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Zymosan promotes proliferation, Candida albicans adhesion and IL-1β production of oral squamous cell carcinoma in vitro

Zymosan promotes proliferation, Candida albicans adhesion and IL-1β production of oral squamous... Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC), and the effect of zymosan (ZYM), a component of the yeast cell wall, on oral cancer remains unclear. The CCK-8 proliferation assay was performed to evaluate the effect of ZYM on the proliferation of the OSCC cell lines WSU-HN4, WSU-HN6 and CAL27, and the potential mechanism was explored by quantitative real-time PCR, immunofluorescence assay and western blot. A cell adhesion assay was conducted to determine the adhesion of Candida albicans to OSCC cells, and the expression of related genes, including TLR2, MyD88, NLRP3, ASC, Caspase-1 and IL-1β, and proteins, including TLR2, MyD88, NF-κBp65, p-NF-κB p65 and E-cadherin was determined. Additionally, the pro-inflammatory cytokines including IL-6, IL-8, TNF-α and IL-1β produced by OSCC cells were detected using a chemiluminescence immunoassay (CLIA). In the current study, the CCK-8 assay showed that ZYM promoted the proliferation of WSU-HN4, WSU-HN6 and CAL27 cells via the TLR2/MyD88 pathway. The cell adhesion assay showed that the number of C. albicans cells per field significantly increased in ZYM-treated OSCC cells compared to controls. When treated with ZYM, OSCC cells secreted significantly more pro-inflammatory cytokine IL-1β, which could enhance inflammation in oral cancer microenvironment. In conclusion, ZYM from the fungal cell wall promotes the proliferation, C. albicans adhesion and IL-1β production in OSCC, as demonstrated by in vitro experiments. Keywords: Zymosan, Candida albicans, Proliferation, Oral cancer, Oral squamous cell carcinoma Introduction metastasis and local recurrence [2]. Recently, imbalanced Oral squamous cell carcinoma (OSCC) is the most com- microbiota or specific microbes have been found to play mon type of head and neck squamous cell carcinoma an indispensable role in tumour initiation, progression (HNSCC), which is the sixth leading cancer type world- and even chemoresistance [3–6]. For example, Helicobac- wide [1]. The most common treatment for oral cancers ter pylori plays a role in inducing or promoting gastric involves radical surgery and chemotherapy, including 5- cancer [7], and different microorganisms, such as Fuso- fluorouracil and cisplatin. However, the 5-year survival bacterium sp., Porphyromonas gingivalis and Candida rate among patients with OSCC is low because of the albicans, have been reported in oral cancers [8–10]. poor prognosis, such as the occurrence of lymph node It has been observed that oral carriage of C. albicans is higher in patients with OSCC or leukoplakia than in those without oral pathology [11, 12], but the association be- * Correspondence: chenfx@sjtu.edu.cn Xu Chen and Qingqiong Luo contributed equally to this work. tween C. albicans and oral cancer remains ambiguous. Department of Clinical Immunology, Shanghai Ninth People’s Hospital, Several reports have proposed different potential pathways Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai by which C. albicans initiates or promotes carcinogenesis, 200011, China Faculty of Medical Laboratory Science, Shanghai Jiao Tong University School such as inducing inflammation [13, 14]. Triggering of of Medicine (SJTUSM), Shanghai 200025, China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 2 of 8 inflammation by inducing TNF-α and IL-8 or production Cell adhesion assay of carcinogenic by-products such as nitrosamine and N- OSCC cell suspension was seeded onto a coverslip nitrosobenzylmethylamine (NBMA) by C. albicans could placed in a 24-well plate, which was disinfected by UV be a risk factor for cancer [15]. Therefore, it is important light, and the cells were incubated for 24 h to attach to to investigate the interaction between microorganisms coverslip, and then stimulated by zymosan or PBS for an and host cells. The adhesion of microorganisms to host additional 24 h. The cells were washed with PBS before cells has been reported to be the first step for bacteria or adding C. albicans suspension in PBS, and were further fungi to influence host cells [8]. However, the adhesion of incubated at 37 °C with 5% CO for 1 h. After incuba- C. albicans to OSCC cells remains unclear. tion, the DMEM with unattached yeast was aspirated, Toll-like receptors (TLRs) and other pattern recognition and each well was washed with PBS and then fixed with receptors (PRRs) play a significant role in the recognition 95% ethanol for 1 h. After that, the coverslip was of pathogens and triggering of innate immune responses, removed for Gram staining. and a group of molecules can act as pathogen-associated molecular patterns (PAMPs), such as glucans, which are Cytokine detection recognized by PRRs [16]. Moreover, the OSCC cells have OSCC cells were seeded into a 12-well plate before treat- been found to express different TLRs, including TLR3 and ment with PBS or zymosan, and then supernatants were TLR4 [17]. Activation of TLRs usually induces recruit- collected to measure the concentrations of IL-1β, IL-6, IL- ment of the adapter molecule myeloid differentiation 8 and TNF-α. These cytokines were measured by chemilu- primary response protein 88 (MyD88), and activation of minescence immunoassay (CLIA) using IMMULITE®1000 TLR/MyD88 typically leads to the activation of NF-κB, (SIEMENS, Germany) with commercial reagents accord- which has been linked to the progression of tumours [18]. ing to the manufacturer’s instructions. However, the role of TLR expressed on OSCC cells in the interaction with microbial components needs to be inves- Quantitative real-time PCR tigated. This study aimed to define the effect of zymosan Total RNA was extracted using TRIzol reagent (Invitro- (a component of the fungal cell wall, ZYM), which is a gen, San Diego, CA, USA), and real-time PCR amplifica- glucan with repeating glucose units connected by β-1,3- tion was carried out by a two-step reaction. First, cDNA glycosidic linkages, on oral cancer by investigating the was synthesized by GeneAmp® PCR system 9700 proliferation and cytokine production of OSCC cells and (Applied Biosystems) with PrimeScript™ RT Reagent kit C. albicans adhesion to cell lines. (TaKaRa, Shiga, Japan), and then real-time PCR was performed in the 7500 system (Applied Biosystems) with SYBR Premix Ex Taq II (TaKaRa). The experiment was Materials and methods repeated in triplicate on independent occasions. The Cell lines and reagents GAPDH gene was used for the normalization of gene ex- In this study, the human OSCC cell lines WSU-HN4, pression, and the relative expression of TLR2, MyD88, WSU-HN6 and CAL27 were cultured in Dulbecco’s NLRP3, ASC, Caspase-1 and IL-1β was determined using −ΔΔCt modified Eagle’s medium (DMEM) supplemented with 10% the 2 method. fetal bovine serum (Gibco, New York, NY, USA) and 1% penicillin-streptomycin. All cells were incubated in a hu- Western blot midified atmosphere containing 5% CO at 37 °C. Zymosan OSCC cells treated with zymosan (100 μg/mL) were col- was purchased from Sangon Biotech (Shanghai, China). C. lected and lysed on ice in RIPA buffer with phosphatase albicans ATCC 90028 was cultured on Sabouraud Dextrose inhibitor (Solarbio, Beijing, China) and phenylmethane- Agar (SDA) at 37 °C in an incubator containing 5% CO . sulfonylfluoride (PMSF). After centrifugation, the pro- tein concentration in supernatants was determined by an Enhanced BCA Protein Assay Kit (Beyotime, Haimen, CCK-8 cell viability assay China), and the protein samples were then incubated at The cell viability assay was performed with CCK-8 100 °C for 10 min. Equal amounts of total protein were (Dojindo, Kumamoto, Japan). OSCC cells (5000 cells/ subjected to SDS-polyacrylamide gel electrophoresis well) were seeded into 96-well plates and cultured for (SDS-PAGE) and then electrophoretically transferred 12–24 h before treatment. The OSCC cells were treated onto a polyvinylidene difluoride (PVDF) membrane with zymosan (10 μg/ml and 100 μg/ml) or with 100 μL (Bio-Rad, Hercules, CA, USA). The membrane was PBS as the control. After 12 h, 24 h or 48 h of treatment, blocked in 5% nonfat milk powder in Tris-buffered sa- 10 μL of CCK-8 reagent was added, and the optical line/Tween 20 (TBST) for 1 h at room temperature and density was read at 450 nm on a microplate reader (Bio- incubated with primary antibody overnight at 4 °C Rad, Hercules, CA, USA) after incubation for 1.5 h. (TLR2, ab16894, Abcam; MyD88, ab133739, Abcam; E- Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 3 of 8 cadherin, ab40772, Abcam; NF-κB p65, #8242, Cell concentration was 10 μg/ml. However, zymosan pro- Signaling Technology; p-NF-κB, #3033, Cell Signaling moted the proliferation of OSCC cells in vitro according Technology and β-actin, A1978, SIGMA) and then incu- to the results of the CCK-8 assay when the concentra- bated with HRP-conjugated secondary antibodies (anti- tion was 100 μg/ml (Fig. 1). The results indicated that a rabbit IgG from Sigma-Aldrich and anti-mouse IgG tumour-promoting effect could be induced by zymosan from Cell Signaling Technology) at a dilution of 1:5000 or other glucan-containing pathogens such as C. for 1 h at room temperature. Protein bands were visual- albicans, at a high concentration. ized using High-sig ECL substrate and a Tanon 5200 CE machine (Tanon, Shanghai, China). The TLR2/MyD88 pathway is involved in the interaction between OSCC and zymosan Immunofluorescence assay To define the receptors of zymosan in OSCC cells, the The OSCC cells treated with PBS or zymosan and placed mRNA expression of receptors including TLR2, TLR6, on glass slides were fixed with 4% paraformaldehyde for Dectin-1 and CR3 was detected by quantitative real-time 20 min, permeabilized with 0.1% Triton X-100 for 3 min PCR (RT-PCR). The results suggested that TLR2 is and blocked with 5% bovine serum albumin for 30 min. expressed on OSCC cells and that TLR2 is more highly The slides were incubated with anti-TLR2 antibody (1: expressed when the cells were treated with zymosan 10, Abcam, MA, USA) at 4 °C overnight. Anti-mouse (Fig. 2a and b). Elevated expression of TLR2 on OSCC IgG antibody with Alexa Fluor 568 served as secondary cells treated with zymosan was also found by immuno- antibody (Thermo Fisher Scientific, USA). After washing fluorescence assay (Fig. 2c). By RT-PCR and western three times with TBST, the cells were stained with DAPI blot, the expression of the downstream adapter molecule for 3 min to visualize the nuclei. Images were taken by MyD88 was also found to be elevated, indicating that an inverted microscope equipped with fluorescence the TLR2/MyD88 pathway is involved in the process of optics (Olympus, Osaka, Japan). stimulation. The activation of NF-κB, which is commonly induced by TLR2/MyD88, has been linked to the progres- Statistical analysis sion of tumours. As shown in Fig. 2b, phosphorylated-NF- The data were displayed as the mean ± standard devi- κB p65 protein levels were significantly increased in OSCC ation, and processed by GraphPad Prism 5 (version 5.01, cells treated with zymosan compared with those without GraphPad Software, CA, USA). Statistical analysis was zymosan. These results indicated that zymosan could conducted by SAS 8.2 (SAS Institute Inc., Cary, NC, facilitate the proliferation of OSCC in vitro by activating USA). Two-tailed P-value less than 0.05 was considered NF-κB via the TLR2/MyD88 pathway. statistically significant and was indicated with * when P <0.05, ** when P<0.01, *** when P < 0.001 and **** when Increased adhesion of C. albicans onto OSCC treated with P < 0.0001. zymosan Clinical observations have indicated an association be- Results tween C. albicans and oral cancers. In the cell adhesion Zymosan from the fungal cell wall promotes the test, the number of C. albicans cells per oil field was proliferation of OSCC significantly increased in ZYM-treated OSCC cells com- To investigate the effect of ZYM on oral cancer cells, we pared to controls (Fig. 3a), suggesting increased adhesion cultured OSCC cell lines (WSU-HN4, WSU-HN6 and of C. albicans onto ZYM-treated OSCC cells. After sum- CAL27) with zymosan at the concentrations of 10 μg/ml marizing the total number of C. albicans cells from 20 and 100 μg/ml for 12 h, 24 h or 48 h. There was no influ- oil fields, a significant increase in C. albicans cells was ence on the growth of OSCC cells when the zymosan found on the surface of ZYM-treated OSCC cells Fig. 1 Proliferation of OSCC cells treated with/without zymosan Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 4 of 8 Fig. 2 Zymosan activated the TLR2/MyD88/NF-κB pathway in OSCC cells Fig. 3 Increased adhesion of C. albicans onto zymosan-treated OSCC cells Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 5 of 8 including WSU-HN4, WSU-HN6 and CAL27 (Fig. 3b). secreted significantly more IL-1β, which was indicative It has been reported that E-cadherin plays an important of an inflammatory response in the OSCC (Fig. 4b). role in the bacterial adhesion [19]. Thus, we determined the expression of E-cadherin by western blot, and the E- NLRP3 inflammasome is activated in zymosan-treated cadherin was more highly expressed in OSCC cells OSCC cells treated with ZYM for 48 h (Fig. 3c). Activation of the NLRP3 inflammasome is the main path to produce IL-1β, and then we determined the expres- Elevated IL-1β production in OSCC cells treated with sion of NLRP3, ASC and caspase-1 by RT-PCR. The zymosan is observed expression of NLRP3, ASC and caspase-1 was enhanced The pro-inflammatory cytokines including IL-6, IL-8, in WSU-HN4 and CAL27 cells (Fig. 5a). The expression TNF-α and IL-1β in the culture supernatant from WSU- of IL-1β was highly enhanced in OSCC cells at both the HN4, WSU-HN6 and CAL27 cell lines treated with/ mRNA and protein levels (Figs. 4b, 5b). These data without zymosan were detected. By statistical analysis, suggested that zymosan could trigger the NLRP3 inflam- there was a significant difference in the level of IL-1β masome and that NLRP3/IL-1β was also activated in produced by ZYM-treated WSU-HN4 (p = 0.0482) and OSCC cells when treated with the fungal cell wall ZYM-treated CAL27 cells (p = 0.0451) compared with component zymosan. controls (Fig. 4a). However, the concentration of IL-1β from WSU-HN6 cells treated with/without zymosan was Discussion below 5 pg/ml. Then, we determined the dynamic level In the present study, we found that zymosan from the of IL-1β in WSU-HN4 and CAL27 cell lines, and the fungal cell wall promotes the proliferation of OSCC cells results showed an increased time-dependent tendency via the TLR2/MyD88/NF-κB signaling pathway. In (Fig. 4b). Moreover, when treated with ZYM, OSCC cells addition, zymosan could promote the expression of E- Fig. 4 Detection of pro-inflammatory cytokine in the culture medium of OSCC cells Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 6 of 8 Fig. 5 Expression of NLRP3/ASC/Caspase-1/IL-1β in OSCC cells cadherin to enhance the adhesion of C. albicans onto the proliferation of OSCC in vitro via the traditional recep- OSCC cells and could further increase IL-1β production tor TLR2. It has been reported that the TLR2-regulated by OSCC cells. These results can provide information to gene signature is associated with tumour growth and that better understand the interaction between C. albicans TLR2-dependent inflammation mediates tumour metasta- and oral cancer (Fig. 6). sis [25, 26]. Moreover, other TLRs, such as TLR3 and As the main type of head and neck squamous cell carcin- TLR4, are also expressed on OSCC [17, 27]. These results oma (HNSCC), oral cancer has been a significant clinical suggested that PMAPs play a pivotal role in the interaction problem worldwide. There is a geographic variation in the of oral cancer and microbiota and that targeting TLRs in incidence of oral cancer, and the countries in South Asia OSCC is a promising therapeutic method. Moreover, an are traditionally considered high-occurrence places [20]. elevated number of C. albicans was observed in OSCC High risk factors for oral cancer include heavy smoking, cells when treated with zymosan, which implied increased excessive alcohol usage and betel chewing. Furthermore, adhesion/interaction between C. albicans and OSCC. This microbiological infections, such as HPV infection and sub- experiment could explain the phenomenon that C. albi- sequent inflammation, may also increase the risk of oral cans is higher in patients with OSCC or leukoplakia than cancer, while other microorganisms such as P. gingivalis, F. in those without oral pathology. E-cadherin was reported nucleatum and C. albicans, play a significant role in the to play a significant role in the adhesion of bacteria onto process of oral cancer [21–24]. However, the detailed host cells [19], and here, we also detected higher levels of mechanism linking these microbes and oral cancer remains E-cadherin in OSCC cells after ZYM treatment for 48 h, unknown. Here, we found that zymosan could promote indicative of E-cadherin in the adhesion of C. albicans onto Fig. 6 Schematic diagram of this study Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 7 of 8 OSCC. However, the detailed role of E-cadherin needs to domains-containing protein 3; IL: Interleukin; 5-FU: 5-Fluorouracil; PCR: Polymerase chain reaction be confirmed in animal models or clinical samples. Recently, it was reported that P. gingivalis promoted Acknowledgments oral carcinogenesis and aggravated the disturption of We appreciated the help from Dr. Yantao Duan, Dr. Wei Wei and Qu Xu in fatty acid metabolism, indicating a close association of P. the experiments. gingivalis, lipid metabolism and oral carcinogenesis [21]. Authors’ contributions With the increased attention on microbiology, increasing Conceived and designed the experiments: XC, QL, FC. Performed the research has shed light on the interaction between experiments: XC, JD, MY, RZ. Analysed the data: XC, QL. Contributed microbiota and cancers. Dejea et al demonstrated a syn- reagents/materials/analysis tools: XC, FC. Wrote the manuscript: X.C, QL, FC. The author(s) read and approved the final manuscript. ergistic interaction between two carcinogenic bacteria with different toxins and established the concept of Funding microbial networks in carcinogenesis [28, 29]. As a key This work was supported by National Natural Science Foundation of China element in oral cancers, inflammation plays a significant (81870762, 81702674), China Scholarship Council (CSC No. 201806230234), Shanghai Sailing Program (19YF1427500), Youth program from Shanghai role in the crosstalk between tumours, immune cells and Municipal Commission of Health and Family Planning (No. 20184Y0025) and microorganisms [30], and infections usually triggers in- fundamental research program funding of Ninth People’s Hospital affiliated flammatory processes. Here, we found that the fungal to Shanghai Jiao Tong University School of Medicine (No. JYZZ013, JYZZ043). cell wall component zymosan was involved in OSCC Availability of data and materials cells secreting significantly more pro-inflammatory All data generated or analyzed during this study are included in this article, cytokine IL-1β through the NLRP3/IL-1β pathway, and the data is also available from the corresponding author on reasonable indicating an inflammatory response in the OSCC. It is request. interesting to find that OSCC cells could secrete pro- Ethics approval and consent to participate inflammatory cytokines (such as IL-1β) and be influ- This study was approved by the Ethics Committee of Shanghai Ninth enced by microbiota or their cell components, which People’s Hospital. indicates a more complex interaction between cancer cells, immune cells and microbiota in tumour micro- Consent for publication Not applicable. environment. Another report from our lab revealed that NLRP3 inflammasome-activated IL-1β promoted 5-FU Competing interests resistance in OSCC both in vitro and in vivo [31], and The authors have declared that no competing interest exists. combining these data, it can be inferred that the pres- Received: 27 April 2020 Accepted: 21 July 2020 ence of C. albicans in oral cancer may influence the effect of chemotherapy by inducing IL-1β production, which was also a potential target for treating oral cancer. References Despite some interesting findings in the current study, 1. Chi AC, Day TA, Neville BW. Oral cavity and oropharyngeal squamous cell carcinoma--an update. CA Cancer J Clin. 2015;65:401–21. there are still some limitations: 1) all the results were 2. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head based on in-vitro experiments; 2) the mechanism and neck cancer. Nat Rev Cancer. 2011;11:9–22. explored in the current study needs further confirmation 3. 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Zymosan promotes proliferation, Candida albicans adhesion and IL-1β production of oral squamous cell carcinoma in vitro

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Abstract

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC), and the effect of zymosan (ZYM), a component of the yeast cell wall, on oral cancer remains unclear. The CCK-8 proliferation assay was performed to evaluate the effect of ZYM on the proliferation of the OSCC cell lines WSU-HN4, WSU-HN6 and CAL27, and the potential mechanism was explored by quantitative real-time PCR, immunofluorescence assay and western blot. A cell adhesion assay was conducted to determine the adhesion of Candida albicans to OSCC cells, and the expression of related genes, including TLR2, MyD88, NLRP3, ASC, Caspase-1 and IL-1β, and proteins, including TLR2, MyD88, NF-κBp65, p-NF-κB p65 and E-cadherin was determined. Additionally, the pro-inflammatory cytokines including IL-6, IL-8, TNF-α and IL-1β produced by OSCC cells were detected using a chemiluminescence immunoassay (CLIA). In the current study, the CCK-8 assay showed that ZYM promoted the proliferation of WSU-HN4, WSU-HN6 and CAL27 cells via the TLR2/MyD88 pathway. The cell adhesion assay showed that the number of C. albicans cells per field significantly increased in ZYM-treated OSCC cells compared to controls. When treated with ZYM, OSCC cells secreted significantly more pro-inflammatory cytokine IL-1β, which could enhance inflammation in oral cancer microenvironment. In conclusion, ZYM from the fungal cell wall promotes the proliferation, C. albicans adhesion and IL-1β production in OSCC, as demonstrated by in vitro experiments. Keywords: Zymosan, Candida albicans, Proliferation, Oral cancer, Oral squamous cell carcinoma Introduction metastasis and local recurrence [2]. Recently, imbalanced Oral squamous cell carcinoma (OSCC) is the most com- microbiota or specific microbes have been found to play mon type of head and neck squamous cell carcinoma an indispensable role in tumour initiation, progression (HNSCC), which is the sixth leading cancer type world- and even chemoresistance [3–6]. For example, Helicobac- wide [1]. The most common treatment for oral cancers ter pylori plays a role in inducing or promoting gastric involves radical surgery and chemotherapy, including 5- cancer [7], and different microorganisms, such as Fuso- fluorouracil and cisplatin. However, the 5-year survival bacterium sp., Porphyromonas gingivalis and Candida rate among patients with OSCC is low because of the albicans, have been reported in oral cancers [8–10]. poor prognosis, such as the occurrence of lymph node It has been observed that oral carriage of C. albicans is higher in patients with OSCC or leukoplakia than in those without oral pathology [11, 12], but the association be- * Correspondence: chenfx@sjtu.edu.cn Xu Chen and Qingqiong Luo contributed equally to this work. tween C. albicans and oral cancer remains ambiguous. Department of Clinical Immunology, Shanghai Ninth People’s Hospital, Several reports have proposed different potential pathways Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai by which C. albicans initiates or promotes carcinogenesis, 200011, China Faculty of Medical Laboratory Science, Shanghai Jiao Tong University School such as inducing inflammation [13, 14]. Triggering of of Medicine (SJTUSM), Shanghai 200025, China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 2 of 8 inflammation by inducing TNF-α and IL-8 or production Cell adhesion assay of carcinogenic by-products such as nitrosamine and N- OSCC cell suspension was seeded onto a coverslip nitrosobenzylmethylamine (NBMA) by C. albicans could placed in a 24-well plate, which was disinfected by UV be a risk factor for cancer [15]. Therefore, it is important light, and the cells were incubated for 24 h to attach to to investigate the interaction between microorganisms coverslip, and then stimulated by zymosan or PBS for an and host cells. The adhesion of microorganisms to host additional 24 h. The cells were washed with PBS before cells has been reported to be the first step for bacteria or adding C. albicans suspension in PBS, and were further fungi to influence host cells [8]. However, the adhesion of incubated at 37 °C with 5% CO for 1 h. After incuba- C. albicans to OSCC cells remains unclear. tion, the DMEM with unattached yeast was aspirated, Toll-like receptors (TLRs) and other pattern recognition and each well was washed with PBS and then fixed with receptors (PRRs) play a significant role in the recognition 95% ethanol for 1 h. After that, the coverslip was of pathogens and triggering of innate immune responses, removed for Gram staining. and a group of molecules can act as pathogen-associated molecular patterns (PAMPs), such as glucans, which are Cytokine detection recognized by PRRs [16]. Moreover, the OSCC cells have OSCC cells were seeded into a 12-well plate before treat- been found to express different TLRs, including TLR3 and ment with PBS or zymosan, and then supernatants were TLR4 [17]. Activation of TLRs usually induces recruit- collected to measure the concentrations of IL-1β, IL-6, IL- ment of the adapter molecule myeloid differentiation 8 and TNF-α. These cytokines were measured by chemilu- primary response protein 88 (MyD88), and activation of minescence immunoassay (CLIA) using IMMULITE®1000 TLR/MyD88 typically leads to the activation of NF-κB, (SIEMENS, Germany) with commercial reagents accord- which has been linked to the progression of tumours [18]. ing to the manufacturer’s instructions. However, the role of TLR expressed on OSCC cells in the interaction with microbial components needs to be inves- Quantitative real-time PCR tigated. This study aimed to define the effect of zymosan Total RNA was extracted using TRIzol reagent (Invitro- (a component of the fungal cell wall, ZYM), which is a gen, San Diego, CA, USA), and real-time PCR amplifica- glucan with repeating glucose units connected by β-1,3- tion was carried out by a two-step reaction. First, cDNA glycosidic linkages, on oral cancer by investigating the was synthesized by GeneAmp® PCR system 9700 proliferation and cytokine production of OSCC cells and (Applied Biosystems) with PrimeScript™ RT Reagent kit C. albicans adhesion to cell lines. (TaKaRa, Shiga, Japan), and then real-time PCR was performed in the 7500 system (Applied Biosystems) with SYBR Premix Ex Taq II (TaKaRa). The experiment was Materials and methods repeated in triplicate on independent occasions. The Cell lines and reagents GAPDH gene was used for the normalization of gene ex- In this study, the human OSCC cell lines WSU-HN4, pression, and the relative expression of TLR2, MyD88, WSU-HN6 and CAL27 were cultured in Dulbecco’s NLRP3, ASC, Caspase-1 and IL-1β was determined using −ΔΔCt modified Eagle’s medium (DMEM) supplemented with 10% the 2 method. fetal bovine serum (Gibco, New York, NY, USA) and 1% penicillin-streptomycin. All cells were incubated in a hu- Western blot midified atmosphere containing 5% CO at 37 °C. Zymosan OSCC cells treated with zymosan (100 μg/mL) were col- was purchased from Sangon Biotech (Shanghai, China). C. lected and lysed on ice in RIPA buffer with phosphatase albicans ATCC 90028 was cultured on Sabouraud Dextrose inhibitor (Solarbio, Beijing, China) and phenylmethane- Agar (SDA) at 37 °C in an incubator containing 5% CO . sulfonylfluoride (PMSF). After centrifugation, the pro- tein concentration in supernatants was determined by an Enhanced BCA Protein Assay Kit (Beyotime, Haimen, CCK-8 cell viability assay China), and the protein samples were then incubated at The cell viability assay was performed with CCK-8 100 °C for 10 min. Equal amounts of total protein were (Dojindo, Kumamoto, Japan). OSCC cells (5000 cells/ subjected to SDS-polyacrylamide gel electrophoresis well) were seeded into 96-well plates and cultured for (SDS-PAGE) and then electrophoretically transferred 12–24 h before treatment. The OSCC cells were treated onto a polyvinylidene difluoride (PVDF) membrane with zymosan (10 μg/ml and 100 μg/ml) or with 100 μL (Bio-Rad, Hercules, CA, USA). The membrane was PBS as the control. After 12 h, 24 h or 48 h of treatment, blocked in 5% nonfat milk powder in Tris-buffered sa- 10 μL of CCK-8 reagent was added, and the optical line/Tween 20 (TBST) for 1 h at room temperature and density was read at 450 nm on a microplate reader (Bio- incubated with primary antibody overnight at 4 °C Rad, Hercules, CA, USA) after incubation for 1.5 h. (TLR2, ab16894, Abcam; MyD88, ab133739, Abcam; E- Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 3 of 8 cadherin, ab40772, Abcam; NF-κB p65, #8242, Cell concentration was 10 μg/ml. However, zymosan pro- Signaling Technology; p-NF-κB, #3033, Cell Signaling moted the proliferation of OSCC cells in vitro according Technology and β-actin, A1978, SIGMA) and then incu- to the results of the CCK-8 assay when the concentra- bated with HRP-conjugated secondary antibodies (anti- tion was 100 μg/ml (Fig. 1). The results indicated that a rabbit IgG from Sigma-Aldrich and anti-mouse IgG tumour-promoting effect could be induced by zymosan from Cell Signaling Technology) at a dilution of 1:5000 or other glucan-containing pathogens such as C. for 1 h at room temperature. Protein bands were visual- albicans, at a high concentration. ized using High-sig ECL substrate and a Tanon 5200 CE machine (Tanon, Shanghai, China). The TLR2/MyD88 pathway is involved in the interaction between OSCC and zymosan Immunofluorescence assay To define the receptors of zymosan in OSCC cells, the The OSCC cells treated with PBS or zymosan and placed mRNA expression of receptors including TLR2, TLR6, on glass slides were fixed with 4% paraformaldehyde for Dectin-1 and CR3 was detected by quantitative real-time 20 min, permeabilized with 0.1% Triton X-100 for 3 min PCR (RT-PCR). The results suggested that TLR2 is and blocked with 5% bovine serum albumin for 30 min. expressed on OSCC cells and that TLR2 is more highly The slides were incubated with anti-TLR2 antibody (1: expressed when the cells were treated with zymosan 10, Abcam, MA, USA) at 4 °C overnight. Anti-mouse (Fig. 2a and b). Elevated expression of TLR2 on OSCC IgG antibody with Alexa Fluor 568 served as secondary cells treated with zymosan was also found by immuno- antibody (Thermo Fisher Scientific, USA). After washing fluorescence assay (Fig. 2c). By RT-PCR and western three times with TBST, the cells were stained with DAPI blot, the expression of the downstream adapter molecule for 3 min to visualize the nuclei. Images were taken by MyD88 was also found to be elevated, indicating that an inverted microscope equipped with fluorescence the TLR2/MyD88 pathway is involved in the process of optics (Olympus, Osaka, Japan). stimulation. The activation of NF-κB, which is commonly induced by TLR2/MyD88, has been linked to the progres- Statistical analysis sion of tumours. As shown in Fig. 2b, phosphorylated-NF- The data were displayed as the mean ± standard devi- κB p65 protein levels were significantly increased in OSCC ation, and processed by GraphPad Prism 5 (version 5.01, cells treated with zymosan compared with those without GraphPad Software, CA, USA). Statistical analysis was zymosan. These results indicated that zymosan could conducted by SAS 8.2 (SAS Institute Inc., Cary, NC, facilitate the proliferation of OSCC in vitro by activating USA). Two-tailed P-value less than 0.05 was considered NF-κB via the TLR2/MyD88 pathway. statistically significant and was indicated with * when P <0.05, ** when P<0.01, *** when P < 0.001 and **** when Increased adhesion of C. albicans onto OSCC treated with P < 0.0001. zymosan Clinical observations have indicated an association be- Results tween C. albicans and oral cancers. In the cell adhesion Zymosan from the fungal cell wall promotes the test, the number of C. albicans cells per oil field was proliferation of OSCC significantly increased in ZYM-treated OSCC cells com- To investigate the effect of ZYM on oral cancer cells, we pared to controls (Fig. 3a), suggesting increased adhesion cultured OSCC cell lines (WSU-HN4, WSU-HN6 and of C. albicans onto ZYM-treated OSCC cells. After sum- CAL27) with zymosan at the concentrations of 10 μg/ml marizing the total number of C. albicans cells from 20 and 100 μg/ml for 12 h, 24 h or 48 h. There was no influ- oil fields, a significant increase in C. albicans cells was ence on the growth of OSCC cells when the zymosan found on the surface of ZYM-treated OSCC cells Fig. 1 Proliferation of OSCC cells treated with/without zymosan Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 4 of 8 Fig. 2 Zymosan activated the TLR2/MyD88/NF-κB pathway in OSCC cells Fig. 3 Increased adhesion of C. albicans onto zymosan-treated OSCC cells Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 5 of 8 including WSU-HN4, WSU-HN6 and CAL27 (Fig. 3b). secreted significantly more IL-1β, which was indicative It has been reported that E-cadherin plays an important of an inflammatory response in the OSCC (Fig. 4b). role in the bacterial adhesion [19]. Thus, we determined the expression of E-cadherin by western blot, and the E- NLRP3 inflammasome is activated in zymosan-treated cadherin was more highly expressed in OSCC cells OSCC cells treated with ZYM for 48 h (Fig. 3c). Activation of the NLRP3 inflammasome is the main path to produce IL-1β, and then we determined the expres- Elevated IL-1β production in OSCC cells treated with sion of NLRP3, ASC and caspase-1 by RT-PCR. The zymosan is observed expression of NLRP3, ASC and caspase-1 was enhanced The pro-inflammatory cytokines including IL-6, IL-8, in WSU-HN4 and CAL27 cells (Fig. 5a). The expression TNF-α and IL-1β in the culture supernatant from WSU- of IL-1β was highly enhanced in OSCC cells at both the HN4, WSU-HN6 and CAL27 cell lines treated with/ mRNA and protein levels (Figs. 4b, 5b). These data without zymosan were detected. By statistical analysis, suggested that zymosan could trigger the NLRP3 inflam- there was a significant difference in the level of IL-1β masome and that NLRP3/IL-1β was also activated in produced by ZYM-treated WSU-HN4 (p = 0.0482) and OSCC cells when treated with the fungal cell wall ZYM-treated CAL27 cells (p = 0.0451) compared with component zymosan. controls (Fig. 4a). However, the concentration of IL-1β from WSU-HN6 cells treated with/without zymosan was Discussion below 5 pg/ml. Then, we determined the dynamic level In the present study, we found that zymosan from the of IL-1β in WSU-HN4 and CAL27 cell lines, and the fungal cell wall promotes the proliferation of OSCC cells results showed an increased time-dependent tendency via the TLR2/MyD88/NF-κB signaling pathway. In (Fig. 4b). Moreover, when treated with ZYM, OSCC cells addition, zymosan could promote the expression of E- Fig. 4 Detection of pro-inflammatory cytokine in the culture medium of OSCC cells Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 6 of 8 Fig. 5 Expression of NLRP3/ASC/Caspase-1/IL-1β in OSCC cells cadherin to enhance the adhesion of C. albicans onto the proliferation of OSCC in vitro via the traditional recep- OSCC cells and could further increase IL-1β production tor TLR2. It has been reported that the TLR2-regulated by OSCC cells. These results can provide information to gene signature is associated with tumour growth and that better understand the interaction between C. albicans TLR2-dependent inflammation mediates tumour metasta- and oral cancer (Fig. 6). sis [25, 26]. Moreover, other TLRs, such as TLR3 and As the main type of head and neck squamous cell carcin- TLR4, are also expressed on OSCC [17, 27]. These results oma (HNSCC), oral cancer has been a significant clinical suggested that PMAPs play a pivotal role in the interaction problem worldwide. There is a geographic variation in the of oral cancer and microbiota and that targeting TLRs in incidence of oral cancer, and the countries in South Asia OSCC is a promising therapeutic method. Moreover, an are traditionally considered high-occurrence places [20]. elevated number of C. albicans was observed in OSCC High risk factors for oral cancer include heavy smoking, cells when treated with zymosan, which implied increased excessive alcohol usage and betel chewing. Furthermore, adhesion/interaction between C. albicans and OSCC. This microbiological infections, such as HPV infection and sub- experiment could explain the phenomenon that C. albi- sequent inflammation, may also increase the risk of oral cans is higher in patients with OSCC or leukoplakia than cancer, while other microorganisms such as P. gingivalis, F. in those without oral pathology. E-cadherin was reported nucleatum and C. albicans, play a significant role in the to play a significant role in the adhesion of bacteria onto process of oral cancer [21–24]. However, the detailed host cells [19], and here, we also detected higher levels of mechanism linking these microbes and oral cancer remains E-cadherin in OSCC cells after ZYM treatment for 48 h, unknown. Here, we found that zymosan could promote indicative of E-cadherin in the adhesion of C. albicans onto Fig. 6 Schematic diagram of this study Chen et al. Infectious Agents and Cancer (2020) 15:51 Page 7 of 8 OSCC. However, the detailed role of E-cadherin needs to domains-containing protein 3; IL: Interleukin; 5-FU: 5-Fluorouracil; PCR: Polymerase chain reaction be confirmed in animal models or clinical samples. Recently, it was reported that P. gingivalis promoted Acknowledgments oral carcinogenesis and aggravated the disturption of We appreciated the help from Dr. Yantao Duan, Dr. Wei Wei and Qu Xu in fatty acid metabolism, indicating a close association of P. the experiments. gingivalis, lipid metabolism and oral carcinogenesis [21]. Authors’ contributions With the increased attention on microbiology, increasing Conceived and designed the experiments: XC, QL, FC. Performed the research has shed light on the interaction between experiments: XC, JD, MY, RZ. Analysed the data: XC, QL. Contributed microbiota and cancers. Dejea et al demonstrated a syn- reagents/materials/analysis tools: XC, FC. Wrote the manuscript: X.C, QL, FC. The author(s) read and approved the final manuscript. ergistic interaction between two carcinogenic bacteria with different toxins and established the concept of Funding microbial networks in carcinogenesis [28, 29]. As a key This work was supported by National Natural Science Foundation of China element in oral cancers, inflammation plays a significant (81870762, 81702674), China Scholarship Council (CSC No. 201806230234), Shanghai Sailing Program (19YF1427500), Youth program from Shanghai role in the crosstalk between tumours, immune cells and Municipal Commission of Health and Family Planning (No. 20184Y0025) and microorganisms [30], and infections usually triggers in- fundamental research program funding of Ninth People’s Hospital affiliated flammatory processes. Here, we found that the fungal to Shanghai Jiao Tong University School of Medicine (No. JYZZ013, JYZZ043). cell wall component zymosan was involved in OSCC Availability of data and materials cells secreting significantly more pro-inflammatory All data generated or analyzed during this study are included in this article, cytokine IL-1β through the NLRP3/IL-1β pathway, and the data is also available from the corresponding author on reasonable indicating an inflammatory response in the OSCC. It is request. interesting to find that OSCC cells could secrete pro- Ethics approval and consent to participate inflammatory cytokines (such as IL-1β) and be influ- This study was approved by the Ethics Committee of Shanghai Ninth enced by microbiota or their cell components, which People’s Hospital. indicates a more complex interaction between cancer cells, immune cells and microbiota in tumour micro- Consent for publication Not applicable. environment. Another report from our lab revealed that NLRP3 inflammasome-activated IL-1β promoted 5-FU Competing interests resistance in OSCC both in vitro and in vivo [31], and The authors have declared that no competing interest exists. combining these data, it can be inferred that the pres- Received: 27 April 2020 Accepted: 21 July 2020 ence of C. albicans in oral cancer may influence the effect of chemotherapy by inducing IL-1β production, which was also a potential target for treating oral cancer. References Despite some interesting findings in the current study, 1. Chi AC, Day TA, Neville BW. Oral cavity and oropharyngeal squamous cell carcinoma--an update. CA Cancer J Clin. 2015;65:401–21. there are still some limitations: 1) all the results were 2. Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head based on in-vitro experiments; 2) the mechanism and neck cancer. Nat Rev Cancer. 2011;11:9–22. explored in the current study needs further confirmation 3. 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Published: Jul 31, 2020

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