Side Effects of Tumor Size Reducing Drugs | Experiment

Side Effects of Tumor Size Reducing Drugs examineManish Kumar Tiwari1. IntroductionObjective Pfizer assimilate developed a new do do do drugsss that appears to reduce the size of it of detail tumours only if be concerned approximately what effect the drug might have on convening tissue. digest how you would use desoxyribonucleic acid technology to address this issue.Cancer disease has plumping complexities in terms of genome variations at cistrontic level and epigenetic level. Immortalization and tumour coevals ar the ii fundamental characteristics of malignant cubicles. This disease is ca utilize by mutations in genes much(prenominal) as oncogenes, deoxyribonucleic acid repairing genes and tumor suppressor genes. Recent researches suggested that more than than one mutations be emergencyed for the thunder mugcers. One of the major drawbacks of the medicines or drugs that are used to treat dirty dogcer is its placement effect on regulation kiosks. The cells which are mostly affected by drugs are quickly dividing cells such as blood cells, fuzz follicles cells, cells found in tract of reproductive organ and digestive system, and cells from immune system. Side stick on standard cells due to chem oppositeapy has become major challenges for researchers. Transcriptome or protein mvictimization profiling for pilecerous cells treated with item drugs may provide usable information nearly possible nerve effectuate on figure cells. When any drugs or medicines are minded(p) for the treatment of any particular tumor disease, it binds with specific receptors (cell surface receptors, Cytoplasmic receptors or nuclear receptors) and leads to musical arrangement and translation touch on and generate specific proteins that dejection be able to stop the cell cycle or initiation of apoptosis. But generally these drugs may as well responsible to translation of unwanted proteins that can cause stead cause on normal tissues.2. App roachMode of action of many drugs that reduces the size of tumor are related to growth cycle (like mutagen, MAP kinase pathway) or desoxyribonucleic acid modifications ( organisation, translation etc.). This system is more equal for in vivo scrutiny in rats or mammal malignant neoplastic diseaseous cell lines which has been described here. The added tumor size reducing drug must(prenominal) bind with specific receptors on the tumor cells. So first step is the identification of pathway via which it acts. In the downstream signaling of the pathway, nigh transcription reckons will be pioneer and will bind to signal shoplifter and reduces the size of tumor. So consequently transcription factors need to be quantified by qPCR as well as the sequence of their takeoff rocket done desoxyribonucleic acid Foot Printing. Now two plasmid DNAs need to be constructed (minimum two plasmids, if there are more transcription factors and promoters, more plasmids with different light fixt ure proteins are needed) containing above identify promoter united with red light protein (RFP) and containing a tumor inducible promoter united with green fluorescent fixture fixture fixture protein (GFP). Now for in vivo testing, mutant mouse are created and transfected with above two plasmids. During the growth, the known tumor inducing compounds/radiation is given to the mouse to induce the tumor. As the drug is added, it will cause trigger of RFP through and through the body but level may be higher(prenominal) in tumor cells but GFP should be induced only in the tumor cells. If GFP is induced in some other normal cells it means that this drug may cause positioning effect on that cells. A fluorescent interpret of mouse will reveal the efficacy and side effects of drug based on RFP and GFP intensity.Figure 1 Schematic intromission of move up. The image of mice is taken from internet which has been used to explain the order.3. MethodThis mode is desirable for in vivo t esting in mice or mammalian cells culture. The briny steps include quantification and identification of transcription factors and promoter sequences respectively, construction of suitable plasmids coupled with red and green fluorescent proteins, transfection of plasmids in mice body, tumor evocation in mice body followed by drug injection and last fluorescent mapping apply fluorescent detector. The instruments and techniques which will be used in this rules are qRT-PCR, DNase Foot Printing assay, Suitable plasmids vector, microinjections, Chemicals, fluorescent proteins (red and green), capillary electrophoresis, tumor inducing cells or chemicals or radiation and fluorescent detector. Validation of this method acting is important so governing body could be possible by using this method for any known drug which side effects on normal cells has been identified completely.3.1. Quantification of Transcription FactorThe exact quantification of transcription factor is the most importa nt part of this method. Micro array or PCR is the favorable technique for quantification of transcription factors but in this method qPCR/QRT-PCR will be give up technique. First step is isolation of cancer cells from mammalian cancerous cell lines. thusly inject target anti-cancer drug and incubate for slightly time because these drugs takes some time to dough their function. later proper incubation, congeries or poly A RNA extraction is the next step. The solution which is used in extraction process should be RNase free other it can degrade our RNA so that exact quantification could non be possible. Sample should be treated with DNase to remove genomic DNA contamination.Flow chart 1 travel involved in quantification of Transcription factorsElectrophoresis and qPCR methods could be used for determination of purity and accurate concentration because these factors are rattling important for proper gene expression profiling. Then C DNA synthesis and validation of C DNA qua lity and quantity could be done by using qRT-PCR. For performing qRT-PCR assay there are two important steps such as selection of appropriate reference genes and designing of PCR primer labeled with fluorescent dye must be needed. For data analysis fluorescent detector can be used to detect transcription factors and their associated genes. Now once genes have been identified by using above method so the identification of their promoter sequence DNA Foot Printing assay will be performed.3.2. Identification of Promoter SequenceDNase Foot printing assay method can be used to identify target promoter sequence. Steps involved in this method is amplification of target DNA through PCR using fluorescent labeled primer at 5 end. Then cleavage of the amplified DNA by using DNase enzyme followed by the capillary electrophoresis. The cleavage pattern will vary due to the presence of transcription factor, because the fertilisation sites are protected by the protein from the cleavage. By using this method we can identify the promoter sequences. By using capillary electrophoresis we can identify the amount and size of DNA fragments and about the bases which are not cleaved by the DNase enzyme.Figure 2 Identification of promoter sequences through DNA Foot Printing assay. The graph between amount and size of DNA fragments in this figure is specifying the bases which are protected by the transcription factor against DNase enzyme.3.3. Construction of Suitable PlasmidsConstruction of suitable expression vectors for mammalian cells, that can carry the desired promoter sequence coupled with fluorescent protein must be needed. The most important characteristics of vectors is presence of all elements that is suitable for expression in host cells. The important elements are promoter, stop and start codon, binding sites for ribosome, ORI region and appropriate selection markers. Some models of vectors like adenoviral, PSV and pCMV are generally used for expression in mammalian cell s. In this method, our expression vectors should contain promoter sequence labeled with red and green fluorescent protein and other important elements. Minimum two type of plasmid vectors need to be constructed. One plasmid should have promoter coupled with RFP which has not induced by the tumor inducible transcription factors. Other plasmid should have tumor inducible promoter coupled with green fluorescent protein. Our main root word is to inject these vectors into the mutated mice body so that we also need to remove the other elements of vectors that can cause any unwanted diseases in mutated mice. The vectors like pED and Pz can be used for the expression in mammalian cells.Figure 3 Construction of plasmids containing promoter coupled with Red and Green fluorescent protein.The very first step for the construction of the recombinant plasmid is the cleavage of both plasmid and target DNA with promoter sequence coupled with fluorescent protein using suitable restriction enzymes. The restriction enzymes creates sticky or blunt ends (depends on type of restriction enzyme used) in both plasmid and target DNA. abutting step is the hybridization of both DNA and plasmid using DNA ligase enzyme. Selection of cells having plasmid with desired sequence is very important so further we need to selection of appropriate vector by using selection markers like antibiotic resistance genes.3.4. TransfectionThe transfer of desired plasmid inside the mice body could be possible through many ship canal such as microinjection, electroporation, shotgun method, through chemicals and viral infections. Transfection through viral infection has some limitations like limited carrying capacity of desired gene and unwanted inflammatory mutations. However, transfection through viral infection have some advantages like easy to handle, easy preparation and slowly monitoring during the process. So, in this method transfection of plasmid in mice should be done directly through microinjecti on into the mice body. One another way for transfection of recombinant plasmids in mice is through recombinant Baculocomputer virus. Baculovirus infects insect cells. Purified budded virus can be isolate from the infected insect cells with recombinant Baculovirus. This purified budded virus can be introduced inside the mice body. For the study of side effects on normal cells in whole body of mice it is very important that this recombinant plasmids will chance upon every parts of body along with tumor affected parts.3.5. installation of Tumor in Micemammalian cancerous cell lines or cell DNA extracted from virally infected cells can be able to induce cancer in mice. Once theses tumorgenic cells is injected inside the mice body it develop specific tumor. After developing cancer in mice body, anti-cancer drug is administered through injection to show the efficacy and side effects on cancerous and non-tumor cells. When drugs binds with specific target receptors, it will induce both pr omoters but with varying intensity. The promoter coupled with RFP will show intensities in both normal and tumor cells but may be higher in tumor cells. But GFP should be induced only in tumor cells if it is inducing in other normal cells with high intensity then it may cause side effects on those normal cells.3.6. Fluorescent MappingAnalysis of fluorescent mapping of these promoters in different locations of the mice body can provide profitable information about possible side effects against designed anti-cancer drugs. For example if GFP will be induced in other cells like vibrissa cells, heart cells, bone marrow cells than we can predict the side effects on these cells because the drug should not induce translational process in normal cells. If this drug induces promoters only in tumor cells then the chances of side effects may be less. We can study possible side effects against various drugs by using this method.Figure 4 This stamp has been limited for illustrating the possibl e results that can be produced by this method. Region B and C in this figure are representing the cancerous cells where GFP has been expressed. Region A is representing the normal cells where GFP has been also expressed so this drug may cause side effects on this cells.3.7. Validation of the MethodThis approach has not been validated because this is the hypothesis only. For the testing of this method whether it is working efficiently or not need to be validated. An efficient approach has been described here. For the validation of this method we need to perform this method on known anti-cancer drugs for specific type of cancers. This method can be apply for known drugs which side effects on normal cells have been identified completely. If fluorescent mapping provide exact location in the body where GFP has been induced and if these locations are related with those areas where this specific drug causes side effects then this method will be validated. But proper validation need to be t ested for various anti-tumor drugs which side effects has been completely known.4. Discussion on that point are so many side effects associated with anti-cancer drugs because these drugs mainly affects rapidly dividing cells and immune system. The drugs or medicines that are currently used have forever and a day some common side effects like typhlitis, diarrhea and hair loss but sometimes these drugs cause serious side effects like colorful damage and cardiac arrest because these drugs are unable(p) to differentiate rapidly growing normal and cancerous cells. So that victimization of proper efficient method for testing possible side effects for any anti-cancer drugs should be developed. In this section a good approach has been described for the identification of possible side effects on normal cells. The idea is based on the role of transcription factors induced by the drug- receptors interactions. As instance certain anti-tumor drugs causes anemia when used for the treatment of specific tumor. ingredientrally the gene called HBB is responsible for anemia because this gene encode genus Beta globins protein. It means that these drugs also induces transcription factor that is responsible for activation of HBB gene. The fluorescent mapping of unknown anti-cancer drug against specific cancer can provides useful information about possible side effects. The figure 4 which has been modified to illustrate the possible results that can be achieved through this method. If the drug is not inducing GFP in normal cells except cancerous cells it means drug will not cause any side effects on normal cells but vice versa if GFP is expressing in other cells along with tumor cells so we can predict possible side effects on those cells because this method is also useful to find out what type of protein or transcription factors are expressed. By using bioinformatics data bases like PDB, gene bank etc, functions of expressed proteins or transcription factors can be easily pre dict. The method which has been described above has not validate yet because this method is only a hypothesis that need further advancement and validation.5. 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