Important role of annexin A2 (ANXA2) in new blood vessel development in vivo and human triple negative breast cancer (TNBC) growth.

Development of new blood vessels in the tumor microenvironment is an essential component of tumor progression during which newly formed blood vessels nourish tumor cells and play a critical role in rapid tumor growth, invasion and metastasis. Nevertheless, how tumor cells develop new blood vessels in the tumor microenvironment (TME) have been enigmatic. Previously, we have shown specific overexpression of ANX A2 in TNBC cells regulates plasmin generation and suspected a role in neoangiogenesis. In this report, we used Matrigel plug model of in vivo angiogenesis and confirmed its role in new blood vessel development. Next, we tested if blocking of ANX A2 in aggressive human breast TME can inhibit angiogenesis and tumor growth in vivo. We showed that aggressive human breast tumor cells growing in nude mice can induce intense neoangiogenesis in the tumor mass. Blocking of ANXA2 significantly inhibited neoangiogenesis and resulted in inhibition of tumor growth. Interestingly, we identified that blocking of ANXA2 significantly inhibited tyrosine phosphorylation (Tyr-P) of ANXA2 implying its involvement in tyrosine signaling pathway and suggesting it may regulate angiogenesis. Taken together, our experimental evidence suggests that ANX A2 could be a novel strategy for disruption of tyrosine signaling and inhibition of neoangiogenesis in breast tumor.

Annexin A2 (ANX A2): An emerging biomarker and potential therapeutic target for aggressive cancers.

ANX A2 is an important member of annexin family of proteins expressed on surface of endothelial cells (ECs), macrophages, mononuclear cells and various types of cancer cells. It exhibits high affinity binding for calcium (Ca++ ) and phospholipids. ANX A2 plays an important role in many biological processes such as endocytosis, exocytosis, autophagy, cell-cell communications and biochemical activation of plasminogen. On the cell surface ANX A2 organizes the assembly of plasminogen (PLG) and tissue plasminogen activator (tPA) for efficient conversion of PLG to plasmin, a serine protease. Proteolytic activity of plasmin is required for activation of inactive pro-metalloproteases (pro-MMPs) and latent growth factors for their biological actions. These activation steps are critical for degradation of extracellular matrix (ECM) and basement proteins (BM) for cancer cell invasion and metastasis. Increased expression of ANX A2 protein/gene has been correlated with invasion and metastasis in a variety of human cancers. Moreover, clinical studies have positively correlated ANX A2 protein expression with aggressive cancers and with resistance to anticancer drugs, shorter disease-free survival (DFS), and worse overall survival (OS). The mechanism(s) by which ANX A2 regulates cancer invasion and metastasis are beginning to emerge. Investigators used various technologies to target ANX A2 in preclinical model of human cancers and demonstrated exciting results. In this review article, we analyzed existing literature concurrent with our own findings and provided a critical overview of ANX A2-dependent mechanism(s) of cancer invasion and metastasis.

Highly atom-economical, catalyst-free, and solvent-free phosphorylation of chalcogenides.

Silica gel promoted, catalyst-free and solvent-free S-P, Se-P and Te-P bond formations are described. A variety of disulfides coupled with diarylphosphine oxides provide the corresponding phosphinothioates in excellent yields. For the first time, diselenides and ditellurides reacted with dialkyl phosphites under catalyst-free conditions to provide the corresponding phosphoroselenoates and phosphorotelluroates, respectively, in good to excellent yields.

Elicitation of Diosgenin Production in Trigonella foenum-graecum (Fenugreek) Seedlings by Methyl Jasmonate.

The effects of methyl jasmonate (MeJA), an elicitor of plant defense mechanisms, on the biosynthesis of diosgenin, a steroidal saponin, were investigated in six fenugreek (Trigonella foenum-graecum) varieties (Gujarat Methi-2, Kasuri-1, Kasuri-2, Pusa Early Branching, Rajasthan Methi and Maharashtra Methi-5). Treatment with 0.01% MeJA increased diosgenin levels, in 12 days old seedlings, from 0.5%-0.9% to 1.1%-1.8%. In addition, MeJA upregulated the expression of two pivotal genes of the mevalonate pathway, the metabolic route leading to diosgenin: 3-hydroxy-3-methylglutaryl-CoA reductase (HMG) and sterol-3-ß-glucosyl transferase (STRL). In particular, MeJA increased the expression of HMG and STRL genes by 3.2- and 22.2-fold, respectively, in the Gujarat Methi-2 variety, and by 25.4- and 28.4-fold, respectively, in the Kasuri-2 variety. Therefore, MeJA may be considered a promising elicitor for diosgenin production by fenugreek plants.

Butanolides from methanolic extract of Litsea glutinosa.

Phytochemical investigations of a MeOH extract obtained from the heartwoods of the Litsea glutinosa (Lauraceae) led to the isolation and characterization of four new butenolides, (3R,4S,5S)-2-hexadecyl-3-hydroxy-4-methylbutanolide 1, litsealactone C (2), and litsealactone D (4), litsealactone G (5), and a new benzoic acid derivative named eusmoside C (3). The structures of these compounds were elucidated on the basis of spectral studies.

Antibody-directed neutralization of annexin II (ANX II) inhibits neoangiogenesis and human breast tumor growth in a xenograft model.

Activation of the fibrinolytic pathway has long been associated with human breast cancer. Plasmin is the major end product of the fibrinolytic pathway and is critical for normal physiological functions. The mechanism by which plasmin is generated in breast cancer is not yet fully described. We previously identified annexin II (ANX II), a fibrinolytic receptor, in human breast tumor tissue samples and observed a strong positive correlation with advanced stage cancer (Sharma et al., 2006a). We further demonstrated that tissue plasminogen activator (tPA) binds to ANX II in invasive breast cancer MDA-MB231cells, which leads to plasmin generation (Sharma et al., 2010). We hypothesize that ANX II-dependent plasmin generation in breast tumor is necessary to trigger the switch to neoangiogenesis, thereby stimulating a more aggressive cancer phenotype. Our immunohistochemical studies of human breast tumor tissues provide compelling evidence of a strong positive correlation between ANX II expression and neoangiogenesis, and suggest that ANX II is a potential target to slow or inhibit breast tumor growth by inhibiting neoangiogenesis. We now report that administration of anti-ANX II antibody potently inhibits the growth of human breast tumor in a xenograft model. Inhibition of tumor growth is at least partly due to attenuation of neoangiogenic activity within the tumor. In vitro studies demonstrate that anti-ANX II antibody inhibits angiogenesis on three dimensional matrigel cultures by eliciting endothelial cell (EC) death likely due to apoptosis. Taken together, these data suggest that selective disruption of the fibrinolytic activity of ANX II may provide a novel strategy for specific inhibition of neoangiogenesis in human breast cancer.

Gender Disparity in Breast Cancer: A Veteran Population-Based Comparison.

BACKGROUND: Male breast cancer (MBC) comprises <1% of all cancers and continues to rise. Because of rarity, there is paucity in the literature; therefore, management of MBC is generalized from female breast cancer (FBC). METHODS: Data from 152 VA Medical Centers were used to analyze the database of Veteran patient with breast cancer diagnosed between 1998 and 2016 using biostatistical software (SAS 9.3). Our primary objective is to compare patient's demographics, breast cancer characteristics, and outcomes for male and female Veterans. FINDING: In total, 8864 patients' records were reviewed;1528 MBC were compared with 7336 FBC with a mean follow up time of 5.5 years (SD 4.17). The mean age at diagnosis was 68.6 years and 57.3 years for MBC and FBC, respectively (P < .0001). Higher numbers of MBC patients (95%) were >50 years of age compared to FBC patients (72%). More MBC patients (16.8 vs. 9.1% and 9 vs. 4%) presented with higher disease stage (III and IV, respectively). Estrogen receptor-positive tumors were more common in MBC (59 versus 52%). Hormonal treatment was received by 27% of MBC versus 19% FBC; chemotherapy 21.3% versus 41.5% and radiation 23.5% versus 60.9%. Forty-two percent MBC and 20% FBC Veterans died during study. Male patients had higher death rate 1.285 (95% CI: 1.150, 1.434, P < .0001) compared to females after adjusting data for age, race, stage, and grade. INTERPRETATION: To the best of our knowledge, this is the largest comparison series of MBC and FBC to date in the Veterans population. The higher mortality rate in MBC patients may be due to late presentation, higher stage at the time of diagnosis and/or tumor biology. Veteran's exposures to hazardous materials during their military deployments as an additional factor for worse prognosis need further investigation.

Breast cancer cell surface annexin II induces cell migration and neoangiogenesis via tPA dependent plasmin generation.

Annexin II, an abundant phospholipids binding cell surface protein, binds tPA and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using human breast cancer model we show that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tissue plasminogen activator (tPA). In vitro both annexin II and tPA interacts which in turn convert zymogen plasminogen to reactive enzyme plasmin. Cell surface produced plasmin inhibited the migration of MDA-MB231 cells. Silencing of annexin II gene in MDA-MB231 cells abolished tPA binding therefore inhibited tPA dependent plasmin generation. These annexin II suppressed MDA-MB231 cells showed reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive human breast cancer cells which correlates with neovascularization of the tumor. Taken together, these data indicate that annexin II may regulate localized plasmin generation in breast cancer. This may be an early event switching breast cancer from the prevascular phase to the vascular phase and thus contributing to aggressive cancer with the possibility of metastasis. The data provide a mechanism explaining the role of annexin II in breast cancer progression and suggest that annexin II may be an attractive target for therapeutic strategies aimed to inhibit angiogenesis and breast cancer.

Effect of vitamin E and selenium supplementation on oxidative stress indices and cortisol level in blood in water buffaloes during pregnancy and early postpartum period.

Pregnancy is a physiology state accompanied by high energy and oxygen demand that may lead to increased level of oxidative stress and development of metabolic and reproductive disorders in pregnant water buffaloes. In the present study, the alterations in serum cortisol and erythrocyte lipid peroxides and superoxide dismutase activities were examined in 28 pregnant water buffaloes supplemented with antioxidant nutrients, Vitamin E and selenium. Another 14 buffaloes did not receive any treatment during pregnancy to serve as negative control. Results of the present study suggested that pregnancy is associated with oxidative stress and supplementation of vitamin E and selenium may be beneficial by alleviating oxidative stress in water buffaloes.

Long-term efficacy and downstream mechanism of anti-annexinA2 monoclonal antibody (anti-ANX A2 mAb) in a pre-clinical model of aggressive human breast cancer.

There is considerable direct evidence that calcium binding protein ANX A2 is a potential target for treating aggressive breast cancer. The most compelling data are based on the finding of ANX A2 overexpression in aggressive triple negative human breast cancer (TNBC) cell lines and in human breast cancer tissues. Previously, we and others reported a unique role of ANX A2 in cancer invasion, including breast cancer. Moreover, we demonstrated that anti-ANX A2 mAb-mediated immunoneutralization of ANX A2 inhibited invasive human breast cancer growth in a xenograft model. We further evaluated the long-term effects of multiple treatments with anti-ANX A2 mAb and its mechanism of inhibition on human breast tumor growth. We now demonstrate that three treatments with anti-ANX A2 mAb led to significant inhibition of breast tumor growth in immunodeficient mice, and that the anti-tumor response was demonstrable from day 94. After treatment, we followed tumor growth for 172 days and demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated that anti-ANX A2 mAb treatment caused significant inhibition of conversion of tissue plasminogen activator (tPA) in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathways in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for treatment of aggressive breast cancer.

Antioxidant activity and protection of pancreatic ß-cells by embelin in streptozotocin-induced diabetes.

OBJECTIVES: The aim of the present study was to evaluate the antioxidant potential of embelin in streptozotocin-induced diabetes. METHODS: Diabetes was induced in rats fasted overnight by the administration of a single dose of streptozotocin, and analyzed for blood, serum, and biological and histological pancreatic tissue parameters in intact control, diabetic, and embelin-treated diabetic rats (n = 9) at the dose levels of 15, 25, and 30 mg/kg/day for 21 days. RESULTS: Diabetes caused highly significant abnormalities in blood, serum, and pancreatic tissue biochemical parameters. Embelin and glibenclamide administration to diabetic rats caused a highly significant decline in the blood glycated hemoglobin and serum glucose levels and nitric oxide activity, with a concomitant increase in the serum insulin level (P < 0.001). Furthermore, embelin and glibenclamide treatment increased the pancreatic antioxidant enzyme status (superoxide dismutase, catalase, reduced glutathione, glutathione peroxidase, glutathione S-transferase, and ascorbic acid), and also decreased the thiobarbituric acid reactive oxygen species contents (P < 0.001). The histoarchitecture of the diabetic rats typically showed a degenerated pancreas with reduced ß-cell counts, while embelin treatment was shown to significantly regenerate islet cells. CONCLUSION: The study proves the potent antioxidant activity of embelin, which has been found to be effective in managing severe hyperglycemia.

The role of apoptosis in immunosuppression of dogs with demodicosis.

The aim of the present study was to evaluate the status of apoptosis in peripheral blood leukocytes of dogs with demodicosis. A total of 26 dogs suffering from demodicosis, and positive for Demodex canis mites by skin scraping, participated in the study, 13 with localized demodicosis (LD) and 13 with generalized demodicosis (GD). A further 13 clinically healthy dogs, all of whom were negative for mites upon skin scraping, were used as controls. The dogs with GD revealed significantly higher (P ≤ 0.0001) percentage of leukocytes with externalization of phosphatidylserine (PS) and depolarized mitochondrial membrane potentials (ΔΨm) as compared with the dogs with LD and healthy controls. These dogs also revealed significantly lower values (P ≤ 0.0001) of hematological parameters viz. hemoglobin, total erythrocytes count total leukocytes count, lymphocytes, monocytes and neutrophils. Significantly higher (P ≤ 0.0001) percentages of leukocytes with externalization of PS and depolarized ΔΨm were also found in dogs with LD as compared with the healthy controls. These dogs also revealed significantly lower values of Hb (P ≤ 0.0001), TEC (P=0.025), TLC (P ≤ 0.0001), lymphocytes (P=0.008), monocytes (P ≤ 0.0001) and neutrophils (P=0.03). It is concluded that premature apoptosis of PBL may be implicated in the immunosuppression of the dogs with demodicosis.

Inflammation, microenvironment, and the immune system in cancer progression.

Since Virchow first proposed in 1863 that tumors could originate from sites of chronic inflammation, it has been well established that chronic inflammation both contributes to cancer progression and predisposes tissue to various types of cancer. Experimental, clinical, and epidemiological studies have all demonstrated the strong association between chronic inflammation and cancer, and many studies have correlated the prolonged presence of the inflammatory milieu with an increased risk for developing cancer. Proinflammatory cytokines, chemokines and adhesion molecules, which regulate the sequential recruitment of leukocytes, are frequently observed in tumor microenvironment. These early desmoplastic changes could stimulate fibroblasts and endothelial cell division and produce components for tissue remodeling and neovascularization, ultimately promoting neoplastic processes. In this review article we overview the current understanding of the role of chronic inflammation in neoangiogenesis, tumor initiation, promotion, and progression.

Roscovitine regulates invasive breast cancer cell (MDA-MB231) proliferation and survival through cell cycle regulatory protein cdk5.

Roscovitine, a purine analogue, has been considered for the treatment of cancer. Anti-cancer therapeutic efficacy is being evaluated in clinical trials. However, the mechanisms remain unclear. In the present study, cyclic-dependent kinase 5 (cdk5) proved to be a molecular target for roscovitine-triggered apoptosis for highly invasive breast cancer cell death. Because our previous studies have shown a potential role of cdk5 in endothelial cell proliferation/apoptosis [Sharma, M.R., Tuszynski, G.P., Sharma, M.C. (2004). Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5. J. Cell Biochem. 91, 398-409], here we not only demonstrate first that Cdk5, p35, and p25 proteins were all expressed in invasive breast cancer cells MDA-MB231 but also showed that cdk5 expression regulates MDA-MB231 cell proliferation. In addition, potent mitogen bFGF up-regulates cdk5 expression. Roscovitine specifically inhibits cdk5 expression/activity in a dose-dependent manner with concomitant inhibition of MDA-MB231 cell proliferation and induction of apoptosis. By contrast, the roscovitine analog olomoucine, a specific inhibitor of cdk4, failed to affect MDA-MB231 cell proliferation and apoptosis which implies the specific involvement of cdk5 in roscovitine-triggered cell death/proliferation. Additionally, roscovitine-mediated inhibition of proliferation is irreversible. These data suggest that cdk5 may have a significant role in the regulation of breast cancer cell proliferation and apoptosis and extend beyond its role in neurogenesis. These results suggest that Cdk5 is a novel player in roscovitine-triggered breast cancer cell apoptosis and inhibition of proliferation, therefore, may be a potential therapeutic target.

The role of annexin II in angiogenesis and tumor progression: a potential therapeutic target.

It is well established that human tumors overproduce plasmin a serine protease that is known to promote angiogenesis, tumor growth and metastasis. However, the mechanism by which endothelial or tumor cells regulate the proteolytic activity of plasmin is not well understood. Cell surface receptors regulate activation of plasminogen to plasmin and its proteolytic activity. Annexin II is one of the well studied receptors for plasminogen and tPA, which binds to plasminogen and converts it to plasmin. Plasmin is a highly reactive enzyme which is physiologically involved in fibrinolysis. Since the proteolytic activity of plasmin is very tightly regulated, uncontrolled production of plasmin can degrade extracellular matrix (ECM) and basement membrane (BM) of the surrounding blood vessels. Thus plasmin plays an important role in neoangiogenesis and cancer invasion and metastasis. Therefore, the receptor which regulates plasmin generation may be an attractive target for the development of anti-cancer/anti-metastatic agents. Angiostatin (AS), internal fragment of plasminogen, has been reported to inhibit human tumor growth and metastasis. We have shown that AS binds to endothelial/cancer cell surface annexin II with high affinity and interferes with plasmin generation suggesting that the role of plasmin/plasminogen system may be more complex than we previously thought. In this review we provide a comprehensive analysis of the literature in context of the role of annexin II in angiogenesis, tumor progression and metastasis. Compelling evidence from the literature and our own findings suggest that annexin II may be a potential target for the development of effective therapeutic strategies for the treatment of cancer and its induced metastasis.

Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action.

Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. epsilon-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.

Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression.

Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies.

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5.

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.

Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells.

Angiostatin(AS), an internal fragment of plasminogen, is one of the most potent specific inhibitors of angiogenesis. Angiostatin treatment has resulted in the complete regression of human tumors implanted subcutaneously into nude mice and has great therapeutic value (O'Reilly et al., Nat. Med. 2, 689-692, 1996). Despite promising therapeutic value in the treatment of cancer, the mechanism of its action is still unknown. We found that angiostatin binds to a 35-kDa protein in bovine aortic endothelial (BAE) cells (Sharma et al., Proc. Am. Assoc. Cancer Res. 42, 568, A3050, 2002). In an attempt to begin to understand angiostatin's mechanism of action, we have purified and characterized this 35-kDa protein from BAE cells. Internal peptide sequence analysis of purified protein demonstrated (SLYYIQQDTK, SYSPYDMLESIK, and ALLYLXGGDD) 100% sequence identity with tyrosine kinase substrate annexin II. Solid phase binding analysis suggests that angiostatin specifically bound to purified annexin II immobilized on 96-well plastic plates. Hundred-fold molar excess of unlabeled AS and anti-annexin II antibody inhibited bindings 85 and 55%, respectively, suggesting specific interaction. Annexin II is a predominant receptor for angiostatin, since neutralizing the angiostatin by soluble receptor (annexin II) effectively blocks angiostatin's anti-EC activity. Similarly, saturating the annexin II receptor by plasminogen in endothelial cells also blocks angiostatin's activity. Both angiostatin and plasminogen bind to purified annexin II in BAE cells saturably with apparent K(d) values of 101 and 164 nM, respectively, for purified annexin II and K(d) values of 83 and 125 nM, respectively, for BAE cells. Anti-annexin II monoclonal antibody inhibited angiostatin and plasminogen binding to endothelial cells by 68 and 62%, respectively, supporting our in vitro studies that annexin II is a receptor for angiostatin. Angiostatin-binding protein/annexin II specifically expressed in endothelial cells but not in fibroblasts suggests its EC-specific function. Epsilon-aminocaproic acid, a lys analogue, effectively blocks angiostatin and annexin II interaction, indicating that the lysine-binding domain of AS is required for binding to annexin II. These results suggest that the antiangiogenic action of angiostatin may be mediated via interaction with annexin II. Identification of annexin II as a receptor for angiostatin provides further evidence that clotting and fibrinolytic pathways are directly involved in the angiogenic process.

Structural modifications of plumieride isolated from Plumeria bicolor and the effect of these modifications on in vitro anticancer activity.

Plumieride was isolated as one of the major components from the biologically active methanolic extract of the bark of Plumeria bicolor (family Apocynaceae). For investigating the effect of substituents on cytotoxic activity it was modified into a series of compounds. Replacing the methyl ester functionality of plumieride with alkyl amides of variable carbon units improved the cytotoxic activity, and a correlation between overall lipophilicity and cytotoxic activity was observed. In plumieride, the glucose moiety was converted into a di- and trisaccharide by following the protection and deprotection approach, and the resulting compounds produced enhanced cytotoxicity. However, these compounds were found to be less effective than plumeiride containing a dodecyl (12 carbon units) amide group. Among all of the derivatives, the naturally occurring plumieride showed the least cytotoxicity (50% cell kill = 49.5 microg/mL), and the dodecyl amide analogue of plumieridepentaacetate produced the best efficacy (50% cell kill = 11.8 microg/mL). The di- and trisaccharide analogues were found to be slightly less effective than the dodecyl derivative (50% cell kill = 15-17 microg/mL). The in vitro cytotoxicity of the plumieride analogues was determined in radiation-induced fibrosarcoma (RIF) tumor cells.