Squamous Cell Lines Research Articles

FP16.01 Development and Characterization of Two Novel Squamous Cell Lung Cancer Mouse Cell Lines

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated with lung cancer is a major Public Health challenge in advanced societies. About 20-30% of all lung cancers are classified as lung squamous cell carcinoma (LUSC), also known as squamous cell carcinoma (SSC or SqCC) or epidermoid carcinoma. The absence of preclinical models makes LUSC disease analysis and research more challenging. The development of well-characterized cellular and molecular tools is essential for the advance in lung cancer research. In the present work, we have performed an exhaustive molecular and functional characterization of a new LUSC research model, recently developed in our laboratory. This model consists of two novel A/J mouse-derived syngeneic cell lines from NTCU-induced LUSC.In our opinion, these cell lines may become a robust tool for the study of squamous cell lung cancer in a reliable and reproducible manner. Carcinogenesis and cell line generation: LUSC tumors were induced by N-nitroso-tris-chloroethylurea (NTCU) (Toronto Research Chemicals) treatment applying 0.04 M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Immunohistochemical staining: Dissected tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were used for tumors immunohistochemical phenotypic characterization. Exome sequencing: Whole exome sequencing was performed on extracted DNA from the tumor-derived cell lines. RNA sequencing analysis: Samples were prepared with the Illumina TruSeq Stranded mRNA kit and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. Tumor immunotherapy experiments: 2x106 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice. When the tumors reached a volume of 75 mm3, mice were randomized and treated intraperitoneally with 3 doses of 100 μg of anti-PD1 or anti-CTLA4. Tumor immune landscape analysis: 2x106 cells were subcutaneously inoculated in one flank of 6 8-week-old female A/J mice . Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis. Metastasis in vivo model: Cells were transduced with a triple modality construct containing a triple fusion protein GFP, luciferase and thymidine kinase. Eight-week old female mice were inoculated in the left cardiac ventricle with 1 × 105 cells. Cells were followed by bioluminescence. We have generated two transplantable LUSC cell lines (UN-SCC679 and UN-SCC680) derived from an N-nitroso-tris-chloroethylurea (NTCU) chemically-induced mouse model in A/J mice. Both cells lines expressed cytokeratins and p40, and lacked thyroid transcription factor 1 (TTF1) expression. Furthermore, we genetically characterized the LUSC cell lines by performing whole exome and RNA sequencing. In addition, we characterized the immune landscape of both tumors in vivo and assessed their response to immunotherapy studies combining the gold-standard checkpoint inhibitors in clinic. Finally, we studied the metastatic potential of each of these two models and confirmed that they reflect the human LUSC organotropism to the brain, bones, liver and adrenal glands. We have generated a very valuable tool for LUSC research that recapitulates the complexity of the human disease.

P68.02 Losartan Enhances Lung Squamous Cell Carcinoma’s Sensitivity to Cisplatin Treatment By Promoting Mesenchymal to Epithelial Transformation

Angiotensin II type 1 receptor (AT1R) overexpression has been associated with poor prognosis in patients with some solid organ cancers. The angiotensin II/AT1R axis is closely related to tumor proliferation, angiogenesis, invasion and metastasis. Platinum resistant non-small cell lung cancer shows significantly higher type I angiotensin II receptor (AGTR1) mRNA expression than their corresponding parent cells. Addition of angiotensin II receptor blocker (ARB) to platinum-based first-line chemotherapy may contribute to prolonged survival in patients with locally advanced squamous cell lung cancer. The TCGA database was analyzed for expression of AGTR1 in tumor tissue from resected lung squamous cell carcinoma (N=497) and correlated with overall survival. Multiple human lung squamous cancer cell lines (SK-MES-1, H520, H1703) with primary mesenchymal phenotype [Fig. 1D] were evaluated invitro for AGTR1 expression [Fig. 1C] and treated with losartan or combination losartan and cisplatin to assess cell survival and EMT expression by western blot. Patients with stage I, II, and III lung squamous cell carcinoma (LUSC) demonstrated improved overall survival with low expression of AGTR1(P =0.0056) [Fig 1A]. When stratified by stage, stage II exhibited low AGTR1 expression and was associated with a better prognosis (P =0.0032). Expression of AGTR1 positively correlated with EMT expression of Vimentin (r=0.57, P <0.0001), ZEB1 (r=0.43, P <0.0001) and ZEB2 (r=0.59, P <0.0001) [Fig 1B]. SK-MES-1, H520 and H1703 express abundant AGTR1. Vimentin expression of SK-MES-1 decreased with losartan treatment [Fig. E] in a dose dependent fashion (0, 0.1, 0.2, 0.5μM). E-cad expression of H520 increased and Vimentin expression decreased with increasing losartan treatment [Fig. F]. Compared to cisplatin treatment alone, combination of Losartan and cisplatin significantly improved the cytotoxic effect in SK-MES-1(P=0.0006), H520 (P =0.0053) and H1703(P =0.0234) [Fig. G]. AGTR1 expression correlates with survival outcomes of lung squamous cell carcinoma in humans and is associated with a more mesenchymal phenotype in vivo and in vitro. Losartan treatment appears to promote a mesenchymal to epithelial transformation and enhances the anti-tumor effect of chemotherapy (cisplatin) compared to chemotherapy alone in lung squamous cancer cell.

Maternally expressed 3 inhibits the biological activity of oral squamous cell carcinoma SCC25 and CAL27 cell lines

The abnormal expression of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is closely related to several tumor diagnosis and progression, such as endometrial carcinoma and ovarian cancer. However, the role of MEG3 in oral squamous cell carcinoma (OSCC) is rarely reported. The current study aimed to evaluate the expression of lncRNA MEG3 in OSCC tissues and cell lines and its effect on the biological behavior of OSCC cell lines. The expression of lncRNA MEG3 in the OSCC tissues and cell lines was detected by reverse transcription-quantitative (RT-q) PCR. The relationship between MEG3 expression and the clinicopathologic characteristics and prognosis of patients with OSCC was analyzed. The lncRNA MEG3 overexpression plasmid and control plasmid were transfected into SCC25 and CAL27 cell lines using the lipofectin method. MTT assay was performed to detect the growth and proliferation of the cell lines. Transwell chamber test was used to detect changes in cell migration and invasion. Flow cytometry was employed to detect changes in apoptosis. Western blotting and RT-qPCR were conducted to detect the expression of the p53 gene. The expression of lncRNA MEG3 in the OSCC tissues and cell lines was significantly compared with normal tissues and cell lines, respectively. The expression level of MEG3 was related to clinical stage, lymph node metastasis, distant metastasis and survival status. Overexpression of lncRNA MEG3 inhibited the proliferation, migration, and invasion of SCC25 and CAL27 cell lines, induced apoptosis and promoted the expression of p53 gene. lncRNA MEG3 played the role of a tumor inhibitor gene and significantly inhibited the biological activity of OSCC cell lines, which may provide a novel idea for molecular targeted therapy of OSCC.

Galangin Enhances Anticancer Efficacy of 5-Fluorouracil in Esophageal Cancer Cells and Xenografts Through NLR Family Pyrin Domain Containing 3 (NLRP3) Downregulation.

BackgroundGalangin is believed to exert antioxidant effects by inhibition of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which has been linked to chemotherapy sensitivity in cancers. In this study, we explored the synergistic effect of galangin in combination with the chemotherapy agent 5-fluorouracil (5-FU) in esophageal cancer cells and xenografts.Material/MethodsThe esophageal squamous epithelium cell line Het-1A and 2 human esophageal cancer cell lines (Eca109, OE19) were used to investigate the effect of galangin with or without 5-FU in vitro through proliferation and invasion analyses, while apoptosis was analyzed in cancer cells. Furthermore, a subcutaneous xenograft tumor model in mice was used to study cancer development in vivo.ResultsCompared with 5-FU monotherapy, combined galangin and 5-FU treatment reduced human esophageal cancer cell growth activities and invasion abilities. The results suggested that galangin had a chemotherapy-sensitized synergistic antitumor effect induced by 5-FU. The susceptibility of cancer cells to apoptosis, which is linked with chemotherapy sensitivity, was induced by 5-FU and further enhanced by galangin. NLRP3 was identified as being significantly activated by 5-FU, but galangin treatment reversed the effect and inhibited NLRP3 expression, which was accompanied by downregulated interleukin-1β levels. Further investigation showed that the induced apoptotic cascade can be mostly reversed by incubation with an NLRP3 activator, irrespective of AKT signaling. Using xenograft mouse models, we found that galangin exposure further restrained cancer development after 5-FU treatment and increased sensitivity to chemotherapy by suppressing the NLRP3 inflammasome pathway.ConclusionsOur results indicated that galangin played a synergistic anticancer role through NLRP3 inflammasome inhibition when paired with FU-5.

Discovery of Oral Anticancer 1,2-Bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine Hybrids That Inhibit Angiogenesis and Induce DNA Cross-Links.

Designing hybrid molecules with dual functions is one approach to improve the therapeutic efficacy of combination treatment. We have previously conjugated phthalazine and bis(hydroxymethyl)pyrrole pharmacophores to form hybrids bearing antiangiogenesis and DNA interstrand cross-linking activities. To improve the bioavailability, we adopted a benzology approach to design and synthesize a new series of 1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazines. These new hybrids retained the dual functions and could be formulated into vehicles for intravenous and oral administration. Among them, we demonstrated that compound 19a with dimethylamine at the C6 position markedly suppressed the tumor growth of human small cell lung cancer cell line H526, squamous lung cancer cell line H520, and renal cancer cell line 786-O in nude mice, implying that compound 19a is a broad-spectrum anticancer agent. Our results implicated that the conjugation of antiangiogenic and DNA cross-linking is likely to be a helpful approach to improving the efficacy of combination therapy.

Abstract 368: Aurora kinase inhibitors cause cell death in HPV+ cells: A plausible treatment option for HPV+ cancers

Abstract Human papilloma virus (HPV) driven cancers are common and lethal. The standard of care chemo-radiation treatment for HPV+ head and neck squamous cell carcinoma (HNSCC) results in permanent, often life-altering, toxicities in a relatively young population (aged ~45-55). Additionally, those who recur have poor outcomes. Therefore, there is an urgent need for better treatment options. No biomarker-driven targeted therapies are available. To address the current unmet need, we hypothesized that a large-scale High Throughput Drug Screen (HTDS) comparing drug efficacy in HPV+ and HPV- cell lines would identify biomarker driven, effective and less toxic options for HPV+ cancers. We tested the drug- sensitivity of 864 unique drugs (0-3.1 µM) in 51 classes based on their targets in 16 HPV+ and 17 HPV- squamous cancer cell lines. Cells were treated for 72 hours and DAPI+ nuclei counted before and after drug treatment. We used the Area Over the Curve_Lethal Dose (AOC_LD) to measure drug sensitivity because it is independent of cell division. We defined effective drugs as those that led to cell death (i.e., less cells at the end of drug treatment than prior to treatment) in at least one cell line. About half of the drugs tested (439) were effective and certain classes of drugs were enriched in either the effective or ineffective categories (Pearson residual). We found that Aurora kinase inhibitors were the most effective class overall. Other effective drug classes included anthracyclines, vinca-alkaloids, and inhibitors of HDAC, proteasome, EGFR, CDC7, and BTK1. Only 30 drugs had a differential effect based on HPV status. All 16 Aurora kinase inhibitors were more effective in HPV+ than in HPV- cells and the difference in sensitivity (dichotomous variable) was statistically significant in 7 of them. To determine if factors other than HPV status predict response to Aurora kinase inhibitors, we compared the expression of 304 proteins (RPPA) and mutational status for the 50 most commonly mutated genes in HNSCC to drug efficacy. The expression of eight proteins were associated with sensitivity to multiple Aurora kinase inhibitors. We validated RPPA data by immunoblotting and observed that only Rb, pRB(S807/811) and p16 correlated with drug sensitivity. For gene mutations, only TP53 mutations correlated with drug resistance with 7 Aurora kinase inhibitors. Interestingly, these two biomarkers, Rb and TP53, corroborate well with HPV status. To validate the screening results, we treated HPV+ cells with two clinically relevant Aurora kinase inhibitors, alisertib and barasertib, and observed significantly more cell death in HPV+ cells in comparison with HPV- cells. Therefore, our present study portrays Aurora kinase inhibitors as an effective strategy for HPV+ cancer cells. Multiple Aurora kinase inhibitors are currently in clinical development making the clinical application of our data possible soon. Citation Format: Tuhina Mazumdar, Li Shen, Wei Xu, Soma Ghosh, Reid T. Powell, Clifford Stephan, Curtis R. Pickering, Jeffery N. Myers, Jing Wang, Faye M. Johnson. Aurora kinase inhibitors cause cell death in HPV+ cells: A plausible treatment option for HPV+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 368.

Model of Human Tongue Squamous Cell Lines Stably Transfected with Human Papillomavirus (HPV)16 E6 and E7 Genes and Biological Characteristic Analysis.

Background Human tongue squamous cell carcinoma (TSCC) is the most common oral cancer with the highest human papillomavirus (HPV) infection rate in oral cancer. The purpose of this study was to research the correlation between HPV and TSCC. Method Plasmid pEGFP/HPV16 E6E7 and plasmid pEGFP/no HPV16 E6E7 were constructed. TSCC cell lines SCC9 and SCC15 were infected by liposome transfection and would be highly selected by antibiotic. Fluorescence imaging, PCR, and Western blot were used to detect the expression of HPV16 E6E7 in cells. The biological characteristics were detected by CCK-8, wound healing assay, qRT-PCR, and Western blot. Result TSCC cell lines transfected with HPV16 E6E7 gene were successfully established and identified. And the proliferation and migration ability of the TSCC cell lines infected with HPV16 E6E7 gene were significantly stronger than that of the blank group. Conclusion TSCC cell lines infected with HPV16 E6E7 with significantly higher ability of proliferation and migration were more malignant than those not infected with HPV16 E6E7.

Forkhead box F1 induces columnar phenotype and epithelial-to-mesenchymal transition in esophageal squamous cells to initiate Barrett's like metaplasia

Multiple genome-wide association studies (GWAS) have linked Forkhead Box F1 (FOXF1) to Barrett's esophagus (BE). Understanding whether FOXF1 is involved in initiation of Barrett's metaplasia could allow FOXF1 to be used for risk stratification and for therapy. Two-dimensional cell cultures and three-dimensional organoid cultures and well-annotated human biopsies were used to determine the role of FOXF1 in BE pathogenesis. Multiple established esophageal squamous and BE cell lines were tested in gain- and loss-of-function studies. Initiation of a BE-like metaplastic change was evaluated by measuring characteristic cytokeratins and global gene expression profiling and by culturing organoids. Epithelial–mesenchymal transition (EMT) was evaluated by immunostaining for E-cadherin, vimentin and Snail, and by cell motility assay. Columnar esophageal epithelium of BE patients exhibited higher expression of FOXF1 compared to normal squamous esophageal epithelium of GERD patients (P < 0.001). Acidic bile salts induced nuclear FOXF1 in esophageal squamous cells. FOXF1 overexpression in normal esophageal squamous cells: (a) increased columnar cytokeratins and decreased squamous cytokeratins, (b) converted squamous organoids to glandular organoids, and (c) switched global gene profiles to resemble that of human BE epithelium (P = 2.1685e − 06 for upregulated genes and P = 8.3378e − 09 for downregulated genes). FOXF1 inhibition in BE cell lines led to loss of BE differentiation markers, CK7, and mucin 2. Also, FOXF1 induced EMT and promoted cell motility in normal esophageal squamous epithelial cells. FOXF1-induced genes mapped to pathways such as Cancer, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair. In conclusion, FOXF1 promotes a BE-like columnar phenotype and cell motility in esophageal squamous epithelial cells, which may have a critical role in BE development. FOXF1 should be studied further as a biomarker for BE and as a target for BE treatment.

Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo.

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA gene-edited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

Synergistic anti-cancer activity of combined 5-fuorouracil and gallic acid-stearylamine conjugate in A431 human squamous carcinoma cell line

Purpose: To evaluate the individual and synergistic anti-cancer effects of 5-fuorouracil (5-FU) and synthesized gallic acid-stearylamine (GA-SA) conjugate in A431 human squamous cancer cell line.&#x0D; Methods: Characterisation of the synthesised conjugate was performed using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The synergistic effect of the combination therapy (5-FU/GA-SA) was assessed by determining their inhibitory concentration (IC30) whereby A431 cells were treated with 5-FU:GA–SA conjugate at various ratios ranging from 5:1 to 1:5.&#x0D; Results: The cytotoxicity of 5-FU was 29 %, while that of the combination of 5-FU with GA–SA conjugate was as high as 60 %. Thus, this combination showed significant synergistic enhancement in cytotoxicity (p &lt; 0.05). The results obtained also revealed that the IC30 values of 5-FU and the GA–SA conjugate were 1 and 10 µg/mL, respectively. The IC30 values of the combination ratios indicated that the dosages used in the study were safe in HaCaT normal cell line.&#x0D; Conclusion: These results indicate that 5-FU/GA–SA conjugate at a ratio of 1:1 is effective against A431 cell line (cancer cells)) but safe in HaCaT cell lines (normal cells).

Hepatitis B X‐interacting protein, involved in increasing proliferation and cell migration, is a prognostic marker in head and neck squamous cell carcinoma

AbstractIntroductionFactors associated with prognosis and molecular mechanisms leading to a malignant phenotype in head and neck squamous cell carcinoma (HNSCC) need to be identified. The focus was on the biological function and clinical impact of hepatitis B X‐interacting protein (HBXIP) in HNSCC.MethodsmRNA expression profiles of HBXIP were compared between tumor and normal tissues in head and neck cancer cases from an online database. The protein expressions of candidate genes were verified by immunohistochemistry (IHC) with tissue microarray blocks designed with 221 cases. Prognostic significance was analyzed using the Kaplan–Meier method and Cox regression analysis. The molecular function of malignant phenotypes was investigated by generating knockdown cell lines of HNSCC with RNA interference.ResultsAnalysis of the online database showed that the mRNA of HBXIP was significantly higher in the HNSCC group than in normal specimens. IHC analysis showed significant differences in the protein expression of HBXIP by clinical stage, T status, and recurrence. Multivariate analysis showed that, in HBXIP IHC‐positive patients, N status, T status, and neural invasion were significantly associated with overall survival. In addition, downregulation of the HBXIP gene in SAS, a tongue squamous cancer cell line, inhibited proliferation and the invasion phenotype.ConclusionsBy increasing the malignant phenotypes of cell proliferation and invasion, upregulation of HBXIP was a prognostic factor in HNSCC.

Electrochemotherapy in Vulvar Cancer and Cisplatin Combined with Electroporation. Systematic Review and In Vitro Studies

Simple SummaryElectrochemotherapy (ECT) is an emerging treatment for solid tumors and an attracting research field due to its clinical results. ECT in association with bleomycin is an effective and safe treatment option in the vulvar cancer palliative setting. With regard to cisplatin (CSP)-based ECT, considering the clear evidence on its efficacy in gynecological tumors, the possibility to improve local control with CSP-based ECT is intriguing and a well-designed randomized clinical trial should be addressed to this issue.Electrochemotherapy (ECT) is an emerging treatment for solid tumors and an attractive research field due to its clinical results. This therapy represents an alternative local treatment to the standard ones and is based on the tumor-directed delivery of non-ablative electrical pulses to maximize the action of specific cytotoxic drugs such as cisplatin (CSP) and bleomycin (BLM) and to promote cancer cell death. Nowadays, ECT is mainly recommended as palliative treatment. However, it can be applied to a wide range of superficial cancers, having an impact in preventing or delaying tumor progression and therefore in improving quality of life. In addition, during the natural history of the tumor, early ECT may improve patient outcomes. Our group has extensive clinical and research experience on ECT in vulvar tumors in the palliative setting, with 70% overall response rate. So far, in most studies, ECT was based on BLM. However, the potential of CSP in this setting seems interesting due to some theoretical advantages. The purpose of this report is to: (i) compare the efficacy of CSP and BLM-based ECT through a systematic literature review; (ii) report the results of our studies on CSP-resistant squamous cell tumors cell lines and the possibility to overcome chemoresistance using ECT; (iii) discuss the future ECT role in gynecological tumors and in particular in vulvar carcinoma.

Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells.

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.

Investigation of the Functional Role of mir-1825 in Laryngeal Squamous Cell Carcinoma Hep-2 Cell Line

Investigation of the Functional Role of mir-1825 in Laryngeal Squamous Cell Carcinoma Hep-2 Cell Line

Acid-electrolyzed functional water-induces Interleukin-1α release from Intracellular Storage Sites in Oral Squamous Cell Carcinoma.

The aim of this study was to examine the acid-electrolyzed functional water (FW)-mediated cytokine release in an oral squamous cell carcinoma-derived cell line (OSCC) following treatment with FW. FW is generated by the electrolysis of a sodium chloride solution and accelerate the burn wound healing. To elucidate the underlying mechanisms, the cytokine/chemokine secretion profile of HSC3 cells was examined using a cytokine array. FW treatment significantly induced interleukin (IL)-1α secretion, which was confirmed by enzyme-linked immunosorbent assay. Subsequently, the HSC3 cells were pre-treated with cycloheximide (CHX) for 1 h prior to FW stimulation to determine whether the augmented IL-1α secretion was due to enhanced protein synthesis. CHX pre-treatment did not affect IL-1α secretion suggesting that the secreted IL-1α might have been derived from intracellular storage sites. The amount of IL-1α in the cell lysate of the FW-treated HSC3 cells was significantly lower than that of the non-treated cells. Immunofluorescence staining using a polyclonal antibody against full-length IL-1α revealed a drastic reduction in IL-1α inside the FW- treated cells. IL-1α is synthesized in its precursor form (pIL-1α) and cleaved to produce pro-piece and mature IL-1α (ppIL-1α and mIL-1α) inside the cells. In the present study, only pIL-1α was detected within the HSC3 cells in its resting state. However, FW stimulation resulted in the release of the 33 kDa and two other smaller forms (about 19 kDa) of the protein. These results indicates that FW treatment induces IL-1α secretion, a typical alarmin, from the intracellular storage in OSCC cells.

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells. Methods: The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3' terminal untranslated region (3' UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively. Results: The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) (P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group (P<0.01) and (1.70±0.20)% of blank control group (P<0.01). The percentage of cells in G(1) phase [(44.74±6.76)%] was decreased while the G(2)/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group (P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups (P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group (P<0.01). Conclusions: The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G(2)/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

Thermo-chemotherapy enhances the immunogenicity of oral squamous cell carcinoma cells by inducing the expression of damage-associated molecular patterns

To investigate whether hyperthermia, chemotherapy and thermo-chemotherapy could trigger the expression of damage-associated molecular patterns (DAMPs). The optimal working concentration of pingyangmycin (PYM) was detected by CCK-8 assay, and temperatures of 39, 42, and 45 ℃ were applied to the oral squamous cell carcinoma CAL27, SCC-15, and Tca8113 cell lines. The effects of different treatments on the apoptosis, calreticulin (CRT) membrane expression and high-mobility group box 1 (HMGB1) secretion of the cells were detected by using Annexin V/propidium iodide (PI), flow cytometry and enzyme-linked immunosorbent assay (ELISA) assay. SPSS 20.0 software was used for statistical analysis. Both hyperthermia and chemotherapy could increase the membrane expression of CRT and the secretion of HMGB1, and furthermore, thermo-chemotherapy group showed significantly increased in apoptosis, CRT membrane expression rate and HMGB1 secretion compared with chemotherapy group, and the difference was statistically significant (P<0.05). Hyperthermia, chemotherapy and thermo-chemotherapy could induce oral squamous cell carcinoma cells succumb to death, and at the same time, they can effectively induce the membrane expression of CRT, and promote the secretion of HMGB1. Moreover, thermo-chemotherapy is significantly better than that of chemotherapy alone in the induction of cell apoptosis and DAMPs expression.

Pseudolaric Acid B Inhibits Proliferation, Invasion, and Angiogenesis in Esophageal Squamous Cell Carcinoma Through Regulating CD147.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common malignant tumor of the digestive system. Studies have shown that pseudolaric acid B (PAB) has several pharmacological effects like anti-microtubule, anti-angiogenesis, and antitumor functions, while the effect and mechanism of PAB on esophageal cancer are still unclear. This study was designed to investigate the effects of PAB on ESCC.MethodsTo study the effects of PAB on the biological function through a series of in vitro and in vivo experiments.ResultsThe results revealed that PAB inhibited the proliferation, invasion, and migration, but promoted the apoptosis of ESCC. Moreover, PAB restrained the growth of cancer cells in vivo and inhibited the angiogenesis of HUVEC in mice with ESCC. CD147 expression was increased in the esophageal squamous cell lines, and interference with CD147 hindered the proliferation, invasion, and migration of ESCC cells, and inhibited the growth and angiogenesis of the esophageal squamous cell line. PAB reduced the expression of CD147 in vivo and in vitro. The expression of MMP2, 3, and 9 was increased after overexpression of CD147, which provided the opportunity to reverse the role of PAB in inhibiting proliferation, invasion, migration, and angiogenesis of ESCC.DiscussionThe results revealed that PAB inhibited the proliferation, invasion, migration, and angiogenesis of ESCC in vitro and in vivo by CD147. PAB is a promising monomer for therapy of ESCC, providing references for future research on ESCC treatment.

Evaluation of Inhibitory Effects of Caffeine on Human Carcinoma Cells

One of the world's most consumed medications is caffeine which is available in the vast majority of beverages. Previously, some effects of caffeine have been evaluated including its inhibitory effect on cancer cells. But, the influence of caffeine on esophagus carcinoma squamous cells (CSC) and head and neck carcinoma cells still has not well understood. Herein, the relation between different amounts of caffeine with the proliferation rate of human esophagus carcinoma squamous cell line KYSE-30 as well as human head and neck carcinoma cell line HN5 was evaluated. Furthermore, concentrations of caffeine were adjusted and their effect on cells were studied. The inhibitory effects of caffeine on cells were measured using the conventional colorimetric MTT assay after 3 and 7 day of incubation. Our findings are suggested that caffeine has a significant inhibitory effect on both cell lines at the concentrations of 20, 50, and 70 milli-mol (mmol). The result shows that caffeine can prevent the proliferation of carcinoma cells and it is a perfect candidate for therapeutic applications.