Characterization of a conjugated polysuccinimide-carboplatin compound.

Carboplatin, an advanced anticancer drug with excellent efficacy against ovarian cancer, was developed to alleviate the side effects that often occur with cisplatin and other platinum-based compounds. Our study reports the in vitro characteristics, viability, and activity of cells expressing the inducible nitric oxide synthase (iNOS) gene after carboplatin was conjugated with polysuccinimide (PSI) and administered in combination with other widely used anticancer drugs. PSI, which has promising properties as a drug delivery material, could provide a platform for prolonging carboplatin release, regulating its dosage, and improving its side effects. The iNOS gene has been shown to play an important role in both cancer cell survival and inhibition. Herein, we synthesized a PSI-carboplatin conjugate to create a modified anticancer agent and confirmed its successful conjugation. To ensure its solubility in water, we further modified the structure of the PSI-carboplatin conjugate with 2-aminoethanol groups. To validate its biological characteristics, the ovarian cancer cell line SKOV-3 and normal ovarian Chinese hamster ovary cells were treated with the PSI-carboplatin conjugate alone and in combination with paclitaxel and topotecan, both of which are used in conventional chemotherapy. Notably, PSI-carboplatin conjugation can be used to predict changes in the genes involved in cancer growth and inhibition. In conclusion, combination treatment with the newly synthesized polymer-carboplatin conjugate and paclitaxel displayed anticancer activity against ovarian cancer cells but was not toxic to normal ovarian cancer cells, resulting in the development of an effective candidate anticancer drug without severe side effects.

Efficient mutation screening for cervical cancers from circulating tumor DNA in blood.

BACKGROUND: Early diagnosis and continuous monitoring are necessary for an efficient management of cervical cancers (CC). Liquid biopsy, such as detecting circulating tumor DNA (ctDNA) from blood, is a simple, non-invasive method for testing and monitoring cancer markers. However, tumor-specific alterations in ctDNA have not been extensively investigated or compared to other circulating biomarkers in the diagnosis and monitoring of the CC. Therfore, Next-generation sequencing (NGS) analysis with blood samples can be a new approach for highly accurate diagnosis and monitoring of the CC. METHOD: Using a bioinformatics approach, we designed a panel of 24 genes associated with CC to detect and characterize patterns of somatic single-nucleotide variations, indels, and copy number variations. Our NGS CC panel covers most of the genes in The Cancer Genome Atlas (TCGA) as well as additional cancer driver and tumor suppressor genes. We profiled the variants in ctDNA from 24 CC patients who were being treated with systemic chemotherapy and local radiotherapy at the Jeonbuk National University Hospital, Korea. RESULT: Eighteen out of 24 genes in our NGS CC panel had mutations across the 24 CC patients, including somatic alterations of mutated genes (ZFHX3-83%, KMT2C-79%, KMT2D-79%, NSD1-67%, ATM-38% and RNF213-27%). We demonstrated that the RNF213 mutation could be used potentially used as a monitoring marker for response to chemo- and radiotherapy. CONCLUSION: We developed our NGS CC panel and demostrated that our NGS panel can be useful for the diagnosis and monitoring of the CC, since the panel detected the common somatic variations in CC patients and we observed how these genetic variations change according to the treatment pattern of the patient.

Combinatory Analysis of Cell-free and Circulating Tumor Cell DNAs Provides More Variants for Cancer Treatment.

BACKGROUND/AIM: Non-invasive biomarker detection using DNA from cell-free circulating DNA (cfDNA) and circulating tumor cells (ctcDNA) are emerging as they can be used for early diagnosis, prognosis and therapeutic target selection for cancer. However, cfDNA and ctcDNA from the same patient have not yet been compared extensively on how different the genetic characteristics of the two are in terms of the overlap between them. MATERIALS AND METHODS: The performance of a customized NGS panel was used to compare the variants found in the 20 pairs of cfDNA and ctcDNA from gynecological cancer patients. RESULTS: A genetic variant analysis revealed that there were only nine common overlapping variants out of 63 between the cfDNA and ctcDNA pairs, while 31 and 22 were unique to cfDNA and ctcDNA, respectively. CONCLUSION: A combinatory analysis of both cfDNA and CTCs from cancer patients can improve the sensitivity of liquid biopsies. These results are expected to provide better genetic target information for guiding clinical strategies for cancer.

Technologies for circulating tumor cell separation from whole blood.

The importance of early cancer diagnosis and improved cancer therapy has been clear for years and has initiated worldwide research towards new possibilities in the care strategy of patients with cancer using technological innovations. One of the key research fields involves the separation and detection of circulating tumor cells (CTC) because of their suggested important role in early cancer diagnosis and prognosis, namely, providing easy access by a liquid biopsy from blood to identify metastatic cells before clinically detectable metastasis occurs and to study the molecular and genetic profile of these metastatic cells. Provided the opportunity to further progress the development of technology for treating cancer, several CTC technologies have been proposed in recent years by various research groups and companies. Despite their potential role in cancer healthcare, CTC methods are currently mainly used for research purposes, and only a few methods have been accepted for clinical application because of the difficulties caused by CTC heterogeneity, CTC separation from the blood, and a lack of thorough clinical validation. Therefore, the standardization and clinical application of various developed CTC technologies remain important subsequent necessary steps. Because of their suggested future clinical benefits, we focus on describing technologies using whole blood samples without any pretreatment and discuss their advantages, use, and significance. Technologies using whole blood samples utilize size-based, immunoaffinity-based, and density-based methods or combinations of these methods as well as positive and negative enrichment during separation. Although current CTC technologies have not been truly implemented yet, they possess high potential as future clinical diagnostic techniques for the individualized therapy of patients with cancer. Thus, a detailed discussion of the clinical suitability of these new advanced technologies could help prepare clinicians for the future and can be a foundation for technologies that would be used to eliminate CTCs in vivo.

Ferulate, an Active Component of Wheat Germ, Ameliorates Oxidative Stress-Induced PTK/PTP Imbalance and PP2A Inactivation.

Ferulate is a phenolic compound abundant in wheat germ and bran and has been investigated for its beneficial activities. The aim of the present study is to evaluate the efficacy of ferulate against the oxidative stress-induced imbalance of protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and serine/threonine protein phosphatase 2A (PP2A), in connection with our previous finding that oxidative stress-induced imbalance of PTKs and PTPs is linked with proinflammatory nuclear factor-kappa B (NF-κB) activation. To test the effects of ferulate on this process, we utilized two oxidative stress-induced inflammatory models. First, YPEN-1 cells were pretreated with ferulate for 1 hr prior to the administration of 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH). Second, 20-month-old Sprague-Dawley rats were fed ferulate for 10 days. After ferulate treatment, the activities of PTKs, PTPs, and PP2A were measured because these proteins either directly or indirectly promote NF-κB activation. Our results revealed that in YPEN-1 cells, ferulate effectively suppressed AAPH-induced increases in reactive oxygen species (ROS) and NF-κB activity, as well as AAPH-induced PTK activation. Furthermore, ferulate also inhibited AAPH-induced PTP and PP2A inactivation. In the aged kidney model, ferulate suppressed aging-induced activation of PTKs and ameliorated aging-induced inactivation of PTPs and PP2A. Thus, herein we demonstrated that ferulate could modulate PTK/PTP balance against oxidative stress-induced inactivation of PTPs and PP2A, which is closely linked with NF-κB activation. Based on these results, the ability of ferulate to modulate oxidative stress-related inflammatory processes is established, which suggests that this compound could act as a novel therapeutic agent.

Kinetics of dopamine release from poly(aspartamide)-based prodrugs.

Preparation of novel biocompatible and biodegradable polymer-based prodrugs that can be applied in complex drug delivery systems is one of the most researched fields in pharmaceutics. The kinetics of the drug release strongly depends on the physicochemical parameters of prodrugs as well as environmental properties, therefore precise kinetical description is crucial to design the appropriate polymer prodrug formula. The aim of the present study was to investigate the dopamine release from different poly(aspartamide) based dopamine drug conjugates in different environments and to work out a kinetic description which can be extended to describe drug release in similar systems. Poly(aspartamide) was conjugated with different amounts of dopamine. In order to alter the solubility of the conjugates, 2-aminoethanol was also grafted to the main chain. Chemical structure as well as physical properties such as solubility, lipophilicity measurements and thermogravimetric analysis has been carried out. Kinetics of dopamine release from the macromolecular prodrugs which has good water solubility has been studied and compared in different environments (phosphate buffer, Bromelain and α-Chymotrypsin). It was found that the kinetics of release in those solutions can be satisfactorily described by first order reaction rate. For poorly-soluble conjugates, the release of dopamine was considered as a result of coupling of diffusion and chemical reaction. Besides the time dependence of dopamine cleavage, a practical quantity, the half-life of the release of loading capacity has been introduced and evaluated. It was found, that dopamine containing macromolecular prodrugs exhibit prolonged release kinetics and the quantitative description of the kinetics, including the most important physical parameters provides a solid base for future pharmaceutical and medical studies. STATEMENT OF SIGNIFICANCE: Poly(aspartamide) based polymer-drug conjugates are promising for controlled and prolonged drug delivery due to their biocompatibility and biodegradability. In this study different poly(aspartamide) based dopamine conjugates were synthesized which can protect dopamine from deactivation in the human body. Since there is no satisfying kinetics description for drug release from covalent polymer-drug conjugates in the literature, dopamine release was investigated in different environments and a complete kinetical description was worked out. This study demonstrates that poly(aspartamide) is able to protect conjugated dopamine from deactivation and provide prolonged release in alkaline pH as well as in the presence of different enzymes. Furthermore, detailed kinetical descriptions were demonstrated which can be used in case of other covalent polymer-drug conjugates.

Silver nanoparticles induce apoptosis through the toll-like receptor 2 pathway.

OBJECTIVE: The objective of this study was to evaluate the effects of silver nanoparticles (AgNPs) on the Toll-like receptor 2 (TLR-2) pathway in cultured cells. STUDY DESIGN: Human chondrocytes, periodontal ligament (PDL) cells, and SCC-9 cells (squamous cell carcinoma from the tongue) were cultured and subjected to cytotoxicity assays. To evaluate the effects of AgNPs on the TLR-2 pathway, TLR-2 small interfering (si) RNA or TLR-2 antibodies were applied to the chondrocytes, followed by the application of AgNPs. RESULTS: AgNPs induced dose-dependent effects on the examined cell types in terms of both cytotoxicity and TLR-2 expression levels. AgNP-mediated apoptosis was reduced after treatment with TLR-2 siRNA in both PDL cells and chondrocytes. Furthermore, functional blocking of TLR-2 with anti-TLR2 antibodies inhibited AgNP-mediated cytotoxicity. AgNPs increased c-Jun phosphorylation, an effect that was reversed after treatment with TLR-2 siRNA. CONCLUSIONS: The results indicate that AgNP-mediated apoptosis most likely occurs via the TLR-2 pathway.

Development of nano-hydroxyapatite graft with silk fibroin scaffold as a new bone substitute.

PURPOSE: This study involves a comparison between the bone regeneration of nano-hydroxyapatite (nHA), as derived from eggshells either with or without silk fibroin scaffolds, and the unfilled control in the rabbit calvarial bony defect model. MATERIALS AND METHODS: Sixteen 4-month-old New Zealand white rabbits, with a mean weight of 2.8 kg (range, 2.5-3.0 kg), were used in this experiment. After the formation of bilateral parietal bony defects (diameter, 8.0 mm), either an nHA or an nHA+silk fibroin combination (nHA+silk) was grafted. The control was unfilled defect. The bone regeneration was evaluated by micro-computed tomography (µCT) and histomorphometric analyses at 4 and 8 weeks. RESULTS: All measured variables of the µCT analysis were significantly higher in the grafted groups (nHA and nHA+silk) than in the unfilled control groups at both 4 and 8 weeks after operation (P < .05). On histomorphometric analysis, there was no significant difference between the groups at 4 weeks after operation. However, the nHA group exerted significantly higher bone regeneration (40.16% ± 8.27%) compared with the unfilled control group (25.66% ± 10.98%) or the nHA+silk group (16.62% ± 3.05%) (P < .05). CONCLUSION: The nHA from eggshells exerted better bone formation than the unfilled control group on both µCT and histomorphometric analyses. Considering the rapid healing in bony defect and easy availability, the nHA from the eggshells could prove to be a good new bone substitute.

Static tensional forces increase osteogenic gene expression in three-dimensional periodontal ligament cell culture.

Orthodontic tooth movement results from the combinational process of both bone resorption and formation in the compressive and tension sides, respectively. However, the genes responsible for new bone formation in tension sides have not been determined. In this study, we used DNA microarray and real-time RT-PCR to identify genes in human periodontal ligament (PDL) cells that undergo significant changes in expression in response to static tensional forces (2 or 12 hours). The genes found were alkaline phospatase (ALP), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and several collagen genes. Furthermore, an ELISA evaluating the expression of VEGF, type IV collagen and MMP-2 found levels significantly increased after 24 and 72 hours (P 0.05). ALP activity was also increased after 24 hours (P 0.05). Collectively, we found the genes up-regulated in our study by the static tensional force are related to osteogenic processes such as matrix synthesis and angiogenesis.

Avemar (wheat germ extract) in cancer prevention and treatment.

Many healthy foods are derived from wheat germ. The molecular composition of these products, however, greatly differs as shown by normal-phase HPLC-mass spectrometry analysis; thus, experimental data obtained by one of them is not necessarily true for the other. Avemar is a nontoxic wheat germ extract registered as a special nutriment for cancer patients in Hungary. It shows potent anticancer activity on cell lines by deeply interfering with glucose metabolism and affecting expressions of several kinases. In in vivo experimental models, Avemar is also effective by enhancing the activity of the immune system such as stimulating NK cell activity (by reducing MHC I molecule expression), enhancing TNF secretion of the macrophages, increasing ICAM 1 molecule expression on the vascular endothelial cells. All of these lead to apoptosis of tumor cells. The wide range of biological activity of Avemar probably cannot be explained by only one active ingredient. Since there are numerous experimental data and the clinical benefit repeatedly confirmed Avemar can be one of the most potent and best researched food supplements available for cancer patients.

Downregulation of matrix metalloproteinases in hyperplastic dental follicles results in abnormal tooth eruption.

In this study, we compared the gene expression profiles of non-syndromic hyperplastic dental follicle (HDF) fibroblasts and normal dental follicle (NDF) fibroblasts using cDNA microarrays, quantitative PCR, and immunohistochemical staining. Microarray analysis showed that several collagens genes were upregulated in the HDFos, including collagen types I, IV, VIII, and XI and TIMP-1, -3, and -4 (fold ratio > 2.0). In contrast, the expression of MMP-1, -3, -10, and -16 together with IL-8 was more than two fold downregulated. The differential expression of the genes encoding alkaline phosphatase, MMP-1, -3, -8, and IL-8 was confirmed by quantitative RT-PCR, while that of 24 HDFs and 18 NDFs was confirmed by immunohistochemical analysis. However, HDFs showed stronger expression of MMP-3 than NDFs (P < 0.001). Collectively, these results indicate that defective regulation of MMPs mediating connective tissue remodeling may be responsible for abnormal tooth eruption.

Effect of estrogen on the expression of cytokines of the temporomandibular joint cartilage cells of the mouse.

PURPOSE: The purpose of this study was to evaluate the effect of estrogen on the synthesis of cytokines of the temporomandibular joint cells. METHODS: Mandibular condyles of 10 mice were resected. Twenty of these condylar cartilages were removed and placed in organ culture for 24 hours with media containing different concentrations of 17beta-estradiol. The chondroblasts from the mandibular condyles of 3 mice were cultured with different concentrations of 17beta-estradiol the same as above. Cytokine concentrations were measured by ELISA. RESULTS: The expression and concentration of IL-1 beta, IL-6, and IL-8 were increased with increasing concentration of 17beta-estradiol. The expression and concentration of IL-4 and IL-10 were not different from the control and experimental group. CONCLUSIONS: These findings suggest that estrogen has the potential to cause temporomandibular joint disease with induction of the proinflammatory cytokines, IL-1 beta, IL-6, and IL-8.

Differential gene expression of periodontal ligament cells after loading of static compressive force.

BACKGROUND: Compressive force is an important mechanical stimulus on the periodontal ligament (PDL) and is closely related to therapeutic tooth movement. In this study, early or late response genes related to the compressive stress in PDL cells were evaluated. Particularly, the expression of interleukin (IL)-6, IL-8, and alkaline phosphatase (ALP) was studied. METHODS: The primary cultured cells from PDL were grown in a three-dimensional collagen gel, and received a continuous static compressive force (1.76 g/cm(2)). The expressed genes were screened by cDNA microarray assays for 2 or 12 hours after the initiation of the mechanical force application. The genes of interest that showed significant changes in expression in the cDNA microarray assay were analyzed further by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunoabsorbent assays (ELISA), and ALP assays. RESULTS: ALP, IL-6, and IL-8 were selected among the genes that significantly changed expression (/M/ >0.7) and subsequently were confirmed by quantitative RT-PCR. The secreted protein concentrations for IL-6, IL-8, and ALP activity were measured at 72 hours after application of continuous static compressive force. The protein level of IL-6 was significantly increased at 72 hours (P <0.001), but there was no significant change in IL-8 (P >0.05). ALP activity was decreased approximately 41.5% compared to the control (P = 0.015). CONCLUSIONS: Considering that IL-6 is a potent osteoclast activator and the compressive side of PDL during orthodontic tooth movement shows the resorption of calcified tissue, the changed expression of IL-6 and ALP in response to the static compressive force in PDL cells may contribute to the orthodontic tooth movement or alveolar bone remodeling.

Ganglion cyst of the temporomandibular joint.

We report a case of cystic lesion in a 37-year-old woman. The patient had an oval-shaped lesion adjacent to the temporomandibular joint. Thick fibrotic tissue and muscle were observed microscopically, but the epithelium lining was not observed. The lesion was diagnosed as a ganglion cyst. The patient's general medical history was non-contributory. High performance liquid chromatography (HPLC) and mass spectrophotometry (MS) revealed some proteins from the fluid in the lesion, such as a filaggrin precursor, dystroglycan, a polyprotein of the hepatitis C virus, and proteins originating from bacteria. The follow-up examinations revealed no recurrence. The probable pathogenesis of the lesion is discussed.