SCC Cell Lines Research Articles

CEPHAELINE SENSITIZES CISPLATIN AND REDUCES ORAL SQUAMOUS CELL CARCINOMA CANCER STEM CELLS

Objective To evaluate the anticancer properties of Cephaeline on Oral Squamous Cell Carcinoma (OSCC) cell lines, including cancer stem cell (CSC) maintenance. Study Design SCC4, SCC9, and SCC25 OSCC cell lines were used. Cell viability was assessed by MTT. The presence of CSC was evaluated by tumorsphere formation. The histone acetylation (H3K9ac) status was determined by immunofluorescence staining. Results A single administration of Cephaeline disrupted CSC by reducing the tumorsphere. Additionally, cephaeline impacted the histone acetylation status, and a concomitant therapy with CDDP reduces the concentration of CDDP required to achieve IC50 in two cell lines (SCC4 and SCC9). Conclusion Our findings suggest that Cephaeline has anti-cancer properties in OSCC, disrupting the ability of cancer cells to generate tumorspheres. Moreover, this may represent an adjunct therapy to conventional treatments for OSCC. In vivo studies are needed to validate these findings. Funding FAPESP 2016/05710-4 To evaluate the anticancer properties of Cephaeline on Oral Squamous Cell Carcinoma (OSCC) cell lines, including cancer stem cell (CSC) maintenance. SCC4, SCC9, and SCC25 OSCC cell lines were used. Cell viability was assessed by MTT. The presence of CSC was evaluated by tumorsphere formation. The histone acetylation (H3K9ac) status was determined by immunofluorescence staining. A single administration of Cephaeline disrupted CSC by reducing the tumorsphere. Additionally, cephaeline impacted the histone acetylation status, and a concomitant therapy with CDDP reduces the concentration of CDDP required to achieve IC50 in two cell lines (SCC4 and SCC9). Our findings suggest that Cephaeline has anti-cancer properties in OSCC, disrupting the ability of cancer cells to generate tumorspheres. Moreover, this may represent an adjunct therapy to conventional treatments for OSCC. In vivo studies are needed to validate these findings.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC)

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

KLK4 affects the biological behavior of oral squamous cell carcinoma through Wnt/β catenin and PI3K/Akt signaling pathways

Purpose: To investigate the effect of kallikrein 4 (KLK4) on oral squamous cell carcinoma (OSCC), and its mechanism of action.
 Method: Human tongue SCC cell line TCA-8113 was cultured, and the interference fragment and irrelevant sequence fragment of KLK4 gene were designed and synthesized. These fragments were constructed into pgcsi-h1 plasmid. Then, the TCA-8113 cells were cultured for 24 h, followed by transfection, after which TCA-8113-NC and TCA-8113-siKLK4 cells were obtained. The mRNA expression of KLK4 was determined using quantitative real-time-polymerase chain reaction (PCR). Proliferative potential was assessed using MTT method, while apoptosis was determined by flow cytometry. Furthermore, cell migration was evaluated by scratch test, and Transwell cell invasion experiment was used to determine cell invasion. Immunoblot assay was applied to determine the expressions of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt.
 Result: Compared with TCA-8113-nc, the expression of KLK4 in TCA-8113-siKLK4 cells was significantly down-regulated, while, relative to TCA-8113-NC, TCA-8113-siKLK4 cells had significantly reduced cell proliferation, invasion and migration, and enhanced apoptosis (p < 0.05). Moreover, there was significant up-regulation of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt in TCA-8113-siKLK4 group, relative to TCA-8113-NC (p < 0.05).
 Conclusion: KLK4 enhances the multiplication, metastasis and invasive potential of OSCC cells, but suppresses apoptotic changes via activation of Wnt/β-catenin/PI3K/Akt signal route. The potential of Kallikrein-4 as biomarker for OSCC cells should be determined in suitable animal models.

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

SOX18 meditates the resistance of Bmi1-expressing cells to cetuximab in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of malignancy in the head and neck region worldwide. The therapeutic strategies for HNSCC remain unsatisfying and limited. Here, we found a population of resistant Bmi1-expressing cells in the presence of cetuximab treatment and reported a novel role of SRY-box transcription factor 18 (SOX18), a member of the SOX family, in promoting HNSCC resistance to cetuximab. This study aimed to investigate the regulatory mechanism of Sox18 in Bmi1-positive cells and to search for better therapeutic targets. We successfully obtained Bmi1CreER, RosatdTomato, and RosaDTA mice and identified Bmi1-expressing cells through lineage tracing. SOX18 expression in HNSCC and normal tissues was analyzed by immunohistochemistry, colocalization of Sox18, and Bmi1-expressing cells was analyzed by immunofluorescence, and SOX18 expression in SCC9 cell lines was quantified by western blotting and quantitative real-time PCR. The investigation of the mechanism of SOX18-mediated cetuximab resistance in Bmi1-positive cells was based on the analysis of single-cell RNA-seq data obtained from the Gene Expression Omnibus (GEO) database. Western blotting was performed to verify the results obtained from the single-cell RNA-seq analysis. In our study, we demonstrated that Bmi1-expressing cells were resistant to cetuximab treatment and that depletion of Bmi1-expressing cells improved cetuximab efficacy in HNSCC. We then discovered that Sox18 mediated the stem cell-like properties of Bmi1-expressing cells and promoted cellular cetuximab resistance through an oxidative phosphorylation pathway. There was a significant downregulation of key genes in the oxidative phosphorylation pathway in Sox18 knockout cell lines. Taken together, the findings of our study suggest that Sox18 mediates the resistance of Bmi1-expressing cells to cetuximab in HNSCC via the oxidative phosphorylation pathway.

Abstract 4936: Activity of carfilzomib in head and neck cancer cell lines in vitro

Abstract Background: Carfilzomib (CF) is a 2nd generation proteasome inhibitor with activity in hematologic cancer, but its study in solid tumors like squamous cell carcinoma of the head and neck (SCCHN) is limited. We investigated its antitumor activity in three SCCHN cell lines. Methods: Cells: We studied two human tongue cell lines (CAL27; SCC-25) and one pharynx line (FaDu); all HPV-negative and p53-mutant. MTT assays: We utilized MTT assays to evaluate cytotoxicity of CF, cisplatin, and their combination. The IC50 of CF and Combination Index (CI) of varying concentrations of combined CF and cisplatin were calculated. Clonogenic assays: Cells were seeded at low density and after 24 hours, cells were treated with varying concentrations of CF. Plates were incubated for 10 days. The cells were fixed and stained with crystal violet dye and colonies counted. Wound Healing assays: After cells reached 100% confluence, a scratch was created using a 200 µL micropipette. Cells were treated with CF and images were acquired at 0, 24, 48, and 72 hours post-scratch. Results: MTT assays: IC50 values as a function of exposure lengths are shown in Table 1. CAL27 was the most sensitive while SCC-25 was the most resistant. In all cell lines, the results of combined CF and cisplatin were complex: at lower doses there was evidence of antagonism, while at higher doses there was synergy. Clonogenic assays: CF significantly inhibited clonogenicity compared to DMSO control (P≤ 0.05). The surviving fraction for CF 2.5, 5, and 10 nM were 0.411. 0.151, 0.051, and 0.5, 0.339, 0.102 in CAL27 and SCC-25 cell lines, respectively. Wound Healing assays: Wound closure % after treatment with CF5 was significantly different from DMSO control (P≤0.05). Wound closure % for CF5 were 30.97, 56.75, and 28.03% compared to 94.65, 75.88, and 42.05% for control in CAL27, FaDu, and SCC-25 cell lines, respectively. Conclusion: CF exhibits antitumor effects in SCCHN in vitro. Our findings support further investigation of CF in combination with conventional agents in vitro and in vivo. IC50 values for carfilzomib in head and neck cancer cell lines Cell line 24 hours 48 hours 72 hours CAL27 18.02 (11.76- 28.22) 9.364 (7.189- 12.45) 6.401 (5.579- 7.373) FaDu 57.34 (41.98-84.90) 16.22 (13.438- 19.446) 8.237 (7.260- 9.365) SCC25 77.78 (67.18- 91.30) 12.87 (11.36- 14.64) 11.49 (10.51- 12.58) Citation Format: Abdullah S. Eldaly, Douglas B. Stairs, Mitchell Machtay. Activity of carfilzomib in head and neck cancer cell lines in vitro. [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 4936.

Abstract 2772: The development of immunotherapy agents from the indigenous herbal plants and microbes used by the tantric practitioner of Vedic Jiva Upakara Cikitsha Tantra (Vedic altruism-based medicine system) of Assam, India

Abstract The ancient India's indigenous tantric community had its unique medicinal philosophy as well as clinical practice, distinct from Ayurveda. One such tantric community was Assam's Vedic Jiva Upakarvada practitioners (1). These practitioners developed an unique health care practice in the silk village of Sualkuchi, and the adjoining area, a part of the Vedic Silk Road (the North-South Silk Road stretching from Tibet to South East Asia via India's Assam state). We have been systematically studying the medical practice of these healing practitioners for the last two decades (1). These practitioners used more than 36 different plant-derived products, as well as soil, plus fecal and urine products rich in diverse microbial communities. They postulated that these agents contain good nigudah (1), microscopic life forms that can activate Avatar Kosha in our body (1). Using our pre-clinical in vitro as well as mouse model of cancer stem cells (2-4), we have screened 12 out of 36 plants, as well as microbial-rich soil of a sacred hill area frequented by tantric practitioners to collect herbal plants, animal feces and soils (the Suad muni ashram area of Sualkuchi). Here we report our preliminary results of the screening of these ingredients for their potential immunotherapy uses. Method: These herbal plants/soils/animal feces items were dissolved in the RPMI culture media using a traditional method using water, as well as special plant-derived juices. The conditioned RPMI culture media was then used to culture CSCs of tumor stemness defense (TSD) phenotype (2). Results: Our preliminary results show that while some of the agents induce apoptosis of TSD, other agents are capable of inducing TSD phenotypes to CSCs of diverse tumors including oral cancer (SCC-25 cell line), breast cancer (MCF-7) and colon cancer cell line (LOVO). Conclusion: The traditional healer used products may have unique anti-cancer including immuno-activating properties that need further exploration. We are now using our mouse models to find out if some of these agents including unique microbes present in these agents may transform cold tumors into hot or immunogenic tumors. 1. https://www.biorxiv.org/content/10.1101/2020.11.14.382572v1.full.pdf 2.PMID: 36248858 3. PMID: 28884113. 3. PMID: 31387932. 4. PMID: 31266772 Citation Format: Lekhika Pathak, Tutumoni Baishya, Partha Saikia, Nihar Ranjan Das, Sailen Baishya, Rupam Das, Pragya Dutta, Chayanika Das, Mallika Maral, Gakul Das, Bikul Das. The development of immunotherapy agents from the indigenous herbal plants and microbes used by the tantric practitioner of Vedic Jiva Upakara Cikitsha Tantra (Vedic altruism-based medicine system) of Assam, India [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 2772.

Abstract 648: Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype

Abstract Oral squamous cell carcinoma (OSCC) is a global health disease affecting developing countries, mainly the South and South East Asian countries. This cancer type is mostly HPV-16 negative and exhibits aggressiveness and resistance to therapies. Our previous studies have shown an oral microbiota (Fusobacterium nucleatum a gram negative anaerobic bacillus) associated with tumour progression in oral squamous cell carcinoma (1). The enhanced degree of stemness involved may confer the CSCs to be highly aggressive, self-sufficient, transient and niche modulatory, the tumour stemness defense (TSD) phenotype (2). The TSD characteristics are expressed as a part of “stem cell niche defence mechanism” (2). The TSD phenotype in ABCG2+ CSCs are characterized by the activation of the MYC-HIF-2alpha stemness pathway (3), as well as altruistic defense mechanism (4). Here we further studied the role of F.N. in reprograming of oral CSCs to TSD phenotype. Methods: SCC-25 cell line containing EpCAM+/ABCG2+ CSCs were treated with saliva samples of oral cancer patients (n=15). The cells were grown in antibiotic-treated media for 72 hours and then grown in serum-free conditioned media obtained from naïve MSC culture (2). The treated cells were then subjected to immunomagnetic sorting to obtain EPCAM+/ABCG2+ CSCs; TSD phenotype of the sorted CSCs were evaluated as described (2). Some cells were lysed and subjected to bacterial culture to detect F.nucleatum. Next, we evaluated the therapy resistance of the saliva-treated CSCs with chemotherapy, targeted therapy, immunotherapy and small molecular inhibitors of stemness pathways. We also obtained primary oral cancer EpCAM+/ABCG2+ of platinum treated patients (n=12), and lysed these cells for bacterial culture to detect F.nucleatum phenotype. Results: We confirmed the previous findings that F.nucleatum can be recovered from the EpCAM+/ABCG2+ of SCC-25 cells treated with patient derived saliva (processed saliva samples from relapsed cases; n=6/16 live bacteria recovered). These EpCAM+/ABCG2+ CSCs exhibited TSD phenotype, including the activation of MYC-HIF-2alpha stemness pathway, as well as TLR2/4/NFkB pathway. Importantly, the intracellular F.nucleatum exhibited an increase in surface proteins Fap 2, FadA, the two proteins involved in oncogenesis. We confirmed the FN induced TSD phenotype by treating SCC-25 cells with laboratory derived FN (ATCC 25586). Importantly, the primary tumor derived EpCAM+/ABCG2+ CSCs of 6/18 subjects exhibited the presence of F.N. Conclusion: Our findings indicate the potential role of oral microbiome especially the F. nulceatum in oral cancer stem cell reprograming to the highly aggressive TSD phenotype. 1. Cancer research. 2018;78(13 Supplement):3064-. 2. PMID: 362488583. 3. PMID: 33887214.4. PMID: 31266772 Citation Format: Partha Jyoti Saikia, Lekhika Pathak, Shirsajit Mitra, Bikul Das. Oral microbiome reprograms oral cancer stem cells to the highly immunosuppressive tumor stemness defense (TSD) phenotype [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 648.

Abstract 4756: NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC)

Abstract Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [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 4756.

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.

Proliferation-associated 2G4 P48 is stabilized by malignant T-cell amplified sequence 1 and promotes the proliferation of head and neck squamous cell carcinoma

Background/purposeProliferation-associated protein 2G4 (PA2G4) has alternative transcriptional and translational initiation. One dominant transcript ENST00000303305 could be translated into two protein isoforms (PA2G4-P42 and PA2G4-P48). In this study, we aimed to explore the effects of PA2G4-P42 and PA2G4-P48 on the proliferation of head and neck squamous cell carcinoma (HNSCC) and the mechanisms regulating PA2G4-P48 stability. Materials and methodsHNSCC cell lines HSC2 and SCC25 with relatively low PA2G4 expression were used for in-vitro cell studies. PA2G4-P42 and PA2G4-P48 overexpression lentiviruses were generated. In vitro cell proliferation was assessed by CCK-8 and colony formation. In vivo tumor cell proliferation was assessed by HSC2 cell-derived xenograft tumors. Liquid chromatography-mass spectrometry (LC-MS)/MS and co-immunoprecipitation (co-IP) assays were applied to check PA2G4-P48 interacting partners. Cycloheximide (CHX) chase and ubiquitin-based co-IP assays were also performed. ResultsPA2G4-P48 was the dominant isoform, with substantially higher expression than PA2G4-P42 in HNSCC. PA2G4-P48 overexpression enhanced HNSCC cell proliferation, but PA2G4-P42 overexpression slowed the proliferation. MCTS1 interacted with PA2G4-P48, but not PA2G4-P42. PA2G4 protein but not its mRNA expression was decreased in cells with MCTS1 knockdown. MG132 treatment abrogated this alteration. MCTS1 overexpression significantly elevated the half-life of PA2G4-P48, while its knockdown drastically reduced the half-life compared with the control cells. In addition, MCTS1 overexpression significantly decreased the polyubiquitination of exogenous flag-tagged PA2G4-P48. MCTS1 overexpression-induced cell proliferation was hampered by knocking down of PA2G4-P48. ConclusionPA2G4-P42 and PA2G4-P48 exert growth-suppressive and growth-promoting effects in HNSCC, respectively. MCTS1 can interact with PA2G4-P48 and prolong its half-life by reducing its poly-ubiquitination.

Effect of Light Emitting Diode and TiO2-decorated Ag Nanoparticles on Skin Cancer A431 Cell Line

Background: Devices based on light-emitting diodes (LEDs) are the newest and safest method for treating a variety of ailments, and dermatologists are now routinely using them. To confirm the effectiveness of this type of treatment, more controlled research are still required. Materials and Methods: A431 Cell line and treatment with blue LED at output light wave intensity 1200-1400 mw/cm. photodynamic therapy (PDT) with TiO2/Ag using human skin cancer cells as experimental model were used. Cell death and a possible mechanism of action were studied. Results: Blue light (wavelength 420–480 nm) exposure caused a rapid and significant decline in viability, which was followed by the death of over half of the cells. The current paper reviews the state of the art in skin LED-based treatment methods. The newest and safest treatment is LED therapy with nanoparticle, the treatment of radioactively irradiated cells with LED causes an increase in cell death. Blue light exposure might open up new possibilities for treating superficial skin cancers in people. Conclusion: The blue light (420–480 nm) of the light-emitting diode had a better anticancer effect on the SCC cell line after 24 hours of incubation. The best results were obtained with a 240-second exposure time in light with a TiO2/Ag concentration of 400 µg/ml.

Expression of Tyrosine Kinase Receptor 2 in Oral Squamous Cell Carcinoma and the Effect on Cell Proliferation and Migration and Epithelial-Mesenchymal Transition Process

To study the expression of tyrosine kinase receptor 2 (Tie2) in oral squamous cell carcinoma (OSCC) and its effect on cell proliferation and migration and the epithelial-mesenchymal transition (EMT) process. Immunohistochemistry (IHC) tests were conducted to examine the expression of Tie2 in OSCC tissues and normal oral mucosa tissues. Western blot was performed to examine the expression of Tie2 in dysplastic oral keratinocyte (DOK) cell line and OSCC cell lines, and the cell line with high Tie2 expression was selected as the experimental cell line. The Tie2-silenced lentiviral vector was successfully transfected onto the experimental cell line for subsequent experiments. Cell proliferation and cloning abilities were examined with CCK-8 and clone formation assays. Cell migration ability was examined with scratch and Transwell assays. The remodeling ability of cytoskeletal F-actin and the expressions of E-cadherin and N-cadherin were examined with confocal laser scanning microscope. Western blot was performed to examine the expression of EMT-related signature proteins, including E-cadherin, N-cadherin, and vimentin, and the expression of the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). IHC results showed that the Tie2-positive rate of the OSCC group (74.5%) was significantly higher than that of the control group (19.4%) ( P<0.0001). The expression of Tie2 was higher in HSC-4 and SCC-9 cell lines compared to that in DOK cells. The lentiviral shRNA-162 group showed the best silencing effect, which was used as the experimental group and applied in subsequent experiments. Compared with those of the control group, the proliferation, cloning and migration capacities of the cells of the experimental group were significantly reduced. Furthermore, the green fluorescence intensity of the cytoskeleton F-actin was reduced, the number of filamentous pseudopods at the leading edge of the cells decreased and their length was shortened, and the expression of E-cadherin was significantly increased, while the expression of N-cadherin and vimentin was significantly reduced in the experimental group in comparison with those of the control group. The expression of p-AKT and p-ERK proteins decreased, while AKT and ERK protein expression increased. Tie2 was highly expressed in most OSCC cells. Silencing Tie2 can inhibit the proliferation, cloning, and migration ability of OSCC cells, inhibit F-actin remodeling, and alter the expression of its EMT-related signature proteins by regulating AKT and ERK signaling pathway, which suggests that Tie2 may be involved in the growth, metastasis and EMT process of OSCC.

Anti-proliferative and immunomodulatory properties of kaffir lime leaves and bioactive compounds on macrophages co-cultured with squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. Late-stage patients have a significant chance of local recurrence and distant metastasis, as well as poor prognosis. Therapeutic goals for patients must be improved and personalized to reduce adverse effects. This study explored the anti-proliferative activity and immunomodulation potential of the constituents of crude kaffir lime leaf extract (lupeol, citronellal and citronellol) under co-culture. Results showed high cytotoxicity to human SCC15 cell line but not to human monocyte-derived macrophages. Treatment with crude extract and the contained compounds also suppressed cell migration and colony formation of SCC15 compared to the untreated control group, while high levels of intracellular ROS production were detected in the treatment group of SCC15. The MuseTM cell analyzer revealed cell cycle arrest at G2/M phase and apoptosis induction. Inhibition of Bcl-2 and activation of Bax, leading to induction of the downstream caspase-dependent death pathway were confirmed by Western blot analysis. Co-culture with activated macrophages, kaffir lime extract and its constituents enhanced the development of pro-inflammatory (M1) macrophages and boosted TNF-α production, resulting in SCC15 apoptosis. Findings revealed novel potential activities of kaffir lime leaf extracts and their constituents in inducing M1 polarization against SCC15, as well as direct anti-proliferative activity.

LncRNA HOXA11-AS maintains the stemness of oral squamous cell carcinoma stem cells and reduces the radiosensitivity by targeting miR-518a-3p/PDK1.

Oral squamous cell carcinoma (OSCC) is the most prevailing oral malignancy. The lncRNA HOXA11-AS shows prominent roles in OSCC. This study explored the effects of lncRNA HOXA11-AS on regulating OSCC stem cell stemness and radiosensitivity by targeting miR-518a-3p/PDK1. Human OSCC cell lines SCC9 and SCC15 were selected. CD133+ cancer stem cells (CSCs) were sorted by immunomagnetic beads. CD133 expression in cells and HOXA11-AS expression in SCC9, SCC15, and CD133+ SCC9, CD133+ SCC15 cells were assessed by flow cytometry and RT-qPCR. HOXA11-AS was silenced/overexpressed in SCC9, SCC15, CD133+ SCC9, and CD133+ SCC15 cells. Cell proliferation, radiosensitivity, invasion, and stem cell sphere formation ability were examined by CCK-8, colony formation, Transwell, and stem cell sphere formation. The levels of stemness-related genes (Oct4, Nanog, Sox2), miR-518a-3p, epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, Vimentin, N-cadherin), and PDK1 were assessed by RT-qPCR and Western blot assay. HOXA11-AS was up-regulated in SCC9, SCC15, CD133+ SCC9, and CD133+ SCC15 cells. HOXA11-AS silencing inhibited OSCC proliferation and invasion and enhanced radiosensitivity. HOXA11-AS maintained CSC stemness in OSCC. HOXA11-AS silencing reduced CD133+ SCC9 and CD133+ SCC15 stem cell sphere formation ability, reduced stem cell stemness-related gene levels, and inhibited EMT. HOXA11-AS regulated OSCC stem cell stemness and radiosensitivity by targeting miR-518a-3p. PDK1 overexpression annulled the regulatory effects of HOXA11-AS silencing on OSCC cell stem cell stemness and radiosensitivity. In vitro lncRNA HOXA11-AS silencing inhibited OSCC stem cell stemness by targeting the miR-518a-3p/PDK1 axis, thus enhancing OSCC cell radiosensitivity.

SEC61 translocon subunit gamma enhances low-dose cisplatin-induced cancer-stem cell properties of head and neck squamous cell carcinoma via enhancing Ca2+-mediated autophagy

Background/purposeHigh SEC61 translocon subunit gamma (SEC61G) expression is associated with an unfavorable prognosis in patients with head and neck squamous cell carcinoma (HNSCC), but the underlying mechanisms remain poorly understood. Materials and methodsHNSCC representative cell lines SCC15 and CAL27 were used to explore the regulation of SEC61G on Ca2+ leak from the endoplasmic reticulum (ER). Ca2+−activated autophagy was monitored by fluorescent labeling of autophagosomes and western blotting assays. CSC marker expression, sphere formation, colony formation, and transwell of invasion were detected to investigate the role of SEC61G in regulating cancer-stem cell (CSC) properties. ResultsAmong the SEC61 complex genes, only SEC61G upregulation is consistently associated with unfavorable progression-free interval and disease-specific survival in patients with HNSCC. Low-dose cisplatin (CDDP) treatment induced SEC61G upregulation in SCC15 and CAL27 cells. SEC61G knockdown significantly impaired CDDP-induced Ca2+ from the ER and the phosphorylation of ERK1/2 and AMPK. CDDP-induced autophagy in HNSCC cells were hampered by SEC61G shRNA, in terms of impaired autophagosome formation, lowered LC3-II/GAPDH ratio and restored p62 expression. CDDP-induced CSC properties, including CSC marker expression, sphere formation, colony formation, and invasive capabilities could be suppressed by shSEC61G and chloroquine, a specific autophagy inhibitor. ConclusionFindings of this study revealed the contribution of SEC61G in promoting cisplatin-induced CSC properties of HNSCC cells via promoting Ca2+-mediated autophagy.

Evaluation of the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata on oral tongue squamous cell carcinoma cell line (SCC-15): An in vitro study.

Oral cancer still represents the leading cause of mortality in India. Due to the drawbacks of current treatment options, a safe, low-cost therapy is the need of the hour. Recently, novel plant extracts with anti-cancer properties have gained greater attention. One among them is Annona muricata and its leaf extract, which has been studied for its anti-cancer effect against various cancers. However, studies on oral cancer cells are very much limited and hence the study. To evaluate the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata (ALEAM) against SCC-15 cell lines through in vitro assays. In vitro assays such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colony formation and wound healing assays were performed. Furthermore, to evaluate the underlying mechanism, gene and protein expression analysis of apoptotic/anti-apoptotic marker genes Bax, P53 and Bcl2, were done using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Student's t-test has been performed for analysis of experimental data. The results showed that ALEAM exhibited significant cytotoxic activity in a dose-dependent manner as well as inhibited colony formation and cell migration. The pro-apoptotic properties were affirmed by a highly significant drop in Bcl-2 gene expression and a highly significant rise in P53 and Bax genes in the study group compared to the control (P < 0.05). The current study provides evidence that ALEAM has the potential to be developed as a novel anti-cancer drug for the treatment of SCC after further clinical studies.

The identification of tumor antigens and immune subtypes based on the development of immunotherapies targeting head and neck squamous cell carcinomas resulting from periodontal disease.

Among cancer treatments, immunotherapy is considered a promising strategy. Nonetheless, only a small number of individuals with head and neck squamous cell carcinoma exhibit positive responses to immunotherapy. This study aims to discover possible antigens for head and neck squamous cell carcinoma, create an mRNA vaccine for this type of cancer, investigate the connection between head and neck squamous cell carcinoma and periodontal disease, and determine the immune subtype of cells affected by head and neck squamous cell carcinoma. To ascertain gene expression profiles and clinical data corresponding to them, an examination was carried out on the TCGA database. Antigen-presenting cells were detected using TIMER. Targeting six immune-related genes (CXCL5, ADM, FGF9, AIMP1, STC1, and CDKN2A) in individuals diagnosed with head and neck squamous cell carcinoma has shown promising results in immunotherapy triggered by periodontal disease. These genes have been linked to improved prognosis and increased immune cell infiltration. Additionally, CXCL5, ADM, FGF9, AIMP1, STC1, and CDKN2A exhibited potential as antigens in the creation of an mRNA vaccine. A nomogram model containing ADM expression and tumor stage was constructed for clinical practice. To summarize, ADM shows potential as a candidate biomarker for predicting the prognosis, molecular features, and immune characteristics of head and neck squamous cell carcinoma cells. Our results, obtained through real-time PCR analysis, showed a significant upregulation of ADM in the SCC-25 cell line compared to the NOK-SI cell line. This suggests that ADM might be implicated in the pathogenesis of HNSC, highlighting the potential of ADM as a target in HNSC treatment. However, further research is needed to elucidate the functional role of ADM in HNSC. Our findings provide a basis for the further exploration of the molecular mechanisms underlying HNSC and could help develop novel therapeutic strategies.

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers.

Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure-activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

A Novel Tongue Squamous Cell Carcinoma Cell Line Escapes from Immune Recognition due to Genetic Alterations in HLA Class I Complex.

Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8+ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)-a component of all HLA class I complex allotypes-was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins' translation and inhibited the activation and killing effect of CD8+ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8+ T cells.