Design, synthesis and structure-activity relationship studies of pyrido[2,3-d]pyrimidin-7-ones as potent Janus Kinase 3 (JAK3) covalent inhibitors.

Aberrantly activated Janus kinase 3 (JAK3) has been constantly detected in various immune disorders and hematopoietic cancers, suggesting its potential of being an attractive therapeutic target for these indications. Clinical benefits of drugs selectively targeting JAK3 versus pan-JAK inhibitors remain unclear. In this study, we report the design and synthesis of a new series of JAK3 covalent inhibitors with a pyrido[2,3-d]pyrimidin-7-one scaffold. After the extensive SAR study, compound 10f emerged to be the most potent JAK3 inhibitor with an IC50 value of 2.0 nM. It showed excellent selectively proliferation inhibitory activity against U937 cells harboring JAK3 M511I mutation, while remained weakly active to the other tested cancer cells. Compound 10f also dose-dependently inhibited the phosphorylation of JAK3 and its downstream signal STAT5 in U937 cells. Taken together, 10f may serve as a promising tool molecule for treating cancers with aberrantly activated JAK3.

MicroRNA-29a Manifests Multifaceted Features to Intensify Radiosensitivity, Escalate Apoptosis, and Revoke Cell Migration for Palliating Radioresistance-Enhanced Cervical Cancer Progression.

Radioresistance remains a major clinical challenge in cervical cancer therapy and results in tumor relapse and metastasis. Nevertheless, the detailed mechanisms are still largely enigmatic. This study was conducted to elucidate the prospective impacts of microRNA-29a (miR-29a) on the modulation of radioresistance-associated cervical cancer progression. Herein, we established two pairs of parental wild-type (WT) and radioresistant (RR) cervical cancer cells (CaSki and C33A), and we found that constant suppressed miR-29a, but not miR-29b/c, was exhibited in RR-clones that underwent a dose of 6-Gy radiation treatment. Remarkably, radioresistant clones displayed low radiosensitivity, and the reduced apoptosis rate resulted in augmented surviving fractions, measured by the clonogenic survival curve assay and the Annexin V/Propidium Iodide apoptosis assay, respectively. Overexpression of miR-29a effectively intensified the radiosensitivity and triggered the cell apoptosis in RR-clones. In contrast, suppressed miR-29a modestly abridged the radiosensitivity and abolished the cell apoptosis in WT-clones. Hence, ectopically introduced miR-29a into RR-clones notably attenuated the wound-healing rate and cell migration, whereas reduced miR-29a aggravated cell mobilities of WT-clones estimated via the in vitro wound-healing assay and time-lapse recording assay. Notably, we further established the in vivo short-term lung locomotion metastasis model in BALB/c nude mice, and we found that increased lung localization was shown after tail-vein injection of RR-CaSki cells compared to those of WT-CaSki cells. Amplified miR-29a significantly eliminated the radioresistance-enhanced lung locomotion. Our data provide evidence suggesting that miR-29a is a promising microRNA signature in radioresistance of cervical cancer cells and displays multifaceted innovative roles involved in anti-radioresistance, escalated apoptosis, and anti-cell migration/metastasis. Amalgamation of a nucleoid-based strategy (miR-29a) together with conventional radiotherapy may be an innovative and eminent strategy to intensify the radiosensitivity and further protect against the subsequent radioresistance and the potential metastasis in cervical cancer treatment.

MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells' Migration in a Parallel-Plate Flow Chamber.

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells' adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells' adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells' adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.

Human Umbilical Cord Mesenchymal Stem Cells Extricate Bupivacaine-Impaired Skeletal Muscle Function via Mitigating Neutrophil-Mediated Acute Inflammation and Protecting against Fibrosis.

Skeletal muscle injury presents a challenging traumatological dilemma, and current therapeutic options remain mediocre. This study was designed to delineate if engraftment of mesenchymal stem cells derived from umbilical cord Wharton's jelly (uMSCs) could aid in skeletal muscle healing and persuasive molecular mechanisms. We established a skeletal muscle injury model by injection of myotoxin bupivacaine (BPVC) into quadriceps muscles of C57BL/6 mice. Post BPVC injection, neutrophils, the first host defensive line, rapidly invaded injured muscle and induced acute inflammation. Engrafted uMSCs effectively abolished neutrophil infiltration and activation, and diminished neutrophil chemotaxis, including Complement component 5a (C5a), Keratinocyte chemoattractant (KC), Macrophage inflammatory protein (MIP)-2, LPS-induced CXC chemokine (LIX), Fractalkine, Leukotriene B4 (LTB4), and Interferon-γ, as determined using a Quantibody Mouse Cytokine Array assay. Subsequently, uMSCs noticeably prevented BPVC-accelerated collagen deposition and fibrosis, measured by Masson's trichrome staining. Remarkably, uMSCs attenuated BPVC-induced Transforming growth factor (TGF)-ß1 expression, a master regulator of fibrosis. Engrafted uMSCs attenuated TGF-ß1 transmitting through interrupting the canonical Sma- And Mad-Related Protein (Smad)2/3 dependent pathway and noncanonical Smad-independent Transforming growth factor beta-activated kinase (TAK)-1/p38 mitogen-activated protein kinases signaling. The uMSCs abrogated TGF-ß1-induced fibrosis by reducing extracellular matrix components including fibronectin-1, collagen (COL) 1A1, and COL10A1. Most importantly, uMSCs modestly extricated BPVC-impaired gait functions, determined using CatWalk™ XT gait analysis. This work provides several innovative insights into and molecular bases for employing uMSCs to execute therapeutic potential through the elimination of neutrophil-mediated acute inflammation toward protecting against fibrosis, thereby rescuing functional impairments post injury.

MicroRNA-29a Exhibited Pro-Angiogenic and Anti-Fibrotic Features to Intensify Human Umbilical Cord Mesenchymal Stem Cells-Renovated Perfusion Recovery and Preventing against Fibrosis from Skeletal Muscle Ischemic Injury.

This study was conducted to elucidate whether microRNA-29a (miR-29a) and/or together with transplantation of mesenchymal stem cells isolated from umbilical cord Wharton's jelly (uMSCs) could aid in skeletal muscle healing and putative molecular mechanisms. We established a skeletal muscle ischemic injury model by injection of a myotoxin bupivacaine (BPVC) into gastrocnemius muscle of C57BL/6 mice. Throughout the angiogenic and fibrotic phases of muscle healing, miR-29a was considerably downregulated in BPVC-injured gastrocnemius muscle. Overexpressed miR-29a efficaciously promoted human umbilical vein endothelial cells proliferation and capillary-like tube formation in vitro, crucial steps for neoangiogenesis, whereas knockdown of miR-29a notably suppressed those endothelial functions. Remarkably, overexpressed miR-29a profitably elicited limbic flow perfusion and estimated by Laser Dopple. MicroRNA-29a motivated perfusion recovery through abolishing the tissue inhibitor of metalloproteinase (TIMP)-2, led great numbers of pro-angiogenic matrix metalloproteinases (MMPs) to be liberated from bondage of TIMP, thus reinforced vascular development. Furthermore, engrafted uMSCs also illustrated comparable effect to restore the flow perfusion and augmented vascular endothelial growth factors-A, -B, and -C expression. Notably, the combination of miR29a and the uMSCs treatments revealed the utmost renovation of limbic flow perfusion. Amplified miR-29a also adequately diminished the collagen deposition and suppressed broad-wide miR-29a targeted extracellular matrix components expression. Consistently, miR-29a administration intensified the relevance of uMSCs to abridge BPVC-aggravated fibrosis. Our data support that miR-29a is a promising pro-angiogenic and anti-fibrotic microRNA which delivers numerous advantages to endorse angiogenesis, perfusion recovery, and protect against fibrosis post injury. Amalgamation of nucleic acid-based strategy (miR-29a) together with the stem cell-based strategy (uMSCs) may be an innovative and eminent strategy to accelerate the healing process post skeletal muscle injury.

Radiation-induced increase in cell migration and metastatic potential of cervical cancer cells operates via the K-Ras pathway.

Radiotherapy is a well established treatment for cervical cancer, the second most common cancer in women worldwide. However, metastasis often circumvents the efficacy of radiotherapy. This study was conducted to elucidate the molecular mechanism of radioresistance-associated metastatic potential of cervical cancer cells. We established three radioresistant cervical cancer cell lines by exposure of cells to a sublethal dose of radiation and screened for lines that exhibited an increased migration phenotype for at least 6 months before undertaking mechanistic studies. Radiation-associated metastatic potential was evaluated using a wound-healing assay, time-lapse recording, and cell locomotion into the lungs of BALB/c nude mice. The radioresistant C33A and CaSki cell lines, but not the radioresistant HeLa cell line, exhibited significantly increased cell migration and wound healing than did wild-type cells. Furthermore, K-Ras played a prometastatic role via the activation of c-Raf/p38, whereas interference of those mediators via either RNA interference-mediated knockdown or the use of chemical inhibitors substantially reversed the radioresistance-associated increase in cell migration. Clinical examination further showed the relative up-regulation of the K-Ras/c-Raf/p38 pathway in locally recurring tumors and distant metastases compared with in the primary cervical tumor. These findings demonstrate that a sublethal dose of radiation can enhance the metastatic potential of human cervical cancer cells via K-Ras/c-Raf/p38 signaling, highlighting the potential development of specific inhibitors for reducing metastatic potential during radiotherapy.

Design, Synthesis, and Bioevaluation of Pyrido[2,3-d]pyrimidin-7-ones as Potent SOS1 Inhibitors.

The use of small molecular modulators to target the guanine nucleotide exchange factor SOS1 has been demonstrated to be a promising strategy for the treatment of various KRAS-driven cancers. In the present study, we designed and synthesized a series of new SOS1 inhibitors with the pyrido[2,3-d]pyrimidin-7-one scaffold. One representative compound 8u showed comparable activities to the reported SOS1 inhibitor BI-3406 in both the biochemical assay and the 3-D cell growth inhibition assay. Compound 8u obtained good cellular activities against a panel of KRAS G12-mutated cancer cell lines and inhibited downstream ERK and AKT activation in MIA PaCa-2 and AsPC-1 cells. In addition, it displayed synergistic antiproliferative effects when used in combination with KRAS G12C or G12D inhibitors. Further modifications of the new compounds may give us a promising SOS1 inhibitor with favorable druglike properties for use in the treatment of KRAS-mutated patients.

Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3.

BACKGROUND: VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. RESULTS: An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. CONCLUSIONS: VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Polymorphonuclear leukocyte transverse migration induces rapid alterations in endothelial focal contacts.

Transmigrated polymorphonuclear leukocytes (PMNs) usually undergo subendothelial transverse migration before penetrating into inner tissue layers. Whether or how endothelial cells (ECs) respond to the PMN migrating underneath them is unknown. A tissue flow chamber was used to establish a fMLP gradient and to observe PMN transverse migration along with its associated endothelial responses in culture (on a collagen gel) or in vascular tissues. Our results indicated that transversely migrating PMNs were in direct contact with the basal side of ECs. Contrasting to focal adhesion kinase (FAK) or proteins with phosphorylated tyrosine, paxillin disappeared rapidly (<1 min) from endothelial focal contacts after encountering the leukocyte's leading edge and soon rejoined them after the PMN had left. In addition, FAK moved away or became dephosphorylated when PMNs remained at the same subendothelial location for longer than 10 min, leaving actin filaments apparently unaltered. Unlike PMN transendothelial migration, PMN transverse migration did not induce any detectable endothelial calcium signaling. Taken together, our findings indicated that PMN transverse migration interrupted endothelial-matrix interactions and induced rapid alterations in endothelial focal contact composition.

Design for Brighter Photon Upconversion Emissions via Energy Level Overlap of Lanthanide Ions.

The perfect energy level overlap of 2H11/2, 4S3/2, and 4F9/2 in Er3+ ions with those of 5F3, 5F4/5S2, and 5F5 in adjacently codoped Ho3+ ions allows efficient interenergy transfer. Therefore, in addition to routine activators, Er3+ or Ho3+ can further act as sensitizers to transfer the upconverted energy to nearby Ho3+ or Er3+, resulting in enhanced upconversion luminescence due to the emission overlap. Proper codoping of Er3+/Ho3+ or Ho3+/Er3+ obviously elevates the maximum doping concentration (thus producing additional upconverted photons) to a level higher than that causing luminescence quenching and significantly enhances upconversion emissions compared with those of singly Er3+ or Ho3+-doped host materials. Indeed, the so-far strongest red upconversion emission under 1532 nm excitation was obtained in LiYF4:Er/Ho@LiYF4 nanoparticles and Ho3+-sensitized Er3+ upconversion emissions excited by 1150 nm laser was simultaneously discovered. With great enhancement compared with that of singly Ho3+ doped counterparts, this work demonstrates the generality and rationality of our design strategy.

Differentiation of Stem Cells From Human Exfoliated Deciduous Teeth Toward a Phenotype of Corneal Epithelium In Vitro.

PURPOSE: The aim of this study was to characterize stem cells from human exfoliated deciduous teeth (SHED) and to investigate the potential of SHED to differentiate toward corneal epithelium-like cells in vitro. METHODS: Mesenchymal and embryonic stem cell markers were analyzed by flow cytometry. The SHED was cocultured in either a transwell noncontact system or in a mixed culture system with immortalized human corneal epithelial (HCE-T) cells to induce the epithelial transdifferentiation. Expression of the mature corneal epithelium-specific marker cytokeratin 3 (CK3) and corneal epithelial progenitor marker cytokeratin 19 (CK19) were detected by immunofluorescence and the reverse transcription-polymerase chain reaction, respectively. RESULTS: SHED strongly expressed a set of mesenchymal stromal cell markers and pluripotency markers including NANOG and OCT-4. Seven days after the transwells were cocultured with HCE-T cells, SHED successfully upregulated epithelial lineage markers CK3 (16.6 ± 7.9%) and CK19 (10.0 ± 4.3%) demonstrating the potential for epithelial transdifferentiation, whereas CK3 and CK19 were barely expressed in SHED when cultured alone. Expression of transcript levels of CK3 and CK19 were significantly upregulated when SHED were transwell cocultured or mixed cultured with HCE-T cells by 7, 14, and 21 days. CONCLUSIONS: We have demonstrated that SHED retain the potential for transdifferentiation to corneal epithelium-like cells by in vitro coculture with immortal corneal epithelium cells. Thus, exfoliated teeth may be an alternative tissue resource for providing stem cells for potential clinical applications in ocular surface regeneration.

Overexpression of galectin-3 enhances migration of colon cancer cells related to activation of the K-Ras-Raf-Erk1/2 pathway.

BACKGROUND: Galectin-3 has been independently correlated with malignant behavior in human colon cancer. The involvement of galectin-3 in the invasiveness of colon cancer cells remains to be determined. We investigated whether galectin-3 was involved in the colon cancer cell migration mediated by certain kinase pathways. METHODS: We studied 2 colon cancer cell lines (DLD-1 and Caco2) and clinical samples. Immunostaining and Western blotting were used to analyze the expression of galectin-3 in vitro and in the clinical samples. Short hairpin RNA and overexpression of galectin-3 were used to study loss- and gain-of-function in a wound-healing assay and a Transwell migration assay, and Western blotting was used to study the Ras-Raf signaling pathway. RESULTS: Galectin-3 was expressed at lower levels in DLD-1 than in Caco2 cells. The lower galectin-3 level in DLD-1 cells was associated with decreased cell migration, in comparison with that of Caco2 cells. Overexpression of galectin-3 increased the migration rate of DLD-1, while knockdown of galectin-3 decreased the migration. Overexpression of galectin-3 was correlated with increased lamellipodia formation and distal lung localization in a mouse model. The galectin-3 enhancement of DLD-1 cell migration was mediated by K-Ras, Raf and Erk1/2 pathway activation, but not the H-Ras, p38, or JNK activation. CONCLUSIONS: Galectin-3 plays an important role in regulating colon cancer cell migration and potential distal localization. The galectin-3 enhancement of cell migration is mediated through the K-Ras-Raf-Erk1/2 pathway. Specific targeting of the K-Ras-Raf-Erk1/2 pathway may be useful for treating colon cancers associated with increased galectin-3 expression.

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation.

Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1ß and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.

Endothelial calcium signaling in rabbit arteries and its local alterations in early-stage atherosclerosis.

This study is to examine whether endothelial calcium signaling is different between atherosclerosis-prone thoracic aortas (TA) and atherosclerosis-resistant carotid arteries (CA) in normal rabbits and how it changes in early-stage atherosclerosis. Local endothelial calcium signaling was examined in arterial segments obtained from rabbits fed with normal or high-cholesterol diet for 1-4 weeks. Contrasting to normal CA, normal TA showed lower endothelial calcium signaling with more concentrated NF-kappaB in the endothelial nuclei. In the same hypercholesterolemic animal, fatty streak formation was much more prominent in TA than in CA. TA endothelial calcium signaling became augmented in the second week of hypercholesterolemia, being most pronounced in smooth regions adjacent to miniature fatty streaks. It was sporadically elevated even in regions away from any detectable TA fatty streak. When the entire TA was covered with fatty streaks in the fourth week of hypercholesteremia, endothelial calcium signaling returned to the original level. In comparison, CA endothelial calcium signaling was reduced around scattered fatty streaks. Reduced calcium signaling happened where CA fatty streaks were 150 microm long (covering 15-30 cells); and it extended to areas adjacent to larger fatty streaks. Moreover, NF-kappaB remained in the cytosol of endothelial cells covering CA fatty streaks. Our results indicate that inter-vascular differences in endothelial calcium signaling may provide partial explanation in their differential susceptibility in atherosclerosis.

Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium.

Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking postfixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere with the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of dissected human umbilical vein tissue. In either experimental model system, both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites. VE-cadherin moved away to different ends of the transmigration site, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 minutes. The relative positions of some junction proteins might rearrange to form a new interendothelial contour after PMNs had transmigrated through multicellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, whereas PECAM-1-containing junctions were opened during PMN transendothelial migration.