Design and biological evaluation of dual tubulin/HDAC inhibitors based on millepachine for treatment of prostate cancer.

In this work, a novel of dual tubulin/HDAC inhibitors were designed and synthesized based on the structure of natural product millepachine, which has been identified as a tubulin polymerization inhibitor. Biological evaluation revealed that compound 9n exhibited an impressive potency against PC-3 cells with the IC50 value of 16 nM and effectively inhibited both microtubule polymerization and HDAC activity. Furthermore, compound 9n not only induced cell cycle arrest at G2/M phase, but also induced PC- 3 cells apoptosis. Further study revealed that the induction of cell apoptosis by 9n was accompanied by a decrease in mitochondrial membrane potential and an elevation in reactive oxygen species levels in PC-3 cells. Additionally, 9n exhibited inhibitory effects on tumor cell migration and angiogenesis. In PC-3 xenograft model, 9n achieved a remarkable tumor inhibition rate of 90.07%@20 mg/kg, significantly surpassing to that of CA-4 (55.62%@20 mg/kg). Meanwhile, 9n exhibited the favorable drug metabolism characteristics in vivo. All the results indicate that 9n is a promising dual tubulin/HDAC inhibitor for chemotherapy of prostate cancer, deserving the further investigation.

Design, synthesis and biological evaluation of CDC20 inhibitors for treatment of triple-negative breast cancer.

The involvement of CDC20 in promoting tumor growth in different types of human cancers and it disturbs the process of cell division and impedes tumor proliferation. In this work, a novel of Apcin derivatives targeting CDC20 were designed and synthesized to evaluate for their biological activities. The inhibitory effect on the proliferation of four human tumor cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and A549) was observed. Among them, compound E1 exhibited the strongest inhibitory effect on the proliferation of MDA-MB-231 cells with an IC50 value of 1.43 µM, which was significantly superior to that of Apcin. Further biological studies demonstrated that compound E1 inhibited cancer cell migration and colony formation. Furthermore, compound E1 specifically targeted CDC20 and exhibited a higher binding affinity to CDC20 compared to that of Apcin, thereby inducing cell cycle arrest in the G2/M phase of cancer cells. Moreover, it has been observed that compound E1 induces autophagy in cancer cells. In 4T1 Xenograft Models compound E1 exhibited the potential antitumor activity without obvious toxicity. These findings suggest that E1 could be regarded as a CDC20 inhibitor deserved further investigation.

Discovery of Novel Isoquinoline Analogues as Dual Tubulin Polymerization/V-ATPase Inhibitors with Immunogenic Cell Death Induction.

Cancer immunotherapy has revolutionized clinical advances in a variety of cancers. Due to the low immunogenicity of the tumor, only a few patients can benefit from it. Specific microtubule inhibitors can effectively induce immunogenic cell death and improve immunogenicity of the tumor. A series of isoquinoline derivatives based on the natural products podophyllotoxin and diphyllin were designed and synthesized. Among them, F10 showed robust antiproliferation activity against four human cancer cell lines, and it was verified that F10 exerted antiproliferative activity by inhibiting tubulin and V-ATPase. Further studies indicated that F10 is able to induce immunogenic cell death in addition to apoptosis. Meanwhile, F10 inhibited tumor growth in an RM-1 homograft model with enhanced T lymphocyte infiltration. These results suggest that F10 may be a promising lead compound for the development of a new generation of microtubule drugs.

Discovery of Novel Potent Covalent Glutathione Peroxidase 4 Inhibitors as Highly Selective Ferroptosis Inducers for the Treatment of Triple-Negative Breast Cancer.

Glutathione peroxidase 4 (GPX4) is a promising target to induce ferroptosis for the treatment of triple-negative breast cancer (TNBC). We designed and synthesized a novel series of covalent GPX4 inhibitors based on RSL3 and ML162 by structural integration and simplification strategies. Among them, compound C18 revealed a remarkable inhibitory activity against TNBC cells and significantly inhibited the activity of GPX4 compared to RSL3 and ML162. Moreover, it was identified that C18 could notably induce ferroptosis with high selectivity by increasing the accumulation of lipid peroxides (LPOs) in cells. Further study demonstrated that C18 covalently bound to the Sec46 of GPX4. Surprisingly, C18 exhibited an outstanding potency of tumor growth inhibition in the MDA-MB-231 xenograft model with a TGI value of 81.0%@20 mg/kg without obvious toxicity. Overall, C18 could be a promising GPX4 covalent inhibitor to induce ferroptosis for the treatment of TNBC.

Discovery of Novel N-Heterocyclic-Fused Deoxypodophyllotoxin Analogues as Tubulin Polymerization Inhibitors Targeting the Colchicine-Binding Site for Cancer Treatment.

Natural products are a major source of anticancer agents and play critical roles in anticancer drug development. Inspired by the complexity-to-diversity strategy, novel deoxypodophyllotoxin (DPT) analogues were designed and synthesized. Among them, compound C3 exhibited the potent antiproliferative activity against four human cancer cell lines with IC50 values in the low nanomolar range. Additionally, it showed marked activity against paclitaxel-resistant MCF-7 cells and A549 cells. Moreover, compound C3 can inhibit tubulin polymerization by targeting the colchicine-binding site of tubulin. Further study revealed that compound C3 could arrest cancer cells in the G2/M phase and disrupt the angiogenesis in human umbilical vein endothelial cells. Meanwhile, C3 remarkably inhibited cancer cell motility and migration, as well as considerably inhibited tumor growth in MCF-7 and MCF-7/TxR xenograft model without obvious toxicity. Collectively, these results indicated that compound C3 may be a promising tubulin polymerization inhibitor development for cancer treatment.

Photoactivatable Adhesive Ligands for Light-Guided Neuronal Growth.

Neuro-regeneration after trauma requires growth and reconnection of neurons to reestablish information flow in particular directions across the damaged tissue. To support this process, biomaterials for nerve tissue regeneration need to provide spatial information to adhesion receptors on the cell membrane and to provide directionality to growing neurites. Here, photoactivatable adhesive peptides based on the CASIKVAVSADR laminin peptidomimetic are presented and applied to spatiotemporal control of neuronal growth to biomaterials in vitro. The introduction of a photoremovable group [6-nitroveratryl (NVOC), 3-(4,5-dimethoxy-2-nitrophenyl)butan-2-yl (DMNPB), or 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP)] at the amino terminal group of the K residue temporally inhibited the activity of the peptide. The bioactivity was regained through controlled light exposure. When used in neuronal culture substrates, the peptides allowed light-based control of the attachment and differentiation of neuronal cells. Site-selective irradiation activated adhesion and differentiation cues and guided seeded neurons to grow in predefined patterns. This is the first demonstration of ligand-based light-controlled interaction between neuronal cells and biomaterials.

Mechanically Reinforced Catechol-Containing Hydrogels with Improved Tissue Gluing Performance.

In situ forming hydrogels with catechol groups as tissue reactive functionalities are interesting bioinspired materials for tissue adhesion. Poly(ethylene glycol) (PEG)⁻catechol tissue glues have been intensively investigated for this purpose. Different cross-linking mechanisms (oxidative or metal complexation) and cross-linking conditions (pH, oxidant concentration, etc.) have been studied in order to optimize the curing kinetics and final cross-linking degree of the system. However, reported systems still show limited mechanical stability, as expected from a PEG network, and this fact limits their potential application to load bearing tissues. Here, we describe mechanically reinforced PEG⁻catechol adhesives showing excellent and tunable cohesive properties and adhesive performance to tissue in the presence of blood. We used collagen/PEG mixtures, eventually filled with hydroxyapatite nanoparticles. The composite hydrogels show far better mechanical performance than the individual components. It is noteworthy that the adhesion strength measured on skin covered with blood was >40 kPa, largely surpassing (>6 fold) the performance of cyanoacrylate, fibrin, and PEG⁻catechol systems. Moreover, the mechanical and interfacial properties could be easily tuned by slight changes in the composition of the glue to adapt them to the particular properties of the tissue. The reported adhesive compositions can tune and improve cohesive and adhesive properties of PEG⁻catechol-based tissue glues for load-bearing surgery applications.

Biological and immunotoxicity evaluation of antimicrobial peptide-loaded coatings using a layer-by-layer process on titanium.

The prevention and control of peri-implantitis is a challenge in dental implant surgery. Dental implants with sustained antimicrobial coating are an ideal way of preventing peri-implantitis. This study reports development of a non- immunotoxicity multilayered coating on a titanium surface that had sustained antimicrobial activity and limited early biofilm formation. In this study, the broad spectrum AMP, Tet213, was linked to collagen IV through sulfo-SMPB and has been renamed as AMPCol. The multilayer AMPCol coatings were assembled on smooth titanium surfaces using a LBL technique. Using XPS, AFM, contact angle analysis, and QCM, layer-by-layer accumulation of coating thickness was measured and increased surface wetting compared to controls was confirmed. Non-cytotoxicity to HaCaT and low erythrocyte hemolysis by the AMPCol coatings was observed. In vivo immunotoxicity assays showed IP administration of AMPCol did not effect serum immunoglobulin levels. This coating with controlled release of AMP decreased the growth of both a Gram-positive aerobe (Staphylococcus aureus) and a Gram-negative anaerobe (Porphyromonas gingivalis) up to one month. Early S. aureus biofilm formation was inhibited by the coating. The excellent long-term sustained antimicrobial activity of this multilayer coating is a potential method for preventing peri-implantitis through coated on the neck of implants before surgery.

Multifocal Langerhans cell histiocytosis in an adult with a pathological fracture of the mandible and spontaneous malunion: A case report.

Langerhans cell histiocytosis (LCH) is rare in the adult population and even rarer with jaw involvement. The current study presents the case of a 39-year-old male who complained of recurrent pain, swelling of the gingiva and an occasional pus-like discharge in the right mandible for one year. The patient was previously prescribed antibiotics, but this did not resolve the problem. An initial panoramic radiograph showed an osteolytic lesion and bone fracture in the right mandible. Eight months later, a new radiograph showed the spontaneous malunion of the fractured mandible. The patient was eventually diagnosed with Langerhans cell histiocytosis by histopathology and immunohistochemistry. Further lesions were found in the ribs and ilium by nuclear bone scanning. The patient was subsequently treated with systemic chemotherapy, and the lesions are currently effectively being controlled. This study is the first to show that spontaneous intralesional bone regeneration may lead to reunification of the mandible fracture caused by LCH in an adult.

Enucleation combined with peripheral ostectomy: its role in the management of large cystic ameloblastomas of the mandible.

AIM: Aggressive resection of a large mandibular cystic ameloblastoma may cause severe deformity and dysfunction, while simple enucleation may carry a high risk of recurrence. The purpose of this study was to evaluate, in terms of both recurrence and the preservation of contour and function, the effectiveness of enucleation combined with peripheral ostectomy (Enu/PO) in managing large mandibular cystic ameloblastomas. MATERIALS AND METHODS: Fourteen patients who had large mandibular cystic ameloblastoma (11 unicystic, 3 multicystic) and had been treated with the Enu/PO were reviewed. RESULTS: The follow-up period was 19-117 months. Using Enu/PO, mandibular continuity was preserved in all patients, and only one patient complained of permanent lip numbness. With bone regeneration, the thinned bone plate significantly thickened and the residual cavity shrank. No pathological fracture occurred. There was recurrence in 3 patients. Two recurrences were treated with radical surgery, and the other with Enu/PO. No new recurrence was found thereafter. All patients were satisfied with their facial appearance. CONCLUSIONS: The Enu/PO technique is excellent in preserving appearance and functions and with a low risk of recurrence, is a reliable option for the management of large mandibular cystic ameloblastomas.

Osteoblast responses to thin nanohydroxyapatite coated on roughened titanium surfaces deposited by an electrochemical process.

PURPOSE: The purpose of this study was to investigate in vitro osteoblast responses to the thin nano-hydroxyapatite (HA) coating on porous implant surfaces. MATERIALS AND METHODS: Surface characteristics of nano-HA coating were evaluated by x-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR). Murine preosteoblast cell (MC3T3-E1) proliferation, alkaline phosphatase (ALP) activity, and osteocalcin release on nano-HA coated surfaces were compared with HA-coated surfaces. RESULTS: The XRD pattern demonstrated that the peak of nano-HA coating matched well with the standard HA patterns. FTIR spectra also showed that the coating consisted of pure HA crystals. Significant increases in cell proliferation, total protein on day 7, ALP activity on day 14 and day 21, and osteocalcin production on day 21 (P < .05) were observed for nano-HA coated surfaces. CONCLUSIONS: It was concluded that thin nano-HA coating, deposited by the electrochemical process, improved proliferation and differentiation of osteoblasts.

Effect of thin nano-hydroxyapatite coating on implant osseointegration in ovariectomized rats.

PURPOSE: The purpose of this study was to investigate the effect of the thin nano-hydroxyapatite (nano-HA) coating on implant osseointegration in an ovariectomized rat model. MATERIALS AND METHODS: Implants were divided into a control group and a test group (nano-HA-coated group). Surface morphology was examined using field-emission scanning electron microscopy (FSEM). Surface roughness of both groups was performed. Sixteen ovariectomized rats randomly received 2 implants in both tibiae. After 12 weeks of implantation, tibias were retrieved and prepared for histomorphometric evaluation and removal torque tests (RTQ). RESULTS: Rodlike crystals uniformly covered the porous surfaces and the surface morphology of the implant was still clear. No significant differences were found in surface roughness between the 2 groups (P > .05). More bone tissue was formed around test implants compared with control implants. Test implants showed a significantly greater BIC, bone area within all threads, and RTQ values compared with control implants (P < .05). CONCLUSIONS: These results indicate the thin nano-HA coating by an electrochemical process has potential benefits to enhance implant osseointegration in ovariectomized rats.

Osteoblast response to porous titanium surfaces coated with zinc-substituted hydroxyapatite.

BACKGROUND: The aims of this study were to deposit a zinc-hydroxyapatite (Zn-HA) coating on titanium surfaces by using the electrochemical process and investigate the cell response to the Zn-HA-coated titanium surface. STUDY DESIGN: Surface characteristics were evaluated by scanning electron microscopy (SEM) and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Murine preosteoblast cell (MC3T3-E1) proliferation, alkaline phosphatase (ALP) activity, and osteocalcin release on Zn-HA-coated surfaces were compared with HA-coated surfaces. RESULTS: Field-emission SEM observation showed rod-like HA crystals with a hexagonal cross-section on the HA-coated surface, although the hexagon of the cross-section of Zn-HA crystals became irregular. ICP-AES analysis showed that Zn was present in the Zn-HA coatings at a Zn/(Ca+Zn) molar ratio of 1.04%. Significant increases in cell proliferation, ALP activity on day 7, and osteocalcin production on day 14 (P < .05) were observed for Zn(2+)-containing HA-coated surfaces. CONCLUSIONS: The present study showed that a Zn-HA coating deposited by using the electrochemical process enhances proliferation and differentiation of osteoblasts, which has the potential benefit to enhance implant osseointegration.

Fabrication and in vitro evaluation of the collagen/hyaluronic acid PEM coating crosslinked with functionalized RGD peptide on titanium.

Surface modification of titanium (Ti) using biomolecules has attracted much attention recently. In this study, a new strategy has been employed to construct a stable and bioactive coating on Ti. To this end, a derivative of hyaluronic acid (HA), i.e. HA-GRGDSPC-(SH), was synthesized. The disulfide-crosslinked Arg-Gly-Asp (RGD)-containing collagen/hyaluronic acid polyelectrolyte membrane (PEM) coating was then fabricated on Ti through the alternate deposition of collagen and HA-GRGDSPC-(SH) with five assembly cycles and subsequent crosslinking via converting free sulphydryl groups into disulfide linkages (RGD-CHC-Ti group). The assembly processes for PEM coating and the physicochemical properties of the coating were carefully characterized. The stability of PEM coating in phosphate-buffered saline solution could be adjusted by the crosslinking degree, while its degradation behaviors in the presence of glutathione were glutathione concentration dependent. The adhesion and proliferation of MC3T3-E1 cells were significantly enhanced in the RGD-CHC-Ti group. Up-regulated bone specific genes, enhanced alkaline phosphatase activity and osteocalcin production, the increased areas of mineralization were also observed in the RGD-CHC-Ti group. These results indicate that the strategy employed herein may function as an effective way to construct stable, RGD-containing bioactive coatings on Ti.

Simvastatin-loaded porous implant surfaces stimulate preosteoblasts differentiation: an in vitro study.

OBJECTIVE: Recent studies demonstrate that simvastatin stimulates bone formation, suggesting the potential application in dental implantology. In this study, our lab developed a simvastatin-loaded titanium porous surface. The aim was to investigate the effect of simvastatin-loaded titanium surfaces on the promotion of osteogenesis in preosteoblasts (MC3T3-E1) in vitro. STUDY DESIGN: The control group consisted of cells cultured on titanium disks without any intervention for different time intervals (4, 7, and 14 days), and the experimental groups (simvastatin-loaded groups) consisted of cells cultured on titanium disks that were preincubated in varying concentration (10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L, and 10(-4) mol/L) of simvastatin for the same time intervals of the control group. Alkaline phosphatase (ALP) activity, type I collagen synthesis, and osteocalcin release were used to measure the cellular osteoblastic activities. RESULTS: All simvastatin-loaded groups showed increased ALP activity compared with the control group at every time point, especially the 10(-7) mol/L group, which significantly increased the activity almost fourfold at 4 days (P < .05). In the type I collagen synthesis assay, all simvastatin-loaded groups showed an increase, and the effect was inverse dose dependent (maximal at 10(-7) mol/L). This stimulatory effect of simvastatin was also observed in the osteocalcin release assay (P < .05; at 10(-7) mol/L, 10(-6) mol/L, maximal at 10(-7) mol/L). CONCLUSION: These results indicate that simvastatin-loaded porous implant surfaces promote accelerated osteogenic differentiation of preosteoblasts, which have the potential to improve the nature of osseointegration.

Enhanced initial proliferation and differentiation of MC3T3-E1 cells on HF/HNO3 solution treated nanostructural titanium surface.

OBJECTIVE: The aim of this study was to investigate the effects of nano- or submicro topography and fluoride ion on the biology of osteoblasts. STUDY DESIGN: Pure Ti plates were sandblasted, etched with an HCl/H(2)SO(4) solution (control surface) and then etched in a diluted HF/HNO(3) solution (test surface). MC3T3-E1 cells attached, spread, and proliferated on both surfaces. RESULTS: The alkaline phosphatase activity was evidently higher for the test surface than for control surface after 4 and 7 days of cell culture. Real-time PCR showed significant increases in type I collagen and osteocalcin gene expression in osteoblast growth on the test surface after 4 days of culture compared with the control surface. CONCLUSION: With nanotopography and fluoride, hydrogen ions might improve MC3T3-E1 cell proliferation and differentiation during the early stages of cell culture.

Osseointegration of titanium implants with a roughened surface containing hydride ion in a rabbit model.

OBJECTIVE: The objective of this study was to investigate the effect of hydride ion on bone formation and bone-bonding strength at the early stage of implantation. STUDY DESIGN: Implants and plates were treated in 2 ways: test implant (implant surface with hydride ion) and control implant (implant surface without hydride ion). Crystal structure of plate surfaces was analyzed by low angle x-ray diffractometry (XRD). Sixty implants (30 test implants and 30 control implants) were inserted into tibias of 10 rabbits. At 2 and 8 weeks postimplantation, tibias were retrieved and prepared for removal torque tests (RTQ) and histomorphometric evaluation, respectively. RESULTS: TiH(2) diffractions appeared on the XRD pattern of the test surface whereas TiH(2) did not appear on the control surface. The test implants showed 51.28% and 64.63% greater RTQ values than did the control implants at 2 and 8 weeks (P = .010, .011, respectively). Histomorphometric evaluation demonstrated the test implants significantly increased bone-implant contact and peri-implant bone formation at 2 and 8 weeks. CONCLUSION: These results suggest the presence of hydride ion in the implant surface may improve bone integration with implant surfaces at the early stage of implantation.

Cell response of titanium implant with a roughened surface containing titanium hydride: an in vitro study.

PURPOSE: The purpose of this study was to investigate the effect of surface chemistry of a sandblasted and acid-etched implant (with and without titanium hydride [TiH(2)]) on cell attachment, proliferation, and differentiation of preosteoblasts (MC3T3-E1). MATERIALS AND METHODS: Sandblasted and dual acid-etched titanium discs comprised the test group, whereas sandblasted, acid-etched, and heat-treated discs comprised the control group. Both groups' discs were sent for surface characterization. MC3T3-E1 cells were cultured on these 2 groups' discs, and then cell attachment, cell proliferation, and cell differentiation were analyzed. RESULTS: Scanning electron microscope analysis showed that the titanium discs in the 2 groups shared the same surface topography; however, x-ray diffraction examination showed that the TiH(2) diffractions only appeared in the test group. Cell attachment and cell proliferation were much better in the test group than in the control group at all time points investigated (P < .05). The expressions of alkaline phosphatase and osteocalcin were significantly higher in the test group than in the control group for both protein and transcription level at every time point (P < .05 or P < .01). CONCLUSIONS: These results suggested that surface chemistry played a significant role in cell response to the sandblasted and acid-etched surface and the presence of TiH(2) might promote the attachment, proliferation, and differentiation of preosteoblasts.

[Study on the effects of overexpression of exogenous Notch1 in tongue squamous cell carcinoma cells on cell growth and expression of epidermal growth factor receptor in vitro].

OBJECTIVE: To investigate the effects of overexpression of exogenous Notch1 in human tongue squamous cell carcinoma (TSCC) cells on cell growth and expression of epidermal growth factor receptor (EGFR) in vitro. METHODS: Human TSCC cell line Tca8113 cells were transiently transfected with the eukaryotic expression plasmid pRAMIC-IRES2-EGFP encoding exogenous intracellular fragment of Notch1 and control plasmid pIRES2-EGFP by Lipofectamine 2000, respectively. Untransfected parental Tca8113 cells served as control. The cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT) assay. The apoptosis was assessed by flow cytometry. The mRNA and protein levels of Notch1 and EGFR in Tca8113 cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The expression of EGFR protein in Tca8113 cells was detected by immunocytochemistry. RESULTS: MTT assay showed that the cell proliferation of Tca8113 cells transfected with pRAMIC-IRES2-EGFP was significantly inhibited as compared with controls (P < 0.05). After transfected with pRAMIC-IRES2-EGFP for 48 h, the apoptosis rate of Tca8113 cells was significantly higher than those of Tca8113 cells transfected with pIRES2-EGFP and untransfected Tca8113 cells (P < 0.05), and Notch1 expression was significantly increased at mRNA (P < 0.05) and protein (P < 0.05) levels, while EGFR expression was significantly decreased at mRNA (P < 0.05) and protein (P < 0.05) levels. CONCLUSION: Overexpression of exogenous Notch1 may inhibit cell growth and down-regulate EGFR expression in TSCC cells.

Biomechanical comparison of biomimetically and electrochemically deposited hydroxyapatite-coated porous titanium implants.

PURPOSE: The purpose of this study was to investigate the effects of biomimetically and electrochemically deposited hydroxyapatite on the fixation of an implant with bone tissue. MATERIALS AND METHODS: Implants were separated into 3 groups: roughened group, biomimetically deposited calcium-phosphorus (BDCaP) group, and electrochemically deposited hydroxyapatite (EDHA) group. We randomly inserted 90 implants into the femurs of 45 rabbits. After 2, 4, and 8 weeks, the femurs were retrieved and prepared for removal torque tests (RTQs) and field-emission scanning electron microscopy observation. RESULTS: During the test period, the EDHA group showed significantly greater RTQ values than did the roughened group and BDCaP group. The BDCaP group failed to increase the RTQ values compared with the roughened group. Field-emission scanning electron microscopy observation showed that the amount of attached bone tissue on the EDHA-coated implant surface was more than that on the roughened and BDCaP-coated implant surfaces during the test period. CONCLUSION: The electrochemical hydroxyapatite coating contributes to the fixation between bone and implant compared with the roughened surface, whereas the biomimetic calcium-phosphorus coating has little effect on the fixation.