The Effect of High-Dose-Rate Pulsed Radiation on the Survival of Clinically Relevant Radioresistant Cells

We demonstrated that low dose pulsed radiation (0.25 Gy) at a high-dose-rate, even for very short intervals (10 s), decreases cell survival to a greater extent than single exposure to a similar total dose and dose rate. The objective of this study was to clarify whether high-dose-rate pulsed radiation is effective against SAS-R, a clinically relevant radioresistant cell line. Cell survival following high-dose-rate pulsed radiation was evaluated via a colony assay. Flow cytometry was utilized to evaluate γH2AX, a molecular marker of DNA double-strand breaks and delayed reactive oxygen species (ROS) associated with radiation-induced apoptosis. Increased cytotoxicity was observed in SAS-R and parent SAS cells in response to high dose rate pulsed radiation compared to single dose, as determined by colony assays. Residual γH2AX in both cells subjected to high-dose-rate pulsed radiation showed a tendency to increase, with a significant increase observed in SAS cells at 72 h. In addition, high-dose-rate pulsed radiation increased delayed ROS more than the single exposure did. These results indicate that high-dose-rate pulsed radiation was associated with residual γH2AX and delayed ROS, and high-dose-rate pulsed radiation may be used as an effective radiotherapy procedure against radioresistant cells.

Particulate Matter (PM10) Promotes Cell Invasion through Epithelial–Mesenchymal Transition (EMT) by TGF-β Activation in A549 Lung Cells

Air pollution presents a major environmental problem, inducing harmful effects on human health. Particulate matter of 10 μm or less in diameter (PM10) is considered an important risk factor in lung carcinogenesis. Epithelial–mesenchymal transition (EMT) is a regulatory program capable of inducing invasion and metastasis in cancer. In this study, we demonstrated that PM10 treatment induced phosphorylation of SMAD2/3 and upregulation of SMAD4. We also reported that PM10 increased the expression and protein levels of TGFB1 (TGF-β), as well as EMT markers SNAI1 (Snail), SNAI2 (Slug), ZEB1 (ZEB1), CDH2 (N-cadherin), ACTA2 (α-SMA), and VIM (vimentin) in the lung A549 cell line. Cell exposed to PM10 also showed a decrease in the expression of CDH1 (E-cadherin). We also demonstrated that expression levels of these EMT markers were reduced when cells are transfected with small interfering RNAs (siRNAs) against TGFB1. Interestingly, phosphorylation of SMAD2/3 and upregulation of SMAD induced by PM10 were not affected by transfection of TGFB1 siRNAs. Finally, cells treated with PM10 exhibited an increase in the capacity of invasiveness because of EMT induction. Our results provide new evidence regarding the effect of PM10 in EMT and the acquisition of an invasive phenotype, a hallmark necessary for lung cancer progression.

EXTH-15. MULTI-FACETED INHIBITION OF TET PATHWAY WITH CELL-PERMEABLE 2HG AND BOBCAT 339 REDUCES PROLIFERATION AND INDUCES APOPTOSIS IN DIPG

Diffuse Intrinsic Pontine Glioma (DIPG) is one of the worst forms of pediatric brain tumors, with a 100% mortality-rate. A prominent mutation observed in them is a lysine to methionine change in histone3 which causes trapping of the repressive-chromatin-complex, leading to genome-level hypomethylation, affecting global epigenetic-regulation. Ten-Eleven Translocation (TET) proteins are alpha-ketoglutarate (α-KG) dependent dioxygenases that mediate the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), a key-step in removing DNA methylation-marks. We previously identified increased levels of 5hmC and TETs in DIPG. In IDH mutant glioma, the R132H-IDH produces an oncometabolite, 2-hydroxyglutarate (2HG), instead of α-KG. 2HG competitively inhibits TET function, resulting in a hypermethylated genome. Bobcat339, a cytosine-analog targets TETs by blocking the cytosine binding-site on TETs. We hypothesized that cell-permeable 2HG and Bobcat339 would synergize to block TET activity in DIPG, restore epigenetic-balance by increasing genomic methylation, and induce cell-death. 2HG induced apoptosis in DIPG cells at concentrations ranging from 100-300uM, as measured by cleaved-PARP western and cleaved-caspase3 immunofluorescence (2-7fold increase in CC3, depending on cell line). Cell-permeable 2HG also decreased proliferation, measured by phospho-RB western in DIPG cells (30-50% reduction in pRB band intensity, depending on cell line). Similarly, Bobcat339 suppressed proliferation at concentrations from 10-50uM, measured by BrdU incorporation (25% reduction in BrdU+ at 50uM, p= 0.02 compared to control). Bobcat339 induced apoptosis, measured by cPARP western and CC3 immunofluorescence (4-10fold more of CC3+ compared to control p< 0.0004). In combination, cell-permeable 2HG (100-200uM) and Bobcat339 (10-20uM) combined to suppress cell-viability, measured by CellTiterBlue assay, producing ZIP scores of ~20 in DIPG cells (indicating high-level synergy). In combination, 2HG and Bobcat339 increased apoptosis in DIPG (3-fold increase in cleaved-PARP band intensity in JHH-DIPG1 cells treated with 20uM Bobcat339 + 100uM 2HG, compared to single-treated cells). Our results may lead to new approaches that target TET pathway in DIPG tumors.

Inhibitory Effects of Cycloheterophyllin on Melanin Synthesis

This study was performed to clarify the inhibitory effects of cycloheterophyllin on melanin synthesis. In order to elucidate the inhibitory effects of cycloheterophyllin on the B16F10 cell line, cell viability, messenger ribonucleic acid (mRNA) expressions, tyrosinase activity assay, and melanin production assay were measured. The effects of cycloheterophyllin on tyrosinase-related protein 1 (TYRP1)/TYRP2/tyrosinase (TYR)/microphthalmia-associated transcription factor (MITF) mRNA expressions and melanin content were determined. Quantitative real-time RT-PCR showed that cycloheterophyllin decreased the mRNA expression level of TYRP1/TYRP2/TYR/MITF genes and melanin production contents than α-MSH-treated B16F10 cells. The tyrosinase activity assay revealed that cycloheterophyllin decreased the melanin production in the B16F10 cells. These data show that cycloheterophyllin increases the whitening effects in the B16F10 cells; thus, cycloheterophyllin is a potent ingredient for skin whitening. Thus, further research on the mechanism of action of cycloheterophyllin for the development of functional materials should be investigated.

Wound Healing Effects of Aloe muth-muth: In Vitro Investigations Using Immortalized Human Keratinocytes (HaCaT)

The traditional use of Aloe spp. for the purpose of wound healing has a long history and is widespread internationally. Recently, a hybrid aloe plant (Aloe muth-muth) has been cultivated by cross pollination between Aloe vera and Aloe ferox. The Aloe muth-muth plant has not yet been investigated for medicinal properties and provides an opportunity for potential biological activity, including wound healing. The aim of this study was to investigate the in vitro wound healing effects of both Aloe muth-muth gel and whole leaf material with the use of the immortalized human keratinocyte (HaCaT) cell line. Cell viability was conducted using methyl thiazolyl tetrazolium (MTT) assays. In vitro wound healing was tested on HaCaT cells using an established scratch assay method. The effect of Aloe muth-muth gel material on HaCaT cell migration was also investigated. Aloe muth-muth gel material exhibited statistically significantly (p < 0.05) higher percentage wound closure compared to the control at all three concentrations investigated. These findings confirm that this newly cultivated species, Aloe muth-muth, also possesses wound healing activity corresponding to that reported for the two species it is derived from, namely, Aloe vera and Aloe ferox. Therefore, Aloe muth-muth has the potential to be used in future wound therapeutics.

Apoptotic Effects of Drug Targeting Conjugates Containing Different GnRH Analogs on Colon Carcinoma Cells

The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.

NHE8 attenuates Ca2+ influx into NRK cells and the proximal tubule epithelium

To garner insights into the renal regulation of Ca2+ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca2+-sensing receptor (CaSR) agonist cinacalcet. This revealed decreased gene expression of Na+/H+ exchanger isoform 8 (NHE8) in response to CaSR activation. These results were confirmed by quantitative real-time PCR. Moreover, administration of vitamin D also decreased NHE8 mRNA expression. In contrast, renal NHE8 protein expression from the same samples was increased. To examine the role of NHE8 in transmembrane Ca2+ fluxes, we used the normal rat kidney (NRK) cell line. Cell surface biotinylation and confocal immunofluorescence microscopy demonstrated NHE8 apical expression. Functional experiments found 5-( N-ethyl- N-isopropyl)amiloride (EIPA)-inhibitable NHE activity in NRK cells at concentrations minimally attenuating NHE1 activity in AP-1 cells. To determine how NHE8 might regulate Ca2+ balance, we measured changes in intracellular Ca2+ uptake by live cell Ca2+ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na+-enhanced Ca2+ influx into NRK cells. Ca2+ influx was mediated by a voltage-dependent Ca2+ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca2+ influx and NHE8 inhibition-augmented Ca2+ influx via a voltage-dependent Ca2+ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca2+ influx in the presence of luminal EIPA at a concentration that would inhibit NHE8. The results of the present study are consistent with NHE8 regulating Ca2+ uptake into the proximal tubule epithelium.

Effects of Quercetin-Loaded Nanoparticles on MCF-7 Human Breast Cancer Cells

Background and objectives: Previous studies have shown anti-tumor activity of quercetin (QT). However, the low bioavailability of QT has restricted its use. This study aimed to assess the toxic effect of QT encapsulated in solid lipid nanoparticles (QT-SLNs) on the growth of MCF-7 human breast cancer cells. Materials and Methods: MCF-7 and MCF-10A (non-tumorigenic cell line) cell lines treated with 25 µmol/mL of QT or QT-SLNs for 48 h. Cell viability, colony formation, oxidative stress, and apoptosis were evaluated to determine the toxic effects of the QT-SLNs. Results: The QT-SLNs with appropriate characteristics (particle size of 85.5 nm, a zeta potential of −22.5 and encapsulation efficiency of 97.6%) were prepared. The QT-SLNs showed sustained QT release until 48 h. Cytotoxicity assessments indicated that QT-SLNs inhibited MCF-7 cells growth with a low IC50 (50% inhibitory concentration) value, compared to the free QT. QT-SLNs induced a significant decrease in the viability and proliferation of MCF-7 cells, compared to the free QT. QT-SLN significantly increased reactive oxygen species (ROS) level and MDA contents and significantly decreased antioxidant enzyme activity in the MCF-7 cells. Following QT-SLNs treatment, the expression of the Bcl-2 protein significantly decreased, whereas Bx expression showed a significant increase in comparison with free QT-treated cells. Furthermore, The QT-SLNs significantly increased apoptotic and necrotic indexes in MCF-7 cells. Viability, proliferation, oxidative stress and apoptosis of MCF-10A cells were not affected by QT or QT-SLNs. Conclusions: According to the results of this study, SLN significantly enhanced the toxic effect of QT against human breast cancer cells.

Application of network diffusion approaches to drug screenings: A perspective on multilayered networks derived from cell lines and drugs

Network diffusion approaches are frequently used for identifying the relevant disease genes and for prioritizing the genes for drug sensitivity predictions. Majority of these studies rely on networks representing a single type of information. However, using multiplex heterogeneous networks (networks with multiple interconnected layers) is much more informative and helps to understand the global topology. We built a multi-layered network that incorporates information on protein-protein interactions, drug-drug similarities, cell line-cell line similarities and co-expressed genes. We applied Random Walk with Restart algorithm to investigate the interactions between drugs, targets and cancer cell lines. Results of ANOVA models show that these prioritized genes are among the most significant ones that relate to drug response. Moreover, the predictive power of the drug response prediction models built using the gene expression data of only the top ranked genes is similar to the models built using all the available genes. Taken together, the results confirm that the multiplex heterogeneous network-based approach is efficient in identifying the most significant genes associated with drug response.