isolation-exosome-enriched-extracellular-vesicles-carrying

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

feeder-free-culture-of-murine-induced-pluripotent-stem-cells

Feeder-Free Culture of Murine Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells, can self-renew and differentiate into all cell types of the adult body. ...

University of Louisville

Sutter Health Shared Laboratory

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12. 

Paraoxonase 2 serves a proapopotic function in mouse and human cells in response to the Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)-homoserine lactone.

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. 

N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.

N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts.

The antigenic similarity between embryos and tumors has raised the idea of using embryonic material as a preventative vaccine against neoplastic disease. Indeed, we have previously reported that a vaccine comprises allogeneic murine embryonic stem cells (ESCs) and murine fibroblasts expressing GM-CSF (to amplify immune responses) successfully blocks the outgrowth of an implantable cancer (Lewis lung carcinoma; LLC) and lung tumors generated in mice using a combination of a mutagen followed by chronic pulmonary inflammation. However, such a vaccine is obviously impractical for application to humans. The use of fibroblasts to generate GM-CSF is needlessly complicated, and intact whole ESCs carry the hazard of generating embryomas/teratomas. Here, we report the successful application of an alternative prophylactic vaccine comprises exosomes derived from murine ESCs engineered to produce GM-CSF. Vaccination of mice with these exosomes significantly slowed or blocked the outgrowth of implanted LLC while control exosomes lacking GM-CSF were ineffective. Examination of tumor-infiltrating immune cells from mice vaccinated with the GM-CSF-expressing exosomes showed robust tumor-reactive CD8 T effector responses, Th1 cytokine responses, and higher CD8 T effector/CD4CD25Foxp3 T regulatory cell ratio in the tumors. We conclude that a similar vaccine derived from GM-CSF- expressing human ESCs can be employed as a preventative vaccine for humans with an increased risk of developing cancer.

Exosomes from GM-CSF expressing embryonic stem cells are an effective prophylactic vaccine for cancer prevention.

The opportunistic bacterium Pseudomonas aeruginosa secretes the quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (C12) to co-ordinate gene expression profiles favorable for infection. Recent studies have demonstrated that high concentrations of C12 impair many aspects of host cell physiology, including mitochondrial function and cell viability. The cytotoxic effects of C12 are mediated by the lactonase enzyme, Paraoxonase 2 (PON2), which hydrolyzes C12 to a reactive metabolite. However, the influence of C12 on host cell physiology at concentrations observed in patients infected with P. aeruginosa is largely unknown. Since the primary site of P. aeruginosa infections is the mammalian airway, we sought to investigate how PON2 modulates the effects of C12 at subtoxic concentrations using immortalized murine tracheal epithelial cells (TECs) isolated from wild-type (WT) or PON2-knockout (PON2-KO) mice. Our data reveal that C12 at subtoxic concentrations disrupts mitochondrial bioenergetics to hinder cellular proliferation in TECs expressing PON2. Subtoxic concentrations of C12 disrupt normal mitochondrial network morphology in a PON2-dependent manner without affecting mitochondrial membrane potential. In contrast, higher concentrations of C12 depolarize mitochondrial membrane potential and subsequently trigger caspase signaling and apoptotic cell death. These findings demonstrate that different concentrations of C12 impact distinct aspects of host airway epithelial cell physiology through PON2 activity in mitochondria.

PON2 mediates mitochondrial dysfunction in tracheal epithelial cells in response to a quorum sensing molecule N-(-3-oxododecanoyl)-l-homoserine lactone.

Our earlier work has shown that a unique stem cell-based vaccine that comprises of murine embryonic stem cells (ESCs) and murine fibroblasts expressing the immunostimulant granulocyte-macrophage colony stimulating factor (GM-CSF) successfully protects mice from the outgrowth of an implantable form of murine lung cancer. The use of live ESCs raises the potential risks of inducing teratomas and autoimmunity. We have attempted to improve the safety and utility of this prophylactic vaccine by employing exosomes derived from murine ESCs engineered to produce GM-CSF (ES-exo/GM-CSF vaccine).

Exosome-based cancer vaccine for prevention of lung cancer.

Paraoxonase 2 (PON2) is a multifunctional intracellular enzyme that has received growing attention for its ability to modulate various aspects of normal and malignant cellular physiology. Recent research has revealed that PON2 is upregulated in tissues from patients with various types of solid tumors and hematologic cancers, likely due to its ability to suppress oxidative stress and evade apoptosis. However, the effects of PON2 on pulmonary oncogenesis are unknown. Here, we conducted studies to investigate how PON2 influences lung cancer cell proliferation in vitro and lung tumorigenesis in vivo using a variety of cellular and animal models. It was found that PON2 expression deficiency hampered the proliferation of cultured lung cancer cells with concomitant cell cycle arrest at the G1 phase. In addition, the loss of endogenous PON2 expression impaired key aspects of oxidative metabolism in lung adenocarcinoma cells. Moreover, we investigated how the interplay between PON2 expression in lung tumors and host mice influences lung tumor initiation and progression. PON2 status in both transplanted tumor cells and mice failed to influence the development of subcutaneously grafted Lewis lung carcinoma (LLC) tumors, orthotopically implanted LLC tumors, and oncogenic Kras-driven primary lung adenocarcinoma tumors. Importantly, the frequencies of tumor-infiltrating myeloid subsets that include myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages were not impacted by PON2 expression in LLC tumor-bearing mice. Overall, our studies indicate that PON2 plays a limited role in murine lung tumorigenesis.

Paraoxonase 2 (PON2) plays a limited role in murine lung tumorigenesis.

Fatal drug overdoses often involve multiple co-intoxicants, including opioids. Hydrocodone, the most prescribed opioid for pain management, is metabolized to the active metabolite hydromorphone by hepatic CYP2D6. Inhibition of CYP2D6 by other compounds can disrupt the analgesic properties of hydrocodone and extend its half-life. Diphenhydramine is an over-the-counter cold medication and is known to inhibit CYP2D6 activity.

CYP2D6 inhibition by diphenhydramine leading to fatal hydrocodone overdose.

To explore pain outcomes in patients prescribed hydrocodone and psychotropic medications with or without CYP2D6 inhibition activity.

Prolonged post-operative hydrocodone usage due to psychotropic drug interaction.

Based on the antigenic similarity between tumor cells and embryonic stem cells (ESCs), several recent studies report the use of intact murine ESCs or exosomes from murine ESCs as cancer vaccines. Since the capacity for self-renewal is one of the most specialized properties shared between ESCs and a subset of tumor cells, cancer stem cells (CSCs), we investigated whether the undifferentiated state of murine ESCs is essential for the prophylactic effectiveness of an ESC-based vaccine. The undifferentiated state of ES-D3, a murine ESC line, was essential for their anchorage-independent growth potential. Importantly, differentiation of ES-D3 cells decreased their efficacy in preventing the outgrowth of implanted lung tumors. Furthermore, the long-term cancer-preventive potential of this vaccine was also inhibited by the differentiation of these cells. To examine the antigenicity of the ESC-derived vaccine, we performed combined affinity chromatography shotgun immunoproteomic experiments to identify antigens specific to the whole-cell ES vaccine as well as to the ESC-derived exosome vaccine. Our data demonstrate that antibodies against several lung cancer-associated keratin members were enriched in the serum of vaccinated mice. In summary, these data suggest that the tumor-preventing efficacy of ESC-based vaccine is reliant on the differentiation properties of these stem cells.

ArticleTotal : 2	Year
Publication title Cited by 12	2021
Publication title	2025

Article	Year
Paraoxonase 2 serves a proapopotic function in mouse and human cells in response to the Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)-homoserine lactone. The Journal of biological chemistry\| PubMed ID: 25627690	2015
N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins. Oncotarget\| PubMed ID: 26758417	2016
N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts. Cellular microbiology\| PubMed ID: 28876505	2018
Exosomes from GM-CSF expressing embryonic stem cells are an effective prophylactic vaccine for cancer prevention. Oncoimmunology\| PubMed ID: 30723593	2019
PON2 mediates mitochondrial dysfunction in tracheal epithelial cells in response to a quorum sensing molecule N-(-3-oxododecanoyl)-l-homoserine lactone. The Biochemical journal\| PubMed ID: 36094147	2022
Exosome-based cancer vaccine for prevention of lung cancer. Stem cell investigation\| PubMed ID: 36742283	2023
Paraoxonase 2 (PON2) plays a limited role in murine lung tumorigenesis. Scientific reports\| PubMed ID: 37337025	2023
CYP2D6 inhibition by diphenhydramine leading to fatal hydrocodone overdose. Drug metabolism and personalized therapy\| PubMed ID: 38741525	2024
Prolonged post-operative hydrocodone usage due to psychotropic drug interaction. Drug metabolism and personalized therapy\| PubMed ID: 39679533	2025
The efficacy of an embryonic stem cell-based vaccine for lung cancer prevention depends on the undifferentiated state of the stem cells. Scientific reports\| PubMed ID: 39739089	2024

Aaron G. Whitt

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