DNA-PKcs as an upstream mediator of OCT4-induced MYC activation in small cell lung cancer.

Small cell lung cancer (SCLC) is a neuroendocrine tumor noted for the rapid development of both metastases and resistance to chemotherapy. High mutation burden, ubiquitous loss of TP53 and RB1, and a mutually exclusive amplification of MYC gene family members contribute to genomic instability and make the development of new targeted agents a challenge. Previously, we reported a novel OCT4-induced MYC transcriptional activation pathway involving c-MYC, pOCT4S111, and MAPKAPK2 in progressive neuroblastoma, also a neuroendocrine tumor. Using tumor microarray analysis of clinical samples and preclinical models, we now report a correlation in expression between these proteins in SCLC. In correlating c-MYC protein expression with genomic amplification, we determined that some SCLC cell lines exhibited high c-MYC without genomic amplification, implying amplification-independent MYC activation. We then confirmed direct interaction between OCT4 and DNA-PKcs and identified specific OCT4 and DNA-PKcs binding sites. Knock-down of both POU5F1 (encoding OCT4) and PRKDC (encoding DNA-PKcs) resulted in decreased c-MYC expression. Further, we confirmed binding of OCT4 to the promoter/enhancer region of MYC. Together, these data establish the presence of a DNA-PKcs/OCT4/c-MYC pathway in SCLCs. We then disruptively targeted this pathway and demonstrated anticancer activity in SCLC cell lines and xenografts using both DNA-PKcs inhibitors and a protein-protein interaction inhibitor of DNA-PKcs and OCT4. In conclusion, we demonstrate here that DNA-PKcs can mediate high c-MYC expression in SCLCs, and that this pathway may represent a new therapeutic target for SCLCs with high c-MYC expression.

Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

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.

MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma.

Despite the improvement in clinical outcome with 13-cis-retinoic acid (13-cisRA) + anti-GD2 antibody + cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients die of recurrent disease. MYCN genomic amplification is a biomarker of aggressive tumors in the childhood cancer neuroblastoma. MYCN expression is downregulated by 13-cisRA, a differentiating agent that is a component of neuroblastoma therapy. Although MYC amplification is rare in neuroblastoma at diagnosis, we report transcriptional activation of MYC medicated by the transcription factor OCT4, functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma in large panels of patient-derived cell lines and xenograft models. We identified novel OCT4-binding sites in the MYC promoter/enhancer region that regulated MYC expression via phosphorylation by MAPKAPK2 (MK2). OCT4 phosphorylation at the S111 residue by MK2 was upstream of MYC transcriptional activation. Expression of OCT4, MK2, and c-MYC was higher in progressive disease relative to pre-therapy neuroblastomas and was associated with inferior patient survival. OCT4 or MK2 knockdown decreased c-MYC expression and restored the sensitivity to 13-cisRA. In conclusion, we demonstrated that high c-MYC expression independent of genomic amplification is associated with disease progression in neuroblastoma. MK2-mediated OCT4 transcriptional activation is a novel mechanism for activating the MYC oncogene in progressive disease neuroblastoma that provides a therapeutic target.

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.

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.

Induction of sestrin 2 is associated with fisetin-mediated apoptosis in human head and neck cancer cell lines.

Fisetin was reported to have an anti-proliferative and apoptotic activity as a novel anti-cancer agent in various cancer cell lines. However, the possible molecular targets for the anti-cancer effect of fisetin in human head and neck cancer (HNCC) have not yet been clarified. In this study, the influence of fisetin on the growth and apoptosis of HNCCs were examined. In HSC3 cells, fisetin treatment reduced the viability and induced apoptosis. Through the results from the screening of the expression profile of apoptosis-related genes, sestrin 2 (SESN2) was functionally involved in fisetin-mediated apoptosis showing the knockdown of SESN2 by siRNA clearly restored fisetin-induced apoptosis. In addition, fisetin reduced the protein expression levels of phospho-mTOR (p-mTOR) and Mcl-1, which are the downstream molecules of SESN2. It also induced PARP cleavage by inducing an increase in the expression levels of SESN2 together with reducing mTOR and Mcl-1 proteins in other three HNCCs (MC3, Ca9.22, and HN22). Taken together, our findings suggest that the anti-cancer effect of fisetin on HNCCs is associated with SESN2/mTOR/Mcl-1 signaling axis.

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

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

Down-regulation and nuclear localization of survivin by sodium butyrate induces caspase-dependent apoptosis in human oral mucoepidermoid carcinoma.

OBJECTIVE: Sodium butyrate (NaBu) is a histone deacetylase inhibitor that possesses an apoptotic ability. However, the molecular mechanism by which NaBu induces apoptosis in human oral mucoepidermoid carcinoma (MEC), a type of salivary gland tumor, remains unclear. MATERIALS AND METHODS: The anticancer effects of NaBu and its related molecular mechanisms were determined by trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, live/dead assay, human apoptosis array, RT-PCR, western blotting, immunocytochemistry, preparation of nuclear fractions, and nude mice tumor xenograft. RESULTS: In this study, we found that NaBu inhibited growth and induced apoptosis in the human oral MEC cell lines MC3 and YD15 with acetylation of histone proteins H2A and H3. NaBu apparently down-regulated survivin protein, as evidenced by the results of the human apoptosis antibody array, and modulated it at the post-translational process. Interestingly, NaBu caused nuclear translocation of survivin protein in both cell lines. NaBu also resulted in decreased expression levels of Bcl-xL mRNA and protein, leading to induction of caspase-dependent apoptosis in human oral MEC cell lines. In addition, NaBu administration inhibited tumor growth in vivo at a dosage of 500 mg/kg/day, but it did not cause any hepatic or renal toxicity. CONCLUSION: This study provides new insights into the molecular mechanism of apoptotic actions by NaBu in human oral MEC and the basis of its clinical application for the treatment of human oral MEC.

Nitidine chloride represses Mcl-1 protein via lysosomal degradation in oral squamous cell carcinoma.

BACKGROUND: We have shown previously that nitidine chloride (NC) induces apoptosis via inhibition of signal transducer and activator of transcription 3 (STAT3). However, its downstream molecules are not fully understood yet. Here, we report that NC as STAT3 inhibitor downregulates myeloid cell leukemia-1 (Mcl-1) protein in HSC-3 and HSC-4 human oral squamous cell carcinoma (OSCC) cells and a nude mouse tumor xenograft model. METHODS: This study investigated the effects of NC on Mcl-1 expression in HSC-3 and HSC-4 cells using Western blotting, RT-PCR, and dual-luciferase assay. Immunohistochemistry was employed to evaluate Mcl-1 expression levels in mouse tumor tissues. Construction of Mcl-1 overexpression vector and transient transfection was done to test the apoptosis of HSC-3 cells. RESULTS: Nitidine chloride did not affect either mRNA level or promoter activity of Mcl-1, and the decrease in Mcl-1 protein by NC was caused by lysosome-dependent degradation, but not proteasome-dependent degradation. The overexpression of Mcl-1 protein in OSCC cell lines was sufficient to block the induction of apoptosis. In addition, NC strongly reduced the expression level of Mcl-1 protein compared with other STAT3 inhibitors such as cryptotanshione and S3I-201 in OSCCs. CONCLUSIONS: Our findings suggest that NC triggers apoptosis via lysosome-dependent Mcl-1 protein degradation and could be chosen as a promising chemotherapeutic candidate against human OSCCs.

In vitro and in vivo anti-cancer activity of silymarin on oral cancer.

Silymarin, a standardized extract from milk thistle fruits has been found to exhibit anti-cancer effects against various cancers. Here, we explored the anti-cancer activity of silymarin and its molecular target in human oral cancer in vitro and in vivo. Silymarin dose-dependently inhibited the proliferation of HSC-4 oral cancer cells and promoted caspase-dependent apoptosis. A human apoptosis protein array kit showed that death receptor 5 may be involved in silymarin-induced apoptosis, which was also shown through western blotting, immunocytochemistry, and reverse transcription-polymerase chain reaction. Silymarin increased cleaved caspase-8 and truncated Bid, leading to accumulation of cytochrome c. In addition, silymarin activated death receptor 5/caspase-8 to induce apoptotic cell death in two other oral cancer cell lines (YD15 and Ca9.22). Silymarin also suppressed tumor growth and volume without any hepatic or renal toxicity in vivo. Taken together, these results provide in vitro and in vivo evidence supporting the anti-cancer effect of silymarin and death receptor 5, and caspase-8 may be essential players in silymarin-mediated apoptosis in 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.

Nitidine chloride acts as an apoptosis inducer in human oral cancer cells and a nude mouse xenograft model via inhibition of STAT3.

Nitidine chloride (NC) is a natural alkaloid compound derived from the plant Zanthoxylum nitidum and is known for its therapeutic anticancer potential. In this study, we investigated the effects of NC on growth and signaling pathways in human oral cancer cell lines and a tumor xenograft model. The apoptotic effects and related molecular targets of NC on human oral cancer were investigated using trypan blue exclusion assay, DAPI staining, Live/Dead assay, Western blotting, Immunohistochemistry/Immunofluorescence and a nude mouse tumor xenograft. NC decreased cell viability in both HSC3 and HSC4 cell lines; further analysis demonstrated that cell viability was reduced via apoptosis. STAT3 was hyper-phosphorylated in human oral squamous cell carcinoma (OSCC) compared with normal oral mucosa (NOM) and dephosphorylation of STAT3 by the potent STAT3 inhibitor, cryptotanshinone or NC decreased cell viability and induced apoptosis. NC also suppressed cell viability and induced apoptosis accompanied by dephosphorylating STAT3 in four other oral cancer cell lines. In a tumor xenograft model bearing HSC3 cell tumors, NC suppressed tumor growth and induced apoptosis by regulating STAT3 signaling without liver or kidney toxicity. Our findings suggest that NC is a promising chemotherapeutic candidate against human oral cancer.

Oridonin induces apoptosis in human oral cancer cells via phosphorylation of histone H2AX.

Oridonin, a natural diterpenoid purified from Rabdosia rubescens, has displayed beneficial biological activities, including anti-proliferation and anti-angiogenesis effects, in various types of cancers. However, the anti-cancer potential of oridonin and its mechanism in oral cancer have never previously been studied. In this study, we assessed the role of oridonin as an inducer of apoptosis in HSC-3 and HSC-4 human oral cancer cells. Our results showed that oridonin reduces the viability of human oral cancer cells and significantly increases the expression of γH2AX, a well-known marker of DNA damage. 4',6-Diamidino-2-phenylindole (DAPI) staining and western blotting showed that oridonin causes nuclear condensation and fragmentation, and induces cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, oridonin-induced γH2AX accumulation was partially abrogated by Z-VAD, a pan-caspase inhibitor. Taken together, our results suggest that oridonin can effectively induce apoptosis by augmenting the expression of γH2AX in response to DNA damage and might be a promising anti-cancer drug candidate for the treatment of oral cancer.

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.

Silymarin and its active component silibinin act as novel therapeutic alternatives for salivary gland cancer by targeting the ERK1/2-Bim signaling cascade.

PURPOSE: Approximately 20% of all salivary gland cancer patients who are treated with current treatment modalities will ultimately develop metastases. Its most common form, mucoepidermoid carcinoma (MEC) is a highly aggressive tumor with an overall 5-year survival rate of ~30%. Until now, several chemotherapeutic drugs have been tested for the treatment of salivary gland tumors, but the results have been disappointing and the drugs often cause unwanted side effects. Therefore, several recent studies have focused on the potential of alternative and/or complementary therapeutic options, including the use of silymarin. METHODS: The effects of silymarin and its active component silibinin on salivary gland cancer-derived MC3 and HN22 cells and their underlying molecular mechanisms were examined using trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead, Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) measurement, quantitative RT-PCR, soft agar colony formation and Western blotting analyses. RESULTS: We found that silymarin and silibinin dramatically increased the expression of the pro-apoptotic protein Bim in a concentration- and time-dependent manner and, concomitantly, induced apoptosis in MC3 and HN22 cells. We also found that ERK1/2 signaling inhibition successfully sensitized these cells to the apoptotic effects of silymarin and silibinin, which indicates that the ERK1/2 signaling pathway may act as an upstream regulator that modulates the silymarin/silibinin-induced Bim signaling pathway. CONCLUSIONS: Taken together, we conclude that ERK1/2 signaling pathway inhibition by silymarin and silibinin increases the expression of the pro-apoptotic Bcl-2 family member Bim which, subsequently, induces mitochondria-mediated apoptosis in salivary gland cancer-derived cells.