Squamous Cell Lines Research Articles

Negative terpinen-4-ol modulate potentially malignant and malignant lingual lesions induced by 4-nitroquinoline-1-oxide in rat model.

Our aim was to verify the modulative TP-4-ol capacity in 4-nitroquinoline-1-oxide induced oral rat cancer. The stereoisomers of TP-4-ol were used against the human tongue squamous cell line and the negative stereoisomer showed lower IC50. Thirty-one Holtzman rats (120-130g) were cancer-induced by 4-nitroquinoline-1-oxide (4-NQO/8weeks/25ppm) and 32 Holtzman rats (120-130g) were used to healthy and TP-4-ol toxicity experiments. Six groups were used, healthy, 0.1nL/g of TP-4-ol, 8nL/g of TP-4-ol, 4-NQO, 4-NQO + 0.1nL/g of TP-4-ol, and 4-NQO + 8nL/g of TP-4-ol. We performed the toxicity analysis by biochemical and histopathological analysis. The biochemistry analysis includes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), urea, and creatinine and the histopathology analysis includes the liver, kidney, lung, and spleen. Specifically, for malign modulation, we performed a macroscopic and microscopic analysis. The group exposed to 0.1nL/g of TP-4-ol demonstrated a reduced risk of malignancy in dysplasia considering the criteria of architecture and cytology. Similarly, a drop of percentual rats with SCC diagnosis was observed in 4-NQO + 0.1nL/g (41.6%) when compared to 4-NQO (87.5%). Moreover, the 4-NQO group presented a median of 2.62 SCC/rat and the 4-NQO + 0.1nL/g demonstrated a median of 0.75 SCC/rat. For toxicity analysis, 4-NQO + 0.1nL/g showed focal necrosis in the kidney and 4-NQO showed lung hemorrhagic areas. The concentration of 0.1nL/g was more effective in reducing the tongue induction of potentially malignant and malignant lesions by 4-NQO. A kidney toxicity was observed in healthy animals exposed to 0.1nL/g of TP-4-ol. The negative isoform of terpinen-4-ol negatively modulates the development of potentially malignant and malignant lesions in rats (Rattus nonverdicts albinos, Holtzman) exposed to 4-NQO. (-)-Terpinen-4-ol reduced the mice percentual with squamous cell carcinoma, 87.5 to 41.6%, and decreased the cancer/rat ratio of 2.62 in 4-NQO to 0.75 in 4-NQO + 0.1nL/g. This represents 52.4% by group and 71.3% in the cancer/rat ratio.

Abstract LB030: Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers

Abstract Small cell lung cancer (SCLC) and other neuroendocrine (NE) lung tumors account for 25% of all lung cancers, they are the most aggressive type and have the poorest prognosis. SCLC does not present driver oncogenes involved in the regulation of kinase signaling, which makes these tumors hard to treat. The few available treatments yield responses early, but relapse occurs and they become resistant. Due to the poor efficacy of therapies, research in SCLC had been moved to the background, with basically no progress over the past decades. Mutations in the RAS-ERK pathway, elevating ERK activity and enhancing tumorigenesis, are common in other types of cancer, including NSCLC. Nevertheless, they are rare in SCLC. Indeed, previous studies demonstrated that ERK activation interferes with growth of SCLC. However, the mechanism by which ERK hinders tumor progression has not been defined yet. One of the most important players in SCLC and other NE tumors is the transcription factor ASCL1, essential for their survival. It has been reported that ASCL1 increases DUSP6 transcription with the consequent ERK activity suppression. Its downregulation affects cell cycle progression and survival. Conversely, inhibition of ERK activation by MEK inhibitors increases ASCL1 mRNA levels. Previous studies in our laboratory suggested that DUSP6 has a major role in a subset of SCLC and NSCLC-NE cell lines. Inhibition of DUSP6 enhances ERK signaling and significantly decreases survival of NE lung tumor cells. We have considered DUSP6 a good candidate to try to understand the mechanism by which ERK interferes with survival. However, hurdles in studying DUSP6 in SCLC include off-target effects of inhibitors and difficulty of gene-edited cell lines. To date, we have been able to generate a DUSP6 KO NSCLC-NE cell line which shows a clear drop in proliferation. Also, the overexpression of ERK or a constitutively active MEK1 mutant in a few SCLC, NE-NSCLC and MEK inhibitor-insensitive squamous lung cancer cell lines render a decrease in survival. DUSP6 is a cytoplasmic anchor for ERK and may reduce the ability of ERK to suppress ASCL1 transcription. For this reason, we are working on studying the effect on survival of ERK localization in the nucleus or the cytoplasm. With our ongoing experiments we are defining the conditions to examine transcriptional impact of ERK on SCLC. Discovering molecular events antagonized by the ERK pathway in SCLC will reveal novel targets controlling the inhibitory mechanisms. One of the SCLC major questions of translational relevance is what are the mechanisms upholding the development of chemoresistance, where the ERK pathway seems to be involved. Hence, the definition of the underlying mechanisms of tumorigenesis and therapy resistance will uncover new potential antitumor targets that currently escape our notice. A better understanding of the molecular biology of SCLC will let us find new therapeutic strategies to overcome the impasse in chemotherapy. Citation Format: Ana Martin-Vega, Svetlana Eartnest, John D. Minna, Jane E. Johnson, Melanie H. Cobb. Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB030.

Abstract 1858: Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels

Abstract Human papilloma virus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is common in young patients (<60 years). All patients experience either permanent toxicity from standard therapy or death from recurrent disease. To address this unmet need for more effective and less toxic therapy, we performed a high throughput drug screen comparing drug efficacy in HPV+ and HPV- cell lines. We tested the drug sensitivity of 864 unique drugs (0-3.1 µM) divided into 51 classes based on their targets in 33 squamous cancer cell lines using a parameter that is independent of cell division. Only one class of drugs, Aurora kinase inhibitors, was more effective in HPV+ than HPV- cell lines. To validate our screening results, we tested two Aurora kinase inhibitors (Alisertib, Barasertib) and observed significant cell death in HPV+ cells, but not in HPV- cells. Alisertib reduced tumor volume in HPV+ PDX mouse models without toxicity. To investigate the underlying mechanism, we compared the expression of ~300 proteins, using RPPA, to drug efficacy and validated the results using immunoblotting. The expression of Rb, pRb(S807/811), and p16 correlated with drug sensitivity. To establish role for Rb, we used a bidirectional approach to manipulate RB1. Reduction in RB1 levels using either shRNA or stable E7 overexpression in HPV- cells resulted in increased Alisertib-induced apoptosis. We also used siRNA to knock down E7 expression in two HPV+ cell lines which resulted in elevated levels of Rb, thereby rescuing the cells from Alisertib-induced apoptosis. Studies have shown Rb-deficient cells have elevated levels of the mitotic-checkpoint protein MAD2, that may impact cell fitness. To test the role of MAD2, we depleted the levels of MAD2 and observed diminishing levels of cyclin B1 accumulation and reduced Alisertib-induced apoptosis. Additionally, we altered the activated levels of MAD2 protein by overexpression of TRIP13 that resulted in partially rescuing cells from undergoing Alisertib-induced apoptosis. These results indicate that Rb- deficient MAD2 overexpressing cells require a finely tuned balance of MAD2 and TRIP13 levels for their mitotic exit. Furthermore, we observed combined inhibition of TRIP13 with Aurora A led to more apoptosis in Rb-deficient cells than the single agents. Our results demonstrate that Rb-deficient cancers are sensitive to Aurora A inhibition due to an imbalance between MAD2 and TRIP13. Based on the above finding, our present study shows that TRIP13 inhibition in combination with Aurora kinase inhibitors leads to more apoptosis and can pave a path for novel therapies in the management of HPV+ tumors. Citation Format: Soma Ghosh, Tuhina Mazumdar, Wei Xu, Reid T. Powell, Clifford Stephan, Li Shen, Curtis R. Pickering, Jing Wang, Faye M. Johnson. Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1858.

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.

Abstract 5407: Targeting NRF2 using specific inhibitor brusatol sensitized esophageal adenocarcinoma cells to cisplatin through inducing ferroptosis

Abstract Background: The incidence rate of the esophageal adenocarcinoma (EAC) has increased rapidly among men during the past four decades in the USA and western countries while the 5 year survival rate is still below 20%. Traditional chemotherapy is still the main stream strategy in treatment of EAC patients. However, chemo-resistance usually develops that finally leads to failure of treatments. NFE2-related factor 2 (NRF2) is the master regulator of cellular anti-oxidant properties that maintains cell viability and cellular homeostasis. Overexpression of NRF2 in human cancers are associated with drug-resistance and poor prognosis. Methods and Results: NRF2 protein levels are higher in several EAC cell lines, as compared to BE cell lines (CPA, BAR10T) and normal esophageal squamous cell lines, using Western blot analysis. We confirmed NRF2 overexpression in primary EAC samples, using immunohistochemistry on a tissue microarray that contained 76 EACs. Brusatol is an NRF2 specific inhibitor originated from natural plant. Administration of Brusatol in EAC cells significantly inhibited NRF2 ARE luciferase activity and reduced gene expression of NRF2 target genes such as GR, HO1, GSTM2, GPX3 and GPX4, resulting in a significant increase in cellular ROS levels, which were further increased by treatment with CDDP with subsequent increase in cancer cell death. Further investigation revealed that Brusatol significantly increased lipid peroxidation levels as detected by immunofluorescence assay and flow cytometry and resulted in significant ferroptosis, in particular in combination with CDDP treatments. We confirmed that Brusatol in combination with CDDP significantly suppressed tumor growth in a xenografting mice model. Conclusion: Constitutive overexpression of NRF2 in EAC cells favors tumor cell survival and drug resistance. NRF2 specific inhibitor may synergize with traditional chemotherapeutic drugs by inducing lipid peroxidation and ferroptosis in EAC. Citation Format: Dunfa Peng, Tianling Hu, Lei Chen, Heng Lu, Shoumin Zhu, Wael El-Rifai. Targeting NRF2 using specific inhibitor brusatol sensitized esophageal adenocarcinoma cells to cisplatin through inducing ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5407.

Fibroblast Growth Factor 3 Is Associated with Tongue Squamous Cell Carcinoma: A Controlled Study.

Objective To explore the effects of fibroblast growth factor 3 (FGF3) on the proliferation, cell cycle, and apoptosis of the tongue squamous cell carcinoma SCC-9 cell line (SCC-9). Methods We measured the proliferation of SCC-9 cells in a control group, an FGF3 intervention group, and a fibroblast growth factor (FGFR) inhibitor intervention group in cholecystokinin octapeptide (CCK-8) experiments. We studied effects of FGF3 on the cell cycle and apoptosis of tongue cancer cells using flow cytometry. We further explored the IRS1/PI3K/AKT signaling pathway by measuring BCL-2 and Bcl-2 Associated X-protein (BAX) mRNA and protein levels with RT-PCR and western blot, respectively. Results Results from the CCK-8 experiment showed that survival rates of cells in the control group, FGF3 intervention group, and FGFR inhibitor intervention group were 100.000% ± 4.026%, 136.330% ± 9.779%, and 83.199% ± 4.954%, respectively; survival rates of SCC-9 cells in all three groups were statistically significant (P < 0.05). Compared with that in the control group, the ratio of cells in G0/G1 phase in the FGFR inhibitor intervention group was higher (P < 0.05) and that in G2/M phase was lower, while the FGF3 intervention group showed opposite results (P < 0.05). The apoptosis rate of tongue cancer cells differed significantly between the FGFR inhibitor intervention and the control groups (P < 0.05). The mRNA and protein expression levels of IRS1, PI3K, and BCL-2 were all increased in the FGF3 intervention group (P < 0.05), while BAX mRNA and protein expression levels were decreased (P < 0.05). The mRNA expression levels of protein kinase B (AKT) showed no differences between groups. The p-AKT protein was overexpressed, while the total amount of AKT protein remained stable (P < 0.05). Conclusion FGF3 contributes to the proliferation of SCC-9 cells by increasing the proportion of cells in G2/M phase. Therefore, appropriately timed inhibition of FGF3 can potentially promote tumor apoptosis through the IRS1/PI3K/AKT signaling pathway. Our results demonstrate the role of FGF3 in the tumor microenvironment in tongue squamous cell carcinoma SCC-9 cells and suggest new therapeutic targets.

The radiosensitizing effect of β-Thujaplicin, a tropolone derivative inducing S-phase cell cycle arrest, in head and neck squamous cell carcinoma-derived cell lines

BackgroundResistance to radiotherapy is a common cause of treatment failure in advanced head and neck squamous cell carcinoma (HNSCC). ß-Thujaplicin, a natural tropolone derivative, acts as an anti-cancer agent and has recently been shown to radiosensitize non-HNSCC cancer cells. However, no data is currently available on its radiosensitizing potential in HNSCC.MethodsTo investigate the effect of ß-Thujaplicin and irradiation in HNSCC cell lines CAL27 and FADU, we performed a cell viability assay, colony forming assay, flow cytometry for cell cycle analysis and a wound healing assay. Drug-irradiation interaction was analyzed using a zero-interaction potency model.ResultsTreatment with ß-Thujaplicin led to a dose-dependent decrease in cell viability and enhanced the effect of irradiation. Clonogenic survival was inhibited with synergistic drug-irradiation interaction. ß-Thujaplicin further led to S-phase arrest and increased the sub-G1 population. Moreover, combined ß-Thujaplicin and irradiation treatment had a higher anti-migratory effect compared to irradiation alone.Conclusionsß-Thujaplicin acts as a radiosensitizer in HNSCC cell lines. Further evaluation of its use in HNSCC therapy is warranted.

COX-2 strengthens the effects of acid and bile salts on human esophageal cells and Barrett esophageal cells

AimsInvestigate the effect and mechanism of COX-2 on viability, intestinal metaplasia, and atypia in human esophageal squamous and Barrett esophageal cell lines.MethodsHuman esophageal squamous and Barrett esophageal cell lines were transfected with a COX-2 expression vector and a COX-2 siRNA, and then were treated with acid, bile salts, and a mixture of both. Cell viability, the expression of COX-2, NF-κB(p65), CDX-2, MUC2, c-myb, and BMP-4, and the morphology and microstructure of cells were then observed.ResultsThe viability of COX-2 overexpressed cells was significantly higher than that of control cells, while the viability of COX-2 siRNA-treated cells was significantly lower than that of control cells. Intestinal metaplasia and atypia were observed in cells overexpressing COX-2. Acid, bile salts, and their mixture inhibited the viability of these two cell lines, but the inhibitory effect of the mixture was stronger than a single treatment in either. SiRNA mediated knockdown of COX-2 strengthened the antiproliferative effects of the mixture on HET-1A and BAR-T cells. The expression of p-p65, CDX-2, and BMP-4 was positively correlated with COX-2 expression, while the expression levels of p65, MUC2, and c-myb remained unchanged.ConclusionCOX-2 may influence the viability, atypia, and intestinal metaplasia of human esophageal cells and Barrett esophageal cells. Activation of the p-p65, CDX-2, and BMP-4 signaling pathways by COX-2 may be part of this mechanism.

CRISPR-mediated knockout of VEGFR2/KDR inhibits cell growth in a squamous thyroid cancer cell line.

Squamous and anaplastic thyroid cancers are the most aggressive and life‐threatening cancer types in humans, with the involvement of lymph nodes in 59% of cases and distant metastases in 26% of cases of all thyroid cancers. The median survival of squamous thyroid cancer patients is < 8 months and therefore is of high clinical concern. Here, we show that both VEGFC and VEGFR2/KDR are overexpressed in thyroid cancers, indicating that VEGF/VEGFR signaling plays a carcinogenic role in thyroid cancer development. Using CRISPR/Cas9, we established a KDR knockout (KO) SW579 squamous thyroid cancer cell line that exhibited dramatically decreased colony formation and invasion abilities (30% and 60% reduction, respectively) when compared to scrambled control cells. To validate the potential of KDR as a therapeutic target for thyroid cancers, we used the KDR RTK inhibitor sunitinib. Protein analysis and live/dead assay were performed to demonstrate that sunitinib significantly inhibited cell growth signal transduction and induced cell apoptosis of SW579 cells. These results suggest that selective targeting of KDR may have potential for development into novel anti‐cancer therapies to suppress VEGF/VEGFR‐mediated cancer development in patients with clinical advanced thyroid cancer.

Presence of CrkI-containing microvesicles in squamous cell carcinomas could have ramifications on tumor biology and cancer therapeutics

Recently, we described a phenomenon whereby apoptotic cells generate and release CrkI-containing microvesicles, which stimulate proliferation in surrounding cells upon contact to compensate for their own demise. We termed these microvesicles “ACPSVs” for Apoptotic Compensatory Proliferation Signaling microvesicles. As immune cells and a majority of current cancer therapeutics destroy tumor cells primarily by apoptosis, we conducted a small pilot study to assess the possibility that ACPSVs may also be generated in squamous cell carcinomas. We first evaluated a primary and a metastatic squamous cell carcinoma cancer cell lines for their ability to produce ACPSVs under normal and apoptotic conditions. We next conducted a pilot study to assess the occurrence of ACPSVs in solid tumors extracted from 20 cancer patients with squamous cell carcinomas. Both cancer cell lines produced copious amounts of ACPSVs under apoptotic conditions. Interestingly, the metastatic squamous cell carcinoma cancer cell line also produced high levels of ACPSVs under healthy condition, suggesting that the ability to generate ACPSVs may be hijacked by these cells. Importantly, ACPSVs were also abundant in the solid tumors of all squamous cell carcinoma cancer patients. Detection of ACPSVs in cancer has potentially important ramifications in tumor biology and cancer therapeutics which warrants further investigation.

CDH3 is associated with poor prognosis by promoting the malignancy and chemoresistance in oral squamous cell carcinoma

CDH3 is recognized as an oncogene in various malignancies. Here, we aim to explore the association of CDH3 expression and prognostic implication in oral squamous cell carcinoma (OSCC). Bioinformatics was used to analyze differentially expressed genes in the TCGA database. The OSCC tissues of 136 cases were used for immunohistochemistry. Cox proportional hazard analysis was used to analyze the relationship between prognostic factors, CDH3 expression and patient survival. Kaplan-Meier analysis was adopted to calculate survival rates. RT-qPCR and Western blot were performed to detect the expression levels of CDH3 in oral squamous cell lines. The cell viability and colony formation abilities were examined by CCK-8 and colony formation assays, respectively. Wound healing assay was performed to examine the invasion ability of cells. CDH3 is up-regulated in oral squamous cell carcinoma and related to bad prognosis. Knock-down of CDH3 limited cell viability, colony formation ability, migration, invasion and chemoresistance of OSCC cells. CDH3 is associated with a poor prognosis through promoting migration, invasion and chemoresistance in oral squamous cell carcinoma.

Stable Reconstitution of Gasdermin-E in a Gasdermin-E-Deficient Head and Neck Squamous Cell Carcinoma Mouse Cell Line Enhances Pyroptosis-Mediated Release of Tumor Immunogenic Molecules

<h3>Purpose/Objective(s)</h3> Gasdermin-E (GSDME) is known to potentiate immunogenic cell death in many tumor types after exposure to anti-cancer agents in a caspase-3 dependent manner. This GSDME-mediated release of immunostimulatory proteins, such as HMGB1, has been shown to increase tumor immunogenicity in mouse model systems of melanoma and colon cancer, resulting in reduced tumor load and lower distal metastasis. We sought to evaluate the role of GSDME-mediated cell death in human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) mouse cell lines. <h3>Materials/Methods</h3> To test whether HNSCC exhibits a GSDME-associated anti-tumor response, two HPV+ HNSCC mouse cell lines, MTEC and TC-1, were screened for GSDME expression by Western blot and then treated with apoptotic agents (raptinal, cisplatin, etoposide, and UV). Primary outcome measures were HMGB1 release (a potent immunostimulatory factor), percent propidium iodide (%PI) uptake (an indicator of plasma membrane damage and pyroptosis) and cell morphology. Using retroviral transduction, MTEC cells were reconstituted with GSDME, and single cell clones expressing comparable levels of recombinant mouse wild type (WT) GSDME and a caspase-3-uncleavable inactive mutant (Aspartate 270/Glutamate) D270A GSDME were selected. Control MTEC transduced with empty vector (EV) were also generated. Single cell clones were tested <i>in vitro</i> with apoptotic agents and evaluated for HMGB1 release, %PI uptake, and cell morphology. <h3>Results</h3> Western blot analysis demonstrated GSDME expression in TC-1 but not MTEC cells. Following treatment with apoptotic agents, TC-1 cells demonstrated HMGB1 release and increased %PI uptake as well as a distinct secondary necrotic phenotype characterized by ballooning of the plasma membrane on morphological analysis. This was not seen in MTEC cells. To examine whether the absence of GSDME in MTEC cells correlated with reduced release of immune-stimulatory molecules, MTEC cells stably reconstituted with GSDME were treated with apoptotic agents. Unlike MTEC cells expressing the uncleavable D270A GSDME or EV control, MTEC cells expressing WT GSDME demonstrated a significant release of HMGB1 and increased %PI uptake. Morphological analysis demonstrated significant ballooning of the plasma membrane only in the WT GSDME MTEC cells. <h3>Conclusion</h3> Treatment with apoptotic agents induces a GSDME-dependent pyroptotic cell death in MTEC and TC-1 cells as evidenced by HMGB1 release, increased %PI uptake, and cell morphology. As pyroptotic cell death of cancer cells induces anti-tumor immunity through the release of HMGB1 and other immunostimulatory factors, promoting GSDME-dependent pyroptosis might be a strategy to overcome treatment resistance in some HNSCC.

Long non-coding RNA LINC00472 inhibits oral squamous cell carcinoma via miR-4311/GNG7 axis

ABSTRACT Emerging studies indicate that long non-coding RNAs play important roles in oral squamous cell carcinoma (OSCC). However, the function of the majority of long non-coding RNAs is still unclear. Recently, LINC00472 has been reported to play crucial roles in multiple cancers. However, the role of LINC00472 in oral squamous cell carcinoma (OSCC) is still not clear. This study found that LncRNA LINC00472 was significantly down-regulated in several squamous cell carcinoma cancer tissues and OSCC cell lines. Over-expression of LINC00472 in OSCC cells inhibited OSCC progression and alleviated OSCC immune responses. Additionally, we confirmed that LINC00472 functioned as an hsa-miR-4311 sponge and regulated the expression of GNG7 (guanine nucleotide-binding protein, gamma 7). Also, we found that LINC00472 over-expression could suppress xenograft tumor growth in vivo. Our study provides evidence that LINC00472 plays an essential role in inhibiting oral squamous cell carcinoma progression and affecting immune responses by directly binding to hsa-miR-4311 to regulate the expression of GNG7 positively.

Knockdown of EphB3 inhibits cell proliferation partly through the AKT signaling pathway and represses epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

BackgroundTo investigate the role and mechanism of erythropoietin-producing hepatocyte receptor B3 (EphB3) in cancer of esophageal squamous cells.MethodsEphB3 expression in esophageal carcinoma squamous tissue and cell lines was determined by immunohistochemistry, western blotting and qRT-PCR. The viability, invasion and migration of cells were assessed by Transwell assay, formation of colonies, CCK-8, and healing of wounds, respectively. Flow cytometry analysis was employed to evaluate the actions of EphB3 on the activity of the cell cycle and the degree of apoptosis. The activity of EphB3 on the growth of tumors in vivo was examined in a mouse xenograft model.ResultsEphB3 was highly expressed in esophageal squamous cell cancer tissue and was positively correlated with cell differentiation, metastasis in lymph node and the TNM stage. Patients with higher EphB3 expression had poorer prognosis in 3-year overall survival rate. EphB3 also overexpressed in esophageal squamous cell cancer cell lines. Knock down of EphB3 expression suppressed proliferation, migration and the invasion of cells in vitro and was shown to delay the growth of tumors in vivo. Silencing of EphB3 reduced the expression of pAKT, cyclinD1 and altered the epithelial-mesenchymal transition (EMT) process. Furthermore, AKT signal pathway agonist SC79 reversed EphB3 downregulation-mediated inhibition of cell proliferation, migration, invasion and EMT process.ConclusionsEphB3 knockdown inhibited the proliferation of esophageal squamous cell cancer partly through the AKT signaling pathway and repressed cell migration and invasion via EMT reversion. The findings of the study suggested that EphB3 might be a novel target for the therapy of esophageal carcinoma.

Scutellarein apoptosis mediated by mitochondria in oral squamous cell carcinomas

Background: The growth of several cancers can be inhibited by naturally occurring medicinal plants. A flavone called Scutellarein found in the perennial herb Scutellaria lateriflora does have a wide range of biological functions. Scutellarein was studied to determine whether it could induce apoptosis and cause cytotoxicity. Methods: On oral squamous cell carcinoma KB cell lines, Scutellarein's cytotoxic activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Scutellarein was added to KB cells at concentrations ranging from 25 to 125 g/mL for 24 h. The real-time polymerase chain reaction was also used to investigate apoptotic induction potential in Scutellarein-incubated KB cell lines by analyzing Bcl-2 protein and gene expression. Results: In KB-cell lines treated with scutellarein, cytotoxicity and anticancer effects were observed, as well as inhibit the growth of cancer cells. In comparison to the cells not treated with scutellarein, KB cells that had been exposed to scutellarein displayed reduced Bcl-2 expression. Conclusion: KB cells were treated with scutellarein to induce apoptosis, suggesting its potential as a chemo preventative agent. This activity appears to be mediated through the modulation of Bcl-2, a cytotoxic gene.

The Effectiveness of a Portable Fluorescence Spectrophotometer for Early Detection of Oral Cancer

Purpose: To investigate whether photodynamic diagnosis (PDD) using a portable fluorescence spectrophotometer (FC-1) can easily and objectively discriminate between normal and tumor cells at the dental chairside, and to further compare it with PDD that requires speculum examination by focusing on protoporphyrin IX (PPIX). Methods: Three cell lines (2 human oral squamous cell carcinoma-derived cell lines, HSC-2 and HSC-3 cells, and oral keratinocytes, HOK cells) were cultured. 5-Aminolevulinic acid hydrochloride (5-ALA) and deferoxamine mesylate (DFO) were mixed in DMEM, and the mixture was set to Control (DMEM only) and PDD (5-ALA+DFO) groups. And then, a fluorescence was measured under room temperature (RT) and 37°C (Incubation) by using FC-1. In this study, the two conditions were combined with the Control and PDD groups to form the Control/RT, Control/Incubate, PDD/RT, and PDD/Incubate groups. Additionally, the amount of singlet oxygen (1O2) generated by irradiation with 405 nm LED was measured using electron spin resonance spectroscopy to detect PPIX in the cell supernatant after 24 hours. Results: In HSC-2 and HSC-3, the fluorescence intensity values increased significantly at 2 hours between the Control/RT and PDD/RT groups. In addition, there was a significant difference between HSC-2 and HSC-3 compared to HOK. In all cell lines, the fluorescence intensity values of the PDD/Incubate group were significantly higher than those of the PDD/Control group. The amount of 1O2 generated by 405 nm LED irradiation was higher in the cell supernatants of all cell lines in the order of Control/RT < Control/Incubate < PDD/RT < PDD/Incubate group, and HSC-3 in the PDD/Incubate group showed a significant increase compared to HOK. Conclusion: It is suggested that PDD using FC-1 can clearly distinguish between normal cells and tumor cells in vitro studies using cell lines at 2 hours under 37°C, and it can detect not only intracellular PPIX, but also extracellular PPIX.

Comprehensive and Integrated Analysis Identifies ZEB1 as a Key Novel Gene in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer with a poor prognosis. Therefore, it is crucial to explore molecular prognostic biomarkers for OSCC. ZEB1 (also known as δEF1) is a member of the zinc finger E-box binding protein family of transcription factors involved in various biological processes, including tumorigenesis, progression, and metastasis. Recent evidence suggests that ZEB1 has a role in the tumorigenicity of oral epithelial cells, although its mode of action needs to be investigated further. To better understand the relationship between ZEB1 and OSCC, we transfected the ZEB1-overexpressing oral squamous cell lines SCC9 and SCC25 with lentivirus and then extracted RNA from the cells for gene expression analysis. Furthermore, the GSE30784 dataset was downloaded from the Gene Expression Omnibus (GEO) database to identify potential biomarkers of OSCC and to assess the potential mechanisms. The criteria for identification of their DEGs were |logFC| > 1 and P < 0.05. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses were also carried out. Integrating the data from the PPI network and survival analysis identified that ZEB1 might be an independent prognostic biomarker in OSCC. In conclusion, integrated bioinformatics and microarray analysis identified the critical gene ZEB1 linked to the overall survival (OS) of patients with OSCC. ZEB1 could be applied as a prognostic biomarker to forecast the survival of patients with OSCC and might indicate innovative therapeutic indicators for OSCC.

Chitinase 3-Like-1 Expression Is Upregulated Under Inflammatory Conditions in Human Oral Epithelial Cells.

Chitinase 3-like-1 (CHI3L1), also known as YKL-40, is a partially secreted glycoprotein and is involved in inflammatory disorders, including inflammatory bowel diseases. CHI3L1 is known to play a role in biological responses such as cell proliferation, tissue remodeling, and inflammation. CHI3L1 forms an immune complex (known as a Chitosome complex) with IL-13 receptor alpha 2 (IL-13 Rα2) and transmembrane protein 219 (TMEM219) to activate the MAPK/ERK and PKB/AKT signaling pathways. The objective of this study is to investigate how the expressions of CHI3L1 and a Chitosome complex in human oral cavity epithelial cells are linked with intraoral inflammatory diseases. CHI3L1 and Chitosome complex mRNA expressions were analyzed using human oral squamous cancer cell lines, HSC3 and HSC4 cells. Signaling activation in HSC4 cells was analyzed by using the western blot technique. Immunohistological analysis was performed using surgical samples obtained from patients with benign oral cavity tumors and cysts. Increased expression of CHI3L1 was observed in both HSC3 and HSC4 cells after TNFα stimulation. The expression of Chitosome complex factors increased as CHI3L1 levels increased, resulting in the activation of a downstream signaling pathway. Among the intraoral tissues, the epithelial cells from inflammatory lesions, but not benign tumors, were found to be intensively stained with the anti-CHI3L1 antibody. It was indicated that the formation of a Chitosome complex is induced during inflammation, leading to the activation of signaling pathways.

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT. Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain. Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

Folic Acid Inhibited Vasculogenic Mimicry in Esophageal Cancer Cell Line Eca-109, the One Target Was EphA2

The degree of vasculogenic mimicry(VM) is correlated with the prognosis of esophageal cancer, and folic acid supplementation could decrease esophagus cancer deaths among populations. This study aimed to explore the effect of folic acid on VM formation of esophageal cancer cell, and the target. Human esophageal squamous cancer cell lines(Eca-109) were cultured with different concentrations of folic acid (0,1,10,100,200,400, 600,800 μg/ml). A cell counting kit-8 (CCK-8) assay was used to measure the cell proliferation. Then, the amount of VM under the effect of different concentrations of folic acid was observed. Target genes were screened out from several possible targets genes including MMP2, MMP9, EphA2, VE-cad or Ln-5γ2 by employing reverse transcription-quantitative polymerase chain reaction(RT-qPCR). Finally, western blot analysis was used to verify the target proteins. In conclusion, this study found that folic acid inhibited the formation of VM in Eca-109 cells, and the one target protein was EphA2.