The phenomenon of life and the open thermodynamic systems may be alternatively described from the existence of (i) a distinct separating border from the surrounding environment (ii) specialized structures inside them, which are functioning with some form of energy exchange with the environment and (iii) the medium in which these sophisticated functions are taking place. In the case of the cell, the distinct border is the cell membrane, the medium is the cytoplasm (which mainly consists of water) with all the cellular organelles, such as mitochondria, lysosomes, endoplasmic reticulum, ribosomes & nucleus, where all basic cellular functions for the homeostasis and reproduction are taking place. Regarding current cancer therapy, pharmaceutical targets of the cancer cell are concerning its border (such as cell membrane & the receptors), intracellular structures (DNA, microtubules) and the cellular functions that occur by these (cell cycle, reproduction).

The research idea of this therapeutic project is focused at the selective deactivation of key cancer proteins (KCPs) intracellular signaling pathways through their stereochemical bounding with individualized, artificially biosynthesized “antisense” polypeptides and deposit of the reaction product as insoluble agglomerates. By applying the principle of complementarity, which in nature creates more thermodynamic stable structures, such as the DNA double helix model described by Professors Watson & Crick [1] and since the time where Professor Louis Pasteur discovered the existence of enantiomers in nature with the separation of tartaric acid in wine and the discovery of racemic mixtures [2], perhaps it is possible, in reverse, to synthesize stereochemically chiral “anti-sense” polypeptides with the KCPs. The chemical reaction of the KCPs with their “anti-sense enantiomeric counterparts” inside the MCC may form insoluble complexes-micceles inside the MCC, resulting to deactivation of intracellular pathways responsible for the appearance & spreading of malignancy, sustaining proliferative signaling, evading growth suppressors, resistance at the apoptotic signals and their ability for angiogenesis, tissue invasion and metastasis [3].

Theoretically, the “anti-sense” proteins may be individually for every patient and based at hers/his malignancy type artificially synthesized, based on the tertiary molecular structure of the cancer protein/s which is/are overexpressed in the MCC or based on proteins which are only expressed (for instance fusion oncoproteins) inside the cancer, but not in the normal cell, expressed in various malignant neoplasms, for instance the fusion oncoprotein PAX8-PPARγ highly expressed in the aggressive type follicular thyroid carcinomas (FTC) [4] or the elf4e, highly expressed in medullary thyroid carcinomas (MTC) and in aggressive variants of papillary thyroid carcinomas (PTC) [5]. This/ese protein/s may be firstly identified by taking biopsy from the malignant tissue and afterwards through specialized diagnostic techniques from Pathology, such as immunohistochemistry, staining and molecular techniques, such as crystallography X-ray diffraction technique and Fourier transforms [6] or nuclear magnetic imaging NMR [7], so as a detailed strereochemical analysis of its/theirs tertiary structure/s to take place. The NMR is currently successfully used for the study of various neoplasms, such as colon, breast, oesophagus, cervix in order to classify tumor grade and type. Subsequently, the amino acid sequence of the KCPs should be identified, such as through mass spectrometry technique and then the design of the “anti-sense” counterparts may follow, such as with the assistance of Computational Methods in Drug Discovery [8], or the Iterative Threading Assembly Refinement (I-ASSER) [9] platforms.

When the amino acid sequence of the KCP is identified, the biosynthesis of the anti-sense enantiomer may follow, with the use of the proteomic technology, such as with the solid phase peptide synthesizer- SPPS [10]. The synthesis of a regiment of enantiomeric, “anti-sense”, polypeptides, where its/theirs tertiary structure/s to be complementary with the tertiary structure of the overexpressed or selectively expressed proteins at the cytoplasm of the cancer cell, correspondingly with the symmetry seen among D- and Lenantiomers. The complementary/ies enantiomeric/s proteins could be be biosynthesized either with natural L-aminoacids [11], either with the use of D-enantiomeric aminoacids from the expanded genetic code [12], or with combination of both forms (L- & D-amino acids). The synthesis technique of the enantiomeric/s polypeptide/s may be accomplished by following the reverse amino acid sequence of the natural polypeptide/s, fact that warrants further research study. Afterwards, it is essential to determine the concentration of the protein/s target/s inside the malignant cell in order to prepare the regiment of the enantiomeric protein/s at a specific concentration, where to reach 1:1 ratio or more with the protein/s target/s in the MCC and a racemic mixture to be formed. Protein concentration determination inside the MCC can be done through the modified Lowry method [13] or through newer ones such as protein quantification ratioing (PQR) technique, which uses a specific fluorescent reporter and quantifies steady-state protein levels within the cell, These may be done without differences in kinetics of the upstream and downstream proteins, such as folding, maturation & turnover rates to interfere significantly with the accuracy of the measurements [14].

Consequently, with the selective introduction of the therapeutic regiment of the “anti-sense” proteins in the MCC, reaching a 1:1 concentration ratio, a racemic solution of enantiomeric polypeptides (KCPs & “anti-sense” polypeptides) inside the MCC will be formed, or most probably a solution with an enantiomeric excess of the one or the other racemate. It is known that in a mixture of a pure enantiomer (R or S) and a racemate (RS), the enantiomeric excess (ee) is defined as the percentage % excess of the one enantiomer over the racemate based, on the type ee=[R-S]/R+Sx100. [15] Enantiomers cocrystallize as racemates on the surface, when the ee of the R enantiomer or S enantiomer remain below a critical value, whereas chiral segregation is achieved and globally homochiral surfaces composed of exclusively one enantiomer and these are obtained as the critical ee is exceeded. The heterochiral-homochiral transition is ascribed to the formation of energetically unfavored homochiral molecular dimmers under the control of the majority rules principle at high ee values. At high ee values, the transition from the insoluble racemate conglomerate complex to the soluble pure enantiomer results due to formation of non-thermodynamically favored dimmers of one or other enantiomer (R or S) which cannot be precipitated as insoluble conglomerates [16].

Τhe initial though was the selective crystallization of the malignant cell cytoplasm, “medium”, by the selective crystallization of the cancer cells which selectively express or overexpress KCPs, responsible for malignancy appear and spread, but not those which are expressed in the healthy ones. Following this, the conception of “anti-sense’’ proteins potential therapy has been tried to adjust to already existing therapies for cancer such as the Hyperthermic Intraperitoneal Chemotherapy (HIPEC) [17], without though any concrete scientific data to support it. Upon further research and collaboration with Heleni and Alan, who directed those previously false assumptions to the correct scientific pathway, the current assumption is to produce agglomerates by the chemical bonding of NCPs with their antisense polypeptide counterparts (Heleni).Also at that time (beginning of the writing of the article) were also to me unknown that cancer DNA modification and editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) [18] & the Trancription activator like effector nucleases (TALENS) [19] are existing therapies (Alan). Τhus the initial theoretic assumption has exluded the genetic editing of the DNA of malignant cancer cell as realistic therapeutic goal and has been moved 2 steps further (translation inhibition and protein aggregation at the post translational level).

Consequently, the research may be firstly redirected at the in vitro study of racemic solutions from KCPs from various malignant neoplasms (solids or liquids) with their chiral counterparts, studying the chemical way of aggregation, the temperature, which this may be accomplished and if those factors are compatible with cellular homeostasis in vivo. If conglomerate formation is only achieved at thermodynamic conditions not compatible with cellular homeostasis, then as a next step it may studied if the formation of a “supercritical” concentration of a racemate with enantiomeric excess from “antisense” polypeptides could lead to insoluble aggregates-precipitates and deactivation of nodal, for the appear and spread of malignancy, intracellular signaling pathways. The achievement of a “supercritical” saturation of KCPs and their enantiomeric counterparts in a solution closely resembles the MCC or in cancer cell lines in vitro and the study of the chemical behavior of the molecules may be done, possible with the method of supercritical fluid chromatography (SFC) [20,21].

In such case, though, the presence of an intermediate binding molecule may be necessary, in order the successfully bonding between KCPs and their chiral polypeptides to occur. This intermediate binding molecule may be attached at the anti-sense polypeptide during administration, so as a spherical final insoluble aggregate to form inside the MCC when react with the KCPs. This intermediate molecule may be derived from the category of intrinsically disorder proteins (IDPs) especially from the subcategory of molecular assemblers, Firstly, the molecular recognition elements (MREs) [22] at the polypeptide structure may be first studied and defined with specialized 2D, 3D Virtual Screening techniques (ligand based methods) [23]. The MREs are very important spots, since they represent the molecular places where the first step of protein interactions occur and where molecular docking of IDPs is taking place [22].

The term “enantiomeric” may not be applicable for proteins, as it is used for more simple inorganic chemical substances. The enantiomeric form of a polypeptide, a mirror image one, as it is inthe shape of Dand L-, chiral, molecules cannot form a spherical structure, when bringing them in close approximation. The concept of chirality, in the case of a molecule with tertiary structure, as with proteins, would be more correct to refer to a molecule with a reverse tertiary structure in all three dimension (x, y, z axes), which, when it comes in close approximation with the KCP to specifically bound and “lock”, such as the key in the keyhole, or such as the enzyme with its substrate, and form an insoluble microsphere. The design should probably care to keep the hydrophobic residual aminoacid groups of the ANP at the outer surface of the polypeptide and probably to have a reverse electric charge from the KCP to facilitate their approximation in the MCC. (Panos)

The research efforts may be focused primarily at the in vitro study of the aggregation type of racemic solutions from natural cancer proteins from various malignant neoplasms with their synthetic enantiomers, with racemic protein crystallography [24], determining the rigid body degrees of freedom (D) between chiral proteins which need to arrange in a particular manner inside the MCC in order to form an insoluble aggregate. In other words, at how many different points of the protein’s tertiary structures may the natural with the chiral proteins to connect in order to form an aggregate and if this may be accomplished in the thermodynamic conditions existing inside the MCCs in vivo. The next step may be the test of the thermodynamic stability of those precipitates, agglomerates or micceles inside a solution which closely resembles the MCC and afterwards the MCC itself in cancer cell lines, such as with the techniques of Differential Scanning Fluorimetry (DSF, nanoDSF) [25], Microscale Thermophoresis (MST) [26] (determination of thermodynamic parameters for interactions and monitor of protein folding) , or with the newest Mastersizer and Microflow Imaging (for molecules<28 μm) platforms. With the latter platforms, it is possible to study the aspect ratio of the molecular complexes, meaning the ratio of the depth with the width of the image of the particle, analyzed at the platform. Based on their aspect ratio, the molecular complexes may be divided as fines, microspheres and agglomerates. The formation of microspheres may carry the most favorable thermodynamic form after reaction of the KCPs with their “anti-sense” enantiomers inside the MCC [27]. Also the study may evaluate which specific thermodynamic conditions are needed in order the KCPs and the “anti-sense” proteins to form a stable and insoluble microsphere.

If the preliminary studies prove the validity of the above theoretic assumptions then phase I trials could commence. A critical question remains the selective delivery of the “anti-sense” proteins inside the MCC in adequate concentration to form the microspheres and deactivating the pathways. The science of nanotechnologynanomedicine can also contribute to this goal with the specialized delivery methods, such as lipid based nanocarriers (liposomes, stealth liposomes, solid lipid nanoparticles) or with the assistance of smart drug delivery systems, such as nano-sized carriers with characteristics size less than 200 nm & more than 10nm , with a neutral charge on the surface or a slight negative zeta potential and coat their surface with polyethylene glycol (PEG) [28], phospholipid bound nanoparticles, intelligent polymeric nanocarriers, electroactive composite nanogels, drug conjugates (antibody-drug, polymer-drug, polymer-protein), viral, or inorganic nanoparticles. Problems with the introduction of mirror image polypeptides into mammalian cells have been currently surmounted with the use of protective antigens (PA) from Bacillus anthracis (anthrax) toxins, attaching the “D-cargo” at the C- terminus of lethal factor (LF) domain of the toxin [29]. Another new, sophisticated method, the spherical nucleic acid (SNA) technology could also offer a solution to the problem of selective delivery. Instead of nucleic acids, at the outer surface of the nanoparticles the individualized “anti-sense” polypeptides could be bounded, which with their introduction in the MCC, they will be detached from the nanoparticle core and subsequently react with the KCPs, providing a potential new method of individualized treatment [30]. Every other scientific method or proposal may be suggested from scientific committees from universities throughout the world, pharmaceutical companies or fellow scientists of the relevant fields or anyone who are more relevant from the writer and can contribute to the implementation of the idea of “anti-sense” proteins for the treatment of patients with malignant neoplastic disease, which may bring our scientific communities and our nations more close with this common goal.

In patients with local advanced (stage III) or generalized metastatic disease (stage IV) in one or multiple organs or in patients with recurrence, when all current anti-neoplastic treatment (medical or surgical) cannot offer any definitive treatment.

The therapeutic rational of complementarity have been first observed at the post-transcriptional modification of the ompF gene [31] and been already successfully applied with the anti-sense nucleotides (mRNAs) treatment, preventing the translation of specific genes in cancer, such as the cytoprotective chaperone protein, clusterin, in advanced cancer such prostatic, breast, transitional renal cell, non-small cell lung [32], where clinical trials are ongoing. An offtarget effect which may be also observed in anti-sense polypeptide treatment, correspondingly with the anti-sense RNA treatment, may also eliminated with strategies, such as concentration optimization with SMART “pooling” technique, bioinformatics and modifications of the anti-sense polypeptide to resist in protease degradation [33] (Reviewer #2). Also, mirror image polypeptides are not recognized by nature’s proteases, are less susceptible to degradation and have also low immunogenic response [34]. If the rational of cancer therapy through anti-sense polypeptides proves to be correct, then perhaps in the future, “libraries” of anti-sense proteins may exist for every malignancy type and only minor adjustments in their polypeptide structure may be done individually for every patient. This may be accomplished with platforms such as the existing Smart Re-profiling platform [35], according the structural analysis of the individualized KCPs, after biopsied the malignant tumor, as it was presented in the article.

Nowadays, with the great evolution of the technology and the scientific methods, as well as the therapeutic possibilities and perspectives that may arise, pose once more as crucial need the redefinition of the limitations of the medical therapeutics and the interventions in patients. It is very important to realize that It is very important to realize that therapeutic intervention is futile and according to the theories of hedonism and desire-satisfaction, described in the literature for a person’s fare well, every possible effort should be made from all health care providers, according to their specialty, to insure patients with terminal illness with adequate pain relief, rehabilitation and sufficient physical and psychological support for them and their families. The clinician must also be aware of their patient’s desires regarding the course of their illness and must also be willing to fulfill them, having also in mind the objective list, which means that there are good interventions for the patients, even though themselves are incapable to recognize them and from this point of view the clinicians should always care for the best interest of their patients. Currently, there has also been a trend towards the balanced patientcentred approach from the classical doctor-centred approach of the medical therapy. It is also very important the patient to actively participate at the final decision of the therapy recommended by the doctor, who always taking into account the personal needs of every patient which is the autonomy, benefiscence, non- maleficence, justice, equitable access, privacy, fiduciary duties and always according to hers/his individual social, cultural, national features as well as hers/his religious and ideological beliefs. “A deeper look in ethical theory is needed where it seems that a Kantian prohibition toward using merely as a means will conflict with a consequentialist perspective that favors an impartial maximization of good outcomes” (Chris) [36,37,38].

To the entire scientific community worldwide, the authors of the references, their citations, their organizations, my parents & all my family my teachers, professors & mentors, my friends, my colleagues, collaborates & reviewers and all those who are working for the health, education, culture & well-being of people.