GLUT5 regulation by AKT1/3-miR-125b-5p downregulation induces migratory activity and drug resistance in TLR-modified colorectal cancer cells.

In cancer, resistance to chemotherapy is one of the main reasons for therapeutic failure. Cells that survive after treatment with anticancer drugs undergo various changes, including in cell metabolism. In this study, we investigated the effects of AKT-mediated miR-125b-5p alteration on metabolic changes and examined how these molecules enhance migration and induce drug resistance in colon cancer cells. AKT1 and AKT3 activation in drug-resistant colon cancer cells caused aberrant downregulation of miR-125b-5p, leading to GLUT5 expression. Targeted inhibition of AKT1 and AKT3 restored miR-125b-5p expression and prevented glycolysis- and lipogenesis-related enzyme activation. In addition, restoring the level of miR-125b-5p by transfection with the mimic sequence not only significantly blocked the production of lactate and intracellular fatty acids but also suppressed the migration and invasion of chemoresistant colon cancer cells. GLUT5 silencing with small interfering RNA attenuated mesenchymal marker expression and migratory activity in drug-resistant colon cancer cells. Additionally, treatment with 2,5-anhydro-d-mannitol resensitized chemoresistant cancer cells to oxaliplatin and 5-fluorouracil. In conclusion, our findings suggest that changes in miR-125b-5p and GLUT5 expression after chemotherapy can serve as a new marker to indicate metabolic change-induced migration and drug resistance development.

Modified TLR-mediated downregulation of miR-125b-5p enhances CD248 (endosialin)-induced metastasis and drug resistance in colorectal cancer cells.

CD248, also called endosialin or tumor endothelial marker-1, is markedly upregulated in almost all cancers, including colon cancers. Changes in microRNA profiles are one of the direct causes of cancer development and progression. In this study, we investigated whether a change in CD248 expression in colon cancer cells could induce drug resistance after chemotherapy, and we explored the relationship between miR-125b-5p levels and CD248 expression in Toll-like receptor (TLR)-modified chemoresistant colon cancer cells. TLR2/6 and TLR5 upregulation in drug-resistant colon cancer cells contributed to miR-125b-5p downregulation and specificity protein 1 (Sp1)-mediated CD248 upregulation via nuclear factor-kappa B (NF-κB) activation. Exposure to specific TLR2/6 or TLR5 ligands enhanced the expression of mesenchymal markers as well as the migratory activity of oxaliplatin- or 5-fluorouracil-resistant colon cancer cells. The transfection of a synthetic miR-125b-5p mimic into chemoresistant cells prevented Sp1 and CD248 activation and significantly impaired invasive activity. Furthermore, Sp1 or CD248 gene silencing as well as miR-125b-5p overexpression markedly reversed drug resistance and inhibited epithelial-mesenchymal transition in colon cancer cells. Taken together, these results suggest that changes in miR-125b-5p levels play an important role in Sp1-mediated CD248 expression and the development of drug resistance in TLR-mutated colon cancer cells.

Gliotoxin Enhances Autophagic Cell Death via the DAPK1-TAp63 Signaling Pathway in Paclitaxel-Resistant Ovarian Cancer Cells.

Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.

Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34+ Cells, Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110δ Pathway.

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.

Delivery of miR-155 to retinal pigment epithelial cells mediated by Burkitt's lymphoma exosomes.

Exosomes are extracellularly secreted vesicles ranging from 40 to 100 nm in diameter that are thought to play important roles in intercellular communication. Exosomes contain numerous proteins, RNA, and lipids that can affect the status of recipient cells under various pathological conditions. MicroRNAs (miRNAs) are small non-coding RNAs that play a major role in post-transcriptional gene silencing by interacting with the 3'-untranslated regions of target genes. Epstein-Barr virus (EBV) has been reported to induce sustained elevation of cellular miRNAs such as miR-155. We hypothesized that miRNAs delivered by exosomes might affect the angiogenesis of retinal pigment epithelial (RPE) cells. Here, we demonstrated that co-culture of EBV-positive Burkitt's lymphoma (BL) cells (Raji) with retinal pigment epithelial (ARPE-19) cells increased the level of miR-155 in recipient cells whereas no major difference was detected for co-culture with EBV-negative BL cells (Ramos). Isolated Raji exosomes increased transcriptional and translational levels of VEGF-A in ARPE-19 cells, which was reversely correlated with von Hippel-Lindau expression. A human umbilical vein endothelial cell tube formation assay showed that delivery of ectopic miR-155 rendered ARPE-19 cells proangiogenic. Our results demonstrate that sustained accumulation of miR-155 mediated by exosomes might affect remote recipient cells such as retinal pigment epithelial cells.

A bioisosteric approach to the discovery of novel N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide-based activators of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation.

Inhibition of translation initiation has emerging implications for the development of mechanism-based anticancer therapeutics. Phosphorylation of eIF2α is recognized as a key target that regulates the translation initiation cascade. Based on the bioisosteric replacement of urea-derived eIF2α phosphorylation activator 1, a novel series of N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide derivatives was designed and synthesized; their effects on the activation of eIF2α phosphorylation was assessed systematically. A brief structure-activity relationship analysis was established by stepwise structural optimization of the squaramide series. Subsequently, the antiproliferative activities of the selected analogues were determined in human leukemia K562 cells. We then identified 10 potent eIF2α phosphorylation activators with considerable anticancer activity. The most promising analogues 19 and 40 possessed higher cancer cell selectivity (SI = 6.16 and 4.83, respectively) than parent 1 (SI = 2.20). Finally, protein expression analysis revealed that compounds 19 and 40 induced eIF2α phosphorylation and its downstream effectors ATF4 and CHOP.

B7-H4 reverse signaling induces the apoptosis of EBV-transformed B cells through Fas ligand up-regulation.

B7-H4 has an inhibitory effect on immune responses via the down-regulation of T cell-mediated immunity, but how the engagement of B7-H4 molecules by counter molecules affects the signaling mechanism of the B7-H4-expressing cells is poorly defined. In this study, we found that B7-H4 expression was enhanced on B cells infected with Epstein-Barr virus (EBV) and that triggering of these molecules induced apoptosis of EBV-transformed B cells. Engagement of B7-H4 initially increased intracellular level of ROS, which then induced the expression of FasL. Engagement of B7-H4 subsequently provoked Fas-mediated and caspase-dependent apoptosis in association with cytochrome c and AIF, and EndoG was released from the mitochondria on EBV-transformed B cells. These results suggest that B7-H4 may be a potential therapeutic target for EBV involved malignancy diseases.

Sequential treatment with celecoxib and bortezomib enhances the ER stress-mediated autophagy-associated cell death of colon cancer cells.

Treatment with celecoxib and bortezomib as single chemotherapeutic agents reduces the viability and proliferation of colorectal cancer cells. The use of these agents in combination with other chemotherapeutic agents is usually associated with adverse effects. In the present study, a combination of celecoxib and bortezomib was investigated for potential synergistic effects in colon cancer cells. The sequential exposure to celecoxib with bortezomib synergistically induced apoptotic death in human colon cancer cells compared with groups treated with a single drug or other drug combinations. c-Jun N-terminal kinase/p38-mitogen-activated protein kinase-induced endoplasmic reticulum (ER) stress through serial exposure to celecoxib and bortezomib may have induced the intracellular Ca2+ release, leading to the generation of autophagosomes in p53-expressing HCT-116 cells. Targeted inhibition of p53 activity or ER stress or treatment with the Ca2+-chelating agent BAPTA-AM suppressed the ER stress-mediated Ca2+ release and apoptosis. Although p53-/- HCT-116 cells were less sensitive to sequential treatment with celecoxib and bortezomib, co-localization of autophagosomes was detected in the absence of CCAAT-enhancer-binding protein homologous protein expression. Treatment of p53-/- HCT-116 cells with BAPTA-AM did not inhibit apoptosis following serial treatment with celecoxib and bortezomib. These results suggest that the order of drug administration is important in treating cancer and that the sequential treatment with celecoxib and bortezomib enhances the ER stress-mediated autophagy-associated cell death of colon cancer cells, regardless of p53 expression.

Transforming growth factor-ß1 regulates human renal proximal tubular epithelial cell susceptibility to natural killer cells via modulation of the NKG2D ligands.

Transforming growth factor-ß (TGF-ß) has a significant role in the response to injury and tissue repair, and it has been detected in various cell types. However, the mechanism by which it regulates the response to ischemia­reperfusion injury (IRI) and manipulates natural killer (NK) cells is not well understood. In the present study, TGF­ß modulated NK cell function, thereby promoting recovery from renal IRI. Human renal proximal tubular epithelial cells (HK­2) treated with TGF­ß exhibited increased surface and intracellular expression of the NK group 2 member D (NKG2D) ligand MICA. This increased surface expression of MICA inhibited NK cell cytotoxicity to the HK­2 cells. In addition, an enzyme­linked immunosorbent assay revealed that TGF­ß treatment evidently increased the amount of soluble MICA released into the culture supernatant from HK­2 cells. Taken together, these findings suggest that TGF­ß­induced release of soluble MICA leads to downregulation of NKG2D, thereby preventing NK cell­mediated cytotoxicity toward renal proximal tubular epithelial cells in renal IRI, which in turn improves the survival of these cells.

Corticotropin-releasing factor induces immune escape of cervical cancer cells by downregulation of NKG2D.

Corticotropin-releasing factor (CRF), a coordinator of the body's responses to stress, is found in various cancer tissues and cell lines. However, the exact abilities of CRF to manipulate natural killer (NK) cells during immune response have not been studied. NKG2D is an activating receptor that is expressed on most NK and CD8+ T cells. MHC class I-related chain A (MICA) and UL16-binding protein (ULBP) 1, 2 and 3 are well-known ligands for NKG2D. In the present study, we reported our findings regarding the role of CRF in cervical cancer cell survival. Human cervical cancer cell line, HeLa cells, had significantly higher intracellular expression of UL16-binding protein 2 (ULBP2) following CRF treatment but had only slightly increased surface expression of ULBP2. Notably, MMPi (pan-metalloproteases inhibitor) blocked the release of ULBP2 molecules from the surface of HeLa cells. Furthermore, incubating NK cells with culture supernatants from CRF-treated HeLa cells, which contained soluble NKG2D ligand, reduced NK cell activity by decreasing surface expression of NKG2D. Collectively, downregulation of NKG2D by CRF-induced soluble NKG2D ligand provides a potential mechanism by which cervical cancer cells escape NKG2D-mediated attack under stress conditions.

IDO metabolite produced by EBV-transformed B cells inhibits surface expression of NKG2D in NK cells via the c-Jun N-terminal kinase (JNK) pathway.

Natural Killer cells are known to play a major role in the innate immune response against viral infections and tumor cells. Several viruses, such as CMV, EBV and HIV-1, have acquired strategies to escape elimination by NK cells. In this study, we observed that EBV infection increased expression of IDO on B cells. To evaluate the function of IDO associated with EBV infection, we investigated whether EBV-induced IDO could modulate expression of NK cell-activation receptor, NKG2D. When NK cells were co-incubated with EBV transformed B cells, surface expression of NKG2D was significantly reduced in NK cells. Incubation with L-kynurenine, an IDO metabolite, down-modulated NKG2D expression in NK cells in a dose- and time-dependent manner. Incubation with the JNK inhibitor SP600125 also inhibited NKG2D expression in NK cells. In addition, we observed that the effect of L-kynurenine was blocked by JNK agonist, anisomycin, suggesting the involvement of the JNK pathway in the signal transduction of L-kynurenine-reduced NKG2D expression. Furthermore, IL-18 significantly reduced L-kynurenine-induced down-regulation of NKG2D expression in NK cells. Taken together, these data indicate that down-regulation of NKG2D by EBV-induced IDO metabolite provides a potential mechanism by which EBV escapes NKG2D-mediated attack by immune cells.