[Paeoniflorin induces apoptosis and cycle arrest in B-cell acute lymphoblastic leukemia cells by inhibiting SENP1/c-Myc signaling pathway].

The effect of paeoniflorin on apoptosis and cell cycle in human B-cell acute lymphoblastic leukemia(B-ALL) and its underlying mechanism were investigated in this study. Nalm-6 and SUP-B15 cells were cultured in vitro and divided into control group(0 µg·mL~(-1)) and experimental groups(200, 400, and 800 µg·mL~(-1) paeoniflorin). Cell counting kit-8(CCK-8) was used to measure the viability of Nalm-6 and SUP-B15 cells, and cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blot was used to detect the protein levels of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase(cleaved PARP), c-Myc, and small ubiquitin-like modifier-specific protease 1(SENP1). The mRNA levels of c-Myc and SENP1 in acute lymphoblastic leukemia(ALL) patients were analyzed based on the Oncomine database. AutoDock was used for molecular docking to analyze the interaction of paeoniflorin with c-Myc and SENP1 proteins. RESULTS:: showed that paeoniflorin inhibited the viability of Nalm-6 and SUP-B15 cells in concentration and time-dependent manners. Compared with the control group, paeoniflorin significantly up-regulated the expression of apoptosis-related proteins cleaved caspase-3 and cleaved PARP to induce apoptosis, evidently increased the proportion of G_2/M phase cells and induced G_2/M phase arrest, and obviously down-regulated the expression of c-Myc and SENP1 proteins in Nalm-6 and SUP-B15 cells. The mRNA levels of c-Myc and SENP1 in ALL patients were higher than those in the normal cell. Molecular docking demonstrated that paeoniflorin had good binding to c-Myc and SENP1 proteins. In summary, paeoniflorin inhibits the proliferation of Nalm-6 and SUP-B15 cells by inducing apoptosis and G_2/M phase arrest, which may be related to the down-regulation of c-Myc and SENP1 proteins.

Protective Cellular Immune Response Induction for Cutaneous Leishmaniasis by a New Immunochemotherapy Schedule.

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo. The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4+ and CD8+ T lymphocytes, IFN-γ levels and reduction of active TGF-ß in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis.

Effectiveness of cysteine proteases on protein/pigment film removal.

OBJECTIVE: Theaflavin (TF) from the black tea can react to human salivary proline-rich proteins (PRPs) to form stains on exposed dental surfaces. Here, we employed a model of protein/pigment film using TF and dephosphorylated bovine ß-casein (Dß-CN), which has an extended conformation, similar to that of salivary PRPs, on a sensor surface to assess the efficacy of cysteine proteases (CPs) including papain, stem bromelain, and ficin, on removing TF bound to Dß-CN and the control TF readsorption on the residual substrate surfaces was also measured. METHODS: The protein/pigment complex film was built by using a quartz crystal microbalance with dissipation (QCM-D). The efficacies of CPs were assessed by Boltzman equation model. The surface details were detected by grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles. RESULTS: The efficacy order of CPs on hydrolyzing protein/pigment complex film is ficin>papain>bromelain. The results from grazing angle infrared spectroscopy spectra, atomic force microscopy images, and contact angles demonstrated that TF bound on the Dß-CN was effectively removed by the CPs, and the amount of TF readsorption on both the residual film of the Dß-CN/TF and the Dß-CN was markedly decreased after hydrolysis. CONCLUSION: This study indicates the potential application of the CPs for tooth stain removal and suggests that these enzymes are worthy of further investigation for use in oral healthcare.

Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes.

The prolamin peptides in wheat gluten and in the homologous storage proteins of barley and rye cause painful chronic erasure of microvilli of the small intestine epithelium in celiac patients. If untreated, it can lead to chronic diarrhea, abdominal distension, osteoporosis, weight-loss due to malabsorption of nutrients, and anemia. In addition to congenital cases, life-long exposure to gluten proteins in bread and pasta can also induce development of celiac sprue in adults. To date, the only effective treatment is life-long strict abstinence from the staple food grains. Complete exclusion of dietary gluten is, however, difficult due to use of wheat in many foods, incomplete labeling and social constraints. Thus, finding alternative therapies for this most common foodborne disease remained an active area of research, which has led to many suggestions in last few years. The pros and cons associated with these therapies were reviewed in the present communication. As different celiac patients are immunogenic to different members of the undigestible proline/glutamine rich peptides of ~149 gliadins and low molecular weight glutenin subunits as well as the six high molecular weight glutenin subunits, an exhaustive digestion of the immunogenic peptides in the stomach, duodenum, jejunum, and ileum of celiacs is required. In view of the above, we evaluated the capacity of cereal grains to synthesize and store the enzymes prolyl endopeptidase from Flavobacterium meningosepticum and the barley cysteine endoprotease B2, which in combination are capable of detoxifying immunogenic gluten peptides in a novel treatment of celiac disease.

Skin-healing activity and toxicological evaluation of a proteinase fraction from Carica candamarcensis.

Previous studies demonstrated that proteinases from latex of C. candamarcensis act as mitogens on fibroblast and epithelial cells and a subsequent report showed their protective, angiogenic and wound healing effects on gastric ulcers. In this study, we present evidence of skin healing activity by the group of proteinases known as P1G10. By using a hairless mouse model, we compared the healing effect following topical application of various concentrations of P1G10. The data confirm that healing actions take place between 0.1 and 1%, without adverse local irritation or systemic toxicological action after a prolonged period of use. The wound healing effect is unaltered when P1G10 is previously inhibited with iodoacetamide. The low permeation of the hydrosoluble formulation Polawax(®) supports the maintenance of the drug at the site of application. These results extend the healing properties of these groups of enzymes in situations of dermatological trauma and open the way to future clinical applications.

The gastric ulcer protective and healing role of cysteine proteinases from Carica candamarcensis.

Latex from Caricaceae contains proteolytic enzymes localized in the fruit, which are used ethnopharmacologically to treat digestive disorders. Some of these proteins display proliferative properties when probed with mammalian cells, suggesting a role in the reconstruction of wounded tissue. We tested the efficacy of a proteolytic fraction derived from Carica candamarcensis, designated as P1G10 in experimental rodent models, to protect and heal chemically induced gastric ulcers. The protective effect of oral administration of P1G10 fraction was analyzed in indomethacin-treated Wistar animals. The healing effect of P1G10 was studied following sub-serous injection of acetic acid in a Wistar rat model. The results show that P1G10 between 0.1 and 10 mg/kg protect indomethacin but not ethanol-induced gastric ulcers. The maximal protection attained was 67% with 10 mg/kg of P1G10. The healing rate by 10 mg/kg of P1G10 using the acetic acid ulcerogenic model is similar to that of omeprazole (10 mg/kg) or ranitidine (100 mg/kg). The effect of P1G10 at 10 mg/kg seems to be mediated by an increase in the mucus content by 25% and stimulation of angiogenesis by 64% in a manner similar to growth factors. These results confirm the protective and healing role of proteinases from C. candamarcensis.

Antitumor effects in vitro and in vivo and mechanisms of protection against melanoma B16F10-Nex2 cells by fastuosain, a cysteine proteinase from Bromelia fastuosa.

In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, and became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes) migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein-chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBS-injected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, and cathepsins B and L cross-reacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies.

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris.

Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.

Debridement of porcine burns with a highly purified, ananain-based cysteine protease preparation.

A novel enzymatic debriding agent was evaluated on experimental full-thickness porcine contact burns. This agent consists of a highly purified, ananain-based, cysteine protease preparation formulated in a hydrophilic cream vehicle. Debridement of full-thickness burns was found to be dependent on several factors including the concentration of enzyme in the vehicle, the duration of treatment, and the hydration status of the burn wound before treatment. With an optimized debridement regimen, burns were consistently debrided of all gelatinized tissue with two 5-hour treatments. Histologic evaluation of the debrided wounds revealed an acellular deeper dermis that was debrided of necrotic cellular debris; however, the collagen matrix of the deeper dermis remained intact. This observation was consistent with a demonstrated in vitro specificity of the ananain-based protease for gelatin over collagen. A direct comparison of debridement efficacy with sutilains ointment, showed the ananain-based, debriding enzyme preparation to provide more rapid debridement of gelatinized tissue. Enzymatically debrided wounds exhibited graft take only after surgical excision of approximately 1 mm of the remaining acellular, avascular dermis. This highly purified enzyme preparation offers the potential for rapid nonsurgical debridement of gelatinized burn tissue, but required additional surgical debridement for graft take in this porcine model.