Shih-Horng Huang

Mechanisms for the magnolol-induced cell death of CGTH W-2 thyroid carcinoma cells.

Magnolol, a substance purified from the bark of Magnolia officialis, inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The aim of this study was to study the effects of magnolol on CGTH W‐2 thyroid carcinoma cells. After 24 h treatment with 80 µM magnolol in serum‐containing medium, about 50% of the cells exhibited apoptotic features and 20% necrotic features. Cytochrome‐c staining was diffused in the cytoplasm of the apoptotic cells, but restricted to the mitochondria in control cells. Western blot analyses showed an increase in levels of activated caspases (caspase‐3 and ‐7) and of cleaved poly (ADP‐ribose) polymerase (PARP) by magnolol. Concomitantly, immunostaining for apoptosis inducing factor (AIF) showed a time‐dependent translocation from the mitochondria to the nucleus. Inhibition of either PARP or caspase activity blocked magnolol‐induced apoptosis, supporting the involvement of the caspases and PARP. In addition, magnolol activated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and inactivated Akt by decreasing levels of phosphorylated PTEN and phosphorylated Akt. These data suggest that magnolol promoted apoptosis probably by alleviating the inhibitory effect of Akt on caspase 9. Furthermore, inhibition of PARP activity, but not of caspase activity, completely prevented magnolol‐induced necrosis, suggesting the notion that it might be caused by depletion of intracellular ATP levels due to PARP activation. These results show that magnolol initiates apoptosis via the cytochrome‐c/caspase 3/PARP/AIF and PTEN/Akt/caspase 9/PARP pathways and necrosis via PARP activation. J. Cell. Biochem. 101: 1011–1022, 2007.

Effects of PPARγ agonists on cell survival and focal adhesions in a Chinese thyroid carcinoma cell line

Peroxisome proliferator‐activated receptor γ (PPARγ) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARγ agonists, ciglitazone and 15‐deoxy‐Δ 12,14‐prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W‐2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time‐ and dose‐dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP‐ribose) polymerase (PARP), and cytosolic cytochrome‐c were noted. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W‐2 cells from the culture substratum. Both the protein levels and immunostaining signals of focal adhesion (FA) proteins, including vinculin, integrin β1, focal adhesion kinase (FAK), and paxillin were decreased after PPARγ agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARγ agonists induced expression of protein tyrosine phosphatase‐PEST (PTP‐PEST), and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since pre‐treatment with orthovanadate prevented PPARγ agonist‐induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W‐2 cells with anti‐integrin β1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin β1 by PPARγ agonists resulted in FA disassembly and might induce apoptosis via anoikis. Our results suggested the presence of crosstalk between apoptosis and integrin‐FA signaling. Moreover, upregulation and activation of PTEN was correlated with reduced phosphorylation of Akt, and this consequence disfavored cell survival. In conclusion, PPARγ agonists induced apoptosis of thyroid carcinoma cells via the cytochrome‐c caspase 3 and PTEN‐Akt pathways, and induced necrosis via the PARP pathway. J. Cell. Biochem. 98: 1021–1035, 2006.

Effects of hyperthermia on the cytoskeleton and focal adhesion proteins in a human thyroid carcinoma cell line.

Hyperthermia is reported to act as a sensitizer to chemotherapeutic drugs in the treatment of cancer. Thyroid follicular carcinoma were used to elucidate the effects of hyperthermic treatment (41–43°C) on cell morphology, cytoskeleton, and the focal adhesion complex. The critical temperature that resulted in inhibition of cell proliferation as the cell number in the same area did not increase over a 23 h time course and irreversible changes in cell morphology was 42–43°C. An immunofluorescence study on heat‐treated cells (43°C, 1–5 h) demonstrated that depolymerization of actin filaments, intermediate filaments, and microtubules accounted for the rounding‐up of cells and detachment from the substratum. Characteristic staining patterns for integrin αv, focal adhesion kinase, and vinculin were noted in untreated cells, but the immunoreactive intensities for these proteins became weaker with time of heat treatment. Anti‐phosphotyrosine staining revealed less immunoreactivity in the focal adhesions in treated cells compared with control cells. The disappearance of integrin αv from the cell surface may result in inhibition of integrin‐mediated activation of focal adhesion kinase, which results in dephosphorylation of focal adhesion components and its disassembly. These results indicate that hyperthermia induces disruption of integrin‐mediated actin cytoskeleton assembly and, possibly, of other integrin‐mediated signaling pathways. J. Cell. Biochem. 75:327–337, 1999.

An acidic extracellular pH induces Src kinase-dependent loss of β-catenin from the adherens junction

Little attention has been paid to the role of adherens junctions (AJs) in acidic extracellular pH (pHe)-induced cell invasion. Incubation of HepG2 cells in acidic medium (pH 6.6) induced cell dispersion from tight cell clusters, and this change was accompanied by downregulation of beta-catenin at cell junctions and a rapid activation of c-Src. Pretreatment with PP2 prevented the acidic pH-induced downregulation of beta-catenin at AJ and in the membrane fractions. The acidic pHe-induced c-Src activation increased tyrosine phosphorylation of beta-catenin and decreased the amount of beta-catenin-associated E-cadherin. The depletion of membrane-bound beta-catenin coincided with enhanced cell migration and invasion, and this acidic pHe-increased cell migration and invasion was prevented by PP2. In conclusion, this study characterizes a novel signaling pathway responsible for acidic microenvironment-promoted migration and invasive behaviors of cancer cells.

Signaling pathway of magnolol-stimulated lipolysis in sterol ester-loaded 3T3-L1 preadipocyes.

The aims of the present study were to examine the effect of magnolol on lipolysis in sterol ester (SE)‐loaded 3T3‐L1 preadipocytes and to determine the signaling mechanism involved. We demonstrate that magnolol treatment resulted in a decreased number and surface area of lipid droplets, accompanied by release of glycerol. The lipolytic effect of magnolol was not mediated by PKA based on the facts that magnolol did not induce an elevation of intracellular cAMP levels, and protein kinase A (PKA) inhibitor KT5720 did not block magnolol‐induced lipolysis. Calcium/calmodulin‐dependent protein kinase (CaMK) was involved in this signaling pathway, since magnolol‐induced a transient rise of intracellular [Ca2+] and Ca2+ influx across the plasma membrane, and CaMK inhibitor significantly abolished magnolol‐induced lipolysis. Moreover, magnolol increased the relative levels of phosphorylated extracellular signal‐related kinases (ERK1 and ERK2). In support of the involvement ERK, we demonstrated that magnolol‐induced lipolysis was inhibited by PD98059, an inhibitor of mitogen‐activated protein kinase kinase (MEK), and PD98059 reversed magnolol‐induced ERK phosphorylation. Further, the relationship between CaMK and ERK was connected by the finding that CaMK inhibitor also blocked magnolol‐induced ERK phosphorylation. Taken together, these findings suggest that magnolol‐induced lipolysis is both CaMK‐ and ERK‐dependent, and this lipolysis signaling pathway is distinct from the traditional PKA pathway. ERK phosphorylation is reported to enhance lipolysis by direct activation of hormone sensitive lipase (HSL), thus magnolol may likely activate HSL through ERK and increase lipolysis of adipocytes.

An acidic extracellular pH disrupts adherens junctions in HepG2 cells by Src kinases‐dependent modification of E‐cadherin

We have previously shown that culturing HepG2 cells in pH 6.6 culture medium increases the c‐Src‐dependent tyrosine phosphorylation of β‐catenin and induces disassembly of adherens junctions (AJs). Here, we investigated the upstream mechanism leading to this pH 6.6‐induced modification of E‐cadherin. In control cells cultured at pH 7.4, E‐cadherin staining was linear and continuous at cell–cell contact sites. Culturing cells at pH 6.6 was not cytotoxic, and resulted in weak and discontinuous junctional E‐cadherin staining, consistent with the decreased levels of E‐cadherin in membrane fractions. pH 6.6 treatment activated c‐Src and Fyn kinase and induced tyrosine phosphorylation of p120 catenin (p120ctn) and E‐cadherin. Inhibition of Src family kinases by PP2 attenuated the pH 6.6‐induced tyrosine phosphorylation of E‐cadherin and p120ctn, and prevented the loss of these proteins from AJs. In addition, E‐cadherin was bound to Hakai and ubiquitinated. Furthermore, pH 6.6‐induced detachment of E‐cadherin from AJs was blocked by pretreatment with MG132 or NH4Cl, indicating the involvement of ubiquitin‐proteasomal/lysosomal degradation of E‐cadherin. An early loss of p120ctn prior to E‐cadherin detachment from AJs was noted, concomitant with a decreased association between p120ctn and E‐cadherin at pH 6.6. PP2 pretreatment prevented the dissociation of these two proteins. In conclusion, pH 6.6 activated Src kinases, resulting in tyrosine phosphorylation of E‐cadherin and p120ctn and a weakening of the association of E‐cadherin with p120ctn and contributing to the instability of E‐cadherin at AJs. J. Cell. Biochem. 108: 851–859, 2009.

Distribution of the cadherin‐catenin complex in normal human thyroid epithelium and a thyroid carcinoma cell line

E‐cadherin is the major cell‐cell adhesion molecule expressed by epithelial cells. Cadherins form a complex with three cytoplasmic proteins, α‐, β‐, and γ‐catenin, and the interaction between them is crucial for anchoring the actin cytoskeleton to the intercellular adherens junctions. The invasive behavior of cancer cells has been attributed to a dysfunction of these molecules. In this study, we examined the distribution of the cadherin‐catenin complex in a Chinese human thyroid cancer cell line, CGTH W‐2, compared with that in normal human thyroid epithelial cells. In the normal cells, using immunofluorescence staining, E‐cadherin and α‐, β‐, and γ‐catenin were found to be localized at the intercellular junction and appeared as 135, 102, 90, and 80 kD proteins on Western blots. In CGTH W‐2 cells, no E‐cadherin and γ‐catenin immunoreactivity was detected by immunofluorescence or Western blotting; α‐ and β‐catenin were detected as 102 and 90 kD proteins on blots but gave a diffuse cytoplasmic immunofluorescence staining pattern in most cells, while β‐catenin was also distributed throughout the cytoplasm in most cells but was found at the cell junction in some, where it colocalized with α‐actinin. The present data indicate that the loss of cell adhesiveness in these cancer cells may be due to incomplete assembly of the cadherin‐catenin complex at the cell junction. However, this defect did not affect the linkage of actin bundles to vinculin‐enriched intercellular junctions. J. Cell. Biochem. 70:330–337, 1998.

Effects of 18β‐Glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells

Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to α‐, β‐, and γ‐catenin, which were seen as continuous, linear staining of cell–cell adherens junction. Treatment with 18β‐glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time‐ and dose‐dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell–cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18β‐glycyrrhetinic acid stimulated a two‐ to three–fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18β‐glycyrrhetinic acid was partially mediated by extracellular signal‐related kinase and calcium/calmodulin‐dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. J. Cell. Biochem. 90: 33–41, 2003.

Impact of previous abdominal surgery on laparoscopic appendectomy for acute appendicitis

BackgroundLaparoscopic appendectomy is one of the most commonly performed laparoscopic procedures. Impact of previous abdominal surgery on laparoscopic appendectomy has not been previously reported.MethodsFrom January 2001 to December 2005, 2029 patients with clinically suspected acute appendicitis underwent laparoscopic surgery in our hospital. Of these, 234 patients (11.5%) were found to have other pathology by intraoperative or histologic findings and were excluded from the study. The 1795 patients who underwent laparoscopic appendectomy for acute appendicitis were divided into three groups: group 1, patients without a history of previous abdominal surgery (n = 1652, 92%); group 2, patients with a history of upper abdominal surgery (n = 20, 1.1%); group 3, patients with a history of lower abdominal surgery (n = 123, 6.8%). Data were collected retrospectively by chart review and analyzed for conversion rate, operative time, intraoperative and postoperative complications, and hospital stay.ResultsOf the 1795 patients, 13 (0.7%) were converted to open appendectomy because of technical difficulty. Overall mean operative time was 57.2 (range, 20–225) min. There was no mortality or intraoperative complications. Overall postoperative complication rate was 10.7% (n = 193): rate of surgical wound infection was 8.2 % (n = 147), surgical wound seroma 1.3% (n = 24), and intra-abdominal abscess 0.8% (n = 14). Overall postoperative hospital stay averaged 3.2 (range, 0–39) days. There were no significant differences between the three groups regarding the conversion rate (0.8% vs. 0% vs. 0%, p = 0.567), operative time (57.3 vs. 55.8 vs. 56.9 min, p = 0.962), postoperative complication rates (10.7 vs. 10 vs. 12.2%, p = 0.863), and hospital stay (3.2 vs. 3.6 vs. 3.1 days, p = 0.673).ConclusionsPrevious abdominal surgery, whether upper or lower abdominal, has no significant impact on laparoscopic appendectomy for acute appendicitis.

Rottlerin inhibits migration of follicular thyroid carcinoma cells by PKCδ‐independent destabilization of the focal adhesion complex

This study examined the effect of rottlerin on the focal adhesion‐mediated cell migration of CGTH W‐2 human follicular thyroid carcinoma cells. Rottlerin (10 µM) resulted in decreased adhesion of CGTH W‐2 cells to matrix substance, which was correlated with metastatic potential. Rottlerin treatment also resulted in a marked reduction in the migration of CGTH W‐2 cells. Protein levels of integrin β1, FAK, and paxillin were decreased by rottlerin. Consistent with this, immunostaining of FAK, vinculin, and paxillin revealed disassembly of the focal adhesions. Disruption of actin stress fibers was noted, which was compatible with reduced expression levels and activities of Rac‐1 and Rho. The effect of rottlerin on cell migration was not attributable to inhibition of PKCδ activity since siRNA knockdown of PKCδ did not recapitulate the effects of rottlerin on cell adhesion and migration. Furthermore, activation of PKCδ by phorbol esters failed to restore the rottlerin‐inhibited migratory ability. The mitochondrial uncoupler, carbonylcyanide‐4‐(trifluoromethoxy)‐phenylhydrazone, was able to mimic several rottlerins effects. In summary, we demonstrated that rottlerin inhibits the migration of CGTH W‐2 cells by disassembly of focal adhesion complexes in a PKCδ‐independent manner, and might play as a mitochondrial uncoupler role in these events. J. Cell. Biochem. 110: 428–437, 2010.