Bin Chuan Ji

Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC-3 cells

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.

Triptolide induced DNA damage in A375.S2 human malignant melanoma cells is mediated via reduction of DNA repair genes.

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.

Induction of apoptosis by curcumin in murine myelomonocytic leukemia WEHI‐3 cells is mediated via endoplasmic reticulum stress and mitochondria‐dependent pathways

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.

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

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

Demethoxycurcumin induces the apoptosis of human lung cancer NCI-H460 cells through the mitochondrial-dependent pathway

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.

Suppression of the migration and invasion is mediated by triptolide in B16F10 mouse melanoma cells through the NF-kappaB-dependent pathway.

Melanoma cancer is one of the major causes of death in humans worldwide. Triptolide is one of the active components of Tripterygium wilfordii Hook F, and has biological activities including induced cell cycle arrest and induction of apoptosis but its antimetastatic effects on murine melanoma cells have not yet been elucidated. Herein, we investigated the effect of triptolide on the inhibition of migration and invasion and possible associated signal pathways in B16F10 murine melanoma cancer cells. Wound healing assay and Matrigel Cell Migration Assay and Invasion System demonstrated that triptolide marked inhibiting the migration and invasion of B16F10 cells. Gelatin zymography assay demonstrated that triptolide significantly inhibited the activities of matrix metalloproteinases‐2 (MMP‐2). Western blotting showed that triptolide markedly reduced CXCR4, SOS1, GRB2, p‐ERK, FAK, p‐AKT, Rho A, p‐JNK, NF‐κB, MMP‐9, and MMP‐2 but increased PI3K and p‐p38 and COX2 after compared to the untreated (control) cells. Real time PCR indicated that triptolide inhibited the gene expression of MMP‐2, FAK, ROCK‐1, and NF‐κB but did not significantly affect TIMP‐1 and ‐2 gene expression in B16F10 cells in vitro. EMSA assay also showed that triptolide inhibited NF‐κB DNA binding in a dose‐dependent manner. Confocal laser microscopy examination also confirmed that triptolide inhibited the expression of NF‐κB in B16F10 cells. Taken together, we suggest that triptolide inhibited B16F10 cell migration and invasion via the inhibition of NF‐κB expression then led to suppress MMP‐2 and ‐9 expressions.

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.