Yuan-Soon Ho

Induction of p53 and p21/WAF1/CIP1 expression by nitric oxide and their association with apoptosis in human cancer cells.

In this study, human and rat cancer cells were used to investigate the expression of p53 and p21/WAF1/CIP1 and their association with apoptosis after exposure to nitric oxide (NO). It was found that NO induced nuclear accumulation of p53 protein in a dose‐ and time‐dependent manner. The level of p53 protein was elevated by about fivefold compared with that of mock‐treated cells 48 h after exposure to 300 ppm NO. The induction of p53 by NO was found by pulse‐chase analysis to be mainly regulated by post‐translational modification. The correlation between p53 status and apoptosis induced by NO in human cancer cells was also investigated in this study. We found that apoptosis was easily induced in cells containing wild‐type p53 (COLO 205 and Hep G2) after exposure to NO. The p21/WAF1/CIP1 protein was induced by NO in cells containing wild‐type p53 (Hep G2) but not in cells without p53 (Hep 3B) or with mutated p53 (HT‐29). Our results indicate that wild‐type p53 and p21/WAF1/CIP1 expression was elevated in human cancer cells by exposure to NO and suggest that this may eventually promote apoptosis.

Suppression of nitric oxide-induced apoptosis by N-acetyl-L-cysteine through modulation of glutathione, bcl-2, and bax protein levels

It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N‐acetyl‐L‐cysteine (LNAC)) and free‐radical spin traps (5,5‐dimethyl‐1‐pyrroline N‐oxide and 2,2,6,6,‐tetramethyl‐1‐piperidinyloxy) against NO‐induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose‐dependent manner, promoted long‐term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl‐2 oncoprotein level in the LNAC‐pretreated cells was significantly elevated in a time‐dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl‐2 protein levels and to its suppression of cellular bax protein in treated cells. Mol. Carcinog. 19:101–113, 1997.

Induction of cyclooxygenase‐2 protein by lipoteichoic acid from Staphylococcus aureus in human pulmonary epithelial cells: involvement of a nuclear factor‐κB‐dependent pathway

This study investigated the role of protein kinase C (PKC) and transcription factor nuclear factor‐κB (NF‐κB) in cyclooxygenase‐2 (COX‐2) expression caused by lipoteichoic acid (LTA), a cell wall component of the gram‐positive bacterium Staphylococcus aureus, in human pulmonary epithelial cell line (A549). LTA caused dose‐ and time‐dependent increases in COX‐2 expression and COX activity, and a dose‐dependent increase in PGE2 release in A549 cells. The LTA‐induced increases in COX‐2 expression and COX activity were markedly inhibited by dexamethasone, actinomycin D or cyclohexamide, but not by polymyxin B, which binds and inactivates endotoxin. The phosphatidylcholine‐phospholipase C (PC‐PLC) inhibitor (D‐609) and the phosphatidate phosphohydrolase inhibitor (propranolol) reduced the LTA‐induced increases in COX‐2 expression and COX activity, while phosphatidylinositol‐phospholipase C inhibitor (U‐73122) had no effect. The PKC inhibitors (Go 6976, Ro 31‐8220 and GF 109203X) and NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), also attenuated the LTA‐induced increases in COX‐2 expression and COX activity. Treatment of A549 cells with LTA caused an increase in PKC activity in the plasma membrane; this stimulatory effect was inhibited by D‐609, propranolol, or Go 6976, but not by U‐73122. Exposure of A549 cells to LTA caused a translocation of p65 NF‐κB from the cytosol to the nucleus and a degradation of IκB‐α in the cytosol. Treatment of A549 cells with LTA caused NF‐κB activation by detecting the formation of NF‐κB‐specific DNA‐protein complex in the nucleus; this effect was inhibited by dexamethasone, D‐609, propranolol, Go 6976, Ro 31‐8220, or PDTC. These results suggest that LTA might activate PC‐PLC and phosphatidylcholine‐phospholipase D to induce PKC activation, which in turn initiates NF‐κB activation, and finally induces COX‐2 expression and PGE2 release in human pulmonary epithelial cell line.

Microtubule damaging agents induce apoptosis in HL 60 cells and G2/M cell cycle arrest in HT 29 cells

Microtubule damaging agents (such as paclitaxel and nocodazole (ND)) have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of these agents in the induction of anti-cancer activity are still unclear. In the present study, we demonstrated that 0.2 microM podophyllotoxin (PDP) induced the occurrence of apoptosis in human leukemic (HL 60) cells and cell cycle arrest at the G2/M phase in HT 29 cells. Our results suggest that the PDP-induced G2/M arrest in HT 29 cells was through the intracellular events including (a) inhibition of normal mitotic spindle formation, (b) elevation of cyclin B1/cdc2 kinase activity, (c) concomitant increases in cdc 25 A phosphatase and cdk 7 kinase activity, and (d) down-regulation of the wee-1 protein expression. On the other hand, activations of the caspases 3, 8, and 9, Bcl-2 hyper-phosphorylation, and increased leakage of cytochrome c from mitochondria into cytosolic fraction were detected in the PDP-treated HL 60 cells. These listed intracellular events were interpreted to lead to the apoptosis observed in PDP-treated HL 60 cells. We further demonstrated that activation of c-jun N-terminal kinase (JNK) signaling pathway may play an important role in the PDP-induced Bcl-2 phosphorylation and apoptosis in HL 60 cells as evidenced by the JNK specific anti-sense oligonucleotide experiment. Our results demonstrated that the occurrence of apoptosis or G2/M cell cycle arrest induced by microtubule damaging agents in different cancer cells was through independent mechanisms. The results from the present study highlight the molecular mechanisms underlying of the PDP-induced anti-cancer activity.

Combination Treatment with Luteolin and Quercetin Enhances Antiproliferative Effects in Nicotine-Treated MDA-MB-231 Cells by Down-regulating Nicotinic Acetylcholine Receptors

Large-scale epidemiological cohort studies performed in the United States indicate that breast cancer risk is associated with active and passive smoking. As of yet, however, there is no direct evidence of antitumor effects by agents that block the effect of tobacco compound nicotine (Nic) on relevant nicotinic receptors (nAChR) involved in breast tumorigenesis. In the present study, the expression profiles of different nAChR subunits in the human breast cancer cell line (MDA-MB-231) were characterized by RT-PCR. Nic (>0.1 microM, 6 h) significantly increased alpha9-nAChR mRNA and protein expression levels in human breast cancer cells (MDA-MB-231 cells). On the other hand, combined treatment with luteolin (Lut, 0.5 microM) and quercetin (Que, 0.5 microM) profoundly decreased MDA-MB-231 proliferation by down-regulating alpha9-nAChR expression. MDA-MB-231 cells were cultured in soft agar to evaluate anchorage-independent colony formation; combined treatment of Lut+Que inhibited Nic-induced MDA-MB-231 colony formation. Interestingly, the number of colonies formed was profoundly reduced in alpha9-nAChR knockdown (Si alpha9) cells in the combined (Lut+Que)-treated group as compared to the relevant control groups. Such results show that Lut- or Que-induced antitransforming activities were not limited to specific inhibition of the alpha9-nAChR receptor. Both alpha5- and alpha9-nAChR appear to be important molecular targets for Lut- and Que-induced antitumor effects in human breast cancer cells.

Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells.

Phloretin (Ph), which can be obtained from apples, apple juice, and cider, is a known inhibitor of the type II glucose transporter (GLUT2). In this study, real‐time PCR analysis of laser‐capture microdissected (LCM) human hepatoma cells showed elevated expression (>5‐fold) of GLUT2 mRNA in comparison with nonmalignant hepatocytes. In vitro and in vivo studies were performed to assess Ph antitumor activity when combined with paclitaxel (PTX) for treatment of human liver cancer cells. Inhibition of GLUT2 by Ph potentiated the anticancer effects of PTX, resensitizing human liver cancer cells to drugs. These results demonstrate that 50–150 µM Ph significantly potentiates DNA laddering induced in Hep G2 cells by 10 nM PTX. Activity assays showed that caspases 3, 8, and 9 are involved in this apoptosis. The antitumor therapeutic efficacy of Ph (10 mg/kg body weight) was determined in cells of the SCID mouse model that were treated in parallel with PTX (1 mg/kg body weight). The Hep G2‐xenografted tumor volume was reduced more than fivefold in the Ph + PTX‐treated mice compared to the PTX‐treated group. These results suggest that Ph may be useful for cancer chemotherapy and chemoprevention.

Involvement of both extracellular signal-regulated kinase and c-jun N-terminal kinase pathways in the 12-O-tetradecanoylphorbol-13-acetate–induced upregulation of p21Cip1 in colon cancer cells

Protein kinase C (PKC), a family of serine‐threonine kinases, has been implicated in the regulation of colon tumorigenesis. However, the specific isoform of PKC involved in this process is not clear. In the present study, we found that treatment of the cultured human colon cancer cell line COLO‐205 with a PKC agonist, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), resulted in cell‐cycle arrest at the G0/G1 phase, decrease in cell number, PKCγ isoform translocation, and upregulation of p21Cip1 protein. Pretreatment of the cells with a PKC inhibitor, staurosporine, prevented the TPA‐induced upregulation of p21Cip1 protein. Based on the findings of the present study including that (a) both extracellular signal‐regulated kinase (ERK) and c‐jun N‐terminal kinase (JNK) were activated in the TPA‐treated COLO‐205 cells, (b) pretreatment with the mitogen‐activated protein kinase kinase inhibitor PD98059 but not with the p38 mitogen‐activated protein kinase inhibitor SB203580 blocked the TPA‐induced p21Cip1 in COLO‐205 cells, and (c) transient transfection of the COLO‐205 cells with dominant negative ERK or JNK plasmid significantly suppressed the TPA‐induced p21Cip1 protein induction, we conclude that both the ERK and JNK pathways are involved in the TPA‐induced upregulation of p21Cip1 protein in the COLO‐205 cells.

Different cell death mechanisms and gene expression in human cells induced by pentachlorophenol and its major metabolite, tetrachlorohydroquinone.

Pentachlorophenol (PCP) and its salt are used extensively as biocide and wood preservative. Due to improper disposal, PCP has become an environmental pollutant and is now considered to be ubiquitos. Metabolic studies carried out in rodents or human liver homogenate have indicated that PCP undergoes oxidative dechlorination to form tetrachlorohydroquinone (TCHQ). The cytotoxicity, cell death mechanisms and gene expression of PCP and TCHQ are investigated in human liver and bladder cells and show that TCHQ induces apoptosis and DNA genomic fragmentation in bladder cells but not liver cells. No apoptotic features could be induced by treatment of PCP in both cell lines. The concentrations of PCP required to cause 50% cell death in T-24 and Chang liver cells were 5-10-fold greater than the concentrations of TCHQ. Several gene products are important in controlling the apoptotic and necrotic processes. Of these, hsp 70, CAS, bcl-2 and bax were studied. The expression of the hsp70 gene increased significantly (2-3-fold) in cells treated with TCHQ. However, no significant change was found in the cells treated with PCP. The expression of CAS gene decreased significantly in T-24 cells treated with both TCHQ and PCP. Whereas, no significant change was found in Chang liver cells with the same treatment. In addition, the expression of the bcl-2/bax protein decreased significantly in these two cell lines treated with TCHQ but not PCP.

MicroRNA-200a and -200b mediated hepatocellular carcinoma cell migration through the epithelial to mesenchymal transition markers

BackgroundMicroRNAs (miRNAs) play an essential role in mediating gene expression in both normal and malignant cells. However, little is known about specific miRNAs during the development of hepatocellular carcinoma (HCC) from well-differentiated to poorly differentiated cells.MethodsWe performed miRNA array analysis of three different HCC cell lines: HepG2, HepJ5, and skHep-1. The expression patterns of miR-200 family members were confirmed by real-time polymerase chain reaction (PCR). We overexpressed miR-200 family members by using a lentivirus system and selected for stably transduced cells using antibiotics. The migration ability of the cells was tested using the Transwell migration assay system.ResultsOur miRNA array and real-time PCR results indicated a decrease in the expression of miR-200 family members in poorly differentiated skHep-1 cells compared with well-differentiated HepG2 cells. We overexpressed miR-200a and miR-200b in both HepJ5 and skHep-1 cells and found that the overexpression of the miR-200 family members did not influence proliferation, although migration was decreased in these cells. We found that overexpression of miR-200 family members led to an upregulation of E-cadherin expression in both HepJ5 and skHep-1 cells. Furthermore, we silenced E-cadherin expression by shRNA in miR200a-HepJ5 cells and found that the migratory ability of these cells was enhanced upon the decrease in E-cadherin expression.ConclusionsMembers of the miR-200 family (miR-200a and miR-200b) play important roles in HCC migration by regulating E-cadherin expression.

Involvement of c-jun N-terminal kinase activation in 15-deoxy-δ12,14-prostaglandin J2–and prostaglandin A1–induced apoptosis in AGS gastric epithelial cells

Cyclopentenone prostaglandins (CyPGs), derivatives of arachidonic acid, have been suggested to exert growth‐inhibitory activity through peroxisome proliferator‐activated receptor (PPAR)‐dependent and ‐independent mechanisms. Here we examined various eicosanoids for growth inhibition and found that the terminal derivative of prostaglandin (PG) J2 metabolism, 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2), and PGA1 markedly inhibited the growth and induced apoptosis in AGS gastric carcinoma cells. There were no significant increases in cell death and DNA‐fragmentation in the cells with overexpression of PPARα or PPARγ, indicating the possibility that 15d‐PGJ2 and PGA1 induced apoptosis through PPAR‐independent pathway. Moreover, 15d‐PGJ2 and PGA1 activated the c‐jun N‐terminal kinase (JNK) and caspase‐3 activity in dose‐ and time‐dependent manners. To examine further the role of JNK signaling cascades in apoptosis induced by 15d‐PGJ2 and PGA1, we transfected dominant‐negative (DN) mutants of JNK plasmid into the cells to analyze the apoptotic characteristics of cells overexpressing DN‐JNK following exposure to 15d‐PGJ2 and PGA1. Overexpression of DN‐JNK significantly repressed both endogenous JNK and caspase‐3 activity, and subsequently decreased apoptosis induced by 15d‐PGJ2 and PGA1. These results suggested that CyPGs, such as 15d‐PGJ2 and PGA1, activated JNK signaling pathway, and that JNK activation may be involved in 15d‐PGJ2‐ and PGA1‐induced apoptosis.