Oral Carcinoma Cell Lines Research Articles

Chemotherapeutic Drug Resistance Associated with Differential miRNA Expression of miR-375 and miR-27 among Oral Cancer Cell Lines.

Recent advances have suggested that non-coding miRNAs (such as miR-21, miR-27, miR-145, miR-155, miR-365, miR-375 and miR-494) may be involved in multiple aspects of oral cancer chemotherapeutic responsiveness. This study evaluated whether these specific miRNAs are correlated with oral cancer responsiveness to chemotherapies, including Paclitaxel, Cisplatin and Fluorouracil (5FU). Commercially available and well-characterized oral squamous cell carcinoma cell lines (SCC4, SCC9, SCC15, SCC25 and CAL27) revealed differing resistance and chemosensitivity to these agents-with SCC9 and SCC25 demonstrating the most resistance to all chemotherapeutic agents. SCC9 and SCC25 were also the only cell lines that expressed miR-375, and were the only cell lines that did not express miR-27. In addition, the expression of miR-375 was associated with the upregulation of Rearranged L-myc fusion (RLF) and the downregulation of Centriolar protein B (POC1), whereas lack of miR-27 expression was associated with Nucleophosmin 1 (NPM1) expression. These data have revealed important regulatory pathways and mechanisms associated with oral cancer proliferation and resistance that must be explored in future studies of potential therapeutic interventions.

The role of p21-activated kinase 4 in the progression of oral squamous cell carcinoma by targeting PI3K-AKT signaling pathway.

Oral squamous carcinoma (OSCC), the most common head and neck malignancy, has a strong propensity for malignant proliferation and metastasis, which will decrease the survival of patients. P21-activated kinase 4 (PAK4), a classical serine/threonine protein kinase with multiple cellular functions, has an essential role in cancer cell migration and invasion. Here, we elucidated the function and possible molecular mechanisms of the effect of PAK4 on the biological behaviors of OSCC. The expression of genes and protein was detected by real-time PCR and western blotting. We used oral squamous carcinoma cell lines, Tca8117, Cal 27, SCC 4, and SCC 9 for validation of our cell function data. Flow cytometry, 3D cultures, and clone formation assay were used to detect proliferation of cells. RNA sequencing and bioinformatic analysis was performed to determine the potential function of PAK4. Immunohistochemistry, western blotting and real-time PCR demonstrated that PAK4 expression was up-regulated in OSCC tissues. Overexpression of PAK4 promoted the proliferation, migration and invasion of OSCC cell lines. RNA sequencing (RNA-seq) for the transcriptome-wide analysis of differential gene expression followed by bioinformatic analysis was performed to determine the potential function of PAK4. Based on the KEGG enrichment analysis and GO analysis of differential expression genes (DEGs) we found that PAK4 promotes the cell-cycle machinery, which associated with 44 regulated genes, thereby promoting cancer cell differentiation. This study demonstrates that the PAK4 regulates the biological behaviors of OSCC by PI3K-AKT signaling pathway, and these findings might provide a novel strategy for OSCC treatment.

In vitro comparative effects of laser photodynamic therapy with methylene blue or aminolevulinic acid on oral squamous cell carcinoma cell line

In vitro comparative effects of laser photodynamic therapy with methylene blue or aminolevulinic acid on oral squamous cell carcinoma cell line

Transduction of miRNA-155 and miRNA-34a in Oral Squamous Cell Carcinoma Cell Line

Transduction of miRNA-155 and miRNA-34a in Oral Squamous Cell Carcinoma Cell Line

Inhibitory Mechanism of IL-6 Production by Orento in Oral Squamous Cell Carcinoma Cell Line CAL27 Stimulated by Pathogen-Associated Molecular Patterns from Periodontopathogenic Porphyromonas gingivalis.

Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.

RIOK2 Contributes to Cell Growth and Protein Synthesis in Human Oral Squamous Cell Carcinoma.

Ribosomes are responsible for the protein synthesis that maintains cellular homeostasis and is required for the rapid cellular division of cancer cells. However, the role of ribosome biogenesis mediators in the malignant behavior of tongue squamous cell carcinoma (TSCC) is unknown. In this study, we found that the expression of RIOK2, a key enzyme involved in the maturation steps of the pre-40S ribosomal complex, was significantly associated with poorer overall survival in patients with TSCC. Further, multivariate analysis revealed that RIOK2 is an independent prognostic factor (hazard ratio, 3.53; 95% confidence interval, 1.19-10.91). Inhibition of RIOK2 expression by siRNA decreased cell growth and S6 ribosomal protein expression in oral squamous cell carcinoma cell lines. RIOK2 knockdown also led to a significant decrease in the protein synthesis in cancer cells. RIOK2 has potential application as a novel therapeutic target for TSCC treatment.

Role Played by Paraoxonase-2 Enzyme in Cell Viability, Proliferation and Sensitivity to Chemotherapy of Oral Squamous Cell Carcinoma Cell Lines.

Oral squamous cell carcinoma represents the most aggressive and frequent form of head and neck cancer. Due to drug resistance, the 5-year survival rate of patients with advanced disease is less than 50%. In order to identify molecular targets for effective oral cancer treatment, we focused on paraoxonase-2 enzyme. Indeed, based on data previously obtained from preliminary immunohistochemistry and Western blot analyses performed on tissue specimens, the enzyme was found to be upregulated in tumor compared with normal oral mucosa. Therefore, paraoxonase-2 gene silencing was achieved in HSC-3 and HOC621 oral cancer cell lines, and the effect on cell proliferation, viability, apoptosis induction and sensitivity to cisplatin and 5-fluorouracil treatment was evaluated. Fourier Transform InfraRed Microspectroscopy analyzed alterations of cellular macromolecules upon treatment. Enzyme level and cell proliferation were also determined in cisplatin-resistant clones obtained from HOC621 cell line, as well as in parental cells. Reported data showed that paraoxonase-2 knockdown led to a reduction of cell proliferation and viability, as well as to an enhancement of sensitivity to cisplatin, together with the activation of apoptosis pathway. Spectroscopical data demonstrated that, under treatment with cisplatin, oxidative damage exerted on lipids and proteins was markedly more evident in cells down-regulating paraoxonase-2 compared to controls. Interestingly, enzyme expression, as well as cell proliferation were significantly higher in cisplatin-resistant compared with control HOC621 cells. Taken together these results seem to candidate the enzyme as a promising target for molecular treatment of this neoplasm.

New substances of Equisetum hyemale L. extracts and their in vivo antitumoral effect against oral squamous cell carcinoma

Ethnobotanical relevanceEquisetum hyemale is used in traditional medicine as an anti-inflammatory, antioxidant, diuretic and anticancer agent. Recent studies have observed antiproliferative activity of this species in some tumor cell lines. Aim of the studyThe aim of this study was to evaluate the antiproliferative activity of the ethanol extract of E. hyemale and its partitions in oral squamous carcinoma cell lines, the death pathways induced by the most active partition, the acute toxicity and therapeutic activity, and the identification of the main compounds. Materials and methodsThe ethanol crude extract was prepared from the stems of E. hyemale and partitions were obtained from this extract with n-hexane, dichloromethane and ethyl acetate. Cytotoxicity assays were performed using MTT on human oral tumor lines SCC-9, SCC4 and SCC-25, and normal primary fibroblasts. The main pathways of programmed cell death were analyzed. Acute toxicity in mice was performed using the most active partition, ethyl acetate. Antitumor activity was accessed in xenotransplants grafts of SCC-9 cells in Balb/nude mice. Phytochemical analysis was performed using UHPLC-MS/MS and dereplication was done using Global Natural Product Social Molecular Networking (GNPS) analysis. ResultsEthanol extract, n-hexane and ethyl acetate partitions showed dose-dependent activity and selectivity towards oral tumor cells, with the ethyl acetate being the most bioactive. This medium polarity partition was shown to induce tumor cell death through apoptosis due to the presence of activated caspase 3/7, DNA fragmentation, chromatin condensation and phosphatidylserine exposure. The ethyl acetate partition also produced low toxicity in mice, provoking mild hepatic changes, but without causing necrosis and significantly reduced tumors volume and weight in xenotransplants of SCC-9 cells. Phytochemical analysis allowed identification of kaempferol glycosides and cinnamic acid derivatives previously described for E. hyemale. In addition it was possible to identify 6 new non-glycolyzed flavonoids 5-Hydroxy-3′,4′,7,8-tetramethoxyflavone (14), 5,4′-Dihydroxy-7,8,3′-trimethoxyflavone (15), 5,7-Dihydroxy-3′,4′-dimethoxyflavone (16), 3′,4,5,7-Tretramethoxyflavone (17), 5-Hydroxy-3′4′,7-trimethoxyflavone (18), and 5,4′-Dihydroxy-3′-7′-dimethoxyflavone (19); besides 5 compounds already determined to be cytotoxic in other species, Isoferulic acid (1), Ferulic acid (2), Atractylenolide III (6), Dihydroxy-3′,4′-dimethoxyflavone (16), and 5-Hydroxy-3′4 ′,7-trimethoxyflavone (18). ConclusionThe results indicate that the E. hyemale extract and partitions inhibited 3 different cell lines of OSCC in a highly selective nontoxic way by inducing apoptosis of the cells. We identified 6 new non-glycosylated flavonoids and 5 other substances in this species.

Activated fibroblasts induce immune escape of TSCC through CCL25/AKT pathway.

Accumulating evidence suggests that activated fibroblasts are the key cells in the T-cell response to tumor immunosuppression. We attempted to investigate the effect of activated fibroblasts on PD-L1 expression and the related immune escape mechanism in tongue squamous cell carcinoma. Western blotting, qPCR, and other techniques were used to study the expression of PD-L1 in tongue squamous cell carcinoma cells and the nude mouse model of transplanted tumors in vivo; clinical tissue samples were verified. In addition, we established a direct coculture model of T cells and tongue squamous cell carcinoma cells explore the mechanisms of immune escape. We found that PDGF-BB induces fibroblast activation by facilitating the oversecretion of chemokine CCL25. Further analysis showed that CCL25 derived from activated fibroblasts activated the Akt signaling pathway to promote PD-L1 expression. The activated fibroblasts inhibited T-cell IFN-γ secretion through the CCL25/Akt/PD-L1 pathway, which indirectly inhibited T-cell proliferation. Activated fibroblasts can induce the high expression of PD-L1 in the oral and tongue squamous cell carcinoma cell line Cal-27 via the CCL25/CCR9/p-Akt axis, to significantly inhibit the proliferation and IFN-γ secretion of T cells and promote the immune escape of tongue squamous cell carcinoma cells.

Introducing a novel chemotherapeutic supplement prepared by silver nanoparticles green-formulated by Salvia officinalis leaf aqueous extract to treat the human oral squamous cell carcinoma

• A clear peak at the wavelength of 448 nm confirms the nanoparticles formation. • The peaks related to the aliphatic O-H and C-H stretching, C=C, C=O and Ag-O are at the wavelengths of 3507, 2913, 1369, 1665 and 462 cm -1 . • The average size of 50-90 nm was recorded. This research displays the synthesis of Silver nanoparticles (Ag NPs) by Salvia officinalis . The structural features of the synthesized nanocomposite were investigated by FT-IR, FE-SEM, UV-Vis studies. MTT assay was used on common human oral squamous cell carcinoma cell lines i.e., KB, HSC-4, HSC-3, HSC-2, and Ca9-22 to survey the cytotoxicity and anti-human oral squamous cell carcinoma effects of the synthesized nanocomposite. The synthesized nanocomposite had very low cell viability and high anti-human oral squamous cell carcinoma activities dose-dependently against KB, HSC-4, HSC-3, HSC-2, and Ca9-22 cell lines without cytotoxicity on the normal cell line (HUVEC). To determine the antioxidant properties of the synthesized nanocomposite, the DPPH test was used in the presence of butylated hydroxytoluene as the positive control. The synthesized nanocomposite inhibited half of the DPPH molecules in the concentration of 181 µg/mL. Maybe significant anti-human oral squamous cell carcinoma potentials of the synthesized nanocomposite against common human oral squamous cell carcinoma cell lines are linked to their antioxidant activities.

Anticancer Effects of Carica papaya L. and Benzyl Isothiocyanate on an Oral Squamous Cell Carcinoma Cell Line: An In Vitro Study.

The study aimed to assess the anticancer effects of leaves of the male and female plant and seeds Carica papaya L. extract and the active compound benzyl isothiocyanate on oral squamous cell carcinoma (OSCC) cell line. Extracts of CO2 strain C. papaya L. seeds were prepared using water, ethanol, and ethanol:water by maceration, and benzyl isothiocyanate was quantified. Alkaloid fractions of leaves of male and female plants of C. papaya L. were prepared and quantified. The anticancer effects of the test substances on the SCC-25 cell line were assessed by MTT, apoptosis assay, cell cycle analysis, and determination of mitochondrial membrane potential. The ethanol:water extract of C. papaya L. (seeds) demonstrated the highest quantity of benzyl isothiocyanate. Male plant leaves demonstrated greater alkaloid content. The leaves of the male plant exhibited apoptosis induction and S-phase arrest, whereas the leaves of the female plant and seeds of C. papaya L. demonstrated G2M-phase arrest and apoptosis induction. C. papaya L. and benzyl isothiocyanate demonstrated anticancer effects. There was a difference in the anticancer effects of leaves of male and female plants of C. papaya L. The anticancer effects of papaya leaves and seeds could be further explored to develop an adjunct therapy for oral cancer to improve prognosis and reduce recurrence rates.

Anti-EGFR monoclonal antibody Cetuximab displays potential anti-cancer activities in feline oral squamous cell carcinoma cell lines.

Feline oral squamous cell carcinoma (FOSCC) is a malignant tumor characterized by an aggressive behavior and poor prognosis, for which no fully effective therapies are available. Studies of comparative oncology suggest that epidermal growth factor receptor (EGFR) may be a therapeutic target in FOSCC, similarly to human head and neck SCC (HNSCC), where the use of anti-EGFR monoclonal antibody Cetuximab has entered the clinical practice. The aim of this study was to assess the efficacy of Cetuximab in three validated preclinical models of FOSCC (SCCF1, SCCF2, SCCF3). Sequencing of tyrosine kinase domain of EGFR in the cell lines revealed a wild-type genotype, excluding the presence of activating mutations. Western blotting experiments demonstrated that Cetuximab inhibited activation of EGFR and its downstream kinase Akt in SCCF1, SCCF2 and SCCF3 along with HNSCC cell line CAL 27 included as control. Importantly, CCK-8 and trypan blue exclusion assays revealed that treatment with Cetuximab caused a decrease in cell proliferation and cell viability in all cell lines, with a general dose- and time-dependent trend. Cell death induced by Cetuximab was associated with cleavage of PARP, indicating occurrence of apoptosis. Taken together, our data suggest that Cetuximab exerts potential anti-cancer activities in FOSCC, paving the way for future translational studies aimed at assessing its employment in the therapy of this lethal cancer of cats.

Echinocystic Acid Bidesmoside Saponins from Microglossa afzelii O. Hoffm and Their Cytotoxic Activity against the CAL-27 Oral Squamous Carcinoma Cell Line.

This paper describes eight new triterpenoid saponins, including afzeliioside A (1), four acetylated afzeliiosides as pairs of inseparable regioisomers, called afzeliiosides B/C (2/3) and D/E (4/5), afzeliiosides F-H (6-8), and a known impatiprin C (9), which were isolated from the n-BuOH fraction of the liana of Microglossa afzelii. Their structures were established mainly by extensive spectroscopic analysis, including 1D and 2D NMR, HRFAB-MS, tandem ESI-MS/MS, and chemical methods, as well as a comparison of their spectral data with those of related compounds. All the isolates were screened for their cytotoxic activity against the CAL-27 oral squamous carcinoma cell line. Only compounds 4/5 (EC50 = 36.0 μg/mL (32.7 μM)) exhibited moderate cytotoxic activity. This work presents the first chemical and biological investigation of Microglossa afzelii and reports, for the first time, on the isolation of saponins in the genus Microglossa.

Over Expression of Cancer Stem Cell Marker CD44 and Its Clinical Significance in Patients with Oral Squamous Cell Carcinoma.

Cancer stem cell marker CD44 is a cell-surface glycoprotein which is involved in various cellular functions such as cell-cell interactions, cell adhesion, haematopoiesis and tumour metastasis. The CD44 gene transcription is partly activated by beta-catenin and Wnt signalling pathway, the later pathway being linked to tumour development. However, the role of CD44 in oral squamous cell carcinoma (OSCC) is not well understood. We investigated the expression of CD44 in peripheral circulation, tumour tissues of oral cancer patients and oral squamous cell carcinoma cell lines by ELISA and quantitative (q)-RTPCR. Relative CD44s mRNA expression was significantly higher in peripheral circulation (p = 0.04), tumour tissues (p = 0.049) and in oral cancer cell lines (SCC4, SCC25 p = 0.02, SCC9 p = 0.03). Circulating CD44total protein levels were also significantly (p < 0.001) higher in OSCC patients that positively correlated with increasing tumour load and loco-regional spread of the tumour. The circulating tumour stem cell marker CD44 appears to be a potent indicator of tumour progression and may be useful for developing suitable therapeutics strategies for patients with oral squamous cell carcinoma.

Geniposide inhibits cell proliferation and migration in human oral squamous carcinoma cells via AMPK and JNK signaling pathways

Iridoids are a special class of cyclopentanoid monoterpenes, which exhibit a wide range of biological effects. The present study aimed to investigate the potential effects of three iridoids genipin, geniposide and geniposidic acid on three human oral squamous cell carcinoma (OSCC) cell lines HSC-2, SCC-9 and A253 in addition to studying the possible underlying mechanisms. Cell viability assay revealed that geniposide treatment significantly suppressed the proliferation of all three cancer cell lines. In addition, geniposide induced SCC-9 cell cycle arrest at the G2/M phase (flow cytometry) through downregulation of cyclin-dependent kinase 2 and Cyclin A2 expression (western blot analysis), whilst also inducing cell apoptosis (flow cytometry and acridine orange/ethidium bromide staining) by dissipating the mitochondrial membrane potential (flow cytometry), and upregulating the expression of cleaved caspase-3 and cleaved poly-ADP ribose polymerase (western blot analysis). A wound-healing assay indicated that geniposide impaired SCC-9 cell migration by increasing the expression of E-cadherin (western blot analysis), whilst suppressing the expression of MMP-2 (western blot analysis). Western blot analysis also demonstrated that geniposide induced autophagy in SCC-9 cells by upregulating the expression of Beclin-1 and light chain 3-II. Mechanistically, geniposide activated the 5'-AMP-activated protein kinase signaling pathway and inhibited the JNK signaling pathway in SCC-9 cells (western blot analysis). The present results indicated that geniposide is able to inhibit the proliferation and migration of the tongue squamous carcinoma cell line SCC-9, suggesting a potential strategy for OSCC treatment.

Poly(rC) Binding Protein 1 Represses the Translation of STAT3 through 5' UTR.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and frequently overexpressed in cancers. However, the regulatory mechanisms of STAT3 expression are not fully understood. Poly(rC)-binding protein1 (PCBP1) is an RNA-binding protein that regulates mRNA stability, splicing, and translation. PCBP1 is a tumor suppressor and can inhibit the translation of several oncogenic genes. We aimed to understand the regulatory mechanisms of STAT3 expression. The 5' UTR or 3' UTR regions of the human STAT3 gene were inserted upstream or downstream of the green fluorescent gene (GFP), respectively, which were used as reporter systems to analyze the inhibitory effects of PCBP1 on the STAT3 gene expression. The deletion and point mutation in 5' UTR were used to search the essential regulatory sequences of the translation inhibition. The mutations of PCBP1 protein were analyzed in the cBioPortal online service. The effects of mutated PCBP1 proteins on STAT3 expression, cancer cell proliferation, and colony formation were analyzed in oral squamous cell carcinoma (OSCC) cell lines. PCBP1 inhibits mRNA translation through a motif in the 5' UTR of STAT3. Moreover, we found two leucine residues (Leu100 and Leu102) of PCBP1 protein frequently mutated in cancers. These mutations abolished the inhibition function of PCBP1 on STAT3 translation. Surprisingly, in contrast to wild-type PCBP1 protein, these mutations can promote the growth and colony formation of cancer cells. Overall, we demonstrate that PCBP1 can inhibit the expression of STAT3 through its 5' UTR, and two leucine residues of PCBP1 protein are essential for its functions.

In Vitro Anticancer Activity of Mucoadhesive Oral Films Loaded with Usnea barbata (L.) F. H. Wigg Dry Acetone Extract, with Potential Applications in Oral Squamous Cell Carcinoma Complementary Therapy.

Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high death rate and an inadequate response to conventional chemotherapeutic drugs. Medical research explores plant extracts’ properties to obtain potential nanomaterial-based anticancer drugs. The present study aims to formulate, develop, and characterize mucoadhesive oral films loaded with Usnea barbata (L.) dry acetone extract (F-UBA) and to investigate their anticancer potential for possible use in oral cancer therapy. U. barbata dry acetone extract (UBA) was solubilized in ethanol: isopropanol mixture and loaded in a formulation containing hydroxypropyl methylcellulose (HPMC) K100 and polyethylene glycol 400 (PEG 400). The UBA influence on the F-UBA pharmaceutical characteristics was evidenced compared with the references, i.e., mucoadhesive oral films containing suitable excipients but no active ingredient loaded. Both films were subjected to a complex analysis using standard methods to evaluate their suitability for topical administration on the oral mucosa. Physico-chemical and structural characterization was achieved by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Pharmacotechnical evaluation (consisting of the measurement of specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) proved that F-UBAs are suitable for oral mucosal administration. The brine shrimp lethality (BSL) assay was the F-UBA cytotoxicity prescreen. Cellular oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBA in blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line were investigated through complex flow cytometry analyses. Moreover, F-UBA influence on both cell type division and proliferation was determined. Finally, using the resazurin-based 96-well plate microdilution method, the F-UBA antimicrobial potential was explored against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019. The results revealed that each UBA-loaded film contains 175 µg dry extract with a usnic acid (UA) content of 42.32 µg. F-UBAs are very thin (0.060 ± 0.002 mm), report a neutral pH (7.01 ± 0.01), a disintegration time of 146 ± 5.09 s, and an ex vivo mucoadhesion time of 85 ± 2.33 min, and they show a swelling ratio after 6 h of 211 ± 4.31%. They are suitable for topical administration on the oral mucosa. Like UA, they act on CLS-354 tumor cells, considerably increasing cellular oxidative stress, nuclear condensation, and autophagy and inducing cell cycle arrest in G0/G1. The F-UBAs inhibited the bacterial and fungal strains in a dose-dependent manner; they showed similar effects on both Candida sp. and higher inhibitory activity against P. aeruginosa than S. aureus. All these properties lead to considering the UBA-loaded mucoadhesive oral films suitable for potential application as a complementary therapy in OSCC.

LC/Q-TOF MS and LC/QQQ MS based bioanalysis of a new ferrocene derivative as a potential anticancer lead with promising drug-like characteristics

Pyrazolopyrimidine ring present in various approved drugs is reported to target the tyrosine kinase receptor. A new pyrazolopyrimidine ferrocene derivative, which targets tumor pyruvate kinase M2 showed an impressive antiproliferative profile against human oral squamous cell carcinoma cell line CAL27 assessed using Alamar blue assay. In line with the lead optimization process, the molecule was studied for physicochemical properties where a bioanalytical method has been developed in plasma on liquid chromatography-mass spectrometry and validated following the USFDA bioanalytical method validation guideline. Plasma stability and plasma protein binding potential of the molecule have been evaluated. All the major metabolites of the compound have been identified through in vitro metabolite study employing rat liver microsome, human liver microsome, and human S9 fractions. The in silico toxicity profile of the metabolites was assessed using ProTox II software. Log P, Log D, and pKa of the molecule were found to be 4.5, 5, and 12, respectively. The molecule was found to be quite stable in plasma and have a moderate affinity towards plasma proteins (about 75 % binding). Four major metabolites have been identified and characterized by UHPLCQ-TOF-MS. The metabolites were found to have a moderate safety profile. The validated bioanalytical method and the metabolic pathway will be useful for future clinical studies and to assess the safety profile of the molecule. The finding of this study may also be useful in analyzing the desired drug-like properties through bioanalysis while designing new chemical entities based on metallocenes.

Inhibition of Ca2+ entry by capsazepine analog CIDD-99 prevents oral squamous carcinoma cell proliferation.

Oral cancer patients have a poor prognosis, with approximately 66% of patients surviving 5-years after diagnosis. Treatments for oral cancer are limited and have many adverse side effects; thus, further studies are needed to develop drugs that are more efficacious. To achieve this objective, we developed CIDD-99, which produces cytotoxic effects in multiple oral squamous cell carcinoma (OSCC) cell lines. While we demonstrated that CIDD-99 induces ER stress and apoptosis in OSCC, the mechanism was unclear. Investigation of the Bcl-family of proteins showed that OSCC cells treated with CIDD-99 undergo downregulation of Bcl-XL and Bcl-2 anti-apoptotic proteins and upregulation of Bax (pro-apoptotic). Importantly, OSCC cells treated with CIDD-99 displayed decreased calcium signaling in a dose and time-dependent manner, suggesting that blockage of calcium signaling is the key mechanism that induces cell death in OSCC. Indeed, CIDD-99 anti-proliferative effects were reversed by the addition of exogenous calcium. Moreover, electrophysiological properties further established that calcium entry was via the non-selective TRPC1 channel and prolonged CIDD-99 incubation inhibited STIM1 expression. CIDD-99 inhibition of calcium signaling also led to ER stress and inhibited mitochondrial complexes II and V in vitro. Taken together, these findings suggest that inhibition of TRPC mediates induction of ER stress and mitochondrial dysfunction as a part of the cellular response to CIDD-99 in OSCC.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines

Objectivesc-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response.Materials and methodsSix OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts.ResultsIrradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression.ConclusionsThese results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.