The anticancer effect of the TLR4 inhibition using TAK-242 (resatorvid) either as a single agent or in combination with chemotherapy: A novel therapeutic potential for breast cancer

Increasing pieces of evidence indicate that inflammatory processes facilitate tumorigenesis; tumor cells simulate the mechanisms by which innate immune cells produce pro-inflammatory cytokines to exploit them for their own survival and proliferation. Toll-like receptor 4 (TLR4), which serves as one of the most well-known receptors on the surface of the immune cells, is often expressed ectopically in the tumor cells resulting in tumor progression, invasion, and chemoresistance. In this study, we examined the anticancer effects of TAK-242, a small molecule inhibitor of TLR4, on different breast cancer cell lines: MCF7, SKBR3, MDA-MB-231, and BT-474. Our results showed that the TLR4 inhibition, as revealed by the downregulation of TLR4 downstream genes, exerted desirable cytotoxicity on the TLR4-expressing cells, at least partly, through the downregulation of EGFR and c-Myc genes. TAK-242 also inhibited the proliferation of anoikis-resistant cells and suppressed the clonal growth of the indicated cells. The results of this study propose a mechanistic pathway by which the inhibition of TLR4 using TAK-242 could augment apoptotic cell death through the alteration of both nuclear factor-кB- and p53-related apoptosis genes in breast cancer cells, especially cells with overexpression of TLR4. Taken together, this study supports the idea that the activation of inflammatory pathways may have a crucial role in breast cancer progression and the inhibition of TLR4 using TAK-242, either as a single agent or in combination, seems to be a novel promising strategy that could be clinically available in foreseeable future.     

HIV-specific T-cells accumulate in the liver in HCV/HIV co-infection

Background and Aims

Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses.

Methods

Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry.

Results

HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4⁺ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8⁺ and CD4⁺ T-cells producing IFN-γ and TNF-α were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8⁺ T-cells produced more of the fibrogenic cytokine, TNF-α. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4⁺ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses.

Conclusion

The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease.     

Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.     

Investigation of the Effects of Phosphate Fertilizer Application on the Heavy Metal Content in Agricultural Soils with Different Cultivation Patterns

The use of phosphate fertilizers is essential in agriculture, because they supply farmland with nutrients for growing plants. However, heavy metals might be included as impurities in natural materials and minerals, so heavy metals can also be present in phosphate fertilizers or other chemical fertilizers. The aim of this work was to assess the heavy metal content and contamination status of agricultural soils in the Hamadan province of Iran used for the cultivation of different crops, including cucumber, potatoes, and sugar beet. Surface soil samples were collected and analyzed to determine the total concentration of specific elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn), before the pollution index was calculated for each element. Soils used for the cultivation of the three types of crop were not contaminated with As, Cr, Cu, Pb, or Zn. However, the pollution indices for Cd were 1.1, 4.4, and 3.8 in cucumber, potato, and sugar beet fields, respectively, which indicated moderate, high, and high levels of contamination, respectively. Soils from potato and sugar beet fields were heavily contaminated with Cd, which may have resulted from long-term overuse of phosphate fertilizers.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Mesenchymal stem cells as the game-changing tools in the treatment of various organs disorders: Mirage or reality?

Recently a growing attention in scientific community has been gathered on potential application of mesenchymal stem cells (MSCs) in various fields of medicine. Owing to the fact that they can be easily isolated from different sources, and simply proliferated in large quantities while keeping their original biological characteristics, they can be successfully used as cell-based therapeutics. Engineering MSCs and other type of stem cells to be carriers of therapeutic agents is a new tactic in the targeted gene and cell therapy of cancers and degenerative diseases. Various useful properties of MSCs including tropism toward tumor/injury site(s), weakly immunogenic, production of anti-inflammatory molecules, and safety against normal tissues have made them prone for regenerative medicine, targeted therapy and treating injured tissues, and immunological abnormalities. In this review, we introduce latest advances, methods, and applications of MSCs in gene therapy of various malignant organ disorders. Additionally, we will cover the problems and challenges which researchers have faced with when trying to translate their basic experimental findings in MSCs research to clinically applicable therapeutics.     

Comparative Evaluation of α-Amylase Refolding Through Two Different Artificial Chaperone Systems

Two different artificial chaperone systems were evaluated in this work using either detergents or CDs as the stripping agents. Upon dilution of urea-denatured α-amylase to a non-denaturing urea concentration in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to remove the detergent molecules. Our results by fluorescence, UV, turbidity measurement, circular dichroism, surface tension and activity assay indicated that the extent of refolding assistance was different due to different inter- and intra- molecular interactions in the two different systems. However, the high activity recovery in the presence of detergents, as the stripping agent, suggests that they can constitute suitable replacement for the more expensive and common stripping agent of cyclodextrins.     

Enhancement of antioxidant enzymes activity and expression of CAT and PAL genes in hazel (Corylus avellana L.) cells in response to low-intensity ultrasound

Induction of antioxidant systems of hazel cells by low-energy ultrasound, the potential role of hydrogen peroxide (H₂O₂) as a signaling molecule in regulation of activity of stress-related enzymes, and expression of catalase (CAT) and phenylalanine ammonialyase (PAL) genes were investigated. Suspension-cultured Corylus avellana L. cells were agitated by an ultrasonic device at 29 kHz with the power of 4 mW/cm², for 8–40 min. The activities of CAT, superoxide dismutase (SOD), and ascorbate peroxidase (APX) of treated cells increased by 4, 1.7 and 7 times of the control ones, respectively. Induction of increase in the expression of CAT gene started 24 h after the treatment with ultrasound. Significant increase also was observed in the expression of PAL gene, 6 h after exposure to ultrasound, which resulted in turn to increase of total contents of soluble phenolics, 24 h of the treatment. Exposure to ultrasound up to 20 min had no adverse effects on cell viability although it slightly increased the accumulation of H₂O₂. However, it is likely that this level of increased H₂O₂ was not deteriorative for hazel cells, but rather triggered antioxidant system and provided hazel cells a sustainable growth after ultrasound treatment.     

Studies on the Refolding Process of Recombinant Horseradish Peroxidase

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl₂ in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.