Yung-Lung Chang

Different roles of p53 in the regulation of DNA damage caused by 1,2-heteroannelated anthraquinones and doxorubicin

The anthracyclin antibiotic agent doxorubicin (DXR) has been widely used as a chemotherapeutic drug for more than 40 years, but its clinical use has been limited by its cardiotoxicity. The mechanism of action of DXR remains uncertain and controversial. A series of 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione compounds were synthesized and their cytotoxicity profiles were analyzed using the National Cancer Institute 60 (NCI 60) platform and human telomerase inhibition assays. In the current study, three of the 1,2-heteroannelated anthraquinones, NSC745795, NSC745885 and NSC745887, were found to differ from each other with respect to their effects on cell cycle regulation, apoptosis, autophagy, senescence and their abilities to induce DNA damage. The differences depended on the presence or absence of a heterocyclic moiety, which suggested that the differences were due, at least in part, to differential effects on specific cellular targets, such as p53. In contrast to DXR, which induced p53 expression, treatment with NSC745885 resulted in the degradation of several proteins, including p53, via proteasome-dependent and -independent pathways in HeLa cells. These results provide insights into the molecular mechanisms governing cell inhibition by 1,2-heteroannelated anthraquinone derivatives and suggest that these mechanisms could serve as the basis for new structure-based drug designs.

Human Mitochondrial NAD(P)+–Dependent Malic Enzyme Participates in Cutaneous Melanoma Progression and Invasion

Cutaneous melanoma is the most life-threatening neoplasm of the skin, accounting for most of the skin cancer deaths. Accumulating evidence suggests that targeting metabolism is an appealing strategy for melanoma therapy. Mitochondrial NAD(P)(+)-dependent malic enzyme (ME2), an oxidative decarboxylase, was evaluated for its biological significance in cutaneous melanoma progression. ME2 mRNA and protein expression significantly increased during melanoma progression, as evidenced by Gene Expression Omnibus analysis and immunohistochemistry on clinically annotated tissue microarrays, respectively. In addition, ME2 knockdown attenuated melanoma cell proliferation in vitro. ME2 ablation resulted in reduced cellular ATP levels and elevated cellular reactive oxygen species production, which activated the AMP-activated protein kinase pathway and inhibited acetyl-CoA carboxylase. Furthermore, ME2 expression was associated with cell migration and invasion. ME2 knockdown decreased anchorage-independent growth in vitro and tumor cell growth in vivo. These results suggested that ME2 might be an important factor in melanoma progression and a novel biomarker of invasion.

Zac1, an Sp1-like protein, regulates human p21WAF1/Cip1 gene expression in HeLa cells

Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21(WAF1/Cip1) gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity by interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21(WAF1/Cip1) gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.

Zac1 functional interactions mediate AP-1 transcriptional activity

A zinc-finger protein which regulates apoptosis and cell cycle arrest 1 (Zac1) is a novel seven-zinc-finger protein that can bind a specific GC-rich DNA element and has intrinsic transactivation activity; therefore, its role as a transcription factor has been proposed. Zac1 not only promotes cell cycle arrest and apoptosis but also acts as a transcriptional cofactor for nuclear receptors and p53. In this study, we examined the functional roles of mouse Zac1 (mZac1) in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate (PMA), a potent Activator protein 1 (AP-1) activator. At first, we found that mZac1 prolonged and enhanced PMA-induced AP-1 activity in both HeLa and HeLa/p53 shRNA cells. We further identified physical and functional interactions between mZac1 and AP-1 proteins (either c-Jun, c-Fos or both). Finally, we showed that Zac1 might function as a selective coactivator of AP-1, demonstrated by AP-1-dependent transcriptional activation of collagenase, c-Fos and p21(WAF1/Cip1) promoter activities. Identification of AP-1 as a specific target for Zac1-mediated transcriptional events not only establishes a direct link between these two pivotal regulatory proteins but also raises the possibility that Zac1 contributes to certain AP-1-dependent biological effects.

Multiple effects of digoxin on subsets of cancer-associated genes through the alternative splicing pathway.

The signaling characteristics of Na(+)/K(+)-ATPase are distinct from its ion pumping activity. Cardiac glycosides modulate the Na(+)/K(+)-ATPase protein complex upon binding, activate downstream signaling pathways and increase [Ca(2+)]i. Recent studies demonstrate that the depletion of p53 and hypoxia-induced factor 1α proteins is caused by cardiac glycosides. However, the detailed mechanisms governing this process are not well known. In this study, we showed that the depletion of p53 proteins by digoxin involved not only inhibition of protein synthesis but also inhibition at the post-transcriptional level. Post-transcriptional regulation occurs via down-regulation of SRSF3, the primary splicing factor responsible for the switch from p53α to the p53β isoform. Digoxin also modulated G2/M arrest, DNA damage and apoptosis through the p53-dependent pathway in HeLa cells. In addition, digoxin was involved in epithelial-mesenchymal-transition progression via E-cadherin reduction and snail induction. Digoxin had similar effects to caffeine, another SRSF3-reduced agent, on the cell cycle profile and DNA damage of cells. Interestingly, combined digoxin and caffeine treatment blocked cell cycle progression and conferred resistance to cell death via snail induction. These findings demonstrate that down-regulation of splicing factor, such as SRSF3, to alter cell cycle progression, cell death and invasion is a potential target for the drug repositioning of cardiac glycosides.

Sirtuin 1 protects the aging heart from contractile dysfunction mediated through the inhibition of endoplasmic reticulum stress-mediated apoptosis in cardiac-specific Sirtuin 1 knockout mouse model

BACKGROUND The longevity regulator Sirtuin 1 is an NAD+-dependent histone deacetylase that regulates endoplasmic reticulum stress and influences cardiomyocyte apoptosis during cardiac contractile dysfunction induced by aging. The mechanism underlying Sirtuin 1 function in cardiac contractile dysfunction related to aging has not been completely elucidated. METHODS We evaluated cardiac contractile function, endoplasmic reticulum stress, apoptosis, and oxidative stress in 6- and 12month-old cardiac-specific Sirtuin 1 knockout (Sirt1-/-) and control (Sirt1f/f) mice using western blotting and immunohistochemistry. Mice were injected with a protein disulphide isomerase inhibitor. For in vitro analysis, cultured H9c2 cardiomyocytes were exposed to either a Sirtuin 1 inhibitor or activator, with or without a mitochondrial inhibitor, to evaluate the effects of Sirtuin 1 on endoplasmic reticulum stress, nitric oxide synthase expression, and apoptosis. The effects of protein disulphide isomerase inhibition on oxidative stress and ER stress-related apoptosis were also investigated. RESULTS Compared with 6-month-old Sirt1f/f mice, marked impaired contractility was observed in 12-month-old Sirt1-/- mice. These findings were consistent with increased endoplasmic reticulum stress and apoptosis in the myocardium. Measures of oxidative stress and nitric oxide synthase expression were significantly higher in Sirt1-/- mice compared with those in Sirt1f/f mice at 6months. In vitro experiments revealed increased endoplasmic reticulum stress-mediated apoptosis in H9c2 cardiomyocytes treated with a Sirtuin 1 inhibitor; the effects were ameliorated by a Sirtuin 1 activator. Moreover, consistent with the in vitro findings, impaired cardiac contractility was demonstrated in Sirt1-/- mice injected with a protein disulphide isomerase inhibitor. CONCLUSION The present study demonstrates that the aging heart is characterized by contractile dysfunction associated with increased oxidative stress and endoplasmic reticulum stress and Sirtuin 1 might have the ability to protect the aging hearts from the inhibition of endoplasmic reticulum-mediated apoptosis.

Procainamide Inhibits DNA Methylation and Alleviates Multiple Organ Dysfunction in Rats with Endotoxic Shock

Excessive inflammatory and oxidative stress lead to circulatory failure, multiple organ dysfunction, and high mortality in patients with sepsis. Microbial infection-induced DNA hypermethylation is associated with the augmentation of inflammation and oxidative stress. In our previous study, the antiarrhythmic drug procainamide inhibits the expression of DNA methyltransferase 1 (DNMT1) and diminishes IL-6 levels in rats with rhabdomyolysis. Thus, we further evaluated the effects of procainamide on the development of circulatory failure and multiple organ dysfunction in rats with endotoxic shock. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS) followed by procainamide administration. The changes of hemodynamics, blood glucose, biochemical variables, and plasma nitric oxide (NO) levels were analyzed during the experimental period. At the end of experiments, animal organs were also obtained for examining superoxide production, neutrophil infiltration, and DNA methylation status. Our results showed that LPS induced circulatory failure, multiple organ dysfunction, and high mortality rate in endotoxemic rats. Overt neutrophil infiltration and superoxide production, accompanied by the elevations of DNMT1 and 5-methylcytosine levels in the lung of endotoxemic rats were also observed. Treatment of endotoxemic animals with procainamide not only inhibited the increased levels of DNMT1 and 5-methylcytosine but also ameliorated neutrophil infiltration and superoxide production in the lung. In addition, the anti-inflammatory gene, IL27RA, was down-regulated in the LPS group and up-regulated in the LPS + Procainamide group. Procainamide also diminished IL27RA methylation in the lung of endotoxemic rat. Moreover, both DNMT inhibitors procainamide and hydralazine improved hypotension, hypoglycemia, and multiple organ dysfunction of LPS-treated rats. Thus, we suggest that the beneficial effects of procainamide could be attributed to the suppression of DNA methylation, neutrophil infiltration, superoxide production, and NO formation. It seems that this old drug may have new potential uses in infectious diseases, in particular, associated with endotoxemia.

CD164 promotes lung tumor-initiating cells with stem cell activity and determines tumor growth and drug resistance via Akt/mTOR signaling

CD164 is a cell adhesion molecule that increases hematopoietic stem cell proliferation, adhesion, and migration via C-X-C chemokine receptor type 4 (CXCR4) signaling. Emerging evidence indicates that elevated CD164 expression is associated with aggressive metastasis, advanced stages, and shorter overall survival in lung cancer. However, no data are available regarding the clinical significance of CD164 expression in lung cancer. This study explores whether CD164 promotes tumor-initiation and drug resistance through the stem cell property. Using tissue microarrays, we determine that CD164 expression is correlated with clinicopathological characteristics in human lung cancer. The CD164 overexpression in normal lung epithelial cells (BEAS2B cells) leads to malignant transformation in vitro, tumorigenicity in xenografted mice, stem cell-like property, and drug resistance through ATP-binding cassette transporters. The CD164 overexpression increases CXCR4 expression and activates Akt/mTOR signaling. Rapamycin, an mTOR inhibitor, hinders cell proliferation along with sphere formation in vitro and impedes tumor growth in vivo. In conclusion, we have provided evidence that CD164 promotes the growth of lung tumor-initiating cells with stem cell properties and induces tumor growth and drug resistance through Akt/mTOR signaling. Therefore, identification of CD164 as a cancer stem cell therapeutic marker may develop an effective therapy in patients with chemoresistant lung cancer.

The subcellular localization and protein stability of mouse alpha-actinin 2 is controlled by its nuclear receptor binding motif in C2C12 cells

Alpha actinin (ACTN) has emerged as a multitasking protein, whose roles range from bundling actin filaments to functioning as a versatile protein interaction platform for proteins involved in structural or signaling aspects. We report here that ACTN2, one of the four ACTN isoforms, may shuttle between the cytoplasm and nucleus where the nuclear exportation takes place in a CRM1-dependent manner. The majority of ACTN2 was found to localize in the cytoplasm and exhibit a lower stability which was demonstrated using either mutants carrying mutated nuclear receptor binding motif or inhibitors against the ubiquitin- and calpain-dependent degradation pathways. Horse serum induced differentiation of C2C12 cells also caused the redistribution of nuclear ACTN2 to the cytoplasm, which subcellular compartment the ACTN2 behaves as an unstable protein. Our data indicated that the model in which ACTN2 functions as a multi-talented coregulator may be controlled by the differential protein stability modulated via nucleo-cytoplasmic trafficking in C2C12 cells.

The regulation of NLRP3 inflammasome expression during the development of cardiac contractile dysfunction in chronic kidney disease

Chronic inflammation plays a crucial role in the long-term complications in patients with chronic kidney disease (CKD). This study aimed to assess the role of NLR pyrin domain-containing protein (NLRP3) inflammasome in cardiac contractile dysfunctions in CKD. The cardiac contractile function was evaluated and the expression of NLRP3 inflammasome and related cytokines in the heart was assessed in a murine sham-operated and 5/6 nephrectomy CKD model in vivo. In vitro, H9c2 cells were treated with uremic toxin indoxyl sulfate (IS), with or without NLRP3 inflammasome inhibition, which was achieved by using small interfering RNA (siRNA)-mediated knockdown of the NLRP3 gene. Moreover, the activation of nuclear factor κB (NF-κB) signaling and apoptosis marker levels were assessed in the IS-treated H9c2 cells. The results demonstrated that CKD can lead to the development of cardiac contractile dysfunction in vivo associated with the upregulation of NLRP3 inflammasome, IL-1β, IL-18, and contribute to the myocardial apoptosis. In vitro experiments showed the upregulation of inflammasome, IL-1β, and IL-18 levels, and cell apoptosis in the IS-treated H9c2 cells through the activation of NF-κB signaling pathway. The transfection of cells with si-NLRP3 was shown to alleviate IL-1β, IL-18, and cell apoptosis. Moreover, decreased cell viability induced by IS was shown to be attenuated by IL-1β or IL-18-neutralizing antibody. In summary, CKD can result in the development of cardiac contractile dysfunction associated with the upregulation of NLRP3 inflammasome/IL-1β/IL-18 axis induced by the uremic toxins.