Squamous Carcinoma Cell Lines Research Articles

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

Evaluation of the Anti-Cancer Effect of Camel Milk on Tongue Squamous Cell Carcinoma Cell Line Using Flow Cytometric Analysis, Nuclear Morphometric Assay, and Western Blotting Technique: An In-Vitro Study

Evaluation of the Anti-Cancer Effect of Camel Milk on Tongue Squamous Cell Carcinoma Cell Line Using Flow Cytometric Analysis, Nuclear Morphometric Assay, and Western Blotting Technique: An In-Vitro Study

Abstract 6971: The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription

Abstract Squamous cell carcinoma, along with several other cancers, has been found to lack expression of lecithin retinol acyltransferase (LRAT), a protein which catalyzes retinol esterification and thereby mediates retinoic acid biosynthesis. When expression of LRAT was reintroduced in squamous cell carcinoma, the protein promoted cell differentiation in addition to retinoid status normalization—whether this effect was dependent on the known enzymatic activity of LRAT in retinol esterification, versus another function, has not yet been established. Cell elasticity differences have also been reported between squamous cell carcinoma and normal keratinocytes. In this study, the role of LRAT expression and activity was evaluated in human squamous cell carcinoma cell line SCC13. LRAT was integrated into the genome of SCC13 via retroviral transduction, both as wild-type LRAT and LRAT with a cysteine-to-serine mutation at the active site for retinol esterification (C161-S). In these transformed cell lines, LRAT promoted formation of a cohesive sheet of tissue which remained intact after its removal from the tissue culture plastic surface, as evaluated at two weeks post-confluence. SCC13 alone produced tissue which easily fragmented during this scraping assay. In an evaluation of cell elasticity with shear modulation force microscopy, relative cell modulus measurements of pre-confluent SCC13 with LRAT expression were found to be significantly greater and consistent with the cell modulus of human keratinocytes (DO33). These effects persisted with mutation of the retinol esterification active site, suggesting that LRAT possesses an additional function which has yet to be described. Variation in transcription levels of multiple genes—urokinase-type plasminogen activator, plasminogen activator inhibitor 1, tissue inhibitor of metalloproteinases 1, and kallikrein 5—was also found based on LRAT expression and level of confluence. These findings suggest that the presence of LRAT influences tissue formation, cell elasticity, and gene transcription in SCC13. Citation Format: Emily Peterson, Kuan-Che Feng, Jay Gao, Miriam Rafailovich, Marcia Simon. The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6971.

Abstract 1990: TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance

Abstract The chemotherapeutic drug Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence are common. About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. 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 orchestrating the transcriptional regulation of several DNA damage response (DDR) genes. Our previous study showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in a manner reliant on the catalytic activity of TIP60. This observation 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. We found that TIP60 levels positively correlated with ΔNp63α stability, protein levels, and cisplatin resistance. Further, silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple cisplatin resistant SCC cell lines. Further, stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α or TIP60 induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Additionally, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α, decreased colony formation, 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 intensified cisplatin-DNA adduct formation and significantly reduced the expression of drug efflux transporters. 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 in SCC. Our findings suggest that the inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers. Citation Format: Akshay Hira, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated acetylation of ΔNp63α is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1990.

Abstract 201: Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin

Abstract Vimentin is recognized as an important marker in the epithelial-mesenchymal transition (EMT) of several cancer cell types. The overexpression of vimentin appears to coincide with increased tumor growth, migration, and poor prognosis. The vulvar cancer cell line, A431, is of squamous cell origin and displays hallmarks of a squamous cell including expression of cytokeratins 8 and 18 as well as E- and P-cadherin. We have found that treatment of A431 cells with the glucocorticoid, clobetasol, results in upregulation of vimentin which is accompanied by change to a more mesenchymal-like morphology. Despite this change in morphology, the cells continue to express cytokeratins 8 and 18. This suggests that the vimentin has a greater impact on cell morphology than does cytokeratins 8 and 18. However, we previously reported that the analogous glucocorticoid, dexamethasone, also downregulates E- and P-cadherin in these cells which may account for the observed change in morphology. Here we examine more closely the impact of vimentin on cellular morphology in the clobetasol treated A431 cells by both immunofluorescence microscopy and Raman imaging. Citation Format: Jani E. Lewis, Kia Haering, Kazushige Yokoyama. Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 201.

Cholesterol depletion induces mesenchymal properties in oral squamous cell carcinoma cell line.

Cholesterol in cell membranes is crucial for cell signaling, adhesion, and migration. Membranes feature cholesterol-rich caveolae with caveolin proteins, playing roles in epithelial-mesenchymal transition and cancer progression. Despite elevated cholesterol levels in tumors, its precise function and the effects of its depletion in oral squamous cell carcinoma remain unclear. The aim of this study was to evaluate the influence of cholesterol depletion in oral squamous cell carcinoma cell line and epithelial-mesenchymal transition process. Cholesterol depletion was induced on SCC-9 cells by methyl-β-cyclodextrin and cell viability, proliferation, apoptosis, and colony formation capacities were evaluated. Gene and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-qPCR) and Western Blot, respectively, and cell sublocalization was assessed by immunofluorescence. Cholesterol depletion resulted in alteration of oral squamous cell carcinoma cell morphology at different concentrations of methyl-β-cyclodextrin, as well as decreased cell proliferation and viability rates. Analysis of CAV1 transcript expression revealed increased gene expression in the treated SCC-9 during the 24 h period, at different concentrations of methyl-β-cyclodextrin: 5 , 7.5, 10, and 15 mM, in relation to parental SCC-9. CAV1 protein expression was increased, with subsequent dose-dependent decrease. A statistically significant difference was observed in samples treated with 5 mM of methyl-β-cyclodextrin (p = 0.02, Kruskal-Wallis test). The immunofluorescence assay showed lower cytoplasmic and membrane labeling intensity in the treated samples for CAV1. These findings indicate the modulation of cholesterol as a possible mechanism underlying the regulation of these molecules and activation of epithelial-mesenchymal transition in oral squamous cell carcinoma.

MicroRNA expression profiling of cutaneous squamous cell carcinomas and precursor lesions

AbstractBackgroundActinic keratoses (AK) are pre‐malignant skin lesions caused by chronic sun exposure. Progression from an AK to intraepidermal carcinoma (IEC) and a cutaneous squamous cell carcinoma (SCC) is well known but the rate of transformation to an invasive SCC is highly variable. Since no definitive biomarkers are available, treatment decisions are made ad hoc.ObjectivesTo fully characterise our AK to SCC progression series, we performed microRNA (miRNA) microarray expression profiling of normal and photodamaged skin, as well as AKs, IEC, and invasive SCCs.MethodsThe study recruited 27 patients who donated fresh biopsies of normal skin, photodamaged skin, AK, IEC, and SCC (n = 67 specimens). All miRbase (v.21) miRNAs were profiled to identify miRNAs related to SCC progression. miRNAs were validated using qRT‐PCR and in vitro phenotypic assays.ResultsThere were 234 robustly expressed miRNAs across the tissue collection, which resulted in 20 miRNA that were differentially expressed ((cor)p ≤ 0.05 and ≥ 10 fold) between normal skin and SCC. Hierarchical clustering all samples illustrated that AKs, IEC, and SCCs were largely indistinguishable, which confirms the premalignant status of an AK. A panel of miRNAs showed significant dysregulation between normal and photodamaged skin and AK. Importantly, we found miR‐34a‐5p and miR‐31‐5p had significant differential expression between AKs and IEC and IEC and SCC respectively. Phenotypic assays determined that the miR‐31 duplex had opposing effects on SCC cell lines which suggests that dysregulation of this duplex may be related to the dynamic control of progression of transformed keratinocytes.ConclusionsThis study confirmed the continuum of AK with IEC and SCC highlighting that miRNA expression plays a role in keratinocyte transformation. Development of our putative miRNA biomarker candidates is warranted to aid in clinical management of patients experiencing high AK load to determine the most appropriate treatment.

Anti-cancer activity of Moringa oleifera Lam. seed oil on oral cancer

BackgroundDespite recent advances in the diagnosis and treatment of oral cancer, relative survival rates have not changed significantly. Moringa oleifera (M. oleifera) Lam. is one such plant with its anticancer properties being proved in its leaves, stem, flowers but no studies are yet reported proving the anticancer property of its seed oil on oral cancer. This study aimed to evaluate the anti-proliferative and cytotoxic effect of M. oleifera seed oil against two Oral squamous cell carcinoma cell lines CAL27 and SCC15 using MTT assay.Results2D GC-TOF Mass spectrometry revealed a total of 199 compounds, among which the majority were alkanes (68.2016%) and fatty acid esters (11.1399%). The MTT assay report showed good dose-dependent activity. A significant reduction in cell viability within 24 h with IC50 value of 17.78 µg/mL and 24.28 µg/mL for all treatment groups was observed for both the cell lines CAL27 and SCC15.ConclusionMTT assay showed a significant decrease in cell viability with an increase in the oil dose, thereby revealing the cytotoxic and anti-proliferative activity of M. oleifera seed oil on oral cancer cell lines namely CAL27and SCC15. The results of this study indicate that M. oleifera seed oil can be used as a potent anti-cancer agent in the treatment of Oral cancer.Graphical abstract

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Histology and Lung Nodule Fluorescence in Intraoperative Molecular Imaging With Pafolacianine

BackgroundIntraoperative molecular imaging (IMI) uses a cancer-targeted fluorescent agent injected into patients to localize tumor nodules. Pafolacianine is a folate receptor (FR)–targeted near-infrared fluorescent probe. Almost 10% of patients have false negative fluorescence findings intraoperatively. We hypothesized that tumor histology explains why lung cancer may not fluoresce. MethodsAdenocarcinoma (AC) (A549, LKR) and squamous cell carcinoma (SCC) (H127, H1264) cell lines were stained with pafolacianine. Near-infrared fluorescent microscopy was used to quantify mean fluorescence intensity. Tissue microarray slides of patients with AC and SCC were evaluated by immunohistochemistry for FR alpha (FRα) and beta (FRβ) expression. Finally, we retrospectively analyzed IMI data from clinical trials of patients with AC and SCC receiving pafolacianine. ResultsAC (intensity 30.31) cell lines have a higher fluorescence intensity than SCC cell lines (intensity 5.4) (P < .001). On slide analysis, 93.8% of ACs expressed FRα compared with 44.4% of SCCs (P = .002). Finally, there were 326 patients enrolled in clinical trials: 211 had lesions localized in vivo, and 134 of these patients had pure AC or SCC. All 9 patients with SCC have a positive smoking history and a mean pack-year of 60.2 (SD 3,6), whereas 76% of patients with AC have a history of smoking and a mean pack-year of 29.3 (P = .02). The odds ratio for fluorescence of (AC/SCC) was 2.05 (P = .004) and 2.01 (P = .02) on univariate and multivariate logistic regression, respectively. ConclusionsDuring IMI with pafolacianine, a nonfluorescent nodule is more likely to be SCC than AC. AC has a high probability of fluorescing because of higher expression of FRα or FRβ, or both.

Inhibiting Effect of Cationic Procyanidin Nanoparticles on Drug-Resistant Oral Squamous Cell Carcinoma Cell Lines

Background: Multidrug resistance (MDR) is one of the primary causes of tumor chemotherapy failure. Therefore, it is essential to uncover new drug treatment protocols. In the current study, employing chitosan (CS) and sodium tripolyphosphate (TPP) as carriers and cross-linking agents, the proanthocyanidins (PHL)/poly (lactic-co-glycolic acid) (PLGA) were encapsulated by emulsion solvent evaporation, through which the cationic CS-PLGA-PHL nanosystem was obtained. The effectiveness of CS-PLGAPHL on the invasion and migration of human oral squamous cell carcinoma cells was discussed, as were their potential mechanisms. Materials and Methods: A CS-PLGA-PHL nanosystem was constructed by emulsion-solvent evaporation. The size distribution, dispersion, and morphology were characterised by the laser particle size analyser and transmission electron microscope. Human oral squamous cell carcinoma drug-resistant cell lines SCC131/R and SCC-15/DDP were cultured in vitro. The two cell types were induced at different concentrations by CS-PLGA-PHL, after which a CCK⁃8 experiment was performed to determine the effect of CS-PLGA-PHL on the proliferation of the two cell lines. The 50% inhibitory concentration (IC50) of CSPLGA- PHL was calculated at different time points. The transwell chamber experiment was performed to identify the effects of CS-PLGA-PHL on the migration and invasion of OSCC cells. The expression levels of MMP-2 and MMP-9 were detected by Western blot and q-PCR. Results: CS-PLGA-PHL is well dispersed. The PDI appeared to be lowest when the mass ratio of chitosan to PLGA equaled 1:15. CS-PLGA-PHL exhibited a marked effect in inhibiting the proliferation of SCC- 131/R and SCC-15/DDP as well as the invasion and migration. CS-PLGA-PHL was able to downregulate the expression of MMP-2 and MMP-9 genes and proteins significantly in drug-resistant cell lines. Conclusion: CS-PLGA-PHL for oral squamous cell carcinoma has been successfully prepared to exert significant inhibition on the proliferation, invasion, and migration of OSCC cells. The mechanism involved was possibly related to the down-regulation of MMP-2 and MMP-9 expression by CS-PLGA-PHL.

Exploring the Role and Prognostic Value of TMEM208 in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the world's eight most common malignancies and a severe hazard to human health. Transmembrane protein 208 (TMEM208) has been reported to be associated with autophagy,which is strongly tied to the onset and development of numerous illnesses, including cancer. For this reason, we investigated the expression and prognostic significance of TMEM208 in HNSCC. To explore the role and molecular mechanism of this gene in HNSCC, we performed a comprehensive analysis of the TMEM208 gene, including gene expression analysis, prognostic analysis, and immune infiltration analysis using the UALCAN, HPA, CVCDAP, DAVID, TIMER, CIBERSORTx, TISIDB, and cBioPortal online databases. It was further validated by in vitro cell culture. Analysis of TCGA data showed that TMEM208 was highly expressed in HNSCC (P < 0.01) and significantly correlated with several clinicopathologic features, and in vitro cellular assays demonstrated that TMEM208 was highly expressed in multiple squamous carcinoma cell lines. Survival analysis showed that high expression of TMEM208 decreased OS (P=0.0088), PFI (P=0.0062), and DSS (P=0.0036) in HNSCC patients. cox regression analysis indicated that high expression of TMEM208 was an independent risk factor for OS in HNSCC patients (P<0.05). In addition, functional enrichment analysis showed that TMEM208 was closely associated with translation, ribosomal and mitochondrial functions, and GSVA analysis showed that TMEM208 was negatively correlated with a variety of immune cell differentiation in HNSCC, with a statistically significant difference. Immunocorrelation analysis showed that TMEM208 could affect immune cell infiltration in HNSCC; in addition, TMEM208 correlated with CD24, CD276, LAG3, and HVEM. In conclusion, TMEM208 holds promise as a prognostic indicator for HNSCC and is closely related to ICI treatment.

Expression and prognosis analysis of TBX2 subfamily in human lung carcinoma.

Lung cancer has a high morbidity and mortality rate of all cancers worldwide. Therefore, there is an urgent need for reliable cancer markers for diagnosis and prognosis of patients with lung cancer. In this study, we used the bioinformatics database to compare the expression of the TBX2 subfamily at the transcriptional and protein levels in non-small cell lung cancer. Then, to confirm our bioinformatics analysis above, we used western bloting to determine the expression of TBX2, TBX3, TBX4 and TBX5 in human lung squamous carcinoma cell lines. Besides, low expression of TBX2 subfamily predicted a poor prognosis of patients with lung cancer. Finally, The methylation database was used to explore the relationship between the low expression of TBX2 subfamily and methylation of gene promoter region. Our data showed a significant decrease of TBX2 subfamily expression in lung cancer tissues of several histological subtypes. Finally, the methylation of TBX2 subfamily members in the promoter region of NSCLC was significantly higher than that in normal tissues. Our research provided sufficient evidence that TBX2 subfamily might play an inhibitory role in malignancy progression of lung cancer, which is promising to shed light on discovering a novel reliable cancer marker for prognosis of lung cancer patients.

In vitro antimetastatic potential of pseudolaric acid B in HSC-3 human tongue squamous carcinoma cell line

ObjectivePseudolaric acid B (PAB) is a novel diterpenoid derived from the traditional Chinese medicinal herb Cortex pseudolaricis that exerts anticancer, anti-inflammatory, and immunomodulatory properties. While the anticancer potential of PAB has been studied, its effects on metastasis have not been well-studied. This study aims to determine the inhibitory effects of PAB on HSC-3 human tongue squamous cell carcinoma (TSCC) cell line. DesignCell viability and soft agar colony formation assays were conducted to assess cellular proliferation and in vitro tumorigenic capacity of TSCC cells, respectively. Additionally, wound healing, transwell migration, and invasion assays were conducted to monitor the aggressive behavior of TSCC cells. Furthermore, Western blotting analysis was conducted to reveal the signaling pathways involved in the modulation of epithelial-mesenchymal transition (EMT). ResultsThe migratory and invasive capacities of HSC-3 cells were suppressed by PAB irrespective of their proliferation states. PAB’s effects on EMT involved upregulation of E-cadherin expression and downregulation of Twist; these were concomitantly accompanied by downregulated phosphorylation of epidermal growth factor receptor (EGFR). ConclusionsPAB suppresses human TSCC in vitro by regulating Twist/E-cadherin through the EGFR signaling pathway. PAB may have potential as a candidate antimetastatic drug for TSCC treatment.

Anticancer effects of punicalagin and 5-fluorouracil on laryngeal squamous cell carcinoma: an &lt;i&gt;in vitro&lt;/i&gt; study

The purpose of this study was to assess the apoptotic effects of punicalagin alone and in combination with 5-fluorouracil (5-FU) on laryngeal squamous cell carcinoma (Hep-2) cell line. Hep-2 cells were cultured and divided into four groups: Group 1 received no therapy and served as control, Group 2 received 5-FU only, Group 3 received punicalagin only, and Group 4 received a combination of 5-FU and punicalagin. After 48 hours of incubation, cellular changes were examined under an inverted microscope. The methyl thiazolyl tetrazolium assay, caspase-3 gene level, and vascular endothelial growth factor (VEGF) level were assessed. The control group showed the highest mean value of cancer cell proliferation rate (1.595±0.58), followed by the punicalagin group (1.263±0.447), then the 5-FU group (0.827±0.256), while the combination group showed the lowest proliferation rate (0.253±0.111). The combination group showed the highest mean value of caspase-3 concentration (3.177±0.736), followed by the 5-FU group (1.830±0.646), and punicalagin group (0.741±0.302), while the control group showed the lowest mean value (0.359±0.117). Regarding VEGF levels, the control group had a statistically significant higher mean value, followed by the punicalagin and 5-FU groups, and finally, the combination group which showed the lowest value. Punicalagin exerts an anticancer effect through anti-proliferative action and induction of apoptosis on Hep-2 cell line. Combining punicalagin with 5-FU potentiates its anti-proliferative, apoptotic, and anti-angiogenic actions. It, further, helps in mitigating the putative side effects of 5-FU by reducing the dose required for its therapeutic effects.

Antioxidant and Antineoplastic Activities of Hibiscus sabdariffa Linn. Petal Extracts against Oral Squamous Cell Carcinoma Cell Line.

To assess the antioxidant and antineoplastic effects of Hibiscus sabdariffa Linn. on oral squamous cell carcinoma cells. Human squamous cell carcinoma HSCC cells were tested for cytotoxicity by a methanol extract of Hibiscus sabdariffa (MEHSP). After 24, 48, and 72 h, the MTT assay and Trypan blue exclusion test were used to determine cell survival and death. 2, 2-diphenyl-1-picrylhydrazyl (DPPH), DNA Protection Assay (DPA), and ferric reducing antioxidant power assay (FRAPA) measured the antioxidant activity of MEHSP. The antioxidant activity (%) ranged from 47.92-82.24 in the DPPH test, 11.61-73.65 in the DPA, and 4.97-52.09 in the FRAPA. The HSCC in-vitro cytotoxicity assay showed dose- and time-dependent cell viability. MEHSP at 5 μg/ml inhibited viable cells, while increasing MEHSP doses decreased cell viability. The Trypan blue exclusion test showed that MEHSP significantly reduced cell viability at 24, 48, and 72 h. Hibiscus sabdariffa contains antioxidant and HSCC-cytotoxic properties.

Novel oncogenic transcriptional targets of mutant p53 in esophageal squamous cell carcinoma.

Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common "hotspot" p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, "non-hotspot" p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic-expression of certain "non-hotspot" mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription-quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild-type and several mutant p53 forms followed by RT-qPCR, chromatin affinity-purification (ChAP), and promoter-luciferase assays indicated the exclusive recruitment of p53 mutants-P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of "non-hotspot" mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor-specific p53 mutations.

Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance.

New evidence has suggested that non-coding microRNAs play a significant role in mediating and modulating chemotherapy resistance, particularly among oral cancers. One recent study found that the upregulation of miR-145 and the downregulation of miR-155 strongly correlated with a limited chemotherapy resistance to Cisplatin, 5-Fluorouracil, and Paclitaxel, although the mechanism(s) responsible for these observations remain unidentified. Using commercially available cell lines of oral squamous cell carcinoma, RNA was isolated, converted into cDNA, and subsequently screened for the expression of downstream targets of miR-145 and miR-155 using qPCR. These results demonstrated the upregulation of miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, as well as the downregulation of miR-140, miR-152, miR-218, miR-221, and miR-224. This screening also confirmed the differential expression and regulation of mir-145 and miR-155 among the cell lines with limited chemotherapy resistance (SCC15). In addition, several downstream targets of these specific microRNAs were upregulated by all oral cancer cell lines, such as MBTD1 and FSCN1, or downregulated in all cell lines, such as CLCN3, FLI-1, MRTFB, DAB, SRGAP1, and ABHD17C. However, three miR-145 downstream targets were identified in the least chemotherapy-resistant cells, exhibiting the differential upregulation of KCNA4 and SRGAP2, as well as the downregulation of FAM135A, with this expression pattern not detected in any of the other oral cancer cell lines. These data strongly support that the differential regulation of these three downstream targets may be related to the chemosensitivity of this oral cancer cell line. The potential involvement of these targets must be further investigated to determine how and whether mechanisms of these cellular pathways may be involved in the observed lack of chemotherapy resistance. These data may be important to design targets or treatments to reduce chemotherapy resistance and improve patient treatment outcomes.

MiR-362-3p inhibited the invasion and metastasis of oral squamous cell carcinoma cells by targeting the regulation of pituitary tumor-transforming gene 1.

This study aimed to explore the effect of pituitary tumor-transforming gene 1 (PTT-G1) on the invasion and proliferation of oral squamous cell carcinoma (OSCC) cell lines under the action of miR-362-3p. The bioinformatics online database was used to query the expression of PTTG1 in head and neck squamous cell carcinoma (HNSCC). The expression of PTTG1 in the Cal-27, HN-30, and HOK cell lines was detected by Western blot. A wound-healing assay was used to determine the effect of PTTG1 on the migration ability of the OSCC cells. The Transwell assay was used to examine the changes in cell-invasion ability. 5-ethynyl-2'-deoxyuridine (EdU) cell-proliferation assay was used to detect changes in cell-proliferation ability. Bioinformatics approach predicted the upstream miRNA of PTTG1. The targeting relationship between miR-362-3p and PTTG1 was examined by the dual luciferase assay, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of miRNA in OSCC tissues. The ENCORI database showed that PTTG1 expression was up-regulated in OSCC tissues. Western blot confirmed that PTTG1 expression was up-regulated in Cal-27 and HN-30 cells than HOK cells. PTTG1 knockout can inhibit the migration, invasion, and proliferation of Cal-27 and HN-30 cells (P<0.05). Bioinformatics prediction websites predicted that the upstream miRNA of PTTG1 was miR-362-3p, and PTTG1 can bind to miR-362-3p. Results of qRT-PCR showed that miR-362-3p expression was downregulated in OSCC tissues compared with normal tissue (P<0.05). Transwell and EdU experiments confirmed that miR-362-3p knockdown can promote the invasion and proliferation of Cal-27 and HN-30 after PTTG1 knockdown. miR-362-3p can inhibit the invasion and proliferation of Cal-27 and HN-30 cells by targeting PTTG1.