Chun Li Su

Mutational Analysis of the Hormone-sensitive Lipase Translocation Reaction in Adipocytes

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093–1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.

Morusin induces apoptosis and suppresses NF-κB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G(1) phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-alpha, IKK-beta and IkappaB-alpha, increased expression of IkappaB-alpha, and suppressed nuclear translocation of NF-kappaB and its DNA binding activity. Dephosphorylation of NF-kappaB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-kappaB.

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase‐8 to increase tBid, disrupted mitochondrial membrane potential (Δψ m), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl‐xL and increased Bax and Bak in mitochondria. Caspase‐8 inhibitor (Z‐IETD) attenuated the justicidin A‐induced disruption of Δψ m. Growth of Hep 3B implanted in NOD‐SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A‐induced apoptosis in these cells proceeds via caspase‐8 and is followed by mitochondrial disruption. Supplementary materials are available at http://myweb.ncku.edu.tw/~a725/.

Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Thus, to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse, at least in part, the gene expression signature of GBM and GSCs, this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here, we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened, thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine, we found that thioridazine induces autophagy in GBM cell lines, and upregulates AMPK activity. Moreover, LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition, thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent, but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties.

Formosanin C‐induced apoptosis requires activation of caspase‐2 and change of mitochondrial membrane potential

Formosanin C is a pure compound isolated from Paris formosana Hayata (Liliaceae). The antitumor efficacy of formosanin C has been observed in cultured cells and animal systems. However, the molecular mechanisms of formosanin C remain unknown. The results of the present study indicate that formosanin C induced apoptosis of HT‐29 cells characterized by exposure of phosphatidylserine, accumulation of cells at the sub‐G1 phase, fragmentation of DNA, and change of nuclear morphology in a time‐ and dose‐related manner. The apoptotic signaling cascades may proceed via proteolytic activation of caspase‐2, change of mitochondrial membrane potential (Δψm), release of cytochrome c and second mitochondria‐derived activator of caspase/direct IAP binding protein with low pI (Smac/DIABLO), activation of caspase‐9 and ‐3, and cleavage of poly(ADP‐ribose) polymerase (PARP). Increase in apoptosis‐inducing factor and endonuclease G expressions in nuclei, and increase in Bax and Bak expressions and decrease in Bcl‐XL expression on mitochondria were also observed in formosanin C‐treated HT‐29 cells. Attenuation of formosanin C‐induced change of Δψm by caspase‐2 inhibitor (Z‐VDVAC) implies that caspase‐2 acts upstream of the mitochondria. Blockage of formosanin C‐induced apoptotic process by using either permeability transition pore inhibitor (cyclosporine A) or caspase‐9 inhibitor (Z‐LEHD) demonstrates the necessity of mitochondria and caspase‐9 in formosanin C‐induced apoptosis of HT‐29 cells. Taken together, the apoptotic mechanism of formosanin C in human colorectal cancer HT‐29 cells involves activation of caspase‐2 and the dysfunction of mitochondria. (Cancer Sci 2009; 100: 503–513)

Involvement of caspases and apoptosis-inducing factor in bufotalin-induced apoptosis of hep 3B cells

Bufotalin is one of the bufadienolides isolated from Formosan Chan Su, which is made of the skin and parotid glands of toads. Ingestion of toad venom results in severe morbidity and high mortality. Although Chan Su is clinically toxic, it has been used as an important traditional Chinese medicine for heart failure and pains. In this study, bufotalin-induced apoptosis in human hepatocellular carcinoma Hep 3B cells was investigated. The results indicate that externalization of phosphatidylserine, accumulation of sub-G(1) cells, fragmentation of DNA, and formation of apoptotic bodies were observed in bufotalin-treated Hep 3B cells. The signaling pathway might be via the activation of caspase-8, increase in mitochondrial tBid, disruption of mitochondrial membrane potential, and translocation of apoptosis-inducing factor (AIF). Active caspase-8 might activate caspase-9 and caspase-3 leading to the cleavage of nuclear PARP. Presence of AIF and cleaved PARP in the nuclei might lead to DNA fragmentation. Caspase-8 inhibitor (Z-IETD) or wide-ranging caspase inhibitor (Z-VAD) significantly suppressed the bufotalin-induced apoptosis, while the anti-Fas neutralization antibody had no effect. These data suggest that bufotalin-induced apoptosis in Hep 3B cells might involve caspases and AIF.

Staphylococcal enterotoxin C1-induced pyrogenic cytokine production in human peripheral blood mononuclear cells is mediated by NADPH oxidase and nuclear factor-kappa B

The staphylococcal enterotoxins produced by Staphylococcus aureus are associated with pyrogenic response in humans and primates. This study investigates the role of NADPH oxidase and nuclear factor‐kappau2003B (NF‐κB) on enterotoxin staphylococcal enterotoxinu2003C1 (SEC1)‐induced pyrogenic cytokine production in human peripheral blood mononuclear cells (PBMC). The results indicate that the febrile response to the supernatant fluids of SEC1‐stimulated PBMC in rabbits was in parallel with the levels of interleukin‐1β and interleukin‐6 in the supernatants. The release of interleukin‐1β and interleukin‐6, nuclear translocation of NF‐κB and its DNA binding activity in the SEC1‐stimulated PBMC were time‐dependent and were completely eliminated by pyrrolidine dithiocarbamate or SN‐50 (NF‐κB inhibitors). The release of reactive oxygen species in the supernatants and translocation of the NADPH oxidase p47phox subunit to the plasma membrane of SEC1‐stimulated PBMC were time‐dependent. Administration of apocynin (NADPH oxidase inhibitor) attenuated the febrile response to the supernatants in rabbits and decreased the translocation of NADPH oxidase p47phox subunit and NF‐κB activity in the SEC1‐stimulated PBMC, and suppressed reactive oxygen species and pyrogenic cytokine production in the supernatants. Taken together, SEC1 may act through an NADPH oxidase mechanism to release reactive oxygen species, which activate NF‐κB in PBMC to stimulate the synthesis of pyrogenic cytokines that trigger a fever response in rabbits.

Justicidin A-Induced autophagy flux enhances apoptosis of human colorectal cancer cells via class III PI3K and Atg5 pathway

Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD‐SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT‐29 cells by conversion of autophagic marker LC3‐I to LC3‐II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent‐tagged LC3 (mRFP‐GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT‐29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double‐strand breaks (representing apoptosis) were also detected in the tumor tissue of HT‐29 cells transplanted into NOD‐SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway‐related molecules p‐PDK1, p‐mTOR, p‐p70S6k/p‐RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5‐Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre‐treatment of the cells with class III PI3K inhibitor 3‐methyladenine or Atg5 shRNA attenuated JA‐induced LC3‐II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre‐treatment with 3‐methyladenine or Atg5 shRNA blocked JA‐induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z‐VAD did not affect JA‐induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer. J. Cell. Physiol. 230: 930–946, 2015.

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA-IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer.

Involvement of apoptosis and autophagy in reducing mouse hepatoma ML-1 cell growth in inbred BALB/c mice by bacterial fermented soybean products

Followed by the results of our previous in vitro report (Food Chem. Toxicol., 2007), the efficacy of the soybean fermentation products containing live bacteria (SCB) was demonstrated using a syngeneic animal model. Murine HBV-related hepatoma ML-1 cells, derived from inbred animals and tumorigenic in BALB/c mice, were implanted subcutaneously to the flank of BALB/c mice on day 0. Three days after implantation, SCB (1.0 or 1.3 ml/mouse/day) or vehicle (water) was orally administrated daily until day 60. The results indicate that SCB significantly reduced (P<0.05) the volumes and weights of tumors during the experimental periods. Examination using TUNEL staining on section of tumors revealed apoptotic phenomenon of nuclear DNA double-strand breaks in the groups of mice received SCB. Immunohistochemistry further revealed an autophagic LC3-II punctate pattern. Of note, SCB induced autophagy in the absence or presence of apoptosis, whereas, apoptosis was observed only in combination with autophagy. In vitro study using autophagy inhibitor indicated that the induction of autophagy promoted apoptosis. These data imply that the suppression in tumor volumes and tumor weights by oral administration of SCB was due to the induction of apoptotic and autophagic cell death, which suggests therapeutic potential of SCB on HBV-related HCC.