HIF-1α Is a Rational Target for Future Ovarian Cancer Therapies.

Ovarian cancer is the eighth most commonly diagnosed cancer among women worldwide. Even with the development of novel drugs, nearly one-half of the patients with ovarian cancer die within five years of diagnosis. These situations indicate the need for novel therapeutic agents for ovarian cancer. Increasing evidence has shown that hypoxia-inducible factor-1α(HIF-1α) plays an important role in promoting malignant cell chemoresistance, tumour metastasis, angiogenesis, immunosuppression and intercellular interactions. The unique microenvironment, crosstalk and/or interaction between cells and other characteristics of ovarian cancer can influence therapeutic efficiency or promote the disease progression. Inhibition of the expression or activity of HIF-1α can directly or indirectly enhance the therapeutic responsiveness of tumour cells. Therefore, it is reasonable to consider HIF-1α as a potential therapeutic target for ovarian cancer. In this paper, we summarize the latest research on the role of HIF-1α and molecules which can inhibit HIF-1α expression directly or indirectly in ovarian cancer, and drug clinical trials about the HIF-1α inhibitors in ovarian cancer or other solid malignant tumours.

Treatment for subtrochanteric fracture and subsequent nonunion in an adult patient with osteopetrosis: A case report and review of the literature.

BACKGROUND: As a congenital metabolic bone disease caused by defective osteoclastic resorption of immature bone, osteopetrosis is characterized by diffused sclerosis of bones, brittle bones, easy fracturing, narrow medullary canals, and a weak fracture healing ability. At present, clear standards and principles for the treatment of fractures in patients with osteopetrosis are lacking. Non-operative treatment can prevent fracture hematoma and preserve the blood supply to the bone fragments, while being associated with frequent failures and higher mortality rates. Meanwhile, closed reduction and internal fixation with intramedullary nail (CRIF + IMN) approaches can also protect blood supply to the fracture site. However, IMN cannot be used for the vast majority of patients with osteopetrosis due to the narrowing of medullary canals. Thus, open reduction and internal fixation with plate remains the most appropriate surgical method for treating fractures in patients with osteopetrosis, but this approach is complicated by the lack of intramedullary hematopoiesis in such patients. Fracture healing primarily depends on the blood supply to the external periosteum. Open reduction can also easily destroy the periosteum and cause delayed fracture healing or even nonunion; however, CRIF may be the most practical approach. As a result, it would be prudent to solve the difficulty of drilling during the operation and the problem of postoperative nonunion. CASE SUMMARY: In 2018, we treated an adult patient with osteopetrosis presenting with a subtrochanteric fracture. The fracture was fixed using a femoral locking compression plate. Because of delayed consolidation, at 12 mo postoperatively the patient was further treated with platelet-rich plasma (PRP) combined with radial extracorporeal shock wave therapy (rESWT). Antero-posterior and lateral radiographs obtained at the latest follow-up (10 mo) showed that the callus had grown at the original fracture site, and the medial fracture line almost disappeared. CONCLUSION: Osteosynthesis remains the first choice of treatment approach for fractures in patients with osteopetrosis, especially peritrochanteric fractures. Preoperative preparation is necessary to avoid risks such as drill bit breakage and iatrogenic fracture during the operation. Moreover, fractures in a patient with osteopetrosis present with a high risk of delayed union and nonunion, which can be potentially cured with PRP + rESWT.

Erratum: Association of Genes Variants in RANKL/RANK/OPG Signaling Pathway with the Development of Osteonecrosis of the Femoral Head in Chinese Population: Erratum.

[This corrects the article DOI: 10.7150/ijms.19124.].

Enhanced Tendon-to-Bone Healing of Chronic Rotator Cuff Tears by Bone Marrow Aspirate Concentrate in a Rabbit Model.

BACKGROUND: To evaluate the influence of bone marrow aspirate concentrate (BMAC) on tendon-to-bone healing in a rabbit rotator cuff model and to characterize the composition of growth factors in BMAC. METHODS: In this in vivo study, 40 rabbits were allocated into five groups: control (C), repair + saline (RS), repair + platelet-rich plasma (PRP; RP), repair + BMAC (RB) and repair + PRP + BMAC (RPB). A tear model was created by supraspinatus tendon transection at the footprint. Six weeks after transection, the torn tendon was repaired along with BMAC or PRP administration. Six weeks after repair, shoulder samples were harvested for biomechanical and histological testing. Ten rabbits were used for processing PRP and BMAC, followed by analysis of blood cell composition and the levels of growth factors in vitro. RESULTS: The ultimate load-to-failure was significantly higher in RPB group compared to RS group (p = 0.025). BMAC-treated groups showed higher values of biomechanical properties than RS group. The histology of BMAC-treated samples showed better collagen fiber continuity and orientation than RS group. BMAC contained significantly higher levels of the several growth factors than PRP. CONCLUSIONS: Locally administered BMAC enhanced tendon-to-bone healing and has potential for clinical applications.

Association of Genes Variants in RANKL/RANK/OPG Signaling Pathway with the Development of Osteonecrosis of the Femoral Head in Chinese Population.

The RANKL/RANK/OPG pathway plays an important role in regulating bone remodeling and bone turnover. However, the association of the genes variants with the risk of ONFH has rarely been reported. Here, we analyzed the correlation of the 10 SNPs polymorphisms of RANKL, RANK, OPG, TRAF6, and NFATC1 genes with the risk and development of ONFH in 200 ONFH patients and 177 health controls of Chinese population with using Mass ARRAY® platform. The results showed that the recessive model of NFATC1rs9518 was significantly associated with increased ONFH risk (OR:8.223, P=0.048); the proportion of stage Ⅳ patients in the rs9518TC genotype carriers was statistically higher than that of stage Ⅲ patients (P=0.03); in the T-C haplotype carriers of Naftac1, the proportion of bilateral hips lesions was also significantly enhanced than that of unilateral hip lesions(P=0.05). In addition, the proportion of idiopathic ONFH in the TT genotype carriers of OPGrs2073617 was significantly higher than that of steroid or alcohol-induced ONFH, respectively, while in the TC genotype carriers of the SNP, the proportion of idiopathic ONFH remarkably decreased compared with that of Alcohol-induced ONFH, P=0.021. Our results were first found that NFATC1rs9518 closely associated with the risk and the development of ONFH, while OPGrs2073617 statistically correlated with the etiological classification of ONFH.

Antitumor activity of lentivirus-mediated interleukin -12 gene modified dendritic cells in human lung cancer in vitro.

OBJECTIVES: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associated antigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whether transduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigen- specific cytotoxic T cells (CTLs) against human lung cancer cells in vitro. METHODS: Peripheral blood monocyte- derived DCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated target of the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCs was measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry. DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag). Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulated by LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methyl thiazolyltetrazolium (MTT). RESULTS: A recombinant lentivirus expressing the IL-12 gene was successfully constructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83 than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstrated good stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and anti- tumor effects. CONCLUSIONS: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have an enhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.

[The effect of Bcl-2 gene silencing on the sensitivity of cell line A549 to chemotherapeutic drugs].

OBJECTIVE: To investigate the effects of miRNA-mediated down-regulation of the Bcl-2 gene on the chemotherapeutic sensitivities and mRNA transcriptions of sensitivity associated genes in human lung adenocarcinoma cell line A549 cells, and therefore to provide experimental data for improving the chemotherapeutic effects on non-small cell lung cancer (NSCLC). METHODS: The miRNA recombinant plasmid targeting to human Bcl-2 gene was designed, synthesized and stably transferred into A549 cells by lipofectin technique as the experiment group. The transcription of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) by agarose gel electrophoresis, real-time PCR, and the protein level of Bcl-2 was measured by Western blot to confirm the function of miRNA plasmid. The cell proliferation was examined by methyl thiazolyl tetrazolium (MTT) assay. Cell cycle was measured by flow cytometry. Drug sensitivities of A549 cells to etoposide, 5-fluorouracil, cisplatin, adriamycin, vincristine, paclitaxel and navelbine were analyzed by MTT assay. The mRNA expressions of excision repair cross-complementing gene 1 (ERCC1), thymidylate synthase (TYMS), Class III ß-tubulin, topoisomerase 2 alpha (TOP2α) genes were detected by RT-PCR and real-time PCR. RESULTS: The recombinant miRNA plasmid was successfully synthesized and stably transferred into A549 cells. The transcription of Bcl-2 mRNA dramatically decreased by 98.1% in the experiment group (RQ = 0.002 ± 0.001) compared to that in the negative control group (RQ = 0.104 ± 0.003) by real-time PCR (t = 98.70, P < 0.05); and the protein level of Bcl-2 in the experiment group decreased by 57.6% by Western blot (t = 7.66, P < 0.05). The cell cycle profile showed that the low expression of Bcl-2 gene led to A549 cell cycle arrest at G1-phase. The results of MTT showed that the growth of A549 cells in the experiment group was markedly inhibited. The sensitivities of A549 cells to etoposide, cisplatin, paclitaxel, and navelbine were significantly enhanced [IC50 values in the experiment group were (107.3 ± 0.1) mg/L, (7.7 ± 0.6) mg/L, (11.5 ± 1.9) mg/L and (10.8 ± 1.6) mg/L; IC50 values in the negative control group were (145.8 ± 0.1) mg/L, (60.7 ± 1.4) mg/L, (80.6 ± 1.7) mg/L and (20.6 ± 1.7) mg/L], the respective t values being 655.33, 108.04, 82.16 and 12.48, all P < 0.05. The mRNA level of ERCC1, TYMS, and TOP2α genes in the experiment group decreased by 99.6%, 92.9% and 96.1% respectively, but Class III ß-tubulin mRNA increased by 122% compared to the negative control group (1.154 ± 0.008, 0.520 ± 0.009), the respective t values being 689.79, 689.37, 768.04 and 160.07, all P < 0.05. CONCLUSION: Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression in A549 cells specifically decreased the mRNA of ERCC1, TYMS, and TOP2α genes, and significantly increased the sensitivities of A549 cells to chemotherapeutic agents such as etoposide, cisplatin, paclitaxel and navelbine.

Human resistin inhibits myogenic differentiation and induces insulin resistance in myocytes.

This study is aimed to investigate the effect of human resistin on myocyte differentiation and insulin resistance. The human resistin eukaryotic expression vector was stable transfected into C2C12 myocyte cells and was transiently transfected into COS7 cells. The effects of human resistin on cell proliferation, cell cycle, and myogenic differentiation of C2C12 cells were examined. Glucose uptake assays was performed on C2C12 myotubes by using [(3)H] 2-deoxy-D-glucose. The mRNA levels of insulin receptor (IR) and glucose transporter 4 (GLUT4) were evaluated by semiquantitative RT-PCR. Results showed by the C2C12 cells transfected with human resistin gene compared with that without transfecting gene are as follows: (1) cell proliferation was significantly promoted, (2) after inducing differentiation, the myotube's diameters and expression of desmin and myoglobin decreased, and (3) glucose uptake ratio was lowered and expression of IR and GLUT4 decreased. However, there was no significant difference in the glucose uptake ratio between C2C12 myotubes treated with a human resistin conditioned medium of COS7 cells and treated with control medium. These results suggest that maybe human resistin has not a direct role on insulin sensitivity of myocytes. However, maybe it impaired the insulin sensitivity of myocytes through suppressing myogenesis and stimulating proliferation of myoblasts.

Effects of SARA on oxygen-glucose deprivation in PC12 cell line.

Ischemic stroke is a major composition of cerebrovascular disease, seriously threatening to human health in the world. Activin A (ActA), belonging to transforming growth factor-beta (TGF-ß) super family, plays an important role in the hypoxic-ischemic brain injury through ActA/Smads pathway. While as an essential phosphorylation assistor in TGF-ß signaling, the functions and mechanisms of smad anchor for receptor activation (SARA) in ischemic brain injury remain poorly understood. To solve this problem and explore the pathological processes of ischemic stroke, we used an Oxygen-Glucose deprivation (OGD) model in nerve growth factor-induced differentiated rattus PC12 pheochromocytoma cells and down regulated the expressions of SARA by RNA interference technology. Our results showed that the repression of SARA before OGD exposure reduced the expressions of Smad2, 3, 4 mRNA and the phosphorylation rate of Smad2 protein, but it did not affect the mRNA expressions of Smad7. After OGD treatment, ActA/Smads pathway was activated and the expression of SARA in the SARA pre-repression group was significantly up-regulated. The pre-repression of SARA increased the sensitivities of nerve-like cells to OGD damage. Moreover, the mRNA expression of Smad7 which was supposed to participate in the negative feedback of ActA/Smads pathway was also elevated due to OGD injury. Taken together, these results suggest a positive role of SARA in assisting the phosphorylation of Smad2 and maintaining the neuron protective effect of ActA/Smads pathway.

Association of sterol regulatory element binding protein 2 and insulin-like growth factor binding protein 3 genetic polymorphisms with avascular necrosis of the femoral head in the Chinese population.

BACKGROUND: Sterol regulatory element binding protein (SREBP)-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways. The insulin-like growth factor binding protein (IGFBP) family regulates growth and metabolism, especially bone cell metabolism, and correlates with osteonecrosis. However, association of their gene polymorphisms with risk of avascular necrosis of the femoral head (ANFH) has rarely been reported. We determined whether SREBP-2 and IGFBP-3 gene polymorphisms were associated with increased ANFH risk in the Chinese population. METHODS: Two single nucleotide polymorphisms of SREBP2 gene, rs2267439 and rs2267443, and one of IGFBP-3 gene, rs2453839, were selected and genotyped in 49 ANFH patients and 42 control individuals by direct sequencing assay. RESULTS: The frequencies of rs2267439 TT and rs2267443 GA of SREBP2 and rs2453839 TT and CT of IGFBP-3 in the ANFH group showed increased and decreased tendencies (against normal control group), respectively. Interaction analysis of genes revealed that the frequency of carrying rs2267439 TT and rs2267443 GA genotypes of SREBF-2 in ANFH patients was significantly higher than in the control group (P < 0.05). Association analysis between polymorphisms and clinical phenotype demonstrated that the disease course in ANFH patients with the rs2453839 TT genotype of IGFBP-3 was significantly shorter than that of CT + CC carriers (P < 0.01). CT + CC genotype frequency in patients with stage III/IV bilateral hip lesions was significantly higher than in those with stage III/IV unilateral lesions and stage II/III bilateral lesions (P < 0.05 - 0.02). CONCLUSIONS: Our results suggested that interaction of SREBP-2 gene polymorphisms and the relationship between the polymorphisms and clinical phenotype of IGFBP-3 were closely related to increased ANFH risk in the Chinese population. The most significant finding was that the CT + CC genotype carriers of IGFBP-3 rs2453839 were highly associated with the development of ANFH.

Poly[[[µ-1,1'-(butane-1,4-di-yl)diimidazole-κN:N](µ-cyclo-hexane-1,4-dicarboxyl-ato-κO,O:O,O)nickel(II)] 0.25-hydrate].

In the title coordination polymer, {[Ni(C(8)H(10)O(4))(C(10)H(14)N(4))]·0.25H(2)O}(n), the coordination of the Ni(II) ion is distorted octa-hedral. The 1,1'-(butane-1,4-di-yl)diimidazole ligand and the cyclo-hexane-1,4-dicarboxyl-ate dianion bridge metal centres, forming a two-dimensional (4,4) network. The network is consolidated by O-H⋯O hydrogen bonds between the statistically occupied water molecules and O atoms of the two carboxylate groups.

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.

BACKGROUND: RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). METHODS: The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant. RESULTS: In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. CONCLUSIONS: shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.