MicroRNAs (miRNAs) are a group of small non-coding RNAs consisting of 18~24 nucleotides in length. Each miRNA is expected to bind a few hundreds of putative target mRNAs, thus inhibiting their translation into protein products mostly by degradation of targets. With its biogenesis extensively deciphered, miRNAs have been implicated in a variety of biological processes, including early development and cellular metabolism. In addition, dysregulation of miRNAs and subsequent alterations in the expression of its target molecules are thought to be linked to the pathophysiology of multiple human illnesses, including cancer. To establish the miRNA-target relationships important for developing a specific disease, it is critical to validate the putative targets of each miRNA suggested by computational methods in vivo. In this review, we will first discuss oncogenic and tumor-suppressive roles of miRNAs in human cancer and introduce computational methods to predict putative targets of miRNAs. Then, the value of Drosophila melanogaster as an alternative model system will be further discussed in studying human cancer and in validating the miRNA-target relationships in vivo. Finally, we will present a possibility of applying the mammals-to-Drosophila-to-mammals approach to study the roles of miRNAs and their targets in the pathophysiology of oral cancer, an intractable type of cancer with poor prognosis and survival rate.

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, was reported to various physiological and pharmacological effects including anti-cancer activity. Nevertheless, there is no report of the anticancer effect of lycorine in oral cancer cells. The effects of lycorine on cell proliferation and apoptosis were examined through trypan blue exclusion assay, 4’-6-diamidino-2-phenylindole (DAPI) stain, Live/Dead assay, Western blot analysis and RT-PCR. Lycorine suppressed cell viability and induced apoptosis in MC3 and HSC-3 cell lines. Lycorine decreased survivin protein but did not affect its mRNA. It regulated survivin through accelerating protein degradation in a time-dependent manner although neither proteasome nor lysosome was not associated with lycorine-mediated protein degradation. Collectively, our results suggest that lycorine may be a potential therapeutic anti-cancer drug candidate for the treatment of human oral cancer.

Tyrosol, a phenylethanoid and a derivative of phenethyl alcohol, possesses various biological properties, such as anti-oxidative and cardioprotective activity. Olive oil is the principal source of tyrosol in the human diet. However, so far the anti-cancer activity of tyrosol has not yet been well defined. This study therefore undertakes to examine the cytotoxic activity and the mechanism of cell death exhibited by tyrosol in KB human oral cancer cells. Treatment of KB cells with tyrosol induced the cell growth inhibition in a concentration- and a time-dependent manner. Furthermore, the treatment of tyrosol induced nuclear condensation and fragmentation of KB cells. Tyrosol also promoted proteolytic cleavage of procaspase-3, -7, -8 and –9, increasing the amounts of cleaved caspase-3, -7, -8 and –9. In addition, tyrosol increased the levels of cleaved PARP in KB cells. These results suggest that tyrosol induces the suppression of cell growth and cell apoptosis in KB human oral cancer cells, and is therefore a potential candidate for anti-cancer drug discovery.

β-carotene is present in carrots, pumpkins, and sweet potatoes. It suppresses many types of cancers by regulating cellular proliferation and apoptosis through a variety of mechanisms. However, the effects of β -carotene on oral cancer cells have not been clearly established. The main goal of this study was to investigate the effects of β-carotene on cell growth and apoptosis in oral cancer cells. Our results demonstrate that treatment with β-carotene induced inhibition of cell growth, and that the effect was dependent on β-carotene treatment time and concentration in KB cells. Furthermore, treatment with β-carotene induced nuclear condensation and fragmentation in KB cells. β-carotene promoted proteolytic cleavage of procaspase-3, -7, -8 and –9 with associated increases in the concentration of cleaved caspase-3, -7, -8 and –9. In addition, the level of cleaved PARP was increased by β-carotene treatment in KB cells. These results suggest that β-carotene can suppress cell growth and induce apoptosis in KB human oral cancer cells, and that it may have potential usefulness in anti-cancer drug discovery efforts.

Terfenadine (TFN) was a second generation histamine receptor antagonist. Although several studies have reported the regulatory effect of H1-histamine receptor antagonists in human cancer cell lines, its effect in oral cancer remains unclear. In this study, we focused on addressing the anti-cancer activity of TFN in human oral cancer cell lines. The anti-cancer activities of TFN were performed by tryphan blue exclusion assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, live/dead assay and Western blot analysis. TFN induced a significant reduction of the growth in three different human oral cancer cell lines (MC3, HSC4 and Ca9.22). TFN markedly induced apoptosis through DNA damage and increase in cytotoxicity. It also accumulated cleaved PARP and caspase 3. This process was due to cleavage of caspase 8 and Bid protein. The results from this study strongly demonstrated that the cleavages of caspase 8 and Bid are required for the apoptotic activity of TFN in human oral cancer cells. Taken together, these findings suggest TFN as a potent anticancer drug candidate for the treatment of oral cancer.

Neuromedin B (NMB) acts as a growth factor or a morphogen and plays a role in cancer progression. Indeed, the NMB receptor (NMB-R) is overexpressed in different types of tumors. In our current study, we investigated the involvement of NMB-R in the proliferation of oral cancer cells. Human oral squamous cell carcinoma (SCC) and human oral cancer cells, SCC-25 cells were found to be NMB-R-positive. The NMB-R antagonist PD168368 inhibited the proliferation of SCC-25 cells and reduced their colony formation capacity. We also found that PD168368 induced the cell cycle arrest and apoptosis of SCC-25 cells in a dose-/time-dependent manner. Overall, this antitumor activity of PD168368 in human oral cancer cells suggests that NMB-R is a potential target for the future prevention and treatment of human cancers.

Dibenzylideneacetone (DBA), an analogue of curcumin has been shown to have anti-cancer activity in a variety of tumor cell lines. However, the anti-cancer activity of DBA and its molecular mechanism in HN22 oral cancer cell line have not been fully explored. The effects of DBA on anti-proliferative and apoptotic activity were evaluated by the trypan blue exclusion assay, 4’-6-diamidino-2-phenylindole (DAPI) staining, Western blot analysis, and reverse transcriptase-polymerase chain Reaction (RT-PCR). Our data showed that the treatment of DBA to HN22 cells exerted anti-proliferative and apoptotic activities and the activity was accompanied by a decrease in Sp1 protein, Sp1 mRNA and its promoter activity. DBA also reduced the expression level of Sp1 protein and caused apoptotic cell death in HN22 cells simultaneouly. Phosphorylation of ERK and JNK were regulated by DBA whereas phosphorylation of p38 was not altered. Overall, our results suggest that the regulation of Sp1 activities and ERK/JNK are involved in DBA-induced apoptosis and DBA can be a promising anticancer drug candidate for the treatment of oral cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing targeted mRNAs. These molecules are about 21-25 nucleotides in length and exert their effects by binding to partially complementary sites in mRNAs, predominantly in the 3'-untranslated region (3'-UTR). Recent evidence has demonstrated that miRNAs can function as oncogenes or tumor suppressors through the modulation of multiple oncogenic cellular processes in cancer development, including initiation, cell proliferation, apoptosis, invasion and metastasis. In our present study, we examined the expression profile of miRNAs related to oral cancer cell growth inhibition using normal human oral keratinocytes (NHOK) and YD-38 human oral cancer cells. By miRNA microassay analysis, 40 and 31 miRNAs among the 1,769 examined were found to be up- and down-regulated in YD-38 cells compared with NHOK cells, respectively. Using qRT-PCR analysis, the expression levels of miR-30a and miR-1246 were found to be increased in YD-38 cells compared with NHOK cells, whereas miR-203 and miR-125a were observed to be decreased. Importantly, the overexpression of miR-203 and miR-125a significantly inhibited the growth of YD-38 cells. This finding and the microarray data indicate the involvement of specific miRNAs in the development and progression of oral cancer.

Angelica decursiva has been used in Korean traditional medicine as an antitussive, an analgesic, an antipyretic and a cough remedy. However, its anti-cancer properties have not yet been well defined. In our current study, we report the cytotoxic activity and the mechanism of cell death induced by ethanol extracts of Angelica decursiva (EEAD) against the human oral cancer cell line, KB. Treatment of KB cells with EEAD induced apoptotic cell death in both a dose- and time-dependent manner as determined by MTT assay and DNA fragmentation. However, no cytotoxic effects of EEAD against human normal oral keratinocytes (HNOK) were evident. By western blot analysis, we found that apoptosis in KB cells is associated with a decrease in procaspase-7 and -9. In addition, the activation of caspase-7 was detectable in living KB cells by fluorescence microscopy. These results suggest that EEAD exhibits anti-cancer activity in KB cells via apoptosis and thus has potential as an anticancer agent in future drug development strategies.

Sulfur is commonly used in Asia as a n herba l medicine to treat infl ammation and cancel‘. and potent chemopreventive effects have been demonstrated in various in vivo and in vitromodels for sulfur-containing compounds found in naturally occun‘ ing products. Here, we report the growth inhibitory and apoptosis-related effects of a newly developedhigh- purity eclible sulfur (ES) on immortali zecl human oral keratinocytes (IHOKs) and on oral cancer cells representing two stages of oral can cer (HN4‘ HN12) basecl on an 3-(4. 5-Dimethylt hiazol-2-yl)-2.5-cliphenyl tetrazolium bromide (MTT) a ssay, Western blotting, cell cycle analysis, ancl nuclear staining. The puri ty of the ES used in th is s tucly was verified by high performance liquid chromat ography (HPLC) , amino acid analysis and energy di spersive spectroscopy (EDS). ES inhibitecl the proliferation of immor talized and malignant oral kerati nocytes in a dose- and time-dependent manner FITC-Annexin V staining. DNA fragmentation testing. and Hoechst 33258 staining revealed that ES inhibits cell growth via apoptosis . ES blocked cell-cycle progression at the sub- Gl phase, with decreased expression 0 1' cyclins Dl, D2, and E, and t heir activating partners cdk2, cdk4, and cdk6‘ and a concomitant induction of p53 and p21/WAF1. Furthermore, ES treatment increased the cytosolic level of cytochrome c a nd resulted in caspase-3 activation‘ and thi s effect was correlated wi th Bax up- regulation and Bcl-2 down- regulation Taken together, these clata suggest that ES is a potential chemopreventive and chemotherapeutic agent for oral cancel

Sulfur is commonly used in Asia as an herbal medicine to treat inflammation and cancer , a nd potent chemo preventi ve effects have been demons tra ted in various in vivo and in vitromodels for s ul fur-containing compounds found in natura l1y occurring product s. Here, we 1'eport the growth inhibitory and apoptosis-related effects of a n ewly developedhigh-puri ty edible sulfur(ES) on immo1'tali zed human o1'al ke1'atinocytes(IHOKs) and on oral cancer cells representing two stages of oral cancer (HN4‘ HN12) based on an 3-(4,5-Dimethylthiazol-2-yl) -2.5- diphe n yltetrazolium bromide(MTI) assay, Western blotting, cell cycle analysis, and nuclear staining. The puri ty of the ES used in thi s study was ve1'ified by high performance liquid chromatog1'aphy (HPLC) , ami no acid analysis and energy dispersive spectroscopy(EDS). ES inhibited the prolife1'ation of imrnortalized and ma lig nant o1'al kerati nocytes in a dose- and time-dependent manne1' FITC-Annex.in V staining, DNA fragmentation t esting. and Hoechst 33258 s taining revealed that ES inhibits cell growth via apoptosis. ES bl ocked cell-cycle prog1'ession at t he sub-Gl phase‘ wi th decreased expression of cyclins Dl, D2‘ and E, and their activating partn ers cdk2‘ cdk4‘ and cdkfì, and a concomitant induction of p53 and p21/WAF1. Furthe1'more, ES treatment in creased the cytosolic level of cytochrome c and resulted in caspase- 3 activation‘ and thi s effect was co1'1'elated with Bax up-regulation and Bcl-2 down-1'egulation Taken together‘ these data suggest that ES is a potential chemopreventive and chemotherapeut ic agent fo r oral ca ncer