The p,p'-DDE disturbs the M1 function without affecting the M2 phenotype nor unstimulated bone marrow-derived macrophages from BALB/c mice.

DDT, a persistent organic pollutant, remains affecting human health worldwide. DDT and its most persistent metabolite (p,p'-DDE) negatively affect the immune response regulation and mechanisms involved in protecting against pathogens Such metabolite decreases the capability to limit intracellular growth of Mycobacterium microti and yeast. However, the effect on unstimulated (M0) and anti-inflammatory macrophages (M2) has been evaluated scanty. Herein, we evaluated the impact of p,p'-DDE at environmentally relevant concentrations (0.125, 1.25, 2.5, and 5 µg/mL) on bone marrow-derived macrophages stimulated with IFNγ+LPS to M1 or with IL-4 +IL-13 to M2. Thus we study whether the p,p'-DDE induces M0 to a specific phenotype or modulates activation of the macrophage phenotypes and explains, at least partly, the reported effects of p,p'-DDE on the M1 function. The p,p'-DDE did not affect the cell viability of M0 or the macrophage phenotypes. In M1, the p,p'-DDE decreased NO•- production and IL-1ß secretion, but increasing cellular ROS and mitochondrial O2•-, but did not alter iNOS, TNF-α, MHCII, and CD86 protein expression nor affect M2 markers arginase activity, TGF-ß1, and CD206; p,p'-DDE, did not affect marker expression in M0 or M2, supporting that its effects on M1 parameters are not dependent on M0 nor M2 modulation. The decreasing of NO•- production by the p,p'-DDE without altering iNOS levels, Arginase activity, or TNF-α, but increasing cellular ROS and mitochondrial O2 suggests that p,p'-DDE interferes with the iNOS function but not with its transcription. The p,p'-DDE decreasing of IL-1ß secretion, without any effect on TNF-α, suggest that an alteration of specific targets involved in IL-1ß secretion may be affected and related to ROS induction. The p,p'-DDE effect on iNOS function and the IL-1ß secretion process, as the NLRP3 activation, deserves further study.

Lactate modulates microglial inflammatory responses after oxygen-glucose deprivation through HIF-1α-mediated inhibition of NF-κB.

Metabolic adaption drives microglial inflammatory responses, and lactate shapes immunological and inflammatory states. However, whether lactate was involved in the regulation of microglial inflammatory responses after cerebral ischemia remains unclear. In this study, the expression of iNOS, arginase-1, phosphorylated NF-κB p65 and IκB-α, and HIF-1α in BV2 cells after oxygen-glucose deprivation (OGD) were detected by western blotting and immunofluorescence. The mRNA levels of microglial responsive markers and inflammatory factors were assessed by real-time-qPCR. The effect of lactate-treated BV2 cells on the survival of primary neurons was observed using transwell co-culture. The results showed that the protein levels of iNOS and arginase-1, the ratio of mRNA levels of iNOS/CD206, CD86/Ym1, IL-6/IL-10, TNF-α/IL-10 and the mRNA levels of IL-6 and TNF-α, as well as the protein levels of phosphorylated NF-κB p65 and IκB-α, were increased after OGD. Lactate treatment inhibited the OGD-induced increase in the protein levels of iNOS, phosphorylated NF-κB p65 and IκB-α, as well as iNOS/CD206, CD86/Ym1, IL-6/IL-10, TNF-α/IL-10, IL-6 and TNF-α mRNA levels in BV2 cells, while promoted arginase-1 protein expression as well as IL-10 and TGF-ß mRNA level. Interestingly, lactate activated HIF-1α and the HIF-1α inhibitor YC-1 reversed the effect of lactate on levels of microglial responsive markers and phosphorylated NF-κB p65 and IκB-α in BV2 cells. Moreover, knockdown of HIF-1α by lentivirus-delivered shRNA also reversed the effect of lactate on phosphorylated NF-κB p65 and IκB-α in BV2 cells after OGD. Finally, and importantly, lactate-treated BV2 microglia increased the viability and decreased the apoptosis of neurons after OGD. These findings revealed that lactate inhibited NF-κB pathway and skewed BV2 microglia toward the protective response through activation of HIF-1α after OGD, thereby improving neuronal survival.

Blueberry Leaf Extract Prevents Lacrimal Hyposecretion in Sjögren's Syndrome-like Model of Non-obese Diabetic Mice.

BACKGROUND/AIM: This study evaluated the effect of blueberry leaf hot water extract (BLEx) on Sjögren's syndrome (SS)-like lacrimal hyposecretion in male non-obese diabetic (NOD) mice. MATERIALS AND METHODS: NOD or BALB/c mice were fed 1% BLEx or control (AIN-93G) for 2 weeks from the age of 4 to 6 weeks. Pilocarpine-induced tear volume was measured using a phenol red-impregnated thread. The lacrimal glands were evaluated histologically by H&E staining. The IL-1ß and TNF-α levels in the lacrimal gland tissue were measured by ELISA. The mRNA expression levels of secretion-related proteins were measured by real-time PCR. LC3 I/II and arginase 1 expression levels were measured by western blot. RESULTS: After feeding with BLEx, pilocarpine-induced tear secretion in NOD mice was increased. In contrast, the mRNA expression levels of the cholinergic muscarinic M3 receptor, aquaporin 5, and ion channels related to lacrimal secretion were not changed by BLEx administration. In addition, the protein expression of arginase 1, which was recently reported to be involved in tear hyposecretion in NOD mice, was also not improved by BLEx administration. Although infiltration in the lacrimal gland of NOD mice was not decreased, the levels of TNF-α and the autophagy-related protein LC3 were significantly suppressed by BLEx treatment. CONCLUSION: BLEx treatment may ameliorate lacrimal hyposecretion in NOD mice by delaying the progression of autoimmune disease by suppressing autophagy in lacrimal glands.

Synergy of interleukin-4 and interferon-γ in arginase-1 production in RAW264.7 macrophages.

BACKGROUND: Interferon (IFN)-γ and interleukin (IL)-4 are considered to be important factors to regulate immune responses. Although the effects of IFN-γ or IL-4 on macrophage functions are well established, their cooperative action is not fully understood. OBJECTIVE: Inducible nitric oxide synthase (iNOS) or arginase (Arg)-1 is a representative marker of M1 or M2 macrophages and plays a role in the acceleration or suppression of inflammatory responses. In the present study, we examined the effect of simultaneous treatment with IFN-γ and IL-4 on macrophage expression of iNOS and Arg-1 using the murine macrophage cell line RAW264.7. METHODS: Protein production and mRNA expression of iNOS and Arg-1 were measured using immunoblotting and reverse transcription-polymerase chain reaction. Cell surface expression of CD86 and programmed death ligand (PD-L) 2 was analyzed using flow cytometry. RESULTS: IFN-γ or IL-4 increased iNOS or Arg-1 protein production, respectively. Of note, IL-4 combined with IFN-γ synergistically increased Arg-1 protein production, whereas IL-4 inhibited IFN-γ-induced iNOS production. This phenomenon was consistent with the mRNA levels. In addition, IL-4 combined with IFN-γ synergistically increased cell surface expression of PD-L2, which is involved in T cell suppression, whereas IL-4 completely inhibited IFN-γ-induced expression of CD86, which is responsible for T cell activation. CONCLUSIONS: In the present study, we found the synergy of IFN-γ and IL-4 in Arg-1 and PD-L2 expression. Thus, macrophages highly expressing Arg-1 and PD-L2 may be induced by both IFN-γ and IL-4 at the inflammatory site, and might play a role in the regulation of inflammatory immune responses.

Mono-PEGylated thermostable Bacillus caldovelox arginase mutant (BCA-M-PEG20) induces apoptosis, autophagy, cell cycle arrest and growth inhibition in gastric cancer cells.

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme-mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.

Mesenchymal stem cell treatment restores liver macrophages homeostasis to alleviate mouse acute liver injury revealed by single-cell analysis.

Acute liver injury (ALI) is characterized by massive hepatocyte necrosis and subsequent recruitment of myeloid cells to liver. Mesenchymal stem cells (MSCs) have therapeutic potential for ALI through their immunoregulation on macrophages, but the mechanism is not completely clear due to the heterogeneity and controversy of liver macrophages. Here, we detected the survival rate, biochemical indexes, histopathology, and inflammatory chemokine levels to assess the efficacy of MSC treatment on CCl4-induced ALI of C57BL/6 mice. Furthermore, flow cytometry and single-cell RNA sequencing (scRNA-Seq) were used to precisely distinguish macrophage populations and reveal the immunoregulation of MSCs. MSC treatment could effectively alleviate ALI and mitigate the recruitment of mononuclear phagocytes. Flow cytometry and scRNA-Seq analyses collectively indicated that there were monocytes with high Ly6C expression and heterogeneous monocyte-derived macrophages (MoMF) with low Ly6C expression in liver. Ly6Chi pro-inflammatory monocytes and Ly6Clo MoMF with powerful phagocytosis dominated during the acute injury period. MSC treatment promoted the transition from Ly6Chi to Ly6Clo population, inhibit the proinflammatory function of monocytes and promote the lysosomal function of MoMF. Furthermore, MSCs attenuated the recruitment of neutrophils by reducing the expression of CXCL2 of MoMF. MoMF with high expression of arginase 1 appeared during the recovery period, and MSCs could increase their expression of arginase 1, which may promote liver repair. To sum up, we demonstrated the characteristics of distinct MoMF during different periods of ALI and revealed their functional changes after MSC treatment, providing immunotherapeutic targets for MSC treatment of ALI.

Human Recombinant Arginase I [HuArgI(Co)-PEG5000]-Induced Arginine Depletion Inhibits Pancreatic Cancer Cell Migration and Invasion Through Autophagy.

OBJECTIVES: Pancreatic cancer is one of the most aggressive solid cancers and the fourth leading cause of cancer death in men and women. We previously showed that arginine depletion, using arginase I [HuArgI(Co)-PEG5000], selectively triggers cell death by autophagy in PANC-1 pancreatic cancer cells. The mechanism of action of [HuArgI(Co)-PEG5000], however, has remained poorly understood. In this study, we investigated the effects of arginine depletion on PANC-1 cell migration, adhesion, and invasion and determined the main molecular targets, which mediate PANC-1 cell response to treatment with HuArgI(Co)-PEG5000. METHODS: This was done through examining 2-dimensional (2D) cell motility assays (wound healing and time lapse), cell adhesion, and cell invasion assays, as well as immunostaining for focal adhesions and invadopodia in cells without or with the treatment with arginase. RESULTS: We demonstrate that arginine depletion decreases PANC-1 2D cell migration, adhesion, and 3D invasion. Moreover, our data suggest that these effects are mediated by autophagy and subsequent decrease in the activation of members of Ras homolog gene family (Rho) GTPase family. CONCLUSIONS: Altogether, these findings uncover the mechanism of action of [HuArgI(Co)-PEG5000] and highlight the promising and selective anticancer potential for arginine depletion in the treatment of pancreatic cancer cells.

Pegylated Recombinant Human Arginase 1 Induces Autophagy and Apoptosis via the ROS-Activated AKT/mTOR Pathway in Bladder Cancer Cells.

Bladder cancer is one of the most commonly diagnosed cancers worldwide, especially in males. Current therapeutic interventions, including surgery, radiation therapy, chemotherapy, and immunotherapy, have not been able to improve the clinical outcome of bladder cancer patients with satisfaction. Recombinant human arginase (rhArg, BCT-100) is a novel agent with great anticancer effects on arginine-auxotrophic tumors. However, the effects of BCT-100 on bladder cancer remain unclear. In this study, the in vitro anticancer effects of BCT-100 were assessed using four bladder cancer cell lines (J82, SCaBER, T24, and 5637), while the in vivo effects were evaluated by establishing T24 nude mice xenograft models. Intracellular arginine level was observed to be sharply decreased followed by the onset of apoptotic events. Furthermore, BCT-100 was found to induce H2O2 production and mitochondrial membrane depolarization, leading to the release of mitochondrial cytochrome c and Smac to the cytosol. Treatment with BCT was observed to upregulate the expression of LC3B and Becllin-1, but downregulate the expression of p62 in a time-dependent manner. Autophagic flux was also observed upon BCT-100 treatment. Besides, the phosphorylation of the AKT/mTOR pathway was suppressed in a time-dependent fashion in BCT-100-treated T24 cells. While N-acetyl-L-cysteine was shown to alleviate BCT-100-induced apoptosis and autophagy, chloroquine, MK-2206, and rapamycin were found to potentiate BCT-100-triggered apoptosis. Finally, BCT-100 was demonstrated to induce autophagy and apoptosis via the ROS-mediated AKT/mTOR signaling pathway in bladder cancer cells.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti-PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

Enzyme-based autophagy in anti-neoplastic management: From molecular mechanisms to clinical therapeutics.

Autophagy is an evolutionarily conserved self-cannibalization process commonly found in all eukaryotic cells. Through autophagy, long-lived or damaged organelles, superfluous proteins, and pathogens are sequestered and encapsulated into the double-membrane autophagosomes prior to fusion with lysosomes for ultimate degradation and recycling. Given that autophagy is deemed both protective and detrimental in malignancies, the clinical therapeutic utilization of autophagy modulators in cancer has attracted immense attentions over the past decades. Dependence of tumor cells on autophagy during amino acid insufficiency or deprivation has prompted us to explore the underlying autophagy regulatory mechanisms to inject amino acid degrading enzymes and enzyme-based strategies into therapeutic maneuvers of autophagy in cancer.

Preclinical safety and antitumor activity of the arginine-degrading therapeutic enzyme pegzilarginase, a PEGylated, cobalt-substituted recombinant human arginase 1.

Metabolic remodeling contributes to the development and progression of some cancers and exposes them to vulnerabilities, including specific nutrient dependencies that can be targeted therapeutically. Arginine is a semiessential amino acid, and several cancers are unable to endogenously synthesize sufficient levels of arginine for survival and proliferation, most commonly due to reduced expression of argininosuccinate synthase (ASS1). Such cancers are dependent on arginine and they can be targeted via enzyme-mediated depletion of systemic arginine. We report the preclinical safety, antitumor efficacy, and immune-potentiating effects of pegzilarginase, a highly potent human arginine-degrading enzyme. Toxicology studies showed that pegzilarginase-mediated arginine depletion is well tolerated at therapeutic levels that elicit an antitumor growth effect. To determine which tumor types are best suited for clinical development, we profiled clinical tumor samples for ASS1 expression, which correlated with pegzilarginase sensitivity in vivo in patient-derived xenograft (PDx) models. Among the histologies tested, malignant melanoma, small cell lung cancer and Merkel cell carcinoma had the highest prevalence of low ASS1 expression, the highest proportion of PDx models responding to pegzilarginase, and the strongest correlation between low or no ASS1 expression and sensitivity to pegzilarginase. In an immune-competent syngeneic mouse model, pegzilarginase slowed tumor growth and promoted the recruitment of CD8+ tumor infiltrating lymphocytes. This is consistent with the known autophagy-inducing effects of arginine depletion, and the link between autophagy and major histocompatibility complex antigen presentation to T cells. Our work supports the ongoing clinical investigations of pegzilarginase in solid tumors and clinical combination of pegzilarginase with immune checkpoint inhibitors.

[HuArgI (co)-PEG5000]-induced arginine deprivation leads to autophagy dependent cell death in pancreatic cancer cells.

In this study, we examined the sensitivity of pancreatic cancer cells to [HuArgI (Co)-PEG5000]-induced arginine deprivation as well as the mechanisms underlying deprivation-induced cell death. [HuArgI (Co)-PEG5000]-induced arginine deprivation was cytotoxic to all cell lines tested with IC50 values in the pM range at 72 h post-treatment. Three of the five cell lines were rescued by the addition of excess L-citrulline and expressed ASS1, indicating partial arginine auxotrophy. The remaining two cell lines, on the other hand, were not rescued by the addition of L-citrulline and did not express ASS1, indicating complete auxotrophy to arginine. In addition, all cell lines exhibited G0/G1 cell cycle arrest, in the surviving cell fraction, at 72 h following arginine deprivation. Analysis of the type of cell death revealed negative staining for annexin V and a lack of caspase activation in all five cancer cell lines, following treatment, indicating that arginine deprivation leads to caspase-independent, non-apoptotic cell death. Finally, we demonstrated that arginine deprivation leads to a marked activation of autophagy and that inhibition of this autophagy greatly decreases cytotoxicity, indicating that arginine deprivation induces autophagic cell death in pancreatic cancer cells. We have shown that pancreatic cancer cells are auxotrophic for arginine and sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation, hence demonstrating that arginine deprivation is a potentially potent and selective treatment for pancreatic cancer. We have also demonstrated that autophagy is activated following arginine-deprivation and that its prolonged activation leads to autophagic cell death.

Human Recombinant Arginase I [HuArgI (Co)-PEG5000]-Induced Arginine Depletion Inhibits Colorectal Cancer Cell Migration and Invasion.

PURPOSE: Colorectal cancer (CRC) is the third most common type of cancer worldwide, and it represents over half of all gastrointestinal cancer deaths. Knowing that cancer cells have a high proliferation rate, they require high amounts of amino acids, including arginine. In addition, several tumor types have been shown to downregulate ASS-1 expression, becoming auxotrophic for arginine. Therefore, Arginine deprivation is one of the promising therapeutic approaches to target cancer cells. This can be achieved through the use of a recombinant human arginase, HuArgI(Co)-PEG5000, an arginine degrading enzyme. METHODS: In this present study, the cytotoxic effect of HuArgI(Co)-PEG5000 on CRC cell lines (HT-29, Caco-2, Sw837) is examined though cytotoxicity assays. Wound healing assays, invasion assays, and adhesion assays were also performed to detect the effect on metastasis. RESULTS: Wound healing and invasion assays revealed a decrease in cell migration and invasion after treatment with arginase. Cells that were treated with arginase also showed a decrease in adhesion, which coincided with a decrease in RhoA activation, demonstrated though the use of a FRET biosensor to detect RhoA activation in a single cell assay, and a decrease in MMP-9 expression. Treating cells with both arginase and L-citrulline, which significantly restores intracellular arginine levels, reversed the effect of HuArgI(Co)-PEG5000 on cell viability, migration, and invasion. CONCLUSION: We can, therefore, conclude that colorectal cancer is partially auxotrophic to arginine and that arginine depletion is a potential selective inhibitory approach for motility and invasion in colon cancer cells.

Contactin 1 modulates pegylated arginase resistance in small cell lung cancer through induction of epithelial-mesenchymal transition.

Drug resistance is a major hurdle in the treatment of small cell lung cancer (SCLC). Previously we demonstrated the potential anticancer effect of pegylated arginase BCT-100 in SCLC cell lines and xenograft models. To facilitate future clinical application of BCT-100 in SCLC treatment, we elucidated the potential mechanisms that underlie acquired drug resistance to BCT-100. H446 and H526 SCLC cells were serially cultured in stepwise increasing concentrations of BCT-100 until stable BCT-100-resistant cell lines emerged (H446-BR and H526-BR). Compared with parent cells, H446-BR and H526-BR displayed stronger migration ability, anoikis resistance and EMT progression. Gene chip assay was employed to select three potential targets (CDH17, CNTN-1 and IGF2BP1). Silencing CNTN-1 rather than CDH17 or IGF2BP1 in H446-BR and H526-BR cells re-sensitized resistant cells to BCT-100 treatment and attenuated the epithelial-mesenchymal transition (EMT) phenotype. The AKT signaling pathway was activated in H446-BR and H526-BR cells accompanied by EMT progression, and AKT inhibitor LY294002 reversed the EMT progression in resistant cells.

Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts.

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

Combined use of arginase and dichloroacetate exhibits anti-proliferative effects in triple negative breast cancer cells.

OBJECTIVES: Drug combination in cancer therapy aims to achieve synergistic therapeutic effect, reduced drug dosage, reduced drug toxicity and minimizes or delays the induction of drug resistance. In the present study, we investigated the anticancer effects of the combination of two metabolic modulators, dichloroacetate (DCA) and bacillus caldovelox arginase (BCA) (or pegyated human arginase (HA)). METHODS: The combination treatments were evaluated in MCF-7 and MDA-MB 231 cells as well as in MDA-MB 231 breast cancer xenograft model. KEY FINDINGS: Dichloroacetate and BCA combination exhibited anti-proliferative effects on MCF-7 cells, which were found to be synergistic. Analysis of the gene expression upon drug treatments revealed that the synergistic anti-proliferative effect on MCF-7 cells was possibly in part due to the activation of the p53 pathway. A similar synergistic anti-proliferative effect was observed in the combined use of DCA and HA on MCF-7 and MDA-MB231 cells, which was due to induction of cell cycle arrest at G2/M phase. Moreover, the combination enhanced anti-tumour activity in a MDA-MB 231 xenograft mouse model. CONCLUSIONS: Our results suggested that dichloroacetate and arginase combination exhibited enhanced anti-cancer effects in preclinical breast cancer models which may offer an additional treatment option for breast cancer.

Arginase-2 protects myocardial ischemia-reperfusion injury via NF-κB/TNF-α pathway.

OBJECTIVE: Arginase-2 exerts an anti-inflammatory potential. However, whether nuclear factor-κB (NF-κB)/TNF-α (tumor necrosis factor-α) pathway is involved in the anti-inflammation effect of arginase-2 has not been fully elucidated. Our study aims to explore the regulatory role of arginase-2 on ischemia-reperfusion injury (IRI) in rats and its underlying mechanism. MATERIALS AND METHODS: 24 male Sprague Dawley (SD) rats were randomly assigned into sham group, IRI group and arginase-2 group, with 8 rats in each group. Electrocardiogram was performed in each rat before and after animal procedures. Serum samples and heart samples of each rat were collected 10 days after animal procedures. Serum levels of CK-MB (creatine kinase-MB) and LDH (lactate dehydrogenase) in each rat were detected using the relative commercial kits. Pathological lesions in rat myocardium were observed by hematoxylin and eosin (HE) staining. Cardiomyocyte apoptosis in rat heart was accessed by TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling) staining. Expression levels of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 in rat myocardium were detected by Western blot and immunohistochemistry. RESULTS: Electrocardiogram showed slower heart rate, lower voltage of QRS wave and longer Q-T interval in rats of IRI group than those of sham group (p < 0.05). A few rats in IRI group even presented arrhythmia. On the contrary, rats in arginase-2 group presented higher heart rate and voltage of QRS wave, as well as shorter Q-T interval compared with those of IRI group (p < 0.05). Rats in arginase-2 group presented lower plasma levels of CK-MB and LDH than those of IRI group. Pathological lesions in rat myocardium and cardiomyocyte apoptosis were alleviated in arginase-2 group in comparison with those of IRI group. Western blot and immunohistochemistry indicated that arginase-2 pretreatment remarkably downregulated expressions of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 in rat myocardium. CONCLUSIONS: Arginase-2 protects myocardial ischemia-reperfusion injury in rats through inhibiting the inflammatory response via suppression of NF-κB/TNF-α pathway.