Opioid Use in Murine Model Results in Severe Gastric Pathology that May Be Attenuated by Proton Pump Inhibition.

Opioids are the gold standard for chronic and acute pain management; however, their consequence on gastric function is relatively understudied. Opioid users have a higher incidence of gastric dysfunction, worse quality of life, increased hospitalizations, and increased use of antiemetic and pain modulator medications. The current study shows that morphine treatment in the murine model results in greater disruption of gastric epithelial cell morphology, increased gastric cell apoptosis, elevated inflammatory cytokines, and matrix metallopeptidase-9 secretion. Morphine treatment also increases gastric acid secretion and causes delays in gastric emptying. Moreover, morphine treatment causes an increase in systemic IL-6 level, which plays an important role in morphine-induced delayed gastric emptying and gastric damage. IL-6 knockout mice show a significant level of reduction in morphine-induced gastric delaying, acid retention, and gastric damage. Thus, morphine-mediated gastric damage is a consequence of the accumulation of acid in the stomach due to increased gastric acid secretion and delayed gastric emptying. Treatment with a proton pump inhibitor resulted in a significant reduction in morphine-induced gastric inflammation, gastric delaying, and improved morphine tolerance. Hence, these studies attribute morphine-mediated induction in gastric acidity and inflammatory cytokines as drivers for morphine-associated gastric pathology and show the therapeutic use of proton pump inhibitors as an inexpensive approach for clinical management of morphine-associated pathophysiology and analgesic tolerance.

Morphine tolerance is attenuated in germfree mice and reversed by probiotics, implicating the role of gut microbiome.

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. We show that morphine analgesic tolerance was significantly attenuated in germfree (GF) and in pan-antibiotic-treated mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacterial communities, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotic therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine's efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of proinflammatory cytokines, which drives morphine tolerance.

Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response.

We studied the effects of gut microbiome depletion by oral antibiotics on tumor growth in subcutaneous and liver metastases models of pancreatic cancer, colon cancer, and melanoma. Gut microbiome depletion significantly reduced tumor burden in all the models tested. However, depletion of gut microbiome did not reduce tumor growth in Rag1-knockout mice, which lack mature T and B cells. Flow cytometry analyses demonstrated that gut microbiome depletion led to significant increase in interferon gamma-producing T cells with corresponding decrease in interleukin 17A and interleukin 10-producing T cells. Our results suggest that gut microbiome modulation could emerge as a novel immunotherapeutic strategy.

NFκB in Pancreatic Stellate Cells Reduces Infiltration of Tumors by Cytotoxic T Cells and Killing of Cancer Cells, via Up-regulation of CXCL12.

BACKGROUND & AIMS: Immunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response. METHODS: Pancreata of C57BL/6 or Rag1-/- mice were given pancreatic injections of a combination of KrasG12D/+; Trp53 R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50-/- mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50-/- fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50-/- PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay. RESULTS: C57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50-/- PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50-/- PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50-/- PSCs, developed the same-size tumors when CD8+ T cells were depleted from C57BL/6 mice or in Rag1-/- mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50-/- PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture. CONCLUSIONS: In studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.

Regulation of angiogenesis by microRNAs in cardiovascular diseases.

Non-coding RNAs are functional RNA molecules comprising the majority of human transcriptome. Only about 1.5% of the human genome is transcribed into messenger RNAs (mRNA) that are translated into proteins. Among the non-coding RNAs, miRNAs are extensively studied and miR targets in endothelial cells, perivascular cells, and angiogenic signaling are relatively well defined. MicroRNAs not only regulate transcripts in situ but also function as paracrine mediators in affecting angiogenesis at distant sites. Exosomal miRs are implicated in modulating endothelial cell function and angiogenesis. Thus miRs have been shown to affect tissue microenvironment in a multitude of ways. A comprehensive analysis of the role of miRs in modulation of angiogenesis and their impact on cardiovascular diseases is presented in this review.

Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis.

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3'-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

Inhibition of epithelial ovarian cancer by Minnelide, a water-soluble pro-drug.

OBJECTIVE: Minnelide is a water-soluble pro-drug of triptolide, a natural product. The goal of this study was to evaluate the effectiveness of Minnelide on ovarian cancer growth in vitro and in vivo. METHODS: The effect of Minnelide on ovarian cancer cell proliferation was determined by real time electrical impedance measurements. Multiple mouse models with C200 and A2780 epithelial ovarian cancer cell lines were used to assess the efficacy of Minnelide in inhibiting ovarian cancer growth. RESULTS: Minnelide decreased cell viability of both platinum sensitive and resistant epithelial ovarian cancer cells in vitro. Minnelide with carboplatin showed additive effects in vitro. Minnelide monotherapy increased the survival of mice bearing established ovarian tumors. Minnelide, in combination with carboplatin and paclitaxel, improved overall survival of mice. CONCLUSIONS: Minnelide is a promising pro-drug for the treatment of ovarian cancer, especially when combined with standard chemotherapy.

Altered gut microbiome drives heightened pain sensitivity in a murine model of metastatic triple-negative breast cancer.

The microbiota residing in the gut environment is essential for host homeostasis. Increasing evidence suggests that microbial perturbation (dysbiosis) regulates cancer initiation and progression at local and distant sites. Here, we have identified microbial dysbiosis with the depletion of commensal bacteria as a host-intrinsic factor associated with metastatic dissemination to the bone. Using a mouse model of triple-negative mammary cancer, we demonstrate that a pre-established disruption of microbial homeostasis using an antibiotic cocktail increases tumor growth, enhanced circulating tumor cells, and subsequent dissemination to the bone. We found that the presence of pathogenic bacteria and loss of commensal bacteria in an antibiotic-induced gut environment is associated with sustained inflammation. Increased secretion of G-CSF and MMP-9 in intestinal tissues, followed by increased neutrophil infiltration and severe systemic inflammation in tumor-bearing mice, indicates the direct consequence of a dysbiotic microbiome. Increased neutrophil infiltration to the bone metastatic niche facilitates extravasation and transendothelial migration of tumor cells. It provides a novel, pre-established, and favorable environment to form an immunosuppressive pre-metastatic niche. The presence of tumor cells in immunosuppressive metastatic tumor niche disrupts the balance between osteoblasts and osteoclasts, promotes osteoclast differentiation, and remodels the bone structure. Excessive bone resorption by osteoclasts causes bone degradation and ultimately causes extreme pain in a bone metastatic mouse model. In clinical settings, bone metastasis is associated with intractable severe pain that severely compromises the quality of life in these patients.

Antibody-Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling.

Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed αEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA sequencing (RNA-seq) of MDA-MB-231-4175 TNBC cells grown in a monolayer (two-dimensional) was compared with cells plated on Matrigel undergoing VM [three-dimensional (3D)]. We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (EGFR-E-P125A). Gene set enrichment analysis demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment.Correlative analysis of the phosphoproteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase Y397 and STAT3 Y705 sites downstream of α5ß1 integrin. Suppression of phosphorylation events downstream of EGFR and α5ß1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5ß1 integrin cross-talk. In vivo, αEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and α5ß1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors. SIGNIFICANCE: αEGFR-E-P125A reduces VM, angiogenesis, tumor growth, and metastasis by inhibiting EGFR and α5ß1 integrin signaling, and is a promising therapeutic agent for TNBC treatment, used alone or in combination with chemotherapy.

Morphine use induces gastric microbial dysbiosis driving gastric inflammation through TLR2 signalling which is attenuated by proton pump inhibition.

BACKGROUND AND PURPOSE: Opioids are the standard drug for pain management; however, their effects on gastric dysfunction are relatively understudied. Opioid users have a higher incidence of gastric pathology leading to increased hospitalization. Herein, we investigated the consequences of morphine use on gastric pathology and the underlying mechanisms. We further investigated the therapeutic benefit of proton pump inhibition to overcome morphine-mediated gastric inflammation. EXPERIMENTAL APPROACH: Mice were implanted with 25 mg slow-release morphine and placebo pellets. Gastric microbiome analyses were performed. Gastric damage was assayed. Gastric pH was measured. Germ-free and TLR2KO mice were used to investigate the mechanisms. Gastroprotective studies were performed with the proton pump inhibitor (PPI) omeprazole. KEY RESULTS: Chronic morphine treatment alters gastric microbial composition and induces preferential expansion of pathogenic bacterial communities such as Streptococcus and Pseudomonas. Morphine causes disruption of the gastric mucosal layer, increases apoptosis, and elevates inflammatory cytokines. Moreover, morphine-mediated gastric pathology was significantly attenuated in germ-free mice, and reconstitution of morphine gastric microbiome in germ-free mice resulted gastric inflammation. In addition, morphine-mediated gastric inflammation was attenuated in TLR2KO mice. Morphine causes a decrease in gastric pH, which contributes to gastric dysbiosis leads to gastric inflammation. Omeprazole treatment inhibits gastric acidity, rescuing morphine-induced gastric dysbiosis and preventing inflammation. CONCLUSION AND IMPLICATIONS: This study attributes morphine-induced gastric acidity as a driver of gastric dysbiosis and pathology and proposes the therapeutic use of PPI as an inexpensive approach for the clinical management of morphine-associated pathophysiology.

Multi-omics analysis revealing the interplay between gut microbiome and the host following opioid use.

Opioid crisis is an ongoing epidemic since the past several decades in the United States. Opioid use-associated microbial dysbiosis is emerging as a key regulator of intestinal homeostasis and behavioral responses to opioid. However, the mechanistic insight into the role of microbial community in modulating host response is unavailable. To uncover the role of opioid-induced dysbiosis in disrupting intestinal homeostasis we utilized whole genome sequencing, untargeted metabolomics, and mRNA sequencing to identify changes in microbiome, metabolome, and host transcriptome respectively. Morphine treatment resulted in significant expansion of Parasuterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and depletion of Lactobacillus johnsonii. These changes correlated with alterations in lipid metabolites and flavonoids. Significant alteration in microbial metabolism (metabolism of lipids, amino acids, vitamins and cofactors) and increased expression of virulence factors and biosynthesis of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) were observed in microbiome of morphine-treated animals. In concurrence with changes in microbiome and metabolome extensive changes in innate and adaptive immune response, lipid metabolism, and gut barrier dysfunction were observed in the host transcriptome. Microbiome depleted mice displayed lower levels of inflammation, immune response and tissue destruction compared to mice harboring a dysbiotic microbiome in response to morphine treatment, thus establishing dysbiotic microbiome as mediator of morphine gut pathophysiology. Integrative analysis of multi-omics data highlighted the associations between Parasutterella excrementihominis, Burkholderiales bacterium 1_1_47, Enterococcus faecalis, Enterorhabdus caecimuris and altered levels of riboflavin, flavonoids, and lipid metabolites including phosphocholines, carnitines, bile acids, and ethanolamines with host gene expression changes involved in inflammation and barrier integrity of intestine. Omic analysis also highlighted the role of probiotic bacteria Lactobacillus johnsonii, metabolites flavonoids and riboflavin that were depleted with morphine as important factors for intestinal homeostasis. This study presents for the first time ever an interactive view of morphine-induced changes in microbial metabolism, strain level gut microbiome analysis and comprehensive view of changes in gut transcriptome. We also identified areas of potential therapeutic interventions to limit microbial dysbiosis and present a unique resource to the opioid research community.

Opioid-induced microbial dysbiosis disrupts irinotecan (CPT-11) metabolism and increases gastrointestinal toxicity in a murine model.

BACKGROUND AND PURPOSE: Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhoea. The chemotherapeutic irinotecan (CPT-11) causes severe gastrointestinal (GI) toxicity due to deconjugation of inactive metabolite SN-38 glucuronide (SN-38G) by bacterial ß-glucuronidases to the active 7-ethyl-10-hydroxycamptothecin (SN-38). Opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11 anti-tumour efficacy and GI toxicity are exacerbated by opioid co-administration. EXPERIMENTAL APPROACH: Eight-week-old C57BL/6 male mice were co-administration with CPT-11 ± opioid. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and quantitative real-time polymerase chain reaction were used to determine the severity of intestinal tissue damage. Human liver microsome In vitro assay was performed to confirm the effects of opioids on CPT-11 metabolism. KEY RESULTS: Gut microbiome analysis showed that morphine treatment induced enrichment of ß-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11 anti-tumour efficacy, this seemed to be microbiome independent. CONCLUSION AND IMPLICATIONS: Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.

Increased expression of collagen prolyl hydroxylases in ovarian cancer is associated with cancer growth and metastasis.

Tumor hypoxia induces collagen deposition and extensive extracellular matrix remodeling, significantly enhancing the processes of invasion and metastasis. Collagen prolyl-4-hydroxylases (P4HA) play a critical role in collagen post-translational modification. The primary objective of this study is to comprehensively assess the role of P4HA in promoting ovarian cancer growth and facilitating metastasis. Human epithelial ovarian cancer cells were transfected with shRNAs to target P4HA1 and P4HA2. The impact of P4HA knockdown on crucial factors such as collagen I deposition, cell proliferation, and migration were examined in vitro. Additionally, in vivo studies involved the injection of both control and P4HA knockdown cells into athymic mice, enabling the assessment of tumor growth and peritoneal metastasis. The relevance of prolyl hydroxylases to clinical outcomes was then determined by analyzing clinical databases. Quantitative RT-PCR showed upregulation of P4HA1 and P4HA2 mRNA in A2780 cells when exposed to hypoxia. ShRNA-mediated downregulation of P4HA1 and P4HA2 significantly reduced the deposition of collagen I. Knockdown of P4HA expression reduced cell proliferation in vitro and peritoneal seeding in vivo. A2780 cells stably transfected with shP4HA1 and shP4HA2 inhibited tumor growth and metastases in athymic mice. Furthermore, our review of the TCGA dataset revealed that increased P4HA1 and P4HA2 mRNA levels are associated with decreased overall survival in patients with ovarian cancer. The increased expression of collagen P4HA has been linked to ovarian cancer growth and metastasis. This evidence highlights their potential as prognostic biomarkers and promising therapeutic targets.

Opioid-Use, COVID-19 Infection, and Their Neurological Implications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an imminent threat to human health and public safety. ACE2 and transmembrane serine protease 2 proteins on host cells provide the viral entry point to SARS-CoV-2. Although SARS-CoV-2 mainly infects the respiratory system, there have been reports of viral neurotropism and central nervous system injury as indicated by plasma biomarkers, including neurofilament light chain protein and glial fibrillary acidic protein. Even with a small proportion of infections leading to neurological manifestation, the overall number remains high. Common neurological manifestations of SARS-CoV-2 infection include anosmia, ageusia, encephalopathy, and stroke, which are not restricted to only the most severe infection cases. Opioids and opioid antagonists bind to the ACE2 receptor and thereby have been hypothesized to have therapeutic potential in treating COVID-19. However, in the case of other neurotropic viral infections such as human immunodeficiency virus (HIV), opioid use has been established to exacerbate HIV-mediated central nervous system pathogenesis. An analysis of electronic health record data from more than 73 million patients shows that people with Substance Use Disorders are at higher risk of contracting COVID-19 and suffer worse consequences then non-users. Our in-vivo and in-vitro unpublished studies show that morphine treatment causes increased expression of ACE2 in murine lung and brain tissue as early as 24 h post treatment. At the same time, we also observed morphine and lipopolysaccharides treatment lead to a synergistic increase in ACE2 expression in the microglial cell line, SIM-A9. This data suggests that opioid treatment may potentially increase neurotropism of SARS-CoV-2 infection. We have previously shown that opioids induce gut microbial dysbiosis. Similarly, gut microbiome alterations have been reported with SARS-CoV-2 infection and may play a role in predicting COVID-19 disease severity. However, there are no studies thus far linking opioid-mediated dysbiosis with the severity of neuron-specific COVID-19 infection.

Morphine mediated neutrophil infiltration in intestinal tissue play essential role in histological damage and microbial dysbiosis.

The gut microbial ecosystem exhibits a complex bidirectional communication with the host and is one of the key contributing factors in determining mucosal immune homeostasis or an inflammatory state. Opioid use has been established to induce gut microbial dysbiosis consistent with increased intestinal tissue inflammation. In this study, we investigated the role of infiltrated immune cells in morphine-induced intestinal tissue damage and gut microbial dysbiosis in mice. Results reveal a significant increase in chemokine expression in intestinal tissues followed by increased neutrophil infiltration post morphine treatment which is direct consequence of a dysbiotic microbiome since the effect is attenuated in antibiotics treated animals and in germ-free mice. Neutrophil neutralization using anti-Ly6G monoclonal antibody showed a significant decrease in tissue damage and an increase in tight junction protein organization. 16S rRNA sequencing on intestinal samples highlighted the role of infiltrated neutrophils in modulating microbial community structure by providing a growth benefit for pathogenic bacteria, such as Enterococcus, and simultaneously causing a significant depletion of commensal bacteria, such as Lactobacillus. Taken together, we provide the first direct evidence that neutrophil infiltration contributes to morphine-induced intestinal tissue damage and gut microbial dysbiosis. Our findings implicate that inhibition of neutrophil infiltration may provide therapeutic benefits against gastrointestinal dysfunctions associated with opioid use.

Morphine suppresses tumor angiogenesis through a HIF-1alpha/p38MAPK pathway.

Morphine, a highly potent analgesic agent, is frequently prescribed for moderate to severe cancer pain. In this study, morphine was administered at a clinically relevant analgesic dose to assess tumor cell-induced angiogenesis and subcutaneous tumor growth in nude mice using mouse Lewis lung carcinoma cells (LLCs). Implantation of mice with a continuous slow-release morphine pellet achieved morphine plasma levels within 250-400 ng/ml (measured using a radioimmunoassay, Coat-A-Count Serum Morphine) and was sufficient to significantly reduce tumor cell-induced angiogenesis and tumor growth when compared with placebo treatment. Morphometric analysis for blood vessel formation further confirmed that morphine significantly reduced blood vessel density (P < 0.003), vessel branching (P < 0.05), and vessel length (P < 0.002) when compared with placebo treatment. Morphine's effect was abolished in mice coadministered the classical opioid receptor antagonist, naltrexone, and in mu-opioid receptor knockout mice, supporting the involvement of the classical opioid receptors in vivo. Morphine's inhibitory effect is mediated through the suppression of the hypoxia-induced mitochondrial p38 mitogen-activated protein kinase (MAPK) pathway. Our results suggest that in vitro morphine treatment of LLCs inhibits the hypoxia-induced nuclear translocation of hypoxia-inducible transcription factor 1alpha to reduce vascular endothelial growth factor transcription and secretion, in a manner similar to pharmacological blockade with the p38 MAPK-specific inhibitor, SB203585. These studies indicate that morphine, in addition to its analgesic function, may be exploited for its antiangiogenic potential.

Immune modulation mediated by extracellular vesicles of intestinal organoids is disrupted by opioids.

Extracellular vesicles (EVs) are effective mediators of intercellular communications between enterocytes and immune cells. The current study showed that EVs isolated from mouse and human intestinal organoids modulated inflammatory responses of various immune cells including mouse bone-marrow derived-macrophages, dendritic cells, microglia cells, and human monocytes. EVs suppressed LPS-elicited cytokine production in these cells while morphine abolished EVs' immune modulatory effects. Microarray analysis showed that various microRNAs, especially Let-7, contributed to EV-mediated immune modulation. Using murine models, we showed that injection of EVs derived from intestinal organoids reduced endotoxin-induced systemic inflammation and alleviated the symptoms of DSS-induced colitis. EVs derived from morphine-treated organoids failed to suppress the immune response in both these models. Our study suggests that EVs derived from intestinal crypt cells play crucial roles in maintaining host homeostasis and opioid use is a risk factor for exacerbating inflammation in patients with inflammatory diseases such as sepsis and colitis.

Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as 'vasculogenic mimicry' (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody-endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear ß-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.