Quercetin induced apoptosis of human oral cancer SAS cells through mitochondria and endoplasmic reticulum mediated signaling pathways.

Oral cancer is a cause of cancer-associated mortality worldwide and the treatment of oral cancer includes radiation, surgery and chemotherapy. Quercetin is a component from natural plant products and it has been demonstrated that quercetin is able to induce cytotoxic effects through induction of cell apoptosis in a number of human cancer cell lines. However, there is no available information to demonstrate that quercetin is able to induce apoptosis in human oral cancer cells. In the present study, the effect of quercetin on the cell death via the induction of apoptosis in human oral cancer SAS cells was investigated using flow cytometry, Annexin V/propidium iodide (PI) double staining, western blotting and confocal laser microscopy examination, to test for cytotoxic effects at 6-48 h after treatment with quercetin. The rate of cell death increased with the duration of quercetin treatment based on the results of a cell viability assay, increased Annexin V/PI staining, increased reactive oxygen species and Ca2+ production, decreased the levels of mitochondrial membrane potential (ΔΨm), increased proportion of apoptotic cells and altered levels of apoptosis-associated protein expression in SAS cells. The results from western blotting revealed that quercetin increased Fas, Fas-Ligand, fas-associated protein with death domain and caspase-8, all of which associated with cell surface death receptor. Furthermore, quercetin increased the levels of activating transcription factor (ATF)-6α, ATF-6ß and gastrin-releasing peptide-78 which indicated an increase in endoplasm reticulum stress, increased levels of the pro-apoptotic protein BH3 interacting-domain death antagonist, and decreased levels of anti-apoptotic proteins B-cell lymphoma (Bcl) 2 and Bcl-extra large which may have led to the decreases of ΔΨm. Additionally, confocal microscopy suggested that quercetin was able to increase the expression levels of cytochrome c, apoptosis-inducing factor and endonuclease G, which are associated with apoptotic pathways. Therefore, it is hypothesized that quercetin may potentially be used as a novel anti-cancer agent for the treatment of oral cancer in future.

Gypenosides induce cell death and alter gene expression in human oral cancer HSC-3 cells.

Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.

Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca2+ , mitochondria membrane potential (ΔΨm ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca2+ production, and decreased the level of ΔΨm and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6ß, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways.

Antitumor effects of deguelin on H460 human lung cancer cells in vitro and in vivo: Roles of apoptotic cell death and H460 tumor xenografts model.

Deguelin, a naturally occurring rotenoid of the flavonoid family, is known to be an Akt inhibitor, to have chemopreventive activities and anti-tumor effect on several cancers. In this study, investigation to elucidate the effect of deguelin on apoptotic pathways in human lung cancer cells and on the anti-tumor effect in lung cancer xenograft nu/nu mice was performed. In vitro studies, found that deguelin induced cell morphological changes, and decreased the percentage of viability through the induction of apoptosis in H460 lung cancer cells. Deguelin triggered apoptosis in H460 cells was also confirmed by DAPI staining, DNA gel electrophoresis, and Annexin V-FITC staining and these effects are dose-dependent manners. It was also found that deguelin promoted the Ca2+ production and activation of caspase-3 but decreased the level of ΔΨm in H460 cells. Western blots indicated that the protein levels of cytochrome c, AIF, and pro-apoptotic Bax and Bak protein were increased, but the anti-apoptotic Bcl-2 and Bcl-x were decreased that may have led to apoptosis in H460 cells after exposure to deguelin. It was also confirmed by confocal laser microscope examination that deguelin promoted the release of AIF from mitochondria to cytosol. In vivo studies, found that in immunodeficient nu/nu mice bearing H460 tumor xenografts showed that the deguelin significantly suppressed tumor growth. Deguelin might be a potential therapeutic agent for the treatment of lung cancer in the future. This finding might fully support a critical event for deguelin via induction of apoptotic cell death and H460 tumor xenografts model against human lung cancer. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 84-98, 2017.

Anticancer effects of cantharidin in A431 human skin cancer (Epidermoid carcinoma) cells in vitro and in vivo.

Cantharidin (CTD), a potential anticancer agent of Traditional Chinese Medicine has cytotxic effects in different human cancer cell lines. The cytotoxic effects of CTD on A431 human skin cancer (epidermoid carcinoma) cells in vitro and in A431 cell xenograft mouse model were examined. In vitro, A431 human skin cell were treated with CTD for 24 and 48 h. Cell phase distribution, ROS production, Ca2+ release, Caspase activity and the level of apoptosis associated proteins were measured. In vivo, A431 cell xenograft mouse model were examined. CTD-induced cell morphological changes and decreased percentage of viable A431 cells via G0/G1 phase arrest and induced apoptosis. CTD-induced G0/G1 phase arrest through the reduction of protein levels of cyclin E, CDK6, and cyclin D in A431 cells. CTD-induced cell apoptosis of A431 cells also was confirm by DNA gel electrophoresis showed CTD-induced DNA fragmentation. CTD reduced the mitochondrial membrane potential and stimulated release of cytochrome c, AIF and Endo G in A431 cells. Flow cytometry demonstrated that CTD increased activity of caspase-8, -9 and -3. However, when cells were pretreated with specific caspase inhibitors activity was reduced and cell viability increased. CTD increased protein levels of death receptors such as DR4, DR5, TRAIL and levels of the active form of caspase-8, -9 and -3 in A431 cells. AIF and Endo G proteins levels were also enhanced by CTD. In vivo studies showed that CTD significantly inhibited A431 cell xenograft tumors in mice. Taken together, these in vitro and in vivo results provide insight into the mechanisms of CTD on cell growth and tumor production. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 723-738, 2017.

Tetrandrine induces programmed cell death in human oral cancer CAL 27 cells through the reactive oxygen species production and caspase-dependent pathways and associated with beclin-1-induced cell autophagy.

Tetrandrine, a bisbenzylisoquinoline alkaloid, is extracted from the root of the Chinese herb Radix Stephania tetrandra S Moore. This compound has antitumor activity in different cancer cell types. In this study, the effects of tetrandrine on human oral cancer CAL 27 cells were examined. Results indicated that tetrandrine induced cytotoxic activity in CAL 27 cells. Effects were due to cell death by the induction of apoptosis and accompany with autophagy and these effects were concentration- and time-dependent manners. Tetrandrine induced apoptosis was accompanied by alterations in cell morphology, chromatin fragmentation, and caspase activation in CAL 27 cells. Tetrandrine treatment also induced intracellular accumulation of reactive oxygen species (ROS). The generation of ROS may play an important role in tetrandrine-induced apoptosis. Tetrandrine triggered LC3B expression and induced autophagy in CAL 27 cells. Tetrandrine induced apoptosis and autophagy were significantly attenuated by N-acetylcysteine pretreatment that supports the involvement of ROS production. Tetrandrine induced cell death may act through caspase-dependent apoptosis with Beclin-1-induced autophagy in human oral cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 329-343, 2017.

Tetrandrine Induces Apoptosis of Human Nasopharyngeal Carcinoma NPC-TW 076 Cells through Reactive Oxygen Species Accompanied by an Endoplasmic Reticulum Stress Signaling Pathway.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy of the head and neck and the incidence is higher in Southeast Asia. Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid, a natural product, and exhibits biological activities including action against many human cancer cell lines. However, the molecular mechanism of TET-induced cell apoptosis in human NPC cells is still unclear. In the present study, we investigated TET-induced apoptotic cell death and associated possible signal pathways on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Phase contrast microscopy was used to examine cell morphology and DAPI staining was used to examine chromatin condensation. Flow cytometry assay was used to measure total viable cells, cell cycle and sub-G1 phase distribution, reactive oxygen species (ROS), Ca2+, and mitochondria membrane potential (ΔΨm) in NPC-TW 076 cells. Results indicate that TET induced cell death through the cell morphological changes, caused G0/G1 phase arrest, increased ROS and Ca2+ production, and finally caused apoptotic cell death in NPC-TW 076 cells. There was no influence on the level of ΔΨm after TET treatment. Western blotting indicated that TET increased endoplasmic reticulum (ER) stress associated protein expression such as GADD153, GRP78, ATF-6α and ATF-6 ßwhich indicated that TET induced cell death through ER stress. ER stress is a potential target in cancer treatment, so the ability of TET to induce ER stress response and to activate programming cell death in NPC-TW 076 cells make this molecule become a promising anticancer agent.

4-Hydroxybutenolide impairs cell migration, and invasion of human oral cancer SCC-4 cells via the inhibition of NF-κB and MAPK signaling pathways.

4-Hydroxybutenolide (K87), a synthetic compound from furfuryl alcohol via photooxidation, was used to investigate whether it can inhibit mobility, migration and invasion of SCC-4 human oral cancer cells in vitro. Cell viability was measured by flow cytometric assay, the enzymatic activities of MMP-2/9 were assayed by gelatin zymography analysis, the protein levels were assayed by western blotting, confocal laser microscopy and EMSA assay, and the gene expression of MMP-2/-7, FAK and ROCK1 mRNA were assayed by PCR. K87 decreased the percentage of viable cells in dose-dependent manner. K87 suppressed cell mobility, migration and invasion of SCC-4 cells dose-dependently. K87 inhibited the enzymatic activities of MMP-2/9 of SCC-4 cells. Western blot analysis revealed that K87 decreased the protein levels in NF-κBp65, COX-2, ROCK1 and Rho A, MMP-1, -2,- 7, -9, VEGF, GRB2, SOS1, PI3K, PKC, PERK, p-PERK, FAK, MEKK3, MKK7, ERK1/2, JNK1/2, p-p38, p38, p-c-Jun, AKT, TIMP2, but increased the protein levels of iNOS, Ras, IRE-1α, p-c-JNK, p-AKT(308), p-AKT(473) and TIMP1. Results from PCR indicated that K87 inhibited the gene expression of MMP-2/-7, FAK and ROCK1 mRNA. Furthermore, confocal laser microscopy was used to confirm that K87 inhibited the translocation of RHOA and ROCK1 in SCC-4 cells. EMSA assay also show that K87 suppressed the nuclear activation of NF-κB and these effects are time-dependent. Western blotting assay indicated that expression of NF-κBp105, NF-κBp50 and NF-κBp65 proteins were decreased and these effects are time-dependent. Based on these observations, we suggest that K87 may be used as a potential agent for anticancer metastasis of human oral cancer in the future.

Allyl Isothiocyanate Induces Cell Toxicity by Multiple Pathways in Human Breast Cancer Cells.

Isothiocyanates (ITCs) occur in many cruciferous vegetables. These compounds, which have significant anticancer actions, can induce apoptosis in different human cancer cell lines. In the present study, we investigated if allyl isothiocyanate (AITC) would induce toxicity in human breast cancer MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) cells. We found that AITC stimulated reactive oxygen species and Ca[Formula: see text] production, and decreased the mitochondrial membrane potential. Activity of caspase-8, -9 and -3 was increased by AITC in both cell lines. AITC also induced mitochondrial-mediated apoptosis, as shown by cytochrome c, AIF and Endo G release from mitochondria, activation of caspase-9 and caspase-3, and formation of DAPI-positive cells. There was a significant reduction in the levels of the anti-apoptotic protein Bcl-2 along with a marked increase in the pro-apoptotic protein Bax in both cell lines. AITC induced apoptosis in human breast cancer MCF-7 cells via AIF and Endo G signaling pathways, but in MDA-MB-231 cells apoptosis occurred via the GADD153 pathway. This study has revealed novel anti-cancer mechanisms of AITC, a compound that is ordinarily present in human diets and may have potential therapeutic effects in various cancers.

Suppression of the migration and invasion is mediated by triptolide in B16F10 mouse melanoma cells through the NF-kappaB-dependent pathway.

Melanoma cancer is one of the major causes of death in humans worldwide. Triptolide is one of the active components of Tripterygium wilfordii Hook F, and has biological activities including induced cell cycle arrest and induction of apoptosis but its antimetastatic effects on murine melanoma cells have not yet been elucidated. Herein, we investigated the effect of triptolide on the inhibition of migration and invasion and possible associated signal pathways in B16F10 murine melanoma cancer cells. Wound healing assay and Matrigel Cell Migration Assay and Invasion System demonstrated that triptolide marked inhibiting the migration and invasion of B16F10 cells. Gelatin zymography assay demonstrated that triptolide significantly inhibited the activities of matrix metalloproteinases-2 (MMP-2). Western blotting showed that triptolide markedly reduced CXCR4, SOS1, GRB2, p-ERK, FAK, p-AKT, Rho A, p-JNK, NF-κB, MMP-9, and MMP-2 but increased PI3K and p-p38 and COX2 after compared to the untreated (control) cells. Real time PCR indicated that triptolide inhibited the gene expression of MMP-2, FAK, ROCK-1, and NF-κB but did not significantly affect TIMP-1 and -2 gene expression in B16F10 cells in vitro. EMSA assay also showed that triptolide inhibited NF-κB DNA binding in a dose-dependent manner. Confocal laser microscopy examination also confirmed that triptolide inhibited the expression of NF-κB in B16F10 cells. Taken together, we suggest that triptolide inhibited B16F10 cell migration and invasion via the inhibition of NF-κB expression then led to suppress MMP-2 and -9 expressions. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1974-1984, 2016.

Tetrandrine induces apoptosis Via caspase-8, -9, and -3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin-1/ LC3-I, II signaling pathways in human oral cancer HSC-3 cells.

Tetrandrine is a bisbenzylisoquinoline alkaloid that was found in the Radix Stephania tetrandra S Moore. It had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC-3 cells in vitro. Treatments of HSC-3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration-dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases-3, -8, and -9, and cleavage of poly (ADP ribose) polymerase (PARP) that were associated with apoptosis, and it also enhanced the expression of LC3-I and -II that were associated with the induction of autophagy in human squamous carcinoma cell line (HSC-3) cells. Tetrandrine induced autophagy in HSC-3 cells was significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre-treatment that confirmed tetrandrine induced cell death may be associated with the autophagy. In conclusion, we suggest that tetrandrine induced cell death may be through the induction of apoptosis as well as autophagy in human oral cancer HSC-3 cells via PARP, caspases/Becline I/LC3-I/II signaling pathways.

Bufalin Inhibits NCI-H460 Human Lung Cancer Cell Metastasis In Vitro by Inhibiting MAPKs, MMPs, and NF-κB Pathways.

Bufalin, a component of Chan Su (a traditional Chinese medicine), has been known to have antitumor effects for thousands of years. In this study, we investigated its anti-metastasis effects on NCI-H460 lung cancer cells. Under sub-lethal concentrations (from 25 up to 100 nM), bufalin significantly inhibits the invasion and migration nature of NCI-H460 cells that were measured by Matrigel Cell Migration Assay and Invasion System. Bufalin also suppressed the enzymatic activity of matrix metalloproteinase (MMP)-9, which was examined by gelatin zymography methods. Western blotting revealed that bufalin depressed several key metastasis-related proteins, such as NF-κB, MMP-2, MMP-9, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-K), phosphorylated Akt, growth factor receptor-bound protein 2 (GRB2), phosphorylated extracellular signal-regulated kinase (ERK), phosphorylated p38, and phosphorylated c-Jun NH2-terminal kinase (JNK). As evidenced by immunostaining and the electrophoretic mobility shift assay (EMSA), bufalin induced not only a decreased cytoplasmic NF-κB production, but also decreased its nuclear translocation. Several metastasis-related genes, including Rho-associated (Rho A), coiled-coil-containing protein kinase 1 (ROCK1), and focal adhesion kinase (FAK), were down-regulated after bufalin treatment. In conclusion, bufalin is effective in inhibiting the metastatic nature of NCI-H460 cells in low, sub-lethal concentrations. Such an effect involves many mechanisms including MMPs, mitogen-activated protein kinases (MAPKs) and NF-κB systems. Bufalin has a potential to evolve into an anti-metastasis drug for human lung cancer in the future.

PEITC inhibits human brain glioblastoma GBM 8401 cell migration and invasion through the inhibition of uPA, Rho A, and Ras with inhibition of MMP-2, -7 and -9 gene expression.

Glioblastoma is the most aggressive primary brain malignancy, and the efficacy of multimodality treatments remains unsatisfactory. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, was found to inhibit the migration and invasion of many types of human cancer cells. In our previous study, PEITC induced the apoptosis of human brain glioblastoma GBM 8401 cells through the extrinsic and intrinsic signaling pathways. In the present study, we first investigated the effects of PEITC on the migration and invasion of GBM 8401 cells. PEITC decreased the migration of GBM 8401 cells in a dose-dependent manner as determined from scratch wound healing and Transwell migration assays. The percentage of inhibition ranged from 46.89 to 15.75%, and from 27.80 to 7.31% after a 48-h treatment of PEITC as determined from the Transwell migration assay and invasion assay, respectively. The western blot analysis indicated that PEITC decreased the levels of proteins associated with migration and invasion, Ras, uPA, RhoA, GRB2, p-p38, p-JNK, p-ERK, p65, SOS1, MMP-2, MMP-9 and MMP-13, in a dose-dependent manner. Real-time PCR analyses revealed that PEITC reduced the mRNA levels of MMP-2, MMP-7, MMP-9 and RhoA in a dose- and time-dependent manner. PEITC exhibited potent anticancer activities through the inhibition of migration and invasion in the GBM 8401 cells. Our findings elucidate the possible molecular mechanisms and signaling pathways of the anti-metastatic effects of PEITC on human brain glioblastoma cells, and PEITC may be considered as a therapeutic agent.

Gallic acid induces DNA damage and inhibits DNA repair-associated protein expression in human oral cancer SCC-4 cells.

Gallic acid (GA), a phenolic compound naturally present in plants, used as an antioxidant additive in food and in the pharmaceutical industry, may have cancer chemopreventive properties. In the present study, we investigated whether GA induced DNA damage and affected DNA repair-associated protein expression in human oral cancer SCC-4 cells. Flow cytometry assays were used to measure total viable cells and results indicated that GA decreased viable cells dose-dependently. The comet assay and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining were used to measure DNA damage, as well as condensation and it was shown that GA induced DNA damage (comet tail) and DNA condensation in a dose-dependent manner. DNA gel electrophoresis was used to examine DNA fragmentation and we found that GA induced DNA ladder (fragmentation). Using western blotting it was shown that GA inhibited the protein expressions of MDC1, O(6)-methylguanine-DNA methyltransferase (MGMT), p-H2A.X, p53, DNA-dependent serine/threonine protein kinase (DNA-PK) and 14-3-3 proteins sigma (14-3-3σ) but increased p-p53, phosphate-ataxia-telangiectasia (p-H2A.X) and ataxia telangiectasia mutated and Rad3-related (p-ATR), phosphate-ataxia telangiectasia mutated (p-ATM) and breast cancer susceptibility protein 1 (BRCA1) in a 24-h treatment. The protein translocation was examined by confocal laser microscopy and results indicated that GA increased the levels of p-H2A.X, MDC1 and p-p53 in SCC-4 cells. In conclusion, we found that GA-induced cell death may proceed through the induced DNA damage and suppressed DNA repair-associated protein expression in SCC-4 cells.

Crude extract of Polygonum cuspidatum promotes immune responses in leukemic mice through enhancing phagocytosis of macrophage and natural killer cell activities in vivo.

Polygonum cuspidatum is a traditional Chinese herbal medicine used in the treatment of various diseases. In the present study, we investigated whether the crude extract of Polygonum cuspidatum (CEPC) could affect immune responses of murine leukemia cells in vivo. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemic mice and then were treated orally with CEPC at 0, 50, 100 and 200 mg/kg for three weeks. Animals were weighed and blood, liver, spleen samples were collected for further analyses. Results indicated that CEPC did not significantly affect the body and liver weight of animals, but reduced the weight of spleen when compared to control groups. Flow cytometric assay demonstrated that CEPC increased the percentage of CD3- (T-cell marker) and CD19- (B-cell marker) positive cells, but reduced that of CD11b-positive ones (monocytes). However, it did not significantly affect the proportion of Mac-3-positive cells (macrophages), compared to control groups. Results indicated that CEPC promoted phagocytosis by macrophages from blood samples at all examined doses but did not affect that of macrophages from the peritoneal cavity. CEPC also promoted natural killer cell activity of splenocytes at 200 mg/kg of CEPC. CEPC promoted B-cell proliferation at 200 mg/kg treatment when cells were stimulated with lipopolysaccharides but did not promote T-cell proliferation at three doses of CEPC treatment on concanavalin A stimulation.

α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells.

α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 µM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro.

Safrole suppresses murine myelomonocytic leukemia WEHI-3 cells in vivo, and stimulates macrophage phagocytosis and natural killer cell cytotoxicity in leukemic mice.

Many anticancer drugs are obtained from phytochemicals and natural products. However, some phytochemicals have mutagenic effects. Safrole, a component of Piper betle inflorescence, has been reported to be a carcinogen. We have previously reported that safrole induced apoptosis in human oral cancer cells in vitro and inhibited the human oral tumor xenograft growth in vivo. Until now, there is no information addressing if safrole promotes immune responses in vivo. To evaluate whether safrole modulated immune function, BALB/c mice were intraperitoneally injected with murine myelomonocytic WEHI-3 leukemia cells to establish leukemia and then were treated with or without safrole at 4 and 16 mg/kg. Animals were sacrificed after 2 weeks post-treatment with safrole for examining the immune cell populations, phagocytosis of macrophages and the natural killer (NK) cells' cytotoxicity. Results indicated that safrole increased the body weight, and decreased the weights of spleen and liver in leukemic mice. Furthermore, safrole promoted the activities of macrophages phagocytosis and NK cells' cytotoxicity in leukemic mice when compared with untreated leukemic mice. After determining the cell marker population, we found that safrole promoted the levels of CD3 (T cells), CD19 (B cells) and Mac-3 (macrophages), but it did not affect CD11b (monocytes) in leukemic mice. In conclusion, safrole altered the immune modulation and inhibited the leukemia WEHI-3 cells in vivo.

Role of human papillomavirus infection in carcinogenesis of oral squamous cell carcinoma with evidences of prognostic association.

BACKGROUND: Betel nut chewing, cigarette smoking and alcohol drinking are thought to be major environmental risk factors responsible for the development of oral squamous cell carcinomas. Oncogenic human papillomavirus infections have a well-established association with uterine cervical carcinoma. However, little is known about the exact role of human papillomavirus infections in oral squamous cell carcinomas. This study is designed to elucidate the role of human papillomavirus infections in cancer development and prognosis of oral squamous cell carcinomas. METHODS: Molecular techniques including in situ hybridization and immunohistochemistry of p16(INK4A) and p53 for evidences of human papillomavirus in tissue micro-arrays were investigated. RESULTS: Twenty-four of 65 cases of oral squamous cell carcinomas were found positive for in situ hybridization and 14 were found positive for p16(INK4A). The majority of cases without the evidence of human papillomavirus were related to p53 over-expression. There were statistically significant correlations between the results of human papillomavirus test and size or extent of the tumor (P = 0.003) or the stage of oral squamous cell carcinomas (P = 0.015). Kaplan-Meier plot analysis demonstrated a tendency of longer survival in cases of oral squamous cell carcinomas with the evidence of human papillomavirus or positive p16 (INK4A). CONCLUSIONS: Human papillomavirus infections may play a unique role in oral carcinogenesis. Our data strongly suggest that human papillomavirus-positive oral squamous cell carcinomas comprise a distinct clinical and pathological disease entity that appears related to a better outcome with longer survival and bears a causally associated relationship different from other carcinogenic mechanisms.

Safrole induces cell death in human tongue squamous cancer SCC-4 cells through mitochondria-dependent caspase activation cascade apoptotic signaling pathways.

Safrole is one of important food-borne phytotoxin that exhibits in many natural products such as oil of sassafras and spices such as anise, basil, nutmeg, and pepper. This study was performed to elucidate safrole-induced apoptosis in human tongue squamous carcinoma SCC-4 cells. The effect of safrole on apoptosis was measured by flow cytometry and DAPI staining and its regulatory molecules were studied by Western blotting analysis. Safrole-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and Bid and down-regulation of the protein levels of Bcl-2 (up-regulation of the ratio of Bax/Bcl-2), resulting in cytochrome c release, promoted Apaf-1 level and sequential activation of caspase-9 and caspase-3 in a time-dependent manner. We also used real-time PCR to show safrole promoted the mRNA expressions of caspase-3, -8, and -9 in SCC-4 cells. These findings indicate that safrole has a cytotoxic effect in human tongue squamous carcinoma SCC-4 cells by inducing apoptosis. The induction of apoptosis of SCC-4 cells by safrole is involved in mitochondria- and caspase-dependent signal pathways.

Diallyl sulfide inhibits murine WEHI-3 leukemia cells in BALB/c mice in vitro and in vivo.

It is well documented that enhanced garlic (Allium sativum) consumption leads to decrease in the cancer incidences. Diallyl sulfide (DAS), one of the components of garlic, induces cytotoxicity and apoptosis in many cancer cell lines. The present studies are focused on the in vivo effects of DAS on leukemia WEHI-3 cells in the BALB/c mice. We examined the effects of DAS on the cytotoxicity of WEHI-3 cells and results indicated that DAS decreased the percentage of viable WEHI-3 cells and these effects are dose-dependent. We examined the effects of DAS on WEHI-3 in vivo and the results indicated that DAS decreased the percentage of Mac-3 and CD11b, indicating that the differentiation of the precursor of macrophage cells was inhibited. DAS stimulated the percentage of CD3 and CD19, indicating that the differentiation of the precursor of T and B cells promoted. The weights of liver and spleen indicated that DAS decreased the weight of these organs after being compared to the control groups. One of the major characteristic of WEHI-3 leukemia is the enlarged spleen in murine after intraperitoneal (i.p.) injection of WEHI-3 cells. In conclusion, DAS affects WEHI-3 cells both in vitro and in vivo.