Interspecific protoplast fusion of atmospheric and room-temperature plasma mutants of Aspergillus generates an L-asparaginase hyper-producing hybrid with techno-economic benefits.

The axenic culture of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase while Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Upon mutagenesis by atmospheric and room-temperature plasma (ARTP), their individual L-asparaginase activities improved 2.3-folds in each of Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E stable mutants. Protoplast fusion of selected stable mutants generated fusant-09 with improved anti-leukemic activity, acrylamide reduction, higher temperature optimum and superior kinetic parameters. Submerged (SmF) and solid-state fermentation (SSF) types were compared; likewise batch, fed-batch and continuous fermentation modes; and fed-batch submerged fermentation was selected on the basis of impressive techno-economics. Fusant L-asparaginase was purified by PEG/Na+ citrate aqueous two-phase system and molecular exclusion chromatography to 69.96 and 146.21-fold, respectively, and characterized by molecular weight, specificity, activity and stability to chemical and physical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 1.667 × 10-3 M, 1666.67 µmol min-1 mg-1 protein, 645.99 s-1 and 3.88 × 105 M-1 s-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants Ile-Thr-Asp-C-180-K and Val-Asp-S-180-E, and from 5.73 and 32.55 from respective original strains. Free-radical scavenging and acrylamide reduction improvements were intermediate. Fusant-09 L-asparaginase is strongly recommended for sustainable economic anti-leukemic and food industry applications.

Radotinib enhances cytarabine (Ara-C)-induced acute myeloid leukemia cell death.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease that frequently relapses after standard chemotherapy. Therefore, there is a need for the development of novel chemotherapeutic agents that could treat AML effectively. Radotinib, an oral BCR-ABL tyrosine kinase inhibitor, was developed as a drug for the treatment of chronic myeloid leukemia. Previously, we reported that radotinib exerts increased cytotoxic effects towards AML cells. However, little is known about the effects of combining radotinib with Ara-C, a conventional chemotherapeutic agent for AML, with respect to cell death in AML cells. Therefore, we investigated combination effects of radotinib and Ara-C on AML in this study. METHODS: Synergistic anti-cancer effects of radotinib and Ara-C in AML cells including HL60, HEL92.1.7, THP-1 and bone marrow cells from AML patients have been examined. Diverse cell biological assays such as cell viability assay, Annexin V-positive cells, caspase-3 activity, cell cycle distribution, and related signaling pathway have been performed. RESULTS: The combination of radotinib and Ara-C was found to induce AML cell apoptosis, which involved the mitochondrial pathway. In brief, combined radotinib and Ara-C significantly induced Annexin V-positive cells, cytosolic cytochrome C, and the pro-apoptotic protein Bax in AML cells including HL60, HEL92.1.7, and THP-1. In addition, mitochondrial membrane potential and Bcl-xl protein were markedly decreased by radotinib and Ara-C. Moreover, this combination induced caspase-3 activity. Cleaved caspase-3, 7, and 9 levels were also increased by combined radotinib and Ara-C. Additionally, radotinib and Ara-C co-treatment induced G0/G1 arrest via the induction of CDKIs such as p21 and p27 and the inhibition of CDK2 and cyclin E. Thus, radotinib/Ara-C induces mitochondrial-dependent apoptosis and G0/G1 arrest via the regulation of the CDKI-CDK-cyclin cascade in AML cells. In addition, our results showed that combined treatment with radotinib and Ara-C inhibits AML cell growth, including tumor volumes and weights in vivo. Also, the combination of radotinib and Ara-C can sensitize cells to chemotherapeutic agents such as daunorubicin or idarubicin in AML cells. CONCLUSIONS: Therefore, our results can be concluded that radotinib in combination with Ara-C possesses a strong anti-AML activity.

Synthesis, Crystallography, and Anti-Leukemic Activity of the Amino Adducts of Dehydroleucodine.

Dehydroleucodine is a bioactive sesquiterpene lactone. Herein, four dehydroleucodine amino derivatives were synthesized using the amines proline, piperidine, morpholine, and tyramine, and spectroscopic methods and single-crystal X-ray diffraction unambiguously established their structures. The cytotoxic activity of these compounds was evaluated against eight acute myeloid leukemia cell lines, and their toxicity to peripheral blood mononuclear cells was also determined. The proline adduct was the most active compound, it showed anti-leukemic activity, upregulated heme oxygenase 1 (HMOX1) and the primary stress-inducible isoform of the heath shock 70 kDa protein 1 (HSPA1A), and downregulated NFkB1 transcription, it was also found to be about 270 times more water soluble than dehydroleucodine.

Antileukemic Activity of Twig Components of Caucasian Beech in Turkey.

Despite the development of a range of anti-cancer agents, cancer diagnoses are still increasing in number, remaining a leading cause of death. Anticancer drug treatment is particularly important for leukemia. We screened Turkish plants and found the unique antileukemic activity of twig components in Turkish Caucasian beech, selectively inducing apoptosis in leukemia cells. This effect is unique among some kinds of beeches, presumably related to oxidative stress. This study would lead to effective use of discarded material, i.e., twig of beech, and a new anti-leukemic drug based on large tree.

Ceramide as a Target of Marine Triterpene Glycosides for Treatment of Human Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous myeloid clonal disorder exhibiting the accumulation of immature myeloid progenitors in the bone marrow and peripheral blood. Standard AML therapy requires intensive combination chemotherapy, which leads to significant treatment-related toxicity. The search for new, low toxic marine agents, inducing the generation of ceramide in leukemic cells is a new approach to improve the therapy of leukemia. This review focuses on the metabolism of sphingolipids, the role of ceramide in treating leukemia, and the antitumor activity, related to ceramide metabolism, of some marine metabolites, particularly stichoposides, triterpene glycosides extracted from sea cucumbers of the family Stichopodiidae.

Synthesis, kinase inhibition and anti-leukemic activities of diversely substituted indolopyrazolocarbazoles.

Pyrazole analogues of the staurosporine aglycone K252c, in which the lactam ring was replaced by a pyrazole moiety, were synthesized. In this series, one or the other nitrogen atoms of the indolocarbazole scaffold was substituted by aminoalkyl chains, aiming at improving protein kinase inhibition as well as cellular potency toward acute myeloid leukemia (AML) cell lines. Compound 19a, substituted at the N12-position by a 3-(methylamino)propyl group, showed high cellular activity in the low micromolar range toward three AML cell lines (MOLM-13, OCI-AML3 and MV4-11) with selectivity over non-cancerous cells (NRK, H9c2). 19a is also a highly potent inhibitor of the three Pim kinase isoforms, Pim-3 being the most inhibited with an IC50 value in the nanomolar range. A selectivity screening toward a panel of 50 protein kinases showed that 19a also potently inhibited PRK2 and to a lower extent AMPK, MARK3, GSK3ß and JAK3. Our results enhance the understanding of the structural characteristics of indolopyrazolocarbazoles essential for potent protein kinase inhibition with therapeutic potential against AML.

Synthesis, DNA binding and anti-leukemic activity of an aminoacyl nucleolipid.

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53-78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, Tm measurements and dynamic light scattering experiments. Moreover, in a single dose (10 µM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.

Production, characterization and techno-economic evaluation of Aspergillus fusant L-asparaginase.

Protoplast fusion is one of the most reliable methods of introducing desirable traits into industrially-promising fungal strains. It harnesses the entire genomic repertoire of fusing microorganisms by routing the natural barrier and genetic incompatibility between them. In the present study, the axenic culture of a thermo-halotolerant strain of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase (L-ASNase) while a xylan-degrading strain of Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Protoplast fusion of the wild strains generated Fusant-06 with improved anti-leukemic and acrylamide reduction potentials. Submerged fed-batch fermentation was preferred to batch and continuous modes on the basis of impressive techno-economics. Fusant-06 L-ASNase was purified by PEG/Na+ citrate aqueous two-phase system (ATPS) to 146.21-fold and global sensitivity analysis report revealed polymer molecular weight and citrate concentration as major determinants of yield and purification factor, respectively. The enzyme was characterized by molecular weight, amino acid profile, activity and stability to chemical agents. Michaelis-Menten kinetics, evaluated under optimum conditions gave Km, Vmax, Kcat, and Kcat/Km as 6.67 × 10-5 M, 1666.67 µmolmin-1 mg-1 protein, 3.88 × 104 min-1 and 5.81 × 108 M-1.min-1 respectively. In-vitro cytotoxicity of HL-60 cell lines by Fusant-06 L-ASNase improved significantly from their respective wild strains. Stability of Fusant-06 L-ASNase over a wide range of pH, temperature and NaCl concentration, coupled with its micromolar Km value, confers commercial and therapeutic value on the product. Free-radical scavenging and acrylamide reduction activities were intermediate and the conferred thermo-halo-stability could be exploited for sustainable clinical and food industry applications.

Bioisosteric replacement of 1H-1,2,3-triazole with 1H-tetrazole ring enhances anti-leukemic activity of (5-benzylthiazol-2-yl)benzamides.

Previously, we discovered that N-(5-benzyl-1,3-thiazol-2-yl)-4-(5-methyl-1H-1,2,3-triazol-1-yl)benzamide possessed a remarkable cytotoxic effect on 28 cancer cell lines with IC50 < 50 µM, including 9 cancer cell lines, where IC50 was in the range of 2.02-4.70 µM. In the present study, we designed a novel N-(5-benzylthiazol-2-yl)amide compound 3d that was synthesized using the original bioisosteric replacement of 1H-1,2,3-triazole ring by the 1H-tetrazole ring. A significantly enhanced anticancer activity in vitro with an excellent anti-leukemic potency towards chronic myeloid leukemia cells of the K-562 line was demonstrated. Two compounds - 3d and 3l - were highly cytotoxic at nanomolar concentrations towards various tumor cells of the following lines: K-562, NCI-H460, HCT-15, KM12, SW-620, LOX IMVI, M14, UACC-62, CAKI-1, and T47D. As a highlight, the compound N-(5-(4-fluorobenzyl)thiazol-2-yl)-4-(1H-tetrazol-1-yl)benzamide 3d inhibited the growth of leukemia K-562 cells and melanoma UACC-62 cells with IС50 of 56.4 and 56.9 nM (SRB test), respectively. The viability of leukemia K-562 and pseudo-normal HaCaT, NIH-3T3, and J774.2 cells was measured by the MTT assay. Together with SAR analysis, it allowed the selection of a lead compound 3d, which demonstrated the highest selectivity (SI = 101.0) towards treated leukemic cells. The compound 3d caused DNA damage (single-strand breaks detected by the alkaline comet assay) in the leukemic K-562 cells. The morphological study of the K-562 cells treated with compound 3d revealed changes consistent with apoptosis. Thus, the bioisosteric replacement in (5-benzylthiazol-2-yl)amide scaffold proved to be a perspective approach in the design of novel heterocyclic compounds with enhanced anticancer potential.

Rhein augments ATRA-induced differentiation of acute promyelocytic leukemia cells.

BACKGROUND: Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid), a natural anthraquinone derivative, is a traditional Chinese herb that has been used as a medication in many Asian countries. It has been used as a laxative and stomach drug for a long time in both China and Korea. It is well-known to have many pharmacological activities, such as anti-cancer, anti-bacterial, anti-fungal, anti-oxidant, anti-atherogenic, anti-angiogenic, anti-fibrosis, anti-inflammatory, hepatoprotective, and nephroprotective properties. However, little is known about how rhein may affect the differentiation activities in acute promyelocytic leukemia (APL) cells. PURPOSE: The present study was designed to examine the anti-leukemic effects of rhein against APL cells and to explore the underlying mechanism. METHODS: Cell viability was investigated by MTS assay. To examine the differentiation activities in APL cells, the cell surface molecules (CD11b, CD14, CCR1 and CCR2), phagocytosis, reactive oxygen species (ROS) were determined by flow cytometry. Also, induction of caspase-3 activity and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. RNA and protein expressions were determined by qRT-PCR and western blotting, respectively. RESULTS: In this study we assessed the role of rhein in treating APL. Interestingly, rhein potentiated all-trans retinoic acid (ATRA)-induced macrophage differentiation in NB4 cells by inducing changes in morphology, expression of the differentiation markers CD11b and CD14, ROS production, phagocytic activity, and expression of CCR1 and CCR2. Signaling through CD11b was found to be dependent on ERK activation. Additionally, rhein induced APL cell death by activating apoptosis and suppressing the mTOR pathway. CONCLUSION: Therefore, we suggest that a combination of rhein and ATRA carries strong therapeutic potential through the beneficial differentiation of APL cells. Moreover, rhein causes cell death via the activation of apoptosis and suppression of survival signals in APL cells. In combination with the ability of rhein to promote functional macrophage differentiation in APL, these properties suggest that a combined treatment of rhein and ATRA has great potential as an anti-leukemic therapy for APL.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.

Large-scale biotechnological production of the antileukemic marine natural product sorbicillactone A.

In the search for novel bioactive compounds from sponge-derived microorganisms, we have recently identified two structurally and biosynthetically unprecedented fungal metabolites, the novel-type alkaloids sorbicillactone A and sorbicillactone B. Sorbicillactone A is active against leukemia cells without showing notable cytotoxicity. Therefore, we have developed an efficient process for its biotechnological production and isolation on a large scale supplying sufficient material for the ongoing preclinical investigations and structure-activity relationship (SAR) studies.

Plant phenyl-propanoids-conjugated silver nanoparticles from edible plant Suaeda maritima (L.) dumort. Inhibit proliferation of K562-human myeloid leukemia cells.

The present study portrays the isolation of four phenylpropanoids - ferulic acid (FA), sinapic acid (SA), caffeic acid (CA), and chlorogenic acid (CHA) from the water extract of Suaeda maritima (L.) Dumort, a phytochemically less explored Indian medicinal plant. Further, synthesis and characterization of silver and gold nanoparticles using the isolated phenylpropanoids were done. The silver nanoparticles synthesized from S. maritima water extract along with silver nano-conjugated forms of the isolated compounds exhibited appreciable anti-leukemic activity against K562 cells (human myeloid leukemia). Especially, the ferulic and CA-conjugated silver nanoparticles showed significant (P < .01) activity against leukemia.

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML.

Radotinib inhibits acute myeloid leukemia cell proliferation via induction of mitochondrial-dependent apoptosis and CDK inhibitors.

Radotinib is a BCR-ABL1 tyrosine kinase inhibitor approved for the second-line treatment of chronic myeloid leukemia. However, effects of radotinib on acute myeloid leukemia (AML) are unclear. In the present study, we observed that radotinib exerted cytotoxic effects on AML cells. Of the various AML cell lines examined (NB4, HL60, HEL 92.1.7, and THP-1), Kasumi-1 was the most sensitive to radotinib. Results of microarray analysis showed that 417 and 595 genes associated with apoptosis and cell cycle regulation, respectively, were differently expressed (i.e., showed >2-fold difference in expression). Radotinib-induced apoptosis involved the mitochondrial pathway. Moreover, radotinib increased the apoptosis of and induced caspase-3 activity in both Kasumi-1 cells and bone marrow cells (BMCs) obtained from patients with AML. Radotinib also increased cleaved caspase-3, caspase-7, and caspase-9 levels and decreased the number of proliferating Kasumi-1 cells and BMCs from patients with AML. In addition, radotinib induced G0/G1 phase arrest by inducing CDKIs p21 and p27 and by inhibiting CDK2, CDK4, and CDK6. These results indicate that radotinib induces caspase-dependent apoptosis and G0/G1 phase arrest in AML cells by regulating CDKI-CDK-cyclin cascade. Moreover, these results indicate that radotinib inhibits AML cell proliferation by inducing mitochondria-dependent apoptosis and CDKIs p21 and p27. To our knowledge, this is the first study to show that radotinib can be potentially used for the anti-leukemic therapy of patients with AML.

Rosmarinic acid potentiates ATRA-induced macrophage differentiation in acute promyelocytic leukemia NB4 cells.

Rosmarinic acid (RA, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid) has a number of biological activities, but little is known about anti-leukemic activities of RA combined with all-trans retinoic acid (ATRA) against acute promyelocytic leukemia (APL) cells. We examined the differentiation marker, CD11b, in bone marrow cells (BMC) of an APL patient, in NB4 cells (APL cell line), and in normal BMC and peripheral blood mononuclear cells (PBMC) of healthy subjects by flow cytometric analysis. ATRA/RA induced expression of CD11b in the BMC of the APL patient and in NB4 cells, but not in normal BMC or PBMC. Therefore, we realized that RA potentiated ATRA-induced macrophage differentiation in APL cells. Further characterization of the induced macrophages showed that they exhibited morphological changes and were able to phagocytose and generate reactive oxygen species. Th also had typical expression of C-C chemokine receptor type 1 (CCR1), CCR2, and intercellular adhesion molecule-1 (ICAM-1). Moreover, the expression of CD11b(+) and CD14(+) cells depended on ERK-NF-κB axis activation. Together, these results indicate that RA potentiates ATRA-induced macrophage differentiation in APL cells. Thus, RA may play an important role as an appurtenant differentiation agent for functional macrophage differentiation in APL. Additionally, the differentiated macrophages might have a normal life span and, they could die. These data indicate that co-treatment with RA and ATRA has potential as an anti-leukemic therapy in APL.

Cytotoxic and apoptogenic effect of Swietenia mahagoni (L.) Jacq. leaf extract in human leukemic cell lines U937, K562 and HL-60.

The apoptogenic activity of Swietenia mahagoni leaf extract (SMLE) was investigated against three human leukemic cell lines - U937, K562 and HL-60. SMLE inhibited cell growth and metabolic activity of the leukemic cells and showed characteristic features of apoptosis. Flow-cytometric analysis showed that SMLE arrested U937 and K562 cell populations in the G2-M phase and the HL-60 cell population in the G1 phase of cell cycle. SMLE induced apoptosis was found to be mediated through mitochondrial intrinsic pathway involving the release of cytochrome c into the cytosol and activation of caspase-9 and caspase-3. Two flavonoids, catechin and quercetin-3-O-glucoside, isolated from SMLE, were found to inhibit the growth and metabolic activity of U937, K562 and HL-60 cells at much lower concentrations thus indicating that these two flavonoids might be the active ingredients responsible for the anti-leukemic activity of SMLE.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.