Potential i-Nos/Arg-1 Switch with NLRP3 and Parasitic Load Down Regulation in Experimental Schistosoma mansoni Infection via Chloroquine Repurposing.

In previous studies, the inhibitory effect of chloroquine on NLRP3 inflammasome and heme production was documented. This may be employed as a double-bladed sword in schistosomiasis (anti-inflammatory and parasiticidal). In this study, chloroquine's impact on schistosomiasis mansoni was investigated. The parasitic load (worm/egg counts and reproductive capacity index [RCI]), i-Nos/Arg-1 expression, splenomegaly, hepatic insult and NLRP3-immunohistochemical expression were assessed in infected mice after receiving early and late repeated doses of chloroquine alone or dually with praziquantel. By early treatment, the least RCI was reported in dually treated mice (41.48 ± 28.58) with a significant reduction in worm/egg counts (3.50 ± 1.29/2550 ± 479.58), compared with either drug alone. A marked reduction in the splenic index was achieved by prolonged chloroquine administration (alone: 43.15 ± 5.67, dually: 36.03 ± 5.27), with significantly less fibrosis (15 ± 3.37, 14.25 ± 2.22) than after praziquantel alone (20.5 ± 2.65). Regarding inflammation, despite the praziquantel-induced significant decrease in NLRP3 expression, the inhibitory effect was marked after dual and chloroquine administration (liver: 3.13 ± 1.21/3.45 ± 1.23, spleen: 5.7 ± 1.6/4.63 ± 2.41). i-Nos RNA peaked with early/late chloroquine administration (liver: 68.53 ± 1.8/57.78 ± 7.14, spleen: 63.22 ± 2.06/62.5 ± 3.05). High i-Nos echoed with a parasiticidal and hepatoprotective effect and may indicate macrophage-1 polarisation. On the flip side, the chloroquine-induced low Arg-1 seemed to abate immune tolerance and probably macrophage-2 polarisation. Collectively, chloroquine synergised the praziquantel-schistosomicidal effect and minimised tissue inflammation, splenomegaly and hepatic fibrosis.

Limited Proteolysis of Cyclooxygenase-2 Enhances Cell Proliferation.

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (ΔNT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the ΔNT- COX-2 protein also interacts in the nucleus with ß-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis.

Substrate-inactivated cyclooxygenase-2 is disposed of by exosomes through the ER-Golgi pathway.

Catalysis of arachidonic acid (AA) by cyclooxygenase-2 (COX-2) gives rise to a single product that serves as a precursor for all prostaglandins, which are central mediators of inflammation. Rapid up-regulation of COX-2 expression in response to pro-inflammatory stimuli is a well-characterized means of generating the large pool of prostaglandins necessary for inflammation. However, an efficient inflammatory process must also terminate rapidly and thus requires cessation of COX-2 enzymatic activity and removal of excess protein from the cell. Previous studies showed that COX-2 that has not been exposed to AA ('naive') degrades in the cellular proteasome. However, continuous exposure to AA induces suicide inactivation of COX-2 and its elimination no longer occurs in neither the proteasomal nor lysosomal machineries. In the present study, we show that either overexpressed or endogenously induced COX-2 is secreted via exosomes through the endoplasmic reticulum-Golgi pathway. We further find that excretion of COX-2 is significantly enhanced by prolonged exposure to AA. Genetic or chemical inhibition of COX-2 enzymatic activity has no effect on its secretion in the absence of substrate, but prevents the additional activity-dependent secretion. Finally, transfer of COX-2 to target cells only occurs in the absence of AA stimulation. Together, these results suggest that exosomal secretion of AA-activated COX-2 constitutes a means to remove damaged inactive COX-2 from the cell.

MBTPS1 regulates proliferation of colorectal cancer primarily through its action on sterol regulatory element-binding proteins.

Among the main metabolic pathways implicated in cancer cell proliferation are those of cholesterol and fatty acid synthesis, both of which are tightly regulated by sterol regulatory element-binding proteins (SREBPs). SREBPs are activated through specific cleavage by membrane-bound transcription factor protease 1 (MBTPS1), a serine protease that cleaves additional substrates (ATF6, BDNF, CREBs and somatostatin), some of which are also implicated in cell proliferation. The goal of this study was to determine whether MBTPS1 may serve as a master regulator in proliferation of colorectal cancer (CRC). Tumors from CRC patients showed variable levels of MBTPS1 mRNA, which were in positive correlation with the levels of SREBPs and ATF6, and in reverse correlation with BDNF levels. Chemical inhibition of MBTPS1 activity in two CRC-derived cell lines resulted in a marked decrease in the levels of SREBPs, but not of its other substrates and a marked decrease in cell proliferation, which suggested that MBTPS1 activity is critical for proliferation of these cells. In accordance, CRISPR/Cas9 targeted knockout (KO) of the MBTPS1 gene resulted in the survival of only a single clone that presented a phenotype of severely attenuated proliferation and marked downregulation of several energy metabolism pathways. We further showed that survival of the MBTPS1 KO clone was dependent upon significant upregulation of the type-1 interferon pathway, the inhibition of which halted proliferation entirely. Finally, rescue of the MBTPS1 KO cells, resulted in partial restoration of MBTPS1 levels, which was in accordance with partial recovery in proliferation and in SREBP levels. These finding suggest that MBTPS1 plays a critical role in regulating colon cancer proliferation primarily through SREBP-associated lipid metabolism, and as such may serve as a possible therapeutic target in CRC.

Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit.

Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression.

Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/- mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.