Physiologic IGFBP7 levels prolong IGF1R activation in acute lymphoblastic leukemia.

Insulin and insulin-like growth factors (IGFs) are mitogenic and prosurvival factors to many different cell types, including acute lymphoblastic leukemia (ALL). Circulating IGFs are bound by IGF binding proteins (IGFBPs) that regulate their action. IGFBP7 is an IGFBP-related protein (IGFBP-rP) that in contrast to other IGFBPs/IGFBP-rPs features higher affinity for insulin than IGFs and was shown to bind the IGF1 receptor (IGF1R) as well. The role of IGFBP7 in cancer is controversial: on some tumors, it functions as an oncogene, whereas in others, it functions as a tumor suppressor. In childhood ALL, higher IGFBP7 expression levels were associated with worse prognosis. Here we show that IGFBP7 exerts mitogenic and prosurvival autocrine effects on ALL cells that were dependent on insulin/IGF. IGFBP7 knockdown or antibody-mediated neutralization resulted in significant attenuation of ALL cell viability in vitro and leukemia progression in vivo. IGFBP7 was shown to prolong the surface retention of the IGF1R under insulin/IGF1 stimulation, resulting in sustained IGF1R, insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) phosphorylation. Conversely, the insulin receptor was readily internalized and dephosphorylated on insulin stimulation, despite IGFBP7 addition. The affinity of homodimeric IGF1R for insulin is reportedly >100 times lower than for IGF1. In the presence of IGFBP7, however, 25 ng/mL insulin resulted in IGF1R activation levels equivalent to that of 5 ng/mL IGF1. In conclusion, IGFBP7 plays an oncogenic role in ALL by promoting the perdurance of IGF1R at the cell surface, prolonging insulin/IGF stimulation. Preclinical data demonstrate that IGFBP7 is a valid target for antibody-based therapeutic interventions in ALL.

Antiproliferative and toxicological properties of drimanes obtained from Drimys brasiliensis stem barks.

Stem barks of Drimys brasiliensis (Winteraceae) are consumed by the population in the form of a condiment. It is widely used to treat gastric and stomach problems and also to treat cancer. The extracts have demonstrated antiproliferative, antileishmanial and antimicrobial activities assigned to drimane sesquiterpenes. This study aimed to optimize the extraction conditions of the drimanes sesquiterpenes identified as 1-ß-(p-coumaroyloxy)-polygodial 1, drimanial 2 and 1-ß-(p-methoxycinnamoyl)-polygodial 3 in stem bark extracts. The HPLC-DAD method was developed and validated for the quantification of drimanes 1-3. The cytotoxic activity of these drimanes in human cancer cells, and the toxicological effects of the hydroethanolic extract, were determined. The extracts were prepared using different extractive conditions (solvents, plant: solvent ratio and time). The cytotoxicity effect was evaluated against leukemia, lymphomas, carcinomas and sarcomas cells using the tetrazolium assay (MTT). Furthermore, the acute toxicity was determined by measuring the biochemical parameters and by histopathological analysis. The hemolytic activity and micronucleus test were also performed. The method was linear, sensitive, precise and accurate for both drimanes 1-3. The best condition for extraction was using dichloromethane with plant: solvent proportion 1:10 (w/v) for six hours under dynamic maceration. Isolated drimanes exhibited cytotoxic effects with IC50 values ​​ranging from 0.13 to 112.67 µM. Compound 1 demonstrated significant results for acute promyelocytic leukemia (NB4) and Burkitt's lymphoma (RAMOS) cells while driamane 3 for Burkitt's lymphoma (RAJI) and acute T cell leukemia (MOLT4) cells. No signs of toxicity was observed and neither was mutagenicity or hemolytic activity.

Antitumor activities of Quercetin and Green Tea in xenografts of human leukemia HL60 cells.

Quercetin is one of the most abundant flavonoids, present in fruits and vegetables and has been shown to have multiple properties capable of reducing cell growth in cancer cells. Green tea is a widely consumed beverage, known for a potential source of free radical scavenging and anti-cancer activities. Herein, we investigate the in vivo antitumor efficacy of quercetin and green tea in human leukemia. Human tumors were xenografted into NOD/SCID mice. Quercetin and green tea reduced tumor growth in HL-60 xenografts accompanied by decreased expression of anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1 and increased expression of BAX, a pro-apoptotic protein. Moreover, caspase-3 was activated to a greater extent after quercetin and green tea treatment. Quercetin and green tea also mediated G1 phase cell cycle arrest in HL-60 xenografts. Treatment with quercetin and green tea induced conversion of LC3-I to LC3-II as well as activation of autophagy proteins, suggesting that quercetin and green tea initiate the autophagic progression. We have provided evidence that quercetin and green tea induces signaling at the level of apoptosis, cell cycle and autophagy which converge to antigrowth effects in HL-60 xenograft mice suggesting that these compounds may be a compelling ally in cancer treatment.

Correction: Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice.

[This corrects the article DOI: 10.1371/journal.pone.0129298.].

Plasma Hsp90 Level as a Marker of Early Acute Lymphoblastic Leukemia Engraftment and Progression in Mice.

Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.

ANKHD1 silencing inhibits Stathmin 1 activity, cell proliferation and migration of leukemia cells.

ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.

Multitarget effects of quercetin in leukemia.

This study proposes to investigate quercetin antitumor efficacy in vitro and in vivo, using the P39 cell line as a model. The experimental design comprised leukemic cells or xenografts of P39 cells, treated in vitro or in vivo, respectively, with quercetin; apoptosis, cell-cycle and autophagy activation were then evaluated. Quercetin caused pronounced apoptosis in P39 leukemia cells, followed by Bcl-2, Bcl-xL, Mcl-1 downregulation, Bax upregulation, and mitochondrial translocation, triggering cytochrome c release and caspases activation. Quercetin also induced the expression of FasL protein. Furthermore, our results demonstrated an antioxidant activity of quercetin. Quercetin treatment resulted in an increased cell arrest in G1 phase of the cell cycle, with pronounced decrease in CDK2, CDK6, cyclin D, cyclin E, and cyclin A proteins, decreased Rb phosphorylation and increased p21 and p27 expression. Quercetin induced autophagosome formation in the P39 cell line. Autophagy inhibition induced by quercetin with chloroquine triggered apoptosis but did not alter quercetin modulation in the G1 phase. P39 cell treatment with a combination of quercetin and selective inhibitors of ERK1/2 and/or JNK (PD184352 or SP600125, respectively), significantly decreased cells in G1 phase, this treatment, however, did not change the apoptotic cell number. Furthermore, in vivo administration of quercetin significantly reduced tumor volume in P39 xenografts and confirmed in vitro results regarding apoptosis, autophagy, and cell-cycle arrest. The antitumor activity of quercetin both in vitro and in vivo revealed in this study, point to quercetin as an attractive antitumor agent for hematologic malignancies.

ANKHD1, a novel component of the Hippo signaling pathway, promotes YAP1 activation and cell cycle progression in prostate cancer cells.

ANKHD1 is a multiple ankyrin repeat containing protein, recently identified as a novel member of the Hippo signaling pathway. The present study aimed to investigate the role of ANKHD1 in DU145 and LNCaP prostate cancer cells. ANKHD1 and YAP1 were found to be highly expressed in prostate cancer cells, and ANKHD1 silencing decreased cell growth, delayed cell cycle progression at the S phase, and reduced tumor xenograft growth. Moreover, ANKHD1 knockdown downregulated YAP1 expression and activation, and reduced the expression of CCNA2, a YAP1 target gene. These findings indicate that ANKHD1 is a positive regulator of YAP1 and promotes cell growth and cell cycle progression through Cyclin A upregulation.

Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin-Darby canine kidney cells.

Bothrops snake venoms cause renal damage, with renal failure being the main cause of death in humans bitten by these snakes. In this work, we investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 microg/mL) significantly (p <0.05) decreased the cellular uptake of neutral red dye after 1 and 3 h. Venom (100 microg/mL) also markedly decreased the transepithelial electrical resistance (RT) across MDCK monolayers. Staining with rhodamine-conjugated phalloidin revealed disarray of the cytoskeleton that involved the stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell-matrix contact region. Feulgen staining showed a significant decrease in the number of cells undergoing mitosis and an increase in the frequency of altered nuclei. Scanning electron microscopy revealed a decrease in the number of microvilli and the presence of cells with a fusiform format. Flow cytometry with annexin V and propidium iodide showed that cell death occurred by necrosis, with little apoptosis, a conclusion supported by the lack of DNA fragmentation characteristic of apoptosis. Pretreating the cells with catalase significantly attenuated the venom-induced loss of viability, indicating a possible involvement of H2O2 in the cellular damage; less protection was observed with superoxide dismutase or N omega-nitro-L-arginine methyl ester. These results indicate that Bothrops alternatus venom is cytotoxic to cultured MDCK cells, possibly via the action of reactive oxygen species. This cytotoxicity could contribute to nephrotoxicity after envenoming by this species.