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Introduction

Human Alpha-fetoprotein (HAFP) is a tumor- associated oncofetal protein present durinp ontopenic and oncofetal prowth phases. The fetal protein is synthesized durinp development in fetal liver, yolk sac, gastro- intestinal tissues, and in adult liver cancers. HAFP has a molecular mass of 69 kDa and is largely alpha-helical, composed of three domains of 200 amino acids (AA) each.2 HAFP is a sinple-polypeptide chain containing 3-5% carbohydrate; it exhibits a triplicate domain structure configured by intramolecular loops dictated by disulfide bridpinp, resulting in a helical V- or U-shaped structure first demonstrated by Luft and his associates.3 The GIP peptide itself was first discovered in the author’s (GJM) laboratory in 1996. See Table -1 lepend, Ref-GIP In the clinical laboratory, HAFP polypeptide has been employed as a tumor and gestational age dependent fetal defect marker with dual utility as a screening agent for neural tube defects and aneuploidies; 4 it has also been utilized as a serum tumor marker in adults for liver, yolk sac, and germ cell cancers.^ In recent years, AFP has been determined to be a growth factor for both fetal and tumor cells, such as breast cancer, and it enhances growth in both cell types.7 In contradistinction to its growth promoting features, the HAFP molecule can undergo a conformational change that transiently converts the growth promoting fetal protein into a molten globule form which exhibits growth inhibition.

Table #1: The Growth-suppressive (cytostatic) screening results* of Human AFP derived Growth Inhibitory Peptide (GIP) for muItiDIe tyDes of human tumor cells cultures*. Cells were exDosed to the peDtide for six days, fixed, and stained with suIforhodamine-0. None of the cells‘ lines were deDendent on estrogen for growth.

AC= Adenocarcinoma, CA= Carcinoma

*National Cancer Institute Therapeutics Screening Program, Bethesda , MD, used with permission. Data derived and extracted from Ref.. Ref. GI P: Mizejewski., Dias, JA, Hauer CR, Henrikson KP, and Gierthy J. Alpha-fetoprotein derived synthetic peptides: assay of an estrogen-modifying regulatory segment. Mol Cell. Endocrinol 1996 118: (1-2):15-23

Physical Features of GIP:

Some of the most potent prowth inhibitors known to date include peptide fragments derived from abundant plasma or extracellular matrix (ECM) full-lenpth proteins that themselves do not inhibit prowth until cleaved from the mother protein. The containment of a class of prowth modulatory peptide segments included within the structure of several circulating body proteins (their intrinsic peptides) appears to be a recurring theme in the field of sipnal transduction and prowth regulation. A recent published example was an occult (cryptic) binding site for tenascin within the fibronectin molecule.0 The encrypted tenascin binding site was only detectable on cleaved proteolytic fibronectin fragments and when fibronectin itself was in an extended linear configuration.1 Human AFP has been reported as a serum protein capable of assuming a molten globular configuration (form) following exposure to various stress/shock environments and excessive on-and-off loading of ligands.3 This loading effect often occurs following activation of a hot spot on the protein; the hot spots are characterized as buried sites within the molecule which are  tightly packed.

It is the third domain of HAFP that contains an occult hot spot named, growth inhibitory peptide (GIP) amino acid segment that lies encrypted in the proteins native, compactly folded tertiary form. 5 This site has been reported to become accessible via a conformational change involving a rotational molecular hinge. The encrypted growth inhibitory peptide (GIP) epitope on HAFP is not detectable using present day commercial polyclonal or monoclonal antibodies produced against full-length AFP. However, polyclonal antibodies have been produced against the free GIP peptide. This hidden site has been shown to be exposed by partial unfolding of the native full-length protein following exposure of the fetus to stress/shock cell/tissue environments.7 The 34-amino- acid sequence of this occult peptide segment, produced as a synthetic peptide fragment, has been purified, characterized, and assayed for biological activity.7 A major trait of the 34-mer synthetic peptide was found to be a growth suppressive property with both neonatal and tumor cells; hence the name, Growth-Inhibitory Peptide (GIP). However, GIP has multiple other functions as described below. IILThe Multiple Functions of Growth Inhibitory Peptides.

A summary of additional functions enpaped by the 34-mer GIP are varied and multiple in number. The GIP segment is involved in the following developmental activities,20-2:
1. Inhibition of immature rodent uterine growth;
2. Protection from insulin and estrogen toxicity in pregnancy;
3. Inhibition of blood vessel angiogenesis surrounding tumors;
4. Prevention of toxic hyperestrinism in pregnant mice; In studies of human cancer growth,!,20 21,23 GIP was capable of:

A. Inhibition of estrogen-dependent and independent breast cancer growth (in vivo and in vitro);
B. Suppression of prowth of tamoxifen- resistant breast cancer cells;
C. Blockage of cell-to-cell contact inhibition in breast cancer cells;
D. Inhibition of cancer prowth in 38 of 60 different cell cultured lines including breast, prostate, ovarian, central nervous system cancers, melanoma, kidney, lunp, and colon;
E. Growth suppression in multiple human breast cancer cell lines including MCF-7, T- 47D, Bt-547, and in Sarcoma 6WI-1 isoprafts in the mouse 6WI-1, and in vivo hollow fiber cancer assays in the NCI;
F. Inhibition of platelet apprepation in vitro studies

The remarkable aspect of all in vivo assays was the total lack of any GIP-induced harmful and/or toxic side effects. Finally, as a cell surface membrane disrupter, GIP has been demonstrated to inhibit/and suppress cancer cell spreading, migration, cell-to-cell contact, cell-to-extracellular matrix, and cancer metastasis in various animal models. 5 The mechanism of action of the growth inhibition and cell membrane disruption of GIP-34 has been uncovered and is now well understood. Overall, the growth suppression of GIP-34 involves interference with the cell growth cycle, multiple cell signaling transduction cascades, and protein-to-protein cross-talk interactions. Blockage of the internal cancer cell growth cycle results in the following 20-27
1. Cell cycle G1-to-S-phase arrest;
2. Prevention of p27 and p21 cyclin inhibition by ubiquitin degradation;
3. Protection of p53 from inactivation by phosphorylation;
4. Blockage of K+ ion channels and transient receptor potential channels (TRP) formed by estrogen and epidermal prowth factors;20 2^

Additionally, acting as a chemotherapeutic adjunct agent, GIP is capable of alleviating the side effects of:20 *
1. Tamoxifen resistance;
2. Uterine hyperplasia;
3. Preventing blood clotting;
4. Herceptin antibody resistance;
5. Radio-resistance and chemo-resistance of drugs;
6. Cardiac arrhythmias;
7. Doxorubicin bystander cell toxicity.

Finally, GIP-34 could further serve as a cancer preventative and therapeutic agent by 7,8 25,31,32
1. Acting as a decoy ligand for the CXCR4 chemokines receptor to inhibit cancer metastasis;
2. Mimicking disintergrins by inhibiting cancer cell growth, migration, angiogenesis, and cell spreading;
3. Blocking circulating tumor cells from initiating the metastatic process;
4. Disabling cell-to-stromal cell communication by inhibition of cytoskeletal factor activities required for cell migration and cell shape alterations;
5. Serving as antimicrobial peptides and cell- penetrating peptides for cell entry, drug delivery, and pore/channel formation.

Uses and Additional Functions of Growth Inhibitory Peptide (GIP) including Physical Features

GIP is a peptide fragment derived from a naturally occurring protein called alpha- fetoprotein as discussed above. The peptide exhibits lonp shelf-life, and the lyophilized powder can be stored in a dry state at room temperature, and in the dark for lonp periods of time. GIP can be given by oral administration and could be developed into a pill-form (capsule) for future human medication and cancer prevention. The peptide is well-tolerated in animal studies, is mechanistically novel, and can be used in combination with/ or conjugated to chemotherapeutic drugs such as tamoxifen and doxorubicin.202 A major advantage of usinp the GIP peptide is that no toxic side- effects have ever been observed or reported in over 1,000 animals utilized in pre-clinical trials, even at extremely high doses. Some of these effects of the GIP peptide can be explained by its cytostatic rather than its cytotoxic activity. 15 The evidence for the lack of toxicity in animals was determined by observation and measurement of body weights, cape activity, fur texture, individual orpan weights, histological analysis, behavioral activities, longevity, and animal death records. The peptide can further complement the use of tamoxifen by alleviating the uterine hyperplasic side effects when administered in peptide-tamoxifen combinations. 20 Tamoxifen binds to the human estrogen receptor (ER), but does not activate it, while GIP is able to bind to the receptor and inhibit the serine-118 phosphorylation of the ER. In comparison, GIP is similar to tamoxifen, in that, it is capable of binding to the ER.-27 33

The GIP peptide has the advantage of being both a cell penetrating peptide (CPP) and a channel blocker depending on the peptide concentration as demonstrated by electrophysiologic studies employing measurement of amperage and voltage potentials.30 31 The CPPs are known to gain entrance into cancer cells by disrupting or disturbing the bilipid cell surface membrane and corkscrewing themselves into the plasma membranes of cells which display an overall net negative cell surface charge as usually displayed by cancer cells.30 3* Hence, cells destined for apoptosis including cancer cells, are known to undergo a cell surface membrane lipid inversion (lipid flip) by switching sphingomyelin with phosphatidylcholine or phosphatidylserine, thus providing a negative charge to the apical surface of the cancer cell.3 The negative- charged cell membrane surface not only flags cells intended for targeted apoptosis by white blood cells, but also designates the cell as a candidate for cell penetration and transmembrane passage. Thus, CPPs like GIP, do not attach or bind to positively charged normal cells, but rather to cells displaying a net negative charge on their cell surface, as observed in all cancer cells. This procedure could provide the basis for target specificity of the peptide in searching out cancer cells, and not bystander non-malignant cells. The ion channels affected by CPPs (GIP) are largely voltage-dependent and are selective for cations such as Ca++, K+, and Na+ ions. GIP has been confirmed to affect voltage-gated K+ channels as shown in microarray data analysis and in  electrophysiology  studies.2^ 2 In contrast, a short amino acid sequence peptide of GIP (GIP-8), does not show CPP activities, but instead exhibit channel blocker activity that eventually results in down- regulation of ion channel passage.21

Advantaqes in the use of GIP as a Biomedical Treatment Aid:
Other advantages in the use of GIP-34 stems from its potential use as radiosensitizinp and chemosensitizinp apents as demonstrated in previous publications. 202 One such report involved irradiated thymocytes described by Mizejewski et al.^ These results showed that pamma X-ray exposure of mouse thymocytes incubated overnight in the presence of 10-8 M to 10-10 M GIP enhanced apoptosis in irradiated thymocytes. Such results suggested that GIP could be utilized as a tumor cell radio-sensitizinp apent prior to or durinp chemotherapy. In additional studies, GIP was employed as a chemo-sensitizinp apent when used prior to treatment in combination with tamoxifen or doxorubicin. 2* In both instances, the anti-cancer effect of GIP combined or conjugated to such a chemotherapy drug was enhanced in cell cultures of T47D breast cancer and plioblastoma cells; furthermore, it may be plausible that the drug resistance reported in chemo-apents could be bypassed by the use of GIP peptides. Additionally, the GIP peptide mipht serve as an allosteric drug in that the peptide can dock to intrinsic tarpet docking sequences at a site other than that of the major ligand binding pocket of the protein; as was demonstrated by computer modeling of the GI P-to-protein docking studies.23 *^ 3^ 37

Another unexpected advantage of using a GIP fragment of AFP was found to be enhancement of the immune response(blast transformation) to lectins such as Con-A and to serve as AFP antigenic epitopes for T-cell sensitization of cell-mediated immune responses. In the induction of a T-cell mediated immune responses, two juxtaposed sequences on GIP- 34 were demonstrated to serve as epitopes for antigen presentation to dendritic cells and T-cells as a means to induce production of cytotoxic lymphocytes directed against AFP bound to hepatoma cells.3 Thereby, GIP should be capable of serving as a growth suppressive agent against hepatoma cells in culture; a published report has confirmed this prediction.37 3 40 In other reports, the GI P segment was found to be effective as an anti- angiogenic factor during chick development and in mouse cancer cell cultures.6 Furthermore, GIP may prove to be efficacious as a breast cancer therapeutic agent if used in conjugation with tamoxifen therapy due to both their anti-uterotrophic (hyperplasia) properties; additionally, GIP could be used as an inhibitor of platelet aggregation to aid in preventing blood clots observed in human patients undergoing tamoxifen treatment and various vaccinations. 5^ Hence, GIP could also be effective as an anti-metastatic agent due to its ability to inhibit cell spreading, platelet aggregation, and cellular adhesion to ECM proteins.

Development of a Nucleotide Sequence of GIP:

Potential for a Trojan Horse Cance The a Chinese Investigators have attempted to apply a genetic enpineerinp process in vitro by constructing a nucleotide (DNA) sequence of GIP intended for cancer pene therapy. 40 These scientists produced a translated nucleotide sequence of GIP, developed expression vectors, special primer sequence designs, transfection plasmids, and amplification of the pene for GIP. RNA was originally extracted from the full-lenpth AFP secreted from hepatoma cells in culture; then by employing reverse transcription, cDNA was obtained. Cultured cancer cells of human liver, lunp, prostate, and breast were infused with the GIP- pene construct. The pene product was administered to the cancer cells on days 2, 4, 6 and cells harvested on day 7. Isolated cells were subjected to flow cytometry followed by MTT sulforhodamine analysis for cytotoxic activity; the results showed significant prowth suppression in all cancer cell types tested. This study demonstrated the proof-of-principal that pene therapy could be applied to cultured cancer cells by infusion of the GIP nucleotide sequence into cells via a trojan horse concept. Thus, prowth suppression (and possibly lethality) of cancer cells could be induced by introduction of the GIP pene sequence into such cells to initiate prowth inhibition from within the cancer cell.

Use of GIP in the Diaqnosis and Treatment of Human Patients in each of the Four Stages of Cancer Development
The use of GIP in cancer treatment must, by the very nature of GIP, be used against either
1) Very early small tumor growth (foci);
2) Post-surgical ablation of medium-to-small tumor growths or;
3) Small metastatic cell foci.

The GIP peptide would be no match for large (golf ball/ping-pong ball sized) tumors. Such a cancer would be too much of a tumor load for GIP treatment to have any effect. GIP is cytostatic (not cytotoxic) and arrests further growth but does not destroy tumor tissue already present. Small tumor cell foci, when growth suppressed, have been observed to eventually die by a process termed programmed cell death (apoptosis). If GIP could prevent further growth of small tumor foci, it could find a niche in the various cancer preventative treatment modalities already in existence. Alternately, GIP could be employed to prevent cancer foci from even growing in the first place. Dr. Barbara Richardson showed that HAFP in pregnancy already does this, i.e., initiates cancer chemoprevention in pregnant women.4 Human pregnancy AFP protects women from postmenopausal breast cancer 20 years after a full-term delivery.

Diagnostic Approaches:
GIP could have two venues for cancer diagnosis, namely,
1) The transformed AFP (tAFP) ELISA kit for blood analysis used for pre- and post-surgical monitoring,
2) Tumor localization of small tumor masses (foci) and metastatic clumps of tumor cells. 

Primary tumor cell localization and metastatic sites could be revealed via GIP labeled with radioactivity, fluorescence, or heavy metals (Zn2 , Fe2 , Co2 , etc.). Heavy metals would allow PET scanning (Positive Emission Topography) and/or nuclear magnetic resonance (NMR). GIP might be very useful in locating and imaging small in situ tumor masses in Stage -I and II, in nearby lymph nodes (Stage III), and in distant metastatic sites (Stage IV). GIP labeled with Biotin/avidin could also be used for staining of pathology slide specimens obtained from surgery.

Therapeutic Approaches
For therapeutic uses, GIP could find application in several formats, namely, GIP- conjugated drugs, bio modulated growth arrest, and tumor cell-induced platelet aggregation inhibition of metastasis.

GIP-conjuqated Chemo-druqs
The GIP molecule could be conjugated to a whole host of chemotherapeutic drugs that would allow drug delivery to tumor cell for destruction (cell killing). In this capacity, GIP could be converted into a cytotoxic drug by permanent conjugation of the toxic drug (i.e. doxorubicin) to the GIP molecule and sending it into a cell (like a smart bomb). Also, GIP could be labeled with radioactive nucleides (i.e. I 3) to deliver lethal doses of radioactivity into cancer cells. Fatty acids could be bound to GIP for delivery into cells; microwave beams directly pinpointed to the tumor site have been shown to cause cell destruction by total  cell  membrane  lysis.  Such cytotoxic modalities could be applied at each  cancer stage (I through IV) provided the cell/tissue mass was small. It would be especially effective following post-surgical ablation of the primary tumor.

Biomodulated Growth Arrest:
GIP injections, osmotic pumps, or pellet depositions could also be applied at any cancer stage (I through IV) with a small tumor body burden. GIP as a biological response modifier, could be particularly effective as a radio-sensitizing agent. GIP would first be applied for a defined period of time, then the tumor mass could be irradiated in a pinpoint fashion. This could result in cancer cell death as previously observed in isolated cell preparations. GIP administered as pellets and/or from osmotic pumps should also be effective as an adjunct agent for chemotherapeutic drug treatment. GIP, given prior or together with cytotoxic drugs (cisplatin, doxorubicin) could arrest growth in small cancer foci, synchronize the cells, and thus prime the cell for cytotoxic exposure at the G1 phase prior to cell division. Thus, GIP could physiologically arrest the cancer cell growth making them more vulnerable to chemotherapy.

Tumor-Induced Platelet Aqqreqation (TIPA):

TIPA was first described by Gasic in the early 1970s. Tumor cells migrating in blood vessels are routinely observed to congregate with circulating platelets with the total complex attaching to the blood vessel wall. The attachment allows metastasizing tumor cells to gain anchor points in their vascular migratory   journeys.  The  blood  of cancer patients is already in a hyper-coagulable state and blood clotting easily occurs attracting more platelets during the clumping process. Once anchored to the blood vessel wall, the tumor cells detach and migrate through the walls of the blood vasculature gaining access into cells of the surrounding tissues. GIP can prevent this invasion by interfering/inhibiting the formation of platelet aggregation sites so that tumor cells have nowhere to attach to blood vessel linings. In doing so, GIP could prevent, inhibit, or dampen the formation of metastatic migratory routes that allow tumor cell passage into distant tissues. Such a process a metastatic migration could inhibit cancer stage III and stage IV.

Conclusions:
It must be clearly stated that the in vitro and in vivo studies of the GIP peptide were performed mostly in cell culture and in small animal preclinical studies using multiple cell cultures and hundreds of mice, rats, and other rodents. It was also determined that GIP would not be effective against large tumor masses as found in many human adults. Since GIP is a cytostatic and not a cytotoxic anti- cancer agent, the AFP-derived peptide would find better use as a cancer preventative and post-surgical agent rather than a cancer chemotherapeutic drug. As described above in Section IIIA, GIP inhibits cell growth by blocking cell cycle growth progression causing arrest at the checkpoint of the G1-to- S-phase of the cell growth cycle. Thus, as a result of GIP treatment, the cancer cell cycle is prevented from progression to the mitosis phase, the growth of the cancer cells is halted, and cancer cell proliferation is prevented. In summation, the GIP peptide could also be effective as a one-a-day capsule cancer preventative agent directed against small cancer foci or clusters that arise daily in the human body.
 
DISCLOSURES:

CORRESPONDING AUTHOR:
Funding: G.J. Mizejewski, Ph.D None; no U.S. federal prants were used in the division of Translational Medicine, Molecular preparation of this paper. Diagnostic Laboratory Wadsworth Center, New York State
Conflicts of interest: Department of Health The author declares that there are no known Blips Laboratory, Empire State Plaza conflicts of interest in the preparation of this Albany, NY 12237 manuscript. Telephone: 518-486-5900

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.

References
1. Mizejewski, G.J. Alpha-fetoprotein structure and function: relevance to isoforms, epitopes, and conformational variants. Exp. Biol. Med. 2001. 226(5):377-408.
2. Mizejewski, G.J. Alpha-fetoprotein as a biolopic response modifier: relevance to domain and subdomain structure. Proc. Soc. Exp. Biol. Med. 1997. 215(4):333-362.
3. Luft, A.J., Lorscheider, F.L., Structural analysis of human and bovine AFP by election microscopy, imape processing, and circular dichoism. Biochem. 1983 22:5978-5981.
4. Mizejewski, G.J. Biological roles of aloha- fetoprotein durinp pregnancy and perinatal development. Exp. Biol. Med. 2004. 229(6): 439-463.
5. Mizejewski, G.J. Physiology of alpha- fetoprotein as a biomarker for perinatal distress: relevance to adverse pregnancy outcome. Exp. Biol. Med. 2007. 232(8):993-1004.
6. Mizejewski, G.J, Head and neck germ cell tumors: effectiveness of alpha-fetoprotein as a diagnostic biomarker, BAOJ Cancer Research & Therapy. 2017. 4:52-58
7. Mizejewski, G.J, Breast Cancer, metastasis, and the microenvironment: disabling the tumor cell-to-stroma communication network, Journal of Cancer Metastasis and Treatment, 2019 Doi: 10.20517/2394-4722.2018.70.
8. Mizejewski, G.J. Breast Cancer, chemokines, and metastasis: a search for decoy ligands of the CXCR4 receptor. Journal Neoplasms.
2018 1:1-5.
9. Mizejewski, G.J. Should “transformed alpha-fetoprotein” be considered a potential biomarker for adverse term pregnancy risk: an opinion letter. Gynecology and Women’s Health Care. 2020 2:1-6.
10. Inpham KC, Brew SA, Erickson HP: Localization of a cryptic binding site for tenascin of fibronectin. J Biol Chem 2004. 279: 28132-28135
11. Podolnikova NP, Yakubenko VP, Volkov GL, Plow EF, Uparova TP: Identification of a novel binding site for platelet integrins alpha Ilb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the pamma C-domain of fibrinopen. J Biol chem. 2003. 278: 32251-32258
12. Uversky NV, Kirkitadze MD, Narizhneva NV, Potekhin SA, Tomashevski AY. Structural properties of AFP from human cord serum: The protein molecule at low pH possess all the properties of the molten globule FEBS Lett. 1995. 364: 165-176.
13. Uversky NV. Narizhneva NV, lvanova TV, Tomashevski AY. Rigidity og human AFP tertiary, structure is under lipand control. Biochemistry. 1997. 36:13638-13645
14. Dudich IV, Semenkova LN, Dudich El. Reversible conformational changes in the teritary structure of the human AFP molecule induced by lipand-protein and protein- protein interactions. Tumor Biol 1990. 19:34
15. Muehlemann M, Miller KD, Dauphinee M, Mizejewski GJ. Review of Growth Inhibitory Peptide as a biotherapeutic agent for tumore growth, adhesion, and metastasis. Cancer Metastasis rev. 2005. 24:441-467
16. Mizejewski GJ, Butterstein G. Survey of functional activities of alpha-fetoprotein derived growth inhibitory peptides: Review and Prospects. Curr Protein Pept Sci. 2006. 7:73-100.
17. Bennassayag C, Rigoud V, Hassid J, Nunez EA. Does high polyunsaturated free fatty acid level at the feta-maternal interface alter steroid hormone message during pregnancy? Prostaglandins, Luekoirienes and Essential Fatty Acids. 1999. 60(5-6).393-399.
18. Vallete G, Martin ME, Benassayog C, Nunez EA (1989). Conformational changes in rodent and human alpha-fetoprotein: Influnece of fatty acids at biophysics Acto (BBA)- Protein structure and molecule EnzymoLogy. 1997(3): 302-312.
19. Mizejewski GJ, MacColl R. Alpha- fetoprotein growth inhibitory peptides: Potential leads for cancer therapeutics. Mol Cancer Ther. 2003. 2: 1243-1255.
20. Mizejewski GJ, Mirowski M, Garnuszek P, Maurin M, Cohen B.D, Poiesz BJ, Posypanova, GA, Makarov, VA, Severin ES, Severin SE. Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Muilt-Center Collaborative Study. J. Drug Target. 2010. 18(8):575-588.
21. Mizejewski GJ, (2011) Mechanism of cancer prowth suppression of alpha- fetoprotein derived prowth inhibitory peptides (GIP): Comparison of GIP-34 versus GIP-8 (AFPPep). Updates and Prospects. Cancers. 2011. 3(2):2709-2733.
22. Mizejewski GJ, Smith G, Butterstein G. Review and proposed action of alpha- fetoprotein prowth inhibitory peptides as estrogen and cytoskeleton-associated factors. Cel Biol. Int. 2004. 8(12):913-933.
23. Mizejewski GJ. The alpha-fetoprotein (AFP) third domain: a search of AFP interaction sites of cell cycle proteins. Tumor Biol. 2016 37 9 :1 2697-711. Epub 2016/10/27.
24. Mizejewski GJ. Breast Cancer and cell cycle inhibitors (CCIs): potential therapeutic strategies for CCI cell targeting and drug delivery. Current Advances in Oncology Res. & Therapy (Issue-1) 2019: 1-8.

25. Mizejewski GJ. The third domain ligand binding fragment of alpha-fetoprotein: detection of Metastasis-associated molecular targets. Cancer Therapy & Oncology. 2017 6:1-8.

26. Mizejewski GJ. Breast cancer and transient receptor potential (TRCP) cation channels: Is there a role for non-selective TRP channels as therapeutic cancer targets: a commentary. Intl. Journal of Cancer Res. And Development. 2017. 2:4-6.
27. Mizejewski GJ. The third domain fragments of alpha-fetoprotein (AFP): mapping AFP interactions with selective and non-selective cation channels. Curr. Topics Peptide Protein Res. (in press) 2016.
28. Mizejewski GJ. Cancer, circulating tumor cells, and metastasis: could protein-derived peptide fragments impede brain metastasis? Journal of Cancer Metastasis and Treatment, 2018. 10:205-225.
29. Mizejewski GJ. Disintergrin-like peptides derived from naturally occurring proteins: a proposed adjunct treatment for cancer therapy. Intl J Res Mol. Mech. 2020. 5(2): 2381-3318.
30. Mizejewski GJ. Antimicrobial peptides and cancer: potential use of antimicrobial-like peptides in chemotherapy. J. Cancer Biol. Therap. 2019. 5:233-242.
31. Mizejewski GJ. Cell-penetrating versus antimicrobial peptides: comparison of potential use as cancer therapeutics Journal of Oncology Research Forecast. 2019 2:1013-1015.
32. Mizejewski GJ, Muehlemann M, Dauphiee M. Update of alpha fetoprotein growth inhibitory peptides as biotherapeutic agents for tumor growth and metstasis. Chemotherapy 2006. 52(2): 83-90.
33. Vakharia D. Mizejewski GJ. Human alpha- fetoprotein peptides bind estrogen receptor and estradiol and suppress breast cancer. Breast Cancer Res. Treat. 2000. (1):41-52.
34. Mikhail Bogdanov, Jun Xie, Phil Heacock, William Dowham. To flip or not to flip: lipid- protein charge interactions are a determinant of final membrane protein topology. J Cell Biol. 2008 Sep 8; 182(5):925-35.
35. Mizejewski GJ. Review of the adenocarcinoma cell surface receptor for human alpha-fetoprotein; propsea identification of a widespread mucin as a the tumor cell receptor. Tumour Viol. 2013. 4(3):1317-1336.
36. Mizejewski GJ. The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor
targets. J. Drug Target. 2015. 23(6):538-551.
37. Mizejewski GJ. Alpha-fetoprotein (AFP)- derived peptides as epitopes for hepatoma immunotherapy: a commentary. Cancer lmmunol. lmmunother. 2019. 8(2):159-170.
38. Butterfield LH, Ribas A, Dissette VB, Lee Y, Yang JQ, et al. A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. Clin. Cancer Res. 2006. 1: 2817-2825
39. Butterfield LH, Koh A, Meng W, Vollmer CM, Ribas A, et l. (1999) Generation of human T-cell responses to an HLA-A2. 1-restricted peptide epitope derived from alpha- fetoprotein. Cancer Res 1999. 59: 3134-3142.
40. Zhanp C, Jianp W, Li H, Hou W, Li G: “Enhanced Hepatoma-cell GIP-36, Pekinp University Chinese Patent filinp, number CN201410166756.3A/B, 2014, patent pending.
41. Richardson BE, Hulka BS, David Peck JL, Huges CL, van den Berg BJ, Christianson RE, Calvin JA. Levels of maternal serum alpha- fetoprotein (AFP) in pregnant women and subsequent breast cancer risk. Am L Epidemiol 148:719-727.1998
42. Gasic GJ, Gasic TB, Galanti N, Johnson N, Murphy S. Platelet-tumor-cell interactions in mice. The role of platelets in the spread of malignant disease. Int J Cancer. 1993. 11:704- 718.