Codium fragile (Suringar) Hariot is an edible green seaweed that belong to the Codiaceae family and has been used in Oriental medicine for the treatment of enterobiasis, dropsy, and dysuria. Methanol extract of codium fragile has anti-oxidant, anti-inflammatory and anti-cancer properties, although the anti-cancer effect on oral cancer has not yet been reported. In this study, we investigated the anti-cancer activity and the mechanism of cell death by methanol extracts of Codium fragile (MeCF) on human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that MeCF inhibits cell viability in a dose-dependent manner, and markedly induced apoptosis, as determined by the MTT assay, Live/Dead assay, and DAPI stain. In addition, MeCF induced the proteolytic cleavage of procaspase -3, -7, -9 and poly(ADP-ribose) polymerase(PARP), and upregulated or downregulated the expression of mitochondrial-apoptosis factor, Bax(pro-apoptotic factor), and Bcl-2(anti-apoptotic factor), . Futhermore, MeCF induced a cell cycle arrest at the G1/S phase through suppressing the expression of the cell cycle cascade proteins, p21, CDK4, CyclinD1, and phospho-Rb. Taken together, these results indicated that MeCF inhibits cell growth, and this inhibition is mediated by caspase- and mitochondrial-dependent apoptotic pathways through cell cycle arrest at the G1/S phase in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, methanol extracts of Codium fragile can be provided as a novel chemotherapeutic drug due to its growth inhibition effects and induction of apoptosis in human oral cancer cells.

Shikonin, a major ingredient in the traditional Chinese herb Lithospermumerythrorhizon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. It has recently been reported that shikonin displays antitumor properties in many cancers. This study was aimed to investigate whether shikonin could inhibit oral squamous carcinoma cell (OSCC) growth via mechanisms of apoptosis and cell cycle arrest. The effects of shikonin on the viability and growth of OSCC cell line, SCC25 cells were assessed by MTT assay and clonogenic assays, respectively. Hoechst staining and DNA electrophoresis indicated that the shikonin-treated SCC25 cells were undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, flow cytometry, MMP activity, and proteasome activity also supported the finding that shikonin induces apoptosis. Shikonin treatment of SCC25 cells resulted in a time- and dose-dependent decrease in cell viability, inhibition of cell growth, and increase in apoptotic cell death. The treated SCC25 cells showed several lines of apoptotic manifestation as follows: nuclear condensation; DNA fragmentation; reduced MMP and proteasome activity; decrease in DNA contents; release of cytochrome c into cytosol; translocation of AIF and DFF40 (CAD) onto the nuclei; a significant shift in Bax/Bcl-2 ratio; and activation of caspase-9, -7, -6, and -3, as well as PARP, lamin A/C, and DFF45 (ICAD). Shikonin treatment also resulted in down-regulation of the G1 cell cycle-related proteins and up-regulation of p27KIP1. Taken together, our present findings demonstrate that shikonin strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins, and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells.

Several studies have shown that curcumin, which is derived from the rhizomes of turmeric, possesses antimicrobial, antioxidant and anti-inflammatory properties. The antitumor properties of curcumin have also now been demonstrated more recently in different cancers. This study was undertaken to investigate the modulation of cell cycle-related proteins and the mechanisms underlying apoptosis induction by curcumin in the SCC25 human tongue squamous cell carcinoma cell line. Curcumin treatment of the SCC25 cells resulted in a time- and dose-dependent reduction in cell viability and cell growth, and onset of apoptotic cell death. The curcumin-treated SCC25 cells showed several types of apoptotic manifestations, such as nuclear condensation, DNA fragmentation, reduced MMP and proteasome activity, and a decreased DNA content. In addition, the treated SCC25 cells showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40/CAD into the nuclei, a significant shift in the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, lamin A/C, and DFF45/ICAD. Furthermore, curcumin exposure resulted in a downregulation of G1 cell cycle-related proteins and upregulation of p27KIP1. Taken together, our findings demonstrate that curcumin strongly inhibits cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via proteasomal, mitochondrial, and caspase cascades in SCC25 cells.

Eugenol is an essential oil found in cloves and cinnamon that is used widely in perfumes. However, the significant anesthetic and sedative effects of this compound have led to its use also in dental procedures. Recently, it was reported that eugenol induces apoptosis in several cancer cell types but the mechanism underlying this effect has remained unknown. In our current study, we examined whether the cytotoxic effects of eugenol upon human melanoma G361 cells are associated with cell cycle arrest and apoptosis using a range of methods including an XTT assay, Hoechst staining, immunocytochemistry, western blotting and flow cytometry. Eugenol treatment was found to decrease the viability of the G361 cells in both a time- and dose-dependent manner. The induction of apoptosis in eugenol-treated G361 cells was confirmed by the appearance of nuclear condensation, the release of both cytochrome c and AIF into the cytosol, the cleavage of PARP and DFF45, and the downregulation of procaspase-3 and -9. With regard to cell cycle arrest, a time-dependent decrease in cyclin A, cyclin D3, cyclin E, cdk2, cdk4, and cdc2 expression was observed in the cells after eugenol treatment. Flow cytometry using a FACScan further demonstrated that eugenol induces a cell cycle arrest at S phase. Our results thus suggest that the inhibition of G361 cell proliferation by eugenol is the result of an apoptotic response and an S phase arrest that is linked to the decreased expression of key cell cycle-related molecules.

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MTT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG (2 μM) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

Chios gum mastic (CGM) is produced from Pistiacia lentiscus L var chia, which grows only on Chios Island in Greece. CGM is a kind of resin extracted from the stem and leaves, has been used for many centuries in many Mediterranean countries as a dietary supplement and folk medicine for stomach and duodenal ulcers. CGM is known to induce cell cycle arrest and apoptosis in some cancer cells. This study was undertaken to investigate the alteration of the cell cycle and induction of apoptosis following CGM treatment of HL-60 cells. The viability of the HL-60 cells was assessed using the MTT assay. Hoechst staining and DNA electrophoresis were employed to detect HL-60 cells undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity analyses were also employed. CGM treatment of HL-60 cells was found to result in a dose- and time-dependent decrease in cell viability and apoptotic cell death. Tested HL-60 cells showed a variety of apoptotic manifestations and induced the downregulation of G1 cell cycle-related proteins. Taken collectively, our present findings demonstrate that CGM strongly induces G1 cell cycle arrest via the modulation of cell cycle-related proteins, and also apoptosis via proteasome, mitochondrial and caspase cascades in HL-60 cells. Hence, we provide evidence that a natural product, CGM could be considered as a novel therapeutic for human leukemia.

The trace element nutrient selenium discharges its well-known nutritional anti-tumor activity. Converging data from epidemiological, ecological and clinical studies have shown that selenium can decrease the risk for some types of human cancers, especially those of the prostate, lung, and colon. Mechanistic studies have indicated that selenium has many desirable attributes of chemoprevention targeting cancer cells through DNA single strand breaks, the induction of reactive oxygen species. However, there is no reports about the relationship between methylseleninic acid (MSeA), one of methylselenol metabolites and cell cycle arrest in LNCaP human prostate cancer cells. Our data showed that MSeA arrested G1/S pahse of cell cycle arrest and inhibited DNA synthesis in LNCaP cells and those cellular events by MSeA were due to the induction of p27 protein which is a well-known cyclin-dependent kinase inhibitor. Taken together, cell cycle arrest occurred by MSeA may contribute to the growth-inhibition of prostate cancer cells.