Ming-Shiou Jan

GSK-3β acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 in ceramide-induced mitochondrial apoptosis

The signaling of glycogen synthase kinase-3β (GSK-3β) has been implicated in stress-induced apoptosis. However, the pro-apoptotic role of GSK-3β is still unclear. Here, we show the involvement of GSK-3β in ceramide-induced mitochondrial apoptosis. Ceramide induced GSK-3β activation via protein dephosphorylation at serine 9. We previously reported that ceramide induced caspase-2 and caspase-8 activation, Bid cleavage, mitochondrial damage, and apoptosis. In this study, we found that caspase-2 activation and the subsequent apoptotic events were abolished by the GSK-3β inhibitors lithium chloride and SB216763, and by GSK-3β knockdown using short interfering RNA. We also found that ceramide-activated protein phosphatase 2A (PP2A) indirectly caused GSK-3β activation, and that the PP2A-regulated PI 3-kinase-Akt pathway was involved in GSK-3β activation. These results indicate a role for GSK-3β in ceramide-induced apoptosis, in which GSK-3β acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 and caspase-8.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Lithium inhibits ceramide- and etoposide-induced protein phosphatase 2A methylation, Bcl-2 dephosphorylation, caspase-2 activation, and apoptosis.

Lithium confers cell protection against stress and toxic stimuli. Although lithium inhibits a number of enzymes, the antiapoptotic mechanisms of lithium remain unresolved. Here, we report a novel role of lithium on the blockage of ceramide- and etoposide-induced apoptosis via inhibition of protein phosphatase 2A (PP2A) activity. Overexpression of PP2A resulted in caspase-2 activation, mitochondrial damage, and cell apoptosis that were inhibited by okadaic acid (OA) and lithium. Lithium and OA abrogated ceramide- and etoposide-induced Bcl-2 dephosphorylation at serine 70. Furthermore, ceramide- and etoposide-induced PP2A activation involved methylation of PP2A C subunit, which lithium suppressed. Lithium caused dissociation of PP2A B subunit from the PP2A core enzyme, whereas ceramide caused recruitment of the B subunit. Taken together, lithium exhibited an antiapoptotic effect by inhibiting Bcl-2 dephosphorylation and caspase-2 activation, which involved, at least in part, a mechanism of down-regulating PP2A methylation and PP2A activity.

GMI, an immunomodulatory protein from Ganoderma microsporum, induces autophagy in non-small cell lung cancer cells

Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.

Mechanism of cell death induced by cationic dendrimers in RAW 264.7 murine macrophage-like cells.

Cationic dendrimers possess attractive nano‐sized architectures that make them suitable as targeted drug/gene delivery systems. However, very little is known about their mechanisms of cell death in cellular systems. In the current study, the apoptotic and necrotic effects of starburst polyamidoamine (PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage‐like cells were investigated. Cationic dendrimer treatment produced a typically dose‐dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with cationic dendrimers. Analysis from flow cytometry demonstrated an increase in hypodiploid DNA population (sub‐G1) and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers. Also, cells treated with DAB dendrimer induced a higher percentage of sub‐G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pre‐treatment of RAW 264.7 cells with the general caspase inhibitor zVAD‐fmk prevented some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involves a caspasedependent pathway. Macrophage cells were also found to be sensitive to induction of apoptosis by dendrimers, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These results could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.

Genetic Requirements for Klebsiella pneumoniae-Induced Liver Abscess in an Oral Infection Model

ABSTRACT Klebsiella pneumoniae is the predominant pathogen of primary liver abscess. However, our knowledge regarding the molecular basis of how K. pneumoniae causes primary infection in the liver is limited. We established an oral infection model that recapitulated the characteristics of liver abscess and conducted a genetic screen to identify the K. pneumoniae genes required for the development of liver abscess in mice. Twenty-eight mutants with attenuated growth in liver or spleen samples out of 2,880 signature-tagged mutants that produced the wild-type capsule were identified, and genetic loci which were disrupted in these mutants were identified to encode products with roles in cellular metabolism, adhesion, transportation, gene regulation, and unknown functions. We further evaluated the virulence attenuation of these mutants in independent infection experiments and categorized them accordingly into three classes. In particular, the class I and II mutant strains exhibited significantly reduced virulence in mice, and most of these strains were not detected in extraintestinal tissues at 48 h after oral inoculation. Interestingly, the mutated loci of about one-third of the class I and II mutant strains encode proteins with regulatory functions, and the transcript abundances of many other genes identified in the same screen were markedly changed in these regulatory mutant strains, suggesting a requirement for genetic regulatory networks for translocation of K. pneumoniae across the intestinal barrier. Furthermore, our finding that preimmunization with certain class I mutant strains protected mice against challenge with the wild-type strain implied a potential application for these strains in prophylaxis against K. pneumoniae infections.

Evaluation of internal consistency and re-test reliability of Bath ankylosing spondylitis indices in a large cohort of adult and juvenile spondylitis patients in Taiwan

The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Score (BAS-G) have been recommended for evaluating function and disability in patients with ankylosing spondylitis (AS). The aim of this study was to develop a Chinese version of the BASDAI, BASFI, and BAS-G and assess their reliability and validity. The Chinese version was obtained after a translation and back-translation process. A total of 447 patients with adult and juvenile AS were assessed using these three instruments. Reliability was tested by internal consistency and test–retest reliability. Internal consistency of the instrument was given as Cronbach’s alpha. Test–retest reliability was assessed by intraclass correlation coefficient. To assess the sensitivity to change, 153 patients were included in an 8-week follow-up study. In our analysis, the reliability of these three instruments—the BASDAI, BASFI, and BAS-G—for a 24-h test–retest showed acceptable intraclass correlation coefficients (0.92–0.94). Our Chinese versions of the BASDAI, BASFI, and BAS-G also showed 0.87, 0.94, and 0.90, respectively, with Cronbach’s alpha coefficient, indicating good reliability. For sensitivity to change in 8-week follow-up, all three instruments showed 5.0 to 5.4% changes. Our Chinese versions of the BASDAI, BASFI, and BAS-G showed adequate reliability, validity, and responsiveness to clinical change. Thus, disease activity and functional status in Chinese-speaking patients with AS may be adequately evaluated with these versions of the original instruments.

Eps8 Protein Facilitates Phagocytosis by Increasing TLR4-MyD88 Protein Interaction in Lipopolysaccharide-stimulated Macrophages

Background: TLR4-mediated p38 MAPK and actin cytoskeleton reorganization are important for macrophage phagocytosis. Results: TLR4-induced Eps8 facilitates TLR4-MyD88 interaction, leading to the activation of p38 MAPK, actin polymerization, and the uptake of bacteria in macrophages. Conclusion: Eps8 is critical in innate immunity. Significance: This is the first report to indicate that Eps8 contributes to efficient phagocytosis in macrophages. Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97Eps8) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.

Protective role of autophagy in branched polyethylenimine (25K)- and poly(L-lysine) (30–70K)-induced cell death

Polyethylenimine (PEI) and poly(L-lysine) (PLL), which are cationic polymers used for gene therapy, are known to be cytotoxic, but their molecular mechanisms of cell death are not fully understood. In this study, we provide evidence that PEI and PLL induced autophagy in HeLa cervical cancer cells. In cells overexpressed with green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) fusion protein, PEI and PLL induced fluorescent puncta formations that represent LC3 recruitment to autophagosomes. In Western blot analysis, conversions of the LC3-I to LC3-II were significant, and p62 degradation was observed in cells treated with PEI and PLL. At higher doses, the ability of endosomal escape by PEI facilitates the conversion of LC3-I to LC3-II without the use of lysosomal protease inhibitors. From the analysis of annexin V-flourescein isothiocyanate (FITC) and propidium iodide (PI) staining by flow cytometry, both apoptosis and necrosis occurred in PEI- and PLL-treated cells. Significant activated caspase-3 expression was detected in PLL- and PEI-treated cells. By applying Z-VAD apoptotic inhibition, apoptosis and autophagy may occur independently or autophagy may be in the upstream of apoptosis on PEI- and PLL-treated cells. The degree of cell death was higher in incubated HeLa cells treated with PEI or PLL plus autophagy inhibitors (3-methyladenine (3-MA) and wortmannin). Treatment with these autophagy inhibitors, however, did not inhibit LC3-II formation specifically. In addition, PEI and PLL induced higher degree of cell death in atg5(-/-) mouse embryonic fibroblast (MEF) cells than in wild-type cells. Autophagy was also induced in PEI- and PLL-treated MEFs, as evidenced by the formation of LC3-II in wild-type-but not in atg5(-/-) MEFs. These results indicate that PEI and PLL can trigger both death and survival pathways simultaneously, and autophagy played a role in cell survival in PEI- and PLL-treated cells. Our study therefore provides deeper insight into the molecular mechanisms of cell death caused by cationic polymers.

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.