Potential Use of Peptides in the Targeting and Therapy of Solid Tumors using a Fetal-derived Peptide: A Review and Prospectus

This article was intended to demonstrate the usage and advantages of employing peptides as therapeutic agents in the treatment of cancer. The concept of using peptides in cancer therapy as a supplement/adjunct to traditional chemotherapy has long been considered. The pharmaceutical industry has been reticent in adopting this mode of cancer therapy possibly due to financial costs of synthesis and/or manufacturing retooling methodologies. As described in the present report, certain peptides could present several advantages over heterocyclic hydrocarbons presently used in the chemotherapeutic drug industry. The benefits of peptide usage in cancer therapy have shown notable cell target specificity, drug cargo transport, short half-lives, highly reduced cell toxicity, and ability to trace the peptide distributions by means of radioisotope, fluorescence, and other scanning techniques. As an example of a candidate peptide for use as a cancer adjunct therapeutic treatment, an Alpha-fetoprotein (AFP) derived growth inhibitory peptide (GIP) was selected due to the many cancers related published reports on GIP over the last two decades. Such reports have encompassed cancer suppressed growth, adhesion, cell spreading, metastasis, and cell-to-cell contact. Other lesser-known GIP reports have encompassed cytoskeleton changes, chemotaxis, cell interaction with the extracellular matrix, tumor cell-to-stroma cell communication networks, signal transduction receptor crosstalk, and interaction with kinase enzymes.

The potential use of peptides in the armamentarium of chemotherapies has recently garnered increased attention in the biomedical field regarding human cancer treatments. The standard modes of therapy for cancer patients following World War II have largely employed the use of radiation and heterocyclic hydrocarbons which often contain cores of benzene ring derivatives containing nitrogenous side chain residues [1]. This classical form of chemotherapy in humans is known to be toxic to all dividing (mitotic) cells in the human body including both normal (non-malignant) cells as well as malignant cancer cells, without exhibiting differences or discrimination. In the course of chemotherapy, normal non-cancer mitotic dividing cells exhibit delirious side effects within organs/tissues such as: hair follicles, inner linings of the digestive tract, and most notably, the bone marrow. These chemotherapeutic adverse side effects result in: hair loss, nausea, vomiting, bloating, loss of appetite, diarrhea, upset stomach, and bone marrow depletion of white blood cells, red blood cells, and platelets [2]. Hence, the search for far less toxic pharmaceutical drugs and compounds for cancer therapies would be highly beneficial and sought after. The use of peptides derived from human naturally-occurring blood proteins would further seem to be a viable adjunct to chemotherapeutic drug agents presently employed in the medical-pharmaceutical field.

The advantages of using peptides as adjunct chemotherapeutic agents in human cancer therapeutic treatments are both many and varied. For example, peptides have been denoted as highly efficient and specific cell targeting agents with short half-lives, and less off-target effects [3]. These latter effects would also avoid their extended lingering presence in the target area. By the use of amphipathic peptides as targeting agents, such peptides can specifically seek out and bind to the cell surface of cancer cells [4]. Such cancer cell homing specificities are possible by taking advantage of the cell membrane “lipid flip” phenomenon which produces a negative cell membrane surface change only on distressed cells targeted for apoptosis such as malignant cells [5]. In this inversion process, phosphatidylserine, which is usually on the inside of the phospholipid leaflet of the bilayer cell membrane, flips to the outside leaflet substituting for phosphoryl ethanolamine (PPE). This switch event produces a net negative charge to the outer surface of the distressed cell membrane [5]. In contrast, normal cells display a net positive surface charge on the outer surface of the plasma cell membrane. As a result of this, amphipathic peptides display a propensity for attraction only to distressed (cancer) cells; this feature presents an enormous advantage over present day classical organic heterocyclic hydrocarbon chemotherapies.

An example of a peptide derived from a naturally-occurring blood protein is herein described by use of an alpha-fetoprotein (AFP)- derived peptide that has been studied and researched for more than three decades by the present author (GJM) [6-9]. AFP itself is a tumor-associated fetal protein (oncofetal protein) from which a 34 amino acid segment has been isolated/purified and termed the “Growth Inhibitory Peptide (GIP)” [8,9].  AFP has served for many years as a biomarker for liver cancer (hepatomas) and malignant tumors of the reproductive and gastrointestinal tracts [10]. When present in fetal shock/stress environments, the full-length AFP molecule undergoes a conformational change which temporarily converts the “growth enhancing protein” into a “growth inhibitory protein.” In this phase, the entire full-length AFP protein is referred to as “transformed AFP” (TAFP) [11]. This structural conformational change exposes a concealed and buried occult AFP peptide segment site named above as GIP [12]. These 34 amino acid segments from AFP have now been synthesized as a free peptide fragment and has since been both biologically and biochemically characterized [9,13]. A recent publication has reported that GIP is capable of suppressing the growth of nine different cancer tumor types [6].

Growth Inhibitory Peptide (GIP) can block the growth and metastasis of solid tumors

In previous studies of GIP administrated to rodent xenograft models of human breast cancer (MCF-7 and GI-101), it was demonstrated that adenosine triphosphate (ATP) energy levels significantly decreased by 7-days post tumor implantation [7]. Thus, cancer cells deplete their ATP energy stores which become considerably reduced. Shortly thereafter, an RNA microarray study in cultured MCF-7 cancer cells, showed that 15 different ATP-associated genes were down-regulated [13] [Table-1].

Table 1: A global RNA microarray analysis was performed on human cultured MCF-7 breast cancer cells treated with Growth Inhibitory Peptide (GIP) for eight days. Transcripts displaying ± 1.0 or larger log fold (log base 2) decreases for RNA analysis shows genes that were downregulated by GIP associated/binding Ca2+ associated proteins and channel-related proteins.

*Global RNA microarray was performed by Affymetrix Genomics Laboratory at Yale/Keck University facility, New Haven, Ct. in collaboration with Dr. Kathleen F. Arcaro, University of Massachusetts, Amherst, MA.

Following an initial injection of GIP into mice bearing a xenograft, GIP circulates in the murine blood vascular circulation and encounters the implanted tumor cells. GIP then attaches to the outer bilayer surface of the tumor plasma cell membrane [13,14]. The AFP-derived peptide penetrates through the cell membrane by both a pore-forming and/or a cell-penetrating mechanism [4]. This event is followed by an increased inward current and a decrease in the cell membrane electrical resistance as demonstrated by Sharps electrode techniques [15,16,17]. After one hour, GIP is observed as diffusely distributed throughout the cell cytoplasm as demonstrated in MCF-7 cultured cells. By two hours, concentrated GIP has become localized (surrounding) in layers around the outer periphery of the nuclear membrane in a perinuclear fashion [9]. It is tempting to speculate that GIP could serve to gate the nuclear membrane and possibly regulate the trans passage of transcription factors and receptors into and out of the nucleus [18,19]. At this point in GIP localization, depletion of the cell’s ATP-energy reserves has decreased as described above [10]. In the course of GIP cell membrane passage, it has been reported that GIP interacts with various K-voltage gated membrane channels such as KV1-1 and KV1.3 ion channels and with Transient Receptor Potential (TRP) channels. These channel activations are capable of producing G1-to-S cell cycle arrest in cultured MCF-7 breast cancers [13,20]. 

It has further been reported that GIP has properties as an amphipathic β-sheet peptide [21]. Such peptides are known to penetrate cell membranes within several minutes of attachment, show no cytotoxicity, and display high solubility in water and saline solutions [22,23]. Since the mechanism of GIP cell membrane trans-passage has now been established, it is known that GIP does not employ cell surface receptors to enter cells and doesn’t participate in the endocytotic process as such. For example, the endocytic process is known to require 30 to 45 minutes to complete [13], while GIP membrane passage requires only minutes. Although β-sheet peptides (i.e., GIP) are not immediately toxic, they are cell membrane disruptors which can eventually produce leaks in the cell membrane that cause slow cell death coincident with depletion of ATP energy stores as reported above.

It has also been reported that GIP can induce cell cytoskeletal shape changes in cancer cells, as well as in platelets [24] [Table 2B]. Thus, GIP is known to induce alterations in the cytoskeletal framework of cancer cells by inducing both actin and tubulin polymerization [25] [Table-3A]. In addition, it was also found that GIP can disrupt the cell membrane bilayers as demonstrated in red blood cell hemagglutination and platelet aggregation studies [8,9]. GIP is also capable of interfering with the process of cell detachment and adhesion to the ECM from a host tumor mass by mimicking adhesion proteins [Table 3C]. Thus, GIP has been shown to bind to cytoskeletal fibers which allows cells to change their shape by affecting structural rigidity changes that prevent cells detaching from a main tumor mass [24]. The cells egressing from a tumor mass enters into blood vessels for subsequent passage and migration to distant migratory destinations. Once in the bloodstream, circulating tumor cells are able to attach to endothelial-adhering intravascular platelet aggregates which shield tumor cells from phagocytic engulfment from neutrophils, monocytes, and various scavenger cells [25]. Thus, by disrupting intravascular platelet aggregation, GIP can prevent circulating tumor cells from seeking refuge and shielding (hiding) within the intravascular platelet clusters adhering to inner blood vessel walls.

Table 2A: GIP was screened for possible kinase enzyme inhibition activity in a kinase proliferation assay performed by the Upstate Bio signaling Corporation, NY. The percent (%) of kinase activity following GIP incubation is listed below. The control assay was 100% and the inhibition or enhancement is listed as percent activity of the control.

*GIP= Growth Inhibitory Peptide; Legend: ASK-1= apoptosis signal regulating kinase-1; BLK= non-receptor tyrosine kinase B-cell ligand; BRK= Breast tumor tyrosine kinase; BTK= Burton’s gammaglobulinemia kinase; cdk3/cyclin-E= cyclin dependent kinase-3 cyclin E; cdk3 P35= cyclin dependent kinase, subunit 35; C5K= C-SRC tyrosine kinase; EGFR= epidermal growth factor receptor tyrosine kinase; GSK-3B= Glycogen synthase kinase-3; HCK= hematopoietic kinase; MKK7B= mitogen activated protein kinase-7; MSK2= mitogen activated protein stress kinase; MIST1= Mitogen activated stress threonine kinase;    PKC-α= Protein Kinase-C alpha; PKC∆= Protein Kinase-C delta; DRAK= Death receptor associated kinase; TBK1= Tank Binding kinase-1 subunit.

Table 2B: The precent of kinase enhancement activity following GIP incubation is listed below. The control assay was 100% and the inhibition or enhancement is listed as percent activity of the control.

* GIP=Growth Inhibitory Peptide; Legend: EpHA4= Ephrin Tyrosine Kinase, A4 subunit; EpHB4= Ephrin tyrosine kinase, B4 subunit; ERb-B4= Epidermal Growth Factor Receptor Kinase-4, EGFR1= Epidermal Growth Factor Recpetor Kinase-1; FGR2= Fibroblast Growth Factor Receptor Kinase 2; FgR= Fibroblast growth factor kinase; IGF-1R= Insulin Growth factor receptor-1; IKK-B= IK-Kapp-B kinase; IR= Insulin Receptor kinase; Met=  Hepatocyte Growth Factor Receptor Kinase; P70S6K= p70 insulin receptor substrate kinase; ZAP-70= Zeta chain-associated kinase.

Table 3 Part A: Amino Acid Sequence matching of Growth Inhibitory Peptide (GIP) is matched to various Extracellular Matrix and Cytoplasmic Cytoskeletal- associated Proteins

Part A: Cytoplasmic Cytoskeletal Associated Proteins

Legend: The amino acid sequences of the Growth Inhibitory Peptide (GIP) were compared and matched with protein sequences derived from the GenBank data bases using the GCG (Wisconsin program FASTA sequence described in Refs 18, 19).

Table 3 Part B: Extracellular Matrix Associated Proteins

*See legend from Table 3A

Table 3 Part C: Amino Acid Sequence matching of Growth Inhibitory Peptide (GIP) with Clotting and Adhesion Associated Proteins

*See legend from Table 3A; Abbreviations: AFP= Alpha Fetoprotein; Attach P= attachment (adhesion) protein; BF= Blood Factor; BMP= bone morphogenetic protein (metalloproteinase); CSP= chondroitin sulfate proteoglycan; Cerebrogly= cerebroglycan; Coll= collage; Collag= collagenase; Collag-SF= collagenase stimulatory factor; Comp= complement component; C/ Eleg- Caenahabitis Elegans; Dros= Drosphilia melanogaster; Fib= fibrogen α-chain; Fibron= fibronectin; G-nexin= glial nexin protease; HUM= human; IP10= interferon-α-induced chemokine; Lysyl= Lysyl hydrocylase; Mus= mouse; MMP= matrix metalloprotease; Prot-P3= Proteinase protein (myeloblastin); PG-IIIa= platelet glycoprotein (fibrinogen receptor); TPA= tissue plasminogen activity; VLA-1= integrin α1, (lamin & collagen receptor); VWF= von Willebrand’s Factor; Xen= xenopus

GIP acting as an integrin antagonist (disintegrin) is capable of blocking integrin receptor activities regarding cell-to-matrix and cell-to-cell contact [26,28]. Protocadherin and Cadherin, which are adhesion proteins, are also downregulated by GIP as observed in MCF-7 RNA microarray results [Table 1] [13]. GIP further induces cell-to-cell contact inhibition by preventing MCF-7 cells from accumulating and piling up in cellular focal mounds, an effect produced by cell exposure to high estrogen levels [27]. Finally, it is of interest that K+ channels such as Kv1.3 (voltage-gated) are known to be positioned in juxtaposition to β1-integrins located within the cell membrane bilayers. GIP itself shows sequence identity (ID) with integrins and can bind to β1 integrins as disintegrin peptide mimics [24,28].

Thirdly, GIP can block the ability of tumor cells to migrate into final nesting destinations in the body distal from the original tumor mass. GIP can block the adherence of tumor cells to extracellular matrix (ECM) proteins at sites distant from the tumor mass [8,14] [Table-3B]. Acting as a disintegrin-like mimic, GIP can block MCF-7 tumor cell adherence to eight different extracellular matrix (ECM) proteins [8,20]. Such ECM proteins include laminin, fibronectin, fibrinogen, collagen, and several others [26,28]. Recent publication has now confirmed that ECM inhibition by GIP can occur [8,9]. Cells metastasizing to distant sites are subject to guidance, attachment, and settling into nesting tissue sites as directed by cytokine proteins of the chemokine family and their receptors [8,20]. An RNA microarray analysis in MCF-7 cells treated with GIP showed that ECM-related proteins such as dystrobrevin are Ca2+ associated and are found to be down-regulated by at least minus 3.0-fold [13]. Since Ca2+ related oscillations and mobilizations are required for cell migration, attachment, and spreading, the microarray data revealed that 14 different Ca2+ associated proteins were down-regulated ranging from -2.0 to -3.9-fold [13]. The PKC enzyme is also a Ca2+ dependent factor whose activity is inhibited by GIP as demonstrated in the upstate kinase screen [see table-2A]. GIP further prevented mouse ascites cancer cells to adhere to multiple body cavity organs confirmed by histo-pathological observations. Such observations demonstrated that GIP could prevent cancer cells from adhering to such organs [8]. Lastly, GIP was shown to produce inhibition of MCF-7 cell migration/spreading in a coverslip in vitro cell culture assay [20]. 

The kinase enzyme screen (KES) by the Upstate Corporation is a “wet chemistry” system was used to analyze which kinase enzymes could interact with GIP [Table 2A, 2B]. The KES is an assay screen system performed in vitro to determine the inhibition and/or enhancement of kinase enzymes with an unknown probe (i.e., GIP). The KES incubation mixture contains only three internal incubated components: 1) Kinase enzyme, 2) substrate, and 3) co-factors. GIP is then added at various concentrations to the kinase incubation mixture. Incidentally, GIP itself has not been reported to serve as a substrate for either Ser/Thr and tyrosine kinase enzymes. A potential interaction of GIP with kinases has 3 possible effects; a) inhibition, b) enhancement, or c) no effect.

Regarding the overall results of kinase screen assays, GIP was screened for possible kinase interaction in multiple assays employing 155 known kinase enzymes in cell-free systems without interference from other biochemical agents [see table 2A]. The GIP screen significantly inhibited 17 kinase enzymes while enhancing the activities of 12 other kinase enzymes. Eleven of the 17 (65%) enzymes inhibited were serine/threonine kinases while the remainder (35%) were tyrosine kinases. Percentages indicated that, GIP enhanced the activity of eight of 12 (66%) tyrosine kinases while 34% of the kinases were of the serine/threonine type [Table 2A, 2B]. These assays indicated that GIP is capable of inhibiting multiple Src-3 type kinases (serine/threonine), while enhancing several Src-2 type kinase (tyrosine) activities. In more detailed confirmation of some of the assays using IC50 curves, 6 kinase enzymes were further assayed by the kinase screen over 9-point titration curve at μMolar concentrations. Thus, certain kinase assays were also confirmed by titration curves which included four serine/threonine and one tyrosine kinase. An inhibition assay of the phospholipase-C (PLC) enzyme together with PKC was highly significant in that these kinases interact with both cell cycle and Transient Receptor Potential (TRP) calcium ion channel regulatory proteins [see below and tables 2A, 2B, and Ref 27,28]. 

The most notable GIP interactions in tables 2A and 2B with various (SRC) kinases included the following: a) TBK (Tank binding kinase; 45% inhibition), b) PKC (alpha/delta kinases; 21-23 inhibition), c) CDK3 and CDK5 kinases (18-28% inhibition), d) ZAP-70 tyrosine kinase (33% enhancement), and e) DRAK kinase (24% inhibition). The tank binding kinase (TBK) is known for its involvement & regulation of cell proliferation, apoptosis, autophagy, and innate and tumor immunity [29]. The PKC alpha/delta kinases are involved with regulation of cell proliferation, survival, cell death, and cancer progression (30). The CDK3 and CDK5 (Ser/Thr) kinases are known to regulate cell cycle G1-to-S phase transition and G2 to mitosis transition, respectfully [31,32]. The Zap-70 tyrosine kinase play a major role in initiating T-cell responses encompassing activities of antigen binding to the T-cell receptor, immunodeficiencies, and cancer (leukemia) progression [33]. Lastly, DRAK kinase is largely involved with cell death and apoptotic signaling [34,35]. As discussed in the above discourse, GIP was capable of inhibiting a multitude of kinases associated with cell proliferation, cell cycle activities, apoptosis, and cancer progression, while enhancing kinase activities regarding tumor immunity and T-cell regulation.

The Protein kinase-C (PKC) alpha assay

The PKC is a cell surface kinase that is a member of the Transient Receptor Potential (TRP) cation channel signaling complex [36]. This signaling system is a 9-member complex scaffold platform termed the “signalplex” [see table 4].  The TRP signalplex is a macromolecular cluster of proteins that function in the non-selective cationic channel signal transduction pathway associated with the cell membrane [3]. The nine members of the TRP ion channel signalplex cell membrane scaffold assembly are enumerated in the following table-4 shown below.

Table 4: List of Proteins in the Signalplex as described in Ref 36

It is noteworthy that many of the nine protein assembly groups are GIP associated with kinase activity or with amino sequence identity [see Table 3].

PKC also binds and associates with phosphatidylserine in the lipid bilayer (leaflet) within the cell membrane. Furthermore, PKC is a cell cycle control enzyme (kinase) that regulates at two levels of the cell cycle, namely, GI progression to S-phase and the G2-to-M transition. Furthermore, it has been reported that GIP down-regulates Cyclin E, up-regulates the retinoblastoma (Rb) protein, and arrests the cell cycle at the G1-to-S phase transition [13]. In light of these studies, it has been further reported that PKCs can regulate cyclins, CDKs, CDK phosphatases, and CIP & KIP cyclin inhibitors [38]. It has been demonstrated that PKC activation causes inhibition of cell cycle growth at the mid-to-late G1 phase [13]. Thus, the overall result of PKC kinase activation is to reduce or diminish Cyclin- E levels, increase retinoblastoma (Rb) levels; and increase p27 and p21 (non-phosphorylated) inhibition levels as shown in previous reports of GIP arrest of the cell cycle [13]. Overall, PKC is one of the kinase pathways that the GIP screening panel detected; PKC also inhibits cell membrane phospholipidase and is involved in TRP signaling events [see table 2A]. It is of interest that tables 2A and 2B both reinforce the concept that various kinase signaling, chemokine events, and apoptosis activities are affected by GIP. Table 2B further demonstrates that the enhanced kinases detected by GIP involves epidermal growth factor receptor signaling, adhesion events, and NFKB-related cellular responses to stresses, cytokines, free radicals, heavy metals, inflammation, and immune response regulation. 

How might GIP be used to treat solid tumors?

GIP has the potential to transport toxic molecules (chemodrugs) as cargo into tumor cells and interfere with intracytoplasmic cell movements within tumor cells [13,14]. Thus, GIP-peptide in capable of confining tumor cells to their original tumor mass, so they cannot detach and migrate (metastasize) to other parts of the body. Also, by means of adherence inhibition, GIP could contribute to making the extracellular cell matrix environment of the metastatic destination sites less accommodating to tumor cells. Finally, the GIP peptide might be able to reduce and confine the migratory potential of cancer cells still residing at the original site of the tumor mass; that is, before detached cells begin to migrate. The present results of a NFKB influence- tank kinase assay screen suggests that this action could occur, and that the RNA global microarray showing downregulation of cytoskeletal and cell adherence proteins support this observation [13].

If GIP could indeed leak and/or allow Ca++ into the cell cytoplasm and activate the TRP channels to promote Ca++ influx, then excess Ca++ accumulation could occur and be toxic to cancer cells; this type of cytotoxicity occurs in a process called “calcium cell toxicity” first reported in 1979 [39,40,41]. The toxic damage results from plasma cell membrane disruption and leakage. Thus, GIP could make potential future metastatic site destinations less accommodating by disrupting the migration and action of the SDF-1 chemokine ligand in combination with its CXCR4 chemokine receptor [42]. This action of preventing attachment of tumor cells to the nesting ECM site destinations could occur in filtration organs such as in liver, lung, and bone marrow. GIP has previously been shown to have AA sequence identity to the chemokine ligands, especially the SDF-1 ligand and could inhibit binding of SDF-1 to its CXCR4 receptor [43].

GIP as a β-sheet cell penetrating peptide could make possible the transport of cargo (toxic drugs) through the cell membrane and into the cytoplasm to deliver chemotherapy into tumor cells. It has been already demonstrated that GIP can transport doxorubicin drugs into tumor cells in culture [14]. Thus, one could attach a toxic cargo chemotherapeutic drug to GIP and have the drug delivered directly into the tumor cell. Presumably, the “lipid-flip membrane” process will allow drug entry exclusively into tumor cell since negatively charged tumor cells are distinguishable from positive charge from normal cells [5].

In the future, GIP could be labeled with 125I- (iodine) for tumor scanning and if attached to Congo-Red, could deliver toxic dyes into amyloid plaques in Alzheimer patients to localize and destroy the plaques (i.e., plaque-busting drug). Labeled GIP could also bind to small clots in blood vessels to localize emboli by radioisotope scintillation cameras. GIP could also be conjugated to drugs such as methotrexate (MTX); this was recently published for a pore-forming peptide; hence the drug conjugate might serve to overcome MTX drug resistance [44,45]. One could attach an ester bond that links the drug to the peptide; so that once inside the cell, the cytoplasm contains non-specific esterase’s that could cleave the drug free from the chemical bond of the carrier peptide. This would allow drug delivery direct exposure into the entire cell cytoplasm so as to bypass drug resistance. In summary, the multiple future therapies for cancer and other diseases treatments described in this report might be employed to include peptide fragments derived from naturally occurring blood proteins of the human body.

Funding

None; no US federal grants were used in the preparation of this paper.

Conflicts of interest

The author declares there are no known conflicts of interest in the preparation of this manuscript.

Acknowledgement

The author extends his thanks and gratitude to Ms. Sarah Andres for her commitment and time expenditure in the skilled typing and processing of the manuscript, references, and tables of this report. Thanks, and gratitude are also extended to Dr. Kathleen F. Arcaro, University of Massachusetts, Amherst, MA for her research collaboration in this study.