Mesenchymal Stem Cells and Extracellular Vesicles Derived from Canine Adipose Tissue Ameliorates Inflammation, Skin Barrier Function and Pruritus by Reducing JAK/STAT Signaling in Atopic Dermatitis.

Canine atopic dermatitis (AD) is a common chronic inflammatory skin disorder resulting from imbalance between T lymphocytes. Current canine AD treatments use immunomodulatory drugs, but some of the dogs have limitations that do not respond to standard treatment, or relapse after a period of time. Thus, the purpose of this study was to evaluate the immunomodulatory effect of mesenchymal stem cells derived from canine adipose tissue (cASCs) and cASCs-derived extracellular vesicles (cASC-EVs) on AD. First, we isolated and characterized cASCs and cASCs-EVs to use for the improvement of canine atopic dermatitis. Here, we investigated the effect of cASCs or cASC-EVs on DNCB-induced AD in mice, before using for canine AD. Interestingly, we found that cASCs and cASC-EVs improved AD-like dermatitis, and markedly decreased levels of serum IgE, (49.6%, p = 0.002 and 32.1%, p = 0.016 respectively) epidermal inflammatory cytokines and chemokines, such as IL-4 (32%, p = 0.197 and 44%, p = 0.094 respectively), IL-13 (47.4%, p = 0.163, and 50.0%, p = 0.039 respectively), IL-31 (64.3%, p = 0.030 and 76.2%, p = 0.016 respectively), RANTES (66.7%, p = 0.002 and 55.6%, p = 0.007) and TARC (64%, p = 0.016 and 86%, p = 0.010 respectively). In addition, cASCs or cASC-EVs promoted skin barrier repair by restoring transepidermal water loss, enhancing stratum corneum hydration and upregulating the expression levels of epidermal differentiation proteins. Moreover, cASCs or cASC-EVs reduced IL-31/TRPA1-mediated pruritus and activation of JAK/STAT signaling pathway. Taken together, these results suggest the potential of cASCs or cASC-EVs for the treatment of chronic inflammation and damaged skin barrier in AD or canine AD.

Expansion of tumor-infiltrating lymphocytes and their potential for application as adoptive cell transfer therapy in human breast cancer.

Adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs) has been successful in treating a considerable proportion of patients with metastatic melanoma. In addition, some patients with several other solid tumors were recently reported to have benefited clinically from such ACT. However, it remains unclear whether ACT using TILs is broadly applicable in breast cancer, the most common cancer in women. In this study, the utility of TILs as an ACT source in breast cancers was explored by deriving TILs from a large number of breast cancer samples and assessing their biological potentials. We successfully expanded TILs ex vivo under a standard TIL culture condition from over 100 breast cancer samples, including all breast cancer subtypes. We also found that the information about the percentage of TIL and presence of tertiary lymphoid structure in the tumor tissues could be useful for estimating the number of obtainable TILs after ex vivo culture. The ex vivo expanded TILs contained a considerable level of central memory phenotype T cells (about 20%), and a large proportion of TIL samples were reactive to autologous tumor cells in vitro. Furthermore, the in vitro tumor-reactive autologous TILs could also function in vivo in a xenograft mouse model implanted with the primary tumor tissue. Collectively, these results strongly indicate that ACT using ex vivo expanded autologous TILs is a feasible option in treating patients with breast cancer.

Suppression of tumorigenesis in mitochondrial NADP(+)-dependent isocitrate dehydrogenase knock-out mice.

The tumor host microenvironment is increasingly viewed as an important contributor to tumor growth and suppression. Cellular oxidative stress resulting from high levels of reactive oxygen species (ROS) contributes to various processes involved in the development and progress of malignant tumors including carcinogenesis, aberrant growth, metastasis, and angiogenesis. In this regard, the stroma induces oxidative stress in adjacent tumor cells, and this in turn causes several changes in tumor cells including modulation of the redox status, inhibition of cell proliferation, and induction of apoptotic or necrotic cell death. Because the levels of ROS are determined by a balance between ROS generation and ROS detoxification, disruption of this system will result in increased or decreased ROS level. Recently, we demonstrated that the control of mitochondrial redox balance and cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2) that supplies NADPH for antioxidant systems. To explore the interactions between tumor cells and the host, we evaluated tumorigenesis between IDH2-deficient (knock-out) and wild-type mice in which B16F10 melanoma cells had been implanted. Suppression of B16F10 cell tumorigenesis was reproducibly observed in the IDH2-deficient mice along with significant elevation of oxidative stress in both the tumor and the stroma. In addition, the expression of angiogenesis markers was significantly down-regulated in both the tumor and the stroma of the IDH2-deficient mice. These results support the hypothesis that redox status-associated changes in the host environment of tumor-bearing mice may contribute to cancer progression.

Silencing of mitochondrial NADP(+)-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity.

Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP-LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

Sensitization of ionizing radiation-induced apoptosis by ursolic acid.

Radiation therapy has been widely used for treating human cancers. However, cancer cells develop radioresistant phenotypes that decrease the efficacy of radiotherapy. Ionizing radiation (IR) induces the production of reactive oxygen species, which play an important role in apoptotic cell death. Therefore, radiation therapy combined with a sensitizer, which modulates cellular redox status, has the potential to enhance therapeutic efficacy in a variety of human cancers. Here, we investigated the radiosensitizing effects of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil. IR-induced apoptosis in cancer cell lines such as DU145, CT26 and B16F10 was significantly enhanced by UA, as reflected by DNA fragmentation, cellular redox status, mitochondrial dysfunction and modulation of apoptotic marker proteins. Additionally, UA combined with IR was also effective for inhibiting tumorigenesis in B16F10 melanoma cells implanted into mice. Taken together, these results suggest that applying UA together with IR may be an effective combination modality for treating cancer.

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells.

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.

Regulation of brefeldin A-induced ER stress and apoptosis by mitochondrial NADP⁺-dependent isocitrate dehydrogenase.

Brefeldin A (BFA), an endoplasmic reticulum (ER)-Golgi transport inhibitor, has been shown to cause accumulation of proteins in the ER, ER stress, and ultimately apoptosis. In this paper, we demonstrate that the knockdown of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm), a mitochondrial NADPH-generating enzyme, by small interfering RNA (siRNA) enhanced BFA-induced apoptosis. However, attenuated IDPm activity results in the suppression of ER stress response, presumably, via the inhibition of the PI3K/Akt pathway. Collectively, our data suggest that the association of IDPm expression and ER stress confers a survival mechanism in A549 cells against BFA-induced apoptosis.

Sensitive to apoptosis gene protein regulates ionizing radiation-induced apoptosis.

Organisms exposed to ionizing radiation (IR) undergo increases in the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. Sensitive to apoptosis gene (SAG) encodes a redox-inducible and apoptosis-protective antioxidant protein. This report demonstrates that the modulation of SAG expression in cultured cells regulates IR-induced apoptosis. A protective role for SAG against IR-induced apoptosis was found in U937 cells transfected with SAG cDNA. A significant decrease in the endogenous production of ROS was also observed in SAG over-expressing cells, compared to control cells, exposed to 2Gy γ-irradiation. These results suggest that SAG plays an important role in regulating IR-induced apoptosis, presumably by maintaining the cellular redox status. Because SAG is over-expressed in many human cancers, targeting SAG expression may have therapeutic value in cancer treatment. Transfection of the pancreatic cancer cell line PC3 with SAG small interfering RNA markedly attenuated the expression of SAG, augmenting their susceptibility to IR-induced apoptosis. The knockdown of SAG expression by RNA interference combined with radiotherapy may be a potential method for radiosensitization.

Modulation of hypoxia-inducible factor-1α expression by mitochondrial NADP+-dependent isocitrate dehydrogenase.

The transcription factor hypoxia-inducible factor-1 (HIF-1) is an important regulator of the tumor response to hypoxia, including increased angiogenesis, glycolytic metabolism, and resistance to apoptosis. In the current study, small interfering RNA (siRNA)-mediated knockdown of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) suppressed hypoxia-induced stimulation of HIF-1α protein expression in PC3 human prostate cancer cells. Treatment with the 26S proteasome inhibitor MG132 failed to abrogate the suppression of HIF-1α accumulation induced by IDPm knockdown, whereas HIF-1α levels were reduced by cycloheximide treatment in both control and IDPm siRNA-transfected cells. These results suggested that the suppression of HIF-1α accumulation by IDPm knockdown in PC3 cells was due to an inhibition of HIF-1α transcription. Inactivation of the phosphoinsotide-3 kinase (PI3K)/Akt pathway decreased HIF-1α expression through inactivation of Sp1. Thus, IDPm siRNA functioned as a potentially useful agent for targeting chemo- and radio-resistant hypoxic cells within solid tumors through inhibition of HIF-1α expression.

Glutathionylation regulates IkappaB.

Although there has been considerable interest in the regulation of NFkappaB activation by glutathionylation, the possibility of IkappaB as a target for glutathionylation has not been investigated. We now report that Cys(189) of IkappaB alpha is a target for S-glutathionylation. This modification is reversed by thiols such as dithiothreitol and GSH. The glutathionylated IkappaB alpha appears to be significantly less susceptible than is native protein to phosphorylation by IkappaB kinase and casein kinase II, as well as to in vitro ubiquitination. This finding suggests that glutathionylation plays a regulatory role, presumably through structural alterations. HeLa cells treated with oxidant inducing GSH oxidation such as diamide showed the accumulation of glutathionylated IkappaB alpha. This mechanism suggests an alternative modification to the redox regulation of cysteine in IkappaB alpha and a possible mechanism in the regulation of NFkappaB activation.

Regulation of heat shock-induced apoptosis by sensitive to apoptosis gene protein.

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates heat shock-induced apoptosis. When we examined the protective role of SAG against heat shock-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed a significant decrease in the endogenous production of reactive oxygen species and oxidative DNA damage in SAG-overexpressed cells compared to control cells on exposure to heat shock. In addition, transfection of PC3 cells with SAG small interfering RNA markedly decreased the expression of SAG, enhancing the susceptibility of heat shock-induced apoptosis. Taken together, these results indicate that SAG may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.

Small interfering RNA-mediated silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor-alpha and anticancer drugs.

Tumor necrosis factor-alpha (TNF-alpha) and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF-alpha and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF-alpha and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

Regulation of ionizing radiation-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase.

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. By supplying NADPH for antioxidant systems, we recently demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are some of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). In this study, we demonstrate that modulation of IDPm activity in U937 cells regulates ionizing radiation-induced apoptosis. When we examined the regulatory role of IDPm against ionizing radiation-induced apoptosis in U937 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. Upon exposure to 2 gray gamma-irradiation, there was a distinct difference between the IDPm transfectant cells in regard to the morphological evidence of apoptosis, DNA fragmentation, cellular redox status, oxidative damage to cells, mitochondrial function, and the modulation of apoptotic marker proteins. In addition, transfection of HeLa cells with an IDPm small interfering RNA decreased the activity of IDPm, enhancing the susceptibility of radiation-induced apoptosis. Taken together, these results indicate that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, and the effect of IDPm small interfering RNA on HeLa cells offers the possibility of developing a modifier of radiation therapy.