New Anticancer Agents: Hormones Made Within the Heart

ANTICANCER RESEARCH 32: 2515-2522 (2012)

Review New Anticancer Agents: Hormones Made within the Heart

DAVID L. VESELY

Departments of Medicine, Molecular Pharmacology and Physiology, and Cardiac Hormone Center, James A. Haley VA Medical Center, and University of South Florida Morsani School of Medicine, Tampa, FL, U.S.A.

Abstract. The heart is a sophisticated endocrine gland involved in blood pressure regulation and maintenance of synthesizing the atrial natriuretic peptide (ANP) plasma volume in animals (4-9) and humans (10-12). These prohormone which contains four peptide hormones, namely cardiac hormones, numbered by their a.a. sequences atrial natriuretic peptide, vessel dilator, kaliuretic peptide beginning at the N-terminal end of the atrial natriuretic and long-acting natriuretic peptide, which decrease up to peptide (ANP) prohormone, consist of the first 30 a.a. of the 97% of human pancreatic, breast, colon, prostate, kidney prohormone, namely, long-acting natriuretic peptide (LANP), and ovarian carcinomas, as well as small-cell and a.a. 31-67 (namely, vessel dilator), a.a. 79-98 (kaliuretic squamous cell lung cancer cells within 24 hours in cell peptide) and a.a. 99-126 (ANP) (13, 14). Each of these culture. In vivo these four cardiac hormones eliminate up to peptide hormones circulates in healthy humans, with the 80% of human pancreatic adenocarcinomas, up to two- concentrations of vessel dilator and LANP in plasma being thirds of human breast cancers, and up to 86% of human 17- to 24-fold higher than that of ANP (15-20). small-cell lung cancers in athymic mice. Their anticancer mechanism(s) target the Rat sarcoma bound guanosine Heart Hormones Eliminate up to triphosphate (RAS)-mitogen activated protein kinase kinase 97% of Cancer Cells In Vitro 1/2 (MEK1/2)-extracellular signal related kinase 1/2 (ERK1/2) kinase cascade in cancer cells. These four cardiac The four cardiac hormones reduce up to 97% of human hormones inhibit up to 95% of the basal activity of Ras, pancreatic, colon, prostate, breast, ovarian and kidney 98% of the phosphorylation of MEK1/2 kinases and 96% of adenocarcinoma cells (21-26), angiosarcoma of the heart the activation of basal activity of ERK1/2 kinases. They also cells (27), melanomas (28), medullary thyroid carcinomas completely block the activity of mitogens such as the ability (29), glioblastomas of the brain (30), as well as small-cell of epidermal growth factor to stimulate ERK and RAS. In (31) and squamous cell lung carcinoma cells (32) in cell addition to inhibiting these mitogen-activated protein culture. There is a 97.4%, 87%, 88% and 89% (p<0.0001 for kinases (MAPKs) they also inhibit MAPK9, i.e. c-Jun-N- each) decrease (namely, elimination) of human prostate terminal kinase 2. These multiple kinase inhibitors are adenocarcinoma cells by vessel dilator, LANP, kaliuretic cytotoxic and cause cell death of cancer cells but not of peptide, and ANP, respectively, within 24 hours at their 1 normal cells. mM concentrations (23). There is no proliferation in the three days following this decrease, induced by the heart The heart is a sophisticated endocrine gland synthesizing a hormones (23). When antibodies to these four cardiac 126 amino acid (a.a.) prohormone which contains four hormones are used, the ability of the hormones to reduce the peptide hormones (1-3). These peptide hormones are number of prostate cancer cells is completely blocked, indicating that their effects are specific, namely not due to some other hormone or substance (23). Dose response curves have revealed that there is a significantly greater (p<0.05) Correspondence to: David L. Vesely, MD, PhD, James A. Haley decrease in the number of cancer cells at each 10-fold Veterans Hospital (151), 13000 Bruce B. Downs Blvd., Tampa, Florida 33612, U.S.A. Tel: +1 8139727624, Fax: +1 8139727623, increase in the concentration of the four cardiac hormones e-mail: [email protected] synthesized by the ANP gene in human breast, colon and prostate cancer cells as well as in small-cell and squamous Key Words: Cancer, natriuretic peptides, metastasis, review. cell carcinoma of lung cells (21-26, 31, 32).

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Table I. Ability of cardiac hormones to eliminate human cancer growing in athymic mice. The numbers in each column are the percentages of human carcinomas eliminated and which never recurred in the primary site in athymic mice, when treated with each of the cardiac hormones for 28 days at 3 nM/kg body weight/minute. VDL, Vessel dilator; LANP, long acting natriuretic peptide; ANP, atrial natriuretic peptide; KP, kaliuretic peptide.

Breast Pancreatic Small-cell cancer adenocarcinoma lung cancer

VDL 67% 33% 71% LANP 50% 20% 86% ANP 33% 80% 43% KP 67% 14% 57%

Figure 1. The atrial natriuretic peptide (ANP) gene in the heart, codes for a 126 amino acid (a.a.) prohormone which through proteolytic processing results in the formation of four cardiac hormones. These four cardiac hormones are: long-acting natriuretic peptide (LANP) consisting of the first 30 a.a. of the 126 a.a. prohormone; vessel dilator (VDL), a.a. 31-67 of the prohormone; kaliuretic peptide (KP), a.a. 79- Heart and Kidney Hormones Eliminate up 98 of this prohormone; and ANP, consisting of a.a. 99-126 of the 126 to 86% of Human Small-Cell Lung a.a. prohormone. Reprinted with permission from reference 47. Carcinomas in Mice

LANP, vessel dilator, KP, and ANP eliminate 86%, 71%, 57%, 43% (p<0.001 for each) of human small-cell lung carcinomas (36). Treated small-cell lung carcinomas that are Heart Hormones Eliminate up to 80% of not eliminated grow rapidly, similar to the untreated Human Pancreatic Adenocarcinomas In Vivo controls, whose volume is 7-fold larger in one week, 18-fold increased in two weeks, 39-fold increased in three weeks, Human pancreatic adenocarcinoma has the lowest 5-year 63-fold increased in one month and 97-fold increased in survival rate of all common types of cancers (33, 34). The 5- volume in six weeks (36). One vessel dilator-treated animal year survival rate of patients with adenocarcinoma of the with small-cell lung carcinoma developed a large tumor pancreas is 1%, with a median survival of only four months (8,428 mm3 volume) upon treatment and this tumor was (33, 34). Current cancer chemotherapy and surgery prolong eliminated utilizing ANP and then LANP, each for four survival by a few months, but the mean survival of four weeks sequentially (36). months, refers to patients who were treated with surgery and/or current cancer chemotherapeutic agents (33, 34). Heart Hormones Eliminate Two-Thirds of When each of the heart hormones are infused Human Breast Carcinomas without any Surgery subcutaneously at 3 nM min–1 kg–1 body weight for 28 days in athymic mice bearing human pancreatic adenocarcinomas, Vessel dilator, LANP, KP and ANP eliminate 67%, 50%, 67% ANP eliminated 80% of the human pancreatic carcinomas and 33% of the human breast adenocarcinomas in athymic (35). Vessel dilator, LANP and KP eliminated the primary mice, when infused subcutaneously for 28 days, with fresh pancreatic carcinomas by 33%, 20%, and 14% in their hormones prepared weekly at 3 nM min–1 kg–1 body weight respective treatment groups (35). In none of the animals in (37). There was no recurrence of the breast cancer at the which the pancreatic adenocarcinomas are eliminated in the primary site and no metastasis, except in the ANP-treated primary site, does a single animal ever have a recurrence in group (37). The natriuretic peptide receptors A and C were the primary site (35). One ANP-treated animal developed a decreased 50% and 31%, respectively, in metastatic versus metastatic lesion and this lesion was eliminated by treatment primary ANP-treated breast adenocarcinomas, possibly with vessel dilator (35). Even in the treated animals which explaining why fewer of the breast carcinomas responded to did not have total elimination of their human pancreatic ANP compared to the other three cardiac hormones, as ANP adenocarcinoma with the four respective heart hormones, works via these receptors while the other peptide hormones tumor volumes decreased to less than 10% (and with vessel have their own specific receptors (2, 3, 37). Thus, the four dilator to less than 2%) compared to those of the untreated hormones made within the heart have the ability to eliminate animals, both during treatment and in a 12-month follow-up human pancreatic, breast and small-cell lung cancer growing period (35). in mice but their respective abilities to eliminate these

2516 Vesely et al: Cardiac Hormones: Anticancer Agents (Review)

Figure 2. Cardiac hormones inhibit five metabolic targets, namely RAS-GTP, MEK1/2, and ERK1/2 of the RAS-MEK1/2-ERK1/2 kinase cascade by 95-98%. These multiple kinase inhibitors are also strong inhibitors (i.e. 91%) of DNA synthesis within cancer cells. Abbreviations: Tyrosine kinase receptor (RTK); Rous sarcoma viral proto-oncogene tyrosine kinase (SRC); Rous sarcoma SH2 C-terminal binding domain adapter protein (SHC); Growth factor receptor-bound protein 2 (GRB2); Son of Sevenless gene (SOS); rat sarcoma bound guanosine diphosphate (RAS-GDP); Rat sarcoma bound guanosine triphosphate (RAS-GTP); Rapidly accelerated fibrosarcoma serine/threonine protein kinase (RAF); Maloney murine sarcoma virus serine/threonine protein kinase (MOS); Mitogen-activated protein kinase kinase 1/2 (MEK1/2); extracellular signal-related kinases 1/2 (ERK1/2); Protein phosphatase 2A (PP2A); Deoxyribonucleic acid (DNA). Reprinted with permission from reference 44.

different types of cancers varies with the type of cancer, as Metabolic Targets of Heart Hormones within illustrated in Table I. Cancer Cells

Mechanism of Action of Heart After binding to their respective receptors on the cancer Hormones: Receptors cells, the cardiac hormones inhibit the RAS mitogen- activated protein kinase (MAPK)/extracellular signal-related Each of the human cancer cell types examined above has kinase-kinase (MEK)-ERK1/2 kinase pathway (Figure 2), natriuretic peptide receptors to mediate the effects of these which is a prototypical signal transduction pathway in cancer peptide hormones (21-32). Thus, when human breast and (38, 39). This pathway is aberrantly activated in many types kidney adenocarcinoma cells were evaluated by western of neoplasms, including prostate and breast cancer, with this blots, natriuretic peptide receptors (NPR) A, B and C found activation being associated with a poor prognosis (38, 39). to be present (22-25). Breast adenocarcinoma cells have These heart hormones inhibit this pathway at several steps developed NPR-A and -C receptors to mediate the effects of (namely, multiple kinase inhibitors), as follows. ANP via a membrane-bound guanylate cyclase, which is part of the NPR-A receptor, and via NPR-C receptor-mediated RAS. Structural alteration in the GTPase RAS occurs in 25% mechanisms, respectively (2, 3). The NPR-C receptor does to 30% of human carcinomas, which allows them to relay not contain guanylate cyclase, which catalyzes the formation mitogen signals in a ligand-independent manner, thereby of the intracellular messenger cyclic GMP (2, 3). obviating the need for ligand activation of growth factor

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Figure 3. Vessel dilator at 10 μM inhibits phosphorylation of mitogen-activated protein kinase kinase (MEK1/2) by 98%. This was maximal at two hours and significant at p<0.00001 when evaluated by analysis of variance. MEK1/2 is at 45 kDa while β-actin (loading control) is at 42 kDa. The relative intensity in the bar graphs is a comparison against untreated MEK1/2 (100% intensity). Modified from reference 45.

receptors that occurs in normal cells (40, 41). Attempts to (43). Stimulation of RAS by mitogens such as epidermal target RAS by perturbing its interaction with either Son of growth factor (EGF) is also inhibited by these four cardiac Sevenless gene (SOS) or with growth factor receptor-bound 2 hormones (44). (GRB2) (Figure 2), have not yielded viable drug development candidates, largely because of the inherent difficulties in MEK1/2 kinases. Vessel dilator and kaliuretic peptide (each disrupting protein–protein interactions with drug-like at 10 μM) inhibited the phosphorylation of MEK1/2 kinases molecules (40). Several drug discovery programs have also by 98% and 81% (both at p<0.0001), respectively [Figure 1; been devoted to finding inhibitors of farnesyltransferase as a (45)]. The inhibition of MEK1/2 lasted for at least two hours, means of preventing the membrane localization of RAS (40). when it was maximal, with both heart hormones (45). Despite the successful identification of several chemical leads LANP and ANP (each at 10 μM) inhibited the that effectively inhibited this prenylation enzyme, tumor cells, phosphorylation of MEK1/2 kinase by 97% and 88% (each have generally been proven to be impervious to the action of at p<0.00001) (46). The inhibition of MEK1/2 was maximal this class of inhibitors (40). at two hours and ceased after four hours with both heart Vessel dilator and kaliuretic peptide (each at 1 μM) hormones (46). The ability of the heart hormones to inhibit inhibited the phosphorylation of RAS by 95% and 90% MEK1/2 is also inhibited by an antibody against cyclic GMP (p<0.0001), respectively. At 0.01 μM of kaliuretic peptide, and cyclic GMP itself inhibited MEK1/2 phosphorylation by the maximal inhibition was 95% (p<0.0001). The inhibition 93% (46). Thus, ANP, vessel dilator, KP and LANP each of RAS lasted for 48 to 72 hours, secondary to both peptides inhibit MEK1/2 kinase mediated via cyclic GMP, as part of (42). Their ability to inhibit RAS was inhibited by an their anticancer mechanism(s) of action (45, 46). antibodyagainst cyclic GMP, and cyclic GMP itself inhibited RAS phosphorylation (89%; p=0.0015) (42). Inhibition of the activation of ERK 1/2 kinases. Vessel dilator ANP and LANP (each at 0.1 μM) inhibited the and kaliuretic peptide (each at 1 μM) inhibited the activation, phosphorylation of RAS by 90% and 83% (both at p<0.0001) namely the phosphorylation, of ERK1/2 kinases by 96% and (43). At 0.01 μM of LANP, the maximal inhibition was 89%, 70% (each at p<0.001) (47). Both have significant effects which occurred within 5 minutes. Both peptide hormones within five minutes at a concentration of 0.01 μM (47). The inhibit RAS for 3 to 4 hours (43). Their ability to inhibit RAS inhibition of ERK1/2 lasted for at least two hours with both is also inhibited an antibody against cyclic GMP and cyclic heart hormones (47). Maximal inhibition of the GMP itself inhibits RAS phosphorylation (72%; p=0.009) phosphorylation of ERK1/2 kinases by ANP and LANP was

2518 Vesely et al: Cardiac Hormones: Anticancer Agents (Review)

94% and 88%, respectively (p<0.0001) (48). The inhibition This study demonstrates that four cardiac hormones cause of ERK1/2 kinases lasted for at least two hours, when it was cytotoxicity in human cancer cells while sparing healthy maximal, with ANP and LANP (48). human cells, when evaluated over a seven-day period. c-Jun-N-terminal kinases. c-Jun-N-terminal kinase (JNK), also Four Heart Hormones Cause Cell Death of known as stress-activated protein kinase 1 alpha (SAPK1α) is Human Cancer Cells But Not of Healthy Cells activated by a variety of extracellular stimuli such as growth factors and environmental stresses (49, 50). Thus, JNK is Nuclear matrix proteins (NMPs) make up the internal structure activated by EGF, tumor necrosis factor, platelet-derived (framework) of the nucleus and are associated with such growth factor and transforming growth factor, as well as by functions as DNA replications, RNA synthesis, and hormone diverse environmental stresses (51-53). Activation of JNK by receptor binding (63, 64). The identification of cell type- EGF is dependent upon H-RAS activation (51, 54). JNK2 is specific NMPs supports their potential contribution to cellular not only associated with cancer development but also with differentiation and tissue development (64). Although the invasion of cancer, e.g. breast cancer (55). Loss of JNK nuclear matrix has been shown to be highly insoluble in vitro, activation coupled with loss of ERK activation promotes cell it is now known that cell death releases soluble NMPs that can death (56). Lung cancer cell growth (53), prostate cancer be detected in the culture supernatant and other fluids proliferation and prostate cancer xenograft growth are containing dead and dying cells (65, 66). Because the level of dependent upon JNK2 (57, 58). Of all the JNK kinases, it is NMP 41/7 detected in the culture supernatant is a function of JNK2, also known as mitogen-activated protein kinase 9 the number of dead or dying cells, the measurement of NMP (MAPK9), that is preferentially required for mediating 41/7 is useful in quantifying cell death (65). proliferation of lung cancer (59) and prostate cancer cells (57). Each of these heart hormones caused death in up to 36% Vessel dilator, LANP, kaliuretic peptide and ANP maximally (p<0.0001) of pancreatic adenocarcinoma cells and in up to reduced the expression of JNK2 by 89%, 88%, 77%, and 89%, 28% (p<0.0001) of prostate cancer cells, over a respectively (each at p<0.0001), in human small-cell lung concentration range of 100 pM to 10 μM (67). There was no cancer cells (60). In human prostate adenocarcinoma cells, cell death of normal human prostate, kidney, or lung cells at JNK2 was maximally decreased by 76%, 84%, 57%, (each at the above concentrations (67). Thus, these four cardiac p<0.0001), and 26% (p<0.01) by vessel dilator, LANP, hormones cause death of pancreatic and prostate cancer cells kaliuretic peptide, and ANP, respectively (60). These results but, at the same concentrations, they do not eliminate normal indicate that the four cardiac hormones are significant prostate, lung, or kidney cells. inhibitors (by up to 89%) of JNK2 in human small-cell lung cancer cells and in human prostate adenocarcinoma cells (up Conclusion to 84%) as part of their anticancer mechanism(s) of action. Four cardiac hormones coded for by one gene (namely, atrial Four Heart Hormones Cause Cytotoxicity of natriuretic peptide) have significant (p<0.0001) anticancer effects Human Cancer Cells But Not of Healthy Cells in reducing up to 97% of all cancer cells tested in cell culture (21-32). Furthermore, these cardiac hormones eliminate up to It would be expected that the heart hormones may be 80% of human pancreatic adenocarcinomas, up to two-thirds of cytotoxic rather than cytostatic as cytostatic agents do not human breast carcinomas and up to 86% of human small-cell cause tumor shrinkage or elimination as these heart lung carcinomas in athymic mice, suggesting that they may have hormones do (33, 36, 37). Cytotoxicity was directly tested very beneficial effects on a number of different types of cancer with a Cyto-Tox-Glo™ Cytotoxicity Assay (Promega, (35-37). Their mechanism(s) of action towards cancer cells is a Madden, WI, USA), which is a cell-based luminescent assay very strong (up to 98%) inhibition of the activity of the RAS- that measures the extracellular activity of a distinct MEK1/2-ERK1/2 kinase cascade mediated via the intracellular intracellular protease (dead-cell protease) when the protease mediator cyclic GMP (42-48). Further clinical studies should is released from membrane-compromised cells (61). The determine their utility in humans with cancer. results of this assay directly correlate with the percentage of cells undergoing cytotoxicity (61). Over a seven-day period, Acknowledgements the four heart hormones caused cytotoxicity of up to 75% on human prostate cancer cells and up to 58% on human This work was supported in part by Merit Review Grants from the United States Department of Veterans Affairs, the James and Esther pancreatic cancer cells, over a concentration range of 100 King Florida Biomedical Research program, the Florida Department pM to 1 μM (62). There was no cytotoxicity towards prostate of Health, and the Mama Mare Breast Cancer Foundation. The and lung cells from healthy individuals exposed to the same contents of this article do not represent the views of the Department concentrations of the cardiac hormones for seven days (62). of Veterans Affairs or of the United States Government.

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Received March 23, 2012 51 Derijard R, Hibi M, Wu IH, Barrett T, Su B, Deng TL, Karin M Revised April 9, 2012 and Davis RJ: Jnk1 – a protein-kinase stimulated by UV-light and Accepted April 10, 2012 Ha-Ras that binds and phosphorylates the c-Jun activation domain.

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