Squamous Carcinoma Cell Lines Research Articles

The development of radioresistant oral squamous carcinoma cell lines and identification of radiotherapy-related biomarkers.

In the treatment of oral squamous cell carcinoma (OSCC), radiation resistance remains an important obstacle to patient outcomes. Progress in understanding the molecular mechanisms of radioresistance has been limited by research models that do not fully recapitulate the biological features of solid tumors. In this study, we aimed to develop novel in vitro models to investigate the underlying basis of radioresistance in OSCC and to identify novel biomarkers. Parental OSCC cells (SCC9 and CAL27) were repeatedly exposed to ionizing radiation to develop isogenic radioresistant cell lines. We characterized the phenotypic differences between the parental and radioresistant cell lines. RNA sequencing was used to identify differentially expressed genes (DEGs), and bioinformatics analysis identified candidate molecules that may be related to OSCC radiotherapy. Two isogenic radioresistant cell lines for OSCC were successfully established. The radioresistant cells displayed a radioresistant phenotype when compared to the parental cells. Two hundred and sixty DEGs were co-expressed in SCC9-RR and CAL27-RR, and thirty-eight DEGs were upregulated or downregulated in both cell lines. The associations between the overall survival (OS) of OSCC patients and the identified genes were analyzed using data from the Cancer Genome Atlas (TCGA) database. A total of six candidate genes (KCNJ2, CLEC18C, P3H3, PIK3R3, SERPINE1, and TMC8) were closely associated with prognosis. This study demonstrated the utility of constructing isogenic cell models to investigate the molecular changes associated with radioresistance. Six genes were identified based on the data from the radioresistant cells that may be potential targets in the treatment of OSCC.

Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

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.

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

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

Abstract 327: Knockdown of VRK1 inhibits the proliferation of small cell neuroendocrine carcinoma of the uterine cervix

Abstract Background: The prognosis of small cell neuroendocrine carcinoma of the uterine cervix (SCNECC) is extremely poor due to the aggressive natural history. Since it is a rare cancer, there are few studies describing its molecular biology. Small cell lung cancer (SCLC) accounts for 95% of small cell carcinomas, therefore, clinically the chemotherapy regimens used for SCNECC have been based on regimens used for SCLC. This study aims to detect therapeutic targets for SCNECC by taking clues from the molecular biological commonalities between SCNECC and SCLC. Methods: We analyzed protein expression of cancer tissue-originated spheroids from lung and cervical cancers using isobaric tags for relative and absolute quantitation (iTRAQ). In vitro, protein expression levels of VRK1 in SCNECC cell lines (TC-YIK and HCSC-1), SCLC cell lines (SBC3 and OS2RA) and cell lines of mucinous adenocarcinoma and squamous cell carcinoma of the uterine cervix (HeLa and ME180) were assessed by western blotting (WB), and the change of cell proliferation induced by siRNA/shRNA-mediated VRK1 knockdown was assessed by WST-8 assay. In vivo, in mice xenograft models of SCNEC, the change of tumor size due to downregulation of VRK1 by shRNA were assessed. Furthermore, to explore the possible role of VRK1 in small carcinoma, pathway enrichment and ontology analysis between VRK1-high and -low-SCLC was performed by Gene Set Enrichment Analysis (GSEA), and it was compared with RNA-seq results of between shRNA-mediated VRK1 knockdown and control SCNECC cell lines. Results: iTRAQ showed that SCNECC and SCLC revealed similar protein expression profiles, rather than organ of origin-specific patterns. In addition, higher expression of VRK1 was detected in small cell carcinomas compared to the others. (SCLC/mucinous adenocarcinoma of the lung=2.86, SCNECC/mucinous adenocarcinoma of the cervix=2.56, SCNECC/squamous cell carcinoma of the cervix=2.17). WB showed that all SCNECC and SCLC cell lines expressed more highly VRK1 than other cervical cancer cell lines. In vitro, siRNA/shRNA-mediated VRK1 knockdown significantly suppressed the cell proliferation of SCNECC and SCLC (-29.0±0.3% in SBC-3 and -33.1±6.7% in TC-YIK, p<0.05, other cell lines were comparable). In vivo, the downregulation of VRK1 significantly suppressed tumor size in mice xenograft models of SCNEC (1726±442mm3 vs 544±233mm3 in TC-YIK, p<0.05, HCSC-1 was comparable). GSEA showed that VRK1 might participate in DNA repair to affect the biological process of SCLC, and the genes contained in this gene set was also detected in RNA-seq of SCNEC, suggesting involvement with VRK1. Conclusion: These findings suggest that VRK1 is involved in the proliferation of SCNECC in relation to DNA repair pathways. Our ongoing research is investigating the functional relationship between VRK1 and genes related to DNA repair and its effect on proliferation of SCNECC in vitro. Citation Format: Mariya Kobayashi, Satoshi Nakagawa, Yuji Kamei, Tatsuo Masuda, Mamoru Kakuda, Kosuke Hiramatsu, Ai Miyoshi, Tomomi Takata, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Tadashi Kimura. Knockdown of VRK1 inhibits the proliferation of small cell neuroendocrine carcinoma of the uterine cervix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 327.

Abstract 4835: EP4 increases the mitochondrial respiration and promotes cell migration in oral cancer

Abstract Introduction: EP4 is one of the prostaglandin E2 (PGE2) receptors. We previously reported that EP4 promoted cell migration via Ca2+ signaling, however the underlying mechanism has remained unclear. In Ca2+ signaling, Store-operated Ca2+entry (SOCE) is a major mechanism of Ca2+ import from the extracellular to the intracellular space. Orai1, one of the selective Ca2+ channels, plays an important role in SOCE. We focused on the correlation with EP4 and Orai1. Additionally, we found that EP4 promoted the phosphorylation of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) and AMP-activated protein kinase (AMPK) located downstream of CaMKK2, i.e., cell energy regulators. AMPK activation can control positively or negatively the cellular adenosine triphosphate (ATP) supply. Therefore, we investigated how EP4 regulated the mitochondrial function via Ca2+ and promoted the cell migration in oral cancer cells. Materials and Methods: HSC-3, human tongue squamous cell carcinoma cell line was used. ONO-AE1-437 was used as EP4 agonist. HSC-3 cells were transduced with Orai1 shRNA, and control shRNA using lentivirus. The migration ability was evaluated by scratch assay. Measurement of intracellular Ca2+ concentration was measured using Fura-2. Immunoprecipitation was performed to evaluate the interaction between EP4 and Orai1. XF Cell Mito Stress Test and XF Real-Time ATP Rate Assay were performed to evaluate the mitochondrial aspiration and the ATP production using Extracellular Flux Analyzer. Results: Scratch assay showed EP4 promoted cell migration in oral cancer cells (n=4, p<0.05). In contrast, Orai1 knockdown negated EP4-induced cell migration. EP4 rapidly increased the intracellular Ca2+ concentration, while Orai1 knockdown negated EP4-indued Ca2+ increase (n=4, p<0.05). Immunoprecipitation showed that EP4 was colocalized and formed complexes with Orai1 (n=4, p<0.05). These results indicated that EP4 promoted the cell migration via Ca2+ signaling through Orai1. EP4 increased the maximal respiration 3h after the stimulation (n=6, p<0.05). ATP production rate shifted mitoATP dominant. These data indicated that EP4 increased the mitochondrial function and the respiratory capacity, potentially regulating its energy supply. These results demonstrated that EP4 promoted mitochondria respiration and then increased the energy production from mitochondria in oral cancer cells. Conclusion: We concluded that EP4 regulated the mitochondrial function via Ca2+ through Orai1 and promoted the cell migration in oral cancer cells. Citation Format: Rina Nakakaji, Masanari Umemura, Kohei Osawa, Soichiro Ishikawa, Kenji Mitsudo, Yoshihiro Ishikawa. EP4 increases the mitochondrial respiration and promotes cell migration in oral cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4835.

Abstract 2392: Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations

Abstract Studying tumor heterogeneity at single cell resolution is crucial for elucidating tumor progression and treatment response. Classically, tumor cells are characterized with a limited set of biomarkers that cannot cover the full extent of the tumor cell heterogeneity. The advent of single cell genomics has enabled scientists to study heterogeneity at high resolution, but gene expression signatures may not translate to readily available biomarkers for functional studies. Multi-dimensional morphology analysis has the potential for higher resolution than cell surface biomarkers while also reducing cell manipulations. The Deepcell platform enables multi-dimensional morphology analysis of unlabeled single cells using artificial intelligence (AI), imaging, and microfluidics, allowing for higher resolution of population heterogeneity beyond protein expression markers. Using the Deepcell platform, we trained an AI model to identify and sort for malignant cells from non-small cell lung cancer cells (NSCLC) tumor biopsies based on morphology. scRNA-seq, CNV, and targeted mutation analysis verified enrichment of carcinoma cells. Sorted cells had gene expression signatures indicative of tumor cells (e.g. EpCAM expression), increased genomic alterations by CNV analysis, and increased allele frequency of P53 and KRAS mutations relative to pre-sorted samples. Overall, the data indicate brightfield images of cells can be used to detect macro-level changes in cell morphology resulting from the molecular events involved in tumorigenesis. In addition, we induced chemotherapeutic drug resistance of lung cancer cell lines in vitro, imaged the resulting cultures on the Deepcell platform, and used the bright-field cell images to generate multi-dimensional morphological embeddings that can be visualized by UMAP. The resulting UMAPs showed distinct clusters of cells for both the parent and resistant cell lines, indicating that there are morphological differences associated with drug resistance. We developed a random forest classifier to identify the top differential morphological features between parental and resistant cell lines. These top features can distinguish between the populations with high accuracy (87%). Finally, we performed multi-dimensional morphology analysis to compare lung adenocarcinoma (LA) and squamous cell carcinoma (SCC) cell lines. The classifier predicted the correct cell type with 75% accuracy, suggesting that differences between these two carcinomas are reflected in their morphology. Together, our data suggest that AI-detected morphological differences between cell populations may show a biologically significant link between morphology and phenotype. As such, multi-dimensional morphology analysis will bolster the understanding of complex disease states and tumor microenvironment, particularly in patient derived biopsies. Citation Format: Andreja Jovic, Kiran Saini, Ryan Carelli, Tiffine Pham, Christian Corona, Jeanette Mei, Michael Phelan, Stephane C. Boutet, Kevn Jacobs, Julie Kim, Manisha Ray, Chassidy Johnson, Nianzhen Li, Mahyar Salek, Maddison Masaeli, Matt Barnes, Cyril Ramathal. Multi-dimensional morphology analysis enables identification and label-free enrichment of heterogeneous tumor cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2392.

Abstract 400: TIP60-mediated regulation of DNp63a is associated with cisplatin resistance

Abstract About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. The chemotherapeutic drug, Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence is common. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by transcriptionally regulating several DNA damage response (DDR) genes. A previous study from our lab showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in manner dependent on the catalytic activity of TIP60. This finding suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. Silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple SCC cell lines, indicating the positive regulation of ΔNp63α by TIP60 is not cell-line specific. Further, TIP60 levels positively correlated with ΔNp63α stability, protein levels and cisplatin resistance. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α and TIP60 induced G2/M arrest, increased cell-death and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α enhanced cisplatin-DNA adduct formation and significantly reduced expression of genes involved in DDR. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance through regulation of genes involved in DNA damage repair. Our findings suggest that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC other epithelial cancers. Citation Format: Akshay Hira, Andrew J. Stacy, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated regulation of DNp63a is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 400.

Ethanolic extract of Ocimum sanctum leaf modulates oxidative stress, cell cycle and apoptosis in head and neck cancer cell lines

Ocimum sanctum Linn. is a medicinal herb that has cytotoxic effects by inducing oxidative stress in some carcinomas. This study aimed to examine the impact of O. sanctum leaf extract on oxidative stress, cell cycle progression, and apoptosis in cell lines of head and neck squamous cell carcinoma (HNSCC). Isogenic primary (HN18/HN30) and metastatic (HN17/HN31) HNSCC cell lines were used. Preparation of the ethanolic extract of O.sanctum leaf (EEOS) was carried out. HNSCC cell lines were exposed to varying concentrations (0.1–0.8 mg/ml) of EEOS for a duration of 72 h, and the MTT assay was utilized to determine the cytotoxic doses. To assess the impact of EEOS on HNSCC cells, the levels of reactive oxygen species (ROS) and malondialdehyde were measured using a fluorometric method. Flow cytometry was utilized to evaluate effects of EEOS on the cell cycle, DNA damage, and apoptosis in HNSCC cells. Caspase-3 and -9 levels in the EEOS-treated HNSCC cells were measured by ELISA. The chemical components in EEOS were detected using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. EEOS exhibited cytotoxicity against the HN18, HN17, HN30 and HN31 cells at minimum concentrations of 0.1, 0.3, 0.2 and 0.2 mg/ml, respectively. Treatment with EEOS resulted in a significant increase in ROS levels in HN18 and HN17 cells. Additionally, EEOS significantly induced the levels of malondialdehyde in HN18 and HN31 cells. Moreover, EEOS arrested the cell cycle in HN30 and HN31 cells, and significantly induced DNA damage and apoptosis in the HN18, HN30, and HN31 cells. EEOS selectively increased caspase-9 in the HN18 cells. However, caspase-3 was activated without apoptosis in the EEOS-treated HN17 cells. The constituents of EEOS were identified as rosmarinic acid, caffeic acid, and apigenin. In conclusion, EEOS exhibits various prooxidative and apoptotic effects between HNSCC cells.

MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH

You have accessJournal of UrologyCME1 Apr 2023MP20-07 NPTX1 IS A NOVEL SERUM BIOMARKER INDUCED BY ARPI TREATED PROSTATE CANCER AND ENRICHED IN LINEAGE PLASTICITY WITH BRN2 HIGH Takeshi Namekawa, Joshua Scurll, Maxim Kobelev, Dwaipayan Ganguli, Olena Sivak, Martin Gleave, Vancouver, Canada, Eva Corey, Colm Morrissey, and Amina Zoubeidi Takeshi NamekawaTakeshi Namekawa More articles by this author , Joshua ScurllJoshua Scurll More articles by this author , Maxim KobelevMaxim Kobelev More articles by this author , Dwaipayan GanguliDwaipayan Ganguli More articles by this author , Olena SivakOlena Sivak More articles by this author , Martin GleaveMartin Gleave More articles by this author , Vancouver Vancouver More articles by this author , Canada Canada More articles by this author , Eva CoreyEva Corey More articles by this author , Colm MorrisseyColm Morrissey More articles by this author , and Amina ZoubeidiAmina Zoubeidi More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen receptor (AR) pathway inhibition is the first-line therapy for metastatic prostate cancer (PCa). However, tumors invariably develop resistance to ARPI. We previously identified a transcription factor Brain-2 (BRN2), encoded by the gene POU3F2, as a master driver of NEPC which is an aggressive form of AR-independent phenotypes treatment resistant PCa. In this study, we identified a surrogate biomarker of BRN2 activity to monitor the progression of treatment resistant PCa. METHODS: To identify BRN2 serum marker, we analyzed BRN2 ChIPseq data from 2 NEPC and 2 neural-type lung squamous cell carcinoma (nLUSC) cell lines with a focus on shared BRN2 peaks. Annotated peaks from enhancers (EnhancerAtlas) and promoters (genes within 20kb) were selected for secreted proteins and were ranked by correlation with BRN2 expression in PCa patients. Validation of protein expression was performed by western blot. R-PLEX Assay kit was used to measure concentrations in culture media from NCI-H660 cells and in serum from mice with NCI-H660 xenograft or PDX tumors and from human PCa patients. RESULTS: We identified 51 candidate serum biomarkers for BRN2 activity (or expression?) using the methods described above. These biomarkers were ranked based on their expression and correlation with BRN2 expression in PCa patient samples across four public data sets. NPTX1 was identified as the top biomarker of BRN2 expression. We further validated association of NPTX1 and BRN2 expression by BRN2 overexpression in MR42D and 22Rv1, indicating that BRN2 positively regulates NPTX1 expression. Next, we showed that NPTX1 is present in culture media from NCI-H660 cells, and that NPTX1 serum concentration positively correlated with tumor volume in mice with NCI-H660 xenografts and with NPTX1 mRNA expression in LuCaP PDX models. Importantly, evaluation of patients’ samples showed that NPTX1 levels were high in 26/35 CRPC serum samples vs levels in serum of healthy volunteers, and that the NPTX1 serum levels were highest in 4/6 patients with high POU3F2 tumor expression. CONCLUSIONS: We identified and validated NPTX1 as a serum marker that can be readily detected in serum from NEPC patients with BRN2high tumours. Source of Funding: U.S. Army Medical Research and Development Command(W81XWH1810689) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e277 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Namekawa More articles by this author Joshua Scurll More articles by this author Maxim Kobelev More articles by this author Dwaipayan Ganguli More articles by this author Olena Sivak More articles by this author Martin Gleave More articles by this author Vancouver More articles by this author Canada More articles by this author Eva Corey More articles by this author Colm Morrissey More articles by this author Amina Zoubeidi More articles by this author Expand All Advertisement PDF downloadLoading ...

Oral Cancer Pain Includes Thermal Allodynia That May Be Attenuated by Chronic Alcohol Consumption.

Oral cancer is one of the most painful cancer types, and is often refractory to existing analgesics. Oral cancer patients frequently develop a tolerance to opioids, the mainstay of current cancer pain therapy, leaving them with limited therapeutic options. Thus, there is a great need to identify molecular mechanisms driving oral cancer pain in an effort to develop new analgesics. Previous reports demonstrate that oral cancer patients experience intense mechanical pain and pain in function. To date, no studies have examined thermal pain in oral cancer patients or the role that alcohol consumption plays in oral cancer pain. This study aims to evaluate patient-reported pain levels and thermal allodynia, potential molecular mechanisms mediating thermal allodynia, and the effects of alcohol consumption on patient-perceived pain. This study evaluated human oral squamous cell carcinoma (OSCC) cell lines for their ability to activate thermosensitive channels in vitro and validated these findings in a rat model of orofacial pain. Patient-reported pain in a south Texas OSCC cohort (n = 27) was examined using a visual analog scale (VAS). Covariant analysis examined variables such as tobacco and alcohol consumption, ethnicity, gender, and cancer stage. We determined that OSCC secretes factors that stimulate both the Transient Receptor Potential Ankyrin type 1 channel (TRPA1; noxious cold sensor) and the Transient Receptor Potential Vanilloid type 1 channel (TRPV1; noxious heat sensor) in vitro and that OSCC-secreted factors sensitize TRPV1 nociceptors in vivo. These findings were validated in this cohort, in which allodynia to cold and heat were reported. Notably, subjects that reported regular alcohol consumption also reported lower pain scores for every type of pain tested, with significantly reduced cold-induced pain, aching pain, and burning pain. Oral cancer patients experience multiple types of cancer pain, including thermal allodynia. Alcohol consumption correlates with reduced OSCC pain and reduced thermal allodynia, which may be mediated by TRPA1 and TRPV1. Hence, reduced pain in these patients may contribute to a delay in seeking care, and thus a delay in early detection and treatment.

Fanconi anemia-isogenic head and neck cancer cell line pairs: A basic and translational science resource.

Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA-associated carcinomas are also therapeutically challenging: they may be multi-focal and stage-advanced at diagnosis, and most individuals with FA cannot tolerate standard-of-care systemic therapies such as DNA cross-linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) to foster new work on the origins, treatment and prevention of FA-associated carcinomas. The FA-CCLR consists of Fanconi-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA-associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient-derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA-dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC-isogenic cell line pairs are available to academic, non-profit and industry investigators via the "Fanconi Anemia Research Materials" Resource and Repository at Oregon Health & Sciences University, Portland OR.

Amphipathic barbiturates as marine product mimics with cytolytic and immunogenic effects on head and neck squamous cell carcinoma cell lines.

The incidence of head and neck squamous cell carcinoma (HNSCC) is increasing and the conventional treatments for this form of cancer can be tough. Despite the success of existing immunotherapies in some HNSCC patients, many do not respond to this type of treatment. Thus, the development of novel anti-cancer therapies should be prioritized. In the current study, the anticancer activity of a panel of novel compounds, herein termed marine product mimics (MPMs), against HNSCC cell lines is explored. The previously reported compound MPM-1, which is structurally related to the novel MPMs, was shown to have promising effects on the HNSCC cell line HSC-3. The results from the current study indicate that the novel MPMs are more potent than MPM-1 but cause a similar type of cell death. The results indicated that the MPMs must cross through the cell membrane to exert their action and that they are lysosomotropic. Further experiments showed that some of the MPMs could induce phosphorylation of eukaryotic initiation factor 2α (eIF2α) in HSC-3 and UT-SCC-24A cells, which indicates that they can activate the integrated stress response that is strongly associated with immunogenic cell death. Cell surface expression of calreticulin and release of HMGB1 and ATP, which are all hallmarks of immunogenic cell death, was also demonstrated in HSC-3 and UT-SCC-24A cells treated with MPMs. This suggests that the MPMs are interesting candidates for future HNSCC cancer therapies.

Enhancement of Anticancer Effects by Combining 5-Fluorouracil with Refametinib in Human Oral Squamous Cell Carcinoma Cell Line

(1) Background: Oral squamous cell carcinoma (OSCC) is a significant health burden worldwide. This study aimed to determine the potentials of Refametinib, an orally bioavailable selective MEK1/2 inhibitor, to increase the effectiveness of 5-Fluorouracil (5-FU), a common cytotoxic drug, in the SCC4 cell line. (2) Methods: SCC4 cells were treated with increasing concentrations of 5-FU, either alone or in combination with Refametinib. The chemosensitivity to treatment was assessed via cell viability assay, microscopic observation, colony formation assay, and detection of apoptotic markers using Western blotting. The whole-cell expression and surface expression of programmed death-ligand 1 (PD-L1), an immune checkpoint protein which contributes to chemoresistance and affects treatment response, were also determined using Western blotting and flow cytometry, respectively. (3) Results: The combined treatment suppressed cell proliferation and promoted apoptosis in a more potent way than 5-FU treatment alone did. Additionally, MEK/ERK inhibition mitigated 5-FU-induced PD-L1 upregulation. (4) Conclusions: This is the first report of an enhanced anticancer effect and reduced PD-L1 expression for the combination of 5-FU with Refametinib in OSCC, suggesting a new promising combination strategy.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines

IntroductionCholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. Materials and methodsCell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-β-cyclodextrin (MβCD - 7.5, 10 or 15 mM) ResultsDecreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein β-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. ConclusionCholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening.

Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer molecules, which, in the presence of oxygen and light, lead to the formation of reactive oxygen species (ROS) and consequent cell death. The selection of the photosensitizer molecule is important for the therapy efficiency; therefore, many molecules such as dyes, natural products and metallic complexes have been investigated regarding their photosensitizing potential. In this work, the phototoxic potential of the DNA-intercalating molecules-the dyes methylene blue (MB), acridine orange (AO) and gentian violet (GV); the natural products curcumin (CUR), quercetin (QT) and epigallocatechin gallate (EGCG); and the chelating compounds neocuproine (NEO), 1,10-phenanthroline (PHE) and 2,2'-bipyridyl (BIPY)-were analyzed. The cytotoxicity of these chemicals was tested in vitro in non-cancer keratinocytes (HaCaT) and squamous cell carcinoma (MET1) cell lines. A phototoxicity assay and the detection of intracellular ROS were performed in MET1 cells. Results revealed that the IC50 values of the dyes and curcumin in MET1 cells were lower than 30 µM, while the values for the natural products QT and EGCG and the chelating agents BIPY and PHE were higher than 100 µM. The IC50 of MB and AO was greatly affected by irradiation when submitted to 640 nm and 457 nm light sources, respectively. ROS detection was more evident for cells treated with AO at low concentrations. In studies with the melanoma cell line WM983b, cells were more resistant to MB and AO and presented slightly higher IC50 values, in line with the results of the phototoxicity assays. This study reveals that many molecules can act as photosensitizers, but the effect depends on the cell line and the concentration of the chemical. Finally, significant photosensitizing activity of acridine orange at low concentrations and moderate light doses was demonstrated.

An In Vitro Pilot Study Investigating the Antineoplastic Effects of GP-2250 on Cutaneous Squamous Cell Carcinoma Cell Lines: Preliminary Results

Advanced cutaneous squamous cell carcinoma (cSCC) can be a life-threatening disease for which effective and safe treatment in advanced stages is very limited. GP-2250 has been recently proven to have—in vitro and in vivo—antineoplastic effects on cancer cells. This study aims to investigate the potential anti-neoplastic effects of GP-2250 on the cSCC cell lines SCC13 and A431 through dose finding assessments, MTT cytotoxicity assays, cell migration assays, BrdU proliferation assays and FCM analysis. Our preliminary results have shown for the first time evidence for anti-neoplastic effects of GP-2250 on cSCC cells, enhancing cytotoxicity, attenuating cancer cell proliferation, inducing apoptosis and reducing tumour cell migration. Further investigations evaluating the modes of action of GP-2250 on cSCC cell lines are warranted in order to justify the use in vivo studies.

LINP1 represses unfolded protein response by directly inhibiting eIF2α phosphorylation to promote cutaneous squamous cell carcinoma

BackgroundEndoplasmic reticulum stress (ER stress) may destroy endoplasmic reticulum homeostasis (ER homeostasis) and leads to programmable cell death. Unfolded protein response (UPR) originally stimulated by ER stress is critical for the survival of tumor cells through trying to re-establish ER homeostasis as an adaption to harsh microenvironment. However, mechanisms involving key regulators in modulating UPR remain underexplored.MethodsThe expression of LINP1 in cutaneous squamous cell carcinoma (cSCC) tissues and cell lines was assessed. Subsequently, LINP1 was knocked out, knocked down or overexpressed in cSCC cells. CCK-8 assays, colony forming assays, transwell migration assays and invasiveness measurement by matrigel-coated transwell were performed to examine the role of LINP1 in cSCC development through gain-of-function and loss-of-function experiments. Transcriptomic sequencing (RNA-Seq) was conducted and indicated the key downstream signaling events regulated by LINP1 including UPR and apoptosis signaling. Furthermore, the direct interaction between LINP1 and eIF2α to modulate UPR and apoptosis was confirmed by RNA pulldown, RNA immunoprecipitation (RIP), ChIP-qPCR and in vitro phosphorylation assays.ResultsIn this study, LncRNA in non-homologous end joining pathway 1 (LINP1) was identified to be one of the top ten highest-expressed LncRNAs in cSCC, the second most common cancer in the world. Functional studies using in vitro and in vivo models revealed that LINP1 functions as an oncogene to promote cell proliferation, colony formation, migration and invasiveness while inhibiting cell apoptosis in cSCC. Transcriptomic sequencing after knockdown of LINP1 indicated LINP1 negatively regulates UPR-related pathways involving key effectors for activating UPR and the apoptosis following the prolonged UPR. Mechanistic study showed LINP1 physically interacts with eIF2α to inhibit its phosphorylation for avoiding unmitigated UPR. Loss of LINP1 followed by enhanced eIF2α phosphorylation led to overactivated UPR and induced DDIT3 expression, contributing to ER stress-induced apoptosis and suppression of cSCC development.ConclusionsOur findings demonstrate a novel regulatory hierarchy of UPR by demonstrating LINP1 as a critical modulator for eIF2α phosphorylation and a suppressor of UPR-mediated apoptosis, which suggests a novel therapeutic target for cSCC treatment.

Isolation, Characterization of Undescribed Alkaloids, and Semisynthetic Modifications of Cytotoxic Pyranoacridone Alkaloids from Glycosmis pentaphylla.

Two undescribed alkaloids (10 and 11), along with nine known alkaloids (1-9), have been isolated from the stem and root bark of Glycosmis pentaphylla. Among them are carbocristine (11), a carbazole alkaloid first time isolated from a natural source, and acridocristine (10), a pyranoacridone alkaloid first time isolated from the genus "Glycosmis". In vitro cytotoxicity of isolated compounds has been analyzed on breast cancer (MCF-7), lung cancer (CALU-3), and squamous cell carcinoma cell lines (SCC-25). The results demonstrated that compounds are moderately active. In order to study the structural activity relationship of majorly isolated compounds, semisynthetic modifications have been done on majorly isolated compounds such as des-N-methylacronycine (4) and noracronycine (1) to synthesize 11 semisynthetic derivatives (12-22) on functionalizable -NH and -OH groups of the pyranoacridone scaffold at 12th and 6th positions. Semisynthetic derivatives are explored on the same cell lines as isolated compounds, and the results exhibit that semisynthetic compounds showed potent cytotoxic activity compared with naturally isolated compounds. In the case of CALU-3, the dimer at -OH position of noracronycine (1), i.e., compound 22, showed 24-fold better activity with an IC50 of 4.49 μM compared with noracronycine (1) with IC50 97.5 μM. In MCF-7, the dimer at -OH position of noracronycine (1), i.e., compound 22, showed 14-fold better activity with an IC50 of 13.2 μM compared with noracronycine (1) with IC50 187 μM.

REST/NRSF Silencing Modifies Neuronal Gene Expression in siRNA-Treated HeLa Cells: A Preliminary Exploration in the Search for Neuronal Biomarkers of Cervical Cancer

Background and Objectives: REST (RE1-silencing transcription factor) diminution is associated with transcriptional relaxation, neuropeptide overexpression, and phenotype redefinition in neuroendocrine cancers, but this effect has barely been studied in cervical cancer (CC). We previously reported reduced expressions of REST in samples with premalignant lesions and CC; however, the transcriptional consequences for neural genes associated with reduced REST expression in CC are unknown. Therefore, the objective of this work was to evaluate the expression of neuronal genes in cancerous cells with reduced expression levels of REST. Materials and Methods: Here, we monitored levels of REST by immunostaining along the premalignant lesions and in invasive cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma (ADC) in tissue samples from female patients from southern Mexico and the derivative cell lines SiHa and HeLa, respectively. Next, we selected REST target genes in silico and explored the effect of REST silencing by RT-PCR in siRNA-treated HeLa cells. Results: The results show a REST diminution in premalignant lesions, SCC, ADC, and cancerous cell lines. Further REST silencing in HeLa cells altered the expression of genes containing the RE1 (Restrictive Element 1) sequence, including CgA (chromogranin A), CHRNβ2 (cholinergic receptor nicotinic β 2 subunit), BDNF (brain-derived neurotrophic factor), CRF (corticotropin-releasing factor), and RASSF1A (Ras association domain family 1). Conclusions: This work provides preliminary evidence of the role of REST loss in the transcriptional regulation of its target genes in HeLa cells, which could have positive implications for the search for new biomarkers of cervical cancer.