A multikinase inhibitor of the Raf ⁄ mitogen-activated protein kinase kinase (MEK) ⁄ extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK ⁄ ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1 ⁄ 2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1 ⁄ 2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK ⁄ ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK ⁄ ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.Abbreviations Akt, protein kinase B; DAPI, 4¢,6-diamidino-

BackgroundMyxozoa is a speciose group of endoparasitic cnidarians that can cause severe ecological and economic effects. Although highly reduced compared to free-living cnidarians, myxozoans have retained the phylum-defining stinging organelles, known as cnidae or polar capsules, which are essential to initiating host infection. To explore the adaptations of myxozoan polar capsules, we compared the structure, firing process and content release mechanism of polar tubules in myxospores of three Myxobolus species including M. cerebralis, the causative agent of whirling disease.ResultsWe found novel functions and morphologies in myxozoan polar tubules. High-speed video analysis of the firing process of capsules from the three Myxobolus species showed that all polar tubules rapidly extended and then contracted, an elasticity phenomenon that is unknown in free-living cnidarians. Interestingly, the duration of the tubule release differed among the three species by more than two orders of magnitude, ranging from 0.35 to 10 s. By dye-labeling the polar capsules prior to firing, we discovered that two of the species could release their entire capsule content, a delivery process not previously known from myxozoans. Having the role of content delivery and not simply anchoring suggests that cytotoxic or proteolytic compounds may be present in the capsule. Moreover, while free-living cnidarians inject most of the toxic content through the distal tip of the tubule, our video and ultrastructure analyses of the myxozoan tubules revealed patterns of double spirals of nodules and pores along parts of the tubules, and showed that the distal tip of the tubules was sealed. This helical pattern and distribution of openings may minimize the tubule mechanical weakness and improve resistance to the stress impose by firing. The finding that myxozoan tubule characteristics are very different from those of free-living cnidarians is suggestive of their adaptation to parasitic life.ConclusionsThese findings show that myxozoan polar tubules have more functions than previously assumed, and provide insight into their evolution from free-living ancestors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1819-4) contains supplementary material, which is available to authorized users.

Toll-like receptor 4 (TLR-4) is crucial in maintaining intestinal epithelial homeostasis, participates in a vigorous signaling process and heightens inflammatory cytokine output. The objective of this study was to determine the effects of glutamine (GLN) on TLR-4 signaling in intestinal mucosa during methotrexate (MTX)-induced mucositis in a rat. Male Sprague–Dawley rats were randomly assigned to one of four experimental groups of 8 rats each: 1) control rats; 2) CONTR-GLN animals were treated with oral glutamine given in drinking water (2%) 48 hours before and 72 hours following vehicle injection; 3) MTX-rats were treated with a single IP injection of MTX (20 mg/kg); and 4) MTX-GLN rats were pre-treated with oral glutamine similar to group B, 48 hours before and 72 hours after MTX injection. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. The expression of TLR-4, MyD88 and TRAF6 in the intestinal mucosa was determined using real time PCR, Western blot and immunohistochemistry. MTX-GLN rats demonstrated a greater jejunal and ileal mucosal weight and mucosal DNA, greater villus height in ileum and crypt depth and index of proliferation in jejunum and ileum, compared to MTX animals. The expression of TLR-4 and MyD88 mRNA and protein in the mucosa was significantly lower in MTX rats versus controls animals. The administration of GLN increased significantly the expression of TLR-4 and MyD88 (vs the MTX group). In conclusion, treatment with glutamine was associated with up-regulation of TLR-4 and MyD88 expression and a concomitant decrease in intestinal mucosal injury caused by MTX-induced mucositis in a rat.

BackgroundArginine (ARG) and nitric oxide maintain the mucosal integrity of the intestine in various intestinal disorders. In the present study, we evaluated the effects of oral ARG supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat.MethodsMale rats were divided into four experimental groups: Control rats, CONTR-ARG rats, were treated with oral ARG given in drinking water 72 hours before and 72 hours following vehicle injection, MTX rats were treated with a single dose of methotrexate, and MTX-ARG rats were treated with oral ARG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. RT-PCR was used to determine bax and bcl-2 mRNA expression.ResultsMTX-ARG rats demonstrated greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-ARG rats (vs MTX) was accompanied by decreased bax mRNA and protein expression and increased bcl-2 protein levels.ConclusionsTreatment with oral ARG prevents mucosal injury and improves intestinal recovery following MTX- injury in the rat.

Treatment with ozone prevents intestinal mucosal damage, stimulates cell proliferation and inhibits programmed cell death following intestinal IR in a rat.