Effect of Streptococcus anginosus on biological response of tongue squamous cell carcinoma cells

Abstract

Background: Streptococcus anginosus(S.anginosus) was reported increased in oral squamous cell carcinoma(OSCC) tissue. The aim of this study was to investigate the response of oral cancer cells in the biological characteristics evoked by the S.anginosus and investigate its potential mechanisms.
Methods: The growth curve and concentration standard curve of S.anginosus were determined , and a series of concentrations of S.anginosus supernatant were applied to OSCC cell lines SCC15,then selected an optimal time and concentration by CCK-8 assay. Then autophagic response, proliferative activity, cell cycle and apoptosis, invasion and migration abilities were evaluated in SCC15.
Results: The results showed that when the ratio of S.anginosus supernatant to cell culture medium was 1:1 and the co-culture time was 16h, the inhibitory effect on SCC15 was the most obvious; Furthermore, the supernatant of Streptococcus upregulated the autophagy activity of SCC15, thus significantly inhibiting its proliferation, migration and invasion ability. Compared with control groups, the cell cycle showed G1 arrest, S and G2 / M phases decreased, and the percentage of apoptotic cells relatively increased(P<0.05).
Conclusion: S.anginosus reduced the proliferation, migration and invasion of SCC15 cells and promoted cell apoptosis; Moreover, autophagy may be one of the mechanisms in this process.

Full text

Page 1/14
Effect of Streptococcus anginosus on biological
response of tongue squamous cell carcinoma cells
Yuan Xu
Chongqing Medical University Stomatology College
Yuhuan Jia
Chongqing Medical University Stomatology College
Liang Chen
Chongqing Medical University Stomatology College
Jing Gao
Chongqing Medical University Stomatology College
Deqin Yang (  yangdeqin@hospital.cqmu.edu.cn )
Chongqing Medical University Stomatology College https://orcid.org/0000-0002-3711-850X
Research article
Keywords: Streptococcus anginosus, oral squamous cell carcinoma, autophagy, proliferation, apoptosis
DOI: https://doi.org/10.21203/rs.3.rs-19115/v4
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

Page 2/14
Abstract
Background:
Streptococcus anginosus
(
S.anginosus
)
was reported increased in oral squamous cell
carcinoma(OSCC) tissue. The aim of this study was to investigate the response of oral cancer cells in the
biological characteristics evoked by the
S.anginosus and investigate its potential mechanisms.
Methods: The growth curve and concentration standard curve of
S.anginosus
were determined , and a
series of concentrations of
S.anginosus
supernatantwere applied to OSCC cell lines SCC15,then selected
an optimal time and concentration by CCK-8 assay. Then autophagic response, proliferative activity, cell
cycle and apoptosis, invasion and migration abilities were evaluated in SCC15.
Results: The results showed that when the ratio of S.anginosus supernatant to cell culture medium was
1:1 and the co-culture time was 16h, the inhibitory effect on SCC15 was the most obvious; Furthermore,
the supernatant of
Streptococcus
upregulated the autophagy activity of SCC15, thus signicantly
inhibiting its proliferation, migration and invasion ability. Compared with control groups, the cell cycle
showed G1 arrest, S and G2 / M phases decreased, and the percentage of apoptotic cells relatively
increased(P<0.05).
Conclusion:
S.anginosus
reduced the proliferation, migration and invasion of SCC15 cells and promoted
cell apoptosis; Moreover, autophagy may be one of the mechanisms in this process.
Background
In the 1990s, the researchers found that there may be a relationship existed between microbes and
tumors [1]. Studies have showed that the primary cause of carcinogenesis was the changes in cellular
metabolism [2]. Microorganisms participate in a series of metabolic activities of the host, and the
metabolites or co-metabolites(produced by the interaction between the host and the microorganisms) of
microorganisms will affect the proliferation and apoptosis of tissue cells and the growth and metastasis
of cells in the tumor microenvironment [3,4]. Currently, the application status of microorganisms in cancer
treatment includes: as a natural medicine database,bacteria provide various active substances with anti-
tumor properties; As a delivery vehicle, bacteria specically target drugs to tumor cellsAs a therapeutic
agentbacteria combining with chemoradiotherapy and immunotherapy synergistically promote the
ecacy of traditional anti-tumor therapy [5].
The human digestive ora is the second set of human genomes, which inhabits a large number of
microorganisms, and the number of which is 10 times the number of their own cells. The micro-ecological
system composed of these bacteria maintains the stability and balance of the microbial community
under their interaction. In recent years, with the development of technologies like metagenomicsand high-
throughput sequencing, research on microbes and human health and diseases has gradually
started.More and more studies have found that in some digestive tract tumors, the composition of the
ora has changed signicantly, and some bacteria have increased or decreased in
specicity.Helicobacter pylori has been identied as an independent risk factor for gastric cancer, while

Page 3/14
studies have also reported that benecial bacteria such as
bidobacteria
can inhibit the occurrence and
development in gastric tumors.Therefore, scholars have proposed that microbes could be regarded as a
double-edged sword in tumors. The metabolism of cancer cells will become a promising therapeutic
target, and the role of microorganisms in cancer treatment is becoming more and more important [6].
  The oral cavity is not only the initiation site of digestive tract but also the predilection site of head and
neck malignant tumors, and 90% of oral cancers are squamous cell carcinoma. Studies have found that
patients with oral squamous cell carcinoma(OSCC) have some signicant changes in the microbial
diversity of cancer tissues and saliva compared with healthy peoplesuch as
Streptococcus,
Porphyromonas, fusobacterium, Veillonella, Actinomyces, Enterobacter
and so on [7,8,9].
S.anginosus
is a
commensal bacterium in the oral cavity, gastrointestinal tract and genitourinary tract. Previous studies
have shown that the detection rate of this strain in esophageal cancer and oral squamous cell carcinoma
is signicantly increased and the pathogenicity in esophageal cancer has been clear [10,11,12]. The oral
cavity connects with esophagus, some researchers considered that this kind of bacterium may involve in
oral squamous cell carcinoma [13,14]. A previous study on the cancer tissues of different sites in oral
cavity estimated that the total detection rate of
S.anginosus
DNA was as high as 88%, and each part was
87% of tongue, 90% of mouth, 83% of cheek, 100% of gums, and this rate was found much less
frequently in healthy people[15].
  In this study, we estimated the changes of SCC15 cells fate by
S.anginosus
and explored the potential
mechanism that reveal the impact of this bacterium on the biological characteristics of oral cancer cells.
Methods
2.1 Baterial growth curve and concentration standard curve
S.anginosus
strain ATCC33397supported by the State Key Laboratory of Oral Disease, Sichuan
Universitywas cultured aerobically at 37℃in THB mediumSolarbioChinaand measure the
absorbance every 2 hours. Then the relationship between concentration of bacteria solution and
absorbance value was obtained byserial dilution plate counting method.
2.2 Cell culture
The oral tongue squamous cell carcinoma cells SCC15 (supported by Chongqing Key Laboratory of Oral
Diseases and Biomedical Sciences)were cultured in DMEM/F12 with 10% fetal bovine serum(Gibco, USA)
containing 100U/ml penicillin and 100 μg/ml streptomycin at 37℃and 5% CO2.
2.3 Co-culture time and concentration
The
S.anginosus
supernatant in stable phases was used for the experiment ,treating with centrifugation
at 4000rpm/min for 20 minutes and ltering twice with 0.22μm lters. Then, the treated supernatant was
called ltrate S and the bacterial medium THB was treated in the same manner as a control, called ltrate

Page 4/14
T. The Subsequent experiments were carried out with ltrate S and ltrate T. A series of concentrations of
ltrate S were applied to SCC15, then an optimal co-culture time and concentration were obtained by CCK-
8 assay.
2.4 Experimental groups and manipulating approaches
Methods to set up SCC15/
S.anginosus
(SCC15/ S) experimental group, SCC15/THB (SCC15/T)
conditional control group, SCC15/S+3-MA conditional control group, SCC15/3-MA negative control group
and SCC15 blank control group.Meanwhile, SCC15 group did not do any treatment, while SCC15/S,
SCC15/T and SCC15/3-MA acted with ltrate S, ltrate T and 3-Methyladenine (3-MA) for 16h
respectively; SCC15/S+3-MA pretreated with 3-MA for 4 hours and then infected with ltrate S for 16h.
2.5Autophagic response measurement
2.5.1 Monodansylcadaverine staining
Monodansylcadaverine(MDC)staining was an independent method to evaluate autophagy. SCC15 cells
were seeded in confocal dishes. After each group was treated accordingly,10μLMDC stain was added and
mixed, and incubated at 37℃for 20 minutesaway from light; Then washed with 300μL 1X wash buffer
twice. Pictures were obtained with the confocal microscope (Thermo, USA).
2.5.2 Real time PCR analysis
Total RNA was isolated from SCC15 cells using Trizol method (Beyotime, China). cDNA was synthesized
by qScript cDNA synthesis kit (Sigma, USA) and primers synthesized by Wuhan Sevier Biotechnology Co.,
Ltd. The sequences are shown in Table 1. Quantitative gene expression was performed for Beclin1, LC3
using one-step RT-PCR kit (Takara, Japan). Expression values were normalized to GAPDH.
2.5.3 Western blot analysis
The cell samples were collected and lysed in RIPA with PMSF (Beyotime, China). Then, protein
concentration was mearsured by BCA protein kit (Beyotime, China). The cell lysates were separated by
10%SDS–PAGE and transferred to the 0.22um PVDF membranes. The bands were detected by enhanced
chemiluminescence (ECL) after the primary antibody at 4℃for a night and second antibody at room
temperature for 1 hour. The antibodies used in this study include: anti-LC3, anti-Beclin1 (Cell Signaling,
USA) and GAPDH (Beyotime, China).
2.6 Cell proliferation analysis
Cells were seeded in a 96-well plate at a density of 5×103/well and incubated at 37℃for 24h ,then each
group was treated accordingly. After that,10μL CCK-8/well with 100 μL DMEM/F12 was added and
incubated at 37℃for 4h. Absorbance was determined at 450nm with a microplate reader.
2.7 Cell cycle and apoptosis analysis

Page 5/14
The cells were digested with trypsin and washed with cold PBS, then xed overnight in 70% ethanol at
-4℃. Ethanol-xed cells were collected and washed with PBS, then cell counting. 106cells of each group
were centrifuged and 0.5 mL PI/RNase stain was added to cell pellets. After 30 minutes at room
temperature in the dark, the cell cycle was analyzed with ow cytometre with a 488nm argon laser.
Cell culture medium of each group was collected respectively and Cells were trypsinized with EDTA-free
trypsin. Then washed cells with cold PBS twice and cell counting. 106cells of each group were
centrifuged and 400 μL 1x AnnexinV binding buffer was added to resuspend cells. Afterwards, added 5μL
FITC and 10 μL PI per group for incubation at -4℃in the dark for 15minutes and 5minutes respectively.
2.8 Cell migration and invasion assay
The cell migration was measured by wound healing assay.Scratched a line with pipette tip in the middle
of each well and washed with PBS 3 times slightly. Then 2 mL of serum-free medium was added to each
well and incubation at 37 ° C for 24 hours. The pictures of each group were recorded at 0h, 6h, and 12h
under microscopy at 40 magnications.
The cell invasion was measured by the transwell system (BD, USA). Treated cells were inoculated in the
upper chamber with free-FBS medium, and F12 culture medium containing 10% FBS was added to the
lower chamber. Additionally, an insert covered with Matrigel was used for invasion measurements. After
24 hours, cells migrated to the opposite side of the insert were stained with DAPI and quantied.
2.9 Statistical analysis
Data analysis was performed by SPSS 25.0 software. All values are calculated and expressed as mean ±
standard deviation (SD). Anaylsis of VarianceANOVAand SNK-q test were used to compare between the
groups. P-value<0.05 was considered statistically signicant.
Results
3.1 S.anginosus
growth curve and concentration standard curve were detected and co-culture time and
concentration were determined
The growth of
S.anginosus
is consistent with the general rule which contains four phases, including: 0h-
4h lag phase, 4h-16h log phase, 16h-24h stable phase, and 24h later as the decay phase(Fig.S1). The
stable phase 20h bacterial solution was selected for experiment because the amount of primary and
secondary metabolites of bacteria will reach the maximum in this period. Then, according to the
concentration standard curve (Fig.S2), the relationship between the concentration and the absorbance
value can be obtained. The bacterial solution at 20h was adjusted to the concentration corresponding to
the growth curve to ensure the same concentration in each test. Furthermore, the results of CCK-8 assay
showed that when a 1:1 relationship between ltrate S and cell culture medium (Fig.S3) and co-cultured

Page 6/14
time was 16 hours (Fig.S4), the effect of inhibition to cells was the most obvious. Therefore, this
concentration and co-cultured time were used for subsequent experiments.
3.2 S.anginosus
up-regulated autophagy activity of SCC15
Autophagy is a physiological compensation process to maintain the homeostasis of eukaryotic cells. It
can not only exert tumor suppressive effects, but also help tumor cells escape the body's metabolism. A
recent study by Chen et al. has supposed that autophagy may inhibited the proliferation of cells [16]. To
investigate whether autophagy participates in the action of
S.anginosus
on SCC15 cells, we rst
established a model system as the negative group by blocking the autophagy pathway with 3-MA ,in
which group autophagy activity was inhibited obviously. Vital staining was performed with MDC dyes,
which a specic uorescent marker for autophagic vacuoles showed that compared with SCC15 group,
the number of autophagosomes was the highest in SCC15/S group, SCC15/S+3-MA group and SCC15/T
group were decreased in turn, and SCC15/3-MA group was the least (Fig.1A). What’s more, the amount of
LC3I protein conversion to LC3II protein has also proved to be well correlated with the degree of
autophagy [17]. We found that the mRNA expression of Beclin1 and LC3 were eciently higher in
SCC15/S group than other groups (p < 0.05) and signicantly decreased in SCC15/3-MA (p < 0.05)
(Fig.1B). Meanwhile, the expression of autophagy-related protein Beclin1 was signicantly enhanced in
SCC15/S group and protein LC3 type II increased, type I decreased, showing the transformation from type
II to type I. while these two proteins Beclin1 and LC3II were both decreased in SCC15/3-MA group
(Fig.1C).
3.3 S.anginosus
inhibits the proliferation and promotes apoptosis of SCC15
We evaluated the proliferative activity of SCC15 cells from 0 to 36 hours by CCK-8 assay. The results
indicated that the SCC15 cells exhibited reduced proliferation after incubation with ltrate S while
SCC15/3-MA group revealed slightly increased (P<0.05). Meanwhile, there was no signicant difference
between SCC15/T group and SCC15/S+3-MA group compared with the blank control group(Fig.2A).
When ow cytometry was used to analyze cell cycle, the results showed that
S.anginosus
abundant
accumulation of SCC15 cells in the G1 phase(Fig.2B). In addition, the number of apoptotic cells in
SCC15/S group was signicantly higher than that in the four control groups(Fig.2C), and the difference
was signicant(P<0.05).
3.4 S.anginosus
was involved in autophagy-mediated inhibition of migration and invasion
The results of wound healing assay showed that the healing area of SCC15/S group was lower than that
of all control groups (P<0.05, Fig.3A), indicating that the mobility of SCC15/S group was decrease
signicantly and highly suggesting that
S.anginosus
was a key factor in the decline of SCC15 cell
motility.
To further evaluate the function of metabolic products of
S.anginosus
on SCC15 cells invasive ability,
transwell chambers coated with matrigel were used. We found that ltrate S of
S.anginosus
resulted in a

Page 7/14
signicant decrease in invasivity compared with control groups(P<0.05). However, opposite result was
dramatically observed when autophagy was inhibited (p < 0.05, Fig.3B).
Discussion
The large number of microbiome in the body affects the susceptibility to cancer, partly because its
metabolites or co-metabolites have important effects on the function of immune cells.Studies report that
15 to 20 percent of cancer cases are associated with microbial infections [18], but the role of
microorganisms in tumor genesis and development is still controversial today.There were some
highlighted questions about whether oral microbiome changes are an important risk factor for oral cancer
development [19]. According to the results, patients with oral leukoplakia (OLK), a lesion with malignant
potential, were more enriched with
Fusobacteria
compared to normal tissue from the same patients [20].
In addition, the opinion that microbiome changes preceded the malignant transformation process was
conrmed, supporting the possible role of microbiome changes in the pathogenesis of disease [9].
Porphyromonas gingivalis
is one of the most studied oral microorganisms in vivo and in vitro. Studies
havefound that continuous stimulation of normal epithelial cells by this kind of bacteria can lead to
tumour-like changes in cells [21]. However, the reverse effect was reported in other study [22]. Some
researchers have summarized the role of oral microbiota in cancer development.The results suggested
that among
streptococci,
S.anginosus
seems to be an especially relevant marker of head, neck, and
esophageal cancers and more common in oral squamous cell carcinoma.They also pointed out three
mechanisms of oral microbiota in cancer pathogenesis. The rst is bacterial stimulation of chronic
inammation.The second is activation of NF-κB and inhibition of cellular apoptosisAnd the third is the
carcinogens produced by bacteria[23].
S.anginosus
,as a human symbiotic bacterium,was rst proposed in 1983 that it may be involved in the
occurrence and development of oral infectious diseases [14].In recent years, many relevant studies have
claimed that the detection rate of this bacterium in OSCC tissues has increased [24], but its actual effect
and mechanism on tumor cells have not been reported. SASAKI et al. isolated and puried a new
bioactive antigen SAA (S.anginosus antigen) from S.anginosus supernatant. They found that SAA
stimulated macrophages to produce high concentrations ofnitric oxide (NO) and various inammatory
factors. NO interacted with O2 or O2- to form reactive nitrogen species (RNS), which leads to a DNA
damage response by DNA base oxidation and nitration.In addition, the β-hemolysin of S.anginosus and
the streptolysin S(SLS) of Streptococcus pyogenes are homologous seriesencoded by a similar gene
cluster;SLS is a highly toxic cytolysin, which help bacteria to cross the epithelial barrier with a tissue
damage and also can against the immune clearance of hosts [25].Based on the reported toxic effects of
metabolites of this bacteria, we selected S.anginosus supernatant of stable period for treatment and
retained the metabolites in the supernatant for related experiments.
Autophagy is a physiological compensation processwhich cell removes damaged proteins and
organelles tomaintain its homeostasis [26]. Stress reactions such as starvation, hypoxia and microbial

Page 8/14
infection can stimulate autophagy. Many literatures have stated the relationship between tumor,
microorganism and autophagy [27,28,29].However, it is not clear whether the decrease in autophagy
activity observed in malignant cells is mechanically signicant or merely incidental to the progression of
malignancy. Invasion and metastasis are the critical markers in the development of cancer, and active cell
migration plays an essential role in the invasion and metastasis cascade of cancers. Our study conrmed
that
S.anginosus
reduced the proliferation, migration and invasion of SCC15 cells and promoted cell
apoptosis; Furthermore, it has been found that Beclin1 located in the cytoplasmic endoplasmic reticulum
is involved in the formation of autophagic vesicles and also an important factor in inducing autophagic
death of tumor cells.Structurally, Beclin1 has the BH3 region that constitutes the apoptotic bcl-2 protein
family, and it is a specic receptor of apoptosis, so it plays an important role in promoting apoptosis [30].
In this study, MDC staining, qPCR and western blot results also conrmed increased autophagy activity
and up-regulated expression of Beclin1 during this process, which was consistent with our results of
increased apoptosis rate.
Picardo SL et al. suggested that microbial regulation may affect the course of the disease [31]. In patients
with advanced cancer treated with immunotherapy, resistance is associated with microbiome
abnormalities and antibiotic treatment. There is certainly evidence that the gut ora plays a key role in the
response of cancer patients to chemoradiotherapy and immunotherapy.Microbiota transplantation (MT),
including fecal microbiota transplantation (FMT) and selective microbiota transplantation (SMT), may
improve the effect of anti-cancer treatment and/or reduce the related side effects [32].Viaud et al.
demonstrated that cyclophosphamide causes discontinuity of the intestinal barrier and subsequently
promotes selective transfer of specic Gram-positive bacteria to secondary lymphoid organs.These
transplanted bacteria can enhance the anti-cancer adaptive immune response of T cells [33].
A new study indicates that the composition of the patient's intestinal ora is an important factor in
regulating the host's response to anti-PD-1 / PD-L1 or anti-CTLA-4 immunotherapy [34]. In addition, the
view that regulation of the microbiome can be used in cancer treatment was proposed [35]. The study in a
mouse model of colonic carcinogenesis showed that oral intake of probiotics containing lactobacilli can
reduce IL-17-producing T cells and inhibit proliferation and tumor formation, which may be achieved by
changing the gut microbiome. National Institute of Health reconstructed the laboratory mice with natural
“wild-type” microbiota, and increased resistance to mutagen and inammation-induced colorectal
tumorigenesis were found [36].Wang et al. observed that
S. anginosus
stimulated peripheral blood of
OSCC patients and healthy people, and found that CD8 T cells were signicantly higher in OSCC patients,
proving that Streptococcus-reactive CD8 T cell responses might contribute to antitumor immunity in
OSCC patients [37].
Above all, microbiome profoundly affects immune development and carcinogenesis. microbiome-
modulating agents are poised to become bona de anti-cancer strategies: immunotherapy.
Immunotherapy derives from the recognition of the synergy between host and microbe. In 1850, several
German physicians found that some cancer patients with active infections showed signs of tumor
regression. In 1900, Coley test bacterial extracted on patients with bone cancer which was one of the rst

Page 9/14
immunotherapies [38]. For the past three decades, several bacteria-based cancer treatments have
emerged, and bacterial vaccines expressing tumor antigens have been shown to be effective in preclinical
studies. Bacillus Calmette-Guerin (BCG) was used in the treatment of non-muscular invasive bladder
cancer, where directly transmitted live bacteria enter the bladder, causing inammation and triggering an
anti-tumor immune response [39]. Synthetic biology approaches to cancer care hold enormous potential,
especially those that make use of bacteria. These methods involve reengineering of bacterial cells for the
delivery biomolecules in host reactions. The concept that microorganisms can invade cancer cells to
target and disrupt critical cancer pathways has been demonstrated. The next step will be to use robust
preclinical models for evaluation.
Oral microora is composed of many microorganisms, among which there are complex
interrelationships. In our study, the outcome of the effect of other microorganisms on the interaction of
S.
anginosus
and the exact clinical role of
S. anginosus
are not clear. Our experiment is only an initial
exploratory experiment. So, the interaction of
S. anginosus
with more OSCC cell line and normal cells and
vivo research should be veried by further experiments.
Conclusions
Our study showed the inhibitory effect of
S.anginosus
on cancer cells. We believe that the results of such
studies, including the present ones, could provide an entry point for future studies of cellular interactions
between
bacteria
and cancer. In addition, future studies should be designed to elucidate the impacts of
S.anginosus
on the chemotherapeutic responses of oral cancer cells, of which outcomes will have a
signicant impact on the clinical treatment of oral cancer.
Abbreviations
S.anginosus
:
Streptococcus anginosus;
OSCC : Oral squamous cell carcinoma;
3-MA : 3-Methyladenine ;
MDC: Monodansylcadaverine;
ANOVA: Anaylsis of Variance;
OLK: Oral leukoplakia;
SAA: Streptococcus anginosus antigen;
NO: NItric oxide;
RNS: Reactive nitrogen species;

Page 10/14
SLS: streptolysin S;
MT: Microbiota transplantation;
FMT: Fecal microbiota transplantation;
SMT: Selective microbiota transplantation;
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable
request.
Competing interests
The authors declare that they have no competing interests
Funding
This work was funded by National Natural Science Foundation of China (No. 31371473 and
No.31571508 to Dq.Y, No.81700958 to L.C.) The National Natural Science Foundation had no role in
design of the study, the collection, analysis, and interpretation of data and writing the manuscript. It
supported the funding of the study only.
Authors’ contributions
YX designed and conducted the experiment, YHJLC and JG performed the literature review and
experimental analysis and DQY supervised the work and reviewed the manuscript, and revised the nal
manuscript as submitted. YX drafted the manuscript. All the authors have read and approved the nal
manuscript.
Acknowledgments
Not applicable.

Page 11/14
References
1. Allavena P, Garlanda C, Borrello MG, Sica A, Mantovani A. Pathways connecting inammation and
cancer. Curr Opin Genet Dev. 2008;18(1):3–10.
2. WARBURG O. On the origin of cancer cells. Science. 1956;123(3191):309–314.
3. Louis P, Hold GL, Flint HJ. The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev
Microbiol. 2014;12(10):661–672.
4. Guglielmi G. How gut microbes are joining the ght against cancer. Nature. 2018;557(7706):482–
484.
5. Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski TF. The microbiome in cancer immunotherapy:
Diagnostic tools and therapeutic strategies. Science. 2018;359(6382):1366–1370.
6. Sullivan LB, Gui DY, Vander Heiden MG. Altered metabolite levels in cancer: implications for tumour
biology and cancer therapy. Nat Rev Cancer. 2016;16(11):680–693.
7. Krogh P, Hald B, Holmstrup P. Possible mycological etiology of oral mucosal cancer: catalytic
potential of infecting Candida albicans and other yeasts in production of N-
nitrosobenzylmethylamine. Carcinogenesis. 1987;8(10):1543–1548.
8. Nagy KN, Sonkodi I, Szöke I, Nagy E, Newman HN. The microora associated with human oral
carcinomas. Oral Oncol. 1998;34(4):304–308.
9. Schmidt BL, Kuczynski J, Bhattacharya A,Huey B, Corby PM,Queiroz EL,et al. Changes in abundance
of oral microbiota associated with oral cancer [published correction appears in PLoS One.
2014;9(8):e106297.
10. Sasaki H, Ishizuka T, Muto M, Nezu M, Nakanishi Y, Inagakl Y, et al. Presence of Streptococcus
anginosus DNA in esophageal cancer, dysplasia of esophagus, and gastric cancer. Cancer Res.
1998;58(14):2991–2995.
11. Morita E, Narikiyo M, Nishimura E, Yano A, Tanabe C, Sasaki H, Tajika S, Kodama Y, Ohya T, et al.
Molecular analysis of age-related changes of Streptococcus anginosus group and Streptococcus
mitis in saliva. Oral Microbiol Immunol. 2004;19(6):386–389.
12. Sasaki M, Yamaura C, Ohara-Nemoto Y, Tajika S, Kodama Y, Ohya T, et al. Streptococcus anginosus
infection in oral cancer and its infection route. Oral Dis. 2005;11(3):151–156.
13. Hamada S, Slade H D. Biology, immunology, and cariogenicity of Streptococcus mutans.
Microbiological Reviews. 1980; 44(2):331-384.
14. Crawford I, Russell C. Streptococci isolated from the bloodstream and gingival crevice of man. J Med
Microbiol. 1983;16(3):263–269.
15. Tateda M, Shiga K, Saijo S, Michiyo S, Tetsuya H, Junkichi Y, et al. Streptococcus anginosus in head
and neck squamous cell carcinoma: implication in carcinogenesis. Int J Mol Med. 2000;6(6):699–
703.
16. Chen N, Eritja N, Lock R, Debnath J. Autophagy restricts proliferation driven by oncogenic
phosphatidylinositol 3-kinase in three-dimensional culture. Oncogene. 2013;32(20):2543–2554.

Page 12/14
17. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 2007;3(6):542–545.
18. Schwabe R F, Jobin C. The microbiome and cancer. Nature Reviews Cancer. 2013; 13(11):800-812.
19. Healy CM, Moran GP. The microbiome and oral cancer: More questions than answers. Oral Oncol.
2019;89:30–33.
20. Amer A, Galvin S, Healy CM, Moran GP. The Microbiome of Potentially Malignant Oral Leukoplakia
Exhibits Enrichment for Fusobacterium, Leptotrichia, Campylobacter, and Rothia Species. Front
Microbiol. 2017;8:2391.
21. Geng F, Liu J, Guo Y, Li C, Wang Hy, Zhao Hj, et al. Persistent Exposure to Porphyromonas gingivalis
Promotes Proliferative and Invasion Capabilities, and Tumorigenic Properties of Human
Immortalized Oral Epithelial Cells. Front Cell Infect Microbiol. 2017;7:57.
22. Cho TJ, Wee SW, Woo VH, Choi JI, Kim SJ, Shin HI, et al. Porphyromonas gingivalis-induced
autophagy suppresses cell proliferation through G1 arrest in oral cancer cells. Arch Oral Biol.
2014;59(4):370–378.
23. Karpiński TM. Role of Oral Microbiota in Cancer Development. Microorganisms. 2019;7(1):20.
24. Sasaki M, Ohara-Nemoto Y, Tajika S, Kobayashi M, Yamaura C, Kimura S. Antigenic characterisation
of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal
exudate cells. J Med Microbiol. 2001;50(11):952–958.
25. Asam D, Mauerer S, Spellerberg B. Streptolysin S of Streptococcus anginosus exhibits broad-range
hemolytic activity. Med Microbiol Immunol. 2015;204(2):227–237.
26. Yoshimori T. Autophagy: a regulated bulk degradation process inside cells. Biochem Biophys Res
Commun. 2004;313(2):453–458.
27. Cosway B, Lovat P. The role of autophagy in squamous cell carcinoma of the head and neck. Oral
Oncol. 2016;54:1–6.
28. Castrejón-Jiménez NS, Leyva-Paredes K, Hernández-González JC, Luna-Herrera J, García-Pérez BE.
The role of autophagy in bacterial infections. Biosci Trends. 2015;9(3):149–159.
29. Garrett WS. Cancer and the microbiota. Science. 2015;348(6230):80–86.
30. Weng J, Wang C, Wang Y, Tang HK, Liang JF, Liu XQ, et al. Beclin1 inhibits proliferation, migration
and invasion in tongue squamous cell carcinoma cell lines. Oral Oncol. 2014;50(10):983–990.
31. Picardo SL, Coburn B, Hansen AR. The microbiome and cancer for clinicians. Crit Rev Oncol Hematol.
2019;141:1–12.
32. Wu X, Zhang T, Chen X, Ji G, Zhang F. Microbiota transplantation: Targeting cancer treatment. Cancer
Lett. 2019;452:144–151.
33. Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillere R, Hannani D, et al. The intestinal microbiota
modulates the anticancer immune effects of cyclophosphamide. Science. 2013;342(6161):971–976.
34. Kroemer G, Zitvogel L. Cancer immunotherapy in 2017: The breakthrough of the microbiota. Nat Rev
Immunol. 2018;18(2):87–88.

Page 13/14
35. Rong J, Liu S, Hu C, Liu C. Single probiotic supplement suppresses colitis-associated colorectal
tumorigenesis by modulating inammatory development and microbial homeostasis. J
Gastroenterol Hepatol. 2019;34(7):1182–1192.
36. Rosshart SP, Vassallo BG, Angeletti D, Hutchinson DS, Morgan AP, Takeda K; et al. Wild Mouse Gut
Microbiota Promotes Host Fitness and Improves Disease Resistance. Cell. 2017;171(5):1015–
1028.e13.
37. Wang J, Sun F, Lin X, Li Z, Mao X, Jiang C. Cytotoxic T cell responses to Streptococcus are
associated with improved prognosis of oral squamous cell carcinoma. Exp Cell Res.
2018;362(1):203–208.
38. Mellman I, Coukos G, Dranoff G. Cancer Immunotherapy Comes of Age. Nature.2011; 480: 480–489.
39. Ingersoll MA, Albert ML. From infection to immunotherapy: host immune responses to bacteria at the
bladder mucosa. Mucosal Immunol. 2013; 6:1041–1053.
Table
Due to technical limitations, Table 1 is only available for download from the Supplementary Files section.
Figures
Figure 2

Page 14/14
S.anginosus promotes apoptosis of SCC15 and inhibits the proliferation through a cell cycle arrest at the
G1/S transition. A, CCK-8 assay was performed to test cell proliferation of SCC15 cells. B, The cell cycle
distributions of SCC15 cells were analyzed with ow cytometry. C, Cell apoptosis rate was measured by
ow cytometry. Data were representative of three independent experiments and shown as mean ± SD, *: P
< 0.05, **: P< 0.01 as versus blank control group.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
SupplementaryFigures2.tif
SupplementaryFigures4.tif