SCC-25 Cells Research Articles

Shikonin Stimulates Mitochondria-Mediated Apoptosis by Enhancing Intracellular Reactive Oxygen Species Production and DNA Damage in Oral Cancer Cells.

Phytotherapy has rendered a new insight towards the treatment of various cancers, including oral cancer with fewer side effects, over the traditional chemotherapeutic drugs to overcome chemoresistance. Shikonin (Shk) is a natural biologically active alkaloid found in the Lithospermum erythrorhizon plant's root. It has potent cytotoxic activities against various cancers. Our study revealed the release time and anticancer potential of Shk on the SCC9 and H357 oral cancer cell lines. We investigated the antiproliferative, antimigratory, cell cycle arresting and apoptosis promoting activity of Shk in oral cancer cells by performing MTT and morphological assay, colony, and tumor sphere formation assay, AO/EtBr and DAPI staining, Annexin V-FITC/PI staining, assay for reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) measurement, comet assay, qRT-PCR, and western blot analysis. We also checked the interaction of DNA and Shk by docking and CD spectroscopy and EtBr displacement assay. As a result, we found that Shk reduced the viability, proliferation, and tumorigenicity of SCC9 and H357 cells in a time and concentration-dependent manner. We obtained half-maximal inhibitory concentration (IC50) at 0.5 µM for SCC9 and 1.25 µM for H357. It promotes apoptosis via overexpressing proapoptotic Bax and caspase 3 via enhancing ROS that leads to MMP depletion and DNA damage and arrests cells at the G2/M & G2/S phase. The antimigratory activity of Shk was performed by analyzing the expression of markers of epithelial-mesenchymal transition like E-cadherin, ZO-1, N-cadherin, and vimentin. These overall results recommended that Shk shows potent anticancer activity against oral cancer cell lines in both in vitro and ex vivo conditions. So, it could be an excellent agent for the treatment of oral cancer.

DEAD/H-box helicase 11 is transcriptionally activated by Yin Yang-1 and accelerates oral squamous cell carcinoma progression.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H-box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC-4 and CAL-27 cells expressing doxycycline-inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang-1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K-AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor-promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment.

CHRDL1 inhibits OSCC metastasis via MAPK signaling-mediated inhibition of MED29

BackgroundCHRDL1 belongs to a novel class of mRNA molecules. Nonetheless, the specific biological functions and underlying mechanisms of CHRDL1 in oral squamous cell carcinoma (OSCC) remain largely unexplored.MethodsRT-qPCR and immunohistochemical staining were employed to assess the mRNA and protein expression levels of the MED29 gene in clinical samples of OSCC. Additionally, RT-qPCR and Western Blot analyses were conducted to investigate the mRNA and protein expression levels of the MED29 gene specifically in OSCC. The impact of MED29 on epithelial-mesenchymal transition (EMT), invasion, and migration of OSCC was evaluated through scratch assay, transwell assay, and immunofluorescence staining. Furthermore, wound healing assay and Transwell assay were utilized to examine whether CHRDL1 influences the malignant behavior of OSCC by modulating MED29 in vitro. The regulatory role of CHRDL1 on MED29 was further elucidated in vivo through a tail vein lung metastasis model in nude mice.ResultsMED29 expression was elevated in tumor tissues of OSCC patients compared with adjacent cancer tissues. Moreover, in CAL27 and SCC25 cell lines, MED29 was upregulated and associated with increased cell migration and invasion abilities. Overexpression of MED29 facilitated EMT in OSCC cell lines, whereas knockdown of MED29 impeded EMT, resulting in diminished cell migration and invasion capacities. CHRDL1 exerted inhibitory effects on the expression of MED29, thereby suppressing EMT progression and consequently restraining the invasion and migration of OSCC cells. Furthermore, CHRDL1 mediated the inhibition of migration of OSCC cell lines to the OSCC through its regulation of MED29.ConclusionsMED29 facilitated the epithelial-mesenchymal transition process in OSCC, thereby promoting migration and invasion. On the other hand, CHRDL1 exerted inhibitory effects on the invasion and metastasis of OSCC by suppressing MED29 through the inhibition of the MAPK signaling pathway.

Pterostilbene suppresses head and neck cancer cell proliferation via induction of apoptosis.

Head and neck cancer (HNC) is one of the most prevalent causes of death worldwide, and so discovering anticancer agents for its treatment is very important. Pterostilbene (PS) is a trans-stilbene reported to be beneficial in managing various cancers. The objective of the study was to evaluate the cytotoxic, antiproliferative, proapoptotic, and antimigrative effect of PS on HEp-2, SCC-90, SCC-9, FaDu, and Detroit-551 cell lines. MTT and live/dead assays were employed to assess cell viability, while a cell migration test was performed to evaluate wound healing capacity. The mRNA, protein, and intracellular expression levels of CASP-3, BAX, and BCL-2 genes were evaluated by real-time PCR, western blotting, and immunofluorescence staining. Annexin V-PI staining was conducted to assess the amounts of viable, apoptotic, and necrotic cells. The results revealed that PS displayed cytotoxic, antiproliferative activity in a dose-dependent manner in HNC cells by upregulating CASP-3 and BCL-2 while downregulating BCL-2 in the apoptotic pathway. The proapoptotic properties were confirmed by the annexin-V-IP results. Moreover, PS displayed a significant suppressive efficacy on the migration capacity of HNC cells. The present study provides proof that PS has the prospective to be improved as an attractive anticancer agent against HNC following advanced studies.

Development and Validation of a Cholesterol-related Gene Signature for Prognostic Assessment in Head and Neck Squamous Cell Carcinoma.

This study seeks to develop a prognostic risk signature for head and neck squamous cell carcinoma (HNSCC) based on cholesterol-related genes (CholRG), aiming to enhance prognostic accuracy in clinical practice. HNSCC poses significant challenges due to its aggressive behavior and limited response to standard treatments, resulting in elevated morbidity and mortality rates.In order to improve prognostic prediction in HNSCC, our study is inspired by the realization that cholesterol metabolism plays a critical role in accelerating the progression of cancer. To this end, we are developing a unique risk signature using CholRG. The aim of this study was to create a CholRG-based risk signature to predict HNSCC prognosis, aiding in clinical decision-making accurately. The TCGA HNSCC dataset, along with GSE41613 and GSE65858, was obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases, respectively. A CholRG-based risk signature was then developed and validated across various independent HNSCC cohorts. Moreover, a nomogram model incorporating CholRG-based risk signature was established. Additionally, functional enrichment analysis was conducted, and the immune landscapes of the high- and low-risk groups were compared. Finally, in vitro experiments were performed using lipid-based transfection to deliver siRNAs targeting ACAT1 to SCC1 and SCC23 cell lines, further examining the effects of ACAT1 knockdown on these cells. Utilizing RNA-seq, microarray, and clinical data from public databases, we constructed and validated a CholRG-based risk signature that includes key genes such as ACAT1, CYP19A1, CYP27A1, FAXDC2, INSIG2, PRKAA2, and SEC14L2, which can effectively predict the clinical outcome of HNSCC. Additionally, our findings were reinforced by a nomogram model that integrates the risk score with clinical variables for more clinically practical prognostic assessment. In addition, patients at high risk show hypoxia and increased oncogenic pathways such as mTORC1 signaling, as well as a suppressed immune microenvironment marked by a reduction in the infiltration of important immune cells. Notably, in vitro experiments showed that ACAT1 depletion significantly suppressed the proliferation, colony formation, and invasion capabilities of HNSCC cells, confirming ACAT1's role in promoting malignancy. Collectively, our study not only underscores the importance of cholesterol metabolism in HNSCC pathogenesis but also highlights the CholRG-based risk signature as a promising tool for enhancing prognostic accuracy and personalizing therapeutic strategies.

Gingerol acts as a potent radiosensitizer in head and neck squamous cell carcinoma

Treatment options for advanced head and neck squamous cell carcinoma (HNSCC) are limited and often cause severe toxicity and debilitating long-term impacts. Developing effective and safer treatments is warranted. Several plant extracts have shown their effectiveness, but a comprehensive comparison between plant extracts in HNSCC has not been reported. Our aim was to investigate the effect of different plant extracts on the proliferation and viability of HNSCC cell lines. In addition, we investigated the efficacy of combining cytotoxic plant extracts with radiation. Since RT is a cornerstone in the treatment and management of HNSCC, it is desirable to enhance its efficacy through combination with cytotoxic agents that have minimal side effects. HNSCC cell lines were treated with various plant extracts at different concentrations. MTT assays were performed to identify the most potent anti-tumor plant extract. Colony-formation assays were performed to determine the radiosensitization effect. To investigate the effect on migration, transwell migration assays were performed. Annexin V staining was performed to analyze cell apoptosis. 6-gingerol resulted in the most significant dose-dependent inhibition in all cell lines compared to other plant extracts. Colony-formation assays showed a significant radiosensitizing effect when 6-gingerol was combined with radiation. In addition, the combination of 6-gingerol with radiation resulted in a significant decrease in HNSCC cell migration. Mechanistically, Annexin V staining showed that the combination of 6-gingerol and RT induces a synergistic apoptotic effect in MOC1, MOC2 and SCC9 cells compared to RT alone. In conclusion, 6-gingerol enhances the effect of radiation in HNSCC cell lines and could be a suitable candidate for combination therapy in HNSCC.

Y27632 induces tongue squamous cell carcinoma cell apoptosis through MAPK-ERK/JNK signal

Abstract Objectives ROCK signaling is considered a therapeutic target for oral squamous cell carcinoma (OSCC). Y27632, a well-established ROCK inhibitor, has previously been reported to block oral squamous cell carcinoma cell growth and has shown cell type dependence in the treatment of other cancers. TP53 is one of the most frequently mutated genes in head and neck cancer. Here, we aim to investigate the role of Y27632 in wild-type and p53 mutant (R175H) SCC9 cells. Methods The p53-mutation (R175H) and p53-null SCC9 cell line were conducted, then, CCK8, colony formation, wound-healing assays, and transwell assay were employed to investigate the role of Y27632 in wtp53 and mutp53 SCC9 cells. The effects of Y27632 in SCC9 cells were also confirmed by the knockdown of ROCK1/2. Additionally, cell cycle and apoptosis were assessed using flow and western blot analysis. The impact of Y27632 on cell senescence was confirmed through the senescence-associated β-gal staining. Furthermore, the inhibition of Y27632 was examined in vivo using tumor-bearing nude mice. Results Our study demonstrates that Y27632 effectively impeded the proliferation of tongue squamous cell carcinoma (TSCC) cells in vitro and in vivo. Additionally, the proliferation and migration of wtp53 and mutp53 SCC9 cells were also significantly suppressed by Y27632 or ROCK siRNA in vitro. Mechanistically, Y27632 induced apoptosis in SCC9 cells via the MAPK-ERK/JNK signaling pathway. Conclusions Our data demonstrated that Y27632 induces apoptosis in SCC9 cells via the MAPK-ERK/JNK signaling pathway, regardless of the presence of p53 mutant variants (R175H). This will provide a potential therapeutic drug for TSCC treatment in the future.

IN VITRO ANTICANCER ACTIVITY OF HISTATIN-1 COMBINATION WITH CISPLATIN IN HEAD AND NECK CANCER CELL LINES.

Chemotherapy of head and neck squamous cell carcinoma (HNSCC) is associated with significant side effects. Antimicrobial peptides (AMPs), which are naturally occurring defense molecules like defensin-1 and LL-37 found in human secretions, have demonstrated potential in prompting tumor cell apoptosis and enhancing the effect of chemotherapeutic agents. However, the anticancer potential of histatin has not yet been thoroughly examined. The aim of the study was to explore the anticancer activity of histatin, an AMP present in human saliva and used alone or in combination with cisplatin in HNSCC cell lines. The gene expression of histatin was evaluated in the HSC4 and SCC25 cell lines by qRT-PCR. Cell proliferation was investigated at different concentrations of histatin peptide (His-1), cisplatin, and their combination using an MTT assay. SCC25 cells expressed both HTN1 (histatin-1) and HTN3 (histatin-3), whereas the HSC4 cell line expressed only HTN1. The combination of exogenous His-1 and cisplatin demonstrated a synergistic anti-proliferative effect against the HNSCC cell lines in a dosedependent manner. The combination of low-dose cisplatin and histatin inhibits HNSCC cell proliferation. His-1 sensitizes tumor cells to the cytotoxic effects of cisplatin potentially allowing for a reduction in its effective concentration.

MMP7, Regulated by c-Jun, is Involved in Oral Squamous Cell Carcinoma and Associated with Cancer-Related Fibroblasts Infiltration.

This study aimed to analyze the expression of Matrix Metalloproteinase 7 (MMP7) and molecular mechanism at the Transcription Factor (TF) level in Oral Squamous Cell Carcinoma (OSCC). MMP7 expression was preliminarily explored in Head and Neck Squamous Cell Car-cinoma (HNSCC) in the online database, followed by functional analysis and prediction of TF of MMP7. IHC was employed to detect MMP7 levels in OSCC samples. SCC9 and 293T cells were used to explore the transcriptional and regulatory effects of predicted TF on MMP7 by reporter double luciferase assay, RT-qPCR, western blotting, and cellular immunofluorescence. Transwell and TUNEL were employed to detect the migration and apoptosis. MMP7 was significantly up-regulated in HNSCC and OSCC tissues. Moreover, MMP7 was positively correlated with CAFs and significantly enriched in the signaling pathway of RNA degradation. The c-Jun pathway was also up-regulated in OSCC tissues, and predicted to be optimal TF of MMP7 with positive regulatory relationship. In OSCC, silencing and over-expression of c-Jun significantly decreased and increased the level of MMP7. Meanwhile, c-Jun affected the behavior of SCC9 cells, which showed that after c-Jun gene silencing, the ability of cell migration was weakened, while apoptosis was enhanced. When c-Jun gene was overexpressed, the migration ability was enhanced, but apoptosis was not significantly affected. MMP7 has been proven to be a key protein in the development of OSCC, and has the potential to become a biological marker and therapeutic target. It has been found that c-Jun could bind to the MMP7 promoter region, and the silencing or overexpression of c-Jun can positively regulate the expression of MMP7.

Vδ1 T Cells Integrated in Full-Thickness Skin Equivalents: A Model for the Role of Human Skin–Resident γδT Cells

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

Regulatory Effects of SLC7A2-CPB2 on Lymphangiogenesis: A New Approach to Suppress Lymphatic Metastasis in HNSCC.

Lymph node metastasis (LNM) is a critical factor affecting the outcomes of head and neck squamous cell carcinoma (HNSCC) and the main reason for treatment failure. This study was designed to examine the effects of the key genes involved in the LNM of HNSCC. Tissue samples (HNSCC) were examined by transcriptome sequencing, and the core genes associated with LNM were detected via bioinformatics analysis. The functions of these core genes were then validated using the TCGA biological database and their effects on the propagation, invasion, and metastasis of HNSCC cells were evaluated through cell culture experiments. Moreover, the effect of core gene expression on the LNM capability of HNSCC was confirmed via a footpad xenograft mice model. In the findings, a key gene involved in the LNM of HNSCC was identified as SLC7A2. It was correlated with adverse clinical prognosis and expressed with low expression in HNSCC tissues. As shown in cell culture experiments, FaDu and SCC15 cell growth, invasion, and migration were inhibited when SLC7A2 was overexpressed. Further, cell apoptosis was stimulated, and lymphangiogenesis was suppressed through the downregulation of CPB2 expression. Animal studies demonstrated that the growth and LNM of HNSCC cells were inhibited by SLC7A2 overexpression. It is concluded that SLC7A2 is involved in HNSCC lymphatic metastasis by controlling CPB2 function. The results are anticipated to offer new directions for the effective treatment of HNSCC.

TROP2 Expression and Therapeutic Implications in Cutaneous Squamous Cell Carcinoma: Insights From Immunohistochemical and Functional Analysis.

Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer, but treatments for advanced cases have limited efficacy. Trophoblast cell-surface antigen 2 (TROP2) is a cell-surface protein that is widely expressed in various tumours, where it exerts significant influence over critical processes such as tumour cell growth, apoptosis, migration, invasion and metastasis. Sacituzumab govitecan, an antibody-drug conjugate (ADC) targeting TROP2, is emerging as a promising strategy for anticancer therapy. In this study, we investigated TROP2 expression in cSCC tissues from 51 patients and evaluated its function in the A431 human SCC cell line. Immunohistochemical analysis revealed TROP2 expression on the plasma membrane of cSCC tissues and A431 cells. A431 cells showed sensitivity to sacituzumab govitecan with a significant concentration-dependent decrease in viable cell number. In addition, Knockdown of TROP2 resulted in decreased expression of cyclin D1 and BCL-2, along with reduced cell viability. Knockdown of TROP2 also resulted in decreased expression of vimentin, along with reduced migratory capacity. These findings suggest that TROP2 plays a crucial role in cSCC cell proliferation and migration, and highlight the potential of sacituzumab govitecan as a promising therapeutic option for cSCC.

The anticancer potential of tetrahydrocurcumin-phytosomes against oral carcinoma progression

BackgroundHerbal medicine combined with nanotechnology offers an alternative to the increasing burden of surgery and/or chemotherapy, the main therapeutics of oral carcinoma. Phytosomes are nano-vesicular systems formed by the interaction between phospholipids and phyto-active components via hydrogen bonding, exhibiting superior efficacy over pure phytocomponents in drug delivery.MethodsTetrahydrocurcumin (THC)-phytosomes were prepared by thin film hydration method. After characterization, in vitro cytotoxicity, antiproliferative capacity, antioxidant potential and full apoptotic workup were paneled on oral squamous cell carcinoma (SCC4) in comparison with native THC-solution and cisplatin (3.58 µg/mL intravenous injection), as positive controls. In addition, we tested the three medications on normal oral keratinocytes and gingival fibroblasts to attest to their tissue-selectivity.ResultsSuccessful preparation of THC-phytosomes using 1:1 molar ratio of THC to phospholipid exhibited significantly increased aqueous solubility, good colloidal properties, and complete drug release after one hour. On SCC4 cells, THC-phytosomes, at their dose-/time-dependency at ~ 60.06 µg/mL escalated cell percentages in the S-phase with 32.5 ± 6.22% increase, as well as a startling 29.69 ± 2.3% increase in apoptotic population. Depletion of the cell colonies survival to 0.29 ± 0.1% together with restraining the migratory rate by -6.4 ± 6.8% validated THC-phytosomes’ antiproliferative capacity. Comparatively, the corresponding results of THC-solution and cisplatin revealed 12.9 ± 0.9% and 25.8 ± 1.1% for apoptosis and 0.9 ± 0.1% and 0.7 ± 0.08% for colony survival fraction, respectively. Furthermore, the nanoformulation exhibited the strongest immuno-positivity to caspase-3, which positively correlated with intense mitochondrial fluorescence by Mitotracker Red, suggesting its implication in the mitochondrial pathway of apoptosis, a finding further explained by the enormously high Bax and caspase-8 expression by RT-qPCR. Finally, the THC groups showed the lowest oxidative stress index, marking their highest free radical-scavenging potential among the test groups.ConclusionsTHC-phytosomes are depicted to be an efficient nanoformulation that enhanced the anticancer efficacy over the free drug counterpart and the conventional chemotherapeutic. Additionally, being selective to cancer cells and less cytotoxic to normal cells makes THC-phytosomes a potential candidate for tissue-targeted therapy.

Okanin Inhibits Cell Growth and Induces Apoptosis and Pyroptosis in Oral Cancer.

Okanin, a flavonoid compound derived from Bidens pilosa L., has garnered attention for its anti-inflammatory properties. Although Bidens pilosa is commonly used in healthcare products and functional foods, the anticancer potential of okanin, particularly in oral cancer, remains underexplored. This study aims to investigate the effects of okanin on oral cancer cell lines and its potential as a therapeutic agent. The study involved assessing the cytotoxic effects of okanin on oral cancer cell lines SAS, SCC25, HSC3, and OEC-M1. The IC50 values were determined using methylene blue assays, and the clonogenic capacity was evaluated through colony formation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis. Caspase-3/7 activity assays and annexin V/7-AAD staining confirmed the induction of apoptosis and pyroptosis. In vivo efficacy was assessed using a SAS xenograft model, and immunohistochemical analysis of xenograft tissue was performed to examine pyroptosis-related markers. Okanin exhibited potent cytotoxic effects with IC50 values of 12.0 ± 0.8, 58.9 ± 18.7, 18.1 ± 5.3, and 43.2 ± 6.2 μM in SAS, SCC25, HSC3, and OEC-M1 cells, respectively. It caused dose- and time-dependent reductions in cell viability and significantly impaired clonogenic capacity. Flow cytometry revealed G2/M cell cycle arrest and increased sub-G1 population, indicating cell cycle disruption and death. Okanin induced both apoptosis and pyroptosis, as confirmed by caspase-3/7 activity and annexin V/7-AAD staining. In vivo, okanin reduced tumor growth and involved pyroptosis-related markers such as CASP1, GSDMC, GSDMD, and GSDME. Okanin demonstrates significant anticancer potential, particularly in oral cancer, by inducing both apoptosis and pyroptosis. Its efficacy in reducing tumor growth in vivo further supports its potential as a novel therapeutic option. Further mechanistic studies are needed to elucidate the pathways involved in okanin-mediated cell death and to explore its clinical applications.

Targeting hexokinase 2 to enhance anticancer efficacy of trichosanthin in HeLa and SCC25 cell models

Background and purpose: Trichosanthin (TCS) is a plant-based ribosome-inactivating protein exhibiting a range of pharmacological properties, including abortifacient and anticancer. However, the routine clinical use in cancer treatment was hampered by its antigenicity. Hexokinase 2 (HK2) is a pivotal regulator of glycolysis, where aberrant expression is observed in many cancers. This study investigates the anticancer effects and mechanisms of TCS in combination with benserazide (Benz), a HK2 inhibitor, in Hela and SCC25 cancer models. Experimental approach: MTT, colony-formation and cell cycle assays were performed to assess the cytotoxic effects of TCS and Benz in HeLa and SCC25 cells. Seahorse assay, western blotting, flow cytometry analysis and RNA sequencing were employed to investigate the pharmacological effects of the combo treatment. SCC25 cell xenograft mouse model was established for in vivo efficacy study. Key results: Combined use of TCS and Benz exhibited synergistic anticancer effects in both Hela and SCC25 cell models. The observed synergistic effects were attributed to the modulation of glycolysis by targeting HK2, leading to reduced lactate production and increased ROS accumulation which further inhibited colony formation and cell cycle progression, as well as triggered apoptosis. Moreover, this combination effectively inhibited NFκB/ERK signalling pathways, which were found to be significantly activated upon single use of TCS. It was found that the combination significantly suppressed the tumour growth in SCC25 cell xenograft mouse model. Conclusion: Our findings suggested that targeting HK2 and modulating glycolysis may offer a promising avenue for improving the therapeutic outcomes of TCS-based anticancer treatments.

CAFs-derived TIAM1 Promotes OSCC Cell Growth and Metastasis by Regulating ZEB2.

Previous studies have suggested that cancer-associated fibroblasts (CAFs) within the tumor microenvironment are a critical factor in tumorigenesis and tumor development. However, the regulatory mechanisms of CAFs on oral squamous cell carcinoma (OSCC) are poorly defined. A CAF-conditioned medium (CAF-CM) was collected and applied to culture OSCC cells. Then, cell viability, proliferation, migration, and invasion were evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and scratch healing assays. T-Lymphoma Invasion and Metastasis 1 (TIAM1), zinc finger E-box-binding homeobox 2 (ZEB2), E-cadherin, and increased N-cadherin protein levels were determined using western blot. TIAM1 and ZEB2 mRNA levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Their interaction was analyzed using Co-immunoprecipitation (Co-IP) assay. SCC25 cells with or without (TIAM1-silencing) CAFs were subcutaneously inoculated in nude mice to assess the effect of TIAM1 in CAFs on OSCC tumor growth in vivo. CAFs expedited OSCC cell proliferation, migration, invasion, and EMT. TIAM1 and ZEB2 expression were upregulated in OSCC patients and OSCC cells, and the TIAM1 level was much higher in CAFs than in OSCC cells. Furthermore, TIAM1 knockdown in CAFs might partly abolish the promotion of CAFs on OSCC cell development, implying that TIAM1 might be secreted by CAFs into the culture medium to exert its effects inside OSCCs. TIAM1 might increase ZEB2 expression, and ZEB2 upregulation might partly reverse the repression of TIAM1 silencing in CAFs on OSCC cell malignant behaviors. In vivo studies confirmed that CAFs accelerated OSCC tumor growth, these effects were partially counteracted by TIAM1 downregulation. Overall, TIAM1 secreted by CAFs could expedite OSCC cell growth and metastasis by regulating ZEB2, providing a promising therapeutic target for OSCC treatment.

Antihypertensive agent losartan promotes tongue squamous cell carcinoma cell proliferation via EGFR/ERK1/2/cyclin D1 signaling axis

ObjectiveTo study the effects of losartan, an angiotensin II receptor blocker, in the SCC4 and SCC25 human tongue squamous cell carcinoma cell lines. MethodsCell proliferation was measured by MTS/PMS activity and trypan blue exclusion assays. The levels of the cell proliferation marker, cyclin D1, were analyzed by western blotting. Apoptosis was assessed by caspase-3 activation and Annexin V-FITC/propidium iodide double staining. Activation of epidermal growth factor receptor (EGFR) and ERK1/2 was validated by western blotting. ResultsModerate concentrations of losartan enhanced the proliferation of SCC4 and SCC25 cells. However, high losartan concentrations induced apoptosis in SCC4 cells. Losartan activated the EGFR/ERK1/2/cyclin D1 signaling axis, which in turn promoted cell proliferation. Afatinib (EGFR inhibitor) and U0126 (ERK1/2 inhibitor) abolished losartan-induced cell proliferation. In contrast, UC2288 (p21 inhibitor) enhanced it. ConclusionsLosartan exhibited dual effects on tongue squamous cell carcinoma cells. Moderate losartan concentrations facilitated cell proliferation, whereas high concentrations induced cytotoxicity in tongue carcinoma cells.

STAT3 interactome predicts presence of proteins that regulate immune system in oral squamous cell carcinoma

ObjectivesSignal transducer and activator of transcription 3 (STAT3) is one of the key proliferation mechanism–related proteins that helps in oral squamous cell carcinoma (OSCC) progression. Immune evasion by STAT3 is mediated by the JAK2/STAT3/PDL1 signaling axis. Based on previous findings, we hypothesized that STAT3-binding partners participate in the inhibition of anti-tumor activity in OSCC. MethodsA 3D cancer-immune co-culture model was constructed using oral cancer cell lines SCC4, SCC9, SCC25, and CAL27 and normal oral cell line OKF6. The cells were co-cultured with natural killer (NK-92) and Jurkat cells. The target protein STAT3 was chosen based on SWATH data, and co-immunoprecipitation (Co-IP)-based proteomics was conducted. The Co-IP LC-MS/MS output was analysed to determine the protein interaction network, gene ontology, pathway analysis, and protein cluster annotation. ResultsSTAT3 in oral cancer cell lines interacts with the epidermal growth factor receptor (EGFR) and other proteins that participate in proliferation and immune mechanisms. Proteome analysis showed that some STAT3-binding proteins found in this study are known immune system regulators. ConclusionOverall, STAT3 interactive proteins regulate the immune system in oral squamous cell carcinoma cells.

14-3-3σ/Stratifin and p21 limit AKT-related malignant progression in skin carcinogenesis following MDM2-associated p53 loss.

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

Induction of Endoplasmic Reticulum Stress and Aryl Hydrocarbon Receptor Pathway Expression by Blue LED Irradiation in Oral Squamous Cell Carcinoma.

Photobiomodulation therapy, as an emerging treatment modality, has been widely used in dentistry. However, reports on blue light therapy for oral cancer are scarce. This study investigated the effects of 457 and 475 nm LED irradiation on SCC-25 cells and explored the potential mechanisms underlying the impact of blue light. Both wavelengths were found to inhibit cell viability, induce oxidative stress, and cause cell cycle arrest without leading to cell death. Notably, the inhibitory effect of 457 nm blue light on cell proliferation was more sustained. Transcriptome sequencing was performed to explore the underlying mechanisms, revealing that blue light induced endoplasmic reticulum stress in SCC-25 cells, with 457 nm light showing a more pronounced effect. Moreover, 457 nm blue light upregulated the expression of the aryl hydrocarbon receptor pathway, indicating potential therapeutic prospects for the combined use of blue light and pharmacological agents.