Porphyromonas gingivalis-Derived Lipopolysaccharide Promotes Glioma Cell Proliferation and Migration via Activating Akt Signaling Pathways

Periodontitis is significantly associated with the risk of cancers in the lung and the digestive system. Emerging evidence shows a plausible link between periodontitis and several types of brain diseases. However, the association between periodontal infection and glioma remains unclear. In the cultured GL261 glioma cells, P. gingivalis lipopolysaccharide (LPS) significantly promoted cell proliferation at concentrations ranging from 10 to 1000 ng/mL. It promoted cell migration at a higher concentration (100 and 1000 ng/mL). Additionally, exposure to 100 ng/mL P. gingivalis LPS induced a significant increase in the expression of TNF-α, TGF-β, MMP2, and MMP9, as well as the phosphorylation level of Akt at Ser473. These changes induced by P. gingivalis LPS were significantly antagonized by the Akt inhibitor. Furthermore, a total of 48 patients with brain tumors were enrolled to investigate their periodontal status before receiving tumor management. Poor periodontal status [probing depth (PD) ≥ 6 mm and attachment loss (AL) >5 mm] was found in 42.9% (9/21) of patients with glioma, which was significantly higher than that in patients with benign tumors and the relevant data in the 4th National Oral Health Survey in China. The glioma patients with both AL > 5 mm and PD ≥ 6 mm had a higher ki-67 labeling index than those with AL ≤ 5 mm or PD < 6 mm. These findings support the association between periodontal infection and glioma progression.


Introduction
Glioma is the most common type of malignant tumor in the brain, accounting for 25.1% of the primary tumors in the central nervous system (CNS) [1]. Gliomas are classified into 4 histological grades based on the malignancy level. Lower-grade gliomas (WHO grades II and III) could evolve into glioblastoma (WHO grade IV), along with tumor progression [2]. Glioblastoma (GBM) is the most common and malignant type of glioma, with an extremely poor prognosis because of its angiogenic, aggressive, and invasive features [3]. Although the mechanism of glioma carcinogenesis is still unclear, several plausible risk factors have been investigated [4].
Chronic inflammation has been widely considered to be involved in tumor progression [5]. Periodontitis, one of the most prevalent oral diseases, is a chronic inflammatory condition characterized by the disruption of the tissues surrounding and supporting the teeth [6]. Patients with periodontal infection exhibit increased concentrations of circulating inflammatory markers, indicating the systemic implications of periodontal infection. With the recent progress in understanding the human microbiome, the association between periodontal infection and tumors has attracted significant attention [7]. Notably, individuals with periodontitis show an increased risk of total cancer compared to those with no history

Cell Culture
Mouse glioblastoma cell line GL261 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultivated in DMEM with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin in an incubator at 37 • C with 5% CO 2 . The cells were subcultured every 4 days, and cells from passages 5-10 were used for experiments.

Cell Counting Kit-8 (CCK-8) Assay
GL261 cells were seeded (5000 cells/well) in 96-well plates and cultured for 24 h. The cells were then treated with different doses of P. gingivalis LPS (Invivogen, San Diego, CA, USA) dissolved in sterile water. After 24, 48, and 72 h of incubation, the cell viability was determined using a CCK-8 assay. Briefly, 10 µL of CCK-8 solution was added to each well and incubated for 4 h. The absorbance at 450 nm was assayed using a microplate reader (BioTek, Winooski, VT, USA).

EdU Immunofluorescence Staining Assay
GL261 cells were seeded (5000 cells/well) in 96-well plates and cultured for 24 h. The cells were then treated with or without P. gingivalis LPS (100 ng/mL) for 24 h. The medium was then removed, and the wells were washed using sterile PBS. The cells were incubated with EdU (10 µM, Beyotime, Haimen, China) for 2 h, and Hoechst was used to stain all the nuclei. Cell images were captured by fluorescence microscopy under ×100 magnification.

Wound-Healing Assay
GL261 cells were seeded into 6-well plates and grown until 90% confluence of the plate bottom was reached. A sterile pipette tip was used to make the scratch wound in each well. After a scratch, the detached cells were removed by a gentle washing, and fresh media containing different doses of P. gingivalis LPS was added to each well. After incubation for 24 h, cell images were collected via an inverted phase-contrast microscope (Nikon, Tokyo, Japan) and analyzed by ImageJ software (version 1.52a, National Institutes of Health, Bethesda, MD, USA).

Transwell Migration Assay
GL261 cells were seeded (10,000 cells/chamber) in the upper chambers in DMEM without FBS, and the bottom chambers were filled with DMEM containing 10% FBS. After 24 h, the top (non-migrated) cells were removed, and the bottom (migrated) cells were fixed and stained with crystal violet to visualize the nuclei. The number of migrating cells was counted under ×100 magnification, and the means for each chamber were measured.

Real-Time qPCR Analysis
The total RNA of the GL261 cells was extracted using TRIzol reagent (Thermo Fisher, Shanghai, China). The isolated RNA was reverse transcribed to cDNA using the cDNA synthesis reagent kit (Toyobo, Japan), and the expression level of the target genes was measured with SYBR Green qPCR Mixes (Bimake, Shanghai, China). The sequences of the primers are listed in Table 1. The real-time qPCR reaction was performed at 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s and 60 • C for 45 s. The expression of the target genes was calculated using the 2-∆∆ct method, and β-actin was used as the endogenous reference. Table 1. RT-qPCR primers.

Forward
Reverse

Western Blot
The total protein of the cells was extracted using RIPA lysis buffer, and the protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher, Waltham, MA, USA). The proteins were separated using 10% SDS-PAGE gels and transferred to the PVDF membranes (Millipore, Chengdu, China). After blocking with 5% milk in Tris-buffer saline for 1 h, the membranes were incubated with primary and secondary antibodies and then visualized using enhanced chemiluminescence (ECL) reagents (VAZYME, Nanjing, China). The following primary antibodies were used: Akt (#9272, Cell Signaling Technology, Danvers, MA, USA), p-Akt (#9271, Cell Signaling Technology), and GAPDH (#8884, Cell Signaling Technology). ImageJ was used to calculate the level of target protein expression, with GAPDH as endogenous control.

Enrollment of Patients with Brain Tumors
Patients diagnosed with brain tumors in the Department of Neurosurgery at Zhongnan Hospital of Wuhan University were recruited between August 2020 and November 2020. The clinical data of 51 patients who underwent oral examinations after hospitalization were collected. One patient with a career history of ionizing radiation exposure and two patients with insufficient oral data were excluded from the study. Finally, 48 patients who received the oral examination were eventually enrolled in the study. The mean ages of patients with glioma and benign tumors were 56.2 and 55.1 years old, respectively. To investigate the prevalence of periodontitis in patients with brain tumors, data on periodontal health status collected in the present study were compared to those in the age-matched population from the 4th National Oral Health Survey in China [27], a cross-sectional oral health survey conducted in all 31 provinces, autonomous regions, and municipalities in China from September 2015 to June 2016.
The study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (NO. 2021043), and all patients were informed in detail concerning the study.

Clinical Examination for Periodontal Health Status
Periodontal examinations were conducted using a manual periodontal probe (Hu-Friedy, Chicago, IL, USA) with 1 mm accuracy. Periodontal health status was recorded according to the probing depth (PD) of periodontal pockets and attachment loss (AL) in each permanent tooth from 6 points, according to the 4th National Oral Health Survey-Examination methods [27]. All oral examinations were performed by the same dental specialist-who had received sufficient training-before the initiation of the study.

Statistical Analysis
The data were expressed as mean ± standard deviation and analyzed using an unpaired t-test, a one-way ANOVA test, a two-way ANOVA test, or a Chi-squared test using Graphpad Prism 8.0. Data were considered statistically significant when the p value was <0.05.

P. gingivalis LPS Promotes the Proliferation and Migration of GL261 Cells
LPS secreted from P. gingivalis is the most common periodontal pathogen [9]. We examined the effects of P. gingivalis LPS on the proliferation and migration of GL261 glioma cells. As shown in Figure 1A, P. gingivalis LPS at doses ranging from 10 to 1000 ng/mL significantly promoted GL261 cell viability. Moreover, the EdU assay indicated that P. gingivalis LPS (100 ng/mL) could promote GL261 cell proliferation ( Figure 1B). The wound-healing assay and transwell migration assay indicated that a high dose (100-1000 ng/mL) of P. gingivalis LPS promoted GL261 cell migration, while a low dose (1-10 ng/mL) had no significant effect on cell migration ( Figure 1C,D). Therefore, 100 ng/mL of P. gingivalis LPS was used for further experiments.

P. gingivalis LPS Activates the Akt Signaling Pathway and Induces Cytokine Secretion of GL261 Cells
The Akt signaling pathway is frequently activated and plays a pro-tumor role in glioblastoma. Therefore, we investigated whether the Akt pathway is involved in the response of GL261 cells to P. gingivalis LPS stimulation. As shown in Figure 2, exposure to 100 ng/mL P. gingivalis LPS induced a significant increase in the phosphorylation level of Akt at Ser473, which indicates the activation of Akt. (two-way ANOVA, n = 5). (B) P. gingivalis LPS (100 ng/mL) increased the rate of EdU positive cells (unpaired t-test, n = 6). (C,D) P. gingivalis LPS promoted cell migration at concentrations ranging from 100 to 1000 ng/mL according to the wound-healing assay (one-way ANOVA, n = 6) and the transwell migration assay (one-way ANOVA, n = 5). ns: no significance; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. untreated cells (0 ng/mL).

P. gingivalis LPS Activates the Akt Signaling Pathway and Induces Cytokine Secretion of GL261 Cells
The Akt signaling pathway is frequently activated and plays a pro-tumor role in glioblastoma. Therefore, we investigated whether the Akt pathway is involved in the response of GL261 cells to P. gingivalis LPS stimulation. As shown in Figure 2, exposure to 100 ng/mL P. gingivalis LPS induced a significant increase in the phosphorylation level of Akt at Ser473, which indicates the activation of Akt. We also examined the P. gingivalis LPS-induced changes in mRNA levels of several tumor-associated cytokines and the invasion-related matrix metalloproteinases (MMPs). As shown in Figure 3, following exposure to P. gingivalis LPS, the mRNA levels of TNF-α and TGF-β were significantly increased, while those of IL-1β, IL-6, and IL-10 remained unchanged. The mRNA levels of MMP-2 and MMP-9 were elevated at 6 h of exposure to P. gingivalis LPS compared to those in the control. We also examined the P. gingivalis LPS-induced changes in mRNA levels of several tumor-associated cytokines and the invasion-related matrix metalloproteinases (MMPs). As shown in Figure 3, following exposure to P. gingivalis LPS, the mRNA levels of TNF-α and TGF-β were significantly increased, while those of IL-1β, IL-6, and IL-10 remained unchanged. The mRNA levels of MMP-2 and MMP-9 were elevated at 6 h of exposure to P. gingivalis LPS compared to those in the control.

P. gingivalis LPS Activates the Akt Signaling Pathway and Induces Cytokine Secretion of GL261 Cells
The Akt signaling pathway is frequently activated and plays a pro-tumor role in g oblastoma. Therefore, we investigated whether the Akt pathway is involved in the sponse of GL261 cells to P. gingivalis LPS stimulation. As shown in Figure 2, exposure 100 ng/mL P. gingivalis LPS induced a significant increase in the phosphorylation level Akt at Ser473, which indicates the activation of Akt. We also examined the P. gingivalis LPS-induced changes in mRNA levels of seve tumor-associated cytokines and the invasion-related matrix metalloproteinases (MMP As shown in Figure 3, following exposure to P. gingivalis LPS, the mRNA levels of TNF and TGF-β were significantly increased, while those of IL-1β, IL-6, and IL-10 remain unchanged. The mRNA levels of MMP-2 and MMP-9 were elevated at 6 h of exposure P. gingivalis LPS compared to those in the control.

Inhibition of Akt Activity Antagonizes P. gingivalis LPS-Induced Cell Proliferation and Migration
To further confirm the involvement of the Akt pathway in the pro-tumor effects of P. gingivalis LPS, the Akt activity in the GL261 cells was inhibited by the Akt inhibitor IV (0.5 µM). Pretreatment with Akt inhibitor IV significantly antagonized P. gingivalis LPS-induced cell proliferation and migration ( Figure 4A-C). Likewise, Akt inhibitor IV pretreatment dramatically diminished the P. gingivalis LPS-induced increase in mRNA levels of TNF-α, TGF-β, MMP-2, and MMP-9 ( Figure 4D). with 0.5 µM AKT inhibitor IV for 1 h, then treated with 100 ng/mL P. gingivalis LPS for 6 h (mRNA assay) or 24 h (proliferation and migration assay). * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA, n = 5.

P. gingivalis LPS Alters DNMTs and TETs Expression in GL261 Cells
Epigenetic modulations are regarded as a profoundly supplementary mechanisms determining the way in which chronic inflammatory diseases affect multiple systemic disorders [28]. Previous studies showed that P. gingivalis LPS could alter the gene expression of the DNA methyltransferase family (DNMTs) and ten-eleven translocation proteins (TETs) in vitro [29][30][31]. Here, we investigated the mRNA expression levels of the two common epigenetic enzymes. As shown in Figure 5, the mRNA levels of DNMTs, TET1, and TET2 were significantly attenuated after exposure to P. gingivalis LPS for 24 h.  , migration (C), and expression of inflammatory cytokines and MMPs (D). GL261 cells were pretreated with 0.5μM AKT inhibitor IV for 1 h, then treated with 100 ng/mL P. gingivalis LPS for 6 h (mRNA assay) or 24 h (proliferation and migration assay). * p < 0.05; ** p < 0.01; *** p < 0.001; oneway ANOVA, n = 5.

P. gingivalis LPS Alters DNMTs and TETs Expression in GL261 Cells
Epigenetic modulations are regarded as a profoundly supplementary mechanisms determining the way in which chronic inflammatory diseases affect multiple systemic disorders [28]. Previous studies showed that P. gingivalis LPS could alter the gene expression of the DNA methyltransferase family (DNMTs) and ten-eleven translocation proteins (TETs) in vitro [29][30][31]. Here, we investigated the mRNA expression levels of the two common epigenetic enzymes. As shown in Figure 5, the mRNA levels of DNMTs, TET1, and TET2 were significantly attenuated after exposure to P. gingivalis LPS for 24 h.

Glioma Patients Have a Higher Prevalence of Poor Periodontal Status
A total of 48 patients, including 21 cases diagnosed with glioma and 27 cases with benign brain tumors (16 meningioma, 6 schwannoma, and 5 pituitary tumors) were enrolled in this study ( Figure 6A). The clinical characteristics of these patients is displayed in Tables 2 and 3. Probing depth (PD) and attachment loss (AL) are two main and essential markers indicating periodontal health status. According to the 4th National Oral Health Survey in China [27], the prevalence of AL > 5 mm and PD ≥ 6 mm in the age-matched Chinese population was 33.4% (1544/4623) and 15.1% (698/4623), respectively. The patients with benign brain tumors showed a comparable proportion of AL > 5 mm (7/27, 25.9%, p = 0.5401) and PD ≥ 6 mm (3/27, 11.1%, p = 0.7880). In contrast, those with gliomas exhibited a significantly higher proportion of AL > 5 mm (12/21, 57.1%, p = 0.0340) and PD ≥ 6 mm (9/21, 42.9%, p = 0.0022) in comparison to the population in the survey (Figure 6B
Next, we investigated whether poor periodontal conditions were associated with glioma malignancy. As shown in Figure 6C, high-grade glioma (WHO grade IV) patients had a slightly higher prevalence of both AL > 5 mm and PD ≥ 6 mm compared to that of patients with lower grade gliomas (58.3% vs. 22.2%, p = 0.1842). Ki-67, a nuclear protein expressed in the proliferating cells, is widely considered to be a tumor proliferation marker, and a high Ki-67 labeling index generally indicates a poor clinical prognosis [32]. Of the patients with gliomas, 19 cases underwent Ki-67 histopathological examination. We further explored the correlation between the Ki-67 labeling index (the fraction of Ki-67-positive tumor cells) and the periodontal conditions in patients with gliomas. We found that patients with both AL > 5 mm and PD ≥ 6 mm had a higher ki-67 labeling index than those with AL≤ 5 mm or PD < 6 mm (41.43% vs. 24.25%, p = 0.0449, Figure 6D). This result indicates that glioma cells in patients with poor periodontal status exhibit a higher proliferation capacity. The representative MRI, histological staining, and oral X-ray images of patients with GBM and benign tumors are shown in Figure 7. 45 46 Male  6  6  Glioblastoma  4  50  46  67 Female  6  8  Glioblastoma  4  50  47  56  Male  7  6  Glioblastoma  4  NA  48  55  Male  8  7 Glioblastoma 4 20 * The values of ALmax and PDmax referred to the maximum of PD and AL of all teeth from one patient. ** The specific Ki-67 indexes of two glioma patients were not available.
Next, we investigated whether poor periodontal conditions were associated with glioma malignancy. As shown in Figure 6C, high-grade glioma (WHO grade IV) patients had a slightly higher prevalence of both AL > 5 mm and PD ≥ 6 mm compared to that of patients with lower grade gliomas (58.3% vs. 22.2%, p = 0.1842). Ki-67, a nuclear protein expressed in the proliferating cells, is widely considered to be a tumor proliferation marker, and a high Ki-67 labeling index generally indicates a poor clinical prognosis [32]. Of the patients with gliomas, 19 cases underwent Ki-67 histopathological examination. We further explored the correlation between the Ki-67 labeling index (the fraction of Ki-67-positive tumor cells) and the periodontal conditions in patients with gliomas. We found that patients with both AL > 5 mm and PD ≥ 6 mm had a higher ki-67 labeling index than those with AL≤ 5 mm or PD < 6 mm (41.43% vs. 24.25%, p = 0.0449, Figure 6D). This result indicates that glioma cells in patients with poor periodontal status exhibit a higher proliferation capacity. The representative MRI, histological staining, and oral X-ray images of patients with GBM and benign tumors are shown in Figure 7.

Discussion
Periodontitis has been linked with various systemic diseases, such as cardiovascular disease, diabetes, and multiple cancers [33,34]. Since oral P. gingivalis has been detected in the brains of patients with several brain diseases, the potential correlation between periodontitis and brain disorders has become a hot research topic [35]. A high prevalence of periodontitis has been identified in patients with several types of brain diseases. A case-control study that included 70 patients with intracranial aneurysms demonstrated that 49% of cases exhibited comorbidity with severe periodontitis [17]. Patients with Down's syndrome and bipolar disorders also have an increased risk of suffering from periodontitis [36,37]. In the present study, we found that the patients with glioma had a higher prevalence of poor periodontal condition (the presence of both AL > 5 mm and PD ≥ 6 mm) than those with benign tumors and the aged-matched Chinese population from the national survey. This comorbidity link suggests that periodontitis likely influences glioma development and progression.
Periodontitis is a chronic inflammatory disease characterized as a subgingival complex microbial pathogen infection. These pathogens can penetrate periodontal tissues into the blood vessels, causing transient bacteremia through toothbrushing and dental procedures [26,38]. Several studies have identified the presence of oral bacteria such as Porphyromonas gingivalis in the vessel wall of intracranial aneurysm specimens [39,40]. The presence of P. gingivalis LPS has also been identified in the postmortem brain specimens of AD patients using immunolabeling and immunoblotting [26]. Additionally, the DNA of P. gingivalis was detected in the cerebrospinal fluid of AD patients [16]. Therefore, these results confirm that P. gingivalis, or at least its LPS, can invade the brain tissue via the blood-brain barrier [26]. Considering the comorbidity of periodontitis and glioma, we speculate that oral P. gingivalis infection influences glioma development and progression. We observed that a low concentration (≥10 ng/mL) of P. gingivalis LPS significantly promoted the proliferation of GL261 cells, whereas only a higher concentration (≥100 ng/mL) promoted the migration of GL261 cells.
LPS from P. gingivalis exerts its effects on various cells by stimulating cytokine secretion [41][42][43][44]. Therefore, we also investigated P. gingivalis LPS-induced changes in the production of inflammation and tumor-associated cytokines. We found that exposure to P. gingivalis LPS significantly increased the mRNA levels of TNF-α, TGF-β, MMP-2, and MMP-9 and had no significant effect on the mRNA levels of IL-1β, IL-6, and IL-10. TNF-α can promote glioma invasion and angiogenesis. TGF-β facilitates glioma growth and immunosuppression and stimulates glioma cell migration and angiogenesis [45]. Furthermore, MMPs are of great importance in facilitating cancer cell migration and invasion by modulating the degradation of the basement membranes and the extracellular matrix. In support of our results, P. gingivalis LPS has been shown to upregulate the expression of MMP2 and MMP9 in oral cancer cells [46]. The increased expression of TNF-α, TGF-β, MMP-2, and MMP-9 might explain how the P. gingivalis LPS exerts pro-tumor effects. Notably, the Akt signaling pathway has been widely demonstrated to take part in the regulation of inflammatory processes and tumor growth. Previous studies showed that P. gingivalis LPS promoted cell growth and cytokine secretion via activating the Akt/NF-κB pathway [46][47][48]. In the present study, treatment with 100 ng/mL P. gingivalis LPS significantly activated Akt, while Akt inhibitor IV attenuated the pro-tumor effects of P. gingivalis LPS. These results indicate that LPS exerts the pro-tumor effects via the Akt pathway. However, further studies are necessary to investigate how P. gingivalis LPS activates Akt and whether other signaling pathways are involved.
Oral pathogens and their virulence are reported to generate epigenetic modulations, thus triggering inflammation [29]. In the present study, P. gingivalis LPS attenuated the gene expression of DNMTs, enzymes facilitating and maintaining the DNA methylation. The modulation of DNMTs corroborates with the previous findings [29,31]. The decreased expression of DNMTs may lead a hypomethylation pattern in P. gingivalis LPS-treated cells, while hypomethylation of the genes is closely linked to transcriptional upregulation [49], indicating that DNMTs might be involved in the cytokine upregulations in P. gingivalis LPS-treated cells. Furthermore, the downregulation of TET1 and TET2 following exposure to P. gingivalis LPS was observed. TETs play a crucial role in erasing the aberrant DNA methylation [28], while the aggregation of aberrant DNA methylation was known to promote cancerization [50,51]. Therefore, the decreased TETs tended to indicate a facilitation for cancer development. However, the epigenetic alterations in cancer cells are complex, and the regional hypermethylation (aberrant DNA methylation) coexists with global hypomethylation [52,53]. Hence, the impact of epigenetic modulation reacting to infectious agents needs to be deeply investigated.
Growing evidence has been emerging in recent years concerning the potential role of gut microbiota in the development and function of CNS. This interaction is termed the gut-brain axis [54]. The oral cavity, full of diverse microbes, is anatomically closer to the cerebrum and shares nearly the same vascular system. It is reasonable to speculate that oral microbial infection may also affect brain disorders. However, few studies have focused on this topic, and most have focused on the correlation between oral microbiota and Alzheimer's disease. Whether oral microbiota plays a role in glioma progression remains unclear. However, a case-control study reported that the oral microbiota composition and gene functions are significantly associated with human brain glioma grade [55]. In support of this, our investigation indicates that LPS derived from oral P. gingivalis exhibits pro-tumor effects on glioma progression.
To date, the causal factors of glioma remain unknown. The low five-year survival rate disappoints the patients and their relatives. Therefore, identifying the potential causes or risk factors is pressing. Periodontitis is a common and reversible disease, and intervention of periodontal infection may benefit the prognosis of patients with glioma.

Conclusions
Periodontal infection with P. gingivalis shows pro-tumor effects on glioma progression via LPS-induced Akt activation. Additionally, patients with glioma have a higher prevalence of exacerbated periodontal conditions. Therefore, further understanding of this microbe-brain tumor link may lead to novel strategies for managing gliomas.