Bisdemethoxycurcumin Induces Cell Apoptosis and Inhibits Human Brain Glioblastoma GBM 8401/Luc2 Cell Xenograft Tumor in Subcutaneous Nude Mice In Vivo.

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Mangiferin induces immune responses and evaluates the survival rate in WEHI-3 cell generated mouse leukemia in vivo.

Mangiferin is a naturally occurring polyphenol, widely distributed in Thymeraceae families, and presents pharmacological activity, including anti-cancer activities in many human cancer cell lines. Mangiferin has also been reported to affect immune responses; however, no available information concerning the effects of mangiferin on immune reactions in leukemia mice in vivo. In the present study, we investigated the effects of mangiferin on leukemia WEHI-3 cell generated leukemia BLAB/c mice. Overall, the experiments were divided into two parts, one part was immune responses experiment and the other was the survival rate experiment. The immune responses and survival rate study, 40 mice for each part, were randomly separated into five groups (N = 8): Group I was normal animals and groups II-V WEHI-3 cell generated leukemia mice. Group II mice were fed normal diet as a positive control; group III, IV, and V mice received mangiferin at 40, 80, and 120 mg/kg, respectively, by intraperitoneal injection every 2 days for 20 days. Leukocytes cell population, macrophage phagocytosis, and NK cell activities were analyzed by flow cytometry. Isolated splenocytes stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A) were used to determine the proliferation of B and T cells, respectively, and subsequently were analyzed by flow cytometry. Results indicated that mangiferin significantly increased body weight, decreased the liver and spleen weights of leukemia mice. Mangiferin also increased CD3 T-cell and CD19 B cell population but decreased Mac-3 macrophage and CD11b monocyte. Furthermore, mangiferin decreased phagocytosis of macrophages from PBMC and peritoneal cavity at 40, 80, and 120 mg/kg treatment. However, it also increased NK cell activity at 40 and 120 mg/kg treatment. There were no effects on T and B cell proliferation at three examined doses. In survival rate studies, mangiferin significantly elevated survival rate at 40 and 120 mg/kg treatment of leukemia mice in vivo.

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca2+ production, levels of ΔΨm and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca2+ productions, decreases the levels of ΔΨm , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells.

Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo.

Bufalin has been shown to be effective against a variety of cancer cells, but its role in lung cancer has never been studied in an animal model. In this study, we evaluated bufalin effects in a human lung cancer cell line NCI-H460 both in vitro and in vivo. Bufalin caused significant cytotoxicity in NCI-H460 cells at a concentration as low as 1 µM. DNA condensation was observed in bufalin-treated cells in a dose-dependent manner. Mitochondrial membrane potential (ΔΨm ) was reduced and reactive oxygen species (ROS) were increased in bufalin-treated NCI-H460 cells. Levels of several proapoptotic proteins such as Fas, Fas-ligand, cytochrome c, apoptosis protease activating factor-1, endonuclease G, caspase-3 and caspase-9 were increased after bufalin treatment. At the same time, anti-apoptotic B-cell lymphoma 2 protein levels were reduced. Bufalin decreased glucose regulated protein-78 gene expression but increased growth arrest- and DNA damage-inducible 153 gene expression. Bufalin injected intraperitoneally in a dose-dependent manner reduced tumor size in BALB/C nu/nu mice implanted with NCI-H460 cells. Bufalin injection did not produce significant drug-related toxicity in experimental animals except at a high dose (0.4 mg kg-1 ). In conclusion, low concentrations of bufalin can induce apoptosis in the human lung cancer cell line NCI-H460 in vitro. Bufalin also reduced tumor size in mice injected with NCI-H460 cells without significant drug-related toxicity. These results indicate that bufalin may have potential to be developed as an agent for treating human non-small cell lung cancer. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1305-1317, 2017.

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.

Alpha-phellandrene-induced apoptosis in mice leukemia WEHI-3 cells in vitro.

Although reports have shown that α-phellandrene (α-PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca2+ and decreased the levels of mitochondrial membrane potential (ΔΨm ) in dose- and time-dependent manners in WEHI-3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α-PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640-1651, 2016.

18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC50 value of 100 µM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (ΔΨm) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.

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.

Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI-3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo.

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI-3 cells in vitro and used WEHI-3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI-3 cells through the G0/G1 phase arrest and induction of caspase-3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI-3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac-3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI-3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo.

The roles of endoplasmic reticulum stress and mitochondrial apoptotic signaling pathway in quercetin-mediated cell death of human prostate cancer PC-3 cells.

Prostate cancer has its highest incidence and is becoming a major concern. Many studies have shown that traditional Chinese medicine exhibited antitumor responses. Quercetin, a natural polyphenolic compound, has been shown to induce apoptosis in many human cancer cell lines. Although numerous evidences show multiple possible signaling pathways of quercetin in apoptosis, there is no report to address the role of endoplasmic reticulum (ER) stress in quercetin-induced apoptosis in PC-3 cells. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human prostate cancer PC-3 cells. Cells were treated with quercetin for 24 and 48 h and at various doses (50-200 µM), and cell morphology and viability decreased significantly in dose-dependent manners. Flow cytometric assay indicated that quercetin at 150 µM caused G0/G1 phase arrest (31.4-49.7%) and sub-G1 phase cells (19.77%) for 36 h treatment and this effect is a time-dependent manner. Western blotting analysis indicated that quercetin induces the G0/G1 phase arrest via decreasing the levels of CDK2, cyclins E, and D proteins. Quercetin also stimulated the protein expression of ATF, GRP78, and GADD153 which is a hall marker of ER stress. Furthermore, PC-3 cells after incubation with quercetin for 48 h showed an apoptotic cell death and DNA damage which are confirmed by DAPI and Comet assays, leading to decrease the antiapoptotic Bcl-2 protein and level of ΔΨm , and increase the proapoptotic Bax protein and the activations of caspase-3, -8, and -9. Moreover, quercetin promoted the trafficking of AIF protein released from mitochondria to nuclei. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade through mitochondrial pathway and ER stress in PC-3 cells.

Phenethyl Isothiocyanate (PEITC) Inhibits the Growth of Human Oral Squamous Carcinoma HSC-3 Cells through G(0)/G(1) Phase Arrest and Mitochondria-Mediated Apoptotic Cell Death.

Phenethyl isothiocyanate (PEITC), an effective anticancer and chemopreventive agent, has been reported to inhibit cancer cell growth through cell-cycle arrest and induction of apoptotic events in various human cancer cells models. However, whether PEITC inhibits human oral squamous cell carcinoma HSC-3 cell growth and its underlying mechanisms is still not well elucidated. In the present study, we evaluated the inhibitory effects of PEITC in HSC-3 cells and examined PEITC-modulated cell-cycle arrest and apoptosis. The contrast-phase and flow cytometric assays were used for examining cell morphological changes and viability, respectively. The changes of cell-cycle and apoptosis-associated protein levels were determined utilizing Western blotting in HSC-3 cells after exposure to PEITC. Our results indicated that PEITC effectively inhibited the HSC-3 cells' growth and caused apoptosis. PEITC induced G(0)/G(1) phase arrest through the effects of associated protein such as p53, p21, p17, CDK2 and cyclin E, and it triggered apoptosis through promotion of Bax and Bid expression and reduction of Bcl-2, leading to decrease the levels of mitochondrial membrane potential (ΔΨ(m)), and followed the releases of cytochrome c, AIF and Endo G then for causing apoptosis in HSC-3 cells. These results suggest that PEITC could be an antitumor compound for oral cancer therapy.

Ouabain Induces DNA Damage in Human Osteosarcoma U-2 OS Cells and Alters the Expression of DNA Damage and DNA Repair-associated Proteins.

BACKGROUND/AIM: Ouabain, isolated from natural plants, exhibits anticancer activities; however, no report has presented its mechanism of DNA damage induction in human osteosarcoma cancer cells in vitro. The aim of this study was to investigate whether ouabain induces DNA damage and repair, accompanied with molecular pathways in human osteosarcoma cancer U-2 OS cells in vitro. MATERIALS AND METHODS: The percentage of viable cell number was measured by flow cytometric assay; DNA damage was assayed by DAPI staining, comet assay, and agarose gel electrophoresis. DNA damage and repair associated protein expressions were assayed by western blotting assays. RESULTS: Ouabain reduced total cell viability, induced chromatin condensation, DNA fragmentation, and DNA damage in U-2 OS cells. Ouabain increased p-ATMSer1981, p-ATRSer428, and p53 at 2.5-10 µM, increased p-p53Ser15 at 10 µM; however, it decreased p-MDM2Ser166 at 2.5-10 µM. Ouabain increased p-H2A.XSer139, MDC-1, and PARP at 2.5-10 µM and BRCA1 at 5-10 µM; however, it decreased DNA-PK and MGMT at 2.5-10 µM in U-2 OS cells at 48 h treatment. Ouabain promoted expression and nuclear translocation of p-H2A.XSer139 in U-2 OS cells and this was confirmed by confocal laser microscopy. CONCLUSION: Ouabain reduced total viable cell number through triggering DNA damage and altering the protein expression of DNA damage and repair system in U-2 OS cells in vitro.

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. MATERIALS AND METHODS: Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. RESULTS: DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. CONCLUSION: DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

Maslinic Acid Induces DNA Damage and Impairs DNA Repair in Human Cervical Cancer HeLa Cells.

BACKGROUND/AIM: Maslinic acid, a natural plant-derived triterpenoid compound, exhibits pharmacological activities, including anti-cancer. In the present study, we investigated the cytotoxic effects of maslinic acid on human cervical cancer HeLa cells in vitro and further investigated the molecular mechanism of maslinic acid-induced DNA damage and repair. MATERIALS AND METHODS: Cell viability was measured by flow cytometry. DNA condensation (apoptotic cell death), DNA damage, and DNA fragmentation (DNA ladder) were assayed by DAPI staining, comet assay, and agarose gel electrophoresis, respectively. The expression of DNA damage and repair proteins was assayed by western blotting. RESULTS: Maslinic acid decreased total cell viability and induced DNA condensation, damage, and fragmentation in HeLa cells. Furthermore, maslinic acid elevated the levels of p-ATMSer1981, p-ATRSer428, p53, p-p53Ser151, p-H2A.XSer139, BRCA1 and PARP at 30-40 µM. However, it decreased the levels of DNA-PK and MGMT. CONCLUSION: Maslinic acid reduced the number of viable HeLa cells by inducing DNA damage and altering the expression of proteins involved in DNA damage and repair.

Ganoderma lucidum extracts inhibited leukemia WEHI-3 cells in BALB/c mice and promoted an immune response in vivo.

Ganoderma lucidum (G. lucidum) is a medicinal mushroom having biological effects such as immunomodulation and anti-tumor actions. In China and many other Asian countries, G. lucidum is used as a folk remedy to promote health and longevity. Although many studies have shown that G. lucidum modulates the immune system, including, for example, antigen-presenting cells, natural killer (NK) cells, and the T and B lymphocytes, the effects of G. lucidum on the WEHI-3 leukemic BALB/c mice are unclear. We attempted to determine whether G. lucidum would promote immune responses in BALB/c mice injected with WEHI-3 leukemia cells. The effects of G. lucidum on the survival rate of WEHI-3 leukemia cells injected into BALB/c mice were examined. It increased the percentages of CD3 and CD19, but decreased the percentages of Mac-3 and CD11b markers, suggesting that differentiation of the precursor of T and B cells was promoted but macrophages were inhibited. It decreased the weight of spleens as compared with control mice. It also promoted phagocytosis by macrophage from peripheral blood mononuclear cell (PBMC) and it also promoted natural killer cell activity. It decreased the percentage of leukemia cells in the spleens of mice before they were injected with WEHI-3 cells. Apparently, G. lucidum affects murine leukemia WEHI-3 cells in vivo.

Gandoderma lucidum extract promotes immune responses in normal BALB/c mice In vivo.

Enhanced fruit and vegetable consumption is closely related to reduced cancer incidence as shown in epidemiological studies. Ganoderma lucidum, one of the most well-known traditional Chinese medicines, has been demonstrated to have pharmacological activities and antitumor effects, in Asian populations. However, the promotion of immune responses in normal BALB/c mice is unclear. In the present study, we investigated the immune responses of BALB/c mice after treatment with G. lucidum extract in vivo. The results demonstrated that G. lucidum extract was able to promote the proliferation of splenocytes under Concanavalin A or lipopolysaccharide stimulation. Compared with the control group, phagocytosis of macrophage was significantly enhanced by intraperitoneal administration of G. lucidum extract at both 3 and 6 mg/kg. Compared with the control group, natural killer cell activity was significantly enhanced by intraperitoneal administration of G. lucidum extract (6 mg/kg). Results of cytometric bead array and flow cytometry indicated that the expressions of interleukin-6 and interferon-gamma also increased (p<0.001) by treatment with G. lucidum extract (3 and 6 mg/kg). In conclusion, the findings of this study implied that G. lucidum extract was able to effectively promote immune responses in BALB/c mice.

Casticin Induces DNA Damage and Impairs DNA Repair in Human Bladder Cancer TSGH-8301 Cells.

BACKGROUND/AIM: Casticin shows anti-cancer effects in many types of cancer. However, there is no information regarding its role in DNA damage in human bladder cancer. The aim of this study was to investigate the effects of casticin on TSGH-8301 cells in vitro. MATERIALS AND METHODS: Viability of cells was assayed by flow cytometry. DNA damage was assayed by DAPI staining, comet assay, and gel electrophoresis. Protein levels were examined by western blotting and confocal laser microscopy. RESULTS: Casticin decreased viability of cells and induced DNA damage. Furthermore, casticin decreased expression of p-ATM, p-ATR, MDC1 and MGMT levels after 48 h of treatment, however, it increased p-ATR and MGMT levels after 12 h. In contrast, casticin increased the levels of p-p53, p-H2A.X, and PARP after 48 h of treatment. As shown by confocal microscopy, casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. CONCLUSION: Casticin decreased viability of human bladder cancer cells through DNA damage.

Gypenosides inhibited invasion and migration of human tongue cancer SCC4 cells through down-regulation of NFkappaB and matrix metalloproteinase-9.

Gypenosides (Gyp), components of Gynostemma pentaphyllum Makino, were found to induce suppression of human tongue squamous cell carcinoma SCC4 cell growth and induce apoptosis in response to overexpression of reactive oxygen species, calcium (Ca(+2)) and to decrease mitochondrial membrane potential in vitro. In this study, the effect of Gyp on cell migration and invasion of human tongue SCC4 cells was examined. SCC4 cells treated in vitro with Gyp migrated and invaded less than cells treated with phosphate-buffered saline (PBS) as a control. Gyp inhibited migration and invasion by down-regulating the production of RAS, NFkappaB, COX2, ERK1/2 and MMP-9 relative to PBS only. These results show that Gyp inhibits invasion and migration of human tongue SCC4 cells by down-regulating proteins associated with these processes, resulting in reduced metastasis.