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.

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.

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.

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.

Pseudolaric Acid B Induces Growth Inhibition and Caspase-Dependent Apoptosis on Head and Neck Cancer Cell lines through Death Receptor 5.

Pseudolaric Acid B (PAB), diterpenoid isolated from the root bark of Pseudolarix kaempferi Gordon tree (Pinaceae), exhibits an anti-proliferative and apoptotic activity in various cancer cell lines but to date, the effects of PAB on head and neck cancer (HNC) cell lines remain to be elucidated. In this study, we showed that PAB significantly inhibited the viability and caspase-dependent apoptosis in HN22 cell line. PAB-induced apoptosis is through inducing death receptor 5 (DR5) together with the increase in the expression of cleaved caspase-8. It also inhibited the proliferations and induced apoptosis through DR5 in other three HNC cell lines (HSC3, Ca9.22, and HSC4). Extending our in vitro findings, we found that ethanol extract of Pseudolarix kaempferi (2.5 mg/kg/day) reduced tumor growth in a xenograft model bearing HN22 cell line without any change in body weight. DR5 were also found to be increased in tumors tissue of PAB-treated mice without any apparent histopathological changes in liver or kidney tissues. Taken together, PAB may be a potential lead compound for chemotherapeutic agents against head and neck cancer.

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

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

Heme Oxygenase-1 is a Key Molecule Underlying Differential Response of TW-37-Induced Apoptosis in Human Mucoepidermoid Carcinoma Cells.

TW-37 is a small-molecule inhibitor of Bcl-2 family proteins, which can induce anti-cancer activities in various types of cancer. In the current study, we investigated the potential molecular mechanism underlying the differential response to TW-37-induced apoptosis in two human mucoepidermoid carcinoma (MEC) cell lines. The differential response and underlying molecular mechanism of human MEC cells to TW-37 was evaluated by trypan blue exclusion assay, western blotting, 4', 6-diamidino-2-phenylindole staining, annexin V/propidium iodide double staining, analysis of the sub-G1 population, human apoptosis array, and measurements of intracellular reactive oxygen species (ROS). TW-37 decreased cell viability and induced apoptosis in YD-15 cells, but not in MC3 cells. Proteome profiling using a human apoptosis array revealed four candidate proteins and of these, heme oxygenase-1 (HO-1) was mainly related to the differential response to TW-37 of YD-15 and MC3 cells. TW-37 also led to a significant increase in intracellular levels of ROS in YD-15 cells, which is associated with apoptosis induction. The ectopic expression of HO-1 recovered YD-15 cells from TW-37-induced apoptosis by reducing intracellular levels of ROS. The expression of HO-1 was reduced through both transcriptional and post-translational modification during TW-37-mediated apoptosis. We conclude that HO-1 is a potential indicator to estimate response to TW37-induced apoptosis in human MEC.

Norcantharidin Suppresses YD-15 Cell Invasion Through Inhibition of FAK/Paxillin and F-Actin Reorganization.

Norcantharidin (NCTD), a demethylated derivative of cantharidin, has been reported to exhibit activity against various types of cancers. However, the anti-invasive effects of NCTD and its molecular mechanism in human mucoepidermoid carcinoma (MEC) remain incompletely elucidated. Clonogenic, wound healing, invasion, zymography, western blotting and immunocytochemistry assays were performed in YD-15 cells to investigate the anti-invasive effect of NCTD and its molecular mechanism of action. The inhibitory effects of NCTD on invasiveness were compared with those of a novel focal adhesion kinase (FAK) kinase inhibitor, PF-562271. NCTD markedly suppressed the colony formation, migration, and invasion of YD-15 cells as well as the activities of MMP-2 and MMP-9. It disrupted F-actin reorganization through suppressing the FAK/Paxillin axis. Moreover, NCTD exhibited a powerful anti-invasive effect compared with that of PF-562271 in YD-15 cells. Collectively, these results suggest that NCTD has a potential anti-invasive activity against YD-15 cells. This study may clarify the impact of NCTD on migration and invasion of human MEC cells.

Antitumor effect of TW-37, a BH3 mimetic in human oral cancer.

TW-37 is a small molecule B cell lymphoma-2 (Bcl-2) homology 3 mimetic with potential anticancer activities. However, the in vivo anti-cancer effect of TW-37 in human oral cancer has not been properly studied yet. Here, we attempted to confirm antitumor activity of TW37 in human oral cancer. TW-37 significantly inhibited cell proliferation and increased the number of dead cells in MC-3 and HSC-3 human oral cancer cell lines. TW-37 enhanced apoptosis of both cell lines evidenced by annexin V/propidium iodide double staining, sub-G1 population analysis and the detection of cleaved poly (ADP-ribose) polymerase and caspase-3. In addition, TW-37 markedly downregulated the expression of Bcl-2 protein, while not affecting Bcl-xL or myeloid cell leukemia-1. In vivo, TW-37 inhibited tumor growth in a nude mice xenograft model without any significant liver and kidney toxicities. Collectively, these data reveal that TW-37 may be a promising small molecule to inhibit human oral cancer.

Apoptosis induced by methanol extract of Potentilla discolor in human mucoepidermoid carcinoma cells through STAT3/PUMA signaling axis.

Potentilla discolor has been used in traditional Chinese medicine for the treatment of hyperglycemia. However, the potential role of Potentilla discolor against cancer and its mode of action remain to be fully elucidated. The present study explored the apoptotic effect of methanol extract of Potentilla discolor (MEPD) in human mucoepidermoid carcinoma (MEC) cell lines of salivary glands. MEPD markedly suppressed the growth and induced apoptotic cell death in MC3 and YD15 cells. MEPD treatment significantly upregulated the expression of PUMA and reduced STAT3 phosphorylation. Overexpression of STAT3 partially recovered the growth of MEC cells inhibited by MEPD. In addition, dephosphorylation of STAT3 by cryptotanshinone (a potent STAT3 inhibitor) was sufficient to inhibit the growth of MEC cells and induce apoptosis via affecting PUMA protein. These results suggest that MEPD has a potential anticancer property via the STAT3/PUMA signaling axis in human MEC cells of salivary gland.

Apoptosis induced by caffeic acid phenethyl ester in human oral cancer cell lines: Involvement of Puma and Bax activation.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE), a natural honeybee product exhibits a spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant and antitumor actions. The purpose of this research was to investigate the anticancer potential of CAPE and its molecular mechanism in human oral cancer cell lines (YD15, HSC-4 and HN22 cells). DESIGN: To determine the apoptotic activity of CAPE and identify its molecular targets, trypan blue exclusion assay, soft agar assay, Western blot analysis, DAPI staining, and live/dead assay were performed. RESULTS: CAPE significantly suppressed transformation of neoplastic cells induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) without inhibiting growth. CAPE treatment inhibited cell growth, increased the cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and augmented the number of fragmented nuclei in human oral cancer cell lines. CAPE activated Bax protein causing it to undergo a conformational change, translocate to the mitochondrial outer membrane, and oligomere. CAPE also significantly increased Puma expression and interestingly Puma and Bax were co-localized. CONCLUSION: Overall, these results suggest that CAPE is a potent apoptosis-inducing agent in human oral cancer cell lines. Its action is accompanied by up-regulation of Bax and Puma proteins.

Gene expression profile in the anterior regeneration of the earthworm using expressed sequence tags.

In order to gain insight into the gene expression profiles associated with anterior regeneration of the earthworm, Perionyx excavatus, we analyzed 1,159 expressed sequence tags (ESTs) derived from cDNA library early anterior regenerated tissue. Among the 1,159 ESTs analyzed, 622 (53.7%) ESTs showed significant similarity to known genes and represented 338 genes, of which 233 ESTs were singletons and 105 ESTs manifested as two or more ESTs. While 663 ESTs (57.2%) were sequenced only once, 308 ESTs (26.6%) appeared 2 to 5 times, and 188 ESTs (16.2%) were sequenced more than 5 times. A total of 803 genes were categorized into 15 groups according to their biological functions. Among 1,159 ESTs sequenced, we found several gene encoding signaling molecules, such as Notch and Distal-less. The ESTs used in this study should provide a resource for future research in earthworm regeneration.

Hox genes in the echiuroid Urechis unicinctus.

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.