Dalton's Lymphoma Tumor Research Articles

Cordyceps militaris: A Comprehensive Study on Laboratory Cultivation and Anticancer Potential in Dalton's Ascites Lymphoma Tumor Model.

Cancer, a predominant cause of mortality, poses a formidable challenge in our pursuit of elevating life expectancy. Throughout history, individuals have sought natural remedies with minimal side effects as an appealing substitute for chemotherapeutic drugs. One such remedy is Cordyceps militaris, a renowned medicinal mushroom deeply entrenched in Asian ethnomedicine. Revered for its rejuvenating and curative attributes, it relied upon for ages. The mushroom's soaring demand outpaced natural availability, necessitating controlled laboratory cultivation as the core focus and exploring the potential of methanolic extracts from harvested Cordyceps militaris fruiting bodies against Dalton's Lymphoma Ascites (DLA) cells in vitro, with a specific emphasis on its anticancer traits. For cultivation, we employed a diverse range of rice substrates, among which bora rice showed promising growth of C. militaris fruiting bodies. To assess DLA cell cytotoxicity, several assays, including trypan blue exclusion assay, MTT assay, and LDH assay, were employed at different time points (24-96 h), which provided valuable insights on DLA cell viability and proliferation, shedding light on its therapeutic potential against cancer. Our studies unveiled that methanolic extract prompts apoptosis in DLA cells via AO/EB dual staining, manifesting consistent apoptosis indicators such as membrane blebbing, chromatin condensation, nuclei fragmentation, and cellular shrinkage at 48-96 h of treatment. Furthermore, these striking repercussions of apoptosis were comprehended by an in silico approach having molecular docking simulation against antiapoptotic proteins like BCL-2, BCL-XL, MCL-1, BFL-1 & HSP100. Methanolic C. militaris extracts exhibited cytotoxicity and apoptotic alterations in DLA cells.

Exploring 2-(benzylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole as antiproliferative agent: Synthesis, single crystal XRD, DFT, Hirshfeld surface analysis, in vitro antiproliferative activity and molecular docking analysis

In this investigation, an oxadiazole derivative namely 2-(benzylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole (2-Btno) was synthesized. The study delves into the exploration of its anticancer efficacy against Dalton's lymphoma (DL) tumor cells, obtained from murine T-cell lymphoma. The compound 2-Btno has been characterized by elemental analysis, IR, UV–Vis., NMR, and single-crystal X-ray diffraction data. The compound 2-Btno crystallizes in a monoclinic system with space group P 21/n. The crystal structure is stabilized by weak CH···O and NH···N hydrogen bonding weak interactions, which are quantitively examined through Hirshfeld surface analysis. The DFT calculations are also performed to verify physiochemical properties of 2-Btno and the results obtained are in good agreement with the experimental results. The HOMO and LUMO energy gap of 3.402 eV for 2-Btno indicates good NLO properties. The cytotoxic activity of 2-Btno against Dalton's lymphoma cells was assessed through the MTT assay. The results indicated notable anticancer efficacy, with an IC50 value of 80 μg/mL. The mode of action of compound 2-Btno was examined through various assays, and the findings indicate that compound 2-Btno functions by downregulating mitochondrial membrane potential and upregulating the reactive oxygen species (ROS) production. Molecular docking study was conducted to establish the correlation between the cytotoxicity of the compound and its binding affinity within the active sites of crucial anti-cancer and anti-apoptotic target proteins MDM2 (PDB: 3JZK), Bcl-XL (PDB: 4QVX) and Bcl-2 (PDB: 6O0K).

Umbelliferone loaded PEGylated liposomes: preparation, characterization and its mitigatory effects on Dalton's ascites lymphoma development.

Umbelliferone (UB) is a phenylpropanoid-based pharmacologically active agent with promising anti-tumor activities. However, complete elucidation of its therapeutic efficacy remains challenging due to low solubility and bioavailability. The present study aimed to develop a liposomal delivery system for UB to enhance its therapeutic efficacy against Dalton's ascites lymphoma tumor model. Umbelliferone-encapsulated nanoliposomes (nLUB) were prepared using the thin-film hydration method and performed a series of characterizations to confirm successful development. The nLUB showed a particle size of 116 ± 3.2nm with a negative surface charge and encapsulation efficiency of 78%. In vitro study results showed that nLUB treatment significantly enhanced cellular uptake and apoptosis induction in lymphoma cells compared to free UB. nLUB treatment significantly stabilized body weight, reduced tumor growth, and improved the serum biochemical and hematological parameters of experimental animals, thereby improving their overall survivability compared to an free UB. Our result indicates that nanoencapsulation enhanced the therapeutic potential of UB, which may find its way to clinical application in the near future.

Mn(II) catalyzed synthesis of 5(4-hydroxyphenyl)-2-(N-phenylamino)-1,3,4-oxadiazole: Crystal structure, DFT, molecular docking, Hirshfeld surface analysis, and in vitro anticancer activity on DL cells

The syntheses and screening of novel synthetic molecules have gained attention as a potential therapeutic agent in the treatment of cancer. In the present study, a new compound 5(4-hydroxyphenyl)-2-(N-phenylamino)-1,3,4-oxadiazole (Hppo) has been synthesized and its anticancer activity is investigated against Dalton's lymphoma (DL) tumor cells derived from murine T-cell lymphoma. The Hppo has been characterized through IR, NMR, and single-crystal X-ray data. The structure of Hppo is stabilized via hydrogen bonding interactions and crystallizes in an orthorhombic system with space group P b c n. The fingerprint plots associated with Hirshfeld surface analysis indicate that there are different types of weak interactions viz. C-H···N, O-H···N and C-H···O. The DFT calculations are also performed to verify physiochemical properties of Hppo and the results obtained are in good agreement with the experimental results. The HOMO and LUMO energy gap of 7.344 eV for Hppo indicates good NLO properties. The cytotoxicity activity of Hppo is tested against Dalton's lymphoma cells using MTT assay which reveals that the compound showed admirable anticancer activity (IC50= 50 µg/mL), which is better than many previously reported compounds. The mechanism of action of Hppo is investigated by performing different biological studies and the results obtained reveal that Hppo acts through down-regulating mitochondrial membrane potential and up-regulating reactive oxygen species production. Molecular docking studies are also performed to obtain more insights on biological activities of Hppo and its mode of action against CYP-19 (PDB: 3EQM), JAK2 (PDB: 5AEP), BCL-2 (PDB: 2O2F), and caspase3 (PDB: 1RE1), and result displayed favorable binding interactions with binding energy -7.43, -7.96, -6.61, and -6.88 Kcal/mol.

Gamma irradiation promotes chemo-sensitization potential of gallic acid through attenuation of autophagic flux to trigger apoptosis in an NRF2 inactivation signalling pathway

Ionizing radiation has the potential to cause structural modification or change in electrochemical properties in parent lead pharmacophores that exhibit enhanced bioactivity. Gallic acid (GA), a triphenolic compound has displayed potent anticancer drug potency due to its withstanding antioxidant propensity. This study uncovered the comparative efficacy of gamma-irradiated gallic acid (GAIR) in the modulation of an antioxidant system for regulation apoptosis and autophagy. GAIR exhibited remarkable anti-proliferative efficacy as shown by MTT, clonogenic survival, and scratch assay. In addition to this, GAIR promoted intrinsic apoptosis through mitochondrial superoxide generation. GAIR decreased the activity of antioxidant enzymes by downregulating nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream effector molecules NAD(P)H Quinone Dehydrogenase 1 (NQO1) and gamma-glutamylcysteine synthetase (GCLC). Simultaneously, GAIR attenuated autophagosome-lysosome fusion without altering the lysosomal activity. Inhibition of autophagic flux resulted in the accumulation of lipid droplets (LDs) such as hexadecanoic acid and oleic acid that fuelled superoxide generation leading to apoptosis. In the meantime, under oxidative upset, conversion of LDs to free fatty acids reduced leading to inhibition of ATP generation that subsequently provoked apoptosis. The effects of autophagy inhibition by GAIR on the therapeutic efficacy of chemotherapeutic drugs was studied and the co-treatment markedly decreased the cell viability and increased apoptosis. Further, GAIR exhibited potent antitumor activity in Dalton's Lymphoma-tumor bearing mice through modulation of apoptosis and autophagy without toxic activity. In conclusion, change in electrochemical properties by gamma radiation enhances the anticancer efficacy of gallic acid through superoxide mediated apoptosis fuelled by inhibition of lipophagy in an NRF2 dependent signaling pathway.

Immune augmentation and Dalton's Lymphoma tumor inhibition by glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus.

With the increase in cancer progression, alternatives in the medicinal field with minimal side effects need to be ascertained. In this context, for the first time novel glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus have been compared for their exquisite property as immunoceuticals. Glucans from both the sources displayed immunological functions which include lymphocyte proliferation, macrophage activation (nitric oxide production, ROS generation, phagocytosis, TNF-α production) as well as macrophage and NK cell mediated cytotoxicity. In vivo studies with Dalton's Lymphoma mice tumor model further enumerated the immune enhancing and tumor regression potential of the two glucan molecules. Highest tumor inhibition of about 75% and 71.4% were observed at 20mg/kg of mycelia and fruit body glucan/glycan treatments. A concomitant increase in the survival period of glucan treated tumor bearing mice was found to be primarily associated with immune boosting and apoptosis of cancerous cells. Both the glucan molecules exhibited similar degree of immune response at the systemic level with only subtle amount of differences in two dimensional in vitro cultures. Efficacy of glucans/glycans as immunomodulators may thereby provide decisive leads in strengthening the immune system along with other therapies.

Biochemical changes associated with ascorbic acid–cisplatin combination therapeutic efficacy and protective effect on cisplatin-induced toxicity in tumor-bearing mice

Cisplatin is one of the well-established anticancer drugs being used against a wide spectrum of cancers. However, full therapeutic efficacy of the drug is limited due to development of various toxicities in the host. This study examines the comparative therapeutic effectiveness and toxicities of cisplatin alone and in combination of dietary ascorbic acid (AA) in ascites Dalton's lymphoma-bearing mice. The findings show that the combination treatment of mice with ascorbic acid plus cisplatin has much better therapeutic efficacy against murine ascites Dalton's lymphoma (DL) in comparison to cisplatin alone and this may involve a decrease in reduced glutathione (GSH), catalase activity and increased lipid peroxidation (LPO) in Dalton's lymphoma tumor cells. At the same time, combination treatment indicates a protective role of ascorbic acid against cisplatin-induced tissue toxicities (side effects) in the hosts. Cisplatin-induced histopathological changes in liver, kidney and testes were decreased after combination treatment. The analysis of renal function test (RFT), liver function test (LFT) and sperm abnormalities also suggest an improvement in these parameters after combination treatment. Therefore, it may be concluded that the increased GSH level, catalase activity and decreased LPO in the tissues, i.e., liver, kidney and testes after combination treatment may be involved in its protective ability against cisplatin-induced tissue toxicities in the host.

DETERMINE THE EFFICACY OF CARBOPLATIN WITH VITAMIN C IN DALTON'S LYMPHOMA TUMOR IN MICE MODEL

Carboplatin, a plat inum - containing anticancer drug treatment cau sed a significant increase in the life span of ascites Dalton’s lymphoma tumor bearing mice. However , as compared to carboplatin alone, combination treatment with vitamin C plus carboplatin resulted in better therapeutic efficacy against Dalton's lymphoma tumor. To investigate difference in resistance mechanism to d i a m i n e ( cyclobutane - 1,1 - di carboxylato (2 - ) - O,O’) platinum (CBDCA) and changes in antioxidant levels in liver tissues in relation to different stages of Dalton’s lymphoma tumor growth and the fol lowing carboplatin treatment. The reduction in intracellular accumulation of CBDCA is thought to be an important factor indicating that CBDCA resistant in DLA cell are mutually cross - resistant. Glutathione level increased in liver of tumor bearing compared to normal mice. I ntracellular accumulation of platinum found to be higher in tissue, after carboplatin treatment the platinum accumulation were significantly decre a sed . Antioxidant defense systems such as superoxide dismutase , glutathione peroxidase gluta thione reductase activities and Glutathione – S - transferase activity w ere determined in tissues of carboplatin treatment at different stages of Dalton’s lymphoma tumor gr owth . Resistance mechanism based on reduction of intracellular drug accumulations and i ncreased activity of GST also depend the level of antioxidant enzymes , GST activities are playing significant role in development of resistance in the presences of tumor under various newly developed platinum derivative factor. Anticancer drugs kill suscep tible cells through induction of apoptosis. Alteration of apoptotic pathways in drug –

Enhanced tumor uptake, biodistribution and pharmacokinetics of etoposide loaded nanoparticles in Dalton′s lymphoma tumor bearing mice

Background:Nanotechnology plays a remarkable role in the field of the treatment of Lymphomas associated with tumor.Objective:The purpose of this study is to determine and to compare the tumor uptake, biodistribution and pharmacokinetics of radiolabeled etoposide and etoposide loaded nanoparticles in Dalton's Lymphoma tumor bearing mice and healthy mice.Materials and Methods:Etoposide loaded nanoparticles were prepared by nanoprecipitation technique using the poly (lactic-co-glycolic) acid (PLGA) in the presence of Pluronic F 68 (F 68) as a stabilizer and characterized by particle size analyzer, zeta potential and transmission electron microscope. Etoposide and etoposide loaded nanoparticles were labeled with Technetium-99m (Tc-99m) by the direct method and various quality control tests were carried out. The labeling parameters like labeling efficiency, stability, etc., were optimized to get high labeling efficiency as well as stability of the labeled formulations. Tc-99m labeled formulations were administered intravenously in Balb C mice and their biodistribution and pharmacokinetics were determined.Results:Mean size of the etoposide loaded PLGA nanoparticles was found to be 105.1 nm. The concentration of both free etoposide and nanoparticles increased with time and showed higher tumor concentrations of both free etoposide and nanoparticles increased with time and showed higher retention, indicating their applicability in effective and prolonged tumor therapy. Nuclear scintigraphic images confirm the presence of labeled complexes at the site of tumor for 24 h at higher concentration than in the normal muscles.Conclusion:This study indicated higher tumor affinity and targeting properties of etoposide loaded nanoparticles than free etoposide.

Cyclophosphamide and ascorbic acid-mediated ultrastructural and biochemical changes in Dalton's lymphoma cells in vivo

Cyclophosphamide, an antineoplastic drug effective against a wide variety of cancers is cytotoxic to normal cells also. Ascorbic acid (vitamin C) at higher concentrations possesses cytotoxic effects and it can also enhance the cytotoxicity of 5-fluorouracil in a dose-dependent manner in mouse lymphoma. In the present study, effect of cyclophosphamide treatment alone and in combination with ascorbic acid in vivo on the ultrastructure and some biochemical changes in Dalton's lymphoma tumor cells were investigated . Cyclophosphamide treatment causes disappearance of cell membrane processes, thickening and reduction in the number of mitochondrial cristae as well as the manifestation of rounded shape of mitochondria. The combination treatment with ascorbic acid plus cyclophosphamide caused further changes in tumor cells showing disintegration in the cell surface membrane, disruption in the nuclear membrane and roundish mitochondria with reduction and disruption in the mitochondrial cristae. The observed ascorbic acid plus cyclophosphamide-mediated decrease in reduced glutathione (GSH) in tumor cells may play an important role in the antitumor activity of cyclophosphamide by weakening cellular antioxidant-mediated defense mechanism, thereby increasing tumor cell's susceptibility to cell death. The cyclophosphamide-mediated decrease in lactate dehydrogenase activity in tumor cells and simultaneous increase in ascites supernatant may possibly indicate alteration in the membrane permeability of tumor cells for lactate dehydrogenase as well as tumor cell injury. Further investigation should determine detailed mechanism(s) involved in cyclophosphamide-induced ultrastructural and biochemical changes in tumor cells.

Abrus abrin derived peptides induce apoptosis by targeting mitochondria in HeLa cells

In our previous study, Abrus abrin derived peptide fraction (ABP) with molecular weight in range of 600-1500Da was shown to have potent antitumor activity in Dalton's lymphoma (DL) tumor bearing mice. The purpose of this study was to elucidate the mechanism of mitochondrial apoptosis induced by the peptide fraction. ABP was found to have selective antiproliferative activity (10ng-100ng/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with a dose of 1000ng/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed DNA fragmentation with appearance of the sub G(0)/G(1) peak indicative of apoptosis. Further investigation results showed that the apoptotic machinery of HeLa induced by ABP was associated with the release of reactive oxygen species, a drop in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3. The peptide fraction was found to target mitochondria of HeLa cells as observed by confocal microscopy. This peptide fraction offers a source of mitochondria penetrating peptides which might have therapeutic induction of apoptosis in cancer cells.

Antitumor and proapoptotic effect of Abrus agglutinin derived peptide in Dalton's lymphoma tumor model

Abrus agglutinin peptide fractions obtained from 10 kD molecular weight cut off membrane permeate (10 kMP), was shown to have selective antiproliferative activity on several tumor cell lines with induction of apoptosis through mitochondrial pathway. The present study was designed to evaluate acute general toxicity and in vivo therapeutic effectiveness of 10 kMPP in Dalton's lymphoma (DL) mice model. The acute toxicity like body weight, peripheral blood cell count, lympho-hematological and biochemical parameters remained unaffected with 1 mg/kg body weight and lower of 10 kMPP. The in vivo antitumor study indicated that there were 27%, 58.5% and 84.5% reduction in DL cell survival in 100, 200 and 500 μg/kg body weight of 10 kMPP, respectively. Analysis of the growth inhibitory mechanism in DL cells revealed nuclear fragmentation and condensation with appearance of the sub G0/G1 peak is indicative of apoptosis. Further, the Western blotting showed apoptosis was mediated by reduction in ratio of Bcl-2 and Bax protein expression, and activation of caspase-3 through release of cytochrome-c in DL cells. Kaplan–Meier survival analysis showed an effective antitumor response (53 ILS%) with dose of 500 μg/kg body weight. Our result showed that the novel peptides present in Abrus agglutinin possess potent antitumor properties which need to be further explored.

Influence of administration route on tumor uptake and biodistribution of etoposide loaded solid lipid nanoparticles in Dalton's lymphoma tumor bearing mice

The study evaluates the capability of tripalmitin nanoparticles in enhancing the tumor uptake of etoposide, and the influence of administration route on the biodistribution and tumor uptake of etoposide loaded tripalmitin (ETPL) nanoparticles in Dalton's lymphoma tumor bearing mice. ETPL nanoparticles were prepared by melt-emulsification and high pressure homogenization followed by the spray drying of nanodispersion. Characterization of the nanoparticles was done by particle size analysis, zeta potential measurement and scanning electron microscopy. The size of ETPL nanoparticles was 387 nm and possessed negative charge. Etoposide and ETPL nanoparticles were radiolabeled with 99mTc with high labeling efficiency. The labeled complexes showed good in vitro stability in the presence of DTPA/cysteine and serum stability. Etoposide and ETPL nanoparticles were injected by subcutaneous, intravenous or intraperitoneal routes and their biodistribution and tumor uptake were determined. Subcutaneous injection reduced the distribution of ETPL nanoparticles to all the tissues studied whereas after intraperitoneal injection, the distribution of ETPL nanoparticles to tissues was higher than free etoposide. The intravenous injection resulted in lower concentrations of ETPL nanoparticles in the organs of RES compared to free etoposide. ETPL nanoparticles experienced significantly high brain distribution after intraperitoneal injection indicating its potential use in targeting etoposide to brain tumors. After subcutaneous injection, the tissue distribution of ETPL nanoparticles increased with time indicating their accumulation at the injection site for a longer time. The tumor uptake of both etoposide and ETPL nanoparticles was significantly high after subcutaneous injection ( P < 0.001) compared to the other routes of administration. The tumor concentration of ETPL nanoparticles after subcutaneous injection was 59 folds higher than that obtained after intravenous and 8 folds higher than after intraperitoneal route at 24 h post-injection. The tumor concentration of ETPL nanoparticles increased with time after subcutaneous injection indicating the slower and progressive penetration from the injection site into the tumor. The study signifies the advantage of incorporating etoposide into tripalmitin nanoparticles in controlling its biodistribution and enhancing the tumor uptake by several folds. The study also reveals that, of the three routes investigated, subcutaneous injection is the route of preference for facilitating high tumor uptake and retention and is likely to have greater antitumor effect resulting in tumor regression.

Synthesis, Structural, and Antitumor Studies of Some 5‐Fluorocytosine and Guanine Complexes

Mixed‐ligand complexes of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with 5‐fluorocytosine (5FC) and guanine (G) have been prepared in aqueous ethanol solution at pH of about 7. The prepared complexes were characterized by various physico‐chemical methods viz. elemental analyses, infrared, UV‐visible, magnetic measurements, and powder x‐ray pattern. On the basis of IR studies, it is suggested that G acts as bidentate ligand coordinating through the N3 and N7 nitrogens whereas 5FC behaves as a monodentate and coordinates to the metal ions through the carbonyl oxygen only. These complexes were subjected to a screening system of Dalton's lymphoma (DL) tumor cells both in vivo and in vitro. Some complexes exhibit significant antitumor activity both in vivo and in vitro.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin.

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 +/- 0.14 vs 7.86 +/- 1.64 micromol/g wet weight, P<or=0.05) but decreased in blood (0.64 +/- 0.10 vs 0.85 +/- 0.09 mg/ml) and testes (9.28 +/- 0.15 vs 10.16 +/- 0.28 micromol/g wet weight, P<or=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 +/- 0.26 micromol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 +/- 0.41 micromol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (approximately 10%) and bone marrow cells (approximately 13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 +/- 1.22, 4.43 +/- 0.26 and 3.28 +/- 0.17 micromol/g wet weight, respectively) decreased significantly (6.04 +/- 0.42, 3.51 +/- 0.32 and 2.17 +/- 0.14 micro mol/g wet weight, P<or=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

SOLID, SOLUTION, AND ANTITUMOR ACTIVITY STUDIES OF MIXED-LIGAND COMPLEXES WITH ADENINE-5-BROMOURACIL BASE PAIR

ABSTRACT Mixed-ligand complexes of Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) with adenine as primary ligand and 5-bromouracil as secondary ligand were prepared in aqueous ethanol solution at pH of about 7 and subjected to a screening system of Dalton's Lymphoma (DL) tumor cells in vitro for the evaluation of viability, 3H-thymidine incorporation and induction of apoptosis in tumor cells. The isolated complexes were characterized by various physico-chemical methods. Solution studies were performed pH-metrically for the interaction of the above metal ions with adenine and 5-bromouracil separately (binary) and in the presence of each other (ternary) at 25±0.1 °C temperature and a constant ionic strength of 0.1 M NaNO3 in aqueous solution.

Purification, characterization and antitumor activity of L-asparaginase isolated from Pseudomonas stutzeri MB-405.

An L-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein-1 with an overall recovery of 27.2%. The apparent M(r) of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38 +/- 0.02. It displayed optimum activity at pH 9.0 and 37 degrees C. The enzyme was very specific for L-asparagine and did not hydrolyze L-glutaminate. The Km of the L-asparaginase was found to be 1.45 x 10(-4) M towards L-asparagine and was competitively inhibited by 5-diazo-4-oxo-L- norvaline (DONV) with a Ki of 0.03 mM. Metal ions such as Mn2+, Zn2+, Hg2+, Fe3+, Ni2+, and Cd2+ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.

Induction of immune rejection of tumors by protein A in mice bearing transplantable solid tissue Dalton's lymphoma tumors.

In a transplantable solid tissue Dalton's lymphoma tumor model in mice we have studied the mode of antitumor action of protein A, a well known biological response modifier. Protein A (15 ug) was administered intravenously in normal and solid tissue Dalton's lymphoma tumor bearing mice on day 3 and 7 after tumor inoculation. Incidence of mortality was more in untreated tumor bearing group than that in PA treated tumor bearers. There was a significant decrease (p less than 0.001) in tumor diameter in PA treated group compared to untreated group. Protein A treatment significantly enhanced the delayed type hypersensitivity (p less than 0.01), T-cell number in spleens (p less than 0.001) and lymph nodes (p less than 0.05) as well as phagocytosis (p less than 0.001) of opsonized SRBC by peritoneal macrophages of tumor bearing animals. Apart from the nonspecific immunopotentiation, Protein A also activates natural Killer (NK) cell activity and also splenic lymphocytes mediated killing of autologous tumor targets in a significant (p less than 0.001) manner. These results suggest that PA treatment activates cellular arc of the immune system in general, and macrophage, T cells and NK cells specifically. In the present communication, we have attempted to provide the information that these immune activations appear to be related to antitumor response induced by Protein A.