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.

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.

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.

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.

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.

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.

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.

Fistein Suppresses Human Osteosarcoma U-2 OS Cell Migration and Invasion via Affecting FAK, uPA and NF-ĸB Signaling Pathway In Vitro.

BACKGROUND/AIM: Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. MATERIALS AND METHODS: The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. RESULTS: Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 µM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3ß and E-cadherin in U-2 OS cells after 48 h of treatment. CONCLUSION: Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.

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 Promotes Immune Responses, Enhances Macrophage and NK Cell Activities, and Increases Survival Rates of Leukemia BALB/c Mice.

Casticin, derived from Fructus Viticis, has anticancer properties in many human cancer cells, however, there is no report to show that casticin promotes immune responses and affects the survival rate of leukemia mice in vivo. The aim of this study is to evaluate the effects of casticin on immune responses and the survival rate of WEHI-3 cells generated in leukemia mice in vivo. Animals were divided into six groups: normal control mice, leukemia control mice, mice treated with ATRA (all-trans retinoic acid), and casticin (0.1, 0.2, and 0.4 mg/kg) treated mice. All animals were treated for 14 days and then measured for body weights, total survival rate, cell markers, the weights of liver and spleen, phagocytosis of spleen cells, NK cell activities and cell proliferation. Results show that casticin did not affect animal appearances, however, it increased body weights and decreased the weights of liver at 0.2 mg/kg and 0.4 mg/kg treatment. Casticin also decreased spleen weight at 0.2 mg/kg and 0.4 mg/kg treatment, increased CD3 at 0.1, 0.2 and 0.4 mg/kg doses and increased CD19 at 0.2 mg/kg treatment but decreased CD11b and Mac-3 at 0.1, 0.2 and 0.4 mg/kg treatment. Casticin (0.1, 0.2 and 0.4 mg/kg) increased macrophage phagocytosis from PBMC (peripheral blood mononuclear cell) and peritoneal cavity. Furthermore, casticin increased NK cells' cytotoxic activity and promoted T cell proliferation at 0.1-0.4 mg/kg treatment with or without concanavalin A (Con A) stimulation, but only increased B cell proliferation at 0.1 mg/kg treatment. Based on these observations, casticin could be used as promoted immune responses in leukemia mice in vivo.

Maslinic Acid Enhances Immune Responses in Leukemic Mice Through Macrophage Phagocytosis and Natural Killer Cell Activities In Vivo.

BACKGROUND/AIM: Maslinic acid (MA), a pentacyclic triterpene extracted from wax-like coatings of olives, has been shown to reduce cancer cell number through induction of autophagy and apoptosis in many human cancer cells including human leukemia HL-60 cells. In the present study, we investigated whether or not MA affects immune responses in a leukemia mouse model. MATERIALS AND METHODS: WEHI-3 cells were intraperitonealIy (i.p.) injected into normal BALB/c mice to develop leukemia. Mice were then treated by i.p. injection with MA at different doses (0, 8, 16 and 32 mg/kg) for 2 weeks. After treatment, all animals were weighed and blood, liver and spleen tissues were weighed. Blood or spleen both were used for determination of cell markers or phagocytosis, natural killer (NK) cell activities and T- and B-cell proliferation, respectively, by using a flow cytometric assay. RESULTS: MA did not significantly affect body, liver, and spleen weights. However, MA increased markers of T-cells (at 16 mg/kg treatment) and monocytes (at 32 mg/kg treatment), but reduced B-cell markers (at 8 mg/kg treatment); MA did not significantly affect cell marker of macrophages. Furthermore, MA increased phagocytosis by macrophages from peripheral blood mononuclear cells and peritoneal cavity at 32 mg/kg treatment and increased NK cell activity at target cell:splenocyte ratio of 25:1 but did not affect B- and T-cell proliferation. CONCLUSION: MA increased immune responses by enhancing macrophage phagocytosis and NK cell activities in leukemic mice.

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.

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.

Bisdemethoxycurcumin Suppresses Migration and Invasion of Human Cervical Cancer HeLa Cells via Inhibition of NF-ĸB, MMP-2 and -9 Pathways.

BACKGROUND/AIM: Bisdemethoxycurcumin (BDMC) exhibits biological activities including anticancer and anti-metastasis in human cancer cell lines, but there is no available information to show whether BDMC suppresses cell migration and invasion of human cervical cancer cells. MATERIALS AND METHODS: Wound-healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of BDMC on HeLa cells in vitro. RESULTS: BDMC reduced the total viable cell number in a dose-dependent manner. The wound-healing assay show BDMC suppressed the movement of HeLa cells. Furthermore, the trans-well chamber assays showed that BDMC suppressed the cell migration and invasion. Gelatin zymograph assay showed that BDMC did not inhibit matrix metalloproteinase-2 (MMP-2) and -9 activities in vitro. However, western blotting assay showed that BDMC significantly reduced protein levels of growth factor receptor-bound protein 2 (GRB2), Ras homolog gene family, member A (Rho A), urokinase-type plasminogen activator (uPA), RAS, MMP-2, and N-cadherin but increased those of phosphor-extracellular-signal related kinase (p-ERK1/2), E-cadherin and nuclear factor-ĸB (NF-ĸB) in HeLa cells. Confocal laser microscopy assay was used to further confirm BDMC increased NF-ĸB when compared to controls. CONCLUSION: BDMC may have potential as a novel anti-metastasis agent for the treatment of human cervical cancer.

Tetrandrine suppresses adhesion, migration and invasion of human colon cancer SW620 cells via inhibition of nuclear factor-κB, matrix metalloproteinase-2 and matrix metalloproteinase-9 signaling pathways.

Tetrandrine (TET) exhibits biological activities, including anticancer activity. In Chinese medicine, TET has been used to treat hypertensive and arrhythmic conditions and has been demonstrated to induce cytotoxic effects on human cancer cell lines. However, to the best of the author's knowledge, no previous studies have revealed that TET affects cell metastasis in SW620 human colon cancer cells. The present study demonstrated that TET decreased the cell number and inhibited cell adhesion and mobility of SW620 cells. Furthermore, a wound healing assay was performed to demonstrate that TET suppressed cell movement, and Transwell chamber assays were used to reveal that TET suppressed the cell migration and invasion of SW620 cells. Western blotting demonstrated that TET significantly reduced protein expression levels of SOS Ras/Rac guanine nucleotide exchange factor 1, phosphatidylinositol 3-kinase, growth factor receptor bound protein 2, phosphorylated (p)-c Jun N-terminal kinase 1/2, p-p38, p38, 14-3-3, Rho A, ß-catenin, nuclear factor-κB p65, signal transducer and activator of transcription-1 and cyclooxygenase-2, in comparison with untreated SW620 cells. Overall, the results of the present study suggested that TET may be used as a novel anti-metastasis agent for the treatment of human colon cancer in the future.

Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro.

Benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) have been demonstrated to induce anticancer function in many human cancer cells and also inhibit cancer cell migration and invasion. However, there are no studies that show BITC and PEITC to inhibit cell migration and invasion in mouse melanoma B16F10 cells. In this study, we investigated anti-metastasis effects of BITC and PEITC in melanoma cancer cells in vitro. Under sub-lethal concentrations (from 1, 2.5 up to 5 µM), BITC and PEITC significantly inhibited cell mobility, migration and invasion nature of B16F10 cells. Gelatin zymography assay also showed that BITC and PEITC inhibited matrix metalloproteinase-2 (MMP-2) activity in B16F10 cells. PEITC reduced MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 but BITC increased those MAPK signaling associated proteins. BITC and PEITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2 but BITC increased FAK at 48 h. Furthermore, PEITC decreased the expression of MMP-2 and tissue inhibitors of matrix metalloproteinases (TIMP) but BITC increased them. PEITC inhibited NF-κB protein levels and DNA binding which was confirmed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that BITC and PEITC can be used in anti-metastasis of melanoma cells in the future.

Cantharidin alters the expression of genes associated with the NKG2D-associated immune response in TSGH-8301 human bladder carcinoma cells.

Cantharidin (CTD) is a natural toxin in beetles of the Mylabris genus (blister beetle), which has been revealed to induce cell death in various types of human cancer cells. However, to the best of our knowledge, no previous studies have investigated the effect of CTD on the expression of genes and their associated signaling pathways in human bladder carcinoma cells. In the present study, CTD-induced cell morphological changes and apoptosis were observed using phase-contrast microscopy and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively, in TSGH-8301 human bladder carcinoma cells. In addition, a complementary DNA microarray analysis demonstrated that CTD treatment led to a >2-fold upregulation of 269 genes. For example, the DNA damage-associated gene DNA-damage-inducible transcript 3 had a 4.75-fold upregulation. Furthermore, another 286 genes were >2-fold downregulated in response to CTD treatment. Matrix-remodeling associated 5, which is associated with cell migration and invasion, was downregulated 7.98-fold.

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.

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.