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| For Oncologists |
ADVANTAGES ON A COMPETITIVE ANALYSIS ON SCIENTIFIC BASE OF OUR TECHNIQUES
Until now the assays that have been in use are the clonogenic assays such as the ATP-TCA, the MTT, and the SRB, among others. These assays are limited to using only the tissue sample, or in the MTT's case the tumor effusion material. The assays are also based only on the quantification analysis of living cells using indirect parameters. For example:
 The SRB assay uses sulforhodamine B, which bonds with total proteins of the cells, and through the colorimetric technique it is possible to find the number of living cells in a cancer cell culture (this is where we place a chemotherapeutic drug in the growth medium).
In the case of MTT, it is based on the tetrazolium assay which depends on the NADP and the NADPH quantities.
Finally, the ATP-TCA assay which measures the production of ATP with the use of fluorometric and photometric techniques (which in turn measure the catalysis of luciferin by the enzyme luciferase), requires mitochondrial ATP to be completed.
Unfortunately, these assays have major disadvantages and as a result do not have widespread clinical use. Specifically:
The MTT assay is affected by the intracellular concentration of glucose and pH. This measurement is time dependent and the time point must be very precise.
In the case of SRB, the assay is much easier and less time consuming than the MTT, however it is based on the fact that sulforhodamine B binds with the intracellular proteins to the amino-end point edge and that is an indirect factor of the cells viability. Also, the SRB, as all other chemo-sensitivity assays cannot provide information quickly and easily about the development of resistant mechanisms, nor about the metastatic ability of the tumor through neo-angiogenesis.
These assays (ATP-TCA, MTT, SRB, Disc, etc) cannot predict the sensitivity or the resistance in a short time frame (the clonogenic assay needs approximately 20 days to complete). The above assays provide only minimal amounts of information about the malignant phenotype of the cancer and do not suggest solutions to avoiding or reversing the resistance mechanisms. Additionally, these assays cannot predict toxicity levels arising from the use of chemotherapeutic drugs, such as 5-FU, nor do they provide any information about such drugs (i.e. the category of biological modifiers).
Remission Labs provides services that overcome the disadvantages mentioned above with the use of micro-array chemo-sensitivity test (CST), which warrants the use of not only the tissue sample from the tumor or tumor effusion material, but also peripheral (whole) blood in patients with cases of advanced stage IV cancer. Using whole blood sample we:
Isolate the circulating cancer cells using gradient solutions and membranes with pores.
Proceed to negative selection using cytospin with anti CD+45 (these antibodies bond to hematological cells and separate them from epithelial cells-malignant).
Move on to the positive selection using EpCam antibody that bond and isolate trough cytospin the epithelial origin cell (cancer cell).
Identify the isolated malignant cells using the immortalization detection kit from Oncogen.
Develop many cell cultures in a 96 well plate and in each slot add to the culture medium a different chemotherapeutic drug.
Then, every 24 hours extract from each culture a small sample and isolate the mRNA.
With the RT-PCR are able to produce the cDNA.
Follow the micro-array hybridization and the colorimetric micro-array analysis (pict.1) using probes for the genes listed 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 known to produce enzymes very crucial for the folate pathway. This pathway is the only biochemical way for the cells to produce thymidine nucleotide which in turn is necessary for the DNA duplication and cell division. When cancer cells are resistant to anti-folates such as methotrexate and 5 fluorouracil, they over-stimulate those genes to the point where the amount of enzymes becomes so big that the inhibitor ahs is in very high concentration and the toxicities become very severe. Also, the mRNA of those genes bounds with the enzymes and it does not allow the inhibitor to bind with them. Additionally, we must mention that especially in the case of 5-FU we test the stimulation of DPD and TP that catalyze the transformation of 5-FU to 5-FUMP, the active product . If the activity of these genes is irregular, the 5FU cannot transform properly and so, the toxicities also become severe.
When it comes to the DNA cross linking and rearranging enzymes, topoisomerase I and II with all their isophorms, they produce enzymes which become targets for the anthraxcyclines, camptothecins, epipodophylins, and adriamycin compounds. These enzymes are crucial during the S phase of the cell cycle (in DNA duplication) and in DNA repair mechanisms. Unfortunately, the resistance mechanisms for the above inhibitors depend only on the over-stimulation of the p glycoprotein pumps MDR1, MRP, and LRP in the lung's tissue.
The genes that produce tubulin a and b and all of their isoforms are crucial to the formation of the nucleus spindle. The alkaloids of Vinka bond along with tubulin B inhibit the formation of the spindle and spot the cell cycle in the metaphases stage. The taxanes bond with the dimmers of tubulin a and b to make the nucleus spindle so stable that it is impossible to de-polymerize . With this mechanism the taxanes stop the cell cycle in the telophasis stage. Unfortunately, both Vinka alkaloids and taxanes become targets of membrane pumps MDR1, MRP1, and LRP or the mutations especially on tubulin b genes, making the molecule unable to bond with the inhibitors.
Additionally, the DNA methyltransferase,O6 acyltransferase, and demethylase of the DNA are genes that are involved in the DNA methylation which regulate the coiling and uncoiling of the genes. With these mechanisms the above genes regulate the other genes' expression to the cells. Specifically in cancer cells, the DNA hypermethylation is one of the mechanisms of resistance and regulation of cancer phenotype and its behavior. Also, those genes are involved in the alkyliating drugs inhibition mechanism.
Finally, the VEGF, FGF and PDGF genes are involved in the angiogenesis procedure. Angiogenesis is necessary for the metastatic procedure. The genes of TGF, EGF, IGF, and somatostatin (somatomedins) are over-stimulated in the cancer cells which triggers again and again the proper membrane receptors that are also over-stimulated which in-turn create a positive feedback mechanism of cell growth and mitosis without ending the start of immortalization. The ecosanoids are also involved as growth factors or transcriptional factors in carcinogenesis and in cancer phenotype of the cells. Particularly, the over-stimulation of cycloxygenase 2 and 5 lipoxygenase is crucial to the above procedure. Studies have already shown that inhibition of these enzymes (coxibs or analogues of triens) leads to the remission of cancer cell growth.
The pharmaceutical substances that are used and are inserted in the culture medium are:
oxaliplatin
cisplatin
cyclophosphamide
ifoshpamide
trophosphamide
treosulphan
melphalan
CCNU
BCNU
ACNU
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mitomycin
procarbazine
dacarbazine
bleomycin
temozolomide
estramustin
doxorubicin
epirubicin
daunorubicin
dactinomycin
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irinotecan
topotecan
idarubicin
etoposide
mitoxandrone
paclitaxel
docetaxel
vincristin
vinblastin
vinorelbin
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taurolidin
5fluoruracil
uraciltegafur
raltitrexed
floxuridine
capecitabine
pemetrexed
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We also check the bio-availability of the cells using a coloring substance (Trypan Blue 0.4%) and taking micro-photos. The Trypan Blue is the most reliable method to measure the viability of a cell culture . Trypan Blue is a unique analogue of aminopterin which can participate in the folate cycle (a chemical pathway that is crucial and overactive in cancer cells). Due to the fact that this aminopterin cannot pass the cell membrane, it colors it in a blue color without coloring 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 a deep blue color. From the 6 samples that we take every 6 days we are able to develop a viability diagram for each chemotherapeutic drug. This procedure continues once every 24 hours for a period of 6 days. The micro-array data as well as the micro-photos of each case are sent to our labs to be analyzed and evaluated by our computers.
We also test 50 of the most popular cancer killing alternative substances. We extract the active ingredients and we test them on the patients cancer as well.*
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 resistance to, quickly and safely.
We also test 50 of the most popular cancer killing alternative substances. We extract the active ingredients and we test them on the patients cancer as well.*
* See sample analysis for more details
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