Antiandrogen Therapy Radiosensitizes Androgen Receptor-Positive Cancers to 18F-FDG.

A subset (35%) of triple-negative breast cancers (TNBCs) expresses androgen receptor (AR) activity. However, clinical trials with antiandrogen drugs have shown limited efficacy, with about a 19% clinical benefit rate. We investigated the therapeutic enhancement of antiandrogens as radiosensitizers in combination with 18F-FDG in TNBC. Methods: We screened 5 candidate drugs to evaluate shared toxicity when combined with either 18F-FDG, x-rays, or ultraviolet radiation, at doses below their respective half-maximal inhibitory concentrations. Cytotoxic enhancement of antiandrogen in combination with 18F-FDG was evaluated using cell proliferation and DNA damage assays. Finally, the therapeutic efficacy of the combination treatment was evaluated in mouse tumor models of TNBC and prostate cancer. Results: Bicalutamide, an antiandrogen drug, was found to share similar toxicity in combination with either 18F-FDG or x-rays, indicating its sensitivity as a radiosensitizer to 18F-FDG. Cell proliferation assays demonstrated selective toxicity of combination bicalutamide-18F-FDG in AR-positive 22RV1 and MDA-MB-231 cells in comparison to AR-negative PC3 cells. Quantitative DNA damage and cell cycle arrest assays further confirmed radiation-induced damage to cells, suggesting the role of bicalutamide as a radiosensitizer to 18F-FDG-mediated radiation damage. Animal studies in MDA-MB-231, 22RV1, and PC3 mouse tumor models demonstrated significant attenuation of tumor growth through combination of bicalutamide and 18F-FDG in the AR-positive model in comparison to the AR-negative model. Histopathologic examination corroborated the in vitro and in vivo data and confirmed the absence of off-target toxicity to vital organs. Conclusion: These data provide evidence that 18F-FDG in conjunction with antiandrogens serving as radiosensitizers has utility as a radiotherapeutic agent in the ablation of AR-positive cancers.

Proliferating cell nuclear antigen directly interacts with androgen receptor and enhances androgen receptor­mediated signaling.

Androgen receptor (AR) and/or its constitutively active splicing variants (AR­Vs), such as AR­V7 and ARv567es, is required for prostate cancer cell growth and survival, and cancer progression. Proliferating cell nuclear antigen (PCNA) is preferentially overexpressed in all cancers and executes its functions through interaction with numerous partner proteins. The aim of the present study was to investigate the potential role of PCNA in the regulation of AR activity. An identical consensus sequence of the PCNA­interacting protein­box (PIP­box) was identified at the N­terminus of human, mouse and rat AR proteins. It was found that PCNA complexes with the full­length AR (AR­FL) and AR­V7, which can be attenuated by the small molecule PIP­box inhibitor, T2AA. PCNA also complexes with ARv567es and recombinant AR protein. The PCNA inhibitors, PCNA­I1S and T2AA, inhibited AR transcriptional activity and the expression of AR target genes in LNCaP­AI and 22Rv1 cells, but not in AR­negative PC­3 cells. The knockdown of PCNA expression reduced dihydrotestosterone­stimulated AR transcriptional activity and abolished the inhibitory effect of PCNA­I1S on AR activity. The PCNA inhibitor, PCNA­I1, exerted additive growth inhibitory effects with androgen deprivation and enzalutamide in cells expressing AR­FL or AR­FL/AR­V7, but not in AR­negative PC­3 cells. Finally, R9­AR­PIP, a small peptide mimicking AR PIP­box, was found to bind to GFP­PCNA at Kd of 2.73 µM and inhibit the expression of AR target genes, AR transcriptional activity and the growth of AR­expressing cells. On the whole, these data strongly suggest that AR is a PCNA partner protein and interacts with PCNA via the PIP­box and that targeting the PCNA­AR interaction may represent an innovative and selective therapeutic strategy against prostate cancer, particularly castration­resistant prostate cancers overexpressing constitutively active AR­Vs.

Effect of Lithium and Aluminum on the Mechanical Properties, In Vivo and In Vitro Degradation, and Toxicity of Multiphase Ultrahigh Ductility Mg-Li-Al-Zn Quaternary Alloys for Vascular Stent Application.

Magnesium alloys are the most widely studied biodegradable metals for biodegradable vascular stent application. Two major issues with current magnesium alloy based stents are their low ductility and fast corrosion rates. Several studies have validated that introduction of Li into the magnesium alloys will significantly improve the ductility while alloying with Al will improve the corrosion resistance and strength. In the present study, we studied the effects of alloying different amounts of Li and Al on the Mg-Li-Al-Zn (LAZ) quaternary alloy system. Rods were made from four different LAZ alloys, namely, LAZ611, LAZ631, LAZ911, and LAZ931 following melting, casting, and then extrusion. Systematic assessment of mechanical properties, in vitro corrosion, cytotoxicity, and in vivo degradation including local and systemic toxicity conducted demonstrated the beneficial effects of Li and Al on the mechanical properties. Our results specifically suggest that alloying with Li significantly improved the ductility while Al enhanced the strength of the LAZ alloys. Four of the LAZ alloys exhibited different corrosion rates in Hank's balanced salt solution depending on the chemical composition. Indirect in vitro cytotoxicity tests also showed lower cytotoxicity for the alloys exhibiting higher corrosion resistance. In vivo corrosion rates in the mouse subcutaneous model showed different corrosion rates compared to the in vitro tests. Nevertheless, all of the four LAZ alloys displayed no local and systemic toxicity based on the histology analysis. This research study, therefore, demonstrated the benefits of using Li and Al as alloying elements in LAZ alloys and the potential use of LAZ alloys for vascular stent application.

Additive effects of a small molecular PCNA inhibitor PCNA-I1S and DNA damaging agents on growth inhibition and DNA damage in prostate and lung cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer.

In Vitro and in Vivo Evaluation of Multiphase Ultrahigh Ductility Mg-Li-Zn Alloys for Cardiovascular Stent Application.

Magnesium alloys have been extensively studied as a novel biodegradable metallic material for cardiovascular stent application. However, the ductility limitation of magnesium alloy has been a key issue for biodegradable stents applications. In this study, two different multiphase ultrahigh ductility Mg-Li-Zn alloys, LZ61 and LZ91, are fabricated in the form of extruded rods and evaluated both in vitro and in vivo. The microstructure, mechanical properties and in vitro degradation are evaluated as well as in vitro cytotoxicity. The in vivo degradation, tissue response, and systematic toxicity are evaluated in a mouse subcutaneous model. Measurements show that LZ61 and LZ91 exhibit more than 40% elongation at fracture without significantly compromising the strength. Both in vitro and in vivo degradation showed low degradation rates for LZ61 but high degradation rate for the LZ91 alloy. Excellent biocompatibility is observed both in vivo and in vitro for LZ61 and LZ91. In summary, this study successfully demonstrates that the ultraductility multiphase Mg-Li-Zn alloy has the potential to be used for stent applications. Compared to LZ91, the LZ61 alloy shows better balance of mechanical properties, corrosion resistance, and biocompatibility, indicating its promise for cardiovascular stent applications.

The Effects of Static and Dynamic Loading on Biodegradable Magnesium Pins In Vitro and In Vivo.

Here we systematically assess the degradation of biodegradable magnesium pins (as-drawn pure Mg, as-cast Mg-Zn-Mn, and extruded Mg-Zn-Mn) in a bioreactor applying cyclical loading and simulated body fluid (SBF) perfusion. Cyclical mechanical loading and interstitial flow accelerated the overall corrosion rate, leading to loss of mechanical strength. When compared to the in vivo degradation (degradation rate, product formation, uniform or localized pitting, and stress distribution) of the same materials in mouse subcutaneous and dog tibia implant models, we demonstrate that the in vitro model facilitates the analysis of the complex degradation behavior of Mg-based alloys in vivo. This study progresses the development of a suitable in vitro model to examine the effects of mechanical stress and interstitial flow on biodegradable implant materials.

Overexpression of secretory phospholipase A2-IIa supports cancer stem cell phenotype via HER/ERBB-elicited signaling in lung and prostate cancer cells.

Resistance to conventional chemotherapies remains a significant clinical challenge in treatment of cancer. The cancer stem cells (CSCs) have properties necessary for tumor initiation, resistance to therapy, and progression. HER/ERBB­elicited signaling supports CSC properties. Our previous studies revealed that secretory phospholipase A2 group IIa (sPLA2­IIa) is overexpressed in both prostate and lung cancer cells, leading to an aberrant high level in the interstitial fluid, i.e., tumor microenvironment and blood. HER/ERBB-PI3K-Akt-NF-κB signaling stimulates sPLA2­IIa overexpression, and in turn, sPLA2­IIa activates EGFR family receptors and HER/ERBB-elicited signaling and stimulates sPLA2­IIa overexpression in a positive feedback manner. The present study determined the molecular mechanisms of sPLA2­IIa in stimulating HER/ERBB-elicited signaling and supporting CSC properties. We found that sPLA2­IIa binds both EGFR and HER3 demonstrated by co-immunoprecipitation experiments and also indirectly interacts with HER2, suggesting that sPLA2­IIa functions as a ligand for both EGFR and HER3. Furthermore, both side population CSCs from non-small cell lung cancer (NSCLC) A549 and H1975 cells and ALDH1­high CSCs from castration-resistant prostate cancer (CRPC) 22Rv1 cells overexpress sPLA2­IIa and produce tumors when inoculated into subcutis of nude mice. Given an aberrant high level of sPLA2­IIa in the tumor microenvironment that should be much higher than that in the blood, our findings support the notion that sPLA2­IIa functions as a ligand for EGFR family receptors and supports CSC properties via HER/ERBB-elicited signaling, which may contribute to resistance to therapy and cancer progression.

In vivo characterization of magnesium alloy biodegradation using electrochemical H2 monitoring, ICP-MS, and XPS.

The effect of widely different corrosion rates of Mg alloys on four parameters of interest for in vivo characterization was evaluated: (1) the effectiveness of transdermal H2 measurements with an electrochemical sensor for noninvasively monitoring biodegradation compared to the standard techniques of in vivo X-ray imaging and weight loss measurement of explanted samples, (2) the chemical compositions of the corrosion layers of the explanted samples by XPS, (3) the effect on animal organs by histology, and (4) the accumulation of corrosion by-products in multiple organs by ICP-MS. The in vivo biodegradation of three magnesium alloys chosen for their widely varying corrosion rates - ZJ41 (fast), WKX41 (intermediate) and AZ31 (slow) - were evaluated in a subcutaneous implant mouse model. Measuring H2 with an electrochemical H2 sensor is a simple and effective method to monitor the biodegradation process in vivo by sensing H2 transdermally above magnesium alloys implanted subcutaneously in mice. The correlation of H2 levels and biodegradation rate measured by weight loss shows that this non-invasive method is fast, reliable and accurate. Analysis of the insoluble biodegradation products on the explanted alloys by XPS showed all of them to consist primarily of Mg(OH)2, MgO, MgCO3 and Mg3(PO4)2 with ZJ41 also having ZnO. The accumulation of magnesium and zinc were measured in 9 different organs by ICP-MS. Histological and ICP-MS studies reveal that there is no significant accumulation of magnesium in these organs for all three alloys; however, zinc accumulation in intestine, kidney and lung for the faster biodegrading alloy ZJ41 was observed. Although zinc accumulates in these three organs, no toxicity response was observed in the histological study. ICP-MS also shows higher levels of magnesium and zinc in the skull than in the other organs. STATEMENT OF SIGNIFICANCE: Biodegradable devices based on magnesium and its alloys are promising because they gradually dissolve and thereby avoid the need for subsequent removal by surgery if complications arise. In vivo biodegradation rate is one of the crucial parameters for the development of these alloys. Promising alloys are first evaluated in vivo by being implanted subcutaneously in mice for 1month. Here, we evaluated several magnesium alloys with widely varying corrosion rates in vivo using multiple characterization techniques. Since the alloys biodegrade by reacting with water forming H2 gas, we used a recently demonstrated, simple, fast and noninvasive method to monitor the biodegradation process by just pressing the tip of a H2 sensor against the skin above the implant. The analysis of 9 organs (intestine, kidney, spleen, lung, heart, liver, skin, brain and skull) for accumulation of Mg and Zn revealed no significant accumulation of magnesium in these organs. Zinc accumulation in intestine, kidney and lung was observed for the faster corroding implant ZJ41. The surfaces of explanted alloys were analyzed to determine the composition of the insoluble biodegradation products. The results suggest that these tested alloys are potential candidates for biodegradable implant applications.

Visual H2 sensor for monitoring biodegradation of magnesium implants in vivo.

A visual sensor for H2 was used to transdermally monitor H2 that originated from biodegrading magnesium (Mg) alloys implanted subcutaneously in mice. The visual sensor consisted of a thin film of H2-sensitive material (MoO3 and Pd catalyst) coated on a flexible plastic sheet that was pressed against the mouse skin directly above the implant. Although the H2 levels permeating through the skin during the degradation process were very low, the sensor changed color to give a three dimensional (3D) visualization of H2 permeation. The correlation between the visual sensor response and measurements made with an electrochemical H2 microsensor on several magnesium alloys demonstrates that the visual sensor has the capability to monitor in real-time the dissolution rate of implants in vivo. This detection method is noninvasive, easy to implement, effective and potentially low cost compared to electrochemical detection. STATEMENT OF SIGNIFICANCE: Biodegradable Mg implants offer advantages over permanent implants such as stainless steel that are used for broken bone repair. Mg alloys gradually dissolve, avoiding the need for removal by a later surgery if complications arise. Here we report a visual H2 sensor that can be used in the research laboratory to monitor the corrosion process in vivo during animal testing of different Mg alloys. The sensor consists of a plastic sheet with a thin coating that changes color in the presence of H2 gas. The sensor is easily used by taping it on the skin over the Mg implant. The color change gives a map of the H2 level permeating from the degrading Mg through the skin above it. This low cost, simple method of monitoring the dissolution of biodegradable implants would greatly facilitate the development of the biodegradable materials, especially in animal studies where in vivo biodegradation is tested.

In vivo monitoring the biodegradation of magnesium alloys with an electrochemical H2 sensor.

UNLABELLED: Monitoring the biodegradation process of magnesium and its alloys in vivo is challenging. Currently, this process is monitored by micro-CT and X-ray imaging in vivo, which require large and costly instrumentation. Here we report a simple and effective methodology to monitor the biodegradation process in vivo by sensing H2 transdermally above a magnesium sample implanted subcutaneously in a mouse. An electrochemical H2 microsensor was used to measure the biodegradation product H2 at the surface of the skin for two magnesium alloys (ZK40 and AZ31) and one high purity magnesium single crystal (Mg8H). The sensor was able to easily detect low levels of H2 (30-400µM) permeating through the skin with a response time of about 30s. H2 levels were correlated with the biodegradation rate as determined from weight loss measurements of the implants. This new method is noninvasive, fast and requires no major equipment. STATEMENT OF SIGNIFICANCE: Biomedical devices such as plates and screws used for broken bone repair are being developed out of biodegradable magnesium alloys that gradually dissolve when no longer needed. This avoids subsequent removal by surgery, which may be necessary if complications arise. A rapid, non-invasive means for monitoring the biodegradation process in vivo is needed for animal testing and point of care (POC) evaluation of patients. Here we report a novel, simple, fast, and noninvasive method to monitor the biodegradation of magnesium in vivo by measuring the biodegradation product H2 with an electrochemical H2 sensor. Since H2 rapidly permeates through biological tissue, measurements are made by simply pressing the sensor tip against the skin above the implant; the response is within 30s.

Target validation and structure-activity analysis of a series of novel PCNA inhibitors.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair. Tumor cells express high levels of PCNA, identifying it as a potentially ideal target for cancer therapy. Previously, we identified nine compounds termed PCNA inhibitors (PCNA-Is) that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, and selectively inhibit tumor cell growth. Of these compounds, PCNA-I1 was most potent. The purpose of this study is to further establish targeting of PCNA by PCNA-I1 and to identify PCNA-I1 analogs with superior potencies. We found that PCNA-I1 does not affect the level of chromatin-associated PCNA harboring point mutations at the predicted binding site of PCNA-I1. Forty-six PCNA-I1 analogs with structures of 1-hydrazonomethyl-2-hydroxy (scaffold A), 2-hydrazonomethyl-1-hydroxy (scaffold B), 2-hydrazonomethyl-3-hydroxy (scaffold C), and 4-pyridyl hydrazine (scaffold D) were analyzed for their effects on cell growth in four tumor cell lines and PCNA trimer stabilization. Compounds in scaffold group A and group B showed the highest trimer stabilization and the most potent cell growth inhibitory activities with a significant potency advantage observed in the Z isomers of scaffold A. The absence of trimer stabilization and growth inhibitory effects in compounds of scaffold group D confirms the essentiality of the hydroxynaphthyl substructure. Compounds structure-activity relationship (SAR)-6 and SAR-24 were analyzed for their effects on and found to reduce chromatin-associated PCNA in tumor cells. This study led to the identification of SAR-24, a compound with superior potencies and potentially improved solubility, which will be used for future development of PCNA-targeting cancer therapies.

Understanding corrosion behavior of Mg-Zn-Ca alloys from subcutaneous mouse model: effect of Zn element concentration and plasma electrolytic oxidation.

Mg-Zn-Ca alloys are considered as suitable biodegradable metallic implants because of their biocompatibility and proper physical properties. In this study, we investigated the effect of Zn concentration of Mg-xZn-0.3Ca (x=1, 3 and 5wt.%) alloys and surface modification by plasma electrolytic oxidation (PEO) on corrosion behavior in in vivo environment in terms of microstructure, corrosion rate, types of corrosion, and corrosion product formation. Microstructure analysis of alloys and morphological characterization of corrosion products were conducted using x-ray computed tomography (micro-CT) and scanning electron microscopy (SEM). Elemental composition and crystal structure of corrosion products were determined using x-ray diffraction (XRD) and electron dispersive x-ray spectroscopy (EDX). The results show that 1) as-cast Mg-xZn-0.3Ca alloys are composed of Mg matrix and a secondary phase of Ca2Mg6Zn3 formed along grain boundaries, 2) the corrosion rate of Mg-xZn-0.3Ca alloys increases with increasing concentration of Zn in the alloy, 3) corrosion rates of alloys treated by PEO sample are decreased in in vivo environment, and 4) the corrosion products of these alloys after in vivo tests are identified as brucite (Mg(OH)2), hydroxyapatite (Ca10(PO4)6(OH)2), and magnesite (MgCO3·3H2O).

A Peptide mimicking a region in proliferating cell nuclear antigen specific to key protein interactions is cytotoxic to breast cancer.

Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo.

Systematic understanding of corrosion behavior of plasma electrolytic oxidation treated AZ31 magnesium alloy using a mouse model of subcutaneous implant.

This study was conducted to identify the differences between corrosion rates, corrosion types, and corrosion products in different physiological environments for AZ31 magnesium alloy and plasma electrolytic oxidation (PEO) treated AZ31 magnesium alloy. In vitro and in vivo tests were performed in Hank's Balanced Salt Solution (HBSS) and mice for 12 weeks, respectively. The corrosion rates of both AZ31 magnesium alloy and PEO treated AZ31 magnesium alloy were calculated based on DC polarization curves, volume of hydrogen evolution, and the thickness of corrosion products formed on the surface. Micro X-ray computed tomography (Micro-CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) were used to analyze morphological and chemical characterizations of corrosion products. The results show that there is more severe localized corrosion after in vitro test in HBSS; however, the thicknesses of corrosion products formed on the surface for AZ31 magnesium alloy and PEO treated AZ31 magnesium alloy in vivo were about 40% thicker than the thickness of corrosion products generated in vitro. The ratio of Ca and P (Ca/P) in the corrosion products also differed. The Ca deficient region and higher content of Al in corrosion product than AZ31 magnesium alloy were identified after in vivo test in contrast with the result of in vitro test.

Antitumor effects of a novel small molecule targeting PCNA chromatin association in prostate cancer.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair. Tumor cells express high levels of PCNA, identifying it as a potentially ideal target for cancer therapy. Previously, we identified nine compounds termed PCNA inhibitors (PCNA-Is) that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, and selectively inhibit tumor cell growth. Of these compounds, PCNA-I1 is most potent. The purposes of this study were to further investigate the effects of targeting PCNA chromatin association on DNA damage and cytotoxicity and to evaluate the therapeutic potential of PCNA-I1 against tumors in mice. Given the important roles of tumor suppressor p53 in regulating sensitivity of tumor cells to chemotherapeutics, we performed studies in two human prostate cancer cell lines differing in p53 expression: LNCaP cells (wild-type p53) and PC-3 cells (p53-null). PCNA-I1 induced DNA damage and apoptosis in both LNCaP and PC-3 cells and enhanced DNA damage and apoptosis triggered by cisplatin. PCNA-I1 also induced autophagy in PC-3 cells. A short-term pretreatment with PCNA-I1 reduced colony formation by 50% in both cell lines. These data suggest that, unlike many other cytotoxic drugs, the effects of PCNA-I1 on tumor cells do not depend on expression of p53. Intravenous administrations of PCNA-I1 significantly retarded growth of LNCaP tumors of in nude mice without causing detectable effects on mouse body weight and hematology profiles. These data provide proof of concept that targeting PCNA chromatin association could be a novel and effective therapeutic approach for treatment of cancer.

Secretory phospholipase A2-IIa upregulates HER/HER2-elicited signaling in lung cancer cells.

Lung cancer is the leading cause of cancer death worldwide. There is an urgent need for early diagnostic tools and novel therapies in order to increase lung cancer survival. Secretory phospholipase A2 group IIa (sPLA2-IIa) is involved in inflammation, tumorigenesis and metastasis. We were the first to uncover that cancer cells secrete sPLA2­IIa. sPLA2­IIa is overexpressed in almost all specimens of human lung cancers examined and is significantly elevated in the plasma of lung cancer patients. High levels of plasma sPLA2-IIa are significantly associated with advanced stage and decreased overall cancer survival. In this study, we further showed that elevated HER/HER2­PI3K-Akt-NF-κB signaling contributes to sPLA2-IIa overexpression in lung cancer cells. sPLA2-IIa in turn phosphorylates and activates HER2 and HER3 in a time- and dose­dependent manner in lung cancer cells. The structure and sequence­based docking analysis revealed that sPLA2-IIa ß hairpin shares structural similarity with the corresponding EGF hairpin. sPLA2-IIa forms an extensive interface with EGFR and brings the two lobes of EGFR into an active conformation. sPLA2-IIa also enhances the NF-κB promoter activity. Anti-sPLA2-IIa antibody, but not the small molecule sPLA2-IIa inhibitor LY315920, significantly inhibits sPLA2­IIa-induced activation of NF-κB promoter. Our findings support the notion that sPLA2-IIa functions as a ligand for the EGFR family of receptors leading to an elevated HER/HER2-elicited signaling. Plasma sPLA2-IIa can potentially serve as lung cancer biomarker and sPLA2­IIa is a potential therapeutic target against lung cancer.

In vitro and in vivo corrosion, cytocompatibility and mechanical properties of biodegradable Mg-Y-Ca-Zr alloys as implant materials.

This study introduces a class of biodegradable Mg-Y-Ca-Zr alloys novel to biological applications and presents evaluations for orthopedic and craniofacial implant applications. Mg-Y-Ca-Zr alloys were processed using conventional melting and casting techniques. The effects of increasing Y content from 1 to 4 wt.% as well as the effects of T4 solution treatment were assessed. Basic material phase characterization was conducted using X-ray diffraction, optical microscopy and scanning electron microscopy. Compressive and tensile tests allowed for the comparison of mechanical properties of the as-cast and T4-treated Mg-Y-Ca-Zr alloys to pure Mg and as-drawn AZ31. Potentiodynamic polarization tests and mass loss immersion tests were used to evaluate the corrosion behavior of the alloys. In vitro cytocompatibility tests on MC3T3-E1 pre-osteoblast cells were also conducted. Finally, alloy pellets were implanted into murine subcutaneous tissue to observe in vivo corrosion as well as local host response through H&E staining. SEM/EDS analysis showed that secondary phase intermetallics rich in yttrium were observed along the grain boundaries, with the T4 solution treatment diffusing the secondary phases into the matrix while increasing the grain size. The alloys demonstrated marked improvement in mechanical properties over pure Mg. Increasing the Y content contributed to improved corrosion resistance, while solution-treated alloys resulted in lower strength and compressive strain compared to as-cast alloys. The Mg-Y-Ca-Zr alloys demonstrated excellent in vitro cytocompatibility and normal in vivo host response. The mechanical, corrosion and biological evaluations performed in this study demonstrated that Mg-Y-Ca-Zr alloys, especially with the 4 wt.% Y content, would perform well as orthopedic and craniofacial implant biomaterials.

In vitro degradation and cytotoxicity response of Mg-4% Zn-0.5% Zr (ZK40) alloy as a potential biodegradable material.

Mg-4 wt.% Zn-0.5 wt.% Zr (ZK40) alloy was studied as a candidate material for biodegradable metallic implants in terms of its biocorrosion resistance, mechanical properties and cytocompatibility. The corrosion characteristics of ZK40 alloy were assessed by potentiodynamic polarization and immersion testing in DMEM+10% FBS solution. Analysis of the degradation characteristics by potentiodynamic polarization measurements shows the corrosion rates of ZK40 alloy in as-cast and solution treatment (T4) condition were slightly higher than those of pure Mg or as-drawn AZ31. Determination of the corrosion rate by the weight loss technique reveals that the as-cast ZK40 resulted in slower degradation than other alloy specimens after 7 days of immersion but exhibited accelerated degradation after 14 and 21 days, respectively. T4-treated ZK40 exhibited stable degradation rates compared to as-cast ZK40 and close to those of pure Mg and AZ31 during immersion testing for 14 and 21 days. In order to examine the in vitro cytocompatibility of ZK40 alloy, live/dead cell viability assay and indirect MTT assay were performed using a murine osteoblast-like cell line (MC3T3). After 3 days of direct culture of MC3T3 on ZK40 alloys the live/dead assay indicated favorable cell viability and attachment. The degradation product of ZK40 also showed minimal cytotoxicity when assessed in indirect MTT assay. The mechanical properties of the as-cast and T4-treated ZK40 alloy were superior to those of pure Mg and comparable to as-drawn AZ31. Solution treatment did not significantly enhance the cytocompatibility and mechanical properties of ZK40 alloy. Overall, the ZK40 alloy exhibited favorable cytocompatibility, biocorrosion, and mechanical properties rendering it a potential candidate for degradable implant applications.

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice.

BACKGROUND: Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. METHODS: LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. RESULTS: All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. CONCLUSION: These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

Small-molecule targeting of proliferating cell nuclear antigen chromatin association inhibits tumor cell growth.

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.