Neck Squamous Carcinoma Cell Lines Research Articles

Engineering Dimeric EGFR-directed IgA Antibodies Reveals a Central Role of CD147 during Neutrophil-mediated Tumor Cell Killing of Head and Neck Squamous Cancer Cells.

Human IgA Abs engage neutrophils for cancer immunotherapy more effectively than IgG Abs. Previous studies demonstrated that engineering approaches improved biochemical and functional properties. In this study, we report a novel, to our knowledge, IgA2 Ab against the epidermal growth factor receptor generated by protein engineering and polymerization. The resulting molecule demonstrated a covalent linkage of L and H chains and an effective polymerization by the joining chain. The engineered dimer outperformed its monomeric variant in functional experiments on Fab-mediated modes of action and binding to the Fc receptor. The capacity to engage neutrophils for Ab-dependent cell-mediated cytotoxicity (ADCC) of adherent growing target cancer cells was cell line dependent. Although the engineered dimer displayed a long-term efficacy against the vulva carcinoma cell line A431, there was a notable in-efficacy against human papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC) cell lines. However, the highly engineered IgA Abs triggered a neutrophil-mediated cytotoxicity against HPV+ HNSCC cell lines. Short-term ADCC efficacy correlated with the target cells' epidermal growth factor receptor expression and the ability of cancer cell-conditioned media to enhance the CD147 surface level on neutrophils. Notably, the HPV+ HNSCC cell lines demonstrated a significant increment in releasing soluble CD147 and a reduced induction of membranous CD147 on neutrophils compared with HPV- cells. Although membranous CD147 on neutrophils may impair proper IgA-Fc receptor binding, soluble CD147 enhanced the IgA-neutrophil-mediated ADCC in a dose-dependent manner. Thus, engineering IgA Abs and impedance-based ADCC assays provided valuable information regarding the target-effector cell interaction and identified CD147 as a putative critical parameter for neutrophil-mediated cytotoxicity.

Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Bioinformatics approach combined with experimental verification reveals OAS3 gene implicated in paclitaxel resistance in head and neck cancer.

This study aimed to identify a candidate gene associated with paclitaxel (PTX) resistance and to evaluate functionally its biological role in the PTX-resistant head and neck squamous cell carcinoma (HNSCC) cell lines and clinical specimens. Microarray data series containing samples of different types of cancers resistant to PTX were analyzed and then a candidate gene associated with PTX resistance was identified using various bioinformatics tools. After the suppression of the target gene expression, changes in cell viability and colony-forming ability were evaluated in PTX-resistant FaDu and SCC-9 cell lines. Bioinformatics analyses of upregulated genes in PTX-resistant cancer cells indicated that OAS3 was associated with PTX resistance. The downregulation of OAS3 expression significantly reduced the viability and colony-forming capacity of PTX-resistant SCC-9 cells by inducing apoptosis and cell cycle arrest at G0/G1 phase. The therapeutic targeting of OAS3 may resensitize PTX-resistant HNSCC cells with high OAS3 expression to PTX treatment.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Abstract B022: A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion

Abstract Our previous results demonstrate an integrin-mediated mechanism of immune evasion in colorectal cancer. Increased levels of latent TGFβ in the experimental tumor environment led to the upregulation of the immune checkpoint marker PD-L1 in HCT116WT cells, facilitating escape from T-cell killing. This effect was rescued in immune co-cultures by pretreating cancer cells with the broad-spectrum integrin inhibitor GLPG-0187. To further elucidate this system, we investigated the TGFβ-integrin axis. Integrin αvβ6 is a major activator of the inert latent TGFβ into the active, immunosuppressive form. With its high specificity within malignant tissue, inhibition of αvβ6 poses an attractive therapeutic target for disrupting TGFβ signaling while avoiding the toxicities of systemic blockade. To identify an endogenous in vitro model of integrin αvβ6-expressing cancer, computational analysis of publicly available mRNA expression data was performed to determine cancer types with high αvβ6 upregulation. This investigation revealed a strong presence of αvβ6 in pancreatic, bladder, head and neck, and cervical cancer tissues. Further in-silico work identified the head and neck squamous cell carcinoma cell lines CAL-27 and FaDu, as well as the pancreatic adenocarcinoma line Capan-2, as suitable models for in vitro study. Flow cytometry of these cell lines was performed to determine the surface expression of integrin αvβ6, revealing that protein expression correlated with the mRNA data from the previous computational studies. Capan-2 and CAL-27 exhibited high levels αvβ6 of expression, while FaDu expressed moderate levels, and HCT116WT exhibited low αvβ6 expression. These cell lines were pretreated with latent TGFβ, simulating sequestered latent TGFβ in the extracellular matrix, and subsequently co-cultured with TALL-104 T-cells. Quantitative fluorescence microscopy revealed that resistance to killing by T-cells correlated with αvβ6 expression across cell lines. These findings are consistent with the hypothesis that upregulated expression of integrin αvβ6 functions as an immunosuppressive resistance mechanism by increasing tumor microenvironment levels of active TGFβ. Analysis of mRNA sequencing data from the TCGA Pancreatic Adenocarcinoma Firehose database revealed that integrin αvβ6 is a strong marker of poor prognosis in pancreatic adenocarcinoma. Future co-culture experiments will investigate the dynamics of TGFβ activation in the tumor microenvironment in relation to αvβ6 expression. Further in vivo studies can facilitate translation of our findings to therapeutic clinical trials. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Vida Tajiknia, Maximilian Pinho-Schwermann, Connor Purcell, Brooke Verschleiser, Wafik S. El-Deiry. A gradient expression of integrin αvβ6 expression in human cancer cells associates with TGFβ mediated immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B022.

Chemopreventive Efficacy of Sulindac Sulfone as a Selective Apoptotic Antineoplastic Drug in Human Head and Neck Squamous Cell Carcinoma Cell Lines: A Systematic Review.

Sulindac sulfone, an active metabolite of sulindac, a non-steroidal anti-inflammatory drug, has good anti-inflammatory potential. The antineoplastic effect of sulindac sulfone is mediated through a cyclooxygenase inhibitory mechanism, followed by apoptosis and inhibition of cell proliferation. Mounting studies have explored the anti-neoplastic effect of sulindac sulfone in various types of cancers in a dose-dependent manner. In this backdrop, we have conducted a systematic review to evaluate the efficacy and dose of sulindac sulfone as an anti-neoplastic agent in human head and neck squamous cell carcinoma cell lines (HNSCCs). In this study, we used a systematic literature review approach, and articles were searched in PubMed, and Medline with the keywords "sulindac sulfone," "anti-neoplastic activity," "chemopreventive," and "head and neck squamous cell carcinoma". A hand-search of journals was also performed. Articles were reviewed and analyzed. The analysis reveals that, based on the in vitro studies on various tumor models, the optimum concentration of sulindac sulfone which elicits anti-neoplastic effects is 200-800 µM. The anti-neoplastic effect is mediated through inhibition of cell proliferation and apoptosis. The results of our systematic review show that the anti-neoplastic activity of pharmacologic Sulindac sulfone is part of its dose-dependent activity, which can be safely employed in the therapy for human HNSCCs and would be responsible for a beneficial outcome of the treatment.

An AI-assisted integrated, scalable, single-cell phenomic-transcriptomic platform to elucidate intratumor heterogeneity against immune response.

We present a novel framework combining single-cell phenotypic data with single-cell transcriptomic analysis to identify factors underpinning heterogeneity in antitumor immune response. We developed a pairwise, tumor-immune discretized interaction assay between natural killer (NK-92MI) cells and patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines on a microfluidic cell-trapping platform. Furthermore we generated a deep-learning computer vision algorithm that is capable of automating the acquisition and analysis of a large, live-cell imaging data set (>1 million) of paired tumor-immune interactions spanning a time course of 24 h across multiple HNSCC lines (n = 10). Finally, we combined the response data measured by Kaplan-Meier survival analysis against NK-mediated killing with downstream single-cell transcriptomic analysis to interrogate molecular signatures associated with NK-effector response. As proof-of-concept for the proposed framework, we efficiently identified MHC class I-driven cytotoxic resistance as a key mechanism for immune evasion in nonresponders, while enhanced expression of cell adhesion molecules was found to be correlated with sensitivity against NK-mediated cytotoxicity. We conclude that this integrated, data-driven phenotypic approach holds tremendous promise in advancing the rapid identification of new mechanisms and therapeutic targets related to immune evasion and response.

The ATR inhibitor berzosertib acts as a radio- and chemosensitizer in head and neck squamous cell carcinoma cell lines

Alterations in the DNA damage response play a crucial role in radio- and chemoresistance of neoplastic cells. Activation of the Ataxia telangiectasia and Rad3-related (ATR) pathway is an important DNA damage response mechanism in head and neck squamous cell carcinoma (HNSCC). Berzosertib, a selective ATR inhibitor, shows promising radio- and chemosensitizing effects in preclinical studies and is well tolerated in clinical studies. The aim of this study was to elucidate the effect of berzosertib treatment in combination with radiation and cisplatin in HNSCC. The HNSCC cell lines Cal-27 and FaDu were treated with berzosertib alone and in combination with radiation or cisplatin. Cell viability and clonogenic survival were evaluated. The effect of combination treatment was evaluated with the SynergyFinder or combination index. Apoptosis was assessed via measurement of caspase 3/7 activation and migration was evaluated using a wound healing assay. Berzosertib treatment decreased cell viability in a dose-dependent manner and increased apoptosis. The IC50 of berzosertib treatment after 72 h was 0.25–0.29 µM. Combination with irradiation treatment led to a synergistic increase in radiosensitivity and a synergistic or additive decrease in colony formation. The combination of berzosertib and cisplatin decreased cell viability in a synergistic manner. Additionally, berzosertib inhibited migration at high doses. Berzosertib displays a cytotoxic effect in HNSCC at clinically relevant doses. Further evaluation of combination treatment with irradiation and cisplatin is strongly recommended in HNSCC patients as it may hold the potential to overcome treatment resistance, reduce treatment doses and thus mitigate adverse events.

Heparan-6-O-Endosulfatase 2 Promotes Invasiveness of Head and Neck Squamous Carcinoma Cell Lines in Co-Cultures with Cancer-Associated Fibroblasts.

Local invasiveness of head and neck squamous cell carcinoma (HNSCC) is a complex phenomenon supported by interaction of the cancer cells with the tumor microenvironment (TME). We and others have shown that cancer-associated fibroblasts (CAFs) are a component of the TME that can promote local invasion in HNSCC and other cancers. Here we report that the secretory enzyme heparan-6-O-endosulfatase 2 (Sulf-2) directly affects the CAF-supported invasion of the HNSCC cell lines SCC35 and Cal33 into Matrigel. The Sulf-2 knockout (KO) cells differ from their wild type counterparts in their spheroid growth and formation, and the Sulf-2-KO leads to decreased invasion in a spheroid co-culture model with the CAF. Next, we investigated whether a fucosylated chondroitin sulfate isolated from the sea cucumber Holothuria floridana (HfFucCS) affects the activity of the Sulf-2 enzyme. Our results show that HfFucCS not only efficiently inhibits the Sulf-2 enzymatic activity but, like the Sulf-2 knockout, inhibits Matrigel invasion of SCC35 and Cal33 cells co-cultured with primary HNSCC CAF. These findings suggest that the heparan-6-O-endosulfatases regulate local invasion and could be therapeutically targeted with the inhibitory activity of a marine glycosaminoglycan.

ΔNp63 silencing, DNA methylation shifts, and epithelial-mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma

TP63 (p63) is strongly expressed in lower-grade carcinomas of the head and neck, skin, breast, and urothelium to maintain a well-differentiated phenotype. TP63 has two transcription start sites at exons 1 and 3′ that produce TAp63 and ΔNp63 isoforms, respectively. The major protein, ΔNp63α, epigenetically activates genes essential for epidermal/craniofacial differentiation, including ΔNp63 itself. To examine the specific role of weakly expressed TAp63, we disrupted exon 1 using CRISPR-Cas9 homology-directed repair in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells having either monoallelic GFP cassette insertion paired with a frameshift deletion allele or biallelic GFP cassette insertion exhibited ΔNp63 silencing. Loss of keratinocyte-specific gene expression, switching of intermediate filament genes from KRT(s) to VIM, and suppression of cell-cell and cell-matrix adhesion components indicated the core events of epithelial-mesenchymal transition. Many of the positively and negatively affected genes, including ΔNp63, displayed local DNA methylation changes. Furthermore, ΔNp63 expression was partially rescued by transfection of the TAp63 knockout cells with TAp63α and application of DNA methyltransferase inhibitor zebularine. These results suggest that TAp63, a minor part of the TP63 gene, may be involved in the auto-activation mechanism of ΔNp63 by which the keratinocyte-specific epigenome is maintained in SCC.

Assessment of Therapeutic Bio-Activity of Cinnamoyl Sulfonamide Hydroxamate in Squamous Cell Carcinoma.

Background Cancer isthe second most common cause of death. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Many treatment modalities such as chemotherapy, radiotherapy, surgery, and immunotherapy are emerging but still, the patients' quality of life is questionable. Despite the advances in therapeutic approaches, the percentages of morbidity and mortality of OSCC have not improved significantly during the last 30 years. Treatment using natural products can act as a potent anti-cancer agent with reduced adverse effects. Cinnamic acid derivatives exhibit anti-cancer potential through histone deacetylase inhibitor (HDAC) enzyme inhibition. Methodology In an experimental study design, cinnamoyl hydroxamate derivatives were prepared. The structure was confirmed using ultraviolet-visible spectroscopy (UV-Vis), nuclear magnetic resonance (NMR), infrared spectroscopy, and mass spectrophotometry. An in-vitro antioxidant assay using nitric oxide scavenging and reducing power assay was done and an in-vitro cytotoxic (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and viability assay were carried out using tryphan blue dye. Results Statistical analysis was performed using SPSS (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp). Cinnamoyl hydroxamate derivatives were obtained and named as compounds3a (E)-N-Hydroxy-3-(4-(N-(phenyl bromo) sulfamoyl) phenyl) acrylamide-)and 3b ((E)-N-Hydroxy-3-(4-(N-(phenyl nitro) sulfamoyl) phenyl) acrylamide). In the nitric oxide scavenging assay, compound 3a showed good antioxidant activity than 3b. Reducing power assay was higher in 3a compared to 3b. Cell viability using tryphan blue exhibited a concentration decrease in % cell viability with an increase in the concentration of human oral cavity squamous cell carcinoma cell line (OECM 1), a unique head and neck squamous carcinoma cellline (UM SCC 6) & human oral squamous cell carcinoma forming metastatic foci (HSC 3) cell lines. Conclusion The results of the present study revealed that the study compounds play a vital role in the up-regulation of apoptotic pathways and regulation of terminal differentiation pathways. The compounds showed good anti-oxidant and anti-cancer activities in lesser concentrations, hence they can be used as a therapeutic agent for oral squamous cell carcinoma.

Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells

Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive. The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells. Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells. SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.

Co-Delivery of Cisplatin and Curcumin Using Mesoporous Silica Nanoparticles to Improve their Anticancer Effects.

This study aimed to prepare and evaluate the physicochemical and anticancer properties of cisplatin and curcumin-loaded mesoporous silica nanoparticles (Cis-Cur-MSNs). In recent years, combination treatment has attained better outcomes than monotherapy in oncology. Cis-Cur-MSNs were prepared by precipitation technique. The objective of the present study was the evaluation of the physicochemical and anticancer properties of cisplatin and curcumin-loaded mesoporous silica nanoparticles (Cis-Cur-MSNs). The prepared materials were assessed in terms of physicochemical methods. The drug release pattern from the MSNs was also evaluated via ultraviolet spectrophotometry. In addition, the porosity and surface area of prepared nanoparticles were determined using the Brunauer-Emmett-Teller (BET) technique. The cytotoxicity of Cis-Cur-MSNs was evaluated on the HN5 cells as head and neck squamous carcinoma cell lines. Furthermore, ROS production of Cis-Cur-MSNs treated cells was evaluated compared with untreated cells. According to the results, prepared nanoparticles displayed nanometer size, rod morphology, and negative surface charge with mesoporous structure belonging to the MCM-41 family (twodimensional hexagonal). Regarding the results of BET adsorption and desorption isotherm analysis for Cis-Cur-MSNs and drug-free MSNs, pore diameter, pore volume, specific surface area, and drug-loaded pore area in MSNs were decreased. In the first 10 days, the prepared nanoparticles exhibited a relatively rapid release pattern for cisplatin and curcumin, and until the 35th day, the release of them from the MSNs continued slowly. The cytotoxic effect of Cis-Cur-MSNs was significantly more than Cur-MSNs and Cis- MSNs in 24 and 48 h incubation time (p < 0.05). The results suggest that Cis-Cur-MSNs may be beneficial in the development of a cancer treatment protocol. Others: The prepared nanoparticle in the present study could be a potential biomaterial for cancer treatment.

Cystatin A Down-regulation in Head and Neck Squamous Cell Carcinoma Cell Lines Decreases Cancer Hallmark Signatures

Abstract Background Cystatin A (CSTA), an endogenous inhibitor of lysosomal cysteine protease, is expressed primarily in epithelial tissues. The expression of CSTA was found to be dysregulated in various cancers and associated with cancer pathogenesis, but its role is reported to be contradictory. Our previous preliminary study found CSTA to be upregulated in the saliva and tissues of patients with head and neck squamous cell carcinoma (HNSCC). In this current study, we have explored the role of CSTA in the pathophysiology of HNSCC. Methods First, we confirmed the upregulation of CSTA in CAL 27 (p = 0.0242) and FaDu (p = 0.0014), two HNSCC cell lines, compared to the normal gingival epithelium. CSTA was then stably knocked down in CAL 27 and FaDu using the lentiviral short hairpin RNA pLKO vector transduction to study the effects of CSTA knockdown on various cancer hallmarks such as cell proliferation ability, invasion, migration, colony formation, and chemotherapy-induced apoptosis. Results CSTA knockdown significantly decreased cell viability, cell migration, transwell invasion, and colony formation in both cell lines. CSTA downregulation also enhanced cisplatin-induced apoptosis. Conclusion Overall, this study suggests the protumorigenic role of CSTA in HNSCC.

Artesunate and cisplatin synergistically inhibit HNSCC cell growth and promote apoptosis with artesunate‑induced decreases in Rb and phosphorylated Rb levels.

In the treatment of head and neck cancer, cisplatin is often used as a therapeutic agent; however, its efficacy is limited and it can cause renal dysfunction as an adverse effect. For this reason, the use of cisplatin is limited in elderly patients with reduced renal function. Recently, artemisinin, which was developed as an antimalarial drug, was found to have antitumor effects and is effective in combination with other anticancer drugs. In the present study, the antitumor effects of artemisinin and its derivatives as well as their combination with cisplatin and iron on head and neck squamous cell carcinoma cell lines, were investigated. Cell viability was determined by a cell viability assay, the cell cycle was analyzed by flow cytometry, cell death was assessed with annexin V and propidium iodide staining, and western blotting was used to analyze retinoblastoma protein (Rb), phosphorylated (p‑)Rb, and other cell cycle‑associated molecules. A total of four artemisinin compounds were examined and it was found that artesunate and dihydroartemisinin had a significant inhibitory effect on growth. It was also identified that the combination of artesunate, cisplatin, and iron inhibited cell proliferation and caused S/G2‑M cell cycle arrest. In addition, western blotting of Rb, a molecule involved in the cell cycle, showed that artesunate induced the loss of not only Rb but also p‑Rb. These results suggested that artesunate is a useful drug in combination with cisplatin.

Multi-Omics Identification of Genetic Alterations in Head and Neck Squamous Cell Carcinoma and Therapeutic Efficacy of HNC018 as a Novel Multi-Target Agent for c-MET/STAT3/AKT Signaling Axis.

Amongst the most prevalent malignancies worldwide, head and neck squamous cell carcinoma (HNSCC) is characterized by high morbidity and mortality. The failure of standard treatment modalities, such as surgery, radiotherapy, and chemotherapy, demands the need for in-depth understanding of the complex signaling networks involved in the development of treatment resistance. A tumor's invasive growth and high levels of intrinsic or acquired treatment resistance are the primary causes of treatment failure. This may be a result of the presence of HNSCC's cancer stem cells, which are known to have self-renewing capabilities that result in therapeutic resistance. Using bioinformatics methods, we discovered that elevated expressions of MET, STAT3, and AKT were associated with poor overall survival in HNSCC patients. We then evaluated the therapeutic potential of our newly synthesized small molecule HNC018 towards its potential as a novel anticancer drug. Our computer-aided structure characterization and target identification study predicted that HNC018 could target these oncogenic markers implicated in HNSCC. Subsequently, the HNC018 has demonstrated its anti-proliferative and anticancer activities towards the head and neck squamous cell carcinoma cell lines, along with displaying the stronger binding affinities towards the MET, STAT3, and AKT than the standard drug cisplatin. Reduction in the clonogenic and tumor-sphere-forming ability displays HNC018's role in decreasing the tumorigenicity. Importantly, an vivo study has shown a significant delay in tumor growth in HNC018 alone or in combination with cisplatin-treated xenograft mice model. Collectively with our findings, HNC018 highlights the desirable properties of a drug-like candidate and could be considered as a novel small molecule for treating head and neck squamous cell carcinoma.

Effect of Curcumin on the Head and Neck Squamous Cell Carcinoma Cell Line HN5.

Curcumin has been isolated from the rhizomes of Curcuma longa. Over the years, it has shown outstanding therapeutic potential in various human disorders, including cancers. The aim is to study curcumin's effects on the apoptosis signaling pathway in the head and neck squamous cell carcinoma (HNSCC) cell line HN5. The cytotoxicity of curcumin on HN5 cells were assessed. In addition, HN5 cells were also treated with curcumin to evaluate its effect on the caspase-8, -9, Bcl-2, Bax, and Stat3 gene expressions. The results exhibited that cell viability reduced following curcumin treatment in a concentration- dependent manner. Curcumin treatment caused decreased expression of Bcl2, with simultaneous upregulation of the Bax/Bcl2 ratio. Curcumin increased caspase-9 expression, did not affect caspase-8, and decreased Stat3 expression. The induction of the mitochondria-dependent apoptosis pathway of curcumin happened by modulating the expression of Bcl2 and Bax genes, resulting in the caspase-9 activation. Furthermore, curcumin decreased the expression of the Stat3 in HN-5 cells. In conclusion, curcumin showed marked anticancer effects in the HN-5 cell line by modulating Stat-3; Bax/Bcl-2 expression in vitro.

Abstract 1411: Different treatment response in several head and neck squamous cell carcinoma(HNSCC) cell lines reflecting underlying genomic &amp; molecular signatures

Abstract 1411: Different treatment response in several head and neck squamous cell carcinoma(HNSCC) cell lines reflecting underlying genomic &amp; molecular signatures

In vitro evaluation of photon and carbon ion radiotherapy in combination with cisplatin in head and neck squamous cell carcinoma cell lines.

Heavy ion radiotherapy, such as carbon ion radiotherapy (CIRT), has multiple advantages over conventional photon therapy. Cisplatin, as a classic anti-tumor drugs, has been tested and discovered as a photon radiosensitizer in several cell lines, including head and neck squamous cell carcinoma (HNSCC). Hence, the aim of our study is to evaluate whether cisplatin can sensitize CIRT towards HNSCC cell lines in vitro. Human nasopharyngeal carcinoma cell line CNE-2, human tongue squamous carcinoma cell line TCA 8113 and human hypopharynx squamous carcinoma cell line FADU were all irradiated with photon beam of 2, 4, 6, 8 Gy (physical dose) and carbon ion beam of 1, 2, 3, 4 Gy (physical dose) and treated with cisplatin. Cell survival was assessed by clonogenic survival assay. CIRT showed significantly stronger cytotoxic effect than standard photon radiotherapy. The relative biological effectiveness (RBE) of carbon ion beam at 10% survival ( ) was calculated 3.07 for CNE-2, 2.33 for TCA 8113 and 2.36 for FADU. Chemoradiotherapy (both photon radiotherapy and CIRT) was more effective than radiotherapy alone. In vitro sensitizer enhancement ratios (SERs) of cisplatin in CNE-2, TCA 8113 and FA DU cell lines after photon irradiation were 1.33, 1.14 and 1.21, while after carbon ion irradiation were 1.02, 1.00 and 0.96, showed that cisplatin sensitized photon irradiation but showed no sensitization effect in carbon ion irradiation in all tested cell lines. In conclusion, high linear energy transfer (LET) CIRT was more effective than photon irradiation to prevent the proliferation of HNSCC cell lines. Additional treatment with cisplatin could sensitize photon irradiation but showed no effect on carbon ion irradiation.