We isolate the epithelial tumor cells from the whole blood or tissue sample.
    The isolated cancer cells are then grown in a culture.

        From the whole blood we isolate the circulating cancer cells using gradient solutions and membranes with pores. After that we proceed to negative selection using cytospin with anti CD+45 (those antibodies bond to hematologic cells and they separate them from epithelial cells-malignant) and after that we proceed to positive selection using EpCam antibody that bond and isolate trough cytospin the epithelial origin cell (cancer cell) . Then we identify that we have actually isolated the malignant cells using the immortalization detection kit from Oncogen.
        After that we develop many cell cultures in a 96 well plate and in each slot we add in the culture medium a different chemotherapeutic drug.
        The pharmaceutical substances that are used and are inserted in the culture medium are:

1. Cytostatics:
   Oxaliplatin, cisplatin, carboplatin, cyclophosphamide, ifoshpamide, trophosphamide, treosulphan, melphalan, CCNU, BCNU, ACNU, mitomycin, procarbazine, dacarbazine, bleomycin, temozolomide, estramustin,bendamustin, doxorubicin, liposomal doxorubicin, epirubicin, daunorubicin, dactinomycin, irinotecan, topotecan, idarubicin, etoposide, mitoxandrone, paclitaxel, docetaxel, vincristin, vinblastin, vinorelbin , taurolidin, 5fluoruracil, uracil-tegafur, raltitrexed, floxuridine, capecitabine, methotrexate, gemcitabine, pemetrexed.
   
   
2. Immunological–modifiers:
   IL-2,IFN,kortisole
   
   
3. Inhibitors–of–growth–factors:
   octreotide, suramin sulfate, cetuximab, rituximab,transtuzumab, imatinib mesylate, coxibs, monteleucast, tamoxifen, raloxifen, leprolide, anastrozole
   
   
4. Inhibitors–of–angiogenesis:
   Bevacizumab,thalidomide
   
   
5. Inhibitors–of–resisanc–mechanism:
   Disulfiram, verapamil, ketoconazol, 5-azacytidine
   
   
6. Holistic–Alternative–substances:
   H2O2, ascorbic acid , carnivora , misteltoe, quercetin , indol-3-carbinol , c-statin , ukrain , polyMVA, co enzyme Q10, essiac tea, modified citrus pectin, IP6 , pacreatic enzymes, salvestrol, Uncaria Tomentosa, carctol, noni juice, annonaceous acetogenins, caesium chloride, reolysin, amygdalin-B17-, artesunate, maitake, lycopene, curcumin, green tee extract, melatonin, ellagic acid, L-methionine, N-acetyl-cystein, Niacin (Vit.B3), L-carnithine, Vitamin E (tocopherol), superoxide dismutase (SOD) , selenium, aloe vera, IFNa2,cytozyme bovine trahea,acemannan, acai berry, avemar pulvis, AHCC-Active Hexose Correlated Compound, silver 32.
           Then every 24 hours we take from each culture a small sample and we isolate the mRNA. Then with the RT-PCR we produce the cDNA. Then we follow the microarray hybridization and the colorimetric microarray analysis using probes for the genes bellow:
           TS ,DHFR , Tubulin a and b , transcription factor about tubulin a, Topoisomerase I and II (a &b), SHMT, DPD, TP, p27, p53, DNA methyltransferase, O6 acyltransferase, DNA deaminase, MRP (I & II), LRP, GST, VEGF, PDGF, EGF, TGFb, IGF, MMP9, nucleotide reductase, COX-2, 5-LOX , SS-r, c-erb-B2.
           To be more precise the thymidylate synthase and DHFR are genes that produce enzymes very crucial for the folate pathway. As you already know this pathway is the only biochemical way for the cells to produce thymidine nucleotide which is necessary for the DNA duplication and cell division. When cancer cells are resisting to antifolates such as methotrexate or 5 fluoruracil ,they over-express those genes so the amount of enzymes becomes so big that the inhibitor ahs to be in very high concentration and the toxicities are very severe. Also the mRNA of those genes bounds with the enzymes and it cannot allow the inhibitor to bind with them. Additionally to that we must mention that especially for 5 FU we test the expression of DPD and TP that catalyze the transformation of 5-FU to 5-FUMP, the active product. If the activity of these genes is not regular the 5FU cannot transform properly and so, the toxicities are very severe also.
           As for the DNA crosslinking rearranging enzymes topoisomerase I and II with all their isophorms, they produce enzymes which become tartgets for the anthracyclines, camptothecins , epipodophylins and adriamycin compounds. These enzymes are crucial on S phase of the cell cycle (in DNA duplication) and on DNA repair mechanisms. Unfortunately, the resistance mechanisms for the above inhibitors depend only on the over-expression of the p glycoprotein pumps MDR1, MRP and LRP in lung's tissue.
           The genes that produce tubulin a and b in all their isoforms are crucial for the formation of the nucleus spindle. The alkaloids of Vinka bond with tubulin B, they inhibit the formation of the spindle and they spot the cell cycle in metaphasis stage. Also the taxanes bond with the dimmers of tubulin a and b and make the nucleus spindle so stable that it is impossible to depolymerize . With this mechanism taxanes stop the cell cycle in telophasis stage. Unfortunately, both Vinka alkaloids and taxanes become targets for membrane pumps MDR1, MRP1 and LRP or the mutations especially on tubulin b genes, make the molecule unable to bond with the inhibitors.
           Additionally, DNA methyltransferase, O6 acyltransferase , and demethylase of DNA are genes that are involved in the DNA methylation and they regulate the coiling and uncoiling of genes. With these mechanisms the above genes are regulating other genes' expression to cells. Especially in cancer cells the DNA hypermethylation is one of the mechanisms of resistance and regulation of cancer phenotype and behavior. Also those genes are involved in alkyliating drugs inhibition mechanism.
           Finally, the VEGF, FGF and PDGF genes are involved to the angiogenesis procedure. As you already know angiogenesis is necessary for the metastatic procedure. Additionally to that the genes of TGF, of EGF, of IGF and somatostatin (somatomedins) are over-expressed in cancer cells and it triggers again and again the proper membrane receptors -that are also over-expressed- and they create a positive feed back mechanism of cell growth and mitosis without ending -the start of immortalization. Also the ecosanoids are involved as growth factors or transcriptional factors in carcinogenesis and in cancer phenotype of the cells. Particularly the over-expression of cycloxygenase 2 and 5 lipoxygenase are crucial for the above procedure. Studies have already shown that inhibition of these enzymes (by coxibs or analogues of triens) leads to inhibition of cancer cell growth.
           We also check the bio-availability of the cells using a coloring substance -Trypan Blue 0.4% - taking micro-photos. The Trypan Blue is the most reliable method to measure the viability of a cell culture . As the specialists know, Trypan Blue is a unique analogue of aminopterin and it can participate to the folate cycle -a chemical pathway that is crucial and overactive in cancer cells. But this aminopterin cannot pass the cell membrane therefore it paints it in blue color but without painting the inner cellular area. In Debris cells (dead cells) it can pass through the broken membrane and it can paint the total cell body in deep blue. From the 6 samples that we take every in 6 days we develop a viability diagram for each chemotherapeutic drug.
           This procedure is made once every 24 hours for 6 days.         The micro-array data as well as the micro-photos of each case are sent to our labs and our computers where they are analyzed and evaluated.
           Once we have collected all of the above mentioned data, we develop a genetic and cytological profile of the patients neoplasmic cancer cells.
   With the above-mentioned data in hand, we develop a safe profile, both genetic & cytological, for the neoplasmic cells in each case.
           As a result, we are able to conclude which specific pharmaceutical substances the cells are sensitive and resistant to, quickly and safely.

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