Novel Gemini vitamin D3 analogs: large structure/function analysis and ability to induce antimicrobial peptide.

We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.

Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF).

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7-30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Ki11502, a novel multitargeted receptor tyrosine kinase inhibitor, induces growth arrest and apoptosis of human leukemia cells in vitro and in vivo.

Ki11502 is a novel multitargeted receptor tyrosine kinase (RTK) inhibitor with selectivity against platelet-derived growth factor receptor alpha/beta (PDGFRalpha/beta). Ki11502 (0.1-1 nM, 2 days) profoundly caused growth arrest, G(0)/G(1) cell-cycle arrest, and apoptosis associated with down-regulation of Bcl-2 family proteins in the eosinophilic leukemia EOL-1 cells having the activated FIP1-like 1/PDGFRalpha fusion gene. Ki11502 decreased levels of p-PDGFRalpha and its downstream signals, including p-Akt, p-ERK, and p-STAT5, in EOL-1 cells. Of note, Ki11502 was also active against imatinib-resistant PDGFRalphaT674I mutant. In addition, Ki11502 inhibited proliferation of biphenotipic leukemia MV4-11 and acute myelogenous leukemia MOLM13 and freshly isolated leukemia cells having activating mutations in FMS-like tyrosine kinase 3 (FLT3). This occurred in parallel with the drug inhibiting FLT3 and its downstream signal pathways, as measured by fluorescence-activated cell sorting using the phospho-specific antibodies. In addition, Ki11502 totally inhibited proliferation of EOL-1 cells growing as tumor xenografts in SCID mice without any noticeable adverse effects. Taken together, Ki11502 has profound antiproliferative effects on select subsets of leukemia including those possessing imatinib-resistant mutation.

Histone deacetylase inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of signaling by nuclear factor kappaB.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease with a poor prognosis in which nuclear factor kappa B (NF-kappaB) is thought to play a role. This study explored the effects of histone deacetylase inhibitors (HDACIs) MS-275, suberoylanilide hydroxamic acid (SAHA), and LBH589 on both human T-cell lymphotropic virus type I (HTLV-1)-infected T cells (MT-1, -2, -4, and HUT102) and freshly isolated ATL cells harvested from patients. HDACIs effectively inhibited the proliferation of these cells. For example, MS-275, SAHA, and LBH589 effectively inhibited the proliferation of MT-1 cells with ED(50s) of 6microM, 2.5microM, and 100nM, respectively, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay on day 2 of culture. In addition, HDACIs induced cell cycle arrest at the G2/M phase and apoptosis of HTLV-1-infected T-cells in conjunction with regulation of apoptosis-related proteins. Electrophoretic mobility shift assay showed that exposure of HTLV-1-infected T-cells to HDACIs for 48h inhibited formation of the NF-kappaB/DNA binding complex. Moreover, we found that HDACIs accumulated NF-kappaB and inhibitory subunit of NF-kappaB in the cytoplasm in conjunction with the down-regulation of NF-kappaB in the nucleus, suggesting that HDACIs blocked nuclear translocation of NF-kappaB. Based on these findings, we believe HDACIs can be useful for treating patients with ATL or other types of cancer in which NF-kappaB plays a role.

A novel treatment strategy targeting Aurora kinases in acute myelogenous leukemia.

The Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. Aberrant expression of these kinases occurs in solid tumors and is associated with aneuploidy and carcinogenesis. We found in this study that Aurora kinase A and B were aberrantly expressed in a variety of types of human leukemia cell lines (n = 15, e.g., PALL-1, PALL-2, HL-60, NB4, MV4-11, etc.), as well as freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n = 44) compared with bone marrow mononuclear cells from healthy volunteers (n = 11), as measured by real-time PCR. ZM447439 is a novel selective Aurora kinase inhibitor. The compound induced growth inhibition, caused accumulation of cells with 4N/8N DNA content, and mediated apoptosis of human leukemia cells as measured by thymidine uptake, cell cycle analysis, and annexin V staining, respectively. Especially profound growth inhibition occurred with the PALL-1 and PALL-2 cells, which possess wild-type p53 gene. In contrast, ZM447439 did not inhibit clonogenic growth of myeloid committed stem cells harvested from healthy normal volunteers. Taken together, inhibition of Aurora kinases may be a promising treatment strategy for individuals with leukemia.

Idiopathic hypereosinophilic syndrome presenting acute abdomen.

We describe a 27-year-old man with hypereosinophilc syndrome (HES) presenting acute abdomen due to acute thrombosis of the mesenteric artery, who had a past history of eosinophilic pneumonia followed by multiple arterial thromboses of the extremities. At the recurrence of eosinophilia, he was treated with high-dose corticosteroids. Immediately after the reduction of peripheral blood eosinophils, he suddenly developed perforation of the intestine due to acute thromboses of mesenteric arteries despite sustained anticoagulation therapy. Molecular analysis demonstrated that the FIP1L1-PDGFRA fusion gene was negative. Histopathology showed thrombi and eosinophilic inflammation of arteries. It is important to recognize that HES could be a cause of acute abdomen.

AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo.

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.

Longitudinal inhibition of PI3K/Akt/mTOR signaling by LY294002 and rapamycin induces growth arrest of adult T-cell leukemia cells.

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.

[Systemic capillary leak syndrome presenting remarkable erythrocytosis].

Systemic capillary leak syndrome (SCLS) is a disorder characterized by hypotension, edema, and an increased hematocrit (Ht) due to sudden leakage of plasma into the extravascular space through some unknown mechanism, in which monoclonal gammopathy is observed. A 30-year-old man visited our emergency department because of abdominal pain, and was admitted to our hematology department because of a markedly increased hemoglobin concentration reaching 26.2 g/dl. The polycythemia was thought to be pseudo-polycythemia due to hemoconcentration, and we diagnosed the patient as having SCLS based on the triad of increased hematocrit, whole-body edema which was especially marked in the lower extremities, and monoclonal gammopathy. The patient recovered after administration of extracellular fluids and albumin, but the attacks recurred. Prophylaxis with terbutaline sulfate, theophylline and corticosteroid reduced the frequency of severe attacks. Because there is possibility that patients with SCLS may be admitted to hematology departments due to severe erythrocytosis, we report this case to increase the awareness of hematologists that SCLS is one of the important differential diagnoses of erythrocytosis.