Abstract
Four cardiac hormones are synthesized by the atrial natriuretic peptide prohormone gene. These hormones, namely, long-acting natriuretic peptide, vessel dilator, kaliuretic peptide, and atrial natriuretic peptide, help regulate blood pressure and blood volume by causing vasodilation, diuresis, and sodium excretion. These cardiac hormones reduce up to 97% of all cancer cells in vitro. These cardiac hormones eliminate up to 86% of human small-cell lung carcinomas, two thirds of human breast cancers, and up to 80% of human pancreatic adenocarcinomas growing in athymic mice. Their anticancer mechanisms of action, after binding to specific receptors on cancer cells, include targeting the Rat sarcoma-bound guanosine diphosphate conversion to RAS guanosine triphosphate (95% inhibition)–mitogen-activated protein kinase kinase 1/2 (98% inhibition)–extracellular signal-related kinase 1/2 (96% inhibition) cascade in cancer cells. They also reduce c-Jun-N-terminal kinase 2 up to 89%. These multiple kinase inhibitors are also inhibitors of vascular endothelial growth factor (VEGF) and its VEGFR2 receptor (up to 89% inhibition). They reduce β-catenin up to 88%. They inhibit the WNT pathway up to 68%, and secreted Frizzled-related protein 3 is decreased up to 84%. AKT, a serine/threonine-protein kinase, is reduced up to 64% by the cardiac hormones. Signal transducer and activator of transcription 3, a final “switch” that activates gene expression that leads to malignancy, is decreased by up to 88% by the cardiac hormones. Of importance, the cross talk between the multiple kinases, VEGF, B-catenin, WNT, and STAT pathways is inhibited by the 4 cardiac hormones.
Keywords
Atrial natriuretic peptide gene in the heart codes for a 126-aa prohormone, which, through proteolytic processing, results in the formation of 4 cardiac hormones. These 4 cardiac hormones are LANP, consisting of the first 30 aa from the N-terminus of the 126-aa prohormone; vessel dilator (VDL), aa 31 to 67 of the prohormone; kaliuretic peptide (KP) aa 79 to 98 of this prohormone; and ANP, consisting of aa 99 to 126 of the 126-aa prohormone.
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The 4 cardiac hormones from the ANP prohormone reduce blood pressure by promoting salt excretion, decreasing blood volume, and vasodilating blood vessels in both animals5–11 and humans.12–14 Although each of the cardiac hormones from the ProANP gene has salt-excreting properties in persons with congestive heart failure (CHF), one of the cardiac hormones, that is, vessel dilator, is better than the rest of the cardiac hormones for treating CHF.14,15 Its effects in the kidney are not blunted like ANP's and BNP's effects.14,15 The reasons that vessel dilator works better than ANP and BNP are the following: (1) vessel dilator enhances sodium excretion via enhancing the synthesis of prostaglandin E2, which, in turn, inhibits Na+-K+ ATPase as first shown by Gunning et al., 7 whereas ANP does not enhance the synthesis of prostaglandin E27,16,17 and (2) ANP's effects in the kidney are blunted, as it is rapidly proteolytically cleaved in the kidney, whereas vessel dilator is not. 18 Vessel dilator is secreted into the urine intact. 18 (3) Vessel dilator's effects last significantly longer than ANP (ie, >6 hours vs 30 minutes).12–14 It is to be noted with respect to treating CHF that vessel dilator is potassium sparing as opposed to ANP in animals 6 and in humans12–14 and, thus, should not lead to hypokalemia in persons with CHF who are treated with vessel dilator.
Cardiac Hormones for the Treatment of Cancer
After the cardiac hormones were shown to help regulate blood volume and blood pressure by enhancing sodium excretion and vasodilating blood vessels,5–15 it was found that they have anticancer effects when used in super physiologic doses.19–38 Thus, the 4 cardiac hormones synthesized by the ANP prohormone reduce up to 97% of a large variety of cancer cells in culture. In human prostate cancer cells, for example, vessel dilator, ANP, LANP, and kaliuretic peptide decrease (ie, eliminate) 97.4%, 89%, 87%, and 88%, respectively, of these cancer cells (each at P < 0.001). 19 Each of the 4 cardiac hormones similarly reduce human pancreatic, 20 colon, 21 breast, 22 renal, 23 small-cell lung, 24 squamous cell lung, 25 and ovarian carcinomas. 26 Less common tumors such as medullary thyroid carcinomas, 27 angiosarcomas of the heart, 28 melanomas, 29 and glioblastomas of the brain 30 are likewise decreased in vitro secondary to the 4 cardiac hormones. ANP also decreases hepatoblastoma cells in culture, but the other cardiac hormones’ effects have not been investigated on hepatoblastoma cells. 31 BNP does not have anticancer effects on any of these cancer cells.19–30
In whole animal studies with human cancers implanted, the 4 cardiac hormones eliminate up to 80% of human pancreatic adenocarcinomas with a one-time treatment subcutaneously via an osmotic pump for 28 days. 32 The human pancreatic carcinomas that are eliminated with the continuous subcutaneous treatment never recur in the primary site when the animals are followed for their entire normal lifespan. 32 These animals die of old age and not of pancreatic cancer. 32 Even in the animals where the pancreatic cancer is not eliminated, these cancers with each of the cardiac hormones is reduced to less than 10% of the volume of the untreated cancers and with vessel dilator to less than 2% of the volume of the untreated cancers. 32 These findings are a marked improvement over the current treatment of pancreatic cancer where the mean survival is 4 months; and this 4-month survival includes persons treated with surgery, current cancer chemotherapy, and/or radiation therapy where these 3 treatments extend a person's life 1 to 2 months.33,34 The 5-year current survival of pancreatic cancers is only 1%.33,34 In the animals with pancreatic cancer treated with the cardiac hormones, it is also important to note if a metastatic lesion develops; it also can be eliminated by using a different cardiac hormone from the one used to treat the primary lesion. 32
The cardiac hormones can also eliminate up to 86% of human small-cell lung carcinomas growing in athymic mice when treated for 28 days via subcutaneous pumps. 35 As with the human pancreatic adenocarcinomas, the human small-cell lung cancers never recur in the primary site once eliminated. 35 The 4 cardiac hormones’ ability to individually eliminate human small-cell lung cancer varies compared to human pancreatic cancer as outlined in Table 1. Vessel dilator and LANP were the most beneficial in treating human small-cell lung cancers and ANP the least beneficial (Table 1). Treatment of pancreatic adenocarcinomas, on the other hand, was best with ANP (Table 1) as opposed to human pancreatic adenocarcinomas. Treated small-cell lung carcinomas that are not eliminated by the cardiac hormones grow rapidly similar to untreated controls. 35 Metastatic human small-cell lung cancers can be eliminated, however, by using the cardiac hormones in a sequential manner, that is, one after another, each for 4 weeks. 35
Ability of Cardiac Hormones to Eliminate Human Cancer Growing in Athymic Mice
As observed in Table 1, vessel dilator and kaliuretic peptide are the best at eliminating human breast adenocarcinomas growing in mice where they each eliminate two thirds of the human breast cancers when treated subcutaneously for 28 days. 36 Again, ANP had the least beneficial effects, with one third of the breast cancers being eliminated. 36 As with the other human cancers, there was no recurrence of the human breast cancers in the primary site during the lifespan of the mice once they were eliminated by one of the cardiac hormones. 36
It would seem that the best way to treat human pancreatic adenocarcinomas is via subcutaneous pumps where the tumor is exposed to the circulating cardiac hormone continuously when this treatment was compared with treating the pancreatic cancer with bolus infusions twice weekly for 4 weeks via a vascular port with vessel dilator or ANP, the bolus infusions eliminated only one third and one sixth, respectively, 37 of the pancreatic cancers versus 80% with ANP continuous infusion. 32 Furthermore, a major difference in these 2 treatments is that the bolus infusion–treated pancreatic cancers that were not eliminated grew very large 37 as opposed to continuous infusion where these same cancers that were not eliminated were reduced to only 2% to 10% of the volume of the untreated cancers. 32
Cardiac Hormones— Anticancer Metabolic Targets
The cardiac hormones have multiple targets within cancer cells that mediate their anticancer effects (Fig. 2). The first step in their mechanism of action is binding to specific receptors.3,38 ANP has 3 receptors, that is, natriuretic peptide receptors (NPRs) A, B, and C.3,38 Each of the cancer cells outlined above have NPR-A, NPR-B and NPR-C receptors.19–22 Vessel dilator, LANP, and kaliuretic peptide have different specific receptors from ANP.5,39,40 Vessel dilator, LANP, and kaliuretic peptide are linear peptide hormones and, thus, do not bind to the NPR receptors, which bind only ring-structured peptides like ANP and BNP, which have a ring formation made by a bond between 2 cysteines in their structures. 3
Cardiac hormones inhibit RAS-GTP, MEK1/2, and ERK1/2 kinases of the RAS-MEK1/2-ERK1/2 kinase cascade by 95% to 98%. These multiple kinase inhibitors are also strong inhibitors (ie, 91%) of DNA synthesis within cancer cells. Other targets, which the cardiac hormones inhibit within cancer cells, are VEGF, the VEGFR-2 receptor, β-catenin, sFrizzled protein-3, JNK, STAT 3, and the WNT pathway. DNA, deoxyribonucleic acid; GRB2, growth factor receptor-bound protein 2; MOS, Malony murine sarcoma virus serine/threonine protein kinase; RAF, rapidly accelerated fibrosarcoma serine/threonine protein kinase; RAS-GTP, rat sarcoma bound guanosine triphosphate; RTK, tyrosine kinase receptor; SHC, Rous sarcoma SH2 C-terminal–binding domain adapter protein; SOS, Son of Sevenless gene; SRC, Rous sarcoma viral proto-oncogene tyrosine kinase. Modified with permission from Sun et al. Eur J Clin Invest. 2010.
Ras-Mek 1/2 - Erk 1/2 Kinase Cascade
Ras
After entering the cell via their specific receptors, each of the 4 cardiac hormones synthesized by the ProANP gene inhibits the conversion of rat sarcoma–bound guanosine diphosphate (RAS-GDP), an inactive form of RAS, to its active form, RAS–guanosine triphosphate.41,42 Vessel dilator, kaliuretic peptide, ANP, and LANP inhibit this conversion to active RAS by 95%, 90%, 90%, and 83%, respectively.41,42 The 4 cardiac hormones also inhibit the enhancement of conversion of inactive RAS to active RAS by mitogens such as epidermal growth factor (EGF) 43 and insulin. 44 The inhibition of the conversion to the active form of RAS by the 4 cardiac hormones seems to be mediated by the intracellular mediator 3’5’ cyclic guanosine monophosphate (GMP) as when an antibody to cyclic GMP is added with the cardiac hormones, it blocks their ability to inhibit RAS.41,42 Cyclic GMP itself inhibits the conversion of RAS-GDP to RAS–guanosine triphosphate.41,42 The ability to effectively inhibit RAS is considered a major therapeutic goal in cancer treatment because previous attempts to inhibit RAS have proved futile.45,46
Mek 1/2 Kinases
In the RAS mitogen-activated protein kinase kinase (MEK) 1/2–extracellular signal-regulated kinase (ERK) 1/2 kinase cascade (Fig. 2) are 2 kinases termed MEK 1 and MEK 2. Mitogen-activated protein kinase kinase (MKK 1) or MEK 1 specifically phosphorylates threonine and tyrosine residues present in the Thr-Glu-Tyr motif of ERK 1/2.47,48 The MEK 2 resembles MEK 1 in phosphorylating ERK 1/2 but is 7 residues longer than MEK 1, with its amino acid sequence being 81%, identical to MEK 1. 48 Vessel dilator, LANP, kaliuretic peptide, and ANP inhibit the phosphorylation of MEK 1/2 kinases by 98%, 97%, 81%, and 88%, respectively.49,50
An antibody against cyclic GMP when added with the 4 cardiac hormones blocks the ability of the cardiac hormones to inhibit MEK 1/2 kinases.49,50 Cyclic GMP itself inhibits MEK 1/2 phosphorylation by 93%, suggesting that cyclic GMP is the mediator of the cardiac hormones’ inhibition of MEK 1 and 2 kinases.49,50
Erk 1/2 Kinases
Extracellular signal-related kinases 1/2 can directly translocate to the nucleus and stimulate the production of nuclear oncogenes such as c-Fos.51,52 Epidermal growth factor (EGF) 52 and vascular endothelial growth factor (VEGF) 52 as well as c-Jun-N-terminal kinases (JNKs) 51 mediate their cancer-causing effects via ERK 1/2 (Fig. 2). Vessel dilator, ANP, kaliuretic peptide, and LANP inhibit the phosphorylation of ERK 1/2 kinases by 96%, 94%, 70%, and 88%, respectively.53,54 Thus, the cardiac hormones are multiple kinase inhibitors inhibiting the basal activity of each step in the RAS-MEK 1/2-ERK 1/2 kinase cascade in human cancer cells as illustrated in Figure 2. In addition to inhibiting the basal activity of ERK 1/2 kinases, the cardiac hormones inhibit the ability of growth factors such as EGF and insulin to stimulate ERK 1/2 kinases. 55
c-Jun-N-terminal Kinases
c-Jun-N-terminal kinase (JNK), as previously mentioned, stimulates ERK 1/2 kinases. 52 Of these kinases, JNK-2 has been associated with the invasion of cancers. 56 Prostate cancer proliferation and prostate cancer growth are dependent on JNK-2 kinase. 57 Likewise, lung cancer growth is dependent on JNK-2. 58 c-Jun-N-terminal kinase 2 is reduced by vessel dilator, ANP, LANP, and kaliuretic peptide by 89%, 89%, 88%, and 77%, respectively, in human small-cell lung cancer cells. 59 These 4 cardiac hormones also decrease JNK-2 up to 84% in human prostate adenocarcinoma cells. 59
Wnt-Signaling Pathway
The name of the WNT pathway comes from a portmanteau of Int (integration 1 gene in breast cancer) and Wg (wingless) Drosophila, which has the best characterized WNT gene. 60 WNT-3a is the protein encoded in humans of this pathway. 61 WNT signaling is stimulated by RAS 62 and VEGF pathways 62 as part of the cross talk between these cancer-promoting pathways. The 4 cardiac hormones reduce WNT-3a up to 68% in human pancreatic cancer cells and reduce WNT-3a up to 53% in human colorectal adenocarcinoma cells. 63
Secreted Frizzled-Related Protein-3
Secreted Frizzled-related protein-3 (sFRP-3), a 300 a.a. glycoprotein,64,65 promotes renal cancer growth when injected into athymic mice. 66 Secreted Frizzled-related protein-3 has been linked to tumor promotion in other types of cancers as well. 67 The Frizzled receptor contains sFRP-3.68,69 Atrial natriuretic peptide activates this receptor. 70 Atrial natriuretic peptide and the Frizzled receptor colocalize on the cell membrane within 30 minutes after ANP addition to culture medium. 70 We have found that vessel dilator, LANP, kaliuretic peptide, and ANP decrease the levels of sFRP-3 by 77% to 78% in human pancreatic cancer cells, 83% to 84% in human colorectal cancer cells and 66% to 68% in human renal cancer cells. 71 Their ability to reduce sFRP-3, which is the cysteine-rich domain of the Frizzled receptor, 65 blocks the propagation of the signals that cause cancer growth.
Akt
The serine/threonine protein kinase AKT (Fig. 2) is important in the growth of many cancers.72–74 AKT is derived from the “AK” mouse strain that develops spontaneous thymic lymphomas, whereas the “t” stands for thymoma. 75 AKT is overexpressed in colorectal cancer cells but not in normal colonic mucosa and hyperplastic polyps. 76 Atrial natriuretic peptide decreases the activation of AKT approximately 2-fold in cell culture. 70 The other cardiac hormones also decrease AKT 77 with their results as follows: Vessel dilator, kaliuretic peptide, and LANP reduce the concentration of AKT by 60%, 61%, and 59% in human pancreatic carcinoma cells; by 47%, 45%, and 46% in human colorectal cancer cells; and by 31%, 32%, and 31% in renal adenocarcinoma cells. 77
Vascular Endothelial Growth Factor
Vascular endothelial growth factor has direct effects of causing cancer cells to grow. 78 This growth is partially due to VEGF's enhancing of blood vessel growth into tumors, which allows oxygen and nutrients to reach the tumor.78–80 Vascular endothelial growth factor's mechanism of action involves stimulating RAS,81,82 MEK 1/2,83,84 and ERK 1/2.85,86 The main receptor involved in VEGF's cancer enhancing effects is the VEGFR2/KDR/Flk-1 receptor.79,80,87 Vessel dilator, LANP, kaliuretic peptide, and ANP decrease the VEGFR2 receptor up to 92% in human prostate cancer cells, up to 83% in human pancreatic cancer cells, and up to 89% in human small-cell lung cancer cells. 88 These 4 cardiac hormones also decrease VEGF's concentrations up to 58%. 88 These cardiac hormones are, thus, the first dual inhibitors of VEGF and its R2 receptor, as there are no compounds that inhibit both, although there are compounds that inhibit VEGF and other compounds that inhibit the VEGFR2 receptor. 88
β-Catenin
Beta-catenin (β-catenin) is one of the downstream targets of VEGF (Fig. 2). 89 β-catenin activation leads to pancreatic,90,91 colon,92,93 renal,94,95 ovarian, 96 endometrial, 96 and gastric cancers. 97 Vessel dilator, LANP, kaliuretic peptide, and ANP reduce β-catenin up to 88% in human pancreatic cancer cells, up to 73% in human renal adenocarcinoma cells, and up to 83% in human colorectal adenocarcinoma cells (Skelton et al., unpublished observation). Atrial natriuretic peptide decreases the expression of β-catenin, which is associated with a redistribution of β-catenin from nuclear and cytoplasmic compartments to cell-to-cell junction sites and is associated with a decrease in proliferation of colon adenocarcinoma cells. 70 Atrial natriuretic peptide also causes a significant down-regulation of c-Myc and cyclin D-1 gene transcription regulated by β-catenin. 70 β-catenin would seem to be a central target of the cardiac hormones’ anticancer effects because the cardiac hormones inhibit upstream RAS kinase, which activates β-catenin, 98 and downstream c-Jun-N-terminal kinase and VEGF, which are activated by β-catenin,89,99 as illustrated in Figure 2.
Signal Transducers and Activators of Transcription
Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that are the final “switches” that activate the gene expression patterns that lead to malignancy.100–102 Signal transducers and activators of transcription 3 of the STATs is important in cancer formation.102,103 Signal transducer and activator of transcription 3 is overexpressed in a variety of human tumors.102,104,105 Epidermal growth factor receptor-mediated growth of squamous carcinoma cells is known to require STAT 3 but not STAT. 104 Targeting STAT 3 is also a strategy for reversing paclitaxel therapy resistance. 106
Vessel dilator, LANP, kaliuretic peptide, and ANP decrease STAT 3 by 88%, 54%, 55%, and 65%, respectively, in human small-cell lung cancers and by 66%, 57%, 70%, and 77% in human pancreatic adenocarcinoma cells. 107 These cardiac hormones do not decrease STAT 1 in either human small-cell lung cancer or pancreatic adenocarcinoma cells. 107 Thus, the 4 cardiac hormones are significant inhibitors of STAT 3 but not of STAT 1, which suggests a specificity for these hormones’ anticancer mechanism(s) of action in human cancer cells. 107
Cross Talk in Cancer Cells
As one observes in Figure 2, there is a significant amount of cross talk between kinases, STAT 3, and VEGF in cancer cells. This cross talk is illustrated in Figure 2 by solid arrows, where one kinase, STAT, or VEGF, stimulates the activation or phosphorylation of another kinase. Vessel dilator, LANP, kaliuretic peptide, and ANP are multiple kinase inhibitors as well as dual inhibitors of VEGF and its VEGFR2 receptor, which allows them to block the cross talk between the various enzymes in cancer cells; and this complex interaction is illustrated by the inhibitory sign (-) in Figure 2.