Phenethyl isothiocyanate and irinotecan synergistically induce cell apoptosis in colon cancer HCT 116 cells in vitro.

Irinotecan (IRI), an anticancer drug to treat colon cancer patients, causes cytotoxic effects on normal cells. Phenethyl isothiocyanate (PEITC), rich in common cruciferous plants, has anticancer activities (induction of cell apoptosis) in many human cancer cells, including colon cancer cells. However, the anticancer effects of IRI combined with PEITC on human colon cancer cells in vitro were unavailable. Herein, the aim of this study is to focus on the apoptotic effects of the combination of IRI and PEITC on human colon cancer HCT 116 cells in vitro. Propidium iodide (PI) exclusion and Annexin V/PI staining assays showed that IRI combined with PEITC decreased viable cell number and induced higher cell apoptosis than that of IRI or PEITC only in HCT 116 cells. Moreover, combined treatment induced higher levels of reactive oxygen species (ROS) and Ca2+ than that of IRI or PEITC only. Cells pre-treated with N-acetyl-l-cysteine (scavenger of ROS) and then treated with IRI, PEITC, or IRI combined with PEITC showed increased viable cell numbers than that of IRI or PEITC only. IRI combined with PEITC increased higher caspase-3, -8, and -9 activities than that of IRI or PEITC only by flow cytometer assay. IRI combined with PEITC induced higher levels of ER stress-, mitochondria-, and caspase-associated proteins than that of IRI or PEITC treatment only in HCT 116 cells. Based on these observations, PEITC potentiates IRI anticancer activity by promoting cell apoptosis in the human colon HCT 116 cells. Thus, PEITC may be a potential enhancer for IRI in humans as an anticolon cancer drug in the future.

Demethoxycurcumin Suppresses Human Brain Glioblastoma Multiforme GBM 8401 Cell Xenograft Tumor in Nude Mice In Vivo.

Demethoxycurcumin (DMC), a derivate of curcumin, has been shown to induce apoptotic cell death in human glioblastoma multiforme GBM 8401 cells via cell cycle arrest and induction of cell apoptosis. However, there is no report showing DMC suppresses glioblastoma multiforme cells in vivo. In the present study, we investigated the effects of DMC on GBM8401 cells in vivo. At first, we established a luciferase-expressing stable clone named GBM 8401/luc2. Second, mice were inoculated subcutaneously with GBM 8401/luc2 cells to generate a xenograft tumor mice model. After inoculation, tumor volume reached 100-120 mm3, and all mice were randomly divided into three groups: Group I was treated with 110 µL phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 30 mg/kg of DMC, and Group III with 60 mg/kg of DMC. Mice from each group were given the oral treatment of DMC by gavage for 21 days. The body weight and tumor volume were recorded every 3 days. DMC significantly decreased the tumor volumes, and 60 mg/kg treatment showed a higher decrease in tumor volumes than that of 30 mg/kg, However, DMC did not affect the body weights. The photons emitted from mice tumors were detected with Xenogen IVIS imaging system, DMC at both doses decreased the total photon flux and 60 mg/kg treatment of DMC has low total photon flux than that of 30 mg/kg. The tumor volumes and weights in 60 mg/kg treatment of DMC were lower than that of 30 mg/kg. Immunohistochemical analysis was used to measure protein expression of tumors and results showed that DMC treatment led to lightly staining with anti-Bcl-2 and -XIAP and 60 mg/kg treatment of DMC has lighter staining with anti-Bcl-2 and -XIAP than that of 30 mg/kg. The higher dose (60 mg/kg) of DMC has higher signals of cleaved-caspase-3 than that of the lower dose (30 mg/kg). Furthermore, the hematoxylin and eosin (H&E) staining of liver tissues showed no significant difference between DMC-treated and control-groups. Overall, these observations showed that DMC suppressed tumor properties in vivo and DMC may be used against human glioblastoma multiforme in the future.

Tetrandrine Suppresses Human Brain Glioblastoma GBM 8401/luc2 Cell-Xenografted Subcutaneous Tumors in Nude Mice In Vivo.

Tetrandrine (TET), a bisbenzylisoquinoline (BBI) alkaloid, is isolated from the plant Stephania tetrandra S. Moore and has a wide range of biological activity, including anticancer properties in vitro and in vivo. At first, we established a luciferase-expressing stable clone that was named GBM 8401/luc2 cells. Herein, the primary results indicated that TET reduced the total cell viability and induced cell apoptosis in GBM 8401/luc2 human glioblastoma cells. However, there is no available information showing that TET suppresses glioblastoma cells in vivo. Thus, we investigated the effects and mechanisms of TET on a GBM 8401/luc2 cell-generated tumor in vivo. After the tumor volume reached 100-120 mm3 in subcutaneously xenografted nude mice, all of the mice were randomly divided into three groups: Group I was treated with phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 25 mg/kg of TET, and Group III with 50 mg/kg of TET. All mice were given the oral treatment of PBS or TET by gavage for 21 days, and the body weight and tumor volumes were recorded every 5 days. After treatment, individual tumors, kidneys, livers, and spleens were isolated from each group. The results showed that TET did not affect the body weights, but it significantly decreased the tumor volumes. The TET treatment at 50 mg/kg had a two-fold decrease in tumor volumes than that at 25 mg/kg when compared to the control. TET decreased the total photon flux, and treatment with TET at 50 mg/kg had a lower total photon flux than that at 25 mg/kg, as measured by a Xenogen IVIS imaging system. Moreover, the higher TET treatment had lower tumor volumes and weights than those of the lower dose. The apoptosis-associated protein expression in the tumor section was examined by immunohistochemical analysis, and the results showed that TET treatment reduced the levels of c-FLIP, MCL-1, and XIAP but increased the signals of cleaved-caspase-3, -8, and -9. Furthermore, the hematoxylin and eosin (H & E) staining of kidney, liver, and spleen tissues showed no significant difference between the TET-treated and control groups. Overall, these observations demonstrated that TET suppressed subcutaneous tumor growth in a nude-mice model via the induction of cell apoptosis.

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL-60 cancer cell xenograft growth in vivo.

Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti-cancer activates. In the present, we investigated the anti-leukemia activity of genistein on HL-60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca2+ production and the level of ΔΨm were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein-induced cell morphological changes, decreased the total viable cells, induced G2 /M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL-60 cells. Genistein promoted ROS and Ca2+ productions and decreased the level of ΔΨm in HL-60 cells. Western blotting assay demonstrated that genistein increased ER stress-associated protein expression such as IRE-1α, Calpain 1, GRP78, GADD153, caspase-7, caspase-4, and ATF-6α at 20-50 µM treatment and increased apoptosis associated protein expression such as pro-apoptotic protein Bax, PARP-cleavage, caspase-9, and -3, but decreased anti-apoptotic protein such as Bcl-2 and Bid in HL-60 cells. Calpain 1, GRP78, and GADD153 were increased in HL-60 cells after exposure to 40 µM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein-treated group. Genistein also increased the expressions of ATF-6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G2 /M phase arrest and the induction of cell apoptosis through ER stress- and mitochondria-dependent pathways in HL-60 cells and suppressed tumor properties 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.

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6ß, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

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.

Bisdemethoxycurcumin-induced S phase arrest through the inhibition of cyclin A and E and induction of apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathways in human lung cancer NCI H460 cells.

Curcuminoids are the major natural phenolic compounds found in the rhizome of many Curcuma species. Curcuminoids consist of a mixture of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although numerous studies have shown that curcumin induced cell apoptosis in many human cancer cells, however, mechanisms of BDMC-inhibited cell growth and -induced apoptosis in human lung cancer cells still remain unclear. Herein, we investigated the effect of BDMC on the cell death via the cell cycle arrest and induction of apoptosis in NCI H460 human lung cancer cells. Flow cytometry assay was used to measure viable cells, cell cycle distribution, the productions of reactive oxygen species (ROS) and Ca2+ , mitochondrial membrane potential (ΔΨm ) and caspase-3, -8 and -9 activity. DNA damage and condension were assayed by Comet assay and DAPI staining, respectively. Western blotting was used to measure the changes of cell cycle and apoptosis associated protein expressions. Results indicated that BDMC significantly induced cell death through induced S phase arrest and induced apoptosis. Moreover, DMC induced DNA damage and condension, increased ROS and Ca2+ productions and decreased the levels of ΔΨm and promoted activities caspase-3, -8, and -9. Western blotting results showed that BDMC inhibited Cdc25A, cyclin A and E for causing S phase arrest, furthermore, promoted the expression of AIF, Endo G and PARP and the levels of Fas ligand (Fas L) and Fas were also up-regulated. Results also indicated that BDMC increased ER stress associated protein expression such as GRP78, GADD153, IRE1α, IRE1ß, ATF-6α, ATF-6ß, and caspase-4. Taken together, we suggest that BDMC induced cell apoptosis through multiple signal pathways such as extrinsic, intrinsic and ES tress pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1899-1908, 2016.

Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1.

Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future.

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.

Different cellular responses of dexmedetomidine at infected site and peripheral blood of emdotoxemic BALB/c mice.

Various sedative agents, including dexmedetomidine (dex), induce immunosuppression, and enhance infection progression. However, there was no information on how anesthetic affects local and systemic cellular immune function. We conducted this study to examine the impact of dex on the differentiation and function of immune cells at site of inflammation and in peripheral blood during endotoxemia of mice. In BALB/c mice with and without endotoxemia, we evaluated the influence of two dosages of 5 and 50 mcg/kg/h intravenous dex on immune cells: including number of T cells (CD3), B cells (CD19), natural killer cells (CD8a), monocytes (CD11b), and macrophages (Mac-3) in peripheral blood, the activities of macrophages in peripheral blood and in peritoneal lavage, and proliferation of B and T cells and of natural killer cells activity in the spleen. Endotoxemia increased the number of CD3 T cells, CD 19 B cells and macrophages in the peripheral blood, augmented macrophage activity in the peritoneum, and increased T cell proliferation and natural killer cell activity in the spleen. Further administration of 5 mcg/kg/h dex attenuated systemic increase in number of T cells, B cells, and macrophages during endotoxemia and 50 mcg/kg/h dex significantly attenuated the increase in activity of macrophages in the peripheral blood during endotoxemia. In the peritoneum, however, 5 mcg/kg/h dex preserved and 50 mcg/kg/h dexmedetomidine enhanced the activity of macrophages during endotoxemia. Increased in proliferation of T cells in spleen during endotoxemia was attenuated by both doses of dex. Last, 50 mcg/kg/h dex enhanced natural killer cells activity during endotoxemia. While preserving the effects of endotoxemia on macrophage's activity in the infection site and natural killer cell's activity in the spleen, dex decreased systemic fulminant immune reaction in endotoxemia, by attenuating the augmented response in the number of T cells, B cells and macrophages, activity of macrophages in the peripheral blood, and proliferation of T cells in spleen during endotoxemia.

The crude extract of Corni Fructus inhibits the migration and invasion of U-2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9, and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells.

Bufalin-inhibited migration and invasion in human osteosarcoma U-2 OS cells is carried out by suppression of the matrix metalloproteinase-2, ERK, and JNK signaling pathways.

Bufalin has been shown to exhibit multiple pharmacological activities, including induction of apoptosis in many types of cancer cell lines. Osteosarcoma is a type of cancer which is difficult to treat and the purpose of this study was to investigate the effects of bufalin on the migration and invasion of human osteosarcoma U-2 OS cells. The wound healing assay and Boyden chamber transwell assay were used for examining the migration of U-2 OS cells. Western blotting and gelatin zymography assays were used for theexpression and activities of metalloproteinase (MMP)-2, MMP-7 or MMP-9 levels. Western blotting analysis also was used for measuring the levels of growth factor receptor-bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), c-Jun N-terminal kinases 1/2 (JNK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 in bufalin-treated U-2 OS cells. Bufalin inhibited the cell migration and invasion of U-2 OS cells in vitro. Moreover, bufalin reduced MMP-2 and MMP-9 enzyme activities of U-2 OS cells. Bufalin also suppressed the protein level of MMP-2 and reduced the levels of mitogen-activated protein kinases (MAPKs) such as JNK1/2 and ERK1/2 signals in U-2 OS cells. Our results suggest that signaling pathways for bufalin-inhibited migration and invasion of U-2 OS cells might be mediated through blocking MAPK signaling and resulting in the inhibition of MMP-2. Bufalin could be a useful agent to develop as a novel antitumor agent by virtue of its ability to inhibit tumor cell migration and invasion.

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species-modulated pathways in U-2 OS human osteosarcoma cells.

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U-2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell-cycle distribution, apoptotic cells in sub-G1 phase, reactive oxygen species (ROS), Ca(2+) levels, and mitochondrial membrane potential (ΔΨm ). Comet assay and 4'-6-diamidino-2-phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis-associated protein levels in U-2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G0/G1 arrest, and apoptosis in U-2 OS cells. CECF-stimulated activities of caspase-8, caspase-9, and caspase-3, ROS, and Ca(2+) production, decreased ΔΨm levels of in U-2 OS cells. CECF increased protein levels of caspase-3, caspase-9, Bax, cytochrome c, GRP78, AIF, ATF-6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell-cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U-2 OS cells via ROS-modulated caspase-dependent and -independent pathways.

Combination Treatment of Sorafenib and Bufalin Induces Apoptosis in NCI-H292 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Lung cancer notably contributes to tumor-associated mortality worldwide, and standard chemotherapy is used for lung cancer patients. However, its therapeutic efficacy remains unsatisfactory. This study aimed to evaluate the effects and molecular mechanisms of sorafenib and bufalin combination therapy on lung cancer cells in vitro. MATERIALS AND METHODS: NCI-H292 cells were treated with sorafenib, bufalin, and sorafenib in combination with bufalin. Cell viability, ROS production, Ca2+ release, and mitochondrial membrane potential were examined by flow cytometric assay. Annexin V/PI staining and chromatin condensation were examined by the apoptosis assays. Finally the molecular mechanism of apoptosis-associated protein expression was investigated by western blotting. RESULTS: NCI-H292 cells treated with sorafenib in combination with bufalin showed significantly decreased viability, enhanced cellular apoptosis, and DNA condensation when compared to that with sorafenib or bufalin alone. Moreover, the combination treatment exhibited higher reactive oxygen species (ROS) production and lower mitochondrial membrane potential (ΔΨm). The combined treatment resulted in higher expression of SOD but lower catalase compared to sorafenib treatment alone. Compared to sorafenib or bufalin treatment alone, the combination treatment resulted in lower Bcl-2 expression but higher Bax, Bad, APAF-1, caspase-3, and caspase-9. CONCLUSION: Sorafenib in combination with bufalin shows more potent cytotoxic effects and cell apoptosis than sorafenib or bufalin treatment alone in NCI-H292 cells. The combined treatment significantly enhanced apoptotic cell death in NCI-H292 lung cancer cells by activating ROS-, mitochondria-, and caspase-signaling pathways in vitro.

Bisdemethoxycurcumin suppresses human osteosarcoma U­2 OS cell migration and invasion via affecting the PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß and MAPK signaling pathways in vitro.

The metastasis of human osteosarcoma (OS) shows a difficult­to­treat clinical scenario and results in decreased quality of life and diminished survival rates. Finding or developing novel treatments to improve the life quality of patients is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was obtained from the rhizome of turmeric (Curcuma longa) and exerts antitumor activities in numerous human cancer cell lines. At present, there is no study showing BDMC effects on OS cell migration and invasion. In the present study, the effects of BDMC on cell migration and invasion of OS U­2 OS cells were investigated in vitro. Cell viability and proliferation were measured by flow cytometric and MTT assays, respectively. Cell motility, MMP­2 and ­9 activity, and cell migration and invasion were assayed by scratch wound healing, gelatin zymography, and Transwell chamber assays, respectively. The protein expression levels were measured by western blotting. BDMC at 20 and 40 µM significantly reduced total cell viability, and BDMC at 5 and 10 µM significantly inhibited cell motility in U­2 OS cells. BDMC significantly suppressed the activities of MMP­2 and MMP­9 in U­2 OS cells. BDMC suppressed cell invasion and migration after 24 h treatment in U­2 OS cells, and these effects were in a dose­dependently manner. Results from western blotting indicated that BDMC significantly decreased the protein expression levels of PI3K/Akt/NF­κB, PI3K/Akt/GSK3ß, and MAPK pathway in U­2 OS cells. Furthermore, BDMC inhibited uPA, MMP­2, MMP­9, MMP­13, N­cadherin, VE­cadherin, and vimentin but increased E­cadherin in U­2 OS cells. Based on these observations, it was suggested that BDMC may be a potential candidate against migration and invasion of human OS cells in the future.

Allyl Isothiocyanate Induces DNA Damage and Impairs DNA Repair in Human Breast Cancer MCF-7 Cells.

BACKGROUND/AIM: Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. MATERIALS AND METHODS: In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. RESULTS: AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 µM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. CONCLUSION: AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.

Ouabain Suppresses Cell Migration and Invasion in Human Gastric Cancer AGS Cells Through the Inhibition of MMP Signaling Pathways.

BACKGROUND/AIM: Ouabain has been shown to induce human cancer cell death via apoptosis. Still, its anti-metastatic effect on cell migration and invasion of human gastric cancer cells has not been addressed. MATERIALS AND METHODS: Cell proliferation and viability were measured by the MTT assay and flow cytometry, respectively. Cell motitlity was analysed by wound healing assay. Cell migration and invasion were analysed by the transwell system. Protein expression was assayed by western blotting. RESULTS: Ouabain decreased AGS cell proliferation, cell viability, and motility. In addition, ouabain inhibited AGS cell migration and invasion. Furthermore, ouabain decreased matrix metalloproteinase-2 (MMP-2) activity at 48 h. Ouabain reduced the levels of proteins associated with PI3K/AKT and p38/MAPK pathways. In addition, ouabain decreased the expressions of N-cadherin, tissue inhibitor of metalloproteinases-1 (TIMP-1), urokinase-type plasminogen activator (c-uPA), and MMP-2 at 48 h. CONCLUSION: Ouabain suppresses cell metastasis through multiple signaling pathways in AGS cells.

Quercetin inhibited murine leukemia WEHI-3 cells in vivo and promoted immune response.

Enhanced flavonoid consumption is closely related with a reduced cancer incidence as shown in epidemiological studies. Quercetin (3,5,7,3',4'-pentahydroxylflavone) is one of the active components of flavonoids which exist in natural plants, particularly in onions and fruits. It was reported that quercetin induced apoptosis in human cancer cell lines, including human leukemia HL-60 cells, but there is no available information as to its effects on leukemia cells in vivo. The purpose of the present studies was to focus on the in vivo effects of quercetin on leukemia WEHI-3 cells. The effects of quercetin on WEHI-3 cells injected into BALB/c mice were examined. Quercetin decreased the percentage of Mac-3 and CD11b markers, suggesting that the differentiation of the precursors of macrophages and T cells was inhibited. There was no effect on CD3 levels but increased CD19 levels. Quercetin decreased the weight of the spleen and liver compared with the olive oil treated animals. Quercetin stimulated macrophage phagocytosis of cells isolated from peritoneum. Quercetin also promoted natural killer cell activity. Based on pathological examination, an effect of quercetin was observed in the spleen of mice previously injected with WEHI-3 cells. Apparently, quercetin affects WEHI-3 cells in vivo.

Tetrandrine inhibits cell migration and invasion in human nasopharyngeal carcinoma NPC-TW 039 cells through inhibiting MAPK and RhoA signaling pathways.

The objective of this study was to investigate the effects of tetrandrine (TET) on cell migration and invasion of nasopharyngeal carcinoma NPC-TW 039 cells in vitro. TET at 1-10 µM did not change cell morphology and also did not decrease the total cell viability and proliferation in NPC-TW 039 cells. It decreased the cell mobility based on decreased wound closure in NPC-TW 039 cells by wound healing assay. TET suppressed the cell migration and invasion using transwell system. TET reduced MMP-2 activities at 1-10 µM and these effects are in dose-dependently. After exposed to various treatments, TET decreased the levels of p-ERK, p-JNK, p-p38, RhoA, and NF-κB at 48 hr. Based on these findings, we may suggest TET-inhibited cell migration and invasion of NPC-TW 039 cells via the suppression of MAPK and RhoA signaling pathways for inhibiting the MMP-2 and -9 expression in vitro. PRACTICAL APPLICATIONS: Tetrandrine (TET), a bis-benzylisoquinoline alkaloid, is obtained from the dried root of Stephania tetrandra. TET has been shown to induce cancer cell apoptosis on human cancer cells but its anti-metastasis effect on cell migration and invasion of nasopharyngeal carcinoma cells has not been investigated. Our results showed that TET significantly repressed the cell mobility, migration, and invasion of NPC-TW 039 cells in vitro that involved in inhibiting RhoA, Ras accompanying with p38/MAPK signaling pathway. We conclude that TET may be the anticancer agents for nasopharyngeal carcinoma therapy in the future.