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.

Trichostatin A induces apoptosis in oral squamous cell carcinoma cell lines independent of hyperacetylation of histones.

AIM OF STUDY: To investigate the apoptotic event of trichostatin A (TSA) and its associated mechanism in oral squamous cell carcinoma (OSCC) lines. MATERIALS AND METHODS: HSC-3 and Ca9.22 cell lines were evaluated using a trypan blue exclusion assay, histone isolation, soft agar assay, live/dead assay, 4%,6-diamidino-2-phenylindole staining, JC-1 mitochondrial membrane potential (MMP) assay, and Western blot analysis to demonstrate the anticancer activity of TSA. RESULTS: TSA decreased OSCC cell viability and proliferation without affecting the histone acetylation. TSA-induced caspase-dependent or -independent apoptosis according to cell types, TSA enhanced the expression levels of Bim protein by dephosphorylating ERK1/2 pathway in HSC-3 cells. TSA also damaged MMP and increased cytosolic apoptosis-inducing factor (AIF) in Ca9.22 cells. CONCLUSION: The present study suggests that TSA may be a potential anticancer drug candidate for the treatment of OSCC through the induction of apoptosis.

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.

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.

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.

Sorafenib potentiates ABT-737-induced apoptosis in human oral cancer cells.

OBJECTIVE: The mimetic BH3 ABT-737, a potent inhibitor of anti-apoptotic Bcl-2 family proteins, has potential as anti-cancer drug in many cancers. Recently, patients treated with ABT-737 have developed drug tolerance during cancer therapy. Therefore, we examined whether ABT-737 is effective in killing MC-3 and HSC-3 human oral cancer cells either alone or in combination with the oncogenic kinase inhibitor, sorafenib. DESIGN: The potentiating activities of sorafenib in ABT-737-induced apoptosis were determined using trypan blue exclusion assay, DAPI staining, cell viability assay and Western blot analysis. RESULTS: Combined use of ABT-737 and sorafenib synergistically suppressed cell viability and induced apoptosis compared with either compound individually. The combination of ABT-737 and sorafenib altered only Bax and Bak proteins and their activations, resulting in mitochondrial translocation of Bax from the cytosol. Additionally, combination treatment-mediated apoptosis may be correlated with ERK and STAT3 pathways. CONCLUSIONS: These results suggest that sorafenib may effectively overcome ABT-737 resistance to apoptotic cell death, which can be a new potential chemotherapeutic strategy against human oral cancer.

Growth-suppressive effect of suberoylanilide hydroxamic acid (SAHA) on human oral cancer cells.

PURPOSE: The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) has been reported to exhibit anticancer activities in various cancer cell types, but as yet there are few reports on the anticancer effects of SAHA in oral squamous cell carcinoma (OSCC)-derived cells and xenograft models. METHODS: The anti-proliferative and apoptotic activities of SAHA were assessed in human HSC-3 and HSC-4 (OSCC)-derived cell lines and JB6 normal mouse skin-derived epidermal cells using histone acetylation, soft agar colony formation, trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead viability/cytotoxicity and Western blot analyses. RESULTS: We found that SAHA treatment resulted in hyperacetylation of histones H2A and H3 and a concomitant decrease in the viability of HSC-3 and HSC-4 cells. SAHA also significantly inhibited the neoplastic transformation of JB6 cells treated with TPA, whereas the viability of these cells was not affected by this treatment. Additionally, we found that SAHA suppressed the anchorage-independent growth (colony forming capacity in soft agar) of HSC-3 and HSC-4 cells. DAPI staining, Live/Dead and Western blot analyses revealed that SAHA can induce caspase-dependent apoptosis in HSC-3 and HSC-4 cells. We also found that SAHA treatment led to inhibition of ERK phosphorylation, and that two MEK inhibitors potentiated SAHA-mediated apoptosis. Okadaic acid treatment inhibited SAHA-mediated apoptosis in both the HSC-3 and HSC-4 cell lines, wheras SAHA induced a profound in vivo inhibition of tumor growth in HSC-3 xenografts. CONCLUSIONS: Our results indicate that the ERK signaling pathway may constitute a critical denominator of SAHA-induced apoptosis in OSCC-derived cells and that SAHA may have therapeutic potential for OSCC.