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.

PW06 suppresses cancer cell metastasis in human pancreatic carcinoma MIA PaCa-2 cells via the inhibitions of p-Akt/mTOR/NF-κB and MMP2/MMP9 signaling pathways in vitro.

PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one], a kind of the carbazole derivative containing chalcone moiety, induced cell apoptosis in human pancreatic carcinoma in vitro. There is no investigation to show that PW06 inhibits cancer cell metastasis in human pancreatic carcinoma in vitro. Herein, PW06 (0.1-0.8 µM) significantly exists in the antimetastatic activities of human pancreatic carcinoma MIA PaCa-2 cells in vitro. Wound healing assay shows PW06 at 0.2 µM suppressed cell mobility by 7.45 and 16.55% at 6 and 24 hours of treatments. PW06 at 0.1 and 0.2 µM reduced cell mobility by 14.72 and 21.8% for 48 hours of treatment. Transwell chamber assay indicated PW06 (0.1-0.2 µM) suppressed the cell migration (decreased 26.67-35.42%) and invasion (decreased 48.51-68.66%). Atomic force microscopy assay shows PW06 (0.2 µM) significantly changed the shape of cell morphology. The gelatin zymography assay indicates PW06 decreased MMP2's and MMP9's activities at 48 hours of treatment. Western blotting assay further confirms PW06 reduced levels of MMP2 and MMP9 and increased protein expressions of EGFR, SOS1, and Ras. PW06 also increased the p-JNK, p-ERK, and p-p38. PW06 increased the expression of PI3K, PTEN, Akt, GSK3α/ß, and E-cadherin. Nevertheless, results also show PW06 decreased p-Akt, mTOR, NF-κB, p-GSK3ß, ß-catenin, Snail, N-cadherin, and vimentin in MIA PaCa-2 cells. The confocal laser microscopy examination shows PW06 increased E-cadherin but decreased vimentin in MIA PaCa-2 cells. Together, our findings strongly suggest that PW06 inhibited the p-Akt/mTOR/NF-κB/MMPs pathways, increased E-cadherin, and decreased N-cadherin/vimentin, suppressing the migration and invasion in MIA PaCa-2 cells in vitro.

Allyl isothiocyanate induces DNA damage and inhibits DNA repair-associated proteins in a human gastric cancer cells in vitro.

Allyl isothiocyanate (AITC) is abundant in cruciferous vegetables and it present pharmacological activity including anticancer activity in many types of human cancer cells in vitro and in vivo. Currently, no available information to show AITC affecting DNA damage and repair-associated protein expression in human gastric cancer cells. Therefore, in the present studies, we investigated AITC-induced cytotoxic effects on human gastric cancer in AGS and SNU-1 cells whether or not via the induction of DNA damage and affected DNA damage and repair associated poteins expressions in vitro. Cell viability and morphological changes were assayed by flow cytometer and phase contrast microscopy, respectively, the results indicated AITC induced cell morphological changes and decreased total viable cells in AGS and SNU-1 cells in a dose-dependently. AITC induced DNA condensation and damage in a dose-dependently which based on the cell nuclei was stained by 4', 6-diamidino-2-phenylindole present in AGS and SNU-1 cells. DNA damage and repair associated proteins expression in AGS and SNU-1 cells were measured by Western blotting. The results indicated AITC decreased nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), glutathione, and catalase, but increased superoxide dismutase (SOD (Cu/Zn)), and nitric oxide synthase (iNOS) in AGS cells, however, in SNU-1 cells are increased HO-1. AITC increased DNA-dependent protein kinase (DNA-PK), phosphorylation of gamma H2A histone family member X on Ser139 (γH2AXpSer139 ), and heat shock protein 90 (HSP90) in AGS cells. AITC increased DNA-PK, mediator of DNA damage checkpoint protein 1 (MDC1), γH2AXpSer139 , topoisomerase II alpha (TOPIIα), topoisomerase II beta (TOPIIß), HSP90, and heat shock protein 70 (HSP70) in SNU-1 cells. AITC increased p53, p53pSer15 , and p21 but decreased murine double minute 2 (MDM2)pSer166 and O6 -methylguanine-DNA methyltransferase (MGMT) in AGS cells; however, it has a similar effect of AITC except increased ataxia telangiectasia and Rad3 -related protein (ATR)pSer428 , checkpoint kinase 1 (CHK1), and checkpoint kinase 2 (CHK2) in SNU-1 cells. Apparently, both cell responses to AITC are different, nonetheless, all of these observations suggest that AITC inhibits the growth of gastric cancer cells may through induction off DNA damage in vitro.

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.

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.

Gefitinib and curcumin-loaded nanoparticles enhance cell apoptosis in human oral cancer SAS cells in vitro and inhibit SAS cell xenografted tumor in vivo.

Curcumin (Cur), a natural product, has been shown to have anti-tumor activities in many human cancer cells. Gefitinib (Gef) is a clinical drug for cancer patients. However, there is no available information to show whether Gef/Cur nanoparticles (NPs) increased cell apoptosis and anti-tumor effects on xenograft mice model in vivo. In this study, γ-polyglutamic acid-coated nanoparticles loaded with Gef and Cur (γ-PGA-Gef/Cur NPs) were developed and its physicochemical properties and antitumor effects were investigated in vitro and in vivo. The γ-PGA-Gef/Cur NPs showed 548.5 ±â€¯93.7 nm in diameter and -40.3 ±â€¯3.87 mV on surface charge. The loading efficiencies of Gef and Cur were 89.5 and 100%, respectively. γ-PGA-Gef/Cur NPs could be internalized into SAS cells and significantly decreased total cell viability of SAS cells. Western blotting results indicated that both free Gef/Cur and γ-PGA-Gef/Cur NPs induced apoptotic cell death via caspase- and mitochondria-dependent pathways. In vivo studies indicated that treatments of PLGA NPs, free Gef/Cur, and γ-PGA-Gef/Cur NPs did not significantly affect appearances and bodyweights of mice. But the γ-PGA-Gef/Cur NPs significantly suppressed tumor size when comparing to free Gef/Cur-treated group. The nanoparticles developed in this study may be used as a potential therapy for oral cancer.

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.

Berberine Inhibits Human Melanoma A375.S2 Cell Migration and Invasion via Affecting the FAK, uPA, and NF-κB Signaling Pathways and Inhibits PLX4032 Resistant A375.S2 Cell Migration In Vitro.

Many studies have demonstrated that berberine inhibited the cell migration and invasion in human cancer cell lines. However, the exact molecular mechanism of berberine inhibiting the cell migration and invasion of human melanoma A375.S2 and A375.S2/PLX (PLX4032 induced resistant A375.S2) skin cancer cells remains unknown. In this study, we investigated the anti-metastasis mechanisms of berberine in human melanoma cancer A375.S2 cells and A375.S2/PLX resistant cells in vitro. Berberine at low concentrations (0, 1, 1.5 and 2 µM) induced cell morphological changes and reduced the viable cell number and inhibited the mobility, migration, and invasion of A375.S2 cells that were assayed by wound healing and transwell filter. The gelatin zymography assay showed that berberine slightly inhibited MMP-9 activity in A375.S2 cells. Results from western blotting indicated that berberine inhibited the expression of MMP-1, MMP-13, E-cadherin, N-cadherin, RhoA, ROCK1, SOS-1, GRB2, Ras, p-ERK1/2, p-c-Jun, p-FAK, p-AKT, NF-κB, and uPA after 24 h of treatment, but increased the PKC and PI3K in A375.S2 cells. PLX4032 is an inhibitor of the BRAFV600E mutation and used for the treatment of cancer cells harboring activated BRAF mutations. Berberine decrease cell number and inhibited the cell mobility in the resistant A375.S2 (A375.S2/PLX, PLX4032 generated resistant A375.S2 cells). Based on these observations, we suggest that the potential of berberine as an anti-metastatic agent in melanoma that deserves to be investigated in more detail, including in vivo studies in future.

Phenethyl Isothiocyanate Inhibits In Vivo Growth of Xenograft Tumors of Human Glioblastoma Cells.

Phenethyl isothiocyanate (PEITC) from cruciferous vegetables can inhibit the growth of various human cancer cells. In previous studies, we determined that PEITC inhibited the in vitro growth of human glioblastoma GBM 8401 cells by inducing apoptosis, inhibiting migration and invasion, and altering gene expression. Nevertheless, there are no further in vivo reports disclosing whether PEITC can suppress the growth of glioblastoma. Therefore, in this study we investigate the anti-tumor effects of PEITC in a xenograft model of glioblastoma in nude mice. Thirty nude mice were inoculated subcutaneously with GBM 8401 cells. Mice with one palpable tumor were divided randomly into three groups: control, PEITC-10, and PEITC-20 groups treated with 0.1% dimethyl sulfoxide (DMSO), and 10 and 20 µmole PEITC/100 µL PBS daily by oral gavage, respectively. PEITC significantly decreased tumor weights and volumes of GBM 8401 cells in mice, but did not affect the total body weights of mice. PEITC diminished the levels of anti-apoptotic proteins MCL-1 (myeloid cell leukemia 1) and XIAP (X-linked inhibitor of apoptosis protein) in GBM 8401 cells. PEITC enhanced the levels of caspase-3 and Bax in GBM 8401 cells. The growth of glioblastoma can be suppressed by the biological properties of PEITC in vivo. These effects might support further investigations into the potential use of PEITC as an anticancer drug for glioblastoma.

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.

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.

Suppression of the TNF-alpha level is mediated by Gan-Lu-Yin (traditional Chinese medicine) in human oral cancer cells through the NF-kappa B, AKT, and ERK-dependent pathways.

Oral cancer is one of the major causes of deaths in the male population of Taiwan. Gan-Lu-Yin (GLY) is used for an adjuvant treatment of Traditional Chinese Medicine in clinical patients. In this study, we investigated the molecular mechanisms in oral cancer cell lines after exposure to GLY. The cytometric bead-based array (CBA) method was used for the examining and analyzing of tumor necrosis factor-alpha (TNF-α) secretion level. TNF-α mRNA expression was determined by real-time PCR analysis. Nuclear factor kappa B (NF-κB) activity and other relative proteins were determined by NF-κB promoter assay, Western blotting, electrophoretic mobility shift assay (EMSA), and immuno-staining analyses. GLY decreased the secretion of TNF-α from the oral cancer CAL 27 cells. Furthermore, 2000 µg/mL of GLY significantly suppressed TNF-α mRNA expression of CAL 27 cells in a time-dependent manner. GLY reduced the levels of proteins, including nuclear NF-κB (p65 and p50), p-IKK (ser176), p-IκB, p-AKT, p-ERK, and nuclear Egr-1 in a time and dose-dependent manner. GLY also suppressed the NF-κB activity and translocation in CAL 27 cells. We suggest that GLY might promote the cure of oral cancer through decreasing the level of TNF-α cytokine, and these actions were mediated partially through the NF-κB, AKT, and ERK-dependent pathways in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1196-1205, 2016.

Bisdemethoxycurcumin induces DNA damage and inhibits DNA repair associated protein expressions in NCI-H460 human lung cancer cells.

Nonsmall cell lung carcinoma (NSCLC) is a devastating primary lung tumor resistant to conventional therapies. Bisdemethoxycurcumin (BDMC) is one of curcumin derivate from Turmeric and has been shown to induce NSCLC cell death. Although there is one report to show BDMC induced DNA double strand breaks, however, no available information to show BDMC induced DNA damage action with inhibited DNA repair protein in lung cancer cells in detail. In this study, we tested BDMC-induced DNA damage and condensation in NCI-H460 cells by using Comet assay and DAPI staining examinations, respectively and we found BDMC induced DNA damage and condension. Western blotting was used to examine the effects of BDMC on protein expression associated with DNA damage and repair and results indicated that BDMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DDR), O6-methylguanine-DNA methyltransferase, DNA repair proteins breast cancer 1, early onset, mediator of DNA damage checkpoint 1 but activate phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Confocal laser systems microscopy was used for examining the protein translocation and results show that BDMC increased the translocation of p-p53 and p-H2A.X (phospho Ser140) from cytosol to nuclei in NCI-H460 cells. In conclusion, BDMC induced DNA damage and condension and affect DNA repair proteins in NCI-H460 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1859-1868, 2016.

Diallyl trisulfide inhibits migration, invasion and angiogenesis of human colon cancer HT-29 cells and umbilical vein endothelial cells, and suppresses murine xenograft tumour growth.

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. We examined the cytotoxic, anti-metastatic, anti-cancer and anti-angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases-2, -7 and -9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary-like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex-vivo angiogenesis. We investigated the anti-tumour effects of DATS against human colon cancer xenografts in BALB/c(nu/nu) mice and its anti-angiogenic activity in vivo. In this in-vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.

Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti-metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9), and cytosolic NF-kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor-AKT (Thr308), Cyclin D, iNOS, COX2, NF-kB p65, p-ERK1/2, p-JNK1/2, p-p38, p-c-jun, MMP-2, MMP-9, MMP-1, MMP-7, MMP-10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti-metastasis agent for the treatment of human colon cancer cells.

Crude extract of Rheum palmatum L induced cell death in LS1034 human colon cancer cells acts through the caspase-dependent and -independent pathways.

Crude extract of Rheum palmatum L (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub-G1 phase (apoptosis), the activities of caspase-8, -9, and -3, reactive oxygen species (ROS) and Ca(2+) levels, and mitochondrial membrane potential (ΔΨm). DNA damage, nuclei condensation, protein expression, and translocation were examined by Comet assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, Western blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration- and time-dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl-2 protein ratio and CERP promoted the activities of caspase-8, -9, and -3. Both ROS and Ca(2+) levels were increased by CERP but the compound decreased levels of ΔΨm in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G, cytochrome c, and GADD153 to induce apoptosis through mitochondrial-dependent pathway.

Diallyl sulfide, diallyl disulfide, and diallyl trisulfide inhibit migration and invasion in human colon cancer colo 205 cells through the inhibition of matrix metalloproteinase-2, -7, and -9 expressions.

Diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are major organosulfur compounds exiting in garlic (Allium sativum). These compounds are reported to exhibit various pharmacological properties such as antibacteria, antiangiogenesis, anticancer, and anticoagulation, and they also induce cytotoxicity and induction of apoptosis in human cancer cells. Although these compounds show wide spectrum of biological activities, there are no reports to show that DAS, DADS, and DATS affected migration and invasion of human colon cancer cells, and their exact molecular mechanisms are not well investigated. Therefore, the purpose of this study was to determine whether DAS, DADS, and DATS affected the invasion and migration abilities of colo 205 human colon cancer cells. The results indicate that DAS, DADS, and DATS at 10 and 25 µM inhibited the migration and invasion of colo 205 cells in the order of DATS < DADS < DAS. DATS is the highest for inhibition of migration and invasion of colo 205 cells. DAS, DADS, and DATS induce downregulation expression of PI3K, Ras, MEKK3, MKK7, ERK1/2, JNK1/2, and p38 and then lead to the inhibition of MMP-2, -7, and -9. DAS, DADS, and DATS inhibited NF-κB and COX-2 for leading to the inhibition of cell proliferation. Taken together, these results demonstrated that application of DAS, DADS, and DATS might serve as potential antimetastatic drugs.

PW06 Triggered Fas-FADD to Induce Apoptotic Cell Death In Human Pancreatic Carcinoma MIA PaCa-2 Cells through the Activation of the Caspase-Mediated Pathway.

Pancreatic cancer has higher incidence and mortality rates worldwide. PW06 [(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2,5-dimethoxyphenyl) prop-2-en-1-one] is a carbazole derivative containing chalcone moiety which was designed for inhibiting tumorigenesis in human pancreatic cancer. This study is aimed at investigating PW06-induced anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro. The results showed PW06 potent antiproliferative/cytotoxic activities and induced cell morphological changes in a human pancreatic cancer cell line (MIA PaCa-2), and these effects are concentration-dependent (IC50 is 0.43 µM). Annexin V and DAPI staining assays indicated that PW06 induced apoptotic cell death and DNA condensation. Western blotting indicated that PW06 increased the proapoptotic proteins such as Bak and Bad but decreased the antiapoptotic protein such as Bcl-2 and Bcl-xL. Moreover, PW06 increased the active form of caspase-8, caspase-9, and caspase-3, PARP, releasing cytochrome c, AIF, and Endo G from mitochondria in MIA PaCa-2 cells. Confocal laser microscopy assay also confirmed that PW06 increased Bak and decreased Bcl-xL. Also, the cells were pretreated with inhibitors of caspase-3, caspase-8, and caspase-9 and then were treated with PW06, resulting in increased viable cell number compared to PW06 treated only. Furthermore, PW06 showed a potent binding ability with hydrophobic interactions in the core site of the Fas-Fas death domains (FADD). In conclusion, PW06 can potent binding ability to the Fas-FADD which led to antiproliferative, cytotoxic activities, and apoptosis induction accompanied by the caspase-dependent and mitochondria-dependent pathways in human pancreatic cancer MIA PaCa-2 cells.

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model.

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9-18 fold) of induction in the microarray data, and its early induction was observed in a 2h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.

Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo.

Numerous studies have shown that rutin has anticancer effects. We have previously reported that rutin induced cell cycle arrest and apoptosis in murine leukemia WEHI-3 cells in vitro and in vivo. However, there are no data showing that rutin inhibits human leukemia HL-60 cells in vivo in a murine xenograft animal model. Human leukemia HL-60 cells were implanted into mice and treated with vehicle (1% DMSO), rutin (120 mg/kg of body weight) or vinblastine (120 µg/kg of body weight). Compounds and agents were injected once every four days intraperitoneally (i.p.) for 36 days. Treatment with 120 mg/kg of rutin or with 120 µg/kg of vinblastine resulted in a reduction of tumor weight and volume when compared with the control groups. Tumor size in xenograft mice treated with 120 mg/kg of rutin was significantly smaller than that in the untreated-control group. These novel findings indicate that rutin inhibits tumor growth in a xenograft animal model. Rutin may be useful in treating leukemia but certainly much more research is needed. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.