Rat Leukemia Research Articles

Pharmacology of a CpG-Linked Anti-Mir-126 Oligonucleotide Targeting Acute Myeloid Leukemia (AML) in Rats and Non-Human Primates (NHP)

Introduction: Acute myeloid leukemia (AML) is a lethal hematopoietic malignancy, characterized by the accumulation of clonal myeloid progenitor cells arrested in development. AML patients have dismal outcomes with a 5-year overall survival (2011 - 2017) of 29.5% (SEER). The persistence of leukemia stem cells (LSCs), a treatment-resistant primitive cell population able to inititiate and maintain AML, is thought to underlie relapse. Even newly approved drugs do not target LSCs efficiently. MicroRNA (miR)-126 is a non-coding regulatory RNA that contributes to LSC homeostasis in the bone marrow niche. Several lines of evidence validate miR-126 as a target in AML. Thus, we designed and made a novel anti-miR-126 oligonucleotide (oligo) linked to a CpG moiety, termed miRisten. miRisten incorporates modifications such as 2'-fluoro and phosphorothioate substituting phosphodiester in the sugar moiety of the nucleotide to enhance in vivo stability. miRisten eradicates LSCs in mouse models of AML and synergizes with FDA-approved AML drugs (PMID: 29505034, 34372909). Here, the results of IND-enabling toxicokinetic (TK) studies of miRisten in rats and NHP are presented. Objective: To investigate the pharmacological properties of miRisten to establish a safe dosing strategy to achieve prolonged knockdown of miR-126 for a first-in-human trial. Experimental Plan and Results: Good laboratory practice (GLP) TK studies were carried out in Sprague-Dawley rats. miRisten was given IV b.i.d. for 19 days at human equivalent doses (HED) of 1.6 and 4 mg/kg. miRisten was measured in plasma after the first dose. Plasma miRisten C max average was 3622 ± 335 and 6010 ± 175 nM post first doses of 1.6 and 4mg/kg, respectively. Plasma t 1/2 averaged 20.0 ± 3.8 minutes across both doses. The area under the curve (AUC 0 - ¥) were 638.9 ± 32 nMxhr (1.6 mg/kg) and 1313.5 ± 439 (4 mg/kg). A parallel toxicology study was undertaken documenting cage-side observations and body weight; complement activation, blood chemistries, urine and hematology were also evaluated. There were no unscheduled deaths in the toxicology segment. Four single NHP exploratory studies were also carried out (Fig. 1: Treatment schema). In each study, miRisten was given IV as a slow bolus (~5') b.i.d. and PK parameters evaluated in plasma. Study #1 & #2: 1 wk at 4mg/kg; #3: 1 wk at 5 mg/kg; #4: 18 days at 4 mg/kg. In all studies, miRisten dosage was ramped up (1, 2, 4 mg/kg for all and 5 mg/kg at dose 4 for Study #3). Solumedrol was given as premedication for the first 3 doses in all studies to prevent known phosphorothioate-mediated complement activation and cytokine release syndrome. Blood draws for PK were carried out immediately following doses 3 and 12 (Studies 1 - 3) and after the last dose on Day 18 (Study 4). The average area under the curve AUC was 3860 ± 1189 (4 mg/kg) and 3954 ± 1569 nMxhr (5 mg/kg) which was greater than observed in mice given the equivalent HED in bolus doses (~6x, 465.0). The t 1/2 of plasma miRisten was 11.7 min on Day 3 and 12.0 min on Day 7, comparable to mouse (9.6 min) and rat (20 min). Cage-side observations, body weight, complement activation, blood chemistries, urine and hematology were also evaluated. There was no terminal sacrifice of NHP however, all animals tolerated the treatment without observable adverse events. Conclusion: The dose ramp-up in NHP combined with pre-medication with solumedrol were undertaken to prevent possible acute inflammatory reactivity leading potentially to death. There were no unscheduled deaths in any species studied. These data are consistent with the safety of miRisten given IV with a dose ramping regimen combined with solumedrol. The greater AUC in NHP may predict better potency in humans. In mice, miRisten at 1.6 mg/kg HED q.d. effectively eradicated LSCs [PMID 29505034].

The potential protective and therapeutic effects of cannabidiol oil on experimental Leukemia induced by DMBA in male rats.

7,12-Dimethylbenzanthracene (DMBA) is a member of the polycyclic aromatic hydrocarbon family. Itis a member of the polycyclic aromatic hydrocarbon family. It is a mutagenic, carcinogenic, and immunosuppressor agent. Cannabidiol (CBD) is a phytocannabinoid. It has anticonvulsant, anti-inflammatory, anti-anxiety, antioxidant, and anti-cancer properties.The purpose of this study was to investigate the possible protective and therapeutic benefits of CBD oil in DMBA-induced leukemia in rats. Experimental animals were divided into six groups of five rats each. Group 1 (normal control) included healthy rats. Group 2 included normal rats thatreceived olive oil. Group 3 included normal rats that received CBD. Group 4 included theDMBA-induced leukemic group. Group 5 (prophylactic group) included rats thatreceived CBD as a prophylaxis before IV injection with DMBA. Group 6 (treated group) included DMBA-induced leukemic rats that received CBD as treatment. Liver functions (total, direct and indirect bilirubin, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), albumin, globulin, and albumin globulin ratio) were measured. Superoxide dismutase (SOD) and catalase (CAT) were also measured. Total RNA extraction followed by-real time qRT-PCR gene expression of LC3-II, Beclin, mTOR, and P62 was performed. Histopathological examination of liver and spleentissues was performed. Administration of CBD in groups 5 and 6 resulted in a significant improvement ofthe levels of liver functions compared to theleukemic untreated rats. Also, the levels of catalase and SOD significantly increased after treatment with CBD compared to the leukemic group. After treatment with CBDin groups 5 and 6, there were downregulations in the expression of all studied genescompared to leukemic untreated rats. Treatment with CBD was more statistically effective than prophylactic use. Administration of CBD resulted in a significant improvement in the biochemical, antioxidant status, morphological, and molecular measures in DMBA-induced leukemia in adult male rats. The therapeutic use was more effective than the prophylactic one.

Possible Protective Effect of Spanish Black Radish on Hematological Parameters Aganist Benzene - Induced Leukemia in Rats

Background: The Spanish black radish, which is grown all over the world and is full of useful nutrients, helps in the therapy of many illnesses. Objective: The aim of this study was to evaluate the possible protective effect of Spanish black radish extracts against benzene-induced leukemia in rats. Materials and Methods: A total of 35 rats were purchased from the animal house at the College of Pharmacy, University of Karbala, Iraq. Benzene was used for the induction of leukemia in experimental animals. An extract of black radish was set after mixing up 50 g of the finely powdered plant material with two solvents (500 mL of 100% ethanol or water). After leukemia induction, rats were randomly divided into seven groups: first group, negative control group; second group, positive control group; third group, (lack radish aqueous extract 100 mg/kg; fourth group, black radish aqueous extract 200 mg/kg; fifth group, black radish ethanol extract 100 mg/kg; sixth group, black radish ethanol extract 200 mg/kg; and seventh group, cyclophosphamide 5 mg/kg. Results: Leukemia induction resulted in a severe increase in the total leukocyte count and a severe decrease in red blood cells, hemoglobin, and platelet levels of the experimental animals. However, there were no significant differences between the negatively treated group and the benzene-treated group regarding platelet count. Posttreatment with Spanish black radish extracts, especially the aqueous plant extract, shows an improvement in blood profiles compared to the benzene-treated group. Conclusion: Our study indicates that Spanish black radish might be a promising natural, antileukemic source.

The Therapeutic and Synergistic Effect of Saussurea costus Extract against Induced Leukemia in Adult Male Rats

The Therapeutic and Synergistic Effect of Saussurea costus Extract against Induced Leukemia in Adult Male Rats

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Phytochemical Screening and In vivo Antileukemic Activity of Methanolic Extract of Seeds of Nigella Sativa L. on Benzene-Induced Leukemia in Wistar Rats

Nigella sativa is exquisite bounty of nature to humankind. It is an annual flowering plant of family Ranunculaceae majorly found in Southwest Asia, seeds are used as flavor food. The present work was directed to evaluate antileukemic effect of N. sativa seeds extract in benzene-induced leukemia in female Wistar rats. Seed powder was extracted using petroleum ether, chloroform, ethyl acetate, and methanol. OECD guidelines 423 were used to study acute oral toxicity study and intravenous injection of benzene was given on every 2nd day up to 21 days to induce leukemia. To assess burden of leukemia, various parameters analyzed are body weight (B.W), organ weight, complete blood picture, serum potassium, phosphate, uric acid, and glutathione. The presence of alkaloids, carbohydrates, cardiac glycosides, tannins, phytosterols, and flavonoids is confirmed by phytochemical screening. Acute oral toxicity study found 2000 mg/kg as a safe dose of N. sativa seed extract. Significant decrease in potassium, phosphate, uric acid, WBC, and increase in B.W, glutathione demonstrated therapeutic efficacy of extract in leukemia. Our results confirmed significant antileukemic effect of N. sativa seed extract, suggesting role of this herb for the treatment of malignancies such as leukemia.

Detection of high antibodies titers against rat leukemia virus in an outbreak of reproductive disorders and lymphomas in Wistar rats.

Young female Wistar rats from a specific pathogen free breeding colony presented an outbreak of infertility along with neurological symptoms and malignant lymphomas. We evaluated the presence and the potential role of the rat leukemia virus (RaLV) in the disease because these clinical signs could be compatible with a retrovirus. RaLV is a mammalian type C endogenous retrovirus initially isolated from in vitro Sprague-Dawley rat embryo cultures. There are no reports of clinical disease in rats associated with this virus, and little is known about its interaction with the host. Using reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, we studied the synthesis of the viral particles and the development of an immune response against the virus in this rat colony. The results showed that healthy and diseased Wistar rats synthetized viral RNA but only diseased animals developed a detectable immune response against RaLV envelop protein. Furthermore, rats with lymphomas tended to have higher titers of antibodies against RaLV epitopes than those with infertility or neurological symptoms. The results suggest that increases in the RaLV infectious particle loads could be involved in the development of lymphomas in young rats. The potential causes of RaLV reactivation are discussed.

Chemoprotection by Kolaviron of Garcinia kola in Benzene-induced leukemogenesis in Wistar rats

ABSTRACT Kolaviron (KV) is a biflavonoid phytoconstituent of defatted Garcinia kola seeds that possessed antiproliferative and pharmacological activities. Benzene is an industrial solvent that, however, constitutes occupation hazard, leading to hematological disturbance and leukemia. Therefore, the potency of kolaviron against benzene-induced hematological and myeloid toxicity leading to leukemia was investigated in a rat model. Preleukemic conditions were induced in Wistar rats by intravenous administration of benzene solution. Following induction, 200 mg/kg kolaviron was administered orally for seven days. Hematological parameters, percentage blast cell occurrence and blood cell morphology were compared between baseline control and leukemic rats with or without kolaviron treatment. Plasma activity of arylesterase of paraoxonase-1, total thiol and advanced oxidation protein products (AOPPs) along with clastogenicity and bone marrow architecture was assessed. Kolaviron restored altered hematology, reduced the occurrence of blasts and improved blood cell morphology. Kolaviron also decreased levels of AOPPs, increased total thiol, improved arylesterase activity and mitigated clastogenicity and dysplasia induced in leukemic rats. In conclusion, kolaviron protected against benzene-induced hematological and myeloid toxicities that are implicated in leukemia.

Oral Intragastric DMBA Administration Induces Acute Lymphocytic Leukemia and Other Tumors in Male Wistar Rats.

BackgroundAnimal models of blood cancer are important tools to study these malignancies and also screen for novel therapeutic agents. Evidence from past research on the carcinogenic properties of 7,12-dimethylbenz[a]-anthracene (DMBA) was provided by a handful of studies. However, recent literature on DMBA carcinogenic activity and the underlying mechanisms is scarce.ObjectiveThe aim of this study was to develop a chemical model of leukemia using DMBA. Male Wistar rats (6 weeks old) were administered 1.5 mg of DMBA dissolved in sesame oil in biweekly doses using oral intragastric intubation.Materials and MethodsFrequent complete blood counts and blood smear morphology assessment were used to assess the development of leukemia, while gross pathology and histopathology staining were used to evaluate malignancy development.ResultsThe results showed that only 4% of rats developed acute lymphocytic leukemia. Interestingly, 36% of the rats developed tumors (parotid tumors [24%] and fibrosarcomas [12%]).ConclusionThese results suggest the pleiotropic potential of DMBA in the induction of multiple types of malignancies, including leukemia. This could be used as a model to validate therapeutic targets for leukemia and other induced malignancies.

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ∼37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.

Anti-leukemogenic Efficacy of Aloe vera Gel on Benzene-induced Leukemia in Wistar Rats

Backgroud: The toxicity of benzene leading to leukemia induction has been well documented in animal model. Aloe vera is a succulent perennial evergreen flowering plant used traditionally in the treatment of jaundice and was found to have potent cytotoxic effect against HL60 human acute myeloid leukemia. The present study investigated the in vivo chemoprotective effects of Aloe vera gel on benzene-induced leukemia in rats.
 Methodology: Leukemia was induced in male Wistar rats of 80-90g weight by intravenously administered 0.2ml benzene solution alternate days for four weeks. Following induction, leukemic rats and normal baseline control rats were randomly assigned into four experimental groups of 6 animals each as follows: Group CTRL (control), normal baseline control rats; Group AVG (Aloe vera gel), normal baseline rats treated with Aloe vera gel (150 mg/kg) for 7 days, Group LKR (leukemic rats), untreated leukemic rats serving as leukemia control and Group LKR + AVG, leukemic rats treated with Aloe vera gel (150 mg/kg) for 7 days.
 Results: Leukemic rats showed altered hematology and morphological deformations such as anisocytosis, poikilocytosis and blast cells occurrence in peripheral blood. Also hypercellularity, severe dysplasia and significantly elevated micronucleated polychromatic erythrocyte were observed in marrow of leukemic rats. Moreover, benzene caused a significant elevation in plasma level of advanced oxidation protein products (AOPPs) with concomitant reduction in total sulfhydryl and arylesterase activity. However, treatment with Aloe vera gel restored blood hematology to near normal and mitigated the deformities in blood cell morphology induced by benzene. Aloe vera supplementation also effected a disappearance of dysplasia and diminution in the frequency of micronucleus in the bone marrow of treated leukemic rats. It also enhanced plasma antioxidant capability by restoring sulfhydryl content and arylesterase activity of the blood and abrogated the increase in plasma content of AOPPs.
 Conclusion: Overall, Aloe vera gel offered chemoprotective effect on Benzene-induced leukemia in rats.

RETRACTED ARTICLE: Chemoprotective Effect of Daphnetin Against Benzene-induced Leukemia via Alteration of CYP2E1.

Leukemia is the overproduction of a large number of immature blood cells entering into the peripheral blood due to the malignancy of blood-forming tissues. The modern treatment mechanisms that are associated with leukemia comprise chemotherapy, allogeneic cell transplantation, and radiation therapy that comes with adverse side effects. Thus, medicinal herbs and their bioactive compounds are gaining more attention nowadays toward the treatment of leukemia, owing to their minimal side effects. The current study was aimed to scrutinize the antileukemic effect of daphnetin against benzene-induced leukemia in rats and explore the underlying mechanism of its action. Benzene was used for the induction of leukemia in experimental rats. The rats were divided into different groups and body weight; hematological parameters, DNA fragmentation, and cell cycle regulatory parameter were also estimated. RT-PCR was used for the estimation of mRNA expression of cytochrome P450 2E1 (CYP2E1), a membrane protein widely found in liver cells. Daphnetin-treated rats showed upregulation of body weight as compared to other groups. Moreover, daphnetin reduced the blasts (67.8%) in leukemic rats. It also altered the hematological parameters such as RBC (69.8%), WBC (54.5%), lymphocytes (47.6%), neutrophils (48.9%), monocytes (44.7%), eosinophils (48.7%), and basophils (43.5%), respectively. Daphnetin-treated rats showed an increased level of proteins p21 and p53 and reduced level of cyclins D1 and E. RT-PCR showed an upregulated mRNA expression of CYP2E1 (48.4%) of the daphnetin-treated group as compared to other groups. The current study shows the antileukemic effect of daphnetin and highlights the possibility of its use in leukemia to minimize the side effect of the usual therapy.

Therapeutic Efficacy of Mcl-1 Antagonism in Large Granular Lymphocyte Leukemia

Introduction: Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes. Immunosuppressive regimens using methotrexate or cyclophosphamide are utilized as front-line treatments in LGL leukemia but are not curative nor are they effective in all patients. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs.We have previously reported that STAT3 is constitutively active in LGL leukemia cells and up to 40% of patients harbor activating mutations in STAT3 within the leukemic clone. We have also reported that the pro-survival Bcl-2-family protein Mcl-1 is upregulated in LGL leukemia patients. Additionally, we have identified MCL1 as a transcriptional target of STAT3 in leukemic LGLs. Recently, novel small molecule inhibitors of Mcl-1 have been identified including marinopyrrole A (maritoclax) and its derivative KS18. These inhibitors effectively induced apoptosis in Mcl-1-dependent cells and suppressed the growth of Mcl-1-dependent hematologic cancer xenografts in nude mice without apparent toxicity. Thus, this study further investigates the role of Mcl-1 in LGL leukemia and the potential therapeutic application of the Mcl-1 antagonists maritoclax and KS18.Results: Knockdown of Mcl-1 in human-derived LGL leukemia cell lines (TL1 and NKL) resulted in decreased cell viability by MTS assay and increased apoptosis by flow cytometry in Mcl-1 siRNA versus scramble siRNA-transfected cells (NKL and TL-1 cells: p < 0.001). Treatment with maritoclax or KS18 decreased cell proliferation and increased apoptosis in NKL, TL1 and RNK16 (rat-derived LGL leukemia cell line) cells (Maritoclax, EC50: 2.45, 1.43 and 1.23 μM respectively) (KS18, EC50: 1.30, 0.60 and 0.31 μM respectively). Ex vivo treatment with maritoclax and KS18 induced selective apoptosis in PBMCs from LGL leukemia patients (NK-LGL, n = 6; T-LGL, n = 12) but not normal donor PBMCs (n = 4) (Maritoclax and KS18, p < 0.0001). Importantly, there was no significant difference in treatment efficacy in patient cells with wild-type STAT3 (n = 9) versus mutant STAT3 (Y640F, n = 5; D661Y, n = 3). We then proceeded to mechanistic studies of Mcl-1 antagonism. Both maritoclax and KS18 induced selective and dose-dependent Mcl-1 degradation by western blot in NKL, TL1, RNK16 and PBMCs from LGL leukemia patients. We further delineated that this effect was proteasome dependent as it is reversible by pre-treatment with MG132, a proteasome inhibitor.Preclinical in vivo studies were then conducted in Fisher F344 rats that were transplanted with primary leukemic LGL cells and monitored for signs of leukemia through serial CBC and body weight measurements. Leukemic rats developed progressive anemia, thrombocytopenia and leukocytosis and significant weight loss. Rats were treated with vehicle or KS18 (2.5 or 5.0 mg/kg i.p. daily) upon detection of leukemic features. In non-leukemic KS18-treated rats, there were no observed toxicities and there was no difference in survival or weight gain versus vehicle-treated non-leukemic rats. However, leukemic KS18-treated rats exhibited findings consistent with tumor lysis syndrome, including sudden death associated with severely increased serum LDH and increased apoptosis in heart and spleen tissues. At necropsy, leuk/KS18 rats exhibited significantly decreased infiltration and elevated apoptosis of leukemic cells in heart and spleen tissues. Conversely, in leuk/veh rats, spleen tissue lacked normal histologic structure with red pulp replaced almost completely by leukemia cells. Ex vivo KS18 treatment also decreased the viability of PBMC and spleen cells from leukemic animals (p < 0.0001). These data indicate that Mcl-1 is a promising therapeutic target as evidenced by potent therapeutic efficacy of KS18 in LGL cell lines and primary patient material and in an aggressive rat model of LGL leukemia. Further studies are required in order to optimize drug dose, timing and concentrations of KS18 in order to prevent potential tumor lysis syndrome and optimize therapeutic benefit. DisclosuresGru:Seattle Genetics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Allium sativum aqueous extract does not have chemo-protective effect on etoposide induced therapy-related DNA damage leading to Acute Myeloid Leukemia in albino-wistar rats.

Therapy-related acute myeloid leukemia (t-AML) is a well-recognized clinical syndrome occurring in a significant fraction of patients who have undergone previous chemotherapy for a solid tumour. We aim to evaluate the effect of aqueous extract of fresh Allium sativum cloves on haematological parameters, bone marrow and DNA of etoposide treated albino wistar rats. Decoction method was used to prepare plant extracts and the rats were weighed and divided into experimental and control groups. Blood and bone marrow sample were analysed and DNA fragment analysis was carried out. There was progressive increase in the weight of animals that received distilled water only for the duration of the experiment while those that received etoposide only showed a sharp decrease in weight by the end of week 3. There was no significant difference in the mean of the haematological parameters in the test and control groups except for platelet count. The bone marrow smears showed no prevention of erythroblast fragmentation by the extract, in the same vein, DNA damage was not abated. Aqueous extract of fresh Allium sativum cloves may not be the option for the prevention of etoposide induced acute myeloid leukemia.

Daphnetin exerts an anticancer effect by attenuating the pro-inflammatory cytokines.

Leukemia is a malignant tissue-forming disease, which induces the overproduction of large numbers of immature blood cells entering the peripheral blood. It is well documented that inflammation plays a crucial role in the expansion of leukemia. Daphnetin has confirmedanti-inflammatory effects against various diseases. In this experimental study, we evaluated the anti-leukemia and anti-inflammatory effect of daphnetin against benzene-induced leukemia in rats and exploredthe underlying mechanism. Benzene was used for inducing leukemia in experimental rats. The rats were divided into different groups and the body weight, hematological parameters, bone marrow cells, cytokines, and inflammatory mediators were estimated. Reverse transcription polymerase chain reaction (RT-PCR)was used for estimating the messenger RNA (mRNA) expression of sphingosine-1-phosphate receptor-1. Daphnetin-treated rats showed upregulationof body weight compared to other groups. Moreover, Daphnetin reduced blasts in leukemic rats. It also altered hematological parameters such as red blood cells, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, monocytes, and basophils, respectively. Daphnetin-treated rats showed a reduction of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β (IL-1β), IL-2, IL-6, and inflammatory mediators includingnuclear factor-κB. RT-PCR showed upregulated mRNA expression of sphingosine-1-phosphate receptor-1 of daphnetin-treated group rats compared to other groups. The current study showed that the anti-inflammatory effect of daphnetin against the benzene-induced leukemia via alteration of cytokines.

Preclinical anticancer studies on the ethyl acetate leaf extracts of Datura stramonium and Datura inoxia

BackgroundCancer is a horrific disease relentlessly affecting human population round the globe. Genus Datura encompasses numerous species with reported medicinal uses. However, its potential as a source of natural anticancer agents is yet to be determined. Datura stramonium (DS) and Datura inoxia (DI) are the two species chosen for this study.MethodsTotal phenolic and flavonoid content (TPC and TFC) as well as antioxidant activity were assessed through colorimetric method. Polyphenolic quantification was done by RP-HPLC. Following extract standardization ethyl acetate leaf extracts of both species (DSL-EA and DIL-EA) were chosen for anticancer studies. In vitro cytotoxicity using various models including cancer cell lines was monitored. Following toxicity studies, benzene (0.2 ml) was used to induce leukemia in Sprague-Dawley rats. Extracts were orally administered to preventive (100 and 200 mg/kg) and treatment (200 mg/kg only) groups. The antileukemic potential of extracts was assessed through haematological, biochemical, endogenous antioxidants and histological parameters.ResultsSignificant TPC and TFC were estimated in DSL-EA and DIL-EA. RP-HPLC quantified (μg/mg extract) rutin (0.89 ± 0.03), gallic acid (0.35 ± 0.07), catechin (0.24 ± 0.02) and apigenin (0.29 ± 0.09) in DSL-EA while rutin (0.036 ± 0.004) and caffeic acid (0.27 ± 0.03) in DIL-EA. Both extracts exhibited significant brine shrimp cytotoxicity (LC50 < 12.5 μg/ml). DIL-EA exhibited greater cytotoxicity against PC-3, MDA-MB 231 and MCF-7 cell lines (IC50 < 3 μg/ml in each case) as well as higher protein kinase inhibitory action (MIC: 25 μg/disc) compared to DSL-EA. Leukemia induced in rats was affirmed by elevated serum levels of WBCs (7.78 ± 0.012 (× 103) /μl), bilirubin (7.56 ± 0.97 mg/dl), Thiobarbituric acid reactive substances (TBARs) (133.75 ± 2.61 nM/min/mg protein), decreased RBCs (4.33 ± 0.065 (× 106)/μl), platelets (344 ± 3.19 (× 103)/μl), total proteins (2.14 ± 0.11 g/dl), Glutathione S-transferases (GST) (81.01 ± 0.44 nM/min/ml), endogenous antioxidant enzymes levels and abnormal liver and kidney functionality in disease control rats. Both species revealed almost identical and significant (p < 0.05) alleviative effects in benzene induced leukemia.ConclusionComprehensive screening divulged the tremendous potential of selected species as potent source of natural anticancer agents in a variety of cancers particularly leukemia. Present study might provide useful finger prints in cancer research and mechanistic studies are prerequisite in logical hunt of this goal.

Whole Genome Sequencing of Spontaneously Occurring Rat Natural Killer Large Granular Lymphocyte Leukemia Identifies JAK1 Somatic Activating Mutation.

Large granular lymphocyte (LGL) leukemia arises spontaneously in elderly Fischer (F344) rats. This rodent model has been shown to emulate many aspects of the natural killer (NK) variant of human LGL leukemia. Previous transplantation of leukemic material into young F344 rats resulted in several strains of rat NK (RNK) primary leukemic cells. One strain, RNK-16, was adapted into the RNK-16 cell line and established as an aggressive NK-LGL leukemia model. Whole genome sequencing of the RNK-16 cell line identified 255,838 locations where the RNK16 had an alternate allele that was different from F344, including a mutation in Jak1. Functional studies showed Jak1 Y1034C to be a somatic activating mutation that mediated increased STAT signaling, as assessed by phosphoprotein levels. Sanger sequencing of Jak1 in RNK-1, -3, -7, and -16 found only RNK-16 to harbor the Y1034C Jak1 mutation. In vivo studies revealed that rats engrafted with RNK-16 primary material developed leukemia more rapidly than those engrafted with RNK-1, -3, and -7. Additionally, ex vivo RNK-16 spleen cells from leukemic rats exhibited increased STAT1, STAT3, and STAT5 phosphorylation compared to other RNK strains. Therefore, we report and characterize a novel gain-of-function Jak1 mutation in a spontaneous LGL leukemia model that results in increased downstream STAT signaling.

Modulation of phospho-proteins by interferon-alpha and valproic acid in acute myeloid leukemia

PurposeValproic acid (VPA) is suggested to be therapeutically beneficial in combination with interferon-alpha (IFNα) in various cancers. Therefore, we examined IFNα and VPA alone and in combinations in selected AML models, examining immune regulators and intracellular signaling mechanisms involved in phospho-proteomics.MethodsThe anti-leukemic effects of IFNα and VPA were examined in vitro and in vivo. We mapped the in vitro phosphoprotein modulation by IFNα-2b and human IFNα-Le in MOLM-13 cells by IMAC/2D DIGE/MS analysis and phospho-flow cytometry, and in primary healthy and AML patient-derived PBMCs by CyTOF. In vivo, IFNα-Le and VPA efficacy were investigated in the immunodeficient NOD/Scid IL2γ−/− MOLM-13Luc+ mouse model and the syngeneic immunocompetent BNML rat model.ResultsIFNα-2b and IFNα-Le differed in the modulation of phospho-proteins involved in protein folding, cell stress, cell death and p-STAT6 Y641, whereas VPA and IFNα-Le shared signaling pathways involving phosphorylation of Akt (T308), ERK1/2 (T202/T204), p38 (T180/Y182), and p53 (S15). Both IFNα compounds induced apoptosis synergistically with VPA in vitro. However, in vivo, VPA monotherapy increased survival, but no benefit was observed by IFNα-Le treatment. CyTOF analysis of primary human PBMCs indicated that lack of immune-cell activation could be a reason for the absence of response to IFNα in the animal models investigated.ConclusionsIFNα-2b and IFNα-Le showed potent and synergistic anti-leukemic effects with VPA in vitro but not in leukemic mouse and rat models in vivo. The absence of IFNα immune activation in lymphocyte subsets may potentially explain the limited in vivo anti-leukemic effect of IFNα-monotherapy in AML.

N-Butylpyridoquinoxaline 1, 4-dioxide alleviates experimentally induced leukemia with up-regulating sphingosine-1-phosphate receptors

Synthesis of Methyl 2-Cinnamamido-3-Hydroxy Propanoate Having Activity against P388 Leukemia CellsTeni Ernawati, Yulia Anita, Puspa Dewi Lotulung and Muhammad Hanafi

Anti-leukemic activity of a four-plant mixture in a leukemic rat model

BackgroundLeukemia is a malignant blood disease caused by the overproduction of a large number of immature blood cells that enter the peripheral blood. Because of the side effects associating the chemotherapy of leukemia, the identification of medicinal herbs, therefore, remains to be an attractive goal to treat leukemia.ResultsIn this study, leukemia was experimentally induced in rats by 7, 12-dimethyl benza[a]anthracene (DMBA) and rats were treated with a water extract of a four-plant (Arctium lappa, Ulmus rubra, Rumex acetosella, and Rheum palmatum) mixture. Application of this four-plant mixture extract successfully recovered weight loss and restored the normal total WBC, lymphocyte and neutrophil counts in a leukemia rat model compared to either the DMSO-treated rats or the leukemic rats before applying the plant mixture. Moreover, this plant mixture decreased the percentage of blasts by two thirds in leukemic rats. By quantitative real-time PCR, sphingosine-1-phosphate receptor-1 mRNA expression in lymphocytes was downregulated in leukemic rats, and this downregulation was significantly alleviated by treating the leukemic rats with the plant mixture.ConclusionThis study investigates, for the first time, the effect of this plant mixture on a chemically induced leukemia rat model. Results further support previous reports about the anti-carcinogenic effect of this plant mixture and highlights the possibility of its use in leukemia treatment to avoid the negative side effects of the usual therapy.