Encapsulating therapeutic cells in RGD-modified agarose microcapsules.

Current cell-based strategies for repairing damaged tissue often show limited efficacy due to low cell retention at the site of injury. Encapsulation of cells within hydrogel microcapsules demonstrably increases cell retention but benefits can be limited due to premature cell escape from the hydrogel microcapsules and subsequent clearance from the targeted tissue. We propose a method of encapsulating cells in agarose microcapsules that have been modified to increase cell retention by providing cell attachment domains within the agarose hydrogel allowing cells to adhere to the microcapsules. We covalently modified agarose with the addition of the cell adhesion peptide, RGD (arginine, glycine, aspartic acid). We then used a microfluidic platform to encapsulate single cells within 50 µm agarose microcapsules. We tracked encapsulated cells for cell viability, egress from microcapsules and attachment to microcapsules at 2 h, 24 h, and 48 h after encapsulation. Many encapsulated cells eventually egress their microcapsule. Those that were encapsulated using RGD-modified agarose adhered to the outer surface of the microcapsule following egress. NIH 3T3 cells showed nearly 45% of egressed cells attached to the outside of RGD modified agarose microcapsules, while minimal cellular adhesion was observed when using unmodified agarose. Similarly, human umbilical vein endothelial cells had up to 33% of egressed cells attached and explant-derived cardiac cells showed up to 20% attachment with the presence of RGD binding domains within the agarose microcapsules.

Cdx2 regulates immune cell infiltration in the intestine.

The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.

Bile Acids Increase Doxorubicin Sensitivity in ABCC1-expressing Tumour Cells.

Tumour cells possess or acquire various mechanisms to circumvent the cytotoxic effects of chemotherapy drugs. One such mechanism involves the overexpression of ABC transporters that facilitate the extrusion of a variety of structurally distinct chemotherapy drugs from the cytoplasm into the extracellular space. While specific ABC transporter inhibitors have been developed, many affect other ABC transporters, particularly at elevated concentrations. It is also unclear whether they show clear efficacy for combatting drug resistance in cancer patients with minimal host toxicity. In this study, we demonstrate the ability of two bile acids [ß-cholanic acid (urso-cholanic acid) and deoxycholic acid] to specifically inhibit ABCC1-mediated drug transport, augmenting doxorubicin accumulation in breast and lung tumour cells selected for doxorubicin resistance through overexpression of the ABCC1 (but not ABCB1) drug transporter. The bile acids could also restore uptake and sensitivity to doxorubicin in human endothelial kidney cells genetically engineered to overexpress the ABCC1 drug transporter. These observations suggest a previously unreported role for bile acids as ABCC1 inhibitors or regulators. Given its additional properties of minimal clinical toxicity in humans and its ability to inhibit aldo-keto reductases involved in anthracycline resistance and anthracycline-induced cardiotoxicity, ß-cholanic acid merits further in vivo and clinical investigation.

Alterations in estrogen signalling pathways upon acquisition of anthracycline resistance in breast tumor cells.

Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression would be expected to reduce the growth rate of the cells, a hypothesis which was confirmed in subsequent cell proliferation experiments. AKR1C3 or AKR1B10 overexpression alone had no effect on doxorubicin sensitivity in MCF-7CC cells, while siRNA-mediated knockdown of AKR1C3 and/or AKR1B10 expression had no significant effect on sensitivity to doxorubicin in MCF-7DOX2 or MCF-7EPI cells. This suggested that enhanced or reduced AKR expression/activity is insufficient to confer anthracycline resistance or sensitivity to breast tumor cells, respectively. Rather, it would appear that AKR overexpression acts in concert with other proteins to confer anthracycline resistance, including reduced E2-dependent expression of both an important apoptosis inhibitor (Bcl-2) and a key protein associated with activation of cell cycle-dependent kinases (cyclin D1).

Role of Drug Metabolism in the Cytotoxicity and Clinical Efficacy of Anthracyclines.

Many clinical studies involving anti-tumor agents neglect to consider how these agents are metabolized within the host and whether the creation of specific metabolites alters drug therapeutic properties or toxic side effects. However, this is not the case for the anthracycline class of chemotherapy drugs. This review describes the various enzymes involved in the one electron (semi-quinone) or two electron (hydroxylation) reduction of anthracyclines, or in their reductive deglycosidation into deoxyaglycones. The effects of these reductions on drug antitumor efficacy and toxic side effects are also discussed. Current evidence suggests that the one electron reduction of anthracyclines augments both their tumor toxicity and their toxicity towards the host, in particular their cardiotoxicity. In contrast, the two electron reduction (hydroxylation) of anthracyclines strongly reduces their ability to kill tumor cells, while augmenting cardiotoxicity through their accumulation within cardiomyocytes and their direct effects on excitation/contraction coupling within the myocytes. The reductive deglycosidation of anthracyclines appears to inactivate the drug and only occurs under rare, anaerobic conditions. This knowledge has resulted in the identification of important new approaches to improve the therapeutic index of anthracyclines, in particular by inhibiting their cardiotoxicity. The true utility of these approaches in the management of cancer patients undergoing anthracycline-based chemotherapy remains unclear, although one such agent (the iron chelator dexrazoxane) has recently been approved for clinical use.

Functional significance of skeletal muscle adiponectin production, changes in animal models of obesity and diabetes, and regulation by rosiglitazone treatment.

Endocrine effects of adipose-derived adiponectin on skeletal muscle have been shown to account, at least in part, for the anti-diabetic effects of this adipokine. Recently, the concept of myokines has gained credence, and the potential for skeletal muscle to produce adiponectin has been suggested. Here we demonstrated an increased level of adiponectin mRNA and protein expression as well as protein secretion in response to rosiglitazone treatment in L6 muscle cells. This correlated with the ability of rosiglitazone to enhance insulin sensitivity for stimulation of protein kinase B (Akt) phosphorylation and glucose transport; rosiglitazone also corrected high-glucose-induced insulin resistance in L6 cells. Overexpression of adiponectin confirmed the functional significance of local production of adiponectin in muscle cells via elevated glucose uptake and increased insulin sensitivity. In obese diabetic db/db mice, there was a change in the adiponectin expression profile in soleus and extensor digitorum longus (EDL) muscle with less high molecular weight (HMW) and more medium (MMW)/low (LMW) molecular weight species detected. Induction of obesity and insulin resistance in rats by feeding a high-fat high-sucrose diet also led to decreased muscle HMW adiponectin content that could be corrected by rosiglitazone treatment. In summary, we show the ability of skeletal muscle cells to produce adiponectin, which can mediate autocrine metabolic effects, thus establishing adiponectin as a bona fide myokine. We also demonstrate that skeletal muscle adiponectin production is altered in animal models of obesity and diabetes and that these changes can be corrected by rosiglitazone.