Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^

Involvement of lipocalin 2 in leukemia and breast cancer

Human lipocalin 2 is described as the neutrophil gelatinase-associated lipocalin (NGAL). The lipocalin 2 gene encodes a small, secreted glycoprotein that possesses a variety of functions, of which the best characterized function is organic iron binding activity. Elevated NGAL expression has been observed in many human cancers including breast, colorectal, pancreatic and ovarian cancers. I focused on the characterization of NGAL function in chronic myelogenous leukemia (CML) and breast cancer. Using the leukemic xenograft mouse model, we demonstrated that over-expression of NGAL in K562 cells, a leukemic cell line, led to a higher apoptotic rate and an atrophy phenotype in the spleen of inoculated mice compared to K562 cells alone. These results indicate that NGAL plays a primary role in suppressing hematopoiesis by inducing apoptosis within normal hematopoietic cells. In the breast cancer project, we analyzed two microarray data sets of breast cancer cell lines ( n = 54) and primary breast cancer samples (n = 318), and demonstrated that high NGAL expression is significantly correlated with several tumor characteristics, including negative estrogen receptor (ER) status, positive HER2 status, high tumor grade, and lymph node metastasis. Ectopic NGAL expression in non-aggressive (ZR75.1 and MCF7) cells led to aggressive tumor phenotypes in vitro and in vivo. Conversely, knockdown of NGAL expression in various breast cancer cell lines by shRNA lentiviral infection significantly decreased migration, invasion, and metastasis activities of tumor cells both in vitro and in vivo . It has been previously reported that transgenic mice with a mutation in the region of trans-membrane domain (V664E) of HER2 develop mammary tumors that progress to lung metastasis. However, we observed that genetic deletion of the 24p3 gene, a mouse homolog of NGAL, in HER2 transgenic mice by breeding with 24p3-null mice resulted in a significant delay of mammary tumor formation and reduction of lung metastasis. Strikingly, we also found that treatment with affinity purified 24p3 antibodies in the 4T1 breast cancer mice strongly reduced lung metastasis. Our studies provide evidence that NGAL plays a critical role in breast cancer development and progression, and thus NGAL has potential as a new therapeutic target in breast cancer.^

Children with acute leukemia: A comparison of outcomes and cost-effectiveness from allogeneic blood stem cell and bone marrow transplantation

The relative merits of PBSCT versus BMT for children with standard and high risk hematologic malignancies remain unclear. In a retrospective single center study, we compared allogeneic peripheral blood stem cell transplantation (PBSCT) (n=30) with bone marrow transplantation (BMT) (n=110) in children with acute leukemia. We studied recipients of HLA matched sibling stem cells, and of stem cells from alternative donors (HLA mismatched and/or unrelated) and determined whether sourcing the stem cells from PB or marrow affected engraftment, incidence of acute and chronic GvHD, and disease-free survival at 1 year. Our results show a modest reduction in time to engraftment from PB stem cells and no greater risk of GvHD, but illustrate that the severity of the underlying disease is by far the greatest determinant of 1 year survival. Patients in the BMT group had a higher treatment success rate and lower costs than the recipients of the PBSCT within the standard but not the high risk disease group, where the treatment success rate and the cumulative costs were lower in the PBSCT group compared to the BMT group. Our current incremental cost-effectiveness ratio and analysis of uncertainty suggest that allogeneic transplantation of bone marrow grafts was a more cost-effective treatment option compared to peripheral blood stem cells in patients with standard risk childhood acute leukemia disease. For high risk disease our data are less prescriptive, since the differences were more limited and the range of costs much larger. Neither option demonstrated a clear advantage from a cost-effectiveness standpoint.^

Folate pathway polymorphisms and neuropsychological impairment after leukemia therapy

Neuropsychological impairment occurs in 20%-40% of childhood acute lymphoblastic leukemia (ALL) survivors, possibly mediated by folate depletion following methotrexate chemotherapy. We evaluated the relationship between two folate pathway polymorphisms and neuropsychological impairment after childhood ALL chemotherapy. Eighty-six childhood ALL survivors were recruited between 2004-2007 at Texas Children's Hospital after exclusion for central nervous system leukemia, cranial irradiation, and age<1 year at diagnosis. Neuropsychological evaluation at a median of 5.3 years off therapy included a parental questionnaire and the following child performance measures: Trail Making Tests A and B, Grooved Pegboard Test Dominant-Hand and Nondominant-Hand, and Digit Span subtest. We performed genotyping for polymorphisms in two folate pathway genes: reduced folate carrier (RFC1 80G>A, rs1051266) and dihydrofolate reductase (DHFR Intron-1 19bp deletion). Fisher exact test, logistic regression, Student's t-test, and ANOVA were used to compare neuropsychological test scores by genotype, using a dominant model to group genotypes. In univariate analysis, survivors with cumulative methotrexate exposure ≥9000 mg/m2 had an increased risk of attention disorder (OR=6.2, 95% CI 1.2 – 31.3), compared to survivors with methotrexate exposure <9000 mg/m2. On average, female survivors scored 8.5 points higher than males on the Digit Span subtest, a test of working memory (p=0.02). The RFC1 80G>A and DHFR Intron-1 deletion polymorphisms were not related to attention disorder or impairment on tests of attention, processing speed, fine motor speed, or memory. These data imply a strong relationship between methotrexate dose intensity and impairment in attention after childhood ALL therapy. We did not find an association between the RFC1 80G>A or DHFR Intron-1 deletion polymorphisms and long-term neuropsychological impairment in childhood ALL survivors.^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^

BIOLOGICAL MECHANISMS AND CLINICAL IMPLICATIONS OF BCR-ABL-INDUCED MITOCHONDRIAL OXIDATIVE STRESS AND CELL SURVIVAL IN CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML), a myeloproliferative disorder, represents approximately 15-20% of all adult leukemia. The development of CML is clearly linked to the constitutively active protein-tyrosine kinase BCR-ABL, which is encoded by BCR-ABL fusion gene as the result of chromosome 9/22 translocation (Philadelphia chromosome). Previous studies have demonstrated that oxidative stress-associated genetic, metabolic and biological alterations contribute to CML cell survival and drug refractory. Mitochondria and NAD(P)H oxidase (NOX) are the major sources of BCR-ABL-induced cellular reactive oxygen species (ROS) production. However, it is still unknown how CML cells maintain the altered redox status, while escaping from the persistent oxidative stress-induced cell death. Therefore, elucidation of the mechanisms by which CML cells cope with oxidative stress will provide new insights into CML leukemogenesis. The major goal of this study is to identify the survival factors protecting CML cells against oxidative stress and develop novel therapeutic strategies to overcome drug resistance. Several experimental models were used to test CML cell redox status and cellular sensitivity to oxidative stress, including BCR-ABL inducible cell lines, BCR-ABL stably transformed cell lines and BCR-ABL-expressing CML blast crisis cells with differential BCL-XL/BCL-2 expressions. Additionally, an artificial CML cell model with heterogenic BCL-XL/BCL-2 expression was established to assess the correlation between differential survival factor expression patterns and cell sensitivity to Imatinib and oxidative stress. In this study, BCL-XL and GSH have been identified as the major survival factors responsive to BCR-ABL-promoted cellular oxidative stress and play a dominant role in regulating the threshold of oxidative stress-induced apoptosis. Cell survival factors BCL-XL and BCL-2 differentially protect mitochondria under oxidative stress. BCL-XL is an essential survival factor in preventing excessive ROS-induced cell death while BCL-2 seems to play a relatively minor role. Furthermore, the redox modulating reagent β-phenethyl isothiocyanate (PEITC) has been found to efficiently deplete GSH and induce potent cell killing effects in drug-resistant CML cells. Combination of PEITC with BCL-XL/BCL2 inhibitor ABT737 or suppression of BCL-XL by BCR-ABL inhibitor Gleevec dramatically sensitizes CML cells to apoptosis. These results have suggested that elevation of BCL-XL and cellular GSH are important for the development of CML, and that redox-directed therapy is worthy of further clinical investigations in CML.

Seasonal aspects of leukemia

An investigation of (a) month/season-of-birth as a risk factor and (b) month/season-of-treatment initation as a prognostic factor in acute lymphoblastic leukemia (ALL) in children, 0-15 years of age, was conducted. The study population used was that of the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute and included children diagnosed and treated for ALL from 1973-1986. Two separate sets of analyses using different exclusion criteria led to similar results. Specifically, the inability to reject the null hypothesis of no significant difference in the variation of monthly/seasonal incidence rates among children residing within the 10 SEER sites using either cosinor analysis or one-way analysis of variance. No association was established between month/season of treatment initiation and survival in ALL among children using either Kaplan-Meier or cosinor analysis. In separate Kaplan-Meier analyses, age, gender, and treatment type were each found to be significant univariate prognostic factors for survival, however. ^

ETIOLOGIC AND CLINICAL FACTORS WHICH DIFFERENTIATE PATIENTS WITH CHROMOSOMAL ABNORMALITIES FROM DIPLOID PATIENTS IN ACUTE NONLYMPHOCYTIC LEUKEMIA

Tumor-specific chromosomal abnormalities have been demonstrated in bone marrow of approximately 50% of newly diagnosed acute nonlymphocytic (ANLL) patients. This study examined two hypotheses: (1) Aneuploid (AA) patients are diagnosed later in the course of their disease than diploid (NN) patients; and (2) AA patients are more likely to have been exposed to environmental agents. Of 324 patients eligible for study, environmental exposure data were obtained for 236 (73%) of them. No evidence was found to suggest that AA patients had more advanced disease than NN patients. Aneuploid patients were more likely than NN patients to: (a) report treatment with cytotoxic drugs for a prior medical condition (odds ratio, adjusted for age, sex and other exposures (OR) = 4.25, 95% confidence intervals, 1.38 to 13.17); (b) smoke cigarettes, OR = 1.82 (1.02, 3.26) and (c) drink alcoholic beverages, OR = 1.91 (1.05, 3.48). No statistically significant associations between aneuploidy and occupational exposures were present, OR = 3.59 (0.76, 17.13). Problems in interpreting these ORs are discussed. ^

Venous thromboembolism in patients with acute leukemia: Prevalence, incidence, recurrence of thrombosis, risk factors, and impact on survival

Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the third most preventable cardiovascular disease and a growing public health problem in the United States. The incidence of VTE remains high with an annual estimate of more than 600,000 symptomatic events. DVT affects an estimated 2 million American each year with a death toll of 300,000 persons per year from DVT-related PE. Leukemia patients are at high risk for both hemorrhage and thrombosis; however, little is known about thrombosis among acute leukemia patients. The ultimate goal of this dissertation was to obtain deep understanding of thrombotic issue among acute leukemia patients. The dissertation was presented in a format of three papers. First paper mainly looked at distribution and risk factors associated with development of VTE among patients with acute leukemia prior to leukemia treatment. Second paper looked at incidence, risk factors, and impact of VTE on survival of patients with acute lymphoblastic leukemia during treatment. Third paper looked at recurrence and risk factors for VTE recurrence among acute leukemia patients with an initial episode of VTE. Descriptive statistics, Chi-squared or Fisher's exact test, median test, Mann-Whitney test, logistic regression analysis, Nonparametric Estimation Kaplan-Meier with a log-rank test or Cox model were used when appropriate. Results from analyses indicated that acute leukemia patients had a high prevalence, incidence, and recurrent rate of VTE. Prior history of VTE, obesity, older age, low platelet account, presence of Philadelphia positive ALL, use of oral contraceptives or hormone replacement therapy, presence of malignancies, and co-morbidities may place leukemia patients at an increased risk for VTE development or recurrence. Interestingly, development of VTE was not associated with a higher risk of death among hospitalized acute leukemia patients.^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.