HSC-3 Cell Lines Research Articles

Clostridium perfringens enterotoxin induces claudin-4 to activate YAP in oral squamous cell carcinomas.

Claudin (CLDN)-4 expression has been associated with malignancy in various cancers. When CLDN4 expression was examined in oral squamous cell carcinoma (OSCC), 22 out of 57 (39%) cases showed immunoreactivity in the nucleus. Nuclear CLDN4-positive cases showed a stronger correlation with cancer progression than the negative cases. Intratumoral anaerobic bacterial DNA examination revealed nuclear CLDN4 expression in 81% of Clostridium perfringens-positive cases. Treatment of human oral squamous cell carcinoma cell lines HSC3 and HSC4 with Clostridium perfringens enterotoxin (CPE), induced CLDN4 nuclear translocation to enhance epithelial-mesenchymal transition (EMT), stemness, cell proliferation and invasive ability. In addition, CPE treatment suppressed phosphorylation of yes-associated protein-1 (YAP1) and promoted YAP1 nuclear translocation, resulting in increased expression of YAP1 target genes; cyclin D1 and connective tissue growth factor. Moreover, it was revealed that the complex of YAP1, CLDN4 and zona occludens-2 (ZO-2) was formed by CPE treatment, further suppressing YAP1 phosphorylation by LATS1 and activating it. Thus YAP activation in OSCC was regarded important in promoting malignant phenotypes. Our research suggested that the control of oral anaerobic bacteria may suppress YAP activation and in turn tumor progression.

CD47-SIRPα Signaling Induces Epithelial-Mesenchymal Transition and Cancer Stemness and Links to a Poor Prognosis in Patients with Oral Squamous Cell Carcinoma.

Background: Oral squamous cell carcinoma (OSCC), with high mortality rates, is one of the most diagnosed head and neck cancers. Epithelial-to-mesenchymal transition (EMT) and the generation of cancer stem cells (CSCs) are two keys for therapy-resistance, relapse, and distant metastasis. Accumulating evidence indicates that aberrantly expressed cluster of differentiation (CD)47 is associated with cell-death evasion and metastasis; however, the role of CD47 in the generation of CSCs in OSCC is not clear. Methods: We investigated the functional roles of CD47 in OSCC cell lines SAS, TW2.6, HSC-3, and FaDu using the bioinformatics approach, immunoblotting, immunofluorescence staining, and assays for cellular migration, invasion, colony, and orosphere formation, as well as radiosensitivity. Results: We demonstrated increased expression of CD47 in OSCC patients was associated with an estimated poorly survival disadvantage (p = 0.0391) and positively correlated with the expression of pluripotency factors. Silencing CD47 significantly suppressed cell viability and orosphere formation, accompanied by a downregulated expression of CD133, SRY-Box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and c-Myc. In addition, CD47-silenced OSCC cells showed reduced EMT, migration, and clonogenicity reflected by increased E-cadherin and decreased vimentin, Slug, Snail, and N-cadherin expression. Conclusion: Of therapeutic relevance, CD47 knockdown enhanced the anti-OSCC effect of radiotherapy. Collectively, we showed an increased CD47 expression promoted the generation of CSCs and malignant OSCC phenotypes. Silencing CD47, in combination with radiation, could provide an alternative and improved therapeutic efficacy for OSCC patients.

Cytotoxic Activities of Extracts and Isolated Compounds of Some Potential Sumatran Medicinal Plants against MCF-7 and HSC-3 Cell Lines

In continuation of our study of Sumatran plants and the bioactivity of their constituents, Stereocaulon halei Lamb (Stereocaulaceae) was extracted and isolated to obtain atranorin ( 1 ), lobaric acid ( 6 ), and methyl- β -orcinol carboxylate ( 9 ). We also prepared methanolic extracts of some Sumatran medicinal plants, i.e. Alpinia denticulata, Alpinia malaccensis, Alpinia submutica, Curcuma zedoaria, Centella asiatia , Mangifera indica, Piper crocatum, Mussaenda frondosa , together with previously isolated compounds, i.e. crocatin A ( 2 ) and crocatin B ( 3 ) from P. crocatum , curcumin ( 4 ) from Curcuma longa , demethoxymatteucinol ( 5 ) from Sphaerostephanos polycarpus , mangiferin ( 7 ) from M. indica , methyl caffeate ( 8 ) from Balanophora elongata , tiliroside ( 10 ) from Guazuma ulmifolia , and usnic acid from Usnea sp ( 11 ). These extracts and compounds were tested for their cytotoxicity against MCF-7 and HSC-3 cell lines by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). It was found that the IC 50 value of atranorin ( 1 ), lobaric acid ( 6 ), methyl- β -orcinol carboxylate ( 9 ), and methanolic extract of A. submutica towards the MCF-7 cell line was 208.20 µM, 172.05 µM, 382.60 µM and 70.95 µg/mL, respectively. The IC 50 value of lobaric acid ( 6 ) and methyl-β-orcinol carboxylate ( 9 ) against the HSC-3 cell line was 88.92 µM and 260.09 µM, respectively. The other extracts did not show significant cytotoxicity.

MiR-126 inhibits cell migration and invasion by targeting ADAM9 in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), the most frequent head and neck cancer, has a high potential for metastasis. MiR-126 plays an important role in the tumorigenesis of many tumors; however, there were little studies in OSCC. The purpose of our study was to explore how miR-126 and ADAM9 worked on migration and invasion in OSCC. The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to detect the mRNA level of miR-126 and ADAM9. The transwell assay was utilized to calculate the migratory and invasive capacities in the OSCC cells. The luciferase report assay was utilized to verify that ADAM9 was a direct target of miR-126. MicroR-126 was downregulated in OSCC tissues and cell lines SCC25 and HSC3. ADAM9 was predicted to be a direct target of miR-126 and was upregulated in the OSCC cells. In addition, miR-126 suppressed the migratory and invasive ability via mediating the expression of ADAM9 by directly targeting its mRNA 3'-noncoding region (UTR), whose partial functions was reversed by ADAM9. MiR-126 inhibited the migratory and invasive capacities of OSCC by directly targeting the 3'-UTR of ADAM9 mRNA. It is suggested that miR-126/ADAM9 axis may play an essential role in inhibiting the abilities of migration and invasion in oral squamous cell carcinoma cells.

Development of a New Cell-Based Oral Model To Study the Interaction of Oral Constituents with Food Polyphenols.

Some polyphenols have unpleasant taste properties such as astringency, which could interfere with consumers' choices. The knowledge on astringency mechanisms points that astringency is a complex phenomenon probably related to more than one physical-chemical mechanism. Thus, this work aims to develop a new and more realistic cell-based model containing human saliva, mucosa pellicle, and an oral cell line (HSC-3) to understand the oral molecular events that could contribute to the overall astringency perception. This model was then used to study the interactions with a food procyanidin fraction (PF) by HPLC. In general, the results revealed higher interaction (synergism) for the model with all the referred oral constituents (mucosa pellicle, salivary proteins, and HSC-3 cell line, HSCMuSp) when compared to the interaction with individual constituents, the PF + cells or PF + saliva. Regarding the procyanidins, a significant interaction was observed for the procyanidin monomer EcG, procyanidin dimers B7 and B2G, and trimer C1.

Gene expression of selected apoptotic markers in human oral squamous carcinoma HSC-3 cell line treated with Myrmecodia pendans plant extract

Introduction: Myrmecodia pendans (M. pendans), also known as Sarang Semut is an epiphyte with potential as a new therapeutic agent. Recently, we reported that M. pendans induced apoptotic activity in human oral squamous carcinoma (HSC-3) cell line. This study aimed to study how M. pendans extract affected apoptotic markers BAX/BCL-2 expression. Materials and methods: M. pendans was purchased from West Papua, Indonesia. The hypocotyl was allowed to dry thoroughly, then extracted aqueously. The extract was kept at -80 °C before freeze drying. The crude extract was used in the experiments. HSC-3 cells were cultured in Dulbecco’s Modified Essential Medium, then exposed to M. pendans at 2.5, 5.0 and 7.5 mg/ml for 24 and 48 h. Doxorubicin (0.005 mg/ml) was used as positive control. BAX and BCL-2 expression was analysed by Quantitative Real Time Polymerase Chain Reaction. Results: BAX and BCL-2 expression in HSC-3 cells was affected by M. pendans. At 24 h, highest levels of BAX and BCL-2 were observed with M. pendans at 2.5 mg/ml, while downregulation was observed at 5 mg/ml. However, after 48 h, BAX and BCL-2 were downregulated at all concentrations. The ratio of BAX/ BCL-2 in HSC-3 cells at 24 h showed significant upregulation at all treatment concentrations, but was downregulated after 48 h. Conclusion: M. pendans extract induced apoptotic activity in HSC-3 cells. This study suggests that induction of apoptosis in HSC-3 cell line is regulated via the pro-apoptotic (BAX) and anti- apoptotic (BCL -2) pathways.

Casticin inhibits invasion and proliferation via downregulation of β-catenin and reversion of EMT in oral squamous cell carcinoma.

Casticin expresses multiple anti-cancer activities, whereas the effect of casticin on oral squamous cell carcinoma (OSCC) is still unclear. β-catenin signaling plays a crucial role in the epithelial-mesenchymal transition which is closely related to tumorigenesis. Herein, we aimed to study the functions of casticin on invasion and migration of OSCC, and clarify whether the effect of casticin on OSCC has a relationship with β-catenin signaling. Human OSCC cell lines UM1 and HSC-3 were treated with different concentrations of casticin. The cell viability was evaluated by MTT and soft agar colony formation. Transwell assay and wound-healing assay were performed to measure the ability of cell invasion and migration. The protein expression was assessed by Western blotting. Casticin displayed inhibitory activities of cell viability, invasion, and migration on OSCC cell lines. Meanwhile, casticin could reverse EMT process and inhibit the expression of β-catenin in OSCC. Knock-down or overexpression of β-catenin could alter the effect of casticin on OSCC. Casticin impaired invasion and migration of OSCC by inhibition of β-catenin and reversal of EMT and could be a potential anti-cancer bioactive agent.

Contribution of p38 MAPK Pathway to Norcantharidin-Induced Programmed Cell Death in Human Oral Squamous Cell Carcinoma.

Norcantharidin (NCTD), a demethylated analog of cantharidin isolated from blister beetles, has been used as a promising anticancer agent; however, the underlying function of NCTD against human oral squamous cell carcinoma (OSCC) has not been fully understood. Here, this study was aimed to investigate the apoptotic effect and molecular targets of NCTD in human OSCC in vitro and in vivo. The anticancer effects of NCTD and its related molecular mechanisms were evaluated by trypan blue exclusion assay, live/dead assay, western blotting, 4-6-Diamidino-2-Phenylindole (DAPI) staining, flow cytometric analysis, Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) assay, and immunohistochemistry. NCTD significantly inhibited cell growth and increased the number of dead cells in HSC-3 and HN22 cell lines. It induced the following apoptotic phenomena: (1) the cleavages of poly (ADP-ribose) polymerase and casepase-3; (2) increase in apoptotic morphological changes (nuclear condensation and fragmentation); (3) increase in annexin V-positive cells or sub-G1 population of cells. NCTD significantly activated the p38 mitogen-activated protein kinase (MAPK) pathway but inactivated the signal transducer and activator of transcription (STAT)3 pathway. A p38 MAPK inhibitor (SB203580) partially attenuated NCTD-induced programmed cell death (apoptosis) in both cell lines, whereas ectopic overexpression of STAT3 did not affect it. NCTD strongly suppressed tumor growth in the tumor xenograft bearing HSC-3 cells, and the number of TUNEL-positive cells increased in NCTD-treated tumor tissues. In addition, NCTD did not cause any histopathological changes in the liver nor the kidney. NCTD induced programmed cell death via the activation of p38 MAPK in OSCC. Therefore, these results suggest that NCTD could be a potential anticancer drug candidate for the treatment of OSCC.

Screening of some Sumatran medicinal plants and selected secondary metabolites for their cytotoxic potential against MCF-7 and HSC-3 cell lines

In continuation of our study of Sumatran plants and bioactivities of their constituents particularly their cytotoxic activities, we re-extracted lichen Stereocaulon graminosum Schaer (Stereocaulaceae), and isolated atranorin (1) and stictic acid (4).. These isolated compounds have been tested for their cytotoxic activities against human breast adenocarcinoma cancer cell (MCF-7) and human oral squamous carcinoma cancer cell (HSC-3) using MTT assay with a slight modification. Some methanolic extracts of the leaves of Elephantopus scaber, Piper betle, Andrographis paniculata, rhizomes of Boesenbergia pandurata, Alpinia galanga, Kaempferia galanga and isolated compounds; ethyl haematommate (2), olivatolic acid (3), deoxyelephantopin (5), andrographolide (6), hydroxychavicol (7), eugenol (8), curcumin (9), pinostrobin (10), pinocembrin (11), alpinetin (12), α-mangostin (13), angiopteroside (14), and stevioside (15) from Sumatran traditional medicinal plants have also been tested.

Abstract 179: M6A demethylation enzyme FTO upregulates LDHA and promotes glycolysis and invasion and migration in oral squamous cell carcinoma

Abstract Objective: To investigate the role of lactate dehydrogenase A (LDHA) in the glycolysis and invasion and metastasis of oral squamous cell carcinoma (OSCC), and to explore the molecular mechanism of fat mass and obesity-associated protein (FTO) regulating LDHA on the glycolysis and invasion and metastasis of OSCC. Methods: Immunohistochemistry and RT-qPCR were performed to detect the expression of LDHA protein and gene in OSCC and adjacent non-cancerous tissues, and their correlation with clinical prognosis and pathological parameters were analyzed. Lentivirus-mediated shRNA silencing of LDHA and lactate dehydrogenase inhibitor (oxamate) were used to establish stable OSCC Cell lines HSC3 and SCC15, Cell Counting Kit-8 and colony formation assay were applied to evaluate cell proliferation, scratch test and Transwell assay were performed to detect cell migration and invasion, Glucose utilization assay and Lactate production assay were carried out to assess cell uptake of glucose and lactate production respectively. The molecular mechanism of FTO regulating LDHA on glycolysis, invasion and metastasis of oral squamous cell carcinoma was further explored, OSCC cell lines HSC3 and SCC15 transfected with siRNAs to silence FTO expression, EpiQuik™ m6A RNA Methylation Quantification Kit was applied to assess the m6A% in total RNA, the effect of FTO on LDHA expression was detected by Western blot and RT-qPCR. The biological function of FTO on oral squamous cell carcinoma cells was demonstrated by cell experiments in vitro. Results: Compared with adjacent non-cancerous tissues, LDHA was highly expressed in OSCC (P<0.001), which was closely related to the differentiation, clinical stage and T stage of OSCC (P<0.05). Silencing LDHA, FTO or treating oxamate in HSC3 and SCC15 cells reduced glucose consumption, lactate production and cell proliferation, invasion and migration (P<0.05). In addition, after FTO silencing, the m6A level of total RNA increased (P<0.05), while the expression level of LDHA protein and mRNA decreased (P<0.01). Conclusion: FTO may promote the expression of LDHA protein and mRNA in a m6A-dependent manner and enhance the ability of glycolysis, proliferation, invasion and metastasis of OSCC. Note: This abstract was not presented at the meeting. Citation Format: Jinsong Hou. M6A demethylation enzyme FTO upregulates LDHA and promotes glycolysis and invasion and migration in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 179.

DOWN-REGULATED YAP INHIBITS PROLIFERATION AND MIGRATION OF ORAL SQUAMOUS CELL CARCINOMA CELLS

Objectives The network including Hippo signaling controls growth, proliferation, differentiation and apoptosis of the cell and tissue which also plays crucial role in organ size control in mammals and drosophila. In this network, tumor suppressor kinases include MST and LATS while YAP and TAZ exist as oncoproteins. Above all YAP is associated with the development of early embryo and the regeneration of the skin wound as well as abnormal growth of cancers in case of over-expression. However, there have been no reports on the effect of down-regulation of YAP in oral cancer cells. And further, research is needed to evaluate the role of YAP in oral squamous cell carcinoma(OSCC) cells. In the current study, we investigated the effects of YAP down-regulation on in vitro proliferation and migration of OSCC cells. Methods We screened 13 OSCC cell lines expression of YAP mRNA and protein were confirmed by PCR and western blot analysis. Among them 2 OSCC cell lines (HSC2, KOSCC11), YAP was expressed high levels comparing with other OSCC cell lines. HSC2 and KOSCC11 cell lines were transfected with sh.RNA compared to sh.RNA Control-transfected cells. Also we performed single cell cloning for cell line's clonal isolation. We checked that down-regulation of YAP. And in vitro cell proliferation and migration assays were used to investigate the effect of YAP down-regulation on cell proliferation and migration. Findings The YAP down-regulated OSCC cells grew significantly slower than the sh.RNA Control transfected cells (p Conclusions These results suggest that down-regulation of YAP induces anti-proliferative and anti-migratory effects in OSCC, and YAP may be a useful target molecule for the treatment of OSCC.

QKI, a miR-200 target gene, suppresses epithelial-to-mesenchymal transition and tumor growth.

The microRNA-200 (miR-200) family plays a major role in specifying epithelial phenotype by preventing expression of the transcription repressors ZEB1 and ZEB2, which are well-known regulators of the epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether miR-200 family members control RNA-binding protein quaking (QKI), a newly identified tumor suppressor that is regulated during EMT. We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset. Forced expression of miR-200b/a/429 inhibited expression of ZEB1/2 and decreased cell migration in OSCC cell lines CAL27 and HSC3. QKI expression was also suppressed by miR-200 overexpression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and protumor effects in both in vitro and in vivo studies of OSCC. Furthermore, high expression of QKI protein is associated with favorable prognosis in surgically resected HNSCC and lung adenocarcinoma. In conclusion, QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis. We suggest that QKI and miR-200 form a negative feedback loop to maintain homeostatic responses to EMT-inducing signals.

Antioxidant Activity of Musa Paradisiaca (Banana) Soft Pith and Its Cytotoxicity Against Oral Squamous Carcinoma Cell Lines

Musa paradisiaca also known as banana possess great medicinal value such as haematopoietic, immunomodulatory, antifungal, anti-diabetic and antioxidant effects. Antioxidant and antiproliferative activities between two different species, banana soft pith (BSP) Awak and Abu were conducted in this study. Briefly, BSP were collected (Baling, Kedah) and extracted by 70% ethanol forming crude extract. The crude extracts were then fractionated by n-hexane, ethyl acetate, butanol and water. All BSP extracts and fractions were subjected to total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging assay, ferric reducing antioxidant potential (FRAP) assay and cytotoxic assay screening (MTT). Fraction ethyl acetate from BSP Abu showed the highest cytotoxic activity towards HSC-4 cell lines (26.95 ± 1.80 µg/mL) and the highest TPC (12.25 ±0.39 mg GAE/g) and FRAP assay (191.03 ± 0.83 mg FeSO4/g) values as compared to other fractions among BSP Abu. However, TPC (13.93 ± 0.28 mg GAE/g) and TFC (441.59 ± 19.31 mg QE/g) values of BSP Awak were found to be higher as compared to BSP Abu.

ABT-263 exhibits apoptosis-inducing potential in oral cancer cells by targeting C/EBP-homologous protein.

ABT-263 is a potent BH3 mimetic that possesses anticancer potential against various types of cancer. In general, this potential is due to its high binding affinity to anti-apoptotic proteins in the Bcl-2 family that disrupt sequestration of pro-apoptotic proteins. In the present study, we sought to identify an alternative regulatory mechanism responsible for ABT-263-mediated anticancer activity in human oral cancer. We investigated the in vitro anti-cancer effects of ABT-263 using a trypan blue exclusion assay, Western blotting, DAPI staining, immunofluorescence staining, a live/dead assay, microarray-based expression profiling, and quantitative real-time PCR. In vivo anti-tumorigenic effects of ABT-263 were examined using a nude mouse tumor xenograft model, a TUNEL assay, and immunohistochemistry. We found that ABT-263 suppressed viability and induced apoptosis in human oral cancer-derived cell lines HSC-3 and HSC-4. Subsequent microarray-based gene expression profiling revealed 55 differentially expressed genes in the ABT-263-treatead group, including 12 genes associated with "endoplasmic reticulum stress and apoptosis." Consistent with the microarray results, the mRNA expression levels of the top four genes (CHOP, TRB3, ASNS, and STC2) were found to be significantly increased. In addition, we found that ABT-263 considerably enhanced the expression levels of the C/EBP-homologous protein (CHOP) and its mRNA, resulting in apoptosis induction in four other human oral cancer-derived cell lines (MC-3, YD-15, HN22, and Ca9.22). Extending our in vitro findings, we found that ABT-263 reduced the growth of HSC-4 cells in vivo at a dosage of 100mg/kg/day without any change in body weight. TUNEL-positive cells were also found to be increased in tumors of ABT-263-treated mice without any apparent histopathological changes in liver or kidney tissues. These results provide evidence that ABT-263 may serve as an effective therapeutic agent for the treatment of human oral cancer.

Dominant-negative p53 mutant R248Q increases the motile and invasive activities of oral squamous cell carcinoma cells.

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.

Preliminary investigations of anti-cancer properties of Myrmecodia Pendens on human oral cancer HSC-3 cell lines

Abstract Myrmecodia pendens (M. pendens) or also known as Sarang semut is an epiphytic plant which might have potential as a new source of alternative therapeutic agent. Recently, it is reported that M. pendens can inhibit the proliferation of HeLa and MCM-B2 cell lines. We report here for the first time the anti-cancer potential of M. pendens on oral cancer cells derived from human oral cavity, HSC-3 cell lines. MTT assay results showed that M. pendens crude extract markedly induced cytotoxicity on HSC-3 cell lines after 24 h (IC50 = 5 mg/mL) and 48 h (IC50 = 3 mg/ml). The apoptotic activity induced by M. pendens was detected in HSC-3 cell lines by flow cytometry within 24 h. These results suggest that M. pendens may be a potential candidate for oral cancer treatment.

Comparison of the in vitro and in vivo toxic effects of thymoquinone using oral cancer HSC-3 and HSC-4 cell lines, oral fibroblasts, HACAT cell line, and Zebrafish embryos

Abstract The discovery and development of anticancer agents were made through long processes from screening new compounds in various cell lines, animal experimental studies, until clinical trials Majority of cancer chemotherapeutic agents have been initially screened in an in vitro cell-based system and then subsequently studied in an in vivo model using small animals destined for pharmacokinetic properties. Zebrafish (Danio rerio) has become one of the most popular animal model systems used for chemical toxicity tests and drug discovery. Thymoquinone (TQ) is the main constituent of black seed (Nigella sativa, spp) essential oil which shows anti-neoplastic activities in various tumor cells. However, to date, studies on the toxicity of TQ on normal cells or animal model are still insufficient. Here we report that TQ is selective toxic to different human cell lines and show negligible effect on the Zebrafish. The MTT assay demonstrated that TQ induced cytotoxicity on oral cancer cells (HSC-3 and HSC-4) but not on the normal cells (human oral fibroblast and HACAT cells). Moreover, no significant morphological change was observed in the Zebrafish model following TQ exposure at the concentration (0.003 mg/mL), similar to those in the cancer cells. The study suggests that TQ as a cancer chemotherapeutic candidate without apparent adverse effects.

Anti-neoplastic potential of ethyl-p-methoxycinnamate of Kaempferia galanga on oral cancer cell lines

Abstract Kaempferia galanga is one of the herbal plant that have many medicinal properties. The plant extract has also been reported to possess in vitro anti-cancer activities on various cancer cell lines such as human colon cancer SW620, cervical cancer C33A, and breast cancer MCF-7 cell lines. Ethyl-p-methoxycinnamate (EPMC), a major constituent of volatile oil of Kaempferia galanga has been attributed to its medicinal benefits. We report here for the first time the anticancer potential of EPMC on oral cancer cells derived from human oral cavity, HSC-3 and Ca922 lines. MTT assay results showed that EPMC markedly induced cytotoxicity on HSC-3 (IC50 0.075 mg/mL) and Ca922 (IC50 0.085 mg/mL) cell lines. The apoptotic activity induced by EPMC was detected in both cell lines by flow cytometry and confirmed with Caspase 3/7 assay. Moreover, quantitative RT-PCR analysis revealed that EPMC-induced apoptosis in both lines elevated the mRNA levels of pro-apoptotic genes caspase-9 and PUMA. These results suggest that EPMC may be a potential candidate for oral cancer treatment.

TGFBI Promotes Tumor Growth and is Associated with Poor Prognosis in Oral Squamous Cell Carcinoma.

Purpose: In a previous study, we found that transforming growth factor beta-induced (TGFBI) is a hub gene strongly associated with oral squamous cell carcinoma (OSCC), using gene chip meta-analysis and PPI network analysis. Thus, the present study was established to explore the role of TGFBI in the pathogenesis of OSCC and to define the underlying mechanisms.Methods: The correlations between TGFBI expression and the clinicopathological features and prognosis of OSCC were analyzed. Then, TGFBI-knockout HSC-3 cell lines were constructed using the CRISPR/Cas9 system. Cell proliferation, migration, and invasion in vitro were determined by cell counting, CCK-8, colony formation, and Transwell assays. Moreover, a xenograft animal study was implemented to determine the tumorigenicity and metastatic ability associated with TGFBI in vivo. The genes and pathways differentially expressed after TGFBI knockout were determined using transcriptional sequencing and bioinformatics.Results: TGFBI expression was significantly higher in OSCC than in normal tissue. Its high expression was also correlated with high stage and was predictive of poor prognosis, as we expected. Knockout of TGFBI inhibited cell proliferation and clone formation, and enhanced cell migration and invasion in vitro. Besides, the xenograft animal study showed that TGFBI knockout suppressed tumor growth and metastasis in vivo. Furthermore, transcriptome sequencing revealed that genes associated with cell proliferation, metastasis, and inflammatory responses exhibited a change of expression upon TGFBI knockout. GO and KEGG analyses indicated that the function of TGFBI is related to responses to bacteria and inflammatory responses.Conclusions: TGFBI overexpression can promote OSCC and is associated with poor prognosis in OSCC patients. TGFBI knockout can inhibit cell proliferation and metastasis in vivo. TGFBI may alter cell responses to bacteria, which causes an imbalance in the immune inflammatory response and promotes the development of OSCC.

Nicotine exposure induces the proliferation of oral cancer cells through the α7 subunit of the nicotinic acetylcholine receptor

Oral cancer and smoking are closely related, because the oral cavity, which is the route of ingestion of tobacco smoke, is in direct contact with the oral mucosa. Nicotine, one of the components of tobacco, can diffuse rapidly to the central nervous system and is responsible for tobacco addiction. Nicotine is present in high concentrations in the bloodstream of smokers; while the addictive effects of this alkaloid have extensively been studied, its effect on tumorigenesis is not clear yet. Therefore, in this study, we examined the effect of nicotine on cell proliferation and the signaling pathways it activates. The human oral squamous cell carcinoma cell line HSC-2 was used as a model system. We demonstrated the correlation between nicotine and epidermal growth factor receptor (EGFR) signaling. Nicotine treatment induced HSC-2 cell proliferation and migration and the phosphorylation of EGFR. Furthermore, nicotine treatment activated the EGFR downstream effectors phosphatidylinositol-3 kinase/AKT and p44/42 mitogen-activated protein kinases (ERK), which, in turn, promoted cell proliferation. Overall, our study suggests that nicotine promotes cell growth and migration through epidermal growth factor (EGF) signaling and plays an important role in oral cancer progression.