Invivo and invitro evaluation of antitumor effects of iron oxide and folate core shell-iron oxide nanoparticles

Nanoparticles are considered viable options in the treatment of cancer. This study was conducted to investigate the effect of magnetite nanoparticles (MNPs) and magnetite folate core shell (MFCS) on leukemic and hepatocarcinoma cell cultures as well as their effect on the animal model of acute myelocytic leukemia (AML). Through current study nanoparticles were synthesized, characterized by various techniques, and their properties were studied to confirm their nanostructure. Invivo study, nanoparticles were evaluated to inspect their cytotoxic activity against SNU-182 (human hepatocellular carcinoma), K562 (human leukemia), and THLE2 (human normal epithelial liver) cells via MTT test. Apoptotic signaling proteins Bcl-2 and Caspase-3 expression were inspected through RT-PCR method. A cytotoxic effect of MNPs and MFCS was detected in previous cell cultures. Moreover, the apoptosis was identified through significant up-regulation of caspase-3, with Bcl-2 down-regulation. Invitro study, AML was induced in rats by N-methyl-N-nitrosourea followed by oral treatment with MNPS and MFCS. Biochemical indices such as aspartate and alanine amino transferases, and lactate dehydrogenase activities, uric acid, complete blood count, and Beta -2-microglubulin were assessed in serum. Immunophenotyping for CD34 and CD38 detection was performed. Liver, kidney, and bone marrow were microscopically examined. Bcl-2 promoter methylation, and mRNA levels were examined. Although, both MNPs and MFCS depict amelioration in biochemical parameters, MFCS alleviated them toward normal control. Anticancer activity of MNPs and MFCS was approved especially for AML. Whenever, administration of MFCS was more effective than MNPs. The present work is one of few studies used MFCS as anticancer agent.

Polymorphism of VDR Gene and the Sensitivity of Human Leukemia and Lymphoma Cells to Active Forms of Vitamin D

The active forms of vitamin D3 (calcitriol and tacalcitol) coupled to the vitamin D receptor (VDR) are known to exhibit anti-cancer properties. However, not all cancer cells are sensitive to the active forms of vitamin D3 and its analogs. The study aimed to determine whether polymorphism of VDR is responsible for the sensitivity of human leukemia and lymphoma cells to calcitriol and tacalcitol. The impact of calcitriol and tacalcitol on the proliferation and morphology of nine different leukemia and lymphoma cell lines was determined. Only MV-4-11, Thp-1, and HL-60 cell lines sensitive to proliferation inhibition by calcitriol and tacalcitol showed morphology changes. Subsequently, the levels of the VDR and 1,25D3-MARRS proteins of calcitriol and tacalcitol binding receptors and the VDR receptor polymorphism in human leukemia and lymphoma cells were ascertained. Contrary to the current understanding, higher levels of VDR are not responsible for the greater sensitivity of cells to calcitriol and tacalcitol. Importantly, we first showed that sensitivity to calcitriol and tacalcitol in leukemias and lymphomas could be determined by the VDR polymorphism. The FokI polymorphism and the presence of the “bat” haplotype were observed only in the sensitive cells.

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.

Caralluma tuberculata N.E.Br Manifests Extraction Medium Reliant Disparity in Phytochemical and Pharmacological Analysis

Solubility of phytoconstituents depends on the polarity of the extraction medium used, which might result in the different pharmacological responses of extracts. In line with this, ethnomedicinally important food plant (i.e., Caralluma tuberculata extracts) have been made in fourteen distinct solvent systems that were then analyzed phytochemically via total phenolic amount estimation, total flavonoid amount estimation, and HPLC detection and quantification of the selected polyphenols. Test extracts were then subjected to a battery of in vitro assays i.e., antioxidants (DDPH scavenging, antioxidant capacity, and reducing power estimation), antimicrobial (antibacterial, antifungal, and antileishmanial), cytotoxic (brine shrimps, THP-1 human leukemia cell lines and normal lymphocytes), and protein kinase inhibition assays. Maximum phenolic and flavonoid contents were computed in distilled water–acetone and acetone extracts (i.e., 16 ± 1 μg/mg extract and 8 ± 0.4/mg extract, respectively). HPLC-DAD quantified rutin (0.58 µg/mg extract) and gallic acid (0.4 µg/mg extract) in methanol–ethyl acetate and methanol extracts, respectively. Water–acetone extract exhibited the highest DPPH scavenging of 36 ± 1%. Total reducing potential of 76.0 ± 1 μg/mg extract was shown by ethanol chloroform while maximum total antioxidant capacity was depicted by the acetone extract (92.21 ± 0.70 μg/mg extract). Maximal antifungal effect against Mucor spp., antileishmanial, brine shrimp cytotoxicity, THP-1 cell line cytotoxicity, and protein kinase inhibitory activities were shown by ethyl acetate-methanol (MIC: 50 µg/disc), n-hexane (IC50: 120.8 ± 3.7 µg/mL), ethyl acetate (LD50: 29.94 ± 1.6 µg/mL), distilled water–acetone (IC50: 118 ± 3.4 µg/mL) and methanol–chloroform (ZOI: 19 ± 1 mm) extracts, respectively. Our findings show the dependency of phytochemicals and bioactivities on the polarity of the extraction solvent and our preliminary screening suggests the C. tuberculata extract formulations to be tested and used in different ailments, however, detailed studies remain necessary for corroboration with our results.

Combination of cytostatic chemotherapy with cisplatin and photodynamic therapy with Radachlorin reduced resistance of K562 and Hela cell lines

In this work were study combination effect of photodynamic therapy and cisplatin on the proliferation activity of K562 human leukemia cells and Hela cervical carcinoma cells. A decrease in cell viability and an increase the fraction of apoptotic cells for combination treatment compared with single therapy were observed. It has been shown that the G2/M-phase of cell cycle decreases compared with cisplatin treatment alone, which demonstrates an increase anti-proliferative effect. The combination index of the photodynamic therapy with Radachlorin and cisplatin was calculated and indicates a synergistic effect.

Enhancement of Anti-Proliferative Activity of the Extracts from Dehulled Adlay by Fermentation with Bacillus subtilis

Dehulled adlay was fermented with Bacillus subtilis BJ3-2, the anti-proliferative activities of the extracts from fermented dehulled adlay were investigated with six types of tumor cells, and then the bioactive components and the anti-proliferative mechanism were primarily explored. Results showed that all the extracts of B. subtilis-fermented dehulled adlay (BDA) and dehulled adlay (DA) had no inhibition effect on human embryonic kidney 239T cells. The anti-proliferative activities of the extracts from BDA against six types of tumor cells were almost always significantly higher than DA. Compared with others, the n-butanol extract of BDA (BDA-Nb) exhibited stronger anti-proliferative activities against human leukemia K562 cells and human non-small cell lung cancer A549 cells. Importantly, the anti-proliferative activity of fermented dehulled adlay against K562 cells was firstly discovered. Meanwhile, BDA-Nb was rich in tetramethylpyrazine, γ-aminobutyric acid, protocatechuic, 2,3,4-trihydroxybenzoic, chlorogenic, p-hydroxybenzoic, caffeic, trans-cinnamic, ferulic acids, and rutin. BDA-Nb induced the proliferative inhibition of K562 and A549 cells due to abnormal cell morphology, the increased cell population in G1 phase and apoptosis rate, the downregulation of Bcl-2, and the upregulation of Bax and caspase-3/8/9. These results indicate that dehulled adlay fermented with B. subtilis could be a potential therapeutic agent for leukemia and lung cancer.

Camel Milk Exosomes Potentiate The Anticancer Effect of Doxorubicin on Multidrug-Resistant Human Leukemia Hl60 Cells in Vitro and in Vivo

Background: Multidrug resistance (MDR) is one of the strategies developed by cancer cells to inhibit the anticancer potential of the majority of chemotherapeutic agents and almost results in treatment failure. Objective: This study aimed to evaluate the therapeutic potential of camel milk exosomes (CME) on multidrug-resistant human acute promyelocytic leukemia HL60 cells (HL60/RS) and to investigate whether this CME could potentiate the anticancer effect of Doxorubicin (DOX) and decrease its side effects. Results: CME alone or combined with DOX significantly induced HL60/RS cell viability loss, apoptosis, and cell cycle arrest at the G0/G1 phase, and downregulated MDR genes (Abcb1, Abcc1, Abcg2) as compared to cells treated with DOX alone. Additionally, CME and DOX co-treated nude mice had the lowest tumor volume, Abcb1, Abcc1, Abcg2, and Bcl2 expression, and the highest Bax and caspase3 expression in HL60/RS xenografts. This combined therapy also decreased DOX adverse effects as revealed by decreased liver damage enzymes and lipid peroxide (MDA) and increased hepatic antioxidant enzymes (SOD, CAT, GPx). Conclusion: CME increased sensitivity of HL60/RS to DOX through, at least in part, reduction of MDR genes, induction of apoptosis, and cell cycle arrest. Thus, CME may be used as safe adjuvants to DOX during cancer treatment. Keywords: Camel milk exosomes; Myeloid leukemia; HL60; Apoptosis; MDR

N-acetylcysteine Can Induce Massive Oxidative Stress, Resulting in Cell Death with Apoptotic Features in Human Leukemia Cells

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•−). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO− from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•− in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation. Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 > 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker. The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group. Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. Disclosures Eguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.