Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3's extracellular domain in sweetener interaction studies.

The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging. This study aimed to address this knowledge gap by expressing an extracellular N-terminal domain encompassing the cysteine-rich domain of human hT1R3 (hT1R3-TMD) in Escherichia coli. The expressed protein was obtained as inclusion bodies, purified by metal affinity chromatography, and refolded using the dilution-refolding method. Through rigorous analysis, we confirmed the successful refolding of hT1R3-TMD and elucidated its structural characteristics using circular dichroism spectroscopy. Notably, the refolded protein was found to exist as either a monomer or a dimer, depending on its concentration. A tryptophan fluorescence quenching assay revealed that the dissociation constants for sucrose, sucralose, and brazzein were >9500 µM, 2380 µM and 14.3 µM, respectively. Our findings highlight the utility of this E. coli expression system for producing functional hT1R3-TMD for investigations and demonstrate the efficacy of the tryptophan fluorescence quenching assay in revealing complex interactions between sweet taste receptors and various sweeteners.

Caffeic acid phenethyl ester: Unveiling its potential as a potent apoptosis inducer for combating hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare form of head and neck cancer that is notorious for its poor prognosis and low overall survival rate. This highlights the need for new therapeutic options for this malignancy. The objective of the present study was to examine the ability of caffeic acid phenethyl ester (CAPE), which is an active compound found in propolis, to combat HSCC tumor growth. CAPE exerted its tumor­suppressive activity in HSCC cell lines through the induction of apoptosis. Mechanistically, the CAPE­mediated apoptotic process was attributed to the perturbation of the mitochondrial membrane potential and the activation of caspase­9. CAPE also modulated survivin and X­linked inhibitor of apoptosis, which are potent members of the inhibitors of apoptosis protein family, either through transcriptional or post­translational regulation, leading to HSCC cell line death. Therefore, the findings of the present study suggested that CAPE is an effective treatment alternative for HSCC via the stimulation of mitochondria­dependent apoptosis.

Apoptotic activity of genipin in human oral squamous cell carcinoma in vitro by regulating STAT3 signaling.

Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin-induced cell death in human oral squamous cell carcinoma (OSCC). The growth-inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit-8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4',6-diamidino-2-phenylindole staining, measurement of the sub-G1 population, and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real-time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase-dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p-STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia-1 (Mcl-1) expression, leading to apoptotic cell death. The genipin-mediated reduction in survivin and Mcl-1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC.

Neuropilin-2 acts a critical determinant for epithelial-to-mesenchymal transition and aggressive behaviors of human head and neck cancer.

PURPOSE: Neuropilin-2 (NRP2) is a multifunctional single-pass transmembrane receptor that binds to two disparate ligands, namely, vascular endothelial growth factors (VEGFs) and semaphorins (SEMAs). It is reportedly involved in neuronal and vascular development. In this study, we uncovered the exact functional role of NRP2 and its molecular mechanism during aggressive behaviors and lymph node (LN) metastasis in human head and neck cancer (HNC) and identified algal methanol extract as a potential novel NRP2 inhibitor. METHODS: In silico analyses and immunohistochemistry were used to investigate the relationship between NRP2 expression and the prognosis of HNC patients. The functional role of NRP2 on the proliferation, migration, invasion, and cancer stem cell (CSC) properties of HNC cells was examined by MTS, soft agar, clonogenic, transwell migration and invasion assays, and sphere formation assays. Signaling explorer antibody array, western blot, and qPCR were performed toward the investigation of a molecular mechanism that is related to NRP2. RESULTS: NRP2 was highly expressed in HNC and positively correlated with LN metastasis and advanced tumor stage and size in patients. Using loss- or gain-of-function approaches, we found that NRP2 promoted the proliferative, migratory, and invasive capacities of human HNC cells. Furthermore, NRP2 regulated Sox2 expression to exhibit aggressiveness and CSC properties of human HNC cells. We demonstrated that p90 ribosomal S6 kinase 1 (RSK1) elevates the aggressiveness and CSC properties of human HNC cells, possibly by mediating NRP2 and Sox2. Zeb1 was necessary for executing the NRP2/RSK1/Sox2 signaling pathway during the induction of epithelial-to-mesenchymal transition (EMT) and aggressive behaviors of human HNC cells. Moreover, the methanol extract of Codium fragile (MECF) repressed NRP2 expression, inhibiting the RSK1/Sox2/Zeb1 axis, which contributed to the reduction of aggressive behaviors of human HNC cells. CONCLUSIONS: These findings suggest that NRP2 is a critical determinant in provoking EMT and aggressive behaviors in human HNC through the RSK1/Sox2/Zeb1 axis, and MECF may have the potential to be a novel NRP2 inhibitor for treating metastasis in HNC patients.

Podophyllotoxin reduces the aggressiveness of human oral squamous cell carcinoma through myeloid cell leukemia­1.

Podophyllotoxin (PPT), which is derived from the podophyllum plant, exhibits marked cytotoxic effects against cancer cells; however, the precise molecular mechanism underlying its activity against human oral squamous cell carcinoma (OSCC) has not been elucidated. In the present study, the mechanism by which PPT induced cytotoxicity in two OSCC cell lines, HSC3 and HSC4, was determined. The effects of PPT on cytotoxicity in HSC3 and HSC4 cells were analyzed using Annexin V/PI double staining, Sub­G1 analysis, soft agar assays, western blotting, and quantitative PCR. The changes in the mitochondrial membrane potential were assessed using a JC­1 assay and cytosolic and mitochondrial fractionation. A myeloid cell leukemia­1 (Mcl­1) overexpression cell lines were also established to study the role of Mcl­1 on apoptosis. The results showed that PPT inhibited the growth of the two human OSCC cell lines and induced apoptosis, which was accompanied by mitochondrial membrane depolarization. Compared with the control, PPT reduced the expression of Mcl­1 in both cell lines through a proteasome­dependent protein degradation process. Overall, these results suggested that targeting of Mcl­1 protein by PPT induced apoptosis, providing a foundation for further pre­clinical and clinical study of its value in the management of OSCC.

Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa.

Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and kcat/Km values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the KmGSH and I50 values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (kcat/KmCDNB) plot was used to estimate the pKa value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pKa value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pKa of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.

Targeting tumor-intrinsic PD-L1 suppresses the progression and aggressiveness of head and neck cancer by inhibiting GSK3ß-dependent Snail degradation.

PURPOSE: PD-L1 is an immune checkpoint protein that allows cells to evade T-cell-mediated immune responses. Herein, we uncover a tumor-intrinsic mechanism of PD-L1 that is responsible for the progression and aggressiveness of HNC and reveal that the extracts of a brown alga can target the tumor-intrinsic signaling pathway of PD-L1. METHODS: The biological functions of PD-L1 in the proliferation and aggressiveness of HNC cells in vitro were examined by metabolic activity, clonogenic, tumorigenicity, wound healing, migration, and invasion assays. The clinical importance of PD-L1 in the prognosis of patients with HNC was analyzed by immunohistochemistry. The relationship between PD-L1 and EMT was confirmed via western blotting, qPCR, and immunocytochemistry. RESULTS: Through our in silico approach, we found that PD-L1 was upregulated in HNC and was correlated with an unfavorable clinical outcome in patients with HNC. PD-L1 was crucial for promoting tumor growth, both in vitro and in vivo. High expression of PD-L1 was closely correlated with LN metastasis in OSCC. PD-L1 facilitated the cytoskeletal reorganization and aggressiveness of HNC cells. Moreover, PD-L1 enhanced the EMT of HNC cells by regulating the Snail/vimentin axis. Consistently, MEIO suppressed the PD-L1/Snail/vimentin axis, thereby inhibiting the aggressiveness of HNC cells. Inhibition of PD-L1 induced by PD-L1 silencing or MEIO treatment caused Snail degradation through a GSK3ß-dependent mechanism. The tumor-intrinsic function of PD-L1 could be attributed to the regulation of the GSK3ß/Snail/vimentin axis. CONCLUSION: The discovery of MEIO targeting the tumor-intrinsic function of PD-L1 may prove particularly valuable for the development of novel and effective anticancer drug candidates for HNCs overexpressing PD-L1.

Inhibition of focal adhesion kinase/paxillin axis by caffeic acid phenethyl ester restrains aggressive behaviors of head and neck squamous cell carcinoma in vitro.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE), one of the components of propolis that is produced by honeybees, reportedly suppresses multiple diseases, including bacterial infection, inflammation, and cancer. We aimed to investigate the inhibitory effects of CAPE on epithelial-mesenchymal transition (EMT) status and aggressive behaviors of human head and neck squamous cell carcinoma (HNSCC) in vitro and the underlying signaling pathway. DESIGN: To examine the cell growth and in vitro tumorigenic potential of HNSCC cells, cell viability and soft agar colony formation assays, respectively, were performed. Transwell migration and invasion assays were conducted to monitor HNSCC cells' aggressive behaviors. Western blotting and immunocytochemistry analyses were done to investigate the signaling pathway responsible for relieving EMT progression and HNSCC cell aggressiveness. RESULTS: CAPE inhibited the in vitro tumorigenic potential of SNU-1041 cells stimulated by epidermal growth factor and suppressed the migratory and invasive capacities of SNU-1041 cells, irrespective of their cell proliferation state. CAPE was, at least partially, capable of inhibiting EMT progression by upregulating E-cadherin expression, which was accompanied by the reduction of phosphorylated focal adhesion kinase (FAK) and Paxillin. The inhibition of the FAK/Paxillin axis by PF-562271 was sufficient to alleviate the EMT progression through the induction of E-cadherin and aggressive behaviors of SNU-1041 cells. CONCLUSIONS: CAPE has a therapeutic potential as an anti-metastatic drug candidate for HNSCC therapy targeting the FAK/Paxillin axis.

Myeloid cell leukemia-1 expression in cancers of the oral cavity: a scoping review.

BACKGROUND: B cell lymphoma-2 (Bcl-2) family members play important roles in cell survival as well as cell death. The role of myeloid cell leukemia-1 (Mcl-1), an important member of the Bcl-2 family, is well established in hematopoietic malignancies. However, the association between Mcl-1 and oral cavity, cancers is not clearly defined. METHODS: A scoping review was conducted until June 30, 2021, using four major databases, PubMed, Scopus, Web of Science, and Embase. Medical subject headings keywords for Mcl-1, along with its other identifiers, and head and neck cancers (only oral cavity tumors) were used to evaluate the expression, function, molecular association, and therapeutic approach of Mcl-1 in oral cavity cancers and precancers. FINDINGS: Mcl-1 expression was associated with the progression of oral cavity cancers. The molecular mechanism and pathways of Mcl-1 in oral cavity cancers established via experimental results have been highlighted in this review. Moreover, the various synthetic and naturally derived therapeutic agents targeting Mcl-1 have been documented. NOVELTY/IMPROVEMENT: Based on our present review, Mcl-1 appears to be an effective anticancer target that can be used in the therapeutic management of oral cancers.

Metabolite profiling of a Sargassum micracanthum methanol extract with in vitro efficacy against human head and neck squamous cell carcinoma aggressiveness.

OBJECTIVE: Extracts from the brown algae Sargassum micracanthum have documented anti-viral, anti-oxidant, and anti-inflammatory activities as well as potential anti-tumor efficacy against several cancer types. Here, we evaluated the inhibitory effect and molecular mechanisms of methanol extract of S. micracanthum (MESM) on the aggressiveness of human head and neck squamous cell carcinoma (HNSCC) using in vitro cell culture-based models. DESIGN: To test the potential efficacy of MESM on the migratory and invasive properties of HNSCC cells, we used wound healing, transwell cell migration and invasion assays. Proteome profiling and functional in silico analysis were applied to investigate the possible modes of action by MESM. We also examined the metabolite profiling of MESM using gas chromatography/mass spectrometry. RESULTS: MESM inhibited the motility of human HNSCC cell lines as well as invasiveness without influencing cell survival. Proteome profiling identified 19 oncogenic proteins significantly downregulated by MESM treatment. Protein-protein interaction network and gene ontology analyses revealed that Tie2 and associated angiogenic signaling pathway components were significantly enriched among these downregulated oncogenic proteins, which was confirmed by validating the reduced Tie2 expression in MESM treatment groups. Metabolite profiling of MESM identified six-carbon sugar alcohols such as D-sorbitol and/or D-mannitol as the main bioactive compounds. D-sorbitol and D-mannitol effectively reduced Tie2 expression and the aggressiveness of human HNSCC cell lines. CONCLUSIONS: These findings suggest that six-carbon sugar alcohols in MESM have promising anti-cancer efficacy for the treatment of human HNSCC and further identify Tie2 signaling components as potential treatment targets.

Effect of PAIP1 on the metastatic potential and prognostic significance in oral squamous cell carcinoma.

Poly Adenylate Binding Protein Interacting protein 1 (PAIP1) plays a critical role in translation initiation and is associated with the several cancer types. However, its function and clinical significance have not yet been described in oral squamous cell carcinoma (OSCC) and its associated features like lymph node metastasis (LNM). Here, we used the data available from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) to analyze PAIP1 expression in oral cancer. The publicly available data suggests that PAIP1 mRNA and protein levels were increased in OSCC. The high PAIP1 expression was more evident in samples with advanced stage, LNM, and worse pattern of invasion. Moreover, the in vitro experiments revealed that PAIP1 knockdown attenuated colony forming, the aggressiveness of OSCC cell lines, decreasing MMP9 activity and SRC phosphorylation. Importantly, we found a correlation between PAIP1 and pSRC through the analysis of the IHC scores and CPTAC data in patient samples. Our findings suggest that PAIP1 could be an independent prognostic factor in OSCC with LNM and a suitable therapeutic target to improve OSCC patient outcomes.

Withaferin A mitigates metastatic traits in human oral squamous cell carcinoma caused by aberrant claudin-1 expression.

Abnormal expression of claudin-1 (CLDN1) has important roles in carcinogenesis and metastasis in various cancers. The role of CLDN1 in human oral squamous cell carcinoma (OSCC) remains unknown. Here, we report the functional role of CLDN1 in metastasis of human OSCC, as a potential target regulated by withaferin A. From gene expression profiling with microarray technology, we found that the majority of notable differentially expressed genes were classified into migration/invasion category. Withaferin A impaired the motility of human OSCC cells in vitro and suppressed metastatic nodule formation in an in vivo metastasis model, both associated with reduced CLDN1. CLDN1 overexpression enhanced metastatic nodule formation in vivo, resulting in severe metastatic lesions in lung tissue. Moreover, CLDN1 expression was positively correlated to lymphatic metastasis in OSCC patients. The impaired motility of human OSCC cells upon withaferin A treatment was restored by CLDN1 overexpression. Furthermore, upregulation of let-7a induced by withaferin A was inversely correlated to CLDN1 expression. Overall, these give us an insight into the function of CLDN1 for prognosis and treatment of human OSCC, substantiating further investigation into the use of withaferin A as good anti-metastatic drug candidate.

Methanol extract of Sedum oryzifolium and its constituent, trehalose, impede the invasiveness of oral squamous cell carcinoma cell lines via downregulation of Slug.

BACKGROUND: Sedum species are reported to possess diverse pharmacological activities in various solid tumors. However, the anticancer functions of Sedum orizyfolium and its constituents have never been determined in human cancers. PURPOSE: The present study focused on addressing the inhibition efficacy of the methanol extract of S. orizyfolium (MESO) and its constituents and the molecular mechanism underlying invasion and epithelial-to-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC) cell lines. STUDY DESIGN/METHODS: After MESO treatment, a wound-healing assay, an invasion assay, and immunocytochemistry were performed in OSCC cell lines, coupled with in silico analysis and immunohistochemistry in OSCC patient samples, to investigate the role of the EMT transcription factor Slug. Trehalose, an active component of MESO, was identified through gas chromatography-mass spectrometry. RESULTS: Among the methanol extracts of 18 various wild plants from South Korea, MESO exhibited the highest anticancer functionality in OSCC cells by downregulating Slug expression. In silico analysis and immunohistochemistry indicated that elevated Slug levels are remarkably associated with tumor progression and invasion in patients with OSCC, suggesting that changes in Slug expression alter EMT progression and invasion in OSCC. Notably, treatment with trehalose, a sugar component of MESO, inhibited invasiveness and Slug expression in OSCC cells. CONCLUSION: Cumulatively, this study highlighted the beneficial role of MESO and trehalose in the inhibition of invasiveness of OSCC cells via suppression of Slug expression and suggested a new design for potential chemotherapeutic drugs against OSCC.

Molecular mechanism underlying the apoptotic modulation by ethanol extract of Pseudolarix kaempferi in mucoepidermoid carcinoma of the salivary glands.

BACKGROUND: Pseudolarix kaempferi is a traditional Chinese natural product that possesses the potential cytotoxic effects against cancer. However, the precise molecular mechanism underlying its cytotoxic effects has not yet been completely elucidated. Here, we clarify the mechanism via which the ethanol extract of P. kaempferi (EEPK) leads to cytotoxicity mediated by apoptosis in mucoepidermoid carcinoma (MEC) originating from the salivary glands. METHODS: We investigated the mechanism underlying the anticancer efficacy of EEPK in human MEC in vitro by assessing mitochondrial dysfunction, mRNA levels, and morphological changes in apoptotic cell nuclei as well as by using a cytotoxicity assay, flow cytometric analysis, and western blotting. RESULTS: EEPK inhibited the growth of two human MEC cells and stimulated the induction of caspase-mediated apoptosis that was accompanied by mitochondrial membrane depolarization. Compared with the vehicle control groups, EEPK decreased myeloid cell leukemia-1 (Mcl-1) expression in both cells whereas it significantly decreased B cell lymphoma-2 (Bcl-2) expression in MC3 cells only. The EEPK-induced altered Mcl-1 expression was caused by translational inhibition and proteasomal degradation. Additionally, EEPK significantly increased p-Bcl-2 (Ser70) expression regardless of its total forms by facilitating the activation of the c-Jun N-terminal kinase (JNK) signaling pathway, which exhibited cell context dependency. Nevertheless, JNK activation following EEPK treatment was, at least in part, required for the proapoptotic efficacy of EEPK in both cells. CONCLUSIONS: This study revealed that EEPK-induced alterations of Mcl-1 inhibition and JNK/Bcl-2 phosphorylation cause apoptosis and provided basic preclinical data for future clinical trials regarding therapy for patients with MEC.

In vitro induction of mitotic catastrophe as a therapeutic approach for oral cancer using the ethanolic extract of Juniperus squamata.

Mitotic catastrophe, a cell death mechanism characterized by abnormal mitosis, has been regarded as a therapeutic approach for the development of anti­cancer drug candidates. The aim of the present study was to investigate the potential effect of the ethanolic extract of Juniperus squamata (EEJS) on the occurrence of mitotic catastrophe in human oral cancer cell lines. The effect of EEJS on the occurrence of mitotic catastrophe was evaluated by measuring cytotoxicity, observing phase­contrast or transmission electron microscope findings, evaluating the appearance of microtubule or chromosome abnormalities, and detecting the phosphorylation of histone H3 (Ser10). The apoptotic effect of EEJS was assessed by detecting cleaved PARP, analyzing the sub­G1 population, Annexin V­FITC/PI double staining, western blot analysis, and the transient transfection of myeloid cell leukemia­1 (Mcl­1) overexpression vectors. EEJS treatment was effective in inhibiting cell proliferation in human oral cancer cell lines. EEJS resulted in the enrichment of enlarged multinucleated cells, the disturbance of microtubule formation, and increased phosphorylation of histone H3 (Ser10), which demonstrates the occurrence of mitotic catastrophe. Additionally, the multinucleated cells underwent apoptotic cell death in a cell context­dependent manner, which was associated with the reduction of Mcl­1 protein levels. Findings of the present study indicate that EEJS could be effective for treating human oral cancer by promoting mitotic catastrophe linked to apoptotic cell death.

SOX7 blocks vasculogenic mimicry in oral squamous cell carcinoma.

BACKGROUND: Vasculogenic mimicry (VM) is the formation of an alternative circulatory system by aggressive tumor cells. The characteristics of VM and its underlying mechanism in oral squamous cell carcinoma (OSCC) remain unclear. This study aims to determine the relationship between VM in OSCC tissues and clinical outcomes and to investigate the biological role of SOX7 in VM in OSCC cells. METHODS: CD31/PAS staining was performed to evaluate VM in OSCC tissue. The relationships between VM and clinicopathological variables, and VM and SOX7 levels were analyzed. The correlation between SOX7 levels and cancer cohorts was investigated using in silico analysis. VM formation assay was performed to observe VM in vitro. To investigate the role of SOX7 in VM formation, SOX7 was transiently over-expressed in SCC-9 cells. VM-modulating genes were identified by Western blotting. RESULTS: We found a positive correlation between VM and lymph node metastasis and patient survival in OSCC (p = 0.003). In silico analysis from The Cancer Genome Atlas and Gene Expression Omnibus database showed that down-regulation of SOX7 expression was significantly correlated with OSCC patients (p = 0.0187) and lymph node metastasis (p = 0.0017). We also found that the presence of VM in OSCC tissue was inversely associated with SOX7 expression (p = 0.020). We observed that overexpression of SOX7 impaired VM formation by reducing the expression of VE-cadherin, thereby inhibiting cell migration and invasion. CONCLUSION: These results suggest that SOX7 plays an important role in the regulation of VM formation and may inhibit OSCC metastasis.

Cryptotanshinone chemosensitivity potentiation by TW-37 in human oral cancer cell lines by targeting STAT3-Mcl-1 signaling.

BACKGROUND: Despite being one of the leading cancer types in the world, the diagnosis of oral cancer and its suitable therapeutic options remain limited. This study aims to investigate the single and chemosensitizing effects of TW-37, a BH3 mimetic in oral cancer, on human oral cancer cell lines. METHODS: We assessed the single and chemosensitizing effects of TW-37 in vitro using trypan blue exclusion assay, Western blotting, DAPI staining, Annexin V-FITC/PI double staining, and quantitative real-time PCR. Mcl-1 overexpression models were established by transforming vector and transient transfection was performed to test for apoptosis. RESULTS: TW-37 enhanced the cytotoxicity of human oral cancer cell lines by inducing caspase-dependent apoptosis, which correlates with the reduction of the myeloid cell leukemia-1 (Mcl-1) expression via transcriptional and post-translational regulation. The ectopic expression of Mcl-1 partially attenuated the apoptosis-inducing capacity of TW-37 in human oral cancer cell lines. Besides, TW-37 decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and nuclear translocation in human oral cancer cell lines at the early time points. Furthermore, TW-37 potentiated chemosusceptibility of cryptotanshinone in human oral cancer cell lines by suppressing STAT3-Mcl-1 signaling compared with either TW-37 or cryptotanshinone alone, resulting in potent apoptosis. CONCLUSIONS: This study not only unravels the single and chemosensitizing effects of TW-37 for treatment of human oral cancer but also highlights the likelihood of TW-37 as a good therapeutic strategy to enhance the prognosis of patients with oral cancer in the future.

Targeting X chromosome-linked inhibitor of apoptosis protein in mucoepidermoid carcinoma of the head and neck: A novel therapeutic strategy using nitidine chloride.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Oridonin induces the apoptosis of mucoepidermoid carcinoma cell lines in a myeloid cell leukemia­1­dependent manner.

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported to exhibit anticancer activities in several tumors. The aim of the present study was to investigate the anticancer effects and molecular mechanisms of oridonin in mucoepidermoid carcinoma (MEC). Treatment with oridonin induced the apoptosis of MC­3 and YD­15 cell and inhibited the expression of myeloid cell leukemia­1 (MCL­1) through the regulation of the protein level through post­translational regulation in these cell lines. Oridonin significantly increased the expression level of truncated Bid (t­Bid) as a downstream target of MCL­1 and subsequently decreased the mitochondrial membrane potential. The ectopic expression of MCL­1 protein was sufficient to reverse the induction of apoptosis and the increased t­Bid expression induced by oridonin in both cell lines. Taken together, these results suggest that oridonin exerts an apoptotic effect through the modulation of MCL­1 and t­Bid in human MEC cell lines and may thus be a potential anticancer drug candidate for the treatment of human MEC.

A new insight into the apoptotic effect of nitidine chloride targeting Checkpoint kinase 2 in human cervical cancer in vitro.

Nitidine chloride (NC), a natural, bioactive, phytochemical alkaloid derived from the roots of Zanthoxylum nitidum, has been reported to exhibit anti-tumor activity against various types of cancer. However, the potential therapeutic role of NC in human cervical cancer has not yet been studied. We are the first to report that NC acts as a potential apoptosis-inducing agent for human cervical cancer in vitro. NC treatment of human cervical cancer cell lines induced caspase-mediated apoptosis, thereby reducing cell viability. Phospho-kinase proteome profiling using a human phospho-kinase array revealed that NC treatment phosphorylated Checkpoint kinase 2 (Chk2) at Thr68, which activates Chk2 in both cell lines. We also found that NC significantly affected the p53/Bim signaling axis, which was accompanied by mitochondrial membrane depolarization and cytochrome c release from the mitochondria into the cytosol. In addition, NC profoundly increased phosphorylation of the histone variant H2AX at Ser139, a typical marker of DNA damage. Taken together, these results provide in vitro evidence that NC can increase Chk2 activation, thereby acting as an attractive cell death inducer for treatment of human cervical cancer.