Impaired DNA double-strand breaks repair by kinesin family member 4A inhibition renders human H1299 non-small-cell lung cancer cells sensitive to cisplatin.

Patients with non-small-cell lung cancer (NSCLC) are routinely treated with the platinum-based chemotherapeutics such as cisplatin. The drug exerts anticancer effects via multiple mechanisms, including DNA double-strand breaks (DSBs). Enhanced DNA DSB repair capacity would be associated with innate or acquired drug resistance. However, despite strong evidence for the role of the chromokinesin kinesin family member 4A (KIF4A) in DSB repair, the relationship between the chromokinesin and cisplatin sensitivity of human NSCLC cells remains unknown. Furthermore, little is known regarding the effect of targeting KIF4A on the function of DSB repair-related proteins in these cells. In the current study, we demonstrated that cisplatin treatment stimulated the expression of KIF4A protein in human NSCLC cells. Depletion of KIF4A by small interfering RNA significantly enhanced cisplatin-induced cell cycle arrest in S and G2/M phases and cytotoxicity in human NSCLC cells. Furthermore, we found that KIF4A inhibition suppressed the ability of cisplatin to induce BRCA2 and Rad51 focus formation and limits the further increase in poly(ADP-ribose) polymerase 1 activity induced by cisplatin treatment in human NSCLC cells. These studies thus identify the chromokinesin KIF4A as a novel modulator of cisplatin sensitivity that is significantly enhanced by the chromokinesin in human NSCLC cells via multiple mechanisms.

"Sandwich-like" structure electrostatic spun micro/nanofiber polylactic acid-polyvinyl alcohol-polylactic acid film dressing with metformin hydrochloride and puerarin for enhanced diabetic wound healing.

A diabetic wound is a typical chronic wound with a long repair process and poor healing effects. It is an effective way to promote diabetic wound healing to design electrospinning nanofiber films with drug-assisted therapy, good air permeability and, a multilayer functional structure. In this paper, a diabetic wound dressing with a "sandwich-like" structure was designed. Metformin hydrochloride, loaded in the hydrophilic PVA inner layer, could effectively promote diabetic wound healing. The drug release was slowed down by osmosis. The laminate film dressing had good mechanical properties, with tensile strength and elongation at break reaching 5.91 MPa and 90.47 %, respectively, which was close to human skin. The laminate film loaded with erythromycin and puerarin in the hydrophobic PLA outer layer had good antibacterial properties. In addition, due to the high specific surface of the electrostatic spun film, it exhibited high water vapor permeability. It facilitates the gas exchange between the wound and the outside world. The cell experiments proved that the laminate film dressing had good biocompatibility. There was no toxic side effect on cell proliferation. In the diabetic animal wound model, it was shown that the closure rate of diabetic wound repair by laminate film reached 91.11 % in the second week. Our results suggest that the "sandwich-like" nanofiber film dressing could effectively promote the healing process and meet the various requirements of diabetic wound dressing as a promising candidate for future clinical application of chronic wound dressings.

Using thermal energy produced by irradiation of Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) for heat-inducible gene expression.

One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we developed a novel heat-inducible gene expression system in which thermal energy generated by Mn-Zn ferrite magnetic nanoparticles (MZF-NPs) under an alternating magnetic field (AMF) was used to activate gene expression. MZF-NPs, obtained by co-precipitation method, were firstly surface modified with cation poly(ethylenimine) (PEI). Then thermodynamic test of various doses of MZF-NPs was preformed in vivo and in vitro. PEI-MZF-NPs showed good DNA binding ability and high transfection efficiency. In AMF, they could rise to a steady temperature. To analyze the heat-induced gene expression under an AMF, we combined P1730OR vector transfection with hyperthermia produced by irradiation of MZF-NPs. By using LacZ gene as a reporter gene and Hsp70 as a promoter, it was demonstrated that expression of a heterogeneous gene could be elevated to 10 to 500-fold over background by moderate hyperthermia (added 12.24 or 25.81 mg MZF-NPs to growth medium) in tissue cultured cells. When injected with 2.6 or 4.6 mg MZF-NPs, the temperature of tumor-bearing nude mice could rise to 39.5 or 42.8 degrees C, respectively, and the beta-gal concentration could increase up to 3.8 or 8.1 mU/mg proteins accordingly 1 day after hyperthermia treatment. Our results therefore supported hyperthermia produced by irradiation of MZF-NPs under an AMF as a feasible approach for targeted heat-induced gene expression. This novel system made use of the relative low Curie point of MZF-NPs to control the in vivo hyperthermia temperature and therefore acquired safe and effective heat-inducible transgene expression.

Up-regulation of OLR1 expression by TBC1D3 through activation of TNFα/NF-κB pathway promotes the migration of human breast cancer cells.

Metastatic spread of cancer cells is the most life-threatening aspect of breast cancer and involves multiple steps including cell migration. We recently found that the TBC1D3 oncogene promotes the migration of breast cancer cells, and its interaction with CaM enhances the effects of TBC1D3. However, little is known regarding the mechanism by which TBC1D3 induces the migration of cancer cells. Here, we demonstrated that TBC1D3 stimulated the expression of oxidized low density lipoprotein receptor 1 (OLR1), a stimulator of cell migration, in breast cancer cells at the transcriptional level. Depletion of OLR1 by siRNAs or down-regulation of OLR1 expression using pomalidomide, a TNFα inhibitor, significantly decreased TBC1D3-induced migration of these cells. Notably, TBC1D3 overexpression activated NF-κB, a major effector of TNFα signaling, while inhibition of TNFα signaling suppressed the effects of TBC1D3. Consistent with this, NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester decreased both TBC1D3-induced OLR1 expression and cell migration, suggesting a critical role for TNFα/NF-κB signaling in TBC1D3-induced migration of breast cancer cells. Mechanistically, TBC1D3 induced activation of this signaling pathway on multiple levels, including by increasing the release of TNFα, elevating the transcription of TNFR1, TRAF1, TRAF5 and TRAF6, and decreasing the degradation of TNFR1. In summary, these studies identify the TBC1D3 oncogene as a novel regulator of TNFα/NF-κB signaling that mediates this oncogene-induced migration of human breast cancer cells by up-regulating OLR1.

Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

[Preparation and characterization of Mn-Zn ferrite oxygene nanoparticle for tumor thermotherapy].

With the sulfate as the materials and NaOH as precipitator, Mn(0.4)Zn(0.6)Fe2O4 nanoparticles were produced, which are proved to be spinel Mn-Zn ferrite analyzed by X-ray diffraction(XRD). Their shapes are approximately global examined by transmission electron microscopy(TEM) and their average diameter is 50 nm measured with image analysis-system. The Curie temperature was measured and in vitro heating test in a alternating magnetic field was carried out. The results show that the Curie temperature is 105. 407 degrees C, While its magnetic fluid could rise to 43 degrees C - 47 degrees C due to different concentration in a alternating magnetic field. The result provide theoretical and practical evidence to select an appropriate material and concentration for tumor

[Preparation and characterization of magnetic nano-particles with radiofrequency-induced hyperthermia for cancer treatment].

Mn0.5Zn0.5Fe2O4 nano-particles were prepared by the chemical co-precipitation, their characteristics were observed with transmission electron microscope (TEM), X-ray diffractometer (XRD) and thermal analysis system, and etc. The temperature changes of the nano-particles of Mn0.5Zn0.5Fe2O4 and its magnetic fluid explored in radiofrequency(RF,200 KHz, 4 KW) were measured. The proliferation ratio of L929 cells cultured in soak of Mn0.5Zn0.5Fe2O4 nano-particles were observed. The experiment indicates that the magnetic particles were about 40 nm diameter in average, round, had strong magnetism, and were proved to be consistent with the standard data of chart of XRD. Its magnetic fluid exposed to RF could be heated up to temperature range from 40 degrees C to 51 degrees C due to the amount of the magnetic nano-particles and intensity of the alternating magnetic field. Magnetic nano-particles were found to have no obvious cytotoxicity to L929 cells.

Effect of phosphorus-32 glass microspheres on human hepatocellular carcinoma in nude mice.

AIM: To study the effects of phosphorus-32 glass microspheres ((32)P-GMS) on human hepatocellular carcinoma in nude mice. METHODS: Human liver cancer cell line was implanted into the dorsal subcutaneous tissue of 40 BALB/c nude mice. Then the 40 tumor-bearing BALB/ c nude mice were allocated into treatment group (n=32) and control group (n=8). In the former group different doses of (32)P-GMS were injected into the tumor mass, while in the latter nonradioactive (31)P-GMS was injected into the tumor mass. The experimental animals were sacrificed on the 14th day. The ultrastructural changes of tumor in both treatment group and control group were studied with transmission electron microscopy (TEM) and stereology. RESULTS: In treatment group, a lot of tumor cells were killed and the death rate of tumor cells was much higher (35-70%). Ultrastructurally, severe nuclear damage was observed in the death cells. The characteristics of apoptosis such as margination of heterochromatin was also found in some tumor cells. Besides, well differentiated tumor cells, degenerative tumor cells and some lymphocytes were seen. The skin and muscle adjacent to the tumor were normal. In control group, the tumor consisted of poorly differentiated tumor cells, in which there were only a few of dead cells(5%). Stereological analysis of ultrastructural morphology showed that Vv of nuclei (53.31+/-3.46) and Vv of nucleoli(20.40+/-1.84) in the control group were larger than those(30.21+/-3.52 and 10.96+/-2.52) in the treatment group respectively (P<0.01), and Vv of RER (3.21+/-0.54) and Vv of mitochondria (4.53+/-0.89) in the control group were smaller than those (8.67+/-1.25 and 7.12+/-0.95) in the treatment group respectively (P<0.01, 0.05). Sv of the membrane of microvilli and canaliculi (27.12 um(2)/100 um(3)+/-11.84 um(2)/100 um(3)) in the control group was smaller than that (78.81 um(2)/100 um(3)+/- 19.69 um(2)/100 um(3)) in the treatment group (P<0.01). But Vv of lipid particles (3.71+/-1.97) and Vv of vacuoles (5.72+/-1.58) were much larger than those (0.30+/-0.16 and 0.35+/-0.15) in the treatment group respectively (P<0.05, P<0.01). CONCLUSION: The experimental results indicate that local administration of (32)P-GMS can produce obvious effect on liver cancer cells and the anticancer effect of (32)P-GMS is directly proportional to the dose administrated. Ultrastructural stereology can also show the effect of (32)P-GMS on the normalization of tumor cells, which is beneficial to the prognosis and treatment of patients. Moreover, local administration of (32)P-GMS is also safe.

Cytoplasmic retention of a nucleocytoplasmic protein TBC1D3 by microtubule network is required for enhanced EGFR signaling.

The hominoid oncogene TBC1D3 enhances epidermal growth factor receptor (EGFR) signaling and induces cell transformation. However, little is known regarding its spatio-temporal regulation and mechanism of tumorigenesis. In the current study, we identified the microtubule subunit ß-tubulin as a potential interaction partner for TBC1D3 using affinity purification combined with mass spectrometry analysis. The interaction between TBC1D3 and ß-tubulin was confirmed by co-immunoprecipitation. Using the same method, we also revealed that TBC1D3 co-precipitated with endogenous α-tubulin, another subunit of the microtubule. In agreement with these results, microtubule cosedimentation assays showed that TBC1D3 associated with the microtubule network. The ß-tubulin-interacting site of TBC1D3 was mapped to amino acids 286∼353 near the C-terminus of the TBC domain. Deletion mutation within these amino acids was shown to abolish the interaction of TBC1D3 with ß-tubulin. Interestingly, the deletion mutation caused a complete loss of TBC1D3 from the cytoplasmic filamentous and punctate structures, and TBC1D3 instead appeared in the nucleus. Consistent with this, wild-type TBC1D3 exhibited the same nucleocytoplasmic distribution in cells treated with the microtubule depolymerizing agent nocodazole, suggesting that the microtubule network associates with and retains TBC1D3 in the cytoplasm. We further found that deficiency in ß-tubulin-interacting resulted in TBC1D3's inability to inhibit c-Cbl recruitment and EGFR ubiquitination, ultimately leading to dysregulation of EGFR degradation and signaling. Taken together, these studies indicate a novel model by which the microtubule network regulates EGFR stability and signaling through tubulin dimer/oligomer interaction with the nucleocytoplasmic protein TBC1D3.