Sodium-Glucose Co-Transporter-2 Inhibitor Empagliflozin Attenuates Sorafenib-Induced Myocardial Inflammation and Toxicity.

Environmental antineoplastics such as sorafenib may pose a risk to humans through water recycling, and the increased risk of cardiotoxicity is a clinical issue in sorafenib users. Thus, developing strategies to prevent sorafenib cardiotoxicity is an urgent work. Empagliflozin, as a sodium-glucose co-transporter-2 (SGLT2) inhibitor for type 2 diabetes control, has been approved for heart failure therapy. Still, its cardioprotective effect in the experimental model of sorafenib cardiotoxicity has not yet been reported. Real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to study the effect of sorafenib exposure on cardiac SGLT2 expression. The impact of empagliflozin on cell viability was investigated in the sorafenib-treated cardiomyocytes using Alamar blue assay. Immunoblot analysis was employed to delineate the effect of sorafenib and empagliflozin on ferroptosis/proinflammatory signaling in cardiomyocytes. Ferroptosis/DNA damage/fibrosis/inflammation of myocardial tissues was studied in mice with a 28-day sorafenib ± empagliflozin treatment using histological analyses. Sorafenib exposure significantly promoted SGLT2 upregulation in cardiomyocytes and mouse hearts. Empagliflozin treatment significantly attenuated the sorafenib-induced cytotoxicity/DNA damage/fibrosis in cardiomyocytes and mouse hearts. Moreover, GPX4/xCT-dependent ferroptosis as an inducer for releasing high mobility group box 1 (HMGB1) was also blocked by empagliflozin administration in the sorafenib-treated cardiomyocytes and myocardial tissues. Furthermore, empagliflozin treatment significantly inhibited the sorafenib-promoted NFκB/HMGB1 axis in cardiomyocytes and myocardial tissues, and sorafenib-stimulated proinflammatory signaling (TNF-α/IL-1ß/IL-6) was repressed by empagliflozin administration. Finally, empagliflozin treatment significantly attenuated the sorafenib-promoted macrophage recruitments in mouse hearts. In conclusion, empagliflozin may act as a cardioprotective agent for humans under sorafenib exposure by modulating ferroptosis/DNA damage/fibrosis/inflammation. However, further clinical evidence is required to support this preclinical finding.

Probing the Internalization and Efficacy of Antibody-Drug Conjugate via Site-Specific Fc-Glycan Labelling of a Homogeneous Antibody Targeting SSEA-4 Bearing Tumors.

Antibody drug conjugates (ADC) are an emerging class of pharmaceuticals consisting of cytotoxic agents covalently attached to an antibody designed to target a specific cancer cell surface molecule followed by internalization and intracellular release of payload to exhibit its anticancer activity. Targeted delivery of cytotoxic payload to a variety of specific cells has been demonstrated to have significant enhancement in clinical efficacy and dramatic reduction in off-target toxicity. Site-specific conjugation of payload to the antibody is highly desirable for development of ADC with well-defined antibody-to-drug ratio, enhanced internalization, reduced toxicity, improved stability, desired pharmacological profile and optimal therapeutic index. Here, we reported a site-specific conjugation strategy for evaluation of antibody internalization and efficacy of ADC designed to target SSEA4 on solid tumors. This strategy stems from the azido-fucose tag of a homogeneous antibody Fc-glycan generated via in vitro glycoengineering approach for site-specific conjugation and optimization of antibody-drug ratio to exhibit optimal efficacy. The ADC consisting of a chimeric anti-SSEA4 antibody chMC813-70, conjugated to the antineo-plastic agent monomethyl auristatin E via both cleavable and non-cleavable linkers showed excellent cytotoxicity profile towards SSEA4-bearing cancer cells. A clear distinction in cytotoxicity was observed among cancer cells with different SSEA4 expression levels.

Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

Stage-specific embryonic antigen-3 (SSEA-3) and ß3GalT5 are cancer specific and significant markers for breast cancer stem cells.

The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. However, the markers currently used for isolation of CSCs are often not selective enough to enrich CSCs for the study of this special cell population. Here we show that the breast CSCs isolated with CD44(+)CD24(-/lo)SSEA-3(+) or ESA(hi)PROCR(hi)SSEA-3(+) markers had higher tumorigenicity than those with conventional markers in vitro and in vivo. As few as 10 cells with CD44(+)CD24(-/lo)SSEA-3(+) formed tumor in mice, compared with more than 100 cells with CD44(+)CD24(-/lo). Suppression of SSEA-3 expression by knockdown of the gene encoding ß-1,3-galactosyltransferase 5 (ß3GalT5) in the globo-series pathway, led to apoptosis in cancer cells specifically but had no effect on normal cells. This finding is further supported by the analysis of SSEA-3 and the two related globo-series epitopes SSEA4 and globo-H in stem cells (embryonic stem cells and induced pluripotent stem cells) and various normal and cancer cells, and by the antibody approach to target the globo-series glycans and the late-stage clinical trials of a breast cancer vaccine.

Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers.

Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical management, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that eight of 11 GBM cell lines are positive for stage-specific embryonic antigen-4 (SSEA-4), and immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4(+) and correlated with high-grade astrocytoma. In addition, an SSEA-4-specific mAb was found to induce complement-dependent cytotoxicity against SSEA-4(hi) GBM cell lines in vitro and suppressed GBM tumor growth in mice. Because SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines.

Plumbagin Induces Reactive Oxygen Species and Endoplasmic Reticulum Stress-related Cell Apoptosis in Human Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC), a major malignancy in Taiwan, is an invasive epithelial neoplasm resulting in a low survival rate. Current treatments do not prevent OSCC progression, and antitumor therapies should be improved. Plumbagin, a natural compound extracted from Plumbago zeylanica L., appears to have antitumor effects in various tumors. The antitumor mechanism of plumbagin in OSCC is still unclear. This study investigated the molecular mechanism through which plumbagin induces apoptosis. MATERIALS AND METHODS: To investigate the antiproliferative and pro-apoptotic effects of Plumbagin on OSCC cells and explore its underlying mechanism, cell counting kit-8, cell cycle analysis, and annexin V/PI assay were conducted. The functions of plumbagin on endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) deficiency were analyzed using flow cytometric analysis. Plumbagin-induced apoptosis-associated proteins were detected using western blotting. RESULTS: Plumbagin induced apoptosis in OSCC cells by suppressing tumor cell proliferation through ROS production, ER stress, mitochondrial dysfunction, and caspases activation. CONCLUSION: Plumbagin is a promising antitumor candidate targeting human OSCC.

Study on antibody Fc-glycosylation for optimal effector functions.

A comprehensive structure-activity relationship study on antibody Fc-glycosylation has been performed using the chimeric anti-SSEA4 antibody chMC813-70 as a model. The α-2,6 sialylated biantennary complex type glycan was identified as the optimal Fc-glycan with significant enhancement in antibody effector functions, including binding to different Fc receptors and ADCC.

Combined Effect of Anti-SSEA4 and Anti-Globo H Antibodies on Breast Cancer Cells.

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

The trend of macrolide resistance and emm types of group A streptococci from children at a medical center in southern Taiwan.

BACKGROUND: Group A streptococcus (GAS) is a common pathogen in children. Macrolide resistance in GAS has been described worldwide. The aims of this study are to analyze macrolide resistance of GAS isolates in southern Taiwan and to clarify the relationship of emm typing and macrolide resistance in the past decade. METHODS: All GAS isolated from patients younger than 18 years at a single tertiary center in southern Taiwan were collected from 2000 to 2012. Antibiotics susceptibility to erythromycin, azithromycin, and clindamycin were determined by agar dilution method, and were interpreted by Clinical and Laboratory Standards Institute (CLSI) standards. emm typing was performed by polymerase chain reaction (PCR). RESULTS: A total of 301 isolates were collected during the period of 13 years. Scarlet fever (38.5%) and acute pharyngitis (32.2%) were the most common diagnosis. Decreased resistance rate of erythromycin from 53.1% in 2000 to 0% in 2010 was found, but it increased rapidly to 65% in 2011. The resistance rate of azithromycin was the lowest (4.2%) in 2005, but was higher than 15% after 2006. The involvement of the erythromycin resistance genes were mefA (53.1%), ermB (35.9%), and ermTR (10.9%). The resistance of clindamycin also increased since 2011. emm12 was the most common serotype and accounted for 44.9% of all isolates. Compared with the non-emm12 group, resistance to erythromycin, azithromycin, and clindamycin were more frequently detected in the emm12 group. CONCLUSION: Increased resistance of GAS to macrolide and clindamycin was found in recent years. emm12 was the main serotype for macrolide resistance.

Childhood tuberculosis in southern Taiwan, with emphasis on central nervous system complications.

BACKGROUND/PURPOSE: Childhood tuberculosis (TB) continues to be a major public health problem in Taiwan. Taiwan remains a highly endemic area despite neonatal Bacillus Calmette-Guérin (BCG) vaccination and the availability of anti-TB therapy. The presentation is highly variable and it is often difficult to make an accurate diagnosis. This study was designed to evaluate the demographic, clinical, and laboratory findings and outcomes of TB in children with emphasis on central nervous system (CNS) complications. METHODS: The medical records of 80 children diagnosed with TB at a medical center in southern Taiwan over the past 24 years (1988-2012) were reviewed. RESULTS: Among them, 48.8% (39/80) had pulmonary TB, 27.5% (22/80) had isolated extrapulmonary TB, and 23.7% (19/80) had disseminated TB. Most infected cases were aged either < 4 years or > 12 years. TB contact history was found in 42.5% (34/80) cases. Fourteen (17.5%) of the cases had CNS involvement. The most common presentations were fever (85.7%), signs of increased intracranial pressure (71.4%), drowsiness (64.3%), and focal neurological signs (57.1%). The major radiological findings were tuberculoma (50%), basilar enhancement (41.6%), infarction (41.6%), hydrocephalus (16.6%), and transverse myelitis (16.6%). The case fatality of CNS TB was 14.3% and 21.4% had neurologic sequelae. CONCLUSION: Findings suggest that positive exposure history and suspicious clinical presentations are important clues for further confirmatory laboratory and image studies in childhood TB. CNS TB usually presented as part of disseminated TB in children. Early diagnosis and treatment may lead to favorable outcomes in CNS TB.

Role of N-linked glycans in the interactions of recombinant HCV envelope glycoproteins with cellular receptors.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma. It infects human liver cells through several cellular protein receptors including CD81, SR-BI, claudin-1, and occludin. Previous reports also show that lectin receptors can mediate HCV recognition and entry. The envelope proteins of HCV (E1 and E2) are heavily glycosylated, further indicating the possible roles of lectin receptor-virus interaction in HCV infection. However, there is limited study investigating the relationship of HCV envelope glycoproteins and lectin as well as non-lectin receptors. Here we used surface plasmon resonance to examine the binding affinity of different glycoforms of recombinant HCV envelope protein to receptors and inspected the infectivity and assembly of HCV pseudoparticles composed of different glycoforms of envelope proteins. Our results indicated that DC-SIGN, L-SIGN, and Langerin had higher affinity to recombinant HCV envelope proteins in the presence of calcium ions than non-lectin receptors, and envelope proteins with Man8/9 N-glycans showed approximate 10-fold better binding to lectin receptors than envelope proteins with Man5 and complex type N-glycans. Interestingly, comparing among glycoforms, recombinant envelope proteins with Man5 N-glycans showed the highest binding affinity when interacting with non-lectin receptors. In summary, the glycans on HCV envelope protein play a modulatory role in HCV assembly and infection and direct HCV-receptor interaction, which mediates viral entry in different cells. Receptors with high affinity to HCV envelope proteins may be considered as targets for development of a therapeutic strategy against HCV.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor.

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galß1-3 or Galα1-4Galß1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.