Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes.

Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. We have developed a Drosophila acute myeloid leukemia model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. We performed both genetic and drug screens using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed that echinomycin, an inhibitor of HIF1A, can effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, we showed that echinomycin treatment can effectively suppress oncogenic RAS-driven leukemia cell proliferation, using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes. This article has an associated First Person interview with the first author of the paper.

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila.

We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1ß, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.

[MIPPO and ORIF for the treatment of elderly proximal humerus fractures of type Neer II:a case control study].

OBJECTIVE: To compare the clinical efficacy of minimally invasive percutaneous plate osteosynthesis(MIPPO)and open reduction and internal fixation (ORIF) in treating senile NEER IIproximal humerus fractures. METHODS: From March 2014 to March 2016, 46 elderly patients with Neer II proximal humerus fractures were retrospectively reviewed. Among them, 20 patients in MIPPO group included 9 males and 11 females with an average age of (70.4±4.4) years old; while 26 patients in ORIF group included 11 males and 15 females with an average age of (70.9±4.0) years old. The length of hospital stay, times of fluoroscopy, beginning time of function rehabilitation, healing time of fracture, Constant Murley score of the shoulder joint at 3, 6, 12 months after operation and complications were observed and compared. RESULTS: All patients were followed up for 12 to 24 months with an average of 16.8±3.7. The healing time of fracture, beginning time of function rehabilitation in MIPPO group were(13.0±0.8) weeks, (3.0±0.9) days respectively and shorter than those in ORIF group which were (13.8±1.4) weeks and(6.8±1.3) days. The times of fluoroscopy in MIPPO group was 19.2±3.7 and more than that in ORIF group which was 12.1±3.4. At 3 and 6 months after operation, Constant Murley score in MIPPO group were 81.3±3.9, 86.6±5.4 and more than that in ORIF group which were 69.5±6.6, 80.5±6.7. There were no differences between two groups in the length of hospital stay, Constant Murley score at 12 months after operation and grading at the final follow-up. There was one fracture redisplacement in each group. And 1 case of axillary nerve injury in MIPPO group, 2 cases of delayed union in ORIF group. No incision infection, screw loosening or plate break was found. CONCLUSIONS: MIPPO and ORIF are both effective in treating Neer II proximal humeral fractures. MIPPO technique has the advantages of faster recovery, earlier rehabilitative exercise and better shoulder function. The disadvantages are more exposure to radiationd and the possibility of axillary nerve injure.

Condyle Head Reimplantation Combined With Vascularized Free Flap for Mandibular Reconstruction.

Due to the anatomic and functional complexity, reconstruction of the condylar process after mandibular tumor extirpation remains a surgical challenge. The aim of this study was to present the results and experience of condyle head reimplantation combined with vascularized free flap for mandibular reconstruction in our institution. In the current study, the authors evaluated the clinical features of condyle head reimplantation combined with vascularized free flap for mandibular reconstruction in 5 patients with benign mandibular intraosseous lesions from December 2013 to March 2015 in our institution. All patients showed nearly normal mouth opening, with minimal mandibular deviation and joint symptoms. The radiograph data showed that 4 patients had no obvious bone resorption of condyles while only 1 patient had prominent condyle resorption. All the condyles were cut above the condylar neck, with the biggest remaining condyle height being 2.8 cm and the smallest one being 1.1 cm. Moreover, the original vertical height of mandibular ramus was decreased by 2 to 3 mm during the surgery. In conclusion, this study revealed that combining the condyle reimplantation with vascularized free flap constitutes a reliable method in condylar reconstruction and a slight reduction of the vertical height of condyles may help to diminish unfavorable outcomes.

A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation.

Clinical studies have identified patients with nephrotic syndrome caused by mutations in genes involved in the biosynthesis of coenzyme Q10 (CoQ10), a lipid component of the mitochondrial electron transport chain and an important antioxidant. However, the cellular mechanisms through which these mutations induce podocyte injury remain obscure. Here, we exploited the striking similarities between Drosophila nephrocytes and human podocytes to develop a Drosophila model of these renal diseases, and performed a systematic in vivo analysis assessing the role of CoQ10 pathway genes in renal function. Nephrocyte-specific silencing of Coq2, Coq6, and Coq8, which are genes involved in the CoQ10 pathway that have been associated with genetic nephrotic syndrome in humans, induced dramatic adverse changes in these cells. In particular, silencing of Coq2 led to an abnormal localization of slit diaphragms, collapse of lacunar channels, and more dysmorphic mitochondria. In addition, Coq2-deficient nephrocytes showed elevated levels of autophagy and mitophagy, increased levels of reactive oxygen species, and increased sensitivity to oxidative stress. Dietary supplementation with CoQ10 at least partially rescued these defects. Furthermore, expressing the wild-type human COQ2 gene specifically in nephrocytes rescued the defective protein uptake, but expressing the mutant allele derived from a patient with COQ2 nephropathy did not. We conclude that transgenic Drosophila lines carrying mutations in the CoQ10 pathway genes are clinically relevant models with which to explore the pathogenesis of podocyte injury and could serve as a new platform to test novel therapeutic approaches.

Association of ATF4 Expression With Tissue Hypoxia and M2 Macrophage Infiltration in Infantile Hemangioma.

Accumulating studies have revealed the hypoxic condition and its crucial role in the distinctive progression of infantile hemangioma (IH), the most common benign tumor in infancy. Activating transcription factor 4 (ATF4), an important gene mediating cellular adaptation to various stress signals, could confer a survival advantage for tumor cells under hypoxia and regulate tumor progression. However, the potential role of ATF4 in IH was still unknown. In this study, the expression of hypoxia inducible factor (HIF)-1α, ATF4, and macrophage colony-stimulating factor (M-CSF) in 27 specimens of IH was measured by immunochemistry and double-labeling immunofluorescence, followed by the Spearman rank correlation test. Our results showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in proliferating IH compared with involuting IH. Meanwhile, HIF-1α and ATF4, in parallel with ATF4 and M-CSF, exhibited positive correlation and synchronous expression. In addition, our in vitro studies demonstrated that hypoxia obviously upregulated the expression of HIF-1α, ATF4, and M-CSF in hemangioma stem cells. Most importantly, their expression was uniformly correlated with the percentage of M2-polarized macrophages in IH. All those results and established evidence indicated that hypoxia-induced ATF4 expression may promote progression of proliferating IH through M-CSF-induced M2-polarized macrophages infiltration.

Magnetic and Folate Functionalization Enables Rapid Isolation and Enhanced Tumor-Targeting of Cell-Derived Microvesicles.

Cell-derived microvesicles (MVs), which are biogenic nanosized membrane-bound vesicles that convey bioactive molecules between cells, have recently received attention for use as natural therapeutic platforms. However, the medical applications of MV-based delivery platforms are limited by the lack of effective methods for the efficient isolation of MVs and the convenient tuning of their targeting properties. Herein, we report the development of magnetic and folate (FA)-modified MVs based on a donor cell-assisted membrane modification strategy. MVs inherit the membrane properties of their donor cells, which allows them to be modified with the biotin and FA on their own membrane. By conjugating with streptavidin-modified iron oxide nanoparticles (SA-IONPs), the MVs can be conveniently, efficiently, and rapidly isolated from the supernatant of their donor cells using magnetic activated sorting. Moreover, the conjugated magnetic nanoparticles and FA confer magnetic and ligand targeting activities on the MVs. Then, the MVs were transformed into antitumor delivery platforms by directly loading doxorubicin via electroporation. The modified MVs exhibited significantly enhanced antitumor efficacy both in vitro and in vivo. Taken together, this study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors, thus facilitating the development of natural therapeutic nanoplatforms.

Characterization of Endothelial Microparticles Induced by Different Therapeutic Drugs for Infantile Hemangioma.

Endothelial microparticles (EMPs) are complex vesicular structures with great significance in vascular pathophysiology. Here, we aimed to determine the impact of therapeutic drugs for infantile hemangioma, a common vascular tumor of infancy, on the biochemical features of EMPs. We exposed human umbilical vein endothelial cells to propranolol (Pro), dexamethasone (Dex), or rapamycin (Rap). Compared with controls, Pro and Rap dramatically augmented EMP release, whereas Dex significantly suppressed EMP generation. Drug-stimulated EMPs could inherit but tended to lose specific endothelial surface antigens from their parental cells. On the one hand, markedly distinct messenger RNA expression patterns were observed within and between drug-stimulated endothelial cells and derived EMPs. On the other hand, Rap-treated endothelial cells and Pro-induced EMPs displayed downregulation of multiple angiogenesis-related molecules at messenger RNA level compared with corresponding controls. Meanwhile, among tested angiogenesis-associated microRNAs, twelve microRNAs were downregulated in drug-induced EMPs, whereas only let-7b and miR-133a were markedly upregulated. Collectively, these data may indicate selective and distinctive package of biomolecules into EMPs depending on specific drugs. Our findings may provide novel insights into the underlying mechanisms of pharmacological therapy for infantile hemangioma.

Hyperbranched-hyperbranched polymeric nanoassembly to mediate controllable co-delivery of siRNA and drug for synergistic tumor therapy.

This study reported a flexible nanoplatform constructed on the pH-dependent self-assembly of two kinds of hyperbranched polymers, and then validated its potency as the controllable siRNA/drug co-delivery vehicle for the combination of chemotherapy with RNA interfering (RNAi) therapy. By virtue of pH-reversible phenylboronate linking, phenylboronic acid-tethered hyperbranched oligoethylenimine (OEI600-PBA) and 1,3-diol-rich hyperbranched polyglycerol (HBPO) can be spontaneously interlinked together into a core-corona nanoconstruction. The special buildup of compactly clustering OEI600-PBA units around hydrophobic HBPO aggregate offered significant advantages over parent OEI600-PBA, including strengthened affinity to siRNA, ability of further loading anticancer drug, easier cellular transport, and acidity-responsive release of payloads. To evaluate the co-delivery capability, Beclin1 siRNA and antitumor DOX were used as the therapeutic models in order to suppress the post-chemotherapy survival of tumor cells caused by drug-induced autophagy. The nanoassembly-mediated single delivery of DOX displayed even better anticancer effects than free DOX, demonstrating the superiority of our pH-responsive nano-design. The nanoassembly-mediated co-delivery of siRNA together with DOX can effectively silence Beclin1 gene, suppress DOX-induced autophagy, and consequently provide strong synergism with a significant enhancement of cell-killing effects in cultured cancerous cells. The in vivo combinational treatment was shown to make the tumor more sensitive to DOX chemotherapy while displaying substantially improved safety as compared with the monochemotherapy.

Macrophages Contribute to the Progression of Infantile Hemangioma by Regulating the Proliferation and Differentiation of Hemangioma Stem Cells.

Macrophage infiltration has been implicated in infantile hemangioma (IH), the most common tumor of infancy. However, the exact role of macrophages in IH remains unknown. This study aims to clarify the functional significance of macrophages in the progression of IH. The distribution of macrophages in human IH was analyzed, and our results revealed that polarized macrophages were more prevalent in proliferating IHs than in involuting IHs, which was consistent with the increased macrophage-related cytokines in proliferating IHs. In vitro results further demonstrated that polarized macrophages effectively promoted the proliferation of hemangioma stem cells (HemSCs) and suppressed their adipogenesis in an Akt- and extracellular signal-regulated kinase 1/2 (Erk1/2)-dependent manner. Moreover, M2- but not M1-polarized macrophages promoted the endothelial differentiation of HemSCs. Furthermore, mixing macrophages in a murine hemangioma model elevated microvessel density and postponed fat tissue formation, which was concomitant with the activation of Akt and Erk1/2 signals. Cluster analysis revealed a close correlation among the macrophage markers, Ki67, vascular endothelial growth factor (VEGF), p-Akt, and p-Erk1/2 in human IH tissues. Collectively, our results suggest that macrophages in IH contribute to tumor progression by promoting the proliferation and endothelial differentiation while suppressing the adipogenesis of HemSCs. These findings indicate that targeting the infiltrating macrophages in IH is a promising therapeutic approach to accelerate IH regression.

KANK deficiency leads to podocyte dysfunction and nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling.

Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA delivery.

Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors.

A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.

Advanced drug delivery systems, which possess post-functionalization feasibility to achieve targetability and traceability, favorable pharmacokinetics with dynamic but controllable stability, and preferable tumor accumulation with prolonged drug residence in disease sites, represent ideal nanomedicine paradigm for tumor therapy. To address this challenge, here we reported a dynamic module-assembly strategy based on reversible boronic acid/1,3-diol bioorthogonality. As a prototype, metastable hybrid nanoself-assembly between hydrophobic hyperbranched diol-enriched polycarbonate (HP-OH) and hydrophilic linear PEG terminated with phenylboronic acid (mPEG-PBA) is demonstrated in vitro and in vivo. The nanoconstruction maintained excellent stability with little leakage of loaded drugs under the simulated physiological conditions. Such a stable nanostructure enabled the effective in vivo tumor accumulation in tumor site as revealed by NIR imaging technique. More importantly, this nanoconstruction presented a pH-labile destruction profile in response to acidic microenvironment and simultaneously the fast liberation of loaded drugs. Accordingly at the cellular level, the intracellular structural dissociation was also proved in terms of the strong acidity in late endosome/lysosome, thus favoring the prolonged retention of remaining drug-loaded HP-OH aggregates within tumor cells. Hence, our delicate design open up a dynamical module-assembly path to develop site and time dual-controlled nanotherapeutics for tumor chemotherapy, allowing enhanced tumor selectivity through prolonged retention of delivery system in tumor cells followed by a timely drug release pattern.

M2-polarised macrophages in infantile haemangiomas: correlation with promoted angiogenesis.

AIMS: The pathogenesis of infantile haemangiomas (IHs) is still far from clear despite the fact that they are common vascular tumours distinctive for their perinatal presentation, rapid growth during the first year of life and subsequent slow involution. AIMS: To determine the role of M2-polarised macrophages in IHs. METHODS: M2-polarised macrophages were initially identified in 20 specimens of IHs by both immunochemistry and immunofluorescence for CD68 and CD163. The immunopositive M2-polarised macrophages in different phases of IHs were quantified, and further analysed for their correlations with the expression levels of Ki67, vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF). RESULTS: The infiltrating macrophages in proliferative IHs were predominantly CD68/CD163, thus of the M2-polarised phenotype, whereas the density of these cells was significantly decreased in the involuting IHs. The high density of M2-polarised macrophages in proliferative IHs was closely correlated with overexpression of M-CSF, one of the cytokines considered to induce macrophages to polarise towards an M2 phenotype. The infiltrating M2-polarised macrophages probably contributed to the proliferation and angiogenesis of haemangioma endothelial cells, as evidenced by their close correlations with the immunoreactivities of Ki67 and VEGF. CONCLUSIONS: Results indicate that the infiltrating M2-polarised macrophages may contribute to the progression of IHs by promoting the angiogenic process.

Occupational exposure to blood and body fluids among dental personnel in a Chinese dental hospital.

OBJECTIVE: To determine the prevalence of occupational exposure (OE) occurring to dentists, dental nurses and senior dental students in a Chinese dental hospital and to assess associated factors with these exposures. METHODS: Data were collected through anonymous questionnaires, which were distributed to dentists, nurses, and senior dental students. RESULTS: All 205 dental personnel who voluntarily participated in the survey completed the questionnaire. Up to 71.2% of respondents experienced at least one OE to blood and body fluids (BBF) one year prior to this study. Dentists had a significantly higher incidence of OE than nurses (82.6% verse 60.8%), and a higher incidence of percutaneous injury than students (63.8% versus 41.7%). Sixty-seven percent of exposed dental personnel did not report the incidents and only 4.1% underwent postexposure blood tests. Although 97.6% of respondents wore gloves, more than half the subjects did not use protective goggles or masks during daily dental practice. CONCLUSION: There was a high level of OE and a significant incidence of underreporting among Chinese dental personnel, particularly in dentists. Inadequate use of personal protective equipment and ignorance about postexposure management were of great concern.

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential.

OBJECTIVES: Adenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC. METHODS: Immunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals. RESULTS: The immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues. CONCLUSIONS: The results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.

Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway.

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.