Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20.

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55 × CD20) that simultaneously targets both CD55 and CD20 to effectively eliminate rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55 × CD20 antibody showed significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, the asymmetric bispecific antibody (SBU-CD55 × CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells compared to other bispecific antibodies with symmetric features. These results demonstrate that enhancing CDC with an asymmetric CD55-binding bispecific antibody could be a new strategy for developing therapeutics to treat patients with relapsed or refractory cancers.

Allomyrina dichotoma larva extract attenuates free fatty acid-induced lipotoxicity in pancreatic beta cells.

BACKGROUD/OBJECTIVES: Allomyrina dichotoma larva (ADL), one of the many edible insects recognized as future food resources, has a range of pharmacological activities. In a previous study, an ADL extract (ADLE) reduced the hepatic insulin resistance of high-fat diet (HFD)-induced diabetic mice. On the other hand, the associated molecular mechanisms underlying pancreatic beta-cell dysfunction remain unclear. This study examined the effects of ADLE on palmitate-induced lipotoxicity in a beta cell line of a rat origin, INS-1 cells. MATERIALS/METHODS: ADLE was administered to high-fat diet treated mice. The expression of apoptosis-related molecules was measured by Western blotting, and reactive oxidative stress generation and nitric oxide production were measured by DCH-DA fluorescence and a Griess assay, respectively. RESULTS: The administration of ADLE to HFD-induced diabetic mice reduced the hyperplasia, 4-hydroxynonenal levels, and the number of apoptotic cells while improving the insulin levels compared to the HFD group. Treatment of INS-1 cells with palmitate reduced insulin secretion, which was attenuated by the ADLE treatment. Furthermore, the ADLE treatment prevented palmitate-induced cell death in INS-1 cells and isolated islets by reducing the apoptotic signaling molecules, including cleaved caspase-3 and PARP, and the Bax/Bcl2 ratio. ADLE also reduced the levels of reactive oxygen species generation, lipid accumulation, and nitrite production in palmitate-treated INS-1 cells while increasing the ATP levels. This effect corresponded to the decreased expression of inducible nitric oxide synthase (iNOS) mRNA and protein. CONCLUSIONS: ADLE helps prevent lipotoxic beta-cell death in INS-1 cells and HFD-diabetic mice, suggesting that ADLE can be used to prevent or treat beta-cell damage in glucose intolerance during the development of diabetes.

Allomyrina dichotoma larval extract attenuates intestinal barrier disruption by altering inflammatory response and tight junction proteins in lipopolysaccharide-induced Caco-2 cells.

Intestinal epithelial cells form a barrier between the intestinal lumen and host connective tissues and play an important role in maintaining intestinal nutrient homeostasis. This study investigated effects of Allomyrina dichotoma (rhinoceros beetle) larval extract (ADLE) on the intestinal barrier damage and explored mechanisms for reversing intestinal barrier dysfunction in lipopolysaccharide (LPS)-stimulated Caco-2, human intestinal epithelial cells. LPS reduced intestinal epithelial barrier function by increasing transepithelial electrical resistance, and this effect was significantly attenuated by ADLE treatment. ADLE also significantly countered the inhibition of tight junction-related protein expression in both LPS-induced Caco-2 cells and intestine from HFD-induced mice. Moreover, ADLE significantly decreased expression and production of inflammatory factors, such as iNOS, cox-2, nitric oxide, and cytokines induced by LPS stimulus. Reduction in phosphorylation of adenosine monophosphate-activated protein kinase was averted by ADLE treatment in LPS treated INS-1 cells. Finally, reactive oxygen stress level was decreased and ATP production was increased by ADLE treatment. ADLE appears to display gut health-promoting effects by reducing inflammation and inducing tight junction proteins in Caco-2 cells. Therefore, ADLE might be useful for preventing or treating intestine cell damage in inflammatory bowel disease.

Upregulation of caveolin-1 and its colocalization with cytokine receptors contributes to beta cell apoptosis.

Caveolin-1 (cav-1), the principal structural and signalling protein of caveolae, is implicated in various signalling events, including apoptotic cell death in type 2 diabetes. However, the precise role of beta cells in apoptosis has not been clearly defined. In this study, we investigated the involvement of cav-1 in cytokine-induced beta cell apoptosis and its underlying mechanisms in the rat beta cell line, INS-1 and isolated islets. Treatment of cytokine mixture (CM, TNFα + IL-1ß) significantly increased the mRNA and protein expression of cav-1, and resulting in increased formation of caveolae. We found that IL-1 receptor 1 and TNF receptor localized to plasma membrane lipid rafts in the control cells and CM treatment recruited these receptors to the caveolae domain. After cav-1 siRNA transfection, CM-dependent NF-κB activation was reduced and consequently downregulated the mRNA expression of iNOS and IL-1ß. Finally, decreased cell viability by CM treatment was ameliorated in both INS-1 cells and isolated islets treated with cav-1 siRNA. These results suggest that increased cav-1 expression and recruitment of cytokine receptors into caveolae contribute to CM-induced beta cell apoptosis.

Allomyrina dichotoma Larva Extract Ameliorates the Hepatic Insulin Resistance of High-Fat Diet-Induced Diabetic Mice.

Allomyrina dichotoma larva is a nutritional-worthy future food resource and it contributes to multiple pharmacological functions. However, its antidiabetic effect and molecular mechanisms are not yet fully understood. Therefore, we investigated the hypolipidemic effect of A. dichotoma larva extract (ADLE) in a high-fat diet (HFD)-induced C57BL/6J mice model. Glucose tolerance and insulin sensitivity in HFD-induced diabetic mice significantly improved after ADLE administration for six weeks. The levels of serum triglyceride (TG), aspartate aminotransferase (AST), alanine transferase (ALT) activity, and lipid accumulation were increased in the liver of HFD-fed mice, but the levels were significantly reduced by the ADLE treatment. Moreover, hepatic fibrosis and inflammatory gene expression in the liver from HFD-treated mice were ameliorated by the ADLE treatment. Dephosphorylation of AMP-activated protein kinase (AMPK) by palmitate was inhibited in the ADLE treated HepG2 cells, and subsequently reduced expression of lipogenic genes, such as SREPBP-1c, ACC, and FAS were observed. The reduced expression of lipogenic genes and an increased phosphorylation of AMPK was also observed in the liver from diabetic mice treated with ADLE. In conclusion, ADLE ameliorates hyperlipidemia through inhibition of hepatic lipogenesis via activating the AMPK signaling pathway. These findings suggest that ADLE and its constituent bioactive compounds are valuable to prevent or treat hepatic insulin resistance in type 2 diabetes.

MicroRNA-181c Inhibits Interleukin-6-mediated Beta Cell Apoptosis by Targeting TNF-α Expression.

We have previously reported that long-term treatment of beta cells with interleukin-6 (IL-6) is pro-apoptotic. However, little is known about the regulatory mechanisms that are involved. Therefore, we investigated pro-apoptotic changes in mRNA expression in beta cells in response to IL-6 treatment. We analyzed a microarray with RNA from INS-1 beta cells treated with IL-6, and found that TNF-α mRNA was significantly upregulated. Inhibition of TNF-α expression by neutralizing antibodies significantly decreased annexin V staining in cells compared with those treated with a control antibody. We identified three microRNAs that were differentially expressed in INS-1 cells incubated with IL-6. In particular, miR-181c was significantly downregulated in IL-6-treated cells compared with control cells and the decrease of miR-181c was attenuated by STAT-3 signaling inhibition. TNF-α mRNA was a direct target of miR-181c and upregulation of miR-181c by mimics, inhibited IL-6-induced increase in TNF-α mRNA expression. Consequently, reduction of TNF-α mRNA caused by miR-181c mimics enhanced cell viability in IL-6 treated INS-1 cells. These results demonstrated that miR-181c regulation of TNF-α expression plays a role in IL-6-induced beta cell apoptosis.

Liquiritigenin prevents palmitate-induced beta-cell apoptosis via estrogen receptor-mediated AKT activation.

Liquiritigenin (LQ) is a major active component of licorice root, which is a flavone used for treating many diseases, including diabetes. LQ has been shown to exhibit a glucose-lowering effect in diabetic mice. Therefore, we investigated the potential of LQ to protect against lipotoxicity-induced beta-cell apoptosis and the underlying molecular mechanisms. Exposure of INS-1 rat insulinoma cells to LQ significantly increased cell viability and blocked palmitate (PA)-induced apoptosis, as evidenced by the reduction of Annexin-V-stained cells, cleaved caspase-3 levels, and poly (ADP-ribose) polymerase (PARP) activity, as well as upregulation of Bcl-2 expression. Moreover, LQ treatment significantly reduced the endoplasmic reticulum (ER) stress response by reducing phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylated eIF-2a, and CHOP expression in PA-treated INS-1 cells. The anti-apoptotic effect of LQ treatment was reversed through co-treatment with fulvestrant, a specific inhibitor of the estrogen receptor. LQ also increased AKT phosphorylation, and inactivation of this molecular event failed to decrease PERK phosphorylation with LQ treatment in PA-treated INS-1 cells. This effect was further accompanied by an inability to recover cell viability. These results suggest that LQ protects INS-1 cells from lipotoxicity-induced apoptosis by suppressing ER stress. We conclude that estrogen receptor-mediated AKT phosphorylation is one of the mechanisms contributing to the anti-apoptotic effect of LQ.