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ABSTRACT 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 (Δ Ψm) 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 Δ Ψm 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, IRE1β, ATF-6α, ATF-6β, 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. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. [ABSTRACT FROM AUTHOR]

Our earlier studies have demonstrated that gallic acid (GA) induced cytotoxic effects including induction of apoptosis and DNA damage and inhibited the cell migration and invasion in human cancer cells. However, GA-affected DNA damage and repair gene expressions in human prostate cancer cells are still unclear. In this study, we investigated whether or not GA induces DNA damage and inhibits DNA repair gene expression in a human prostate cancer cell line (PC-3). The results from flow cytometric assay indicated that GA decreased the percentage of viable PC-3 cells in a dose- and time-dependent manner. PC-3 cells after exposure to different doses (50, 100, and 200 μM) of GA and various periods of time (12, 24, and 48 h) led to a longer DNA migration smear (comet tail) occurred based on the single cell gel electrophoresis (comet assay). These observations indicated that GA-induced DNA damage in PC-3 cells, which also confirmed by 4,6-diamidino-2-phenylindole dihydrochloride staining and DNA agarose gel electrophoresis. Alternatively, results from real-time polymerase chain reaction assay also indicated that GA inhibited ataxia telangiectasia mutated, ataxia-telangiectasia and Rad3-related, O6-methylguanine-DNA methyltransferase, DNA-dependent serine/threonine protein kinase, and p53 mRNA expressions in PC-3 cells. Taken together, the present study showed that GA caused DNA damage and inhibited DNA repair genes as well as both effects may be the critical factors for GA-inhibited growth of PC-3 cells in vitro. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 579-587, 2013. [ABSTRACT FROM AUTHOR]

Curcumin, derived from the food flavoring spice turmeric ( Curcuma longa), has been shown to exhibit anticancer activities and induce apoptosis in many types of cancer cell lines. In our previous study, curcumin was able to inhibit murine myelomonocytic leukemia WEHI-3 cells in vivo. However, there is no report addressing the cytotoxic responses and the mechanisms underlying curcumin-induced apoptotic cell death in WEHI-3 cells. Therefore, we hypothesized that that curcumin affected WEHI-3 cells and triggered cell death through apoptotic signaling pathways. The effects of curcumin on WEHI-3 cells were investigated by using flow cytometric analysis, comet assay, confocal laser microscopy and Western blotting. In this study, we found that curcumin induced apoptosis in WEHI-3 cells in a dose-dependent (5-20 μM) manner. Interestingly, curcumin enhanced the level of the antiapoptotic protein Bcl-2 which might show that curcumin-induced apoptosis is done through the ER stress signaling pathways based on the increase of CIEBP homologous protein (CHOP), activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE1), and caspase-12 in WEHI-3 cells. Moreover, curcumin increased the reactive oxygen species (ROS) production and cytosolic Ca2+ release, and induced DNA damage, but decreased the level of mitochondrial membrane potential (ΔΨm) in WEHI-3 cells. In conclusion, curcumin-induced apoptosis occurs through the ROS-affected, mitochondria-mediated and ER stress-dependent pathways. The evaluation of curcumin as a potential therapeutic agent for treatment of leukemia seems warranted. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013. [ABSTRACT FROM AUTHOR]

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., (© 2015 Wiley Periodicals, Inc.)

Lung cancer is the most common cause of cancer-related mortality in the US as well as other regions of the world. Curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) are the major components of Curcuma longa L. It has been reported that curcumin inhibits the growth of various types of cancer cells in vitro and in vivo. However, the mechanisms involved in the inhibition of cell growth and induced apoptosis by DMC in human lung cancer cells remain unclear. In the present study, we investigated the effect of DMC on cell death via the induction of apoptosis in NCI-H460 human lung cancer cells. Flow cytometric assay was used to examine the total percentage of viable cells, the population of cells in the sub-G1 phase of the cell cycle, the level of reactive oxygen species (ROS), Ca²⁺ production, mitochondrial membrane potential (ΔΨm) and caspase activity. Western blotting was used to examine the changes in the expression of cell cycle- and apoptosis-associated proteins. Confocal microscopy was used to examine the translocation of apoptosis-associated proteins. The results indicated that DMC significantly induced cell morphological changes and decreased the percentage of viable NCI-H460 cells and DMC induced apoptosis based on the cell distribution in the sub-G1 phase. Moreover, DMC promoted ROS and Ca²⁺ production and decreased the level of ΔΨm and promoted the activities of caspase-3, -8 and -9. The Western blotting results showed that DMC promoted the expression of AIF, Endo G and PARP. The levels of Fas ligand (Fas L) and Fas were also upregulated. Furthermore, DMC promoted expression of ER stress-associated proteins such as GRP78, GADD153, IRE1β, ATF-6α, ATF-6β and caspase-4. Based on the findings, we suggest that DMC may be used as a novel anticancer agent for the treatment of lung cancer in the future.

Demethoxycurcumin (DMC) is a key component of Chinese medicine (Turmeric) and has been proven effective in killing various cancer cells. Its role in inducing cytotoxic effects in many cancer cells has been reported, but its role regarding DNA damage on lung cancer cells has not been studied in detail. In the present study, we demonstrated DMC-induced DNA damage and condensation in NCI-H460 cells by using the Comet assay and DAPI staining examinations, respectively. Western blotting indicated that DMC suppressed the protein levels associated with DNA damage and repair, such as 14-3-3σ (an important checkpoint keeper of DNA damage response), DNA repair proteins breast cancer 1, early onset (BRCA1), O6-methylguanine-DNA methyltransferase (MGMT), mediator of DNA damage checkpoint 1 (MDC1), and p53 (tumor suppressor protein). DMC activated phosphorylated p53 and p-H2A.X (phospho Ser140) in NCI-H460 cells. Furthermore, we used confocal laser systems microscopy to examine the protein translocation. The results showed that DMC promotes the translocation of p-p53 and p-H2A.X from the cytosol to the nuclei in NCI-H460 cells. Taken together, DMC induced DNA damage and affected DNA repair proteins in NCI-H460 cells in vitro., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Gallic acid (GA), a phenolic compound naturally present in plants, used as an antioxidant additive in food and in the pharmaceutical industry, may have cancer chemopreventive properties. In the present study, we investigated whether GA induced DNA damage and affected DNA repair-associated protein expression in human oral cancer SCC-4 cells. Flow cytometry assays were used to measure total viable cells and results indicated that GA decreased viable cells dose-dependently. The comet assay and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining were used to measure DNA damage, as well as condensation and it was shown that GA induced DNA damage (comet tail) and DNA condensation in a dose-dependent manner. DNA gel electrophoresis was used to examine DNA fragmentation and we found that GA induced DNA ladder (fragmentation). Using western blotting it was shown that GA inhibited the protein expressions of MDC1, O(6)-methylguanine-DNA methyltransferase (MGMT), p-H2A.X, p53, DNA-dependent serine/threonine protein kinase (DNA-PK) and 14-3-3 proteins sigma (14-3-3σ) but increased p-p53, phosphate-ataxia-telangiectasia (p-H2A.X) and ataxia telangiectasia mutated and Rad3-related (p-ATR), phosphate-ataxia telangiectasia mutated (p-ATM) and breast cancer susceptibility protein 1 (BRCA1) in a 24-h treatment. The protein translocation was examined by confocal laser microscopy and results indicated that GA increased the levels of p-H2A.X, MDC1 and p-p53 in SCC-4 cells. In conclusion, we found that GA-induced cell death may proceed through the induced DNA damage and suppressed DNA repair-associated protein expression in SCC-4 cells., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Cancer metastasis is the major cause of cancer patient death. Melanoma is a highly important metastasis in human cancer. Cantharidin (CTD), identified as an active component of natural mylabris (Mylabris phalerata Pallas), induces apoptosis in many human cancer cells. In the present study, we investigated the anti-metastasis effects of CTD in human melanoma cancer A375.S2 cells. Flow cytometry was used to measure CTD-induced cytotoxic effects in A375.S2 cells. Wound healing assay indicated that CTD suppressed the migration of A375.S2 cells in a dose-dependent manner. The Matrigel Transwell Assay was used for cell migration and invasion examination and the results showed that CTD inhibited both. Gelatin zymography was used to investigate the activities of MMP-2/9 and the results indicated that CTD inhibited the enzymatic activities of MMP-2/9 in A375.S2 cells. The protein expression of A375.S2 cells following incubation with CTD was examined by western blotting and the results showed that CTD decreased the expression of ERK1/2, PI3K, FAK, MMP-2, -9, COX-2, NF-κB p65, TIMP 1, TIMP 2, VEFG, uPA, Rho A, GRB2, ROCK-1 and Ras, but increased the expressions of p38, JNK, p-c-jun and PKC. Based on those observations, we suggest that CTD may be used as a novel anti-cancer metastasis agent of human melanoma cancer in the future., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Numerous studies have shown that phenethyl isothiocyanate (PEITC) induces apoptosis of different types of human cancer cell lines, however, there are no reports showing that PEITC inhibits tumor growth in a xenograft model of melanoma in nude mice. We investigated effects of PEITC on the growth of xenografted A375.S2 cell tumors in nude BALB/c mice. A375.S2 cancer cells were inoculated subcutaneously into the lower flanks of mice. Seven days post-inoculation, mice having one palpable tumor were randomly divided into three groups and injected intraperitoneally with PEITC (0, 20 and 40 mg/kg). PEITC reduced tumor weight but total body weight was unaffected. These in vivo results provide support for further investigations to determine the potential use of PEITC as an anticancer drug., (Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Wogonin, a natural and biologically-active flavonoid found in plants, has been reported to exhibit anticancer effects on several cancer cell types. However, there is no available information regarding the responses to wogonin in leukemia mouse models. At concentrations of 10-200 μM, wogonin reduced the percentage of viable WEHI-3 cells in a concentration-dependent manner. In an in vivo study, WEHI-3 cells were intraperitoneally injected into normal BALB/c mice for establishing leukemic BALB/c mice to determine the anti-leukemia activity of wogonin. Wogonin increased the survival rate and the body weight of leukemic mice when compared to vehicle (olive oil)-treated groups. Furthermore, the results also revealed that wogonin increased the percentage of cluster of differentiation-3 CD3 (T-cell marker) and CD19 (B-cell marker) but reduced that of Mac-3 (macrophages) and CD11b (monocytes) cell surface markers in treated mice as compared with the untreated leukemia group. Based on these observations, wogonin might exhibit anti-leukemia effects on murine WEHI-3 cell line-induced leukemia in vivo.

A number of experiments have demonstrated that benzyl-isothiocyanate (BITC) induces cytotoxic cell death through the induction of apoptosis in various human cancer cell lines. In the present study, we investigated the effects of BITC on the growth of A375.S2 cell xenograft tumors in nude BALB/c mice in vivo. The A375.S2 cancer cells were inoculated subcutaneously into the lower flanks of each nude mouse. After cancer cell inoculation, all animals were maintained in the animal room for seven days and all mice produced one palpable tumor. Animals were randomly divided into two groups, each mouse was individually given intraperitoneal injections of BITC (20 mg/kg) or not (control). Results from the in vivo experiments indicated that BITC did not significantly affect the body weight of nude BALB/c mice bearing xenograft A375.S2 cell tumors but did significantly decrease the tumor weight.

Numerous studies have demonstrated that triptolide induces cell cycle arrest and apoptosis in human cancer cell lines. However, triptolide-induced DNA damage and inhibition of DNA repair gene expression in human skin cancer cells has not previously been reported. We sought the effects of triptolide on DNA damage and associated gene expression in A375.S2 human malignant melanoma cells in vitro. Comet assay, DAPI staining and DNA gel electrophoresis were used for examining DNA damage and results indicated that triptolide induced a longer DNA migration smear based on single cell electrophoresis and DNA condensation and damage occurred based on the examination of DAPI straining and DNA gel electrophoresis. The real-time PCR technique was used to examine DNA damage and repair gene expression (mRNA) and results indicated that triptolide led to a decrease in the ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA-1), p53, DNA-dependent serine/threonine protein kinase (DNA-PK) and O6-methylguanine-DNA methyltransferase (MGMT) mRNA expression. Thus, these observations indicated that triptolide induced DNA damage and inhibited DNA damage and repair-associated gene expression (mRNA) that may be factors for triptolide-mediated inhibition of cell growth in vitro in A375.S2 cells.

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.

It is well known that the response of cancer cells to chemotherapeutic drugs involves the activation of apoptotic pathways. Benzyl isothiocyanate (BITC) is an important compound found in plant food and has been shown to have anti-cancer effects on human cancer cells, but its effect on prostate cancer cells in vitro remains unknown. The aim of the present study was to investigate the effects of BITC on DU 145 human prostate cancer cells in order to clarify whether a time/concentration range for optimal BITC-induced apoptosis exists and to find the associated signaling pathway. Cell morphological changes, percentage of cell viability, DNA damage and apoptosis in DU 145 cells were examined by phase-contrast microscopy, flow cytometric assay, 4',6-diamidine-20-phenylindole dihydrochloride staining, comet assay and Western blotting analysis. The results indicate that BITC induces cell morphological changes, decreases the percentage of viable cells (induction of cell cytotoxicity), and induces DNA damage and apoptosis in DU 145 cells in a time- and dose-dependent manner. Flow cytometric assays indicated that BITC promoted reactive oxygen species and Ca2+ productions and decreased the levels of mitochondrial membrane potential (ΤYm), while the pre-treatment with N-acetylcysteine caused an increase in the percentage of viable cells. BITC also promoted caspase-3, -8 and -9 activities. Furthermore, when cells were pre-treated with the caspase-3 inhibitor and then treated with BITC, this led to an increase in the percentage of viable cells. Confocal laser microscopy examination indicated that BITC promoted the expression of AIF and Endo G, which were released from the mitochondria in DU 145 cells. In conclusion, BITC induces apoptosis in DU 145 cells through the release of AIF and Endo G from the mitochondria and also promotes caspase-3 activation.