Shyr Yi Lin

In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter.

Phloretin (Ph), a natural product found in apples and pears with glucose transporter (GLUT) inhibitory activity, exerts antitumor effects. However, little is known about its effects on human liver cancer. The purpose of this study is to test the cytotoxic effects of Ph on HepG2 cells and to identify the underlying molecular pathways. Human hepatocellular carcinoma specimens and HepG2 show a high level of GLUT2 transporter activity in the cell membrane. Real‐time PCR and MTT assays demonstrate that Ph‐induced cytotoxicity correlates with the expression of GLUT2. Flow cytometry and DNA fragmentation studies show that 200 μM Ph induces apoptosis in HepG2, which was reversed by glucose pretreatment. GLUT2 siRNA knockdown induced HepG2 apoptosis, which was not reversed by glucose. Western blot analysis demonstrates that both intrinsic and extrinsic apoptotic pathways in addition to Akt and Bcl‐2 family signaling pathways are involved in Ph‐induced cell death in HepG2 cells. Furthermore, using flow cytometry analysis, a mitochondrial membrane potential assay and Western blot analysis, we show that cytochalasin B, a glucose transport inhibitor, enhances the Ph‐induced apoptotic effect on HepG2 cells, which was reversed by pretreatment with glucose. Furthermore, we found significant antitumor effects in vivo by administering Ph at 10 mg/kg intraperitoneally to severe combined immune deficiency mice carrying a HepG2 xenograft. A microPET study in the HepG2 tumor‐bearing mice showed a 10‐fold decrease in 18F‐FDG uptake in Ph‐treated tumors compared to controls. Taken together, these results suggest that Ph‐induced apoptosis in HepG2 cells involves inhibition of GLUT2 glucose transport mechanisms.

Molecular mechanisms of apoptosis induced by magnolol in colon and liver cancer cells

Magnolol has been reported to have anticancer activity. In this study we found that treatment with 100 μm magnolol induced apoptosis in cultured human hepatoma (Hep G2) and colon cancer (COLO 205) cell lines but not in human untransformed gingival fibroblasts and human umbilical vein endothelial cells. Our investigation of apoptosis in Hep G2 cells showed a sequence of associated intracellular events that included (a) increased cytosolic free Ca2+; (b) increased translocation of cytochrome c (Cyto c) from mitochondria to cytosol; (c) activation of caspase 3, caspase 8, and caspase 9; and (d) downregulation of bcl‐2 protein. Pretreatment of the cells with the phospholipase C inhibitor 1‐[6‐[[(17β)‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino]hexyl]‐1 H‐pyrrole‐2,5‐dione (U73122) or the intracellular chelator of Ca2+ 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA/AM) inhibited the subsequent magnolol augmentation of [Ca2+]i and also the activation of caspase‐8 and caspase‐9, so that the occurrence of apoptosis in those cells was greatly reduced. Pretreatment of the cells with ZB4 (which disrupts the Fas response mechanism) also decreased the subsequent magnolol‐induced caspase‐8 activation and reduced the occurrence of apoptosis. We interpreted these findings to indicate that the above‐listed sequence of intracellular events led to the apoptosis seen in Hep G2 cells and that [Ca2+]i, Cyto c, and Fas function as intracellular signals to coordinate those events.

Tobacco-specific carcinogen enhances colon cancer cell migration through α7-nicotinic acetylcholine receptor

Objective:To study the mechanism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-enhanced migration of colon cancer cells. Background Data:Long-term cigarette smoking increases the risk of colorectal cancer mortality. Tobacco-specific carcinogen, NNK, was reported to increase DNA synthesis of colon cancer cells. Since metastasis is the major cause of cancer death, the influence of NNK on the migration of colon cancer cells remains to be determined. Methods:Receptor for NNK in colon cancer cells was identified by polymerase chain reaction (PCR) and real-time PCR. The influence of NNK on migration of colon cancer cells was evaluated by transwell and wound-healing assay. Receptor-mediated migration was studied by both inhibitor and small interfering RNA. Results:α7 nicotinic acetylcholine receptor, α7-nAChR, was identified in 2 colon cancer cell lines, HT29 and DLD-1. NNK enhanced HT29 cell migration in both transwell and wound-healing assays. NNK also enhanced DLD-1 cell migration in dose dependent manner. We used inhibitor and siRNA to demonstrate that α7-nAChR mediated NNK-enhanced colon cancer cell migration and downregulation of E-cadherin were involved in NNK-enhanced migration of colon cancer cells. Furthermore, Snail and ZEB1, 2 major transcription repressors of E-cadherin in colon cancers, were induced by NNK treatment. Conclusions:Tobacco specific carcinogen, NNK, enhanced colon cancer metastasis through α7-nAChR and E-cadherin—one of the hallmarks of epithelial mesenchymal transition—and its transcription repressors. Therefore, smoking should be avoided in the patients with colorectal cancer.

In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53‐associated cell cycle arrest

Terbinafine (TB) (Lamisil®), a promising oral antifungal agent used worldwide, has been used in the treatment of superficial mycosis. In our study, we demonstrated that TB dose‐dependently decreased cell number in various cultured human malignant cells. Flow cytometry analysis revealed that TB interrupts the cell cycle at the G0/G1 transition. The TB‐induced cell cycle arrest in colon cancer cell line (COLO 205) occurred when the cyclin‐dependent kinase (cdk) system was inhibited just as the levels of p53, p21/Cip1 and p27/Kip1 proteins were augmented. In the TB‐treated COLO 205, the binding between p53 protein and p53 consensus binding site in p21/Cip1 promoter DNA probe was increased. Pretreatment of COLO 205 with p53‐specific antisense oligodeoxynucleotide decreased the TB‐induced elevations of p53 and p21/Cip1 proteins, which in turn led to arrest in the cell cycle at the G0/G1 phase. Moreover, in the p53 null cells, HL60, TB treatment did not induce cell cycle arrest. Taken together, these results suggest an involvement of the p53‐associated signaling pathway in the TB‐induced antiproliferation in COLO 205. We further examined whether administration of TB could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. COLO 205 cells implanted subcutaneously in nude mice formed solid tumor; subsequent intraperitoneal injections of TB (50 mg/kg) led to obvious decline in tumor size, up to 50–60%. In these tumors, increases in the p21/Cip1, p27/Kip1 and p53 proteins and the occurrence of apoptosis were observed. Combined treatment with TB and nocodazole (ND), a clinically used anticancer agent, potentiated the apoptotic effect in COLO 205. These findings demonstrate for the first time that TB can inhibit the proliferation of tumor cells in vitro and in vivo.

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells.

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G0/G1 phase cell‐cycle arrest. The minimal dose of TF induced significant G0/G1 arrest in these cells was 1–3 μM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin‐dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF‐treated cells. On the other hand, significant apoptosis, but not G0/G1 arrest, was induced in the HL 60 (p53‐null) or Hep 3B (with deleted p53) cells when treated with TF (3–5 μM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G0/G1 arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF‐treated group was clearly attenuated by pretreatment with p27/Kip1–specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF‐induced apoptosis. We also demonstrated that the TF‐induced G0/G1 cell‐cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF‐treated normal human fibroblast, even at a dose as high as 25 μM. Our study showed the molecular mechanisms for TF‐induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.

Liposome encapsulation reduces cantharidin toxicity

Several reports have demonstrated that cantharidin is a strong anticancer compound in vitro; however, its in vivo usefulness is often limited due to its high systemic toxicity. In this study, we encapsulated cantharidin into pegylated liposomes and studied its activity against human breast cancer MCF-7 cells in vitro and its systemic toxicity in mice. Another two methods were also used to reduce the dosage of cantharidin, including labeling liposomal cantharidin with octreotide and exposing cells to hyperbaric oxygen. The cytotoxic activity of pegylated liposomal cantharidin was drastically reduced compared with free cantharidin in vitro. Octreotide-labeled pegylated liposomal cantharidin induced cell death by specifically targeting somatostatin receptors in MCF-7 cells. Cell death was augmented with a low dose of cantharidin under hyperbaric oxygen. Liposomal cantharidin had significantly less systemic toxicity than free cantharidin in vivo and also exhibited a high efficacy against antitumor growth in nude mice. These results suggest that the systemic toxicity of cantharidin can be mitigated by liposome encapsulation; however, that did not decrease its antitumor activity.

Involvement of Ras/Raf-1/ERK actions in the magnolol-induced upregulation of p21 and cell-cycle arrest in colon cancer cells

Previously, we showed that magnolol induces cell‐cycle arrest in cultured colon and liver cancer cells through an upregulation of the p21 protein [ 1 ]. The aim of this study was to delineate the molecular mechanism underlying this magnolol‐induced increase of p21 protein. Thus our RT‐PCR analysis demonstrated that the mRNA levels of p21 were increased at 1 h after magnolol treatment and sustained for at least 24 h. The p21 promoter activity was also increased by magnolol treatment. Western blot analysis demonstrated that treatment of COLO‐205 cells with magnolol increased the levels of phosphorylation of extracellular signal‐regulated kinase (ERK). Pretreatment of the cells with PD98059 abolished the magnolol‐induced upregulation of p21 protein, suggesting the involvement of an ERK pathway in the magnolol‐induced upregulation of p21 in COLO‐205 cells. Ras inhibitor peptide abolished the magnolol‐induced increase of phosphorylated ERK protein levels, increase of p21 protein, and decrease of thymidine incorporation. Moreover, treatment of COLO‐205 with magnolol increased the phosphorylated Raf‐1 protein (the Ras target molecule). Pretreatment of the cells with Raf‐1 inhibitor reversed the magnolol‐induced decrease in thymidine incorporation. Treatment of the cells with CaM kinase inhibitor, but not protein kinase A (PKA) inhibitor or phosphatidylinosital 3‐kinase (PI3K) inhibitor, abolished the magnolol‐induced activation of ERK and decrease of thymidine incorporation. Taken together, our results suggest that magnolol activates ERK phosphorylation through a Ras/Raf‐1‐mediated pathway. Subsequently, p21 expression is increased, and finally thymidine incorporation is decreased.

Checkpoint kinase 1-mediated phosphorylation of Cdc25C and bad proteins are involved in antitumor effects of loratadine-induced G2/M phase cell-cycle arrest and apoptosis

In this study, we first demonstrated that loratadine (LOR), a promising world widely used oral anti‐histamine, effectively inhibits growth of tumors derived from human colon cancer cells (COLO 205) in an in vivo setting. In vitro study demonstrated that the anti‐tumor effects of LOR in COLO 205 cells were mediated by causing G2/M phase cell growth cycle arrest and caspase 9‐mediated apoptosis. Cell‐cycle arrest induced by LOR (75 µM, 24 h) was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr‐15‐phosphorylated Cdc2 (inactive form). Interestingly, LOR (75 µM, for 4 h) treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser‐216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14‐3‐3. We further demonstrated that the LOR‐induced Cdc25C (Ser‐216) phosphorylation was blocked in the presence of checkpoint kinase 1 (Chk1) specific inhibitor (SB‐218078). The cells treated with LOR in the presence of Chk1 specific inhibitor (SB‐218078) were then released from G2/M arrest into apoptosis. These results implied that Chk1‐mediated phosphorylation of Cdc25C plays a major role in response to LOR‐mediated G2/M arrest. Although the Chk1‐mediated cell growth arrest in response to DNA damage is well documented, our results presented in this study was the first report to describe the Chk1‐mediated G2/M cell‐cycle arrest by the histamine H1 antagonist, LOR.

MCPIP1 Suppresses Hepatitis C Virus Replication and Negatively Regulates Virus-Induced Proinflammatory Cytokine Responses

Human MCP-1–induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.

NF‐κB pathway is involved in griseofulvin‐induced G2/M arrest and apoptosis in HL‐60 cells

Griseofulvin (GF), an oral antifungal agent, has been shown to exert antitumorigenesis effect through G2/M cell cycle arrest in colon cancer cells. But the underlying mechanisms remained obscure. The purpose of this study is to test the cytotoxic effect of GF on HL‐60 and HT‐29 cells and elucidate its underlying molecular pathways. Dose‐dependent and time‐course studies by flow cytometry demonstrated that 30 to 60 µM GF significantly induced G2/M arrest and to a less extend, apoptosis, in HL‐60 cells. In contrast, only G2/M arrest was observed in HT‐29 cells under similar condition. Pretreatment of 30 µM TPCK, a serine protease inhibitor, completely reversed GF‐induced G2/M cell cycle arrest and apoptosis in HL‐60 cells but not in HT‐29 cells. The GF‐induced G2/M arrest in HL‐60 cells is reversible. Using EMSA and super‐shift analysis, we demonstrated that GF stimulated NF‐κB binding activity in HL‐60 cells, which was completely inhibited by pretreatment of TPCK. Treatment of HL‐60 with 30 µM GF activated JNK but not ERK or p38 MAPK and subsequently resulted in phosporylation of Bcl‐2. Pretreatment of TPCK to HL‐60 cells blocked the GF‐induced Bcl‐2 phosphorylation but not JNK activation. Time course study demonstrated that activation of cdc‐2 kinase activity by GF correlated with Bcl‐2 phosphorylation. Taken together, our results suggest that activation of NF‐κB pathway with cdc‐2 activation and phosphorylation of Bcl‐2 might be involved in G2/M cell cycle arrest in HL‐60 cells. J. Cell. Biochem. 101:1165–1175, 2007.