Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

Design of Polyamine-Grafted Starches for Nucleotide Analogue Delivery: In Vitro Evaluation of the Anticancer Activity

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Involvement of Mitochondrial Activity and OXPHOS in ATP Synthesis During the Motility Phase of Spermatozoa in the Pacific Oyster, Crassostrea gigas

In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied 1) changes in spermatozoal mitochondrial activity, that is, mitochondrial membrane potential (MMP), and intracellular ATP content in relation to motion parameters and 2) the involvement of OXPHOS for spermatozoal movement using carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoal ATP content decreased steadily during the first 9 h postactivation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that the ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or from potential phosphagens during the first 2 h of movement as deduced by the decoupling by CCCP of OXPHOS. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyze intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis , in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii (SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Leishmanicidal and cytotoxic activity of extracts and saponins from Ilex laurina (Aquifoliaceae)

Purpose: To evaluate the leishmanicidal and cytotoxic activity of alcohol and non-alcohol extracts and saponins from Ilex laurina . Methods: Extracts were obtained by percolation with solvents of different polarities: hexane, dichloromethane, ethyl acetate and ethanol. The ethyl acetate extract was subjected to silica gel column chromatography eluting with a step gradient of dichloromethane-methanol. All products were evaluated in vitro for leishmanicidal activity against amastigotes of leishmania panamensis and cytotoxicity on U- 937 cells. Results: Two saponins were isolated from the ethyl acetate extract. The ethyl acetate extract showed high leishmanicidal activity against intracellular amastigotes of L. panamensis (EC50, 7.5 ± 1.5 μg/mL) and low activity against axenic amastigotes (EC50, 52.8 ±1.6 μg/mL); this extract showed also high cytotoxicity (LC50, 57.7 ± 12.1 μg/mL). Saponin 2 exhibited high activity against intracellular amastigotes (EC50, 5.9 ± 0.5 μg/mL) but also showed high cytotoxicity on U-937 cells (EC50, 25.7 ± 6.1 μg/mL). This compound showed similar leishmanicidal activity and cytotoxicity to meglumine antimoniate and amphotericin B, respectively, drugs currently used for the treatment of leishmaniasis. Conclusions: Based on these results, Ilex laurina is a potential source of compounds that can lead to the development of new therapeutic alternatives against leishmaniasis.

Licochalcone A exerts antitumor activity in bladder cancer cell lines and mice models

Purpose: To investigate the effect of licochalcone A (LA) on the inhibition of cell proliferation and ERK1/2 phosphorylation in bladder carcinoma cell lines. Methods: Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Dye-binding method was used to examine the concentration of proteins. Lymphocytes were extracted from mice and after surface staining were subjected to BD fixation and permeabilization for intracellular staining. Flow cytometry was used to measure cellular fluorescence. Results: MTT results revealed a significant decrease in the proliferation of UM-UC-3, J82 and HT-1197 cell lines on treatment with LA. LA also induced reduction in phosphorylation of ERK1/2 in all three carcinoma cell lines. In the mouse model, licochalcone A treatment via intraperitoneal (ip) administration induced a significant decrease in the level of regulatory T cells (Tregs). Comparison of the mouse interferon-α (IFN-α)-treated and LA-treated groups revealed that LA treatment caused enhancement of cytotoxic T lymphocyte (CTL) activity similar to that of IFN-α. Administration of UM-UC-3 cells in C3H/HeN mice resulted in marked reduction in the counts for splenocytes and CD4+ CD25+ Foxp3+ T (regulatory T cells) cell proportion in LA-treated mice compared to untreated control group. Conclusion: Licochalcone A may be of therapeutic importance for the prevention of bladder carcinoma. However, studies are required to ascertain the compound’s usefulness in this regard.

Intracellular signaling and trafficking in cancer: Role of Rab5-GTPases in migration and invasion of breast cancer cells

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small signaling molecules and critical in the regulation of many cellular processes including cell migration, growth via the endocytic pathway. This research involves the role of Rab GTPases, specifically Rab5 and its guanine exchange factors (GEFs), in the promotion of cancer cell migration and invasion. Two important questions abound: Are IGFR stimulation and downstream effect involved the endocytic pathway in carcinogenesis? What role does Rab5 play in cell migration and invasion of cancer cells? The hypothesis is that growth factor signaling is dependent on Rab5 activity in mediating the aggressiveness of cancer cells. The goal is to demonstrate that IGF-1 signaling is dependent on Rab5 function in breast cancer progression. Here, the results thus far, have shown that while activation of Rab5 may mediate increased cell proliferation, migration and invasion in breast cancer cells, the Rab5 GEF, RIN1 interacts with the IGFR thereby facilitating migration and invasion activities in breast cells. Furthermore, endocytosis of the IGFR in breast cancer cells seems to be caveolin dependent as the data has shown. This taken together, the data shows that IGF-1 signaling in breast cancer cells relies on IGF-1R phosphorylation, caveolae internalization and sequestration to the early endosome RIN1 function and Rab5 activation.^