression in tumor tissues and corresponding adjacent normal tissues of ESCC patients by immunohistochemistry (IHC). The association between P. gingivalis infection and Beclin1 expression was preliminarily examined, and the correlations between P. gingivalis infection and low Beclin1 expression with clinicopathological parameters and 5-year survival time were assessed. Moreover, we successfully established Beclin1-downregulated KYSE150 and KYSE30 ESCC cells, and compared these cells with wild type ESCC cells in terms of cell proliferation, migration and antiapoptotic abilities under P. gingivalis infection. Our findings reveal that P. gingivalis infection and low Beclin1 expression can promote the development and progression of ESCC, demonstrate that the autophagy-related protein Beclin1 is involved in the oncogenic pathogenesis of P. gingivalis, and yield new insights into preventation and targeted treatment strategies for ESCC.

Materials and Methods

Patients and Tissue Specimens

For this study, with approval of the First Affiliated Hospital of Henan University of Science and Technology Ethics Committee (approval ID: HAUST-ETHICS-2021-03-B028, date of approval:28th June), a total of 383 patients with ESCC from the First Affiliated Hospital of Henan University of Science and Technology (Luoyang, Henan, China) and Anyang Tumor Hospital (Anyang, Henan, China) were enrolled after radical resection. The inclusion and exclusion criteria of this study are shown in Figure 1. Patients who had consumed alcohol at least once a week for a minimun of 1 year were classified as drinkers and the rest were classified as nondrinkers. The definition of smoking habits of patient is similar to that on alcohol, those who smoked at least once a week for a minimun of 1 year were defined as smokers and the rest as non-smokers [23]. The postoperative status of each patient was updated via telephone follow-up every 3 months for up to 60 months. The overall survival (OS) of all enrolled patients was calculated from the day of surgery to death caused by ESCC as the endpoint. Patients who died due to other causes were excluded. 370 ESCC patients were ultimately included in the study.

FIGURE 1

The inclusion and exclusion criteria of this study.

Immunohistochemistry

The paraffin-embedded ESCC and corresponding paracancerous tissues were serially sectioned at a thickness of 3 μm, and an immunohistochemical kit (Zhongshan Golden Bridge Bio-technology,Beijing China) was used, according to the instructions and related Ref. [24], Serial sections were incubated with a P. gingivalis rabbit polyclonal antibody (1:1000 dilution, Stomatological College of St. Louis, MO, United States) [25] and a Beclin1 rabbit monoclonal antibody (1:100, Abcam) to detect P. gingivalis infection and Beclin1 expression in each tissue. The average value of five field scores at high magnification (×400) was taken as the final immunohistochemical score. The immunohistochemical scoring criteria of P. gingivalis were according to a related Ref. [9], An immunohistochemical score: grade 0 (0–10% tumor cells stained by the P. gingivalis antibody); grade 1 (10–30% tumor cells stained by the P. gingivalis antibody); grade 2 (30–60% tumor cells stained by the P. gingivalis antibody), and grade 3 (over 60% tumor cells stained by the P. gingivalis antibody), For statistical analysis, with the scores of ≤1 indicated negative staining and with the scores of ≥2 were classified as positive staining of P. gingivalis. The immunohistochemical scoring criteria of Beclin1 was according to a related Ref. [26], The immunohistochemical score: grade 0 (0–10% tumor cells stained by the Beclin1 antibody); grade 1 (11–30% tumor cells stained by the Beclin1 antibody); grade 2 (31–70% tumor cells stained by the Beclin1 antibody), and grade 3 (over 70% tumor cells stained by the Beclin1 antibody), For statistical analysis, with the scores of ≤1 were classified as low level expression of Beclin1 and with the scores of ≥2 and ≤3 were classified as high level expression of Beclin1. Two senior pathologists jointly identified the positive sections that were used as positive controls, and PBS buffer instead of the primary antibody was used as negative controls. Conflicting results were resolved using a multihead microscope or revalidation.

Bacteria Strain and Cell Infection

P. gingivalis strain ATCC 33277 was donated by Richard J. Lamont and cultured in trypticase soy broth supplemented with yeast extract (1 mg/ml), and hemin (5 μg/ml), at 37°C under anaerobic conditions with 85% N2, 10% H2, and CO2 [27]. The culture optical density was measured by ultraviolet spectrophotometer reached an optical density at 600 nm of 1.0, which corresponded to −1 × 109 colony-forming units/ml. For P. gingivalis infection of ESCC cells, cell were seeded in 6-well plates at 1 × 105 cells per well and culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70–80%. Cells were then inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10 [10], The required amount of P. gingivalis was measured and calculated by ultraviolet spectrophotometry, and the corresponding volume of bacterial liquid was then centrifuged at 12,000 rpm at 4°C and resuspended in cell culture medium to infect ESCC cells.

Cell Culture and Transfection Reagents

We used human KYSE-150 and KYSE30 cells (purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) as the ESCC cell line, which were cultured in 10% FBS supplemented RPMI-1640 (Gibco) at 37°C in an incubator with 95% air and 5% carbon dioxide[27], The cell culture medium was replaced once every 2 days and treated with EDTA-free trypsin (Gibco) when the density of ESCC cells was 70–80%, The supernatant was obtained and centrifuged at 150 g for 5 min to collect cells. Cells were then seeded in a 6-well plate at a density of 1 × 105 cells per well and 70–80% confluency the next day. Lipofectamine RNAiMAX Reagent (Invitrogen) was diluted with Opti-MEM™ (Gibco) (dilution ratio1:16) and Beclin1 siRNA (Invitrogen) was diluted to 30 pmol with Opti-MEM™ according to the manufacturer’s instructions. Diluted siRNA was added to diluted Lipofectamine® RNAiMAX Reagent (dilution ratio1:1) and incubated for 5 min at room temperature. The siRNA-Lipofectamine complex was added to cells for 24 h, and the knockdown efficiency was detected by Western blotting.

Western Blot

Protein was extracted from cells using radioimmunoprecipitation assay lysis buffer (Thermo) according to the manufacturer’s instructions. The cell culture medium was removed, and lysis buffer was added to the cells for protein extraction. The concentration of protein was measured and quantified by the BCA Protein Quantitative Kit (Solarbio). A total of 30 μg protein was electrophoresed and transferred to polyvinylidene fluoride membranes, then blocked with fat-free milk and incubated with Beclin1 (diluted 1:1000 in TBST, Abcam) and GAPDH (diluted 1:2000 in TBST, Abcam) primary antibodies. Goat anti-rabbit IgG (diluted 1:2000 in TBST, Abcam) was incubated. The signals were detected using Immobilon Western Chemilum HRP Substrate (Millipore), and the images were captured by a gel imaging system (Bio-Rad), The density of corresponding protein bands was measured quantitatively by Image Lab software (Version 3.0), and calculated by the ratio of the density value of the target protein band and the internal reference protein band. The experiments were performed in triplicate and the representative plates are shown.

Cell Proliferation Assay

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, GLPBIO) assay according to the manufacturer’s instructions. Briefly, ESCC cells were seeded in triplicate in 96-well plates with a density of 2,000 cells per well, and infected with P. gingivalis and transfected with Beclin1 siRNA under the conditions described above. Then, 100 μl culture medium was added to each well, and the cells were cultured in an incubator with 5% carbon dioxide at 37°C for 24 h. After fresh culture medium was changed, 10 μl of CCK-8 reagent was added to each experimental well, followed by incubation under the above culture conditions and the optical density (OD) value was measured using a microplate spectrophotometer (PerkinElmer, Waltham, MA, United States) at 490 nm. The cell viability rate was calculated by the discrepancy between the OD value of the experimental well and the control well after removing the OD value of the blank well. The experimental wells were mixed with infected P. gingivalis or transfected with Beclin1 siRNA ESCC cells, cell culture medium and CCK-8 reagent; The control wells were mixed with wild type ESCC cells, cell culture medium and CCK-8 reagent; The blank wells were mixed with cell culture medium and CCK-8 reagent; The experiments were performed in triplicate with three parallel samples for each and the representative results are shown.

Clonogenic Assay

ESCC cells were infected with P. gingivalis for 24 h, and cells and siBeclin1 were added to 6-well plates at 1,000 cells per well and placed in an incubator for 10–15 days. The cell culture medium was changed three times a week. On day 14, the cell culture medium was removed and the 1% crystal violet added to the cells for colonie staining, and images were captured. Individual colonies were counted and the quantified results are shown in the histogram. The experiments were performed in triplicate with three parallel samples for each and the representative results are shown.

Wound-Healing Assay

ESCC cells were infected with P. gingivalis for 24 h, and cells with siBeclin1 were seeded in 6-well plates at 1 × 105 cells per well and placed in an incubator until the cells were 70–80% confluent. A sterile pipette tip was used to scratch a gap in each well, and the position of the scratch was recorded. Cell culture medium (serum-free) was added to each well, and the cells were placed in an incubator for routine culture. Photographs were captured at the same location of cells in each well every 6 h. ImageJ software (Version 1.52) was used to measure the size of the blank area in each well at different time points to calculate the migration area. The experiments were performed in triplicate with three parallel samples for each and the representative results are shown.

Apoptosis Assay

ESCC cells were infected with P. gingivalis for 24 h, and cells with siBeclin1 RNA were seeded in 6-well plates at 1 × 105 cells and placed in an incubator until the cells were 70–80% confluent. After induction with 1 μmol/L paclitaxel (Selleck) for 48 h, the cells were treated with EDTA-free trypsin, and the supernatant was centrifuged at 150 g for 5 min to collect cells. Annexin V binding buffer (500 μl), 5 μl Annexin V-FITC, and 5 μl propidium iodide were added according to the instructions for the Annexin V-FITC/PI kit (Solarbio). After mixing and incubation at room temperature away from light for 10 min, the apoptosis rate was detected by flow cytometry. The experiments were performed in triplicate with three parallel samples for each and the representative results are shown.

Statistical Analysis

The experimental results were analysed by SPSS 26.0 software (SPSS Inc., Chicago, IL, United States). Cohen’s kappa coefficient was used for correlation analysis, and the chi-squared test was adopted to illustrate the relationship between categorical variables [28]. Experimental results are presented as the means ± standard deviation , and the differences between each group were analysed using the t-test. The Kaplan-Meier method was used to draw the survival curve, and the log-rank method was used to test the difference in survival time. A total of 370 ESCC patients were followed up for 60 months, patients who survived after 60 months of follow-up were censored data, and patients who died due to ESCC were included. p < 0.05 was considered statistically significant.

Results

P. gingivalis Infection is Negatively Associated With Expression of the Autophagosomal Protein Beclin1 in ESCC Tissues

To evaluate the correlation between P. gingivalis infection and Beclin1 protein expression, 370 patients with ESCC were included. Serial sections of a patient’s tumor and corresponding adjacent normal paraffin-embedded tissues were assessed using IHC (Figure 2). In the serial sections, yellow or brown colouring was observed in the cytoplasm of tumor cells in tumor tissues, indicating a positive P. gingivalis infection status, and most adjacent normal tissues were negative for P. gingivalis infection (p = 0.0003, Table 1). Yellow or brown colouring in the cytoplasm of esophageal epithelial cells in adjacent normal tissues indicated positive Beclin1 protein expression, and Beclin1 expression was mostly negative in the tumor tissues (p = 0.0001, Table 2). Beclin1 expression was negatively correlated with P. gingivalis infection in tumor tissues. (Kappa = 0.398, p = 0.0001, Table 3).

FIGURE 2

IHC detection of P. gingivalis infection and Beclin1 expression in ESCC samples (×400). (A,C) The infection status of P. gingivalis in ESCC tissues. (B,D) The expression of Beclin1 in ESCC tissues. (E) The infection status of P. gingivalis in normal esophageal epithelium tissues. (F) The expression of Beclin1 in normal esophageal epithelium tissues. Samples with P. gingivalis infection and low expression of Beclin1 simultaneously in two serial sections of an ESCC patient were classified as the “Positive group,” and samples that did not satisfy these conditions were as the “Negative group.”

TABLE 1

Comparison of the positive rate of P. gingivalis infection in tumor tissues and corresponding adjacent normal tissues from 370 ESCC patients.

Parameter		ESCC n (%)
		P.gingivalis (+)	P.gingivalis (−)	χ 2	P
Adjacent normal tissue	P.gingivalis (+)	14 (3.8)	0 (0.0)	20.869	0.0003
	P.gingivalis (−)	138 (37.3)	218 (58.9)

TABLE 2

Comparison of the positive rate of Beclin1 expression in tumor tissues and corresponding adjacent normal tissues from 370 ESCC patients.

Parameter		ESCC n (%)
		Beclin1 (+)	Beclin1 (−)	χ 2	P
Adjacent normal tissue	Beclin1 (+)	124 (33.5)	172 (46.5)	46.626	0.0001
	Beclin1 (−)	0 (0.0)	74 (20.0)

TABLE 3

Cohen’s kappa coefficient analysis of the consistency between P. gingivalis positivity and Beclin1 negativity in tumor tissues.

Parameter		P.gingivalis n (%)
		+	−	Kappa	P
Beclin1	+	12 (3.2)	112 (30.3)	0.398	0.0001
	−	140 (37.8)	106 (28.7)

*Kappa value > 0.7, excellent; = 0.4–0.7, good; <0.4, poor agreement.

Associations of P. gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters in 370 ESCC Patients

P. gingivalis infection and Beclin1 expression was positively associated with age, smoking, alcohol, sex, tumor differentiation, depth of invasion, lymphatic metastasis and clinical stages in ESCC patients. As for ESCC patients with simultaneous P. gingivalis infection and low Beclin1 expression, there were significantly more males, smokers, alcoholics and less elders with poorer tumor differentiation, deeper invasion, significantly more lymphatic metastasis and more frequent occurrences of advanced clinical stages, as presented in Table 4.

TABLE 4

Correlation of Porphyromonas gingivalis infection and low Beclin1 expression with clinicopathological parameters in 370 ESCC patients.

Factors	n	P.gingivalis infection status		χ2	P	Beclin1 expression status		χ2	P	Simultaneous P.gingivalis infection and low Beclin1 expression		χ2	P
		Infected	Uninfected			Positivity	Negativity			Positive group	Negative group
Gender
Male	255	131 (35.41)	124 (33.51)	35.901	0.0001	61 (16.50)	194 (52.40)	33.877	0.0001	124 (33.50)	131 (35.40)	40.607	0.0001
Female	115	21 (5.68)	94 (25.40)			63 (17.00)	52 (14.10)			16 (4.30)	99 (26.80)
Age (years)
≤60	163	82 (22.16)	81 (21.90)	10.245	0.0015	26 (7.00)	137 (37.00)	40.331	0.0001	68 (18.40)	95 (25.60)	1.865	0.1953
>60	207	70 (18.92)	137 (37.02)			98 (26.50)	109 (29.50)			72 (19.50)	135 (36.50)
Smoking
Yes	200	104 (28.11)	96 (25.95)	21.441	0.0005	43 (11.60)	157 (42.40)	28.195	0.0001	100 (27.00)	100 (27.00)	27.375	0.0001
No	170	48 (12.97)	122 (32.97)			81 (21.90)	89 (24.10)			40 (10.80)	130 (35.20)
Alcohol
Yes	204	108 (29.19)	96 (25.95)	26.424	0.0001	40 (10.80)	164 (44.30)	39.460	0.0001	104 (28.10)	100 (27.00)	33.391	0.0001
No	166	44 (11.89)	122 (32.97)			84 (22.70)	82 (22.20)			36 (9.70)	130 (35.20)
Differentiation Type
Poorly differentiated	59	41 (11.08)	18 (4.86)	45.392	0.0001	12 (3.20)	47 (12.70)	33.466	0.0001	48 (13.00)	11 (3.00)	82.188	0.0001
Moderately differentiated	185	86 (23.24)	99 (26.76)			45 (12.20)	140 (37.80)			76 (20.50)	109 (29.50)
Well differentiated	126	25 (6.76)	101 (27.30)			67 (18.20)	59 (15.90)			16 (4.30)	110 (29.70)
Depth of Invasion
≥Serous membrane	270	133 (35.95)	137 (37.02)	27.605	0.0001	67 (18.20)	203 (54.80)	33.925	0.0001	120 (32.40)	150 (40.60)	18.538	0.0012
<Serous membrane	100	19 (5.14)	81 (21.89)			57 (15.40)	43 (11.60)			20 (5.40)	80 (21.60)
Lymph Node Metastasis
Yes	190	124 (33.50)	66 (17.80)	94.357	0.0001	14 (3.80)	176 (47.60)	119.815	0.0001	112 (30.20)	78 (21.10)	73.992	0.0001
No	180	28 (7.60)	152 (41.10)			110 (29.70)	70 (18.90)			28 (7.60)	152 (41.10)
Clinical Stages
I/II	202	29 (7.84)	173 (46.76)	131.272	0.0001	112 (30.30)	90 (24.30)	96.039	0.0001	28 (7.60)	174 (47.0)	108.733	0.0001
III/IV	168	123 (33.24)	45 (12.16)			12 (3.20)	156 (42.20)			112 (30.30)	56 (15.10)

Samples with P. gingivalis infection and low expression of Beclin1 simultaneously in two serial sections of an ESCC patient were classified as the “Positive group,” and samples that did not satisfy these conditions were as the “Negative group.”

Associations of P. gingivalis Infection and Low Beclin1 Expression With the Prognosis of 370 ESCC Patients

The 5-year overall survival rate and median survival time of the 370 ESCC patients were 23.24% and 30.000 ± 1.570 months, respectively (Table 5 and Figure 3A). The 5-year survival rate and median survival time of ESCC patients with the presence of P. gingivalis infection were 13.1% and 22 ± 1.849 months, respectively, which were significantly shorter than those of the patients with absence of P. gingivalis infection (30.1% and 35 ± 1.99) (p = 0.0003), as presented in Table 5 and Figure 3B. The 5-year survival rate and median survival time of ESCC patients with low Beclin1 expression were 10.5% and 22 ± 1.56 months, respectively, which were significantly shorter than those of the patients with high Beclin1 expression (48.3% and 53 ± 1.57) (p = 0.0001), as presented in Table 5 and Figure 3C. The 5-year survival rate and median survival time of patients with simultaneous P. gingivalis infection and low Beclin1 expression (Positive group) were 14.2% and 21 ± 2.22 months, respectively, which were significantly shorter than those of nonsimultaneous P. gingivalis infection and low Beclin1 expression (Negative group) (28.69% and 34 ± 1.71 months) (p = 0.0002), as presented in Table 5 and Figure 3D.

TABLE 5

The mean and median survival times (months) of ESCC patients with P. gingivalis infection and low Beclin1 expression.

Factors	Mean survival time	Std. Error	95% Confidence interval		Median survival time	Std. Error	95% Confidence interval		χ 2	P
			Lower bound	Upper bound			Lower bound	Upper bound
P. gingivalis infected	26.47	1.52	23.51	29.45	22.00	1.85	18.37	25.62	26.808	0.0003
P. gingivalis uninfected	37.38	1.28	34.87	39.88	35.00	1.99	31.11	38.89
Beclin1 positivity	45.68	1.44	42.85	48.51	53.00	1.57	49.72	56.28	76.13	0.0001
Beclin1 negativity	26.46	1.14	24.22	28.69	22.00	1.56	18.93	25.07
Positive group	26.43	1.62	23.25	29.60	21.00	2.22	16.65	25.35	22.34	0.0002
Negative group	36.84	1.23	34.42	39.25	34.00	1.71	30.64	37.36
Overall	32.90	1.02	30.91	34.89	30.00	1.57	26.93	33.07

“Std.” are the abbreviations of “standard.” Samples with P. gingivalis infection and low expression of Beclin1 simultaneously in two serial sections of an ESCC patient were classified as the “Positive group,” and samples that did not satisfy these conditions were as the “Negative group.”

FIGURE 3

Kaplan-Meier curves for 5-year survival after surgery in 370 ESCC patients. (A) Kaplan-Meier curve for 5-year survival after surgery for ESCC patients. (B) Kaplan-Meier curve for 5-year survival after surgery in ESCC patients with P. gingivalis infection. (C) Kaplan-Meier curve for 5-year survival after surgery in ESCC patients with Beclin1 expression. (D) Kaplan-Meier curves for 5-year survival after surgery for ESCC patients in the Positive and Negative groups. Samples with P. gingivalis infection and low expression of Beclin1 simultaneously in two serial sections of an ESCC patient were classified as the “Positive group,” and samples that did not satisfy these conditions were as the “Negative group.”

Effects of P. gingivalis Infection and Beclin1 Downregulation on the Proliferation, Migration and Antiapoptosis of ESCC Cells In Vitro

To investigate the effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptosis of ESCC cells (KYSE-150 and KYSE-30) in vitro, we infected ESCC cells with P. gingivalis for 24 h and compared them with ESCC cells transfected with Beclin1 siRNA. Western blot results (Figures 4A,B) showed that Beclin1 expression decreased with the prolongation of P. gingivalis infection in ESCC cells, which were consistent with the clinical ESCC patient’s IHC results (Figure 2 and Table 3). Compared with the control, the ability of proliferation (Figures 4C–E) and migration (Figures 4F,G) in P. gingivalis infected or Beclin1-downregulated ESCC cells were significantly enhanced, and the efficiency of paclitaxel-induced apoptosis (Figures 4H,I) were significantly decreased in vitro. These findings suggest that the effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptosis of ESCC cells were consistent.

FIGURE 4

Effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptosis of ESCC cells (KYSE-150 and KYSE-30) in vitro. (A) The effects of P. gingivalis infection and Beclin1 siRNA on Beclin1 expression in ESCC cells were detected using Western blotting. (B) The expression of Beclin1 was analysed by t-tests. (C,D) Cell proliferation and clonogenic ability in vitro were detected using CCK-8 and colony formation assays, respectively. (E) The difference in cell proliferation ability was analysed using t-tests. (F) The migration ability was detected using a wound-healing assay. (G) The difference in cell migration area was analysed using t-tests. (H) Apoptosis inhibition was detected using an Annexin V-FITC apoptosis kit. (I) A t-test was used to analyse the difference in apoptosis rates. These experiments were independently repeated 3 times, and the mean of three experiments (n = 3) is shown. Results are presented as the mean ± SEM *p < 0.05, **p < 0.01.

Discussion

ESCC is a common gastrointestinal malignancy with significant regional characteristics [29] and a low 5-year survival rate. With continuous advancements in clinical medicine, the treatment outcomes for ESCC have obviously improved, but the prognosis remains poor [30]. Therefore, it is important to find an accurate indicator to provide an effective measure for the early diagnosis and treatment of esophageal cancer. Chronic infection with pathogenic microorganisms was not discussed in the context of cancer research. Pathogenic microorganisms are associated with the occurrence and development of malignancies. The gram-negative anaerobe P. gingivalis widely colonizes in the oral cavity [31] and upper digestive tract [9], and it was identified as a toxic opportunistic pathogen in recent years [32]. Chronic P. gingivalis infection and long-term colonization promote the progression of oral squamous cell carcinoma [33], lung cancer [34], and colon cancer [35]. Our previous study confirmed that the colonization rate of P. gingivalis in ESCC tissues was much higher than that in the corresponding adjacent normal tissues, and the OS of ESCC patients with P. gingivalis infection was significantly shortened [8]. As a result, P. gingivalis plays a great role in the development and progression of ESCC, but its specific pathogenic mechanism has not been completely ascertained. Recent research reports that pathogenic microorganisms supply energy for their own growth and metabolism via regulation of the autophagy process of host cells [36]. SARS-CoV-2 virus and human parainfluenza virus block autophagosome and lysosome fusion by inhibiting the formation of the Beclin-1-vps34-UVrag complex or the interaction between SNAP29 and Syntaxin17, which leads to the accumulation of autophagosomes and the internal components of autophagosomes are degraded to support virus long-term viral colonization and proliferation [37, 38]. P. gingivalis also interferes with the process of autophagosome and lysosome fusion via its virulence factors (gingipain) to support the colonization of endothelial cells [39]. FimA is the most representative fimbrillin of P. gingivalis, and it plays an important role in mediating the pathogenic ability of P. gingivalis for its colonization in host cells [40] via the targeting of TLR2 and promotion of autophagic destruction of P. gingivalis [41]. Our previous study confirmed that the infected with wild-type P. gingivalis ESCC cells have better invasion and metastasis abilities than the cells infected with FimA-deficient P. gingivalis cells [27]. These results suggest that there are many different complex mechanisms involved in the pathogenesis of P. gingivalis, and autophagy may be an important factor for P. gingivalis virulence in host cells.

Beclin1 is a key regulator during the process of autophagy and regulates autophagosome synthesis and maturation [42-44]. Microbes are likely to acquire host nutrients from interfering with the process of autophagy by interacting with Beclin1 [45]. The expression of Beclin1 is also closely related to the development and progression of several malignancies. Patients with high Beclin1 expression in ovarian cancer generally have significantly longer survival than patients with low Beclin1 expression [46]. Beclin1 inhibits the rate of proliferation and increases the apoptosis ability of cervical cancer cells, and it also increases the chemosensitivity of cervical cancer patients [47]. Patients with Beclin1 deletion of tongue squamous cell carcinoma exhibit poor differentiation, early lymphatic metastasis and poor prognosis [21]. The overexpression of Beclin1 inhibits the tongue squamous cell carcinoma cells ability of cell proliferation, migration and invasion [48]. Most research has confirmed that Beclin1 has been evaluated as an important prognostic marker in several malignancies, and low expression of Beclin1 is associated with unfavorable prognosis in ESCC [49], colorectal cancer [50]and lung cancer [51]. Targeting Beclin1 to limit the development and progression of malignancies could provide a possible therapeutic intervention for prolonging the survival of cancer patients.

The present study illustrated that the colonization of P. gingivalis in ESCC tissues was much higher than that in the corresponding adjacent normal tissues, and the expression of Beclin1 in tumor tissues was much lower than that in paracancerous tissues. P. gingivalis infection in tumor tissues was highly consistent with low Beclin1 expression and the Beclin1 expression in ESCC cells was gradually decreased with the prolongation of P. gingivalis infection. These results suggest that P. gingivalis infection is associated with low expression of the autophagosomal protein Beclin1 in ESCC cells and tissues. The correlation of P. gingivalis infection and low Beclin1 expression with the clinicopathological parameters in ESCC patients was analysed by the chi-squared test and the results showed that it was related to sex, age, smoking and alcohol consumption, which suggests that the oral microenvironment of older male ESCC patients with smoking and alcohol intake was worse, Patients with these features were more susceptible to persistent P. gingivalis colonization and exhibited low Beclin1 expression. Moreover, The P. gingivalis infection and low Beclin1 expression also correlated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis. We also confirmed this in vitro, and the results showed that the effect of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptosis of ESCC cells were consistent, while these results indicate that the P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC.

Autophagy, as an important intracellular physiological activity, plays a bidirectional role in promoting and inhibiting tumor development in the complex tumor microenvironment [14]. Autophagy can prevent normal endothelial cells from necrosis and inflammation to evade the tumorigenesis [52], In addition, autophagy can also provide nutrition and energy for tumor development and progression [14]. Similarly, Autophagy is also a double-edged sword during the invasion of pathogenic microorganisms [53]. Xenophagy can be activated as an innate immune response to degrade intracellular microbes when cells are being invaded [54]. With the persistent infection of pathogenic microbes, the virulence factors from pathogenic microbes can evolve into a variety of mechanisms to evade the elimination of autophagy via blocking autophagic flux, and the cellular components of autophagosome was degraded as nutrition and energy for their persistent growth and colonization [55]. Therefore, xenophagy is a highly dynamic and multifactor process during the pathogenic microbe invasion [11]. Fusobacterium nucleatum (F.nucleatum), as a highly virulent bacteria [56], has been reported as a potentially influential driver of colorectal cancer [57]. Beclin1 expression was inversely associated with F. nucleatum infection in colorectal cancer tissue [58], and another study proved a different result that Beclin1 expression was gradually increased with the prolongation of F. nucleatum infection and these effects were not found in Bifidobacterium and Escherichia coli infection [59]. These differences may be due to the different types of cell or bacterial strains and different MOIs or conditions of infection used by the researcher, and they also reveal that after highly virulent bacterial infection and caused cellular stress, the regulation of autophagy has become a more intricate process than before [36]. Recent research has reported that P. gingivalis infection can activate autophagy in cementoblasts [60], human umbilical vein endothelial cells [61] and periodontal ligament fibroblasts [62], and subsequently generate damaging effects through inflammatory pathways, 3-MA and CQ may attenuate the inflammatory injury by suppressing autophagy activity and Beclin1 expression [63]. These studies proved a positive association between the occurrence and development of P. gingivalis-induced autophagy and inflammatory injury, However, the P. gingivalis-induced autophagy pathway can also prevent inflammation by regulating inflammatory signalling [64]. Similar to F. nucleatum, P. gingivalis is a highly virulent carcinogenic bacterium, and its pathogenic virulence can also regulate autophagy via different pathways [65]. However, it has been reported that P. gingivalis generally disturbs host autophagy by its pathogenic virulence for its long-term colonization and proliferation in the host cell [64, 66], and more specific and comprehensive autophagy-related oncogenic pathogenesis of P. gingivalis remains to be further explored. In this study, we revealed that P. gingivalis infection is negatively associated with expression of the autophagosomal protein Beclin1 in ESCC tissues and illustrated that P. gingivalis infection and low Beclin1 expression are correlated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis. Moreover, we also found that P. gingivalis infection and Beclin1 downregulation potentiates the proliferation, migration and antiapoptosis of ESCC cells. Our findings provide evidence that the autophagosomal protein Beclin1 is involved in the oncogenic pathogenesis of P. gingivalis.

We acknowledge the potential limitations of the current study. On the one hand, autophagy is a highly dynamic process and multiple proteins are involved in the autophagic flux, the association of P. gingivalis infection with other autophagy-related proteins in ESCC still remains to be explored. In addition, the occurrence and progression of ESCC is a multifactor and multistep evolutionary process and the tumor microenvironment of ESCC patients is highly complex [67], Consequently, our results should be further validated by a xenograft model and clinical trial to explore the specific autophagy-related oncogenic pathogenesis of P. gingivalis.

In summary, the present research preliminarily demonstrates an autophagic pathogenic mechanism of P. gingivalis in ESCC and highlights an association of P. gingivalis infection and low Beclin1 expression with progression of ESCC. Our findings indicate that disruption of permanent P. gingivalis colonization and effective restoration of Beclin1 expression in ESCC patients may contribute to the preventation and targeted treatment, and yield new insights into the aetiological research of ESCC.