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Anti-Cancer Drug Design and Discovery - Science topic

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Please have look on our(Eminent Biosciences (EMBS)) collaborations.. and let me know if interested to associate with us
Our recent publications In collaborations with industries and academia in India and world wide.
EMBS publication In association with Universidad Tecnológica Metropolitana, Santiago, Chile. Publication Link: https://pubmed.ncbi.nlm.nih.gov/33397265/
EMBS publication In association with Moscow State University , Russia. Publication Link: https://pubmed.ncbi.nlm.nih.gov/32967475/
EMBS publication In association with  Icahn Institute of Genomics and Multiscale Biology,, Mount Sinai Health System, Manhattan, NY, USA. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/29199918
EMBS publication In association with  University of Missouri, St. Louis, MO, USA. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30457050
EMBS publication In association with  Virginia Commonwealth University, Richmond, Virginia, USA. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27852211
EMBS publication In association with  ICMR- NIN(National Institute of Nutrition), Hyderabad Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/23030611
EMBS publication In association with  University of Minnesota Duluth, Duluth MN 55811 USA. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27852211
EMBS publication In association with  University of Yaounde I, PO Box 812, Yaoundé, Cameroon. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30950335
EMBS publication In association with  Federal University of Paraíba, João Pessoa, PB, Brazil. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30693065
Eminent Biosciences(EMBS) and  University of Yaoundé I, Yaoundé, Cameroon. Publication Link: https://pubmed.ncbi.nlm.nih.gov/31210847/
Eminent Biosciences(EMBS) and  University of the Basque Country  UPV/EHU, 48080, Leioa, Spain. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27852204
Eminent Biosciences(EMBS) and  King Saud University, Riyadh, Saudi Arabia. Publication Link: http://www.eurekaselect.com/135585
Eminent Biosciences(EMBS) and  NIPER , Hyderabad, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/29053759
Eminent Biosciences(EMBS) and  Alagappa University, Tamil Nadu, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30950335
Eminent Biosciences(EMBS) and  Jawaharlal Nehru Technological University,  Hyderabad , India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/28472910
Eminent Biosciences(EMBS) and  C.S.I.R – CRISAT, Karaikudi, Tamil Nadu, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30237676
Eminent Biosciences(EMBS) and  Karpagam academy of higher education, Eachinary, Coimbatore , Tamil Nadu, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30237672
Eminent Biosciences(EMBS) and  Ballets Olaeta Kalea, 4, 48014 Bilbao, Bizkaia, Spain. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/29199918
Eminent Biosciences(EMBS) and  Hospital for Genetic Diseases, Osmania University, Hyderabad - 500 016, Telangana, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/28472910
Eminent Biosciences(EMBS) and  School of Ocean Science and Technology, Kerala University of Fisheries and Ocean Studies, Panangad-682 506, Cochin, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27964704
Eminent Biosciences(EMBS) and  CODEWEL Nireekshana-ACET, Hyderabad, Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/26770024
Eminent Biosciences(EMBS) and  Bharathiyar University, Coimbatore-641046, Tamilnadu, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27919211
Eminent Biosciences(EMBS) and  LPU University, Phagwara, Punjab, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/31030499
Eminent Biosciences(EMBS) and  Department of Bioinformatics, Kerala University, Kerala. Publication Link: http://www.eurekaselect.com/135585
Eminent Biosciences(EMBS) and  Gandhi Medical College and Osmania Medical College, Hyderabad 500 038, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27450915
Eminent Biosciences(EMBS) and  National College (Affiliated to Bharathidasan University), Tiruchirapalli, 620 001 Tamil Nadu, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/27266485
Eminent Biosciences(EMBS) and  University of Calicut - 673635, Kerala, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/23030611
Eminent Biosciences(EMBS) and  NIPER, Hyderabad, India. ) Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/29053759
Eminent Biosciences(EMBS) and  King George's Medical University, (Erstwhile C.S.M. Medical University), Lucknow-226 003, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/25579575
Eminent Biosciences(EMBS) and  School of Chemical & Biotechnology, SASTRA University, Thanjavur, India Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/25579569
Eminent Biosciences(EMBS) and  Safi center for scientific research, Malappuram, Kerala, India. Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/30237672
Eminent Biosciences(EMBS) and  Dept of Genetics, Osmania University, Hyderabad Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/25248957
EMBS publication In association with  Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad Publication Link: https://www.ncbi.nlm.nih.gov/pubmed/26229292
Sincerely,
Dr. Anuraj Nayarisseri
Principal Scientist & Director,
Eminent Biosciences.
Mob :+91 97522 95342
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The extensive successful use in people of mRNA vaccines for Covid-19 suggests they mark a key scientific breakthrough with broad impacts. These breakthrough vaccines are notable for combining two advances examined for years in the laboratory: pseudouridine modified mRNA and its encapsulation in lipid nanoparticles. Pseudouridine has been used in nucleotides for years to stabilize and study key protein-nucleotide complexes (e.g. Parikh et al. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5083-8. doi: 10.1073/pnas.97.10.5083. PMCID: PMC25785 ). In fact, Pseudouridine (Ψ) is the most abundant RNA modification and has been termed the 5th base of RNA (Front. Cell Dev. Biol., 16 March 2021 https://doi.org/10.3389/fcell.2021.628415). Similarly Lipid nanoparticles have been used and studied for over 20 years (e.g. Puri et al. Crit Rev Ther Drug Carrier Syst. 2009; 26(6): 523–580. PMCID: PMC2885142). Yet the combination of mRNA modification by N1-methylpseudouridine to increase safety and efficacy with lipid nanoparticles to encapsulate mRNA for its protection and delivery made the Covid-19 mRNA vaccines feasible. Importantly, this transformative combination seems applicable to other vaccines and approaches suggesting it is worth considering the many possible applications that may advance biotechnology and human health.
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I think the effect will be significant on the progress of treatment, and the world will witness the end of cancer in the future
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I am trying to grow cancer cells and do MTT assay for drug screening. Are there any alternatives to FBS? I know that FBS and FCS are widely used for cell culturing, but I am looking for other options.
I found one online and got it ordered:
But the cells are not growing properly in this.
Any suggestions in this regard?
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You can try thermofisher product for serum free media
https://www.thermofisher.com/in/en/home/life-science/cell-culture/mammalian-cell-culture/serum-free-media. As Adam Shapiro has mentioned, you have to slowly adapt the cultures to serum free conditions. You can also try using DMEM/Ham's F-12 (50:50) supplemented with growth factors and hormones.
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There are some studies on anti-tumor effect of NK cells with conflicting results. We want to evaluate the anti tumor effect of NK cells in vitro and in vivo. What are the key biomarkers or surface proteins which would determine the effectiveness of NK cells against tumors?
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For my knowledge, tumor cells can induce exhaustation in NK cells, that is a condition characterized by impaired function and an inhibited phenotype with the expression of receptors such as PD-1 and Tim-3. Also, tumor cells can overexpress and/or shed NK cell ligands, which in turn causes receptor internalization.
However, exhaustation is often mediated by other immune cells recruited in the tumor microenvironment rather by tumor cells themselves, so it is very complex to model it in vitro
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Specifically as a polivitaminic compound, and as an anti-tumoral agent.
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Hi
My colleagues in next lab assumes that they purified  new biosrfactant and they want to know its anti cancer properties or its cytotoxicity toward cancer cells..
I've exposed this biosrfactant to cancer cell lines and waiting for the results
What are your thoughts about effect of biosrfactant  did they will affect on cancer cells or not ?  And on which pathway? 
Thanks 
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Some notable scientists have been claiming that amygdalin (Laetrile) is effective against cancer based on a cyanogenic compound specific for cancer cells.
Please does any one have solid article supporting this claim or any scientific research that refute this claim?
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Role of laetrile (also known as vitamin B17) in cancer treatment has not been yet scientifically proven. Please go through the following RG links.
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I am using MDA-MB-231 and MCF7 for doing experiment. We have CO2 independent medium. Could you tell me how should I add other components (FBS, Glutamin/max, Penicillin, Insulin..). If I need 1ml medium, how should I dilute these components?
For FBS, Glutamin.. should I store in refrigerator (2-8 degree) for long time or I have to freeze them?
I look forward to hearing from you.
Thank you so much for your help.
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You must add the additive based on the final amount of the material in the culture medium. For example, the 1. The serum concentration at the final concentration should be 10%, it is easy to calculate the serum required by a simple mathematical procedure (10 cc for 90 cc and 50 cc for 450 cc, volume Your initial culture is 500 cc, so you need to add 5 cc to 50 cc, so you need to add 55 cc of the serum to your crop for the 500 cc medium. Note that in solutions of grams / volume, the amount of material is dissolved in 100 cc of solvent, for example 10% glucose, 10 g glugose per 100 cc solvent, but in volume / volume solutions such as 30% alcohol 30 cc of alcohol is dissolved in 70 cc distilled water. You should filter the additive material in the addition of materials to the cell culture medium; besides antibiotics .
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I have tested a compound ( having anticancer potential) on PBMC for 24 hour, is there any need to test for 48 hour in vitro ?
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Hi Yogesh!
The minimum incubation time for measurement of cytotoxicity is 24 h, but however you may get lover LC50 concentration by increasing the drug exposure time.
Further PBMC is not cancerous cell line, then how would you be able to determine anticancer potential on it? You may have to choose some cancer cell lines to study the said effect. Again you may test cytotoxicity of your drug on PBMC in order to determine harmful activity of your drug on normal healthy cell lines (PBMC).
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is Anti-Cancer Drug 'Curcumin' present in injectable or liquid form in INDIA ? if yes, then Can any one give me its details ?
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Yes, Curcumin emulsion is available with synthite industries Ltd. We are working on its charecterization @ our facility.
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Hello, I'm curious if anyone has a good method of approximating human dose equivalents based on in-vitro studies of novel cancer drugs? I have only found literature to help convert in-vivo mouse doses to human doses. Thanks in advance!
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Great, this is very helpful. Thanks for your insights!
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i am working on anticancer drugs that may cause oxidative stress in cancer and normal cells.my interest is, treatment of that drug to RBC cells in invitro  and to check the ROS generation on the cells.
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There may be a problem with monitoring fluorescence in red cells as the haemoglobin will quench both the excited and emitted light better to stick with glutathione and oxidized haemoglobin with spectroscopy.
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Dear All,
Greeting!
I am have developed 5FU loaded nanogels and want to evaluate and compare anti- cancer efficacy using EAC liquid tumor model?
I have focused to study Weight variation, Hem-analysis and Histopathology(heart, liver, spleen, lung and kidney) in normal, control, STD(5-FU) and 5FU- Nanogel groups. 
I would like to know is there any other parameters that could be evaluated?
Also is there any protocol where i can remove know volume of liquid tumour cells diluted in known volume PBS/saline and count and compare with each other (std, control and test group)? If yes can you please share the protocol with me. 
Thank You
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Thank You Manoj. I have focused to study Weight variation, Hem-analysis, Histopathology(heart, liver, spleen, lung and kidney) and MST. Thanks again
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We prepared 10 mM solution  for MTT assay of a compound supposed to have M.wt 424 g/mol. We got IC 50 value 22 micro L but latter we realize that the actual mass of the compound was 648 g/mol and not 424. How can we correct the Ic50 value with out repeating the experiment
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10mM (based on the wrong MW  ,  424)  is  equivalent to 6.54 mM ( based on the new MW 648)
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I have some chemical products which need to be tested for their anti-tumoral property. For this I dissolved them in 100% DMSO and later diluted them in Medium cell culture  (0,1 nM to 100µM). I use the same concentration of DMSO (0,1nM to 200µM) , as control. after 72H I do my MTT essay and I calculate cell survival (%) = treated cell/ Treated cell by the same concentration of  DMSO)*100.
The problem that my results are the same of DMSO. 
Can I get some Idea to improve my results. Can I get another experimental ideas.
Your suggestions will be highly appreciated.
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Hi Zahira, I agree with Mark's comment above.  My rule of thumb is to try to use 1 ul of DMSO per 1ml.  So, when making my drug stock I make sure the concentration is high enough that I'll be using around 1ul per mL.  My control for my assay is then 1ul per mL or 0.1%.  The amount of DMSO you are using is very high and is enough to kill the cells by itself.  I tested the IC50 of DMSO in my cell lines and it was between 1.5-3.5% in about 8 cell lines I tested (ewing sarcoma and rhabdomyosarcoma cell lines).
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suppose you have a compound (structure is derived) then what is the most important features of the compound that makes it suitable for an anticancer activity ?
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Not at all Krishnanand!
A compound to be an anticancer agent must not be in any case toxic for cells. These are a subgroup, and only a subgroup, of anticancer compounds named cytotoxic anticancer drugs.
All the best
Robert
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I would be interested.
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Can a compound protect DNA from oxidizing agent in DNA protection assay ?
and same compound having anticancer potential can cause DNA damage in cancer cell line ?
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@Antonio
I mean to say if a compound is anticancer, it induces apoptosis that cause DNA fragmentation (stained by DAPI ) in cancer cell lines.
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A joint call for research project proposals between Portugal and Algerian scientists / researchers is now available, and invite all local scientists / researchers wishing to participate, to submit their proposals.
The coordination of the program’s activities and its piloting is placed under the responsibility of two focal points: Thematic Agency of Research in Sciences and Technology for the Algerian part and Foundation for Science and Technology for the Portuguese part.
I'm interested to develop a mutual project in the domainof oncology.
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What about including a Belgian guy in your (hopefully wining) team?
I have (and still being) already successfully collaborating with Portugal (see the attached articles) ...
I have (and still being) already successfully collaborating with countries "more or less" in fact "not so far" from Algeria (see the attached articles).
I am eagerly waiting to successfuly enter with a fruitfull Algerian collaboration!
Best regards
Robert
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One of the anti-cancer molecules I am working with activates these receptors. This molecule causes robust cytotoxicity in different breast and colon cancer cell-lines as well as in different models of leukemia. However, this molecule fails to kill A549. I will deeply appreciate a reply.
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Thanks Robert.
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While validating the mode of action of a novel anti-cancer compound, we have options of using small interference RNA or pharmacological inhibitors of a particular signaling molecule.
In several papers we usually found that in the presence of a pharmacological inhibitor of a signaling component (e.g. MEK1/2), the observed biological activity of test compound is almost null, which validates that the test compound is down regulating that particular signaling pathway leading to its anti-cancer activity. Thats convincing to me..
But, there are also papers which reports that in the presence of a pharmacological inhibitor of a signaling component (e.g. MEK1/2), the observed biological activity of test compound is elevated. And, in those papers also the authors claim that the mode of action of test compound is through that signaling pathway. Is it true? 
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I agree with Robert Kiss. A dogma set only on mutations led us out of the way for long time. Cancer is a complex disease, where the same type of cancer has phenotypic characteristics very different even in different persons. It can rightly be regarded as a new tissue produced by an environment characterized by a lasting chronic inflammation. What usually is omitted in the scientific considerations on cancer, is a matter related to the human metabolism that is not adequately considered, either for poor familiarity of many, either because of the complexity it introduces. It is now well known that at least 40% of the proteins present in eukaryotic cells are intrinsically disordered (IDPs). The most important property of these proteins is of not possessing an organized structure over time under physiological conditions. But the most important consequence is their enormous capacity to interact with many and different molecular partners paving the way to different functional activities. This ability is even more amazing when you consider that interactomic studies have clearly shown that they have a key role in the metabolism where they occupy key points (Hub proteins) as connectors to other metabolic pathways. They are present in all cellular compartments, including nucleus.We still do not know exactly when and how a biological function is changed through a change of IDP partners, but this makes the metabolic environment indeterminate. In fact, on the large-scale, a metabolic perturbation cannot be adequately understood by means of the usual deterministic logic that is based on cause and effect. When a cell is metabolically stressed by internal or external perturbations, the metabolic response is predictable only on small scale (at enzyme level, or single signaling pathway) whereas the general metabolic vision is blurred  just because it lacks the knowledge of the new functional behavior of the IDP.  Most likely in cancer we are dealing with impairment of the IDPs, which, therefore, are the targets of choice for a drug that can effectively combat and treat cancer. Until now, the facts show that many drugs are ineffective and at best they allow some survival situation with strong side effects and a big useless waste of money.
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For Anti-cancer activity assessment, I need to take a standard group. For this reason, I need to know the dosage of a standard anticancer drug. I have to administer the drug to mice for 10 days according to the in vivo Anti-cancer protocol.
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May be this article can be of help for you.
Best regards
Robert
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Hi,
 I am working on the research of anti-tumor drugs, I want to know is there any formula for the dose of chemotherapeutic drugs between the cytology test and the animal experiment ?
Thanks a lot!
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I think it is depend on what is the IC50 value of the drug, start with 1/4 or 1/8 from the ic50 value on mice and than establish ED50 LD50 and therapeutics index for further testing.
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Hi ! I am working on 3D culture of cancer cells forming spheroids in hydrogels for drug discovery and screening. Do you think that these small tumors are the best model for testing anti-cancer drugs?
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As others have alluded, obviously there's no "the best" model, short of a phase 3 clinical trial.  
I think these kinds of models are interesting, as they allow you to ask certain questions that aren't accessible in 2D culture. For example, there has been some very interesting work in blood vessel formation in these types of hydrogel cultures.  You can image them, and measure their rate of growth.  So if you're a believer in the anti-angiogenesis approach, you might consider that.  (which I'm not, in the light of the well known absence of efficacy of certain overpriced blockbuster drugs, but that's a different story...).  But important and deep-pocketed people still do, so if your model is selling it to them, why not. 
In general, look for things that you can do with your model, that are difficult to do in other models.  I'm sure there are many legitimate uses. 
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azo compounds are considered carcinogenic, but some of them show anti microbial activity. However, I did not find sufficient work recently published that reports  anti cancer activity of azo compounds. 
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Ali
Please, read the enclosed attachment from SciFinder search
Good luck
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I need to chemically synthesize this purine derivative. Also,  a microwave-assisted synthesis is an acceptable suggestion.
thanks in advance.
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Dear Ms. Maaz,
There are many methods from which the given compound could be synthesized. Some of them are portrayed in the attachment.
Best wishes
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I am looking for the compounds having potent effect on other cancer cell lines with the IC50 value (< 50 micromolar). I would like to test that compound against kidney cancer cell lines and later on in clinical trials.
Thanks in advance.
Please, anyone have idea then I am eagerly waiting for the answer. 
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Would you like to suggest me some Pr. Dipak
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I have isolated a protein from algae and want to subjected it for anti-cancer activity
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Hi Kishore,
First, what type of cancer would you like your algae be tested? If you would like, you may want to try HeLa cells, one of the most common cancer cell line, and MCF-7, a breast cancer cell line. Usually, you would want to know if your natural compound could inhibit growth or proliferation of these cancer cells. However, you should start determining the IC50 of your compound in healthy cell lines first. In this way, you'll be able to know what concentration of your compound you would use. Ideally, at that particular concentration, the healthy cells continue to grow and proliferate while cancer cells are inhibited. I hope the following outline will help you:
Cytotoxicity Assay
1. Determine water solubility of your compound. This will be helpful in solubilizing your compound in culture medium. If insoluble in water, you have to decide if what solvent can be used e.g., DMSO, ethanol, that suits your protein without losing activity
2. Find an available healthy cell line that originated from a similar tissue where the cancer line was derived e.g., HeLa cells = cervical cancer cell line, so I hope you find a healthy cervical cell line (most likely a primary cell line)
3. At various concentration of your protein, usually at 10-fold dilution, treat your healthy monolayer of cells with your compound. Include untreated (negative) control or vehicle control (for compound that was water insoluble). *well plates Highest concentration may start at the max amount of your compound soluble in your medium. This is arbitrary.
4. Monitor cell proliferation (under microscope) and cell viability (MTT assay). You may want to try RealGlo from Promega, where this reagent can be added from seeding the cultures and will develop color over time if cells proliferate. Reagent was claimed not be cytotoxic. This can be measured by spectrophotometer plate reader.
4. Generate curves by plotting absorbance values. Determine IC50 and the concentration of compound where your cells proliferate well, or with least cytotoxic effect. Compare with your control.
5. Use this concentration of your compound to start with. You may now do another round of culture this time with the cancer cell line of interest. You may follow the steps provided with modifications e.g., cancer cell instead of healthy cells, highest concentration to start with = the least cytotoxic concentration, inclusion of anti-cancer compounds as positive controls, etc.
I hope this outline would help you in your experiment. All the best!
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I am testing various anticancer drugs onto several cell lines. I test the cytotoxicity based on 72hr incubation with drugs and assayed via MTS. I keep on getting low absorbance for negative control compared to treated cell lines at low dose of anticancer drugs, I tried PrestoBlue and yielded the same results. My question is; how to explain this phenomena where cell line treated with low dose of anticancer drugs has higher absorbance than negative control, non-treated cells? The expected result is the sigmoidal curve while I obtained a higher peak at low dose (more than control). I tried to find journals explaining this but I could not find one as most graphs in journals managed to get good sigmoidal curve with best fit line at R2 at least 0.9 whereas mine below 0.9. I am using graphpad and excel for comparison.
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I have another comment which is not related to your recent question.
You mentioned your volume is 200 µL in each 96 well. As you know the total volume for a flat bottom well is 360 µL and the recommended working volume is 75 to 200 µL; and you are using the maximum recommended working volume of a well in a 96 well plate. I think I know why; you are not renewing your drug and medium at all. Am I right? Please check the half-life (the duration of action of a drug is known as its half-life) of each drug that you are using. For example some drugs should be renewed each 24 hours (or less and more) for cell treatment.
I just want you to be sure you are doing the right thing for each drug. So check it.
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RAS is oncogene protein in human cancer. I want to design kind pf drug with nanaoparticule to destroy combination pf RAF-ROK protein and activate cell differentiation.
I need to know which kind of coating subtitle and know about the shape, size, and nanoparticle morphology.
Could you please help me?   
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Dear Neda,
Nice to meet you!
As you know, Ras protein and Ras superfamily mediate membrane association and serve as lipid anchors. So their functions are limited to the cytoplasmic side of the cell membrane, where they receive specific signals and transmit them further.
Thus, if you are going to design a Ras inhibitor peptide and corresponding nano-delivery system, first of all it must be able to penetrate the target cells! and this requires the drug and/or delivery system be actively targeted to the cancer cells. There are good tumor penetrating peptides and motifs serving also as receptor targeting ligands...
In respect of the delivery system, there are different options: Liposomes, Polymeric nanoparticles, dendrimers, targeted drug conjugates, etc. Selection depends on the research purpose and design.. I'm myself working on PLGA-b-PEG copolymer nanoparticles.
Regarding the peptide design, you should do some bioinformatics stuff, modeling and protein-protein docking. You also may go through "phage display technology" to select a specific peptide instead of de novo design. However, first of all you need to determine your very target. You may target Ras protein directly and design a peptide that specifically binds to and triggers conformational changes in "inactive Ras" (GDP-state-Ras), so that the protein is inhibited from activation; Or you may design a peptide that specifically binds to GEF(Guanine nucleotide exchange factor) which is the activator of Ras, and hence prevents from Ras activation(converting to GTP-state-Ras). 
Any way, I wish you success dear compatriot !
Sincerely,
Farzad.
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Induction of a mammary gland tumor in female Sprague-dawley rats by inoculation of rat mammary gland tumor cell line, LA7. I would like to get some advice : Is this model reliable for assessment of anti-tumor action? I got some references for this model.
 1.    Evaluation of cytotoxic and chemotherapeutic properties of boldine in breast cancer using in vitro and in vivo models (Drug Design, Development and Therapy 2014:8 719–733)
2.    Induction of mammary gland tumor in female Sprague-Dawley rats with LA7 cells (African Journal of Biotechnology Vol. 9(28), pp. 4491-4498, 12 July, 2010)
  I'm  looking to find a reliable tumor model with cell-line as cost and time are limiting factors. Thank you for your attention.
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Currently, during preclinical testing in vitro are two critical positions. Firstly, it is desirable to use human cell line culture. And secondly, it is necessary to apply the model of 3D cell culture. It may be linear or culture cells of the primary cell culture. Both of these requests are directed to the maximum approximation test conditions to natural conditions characteristic of a person (including a three-dimensional principle of cell populations at the level of the whole organism).
Please, try to open the link below.
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Does the phosphorylation of MAPK proteins alter over time during drug treatment in cancer models? For instance, if I treat melanoma cells with current line of therapy, then do the pERK and pMEK changes at early time like 2hrs, 6hrs, 12hrs, 24hrs, 48hrs upon treatment ? Does it conclude anything?
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Thank you so much for your comment guys. I will consider this for my future experiments :)  
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I am doing colony formation assay using methlycellulose media on RPMI8226 and U266 cells. In the treated samples, number of colonies is reduced compared to the control samples but size of the colonies is increased. Any explanation for this?
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As Dr, Marie Boyd suggested, there is a great problem whether or not drug resistant phenotype is homogeneously or heterogeneously recognized. Furthermore, you have to keep in mind that in the experiment of colony-formation assay and related experiments, colony size and its numbers should be distinguished and they do not necessarily show the consistent increase/decreased change. In the case of your experiments, the colony of U266 cells is composed of heterogeneous cellular population between central and peripheral region. In addition, the tendency to entry into G0 dormant status in the hypoxic microenvironment as compared with its periphery should be evaluated in terms of cell-cycle analysis of U266 cells in each colony.
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We have synthesized some 4-aminoquinoline-pyrimidine conjugates, few of which were selected by NCI for Five dose studies on 60 cancer cell lines and were showing some interesting results. For further biochemical studies, we want collaboration. If anyone is interested, please let me know.
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 4-aminoquinoline-pyrimidine seems to be a promising antimalarial drug.
is there a known target in case of cancer cells?
More information would be appreciated.
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I was collecting information about the list of drugs designed/discovered using Computational Chemistry. Any inputs?
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For downloading the clinical drugs you can go thorough the DrugBank webpage.
The link is as follows:
Here, you can see different sets of drug molecules, classified as:
Approved Drugs, Experimental  Drugs, Nutraceutical Drugs, Illicit Drugs and Withdrawn Drugs. 
The downloaded molecules are in SDF format and can be opened via different molecular display softwares such as MolView or Discovery Studio. 
I hope it helped. 
Ahmad, 
Assistant Professor of Computer-Aided Molecular Design, 
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PBD structure is available for the competitive binding site of GSK3 with various ligand and its well defined.  there are some inhibitor know which bind other than ATP binding site such as TDZD-8 () and manzamineA these are non competitive inhibitor of GSk3b
1. I want to know the binding site (amino acid) of these inhibitors.
2. Weather crystal structure is available for non ATP binding inhibitor of GSK3
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Dear Abdul I want the exact binding site (amino acid ) reported in literature of TDZD-8 and manzamineA. So i can docked newly design ligand in non competitive binding site.
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Very recently, German Volkswagen Stiftung published its call:
"Call for trilateral partnerships/projects of scientists from Ukraine, Russia, and Germany"
Together with some collegues from the Cologne/Bonn area, I would be interested to file a grant application on cancer drug resistance.
If there are collegues out there interested in this field from Russia and the Ukraine, feel free to contact me.
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ok thankyou, in future if any I shall happy to participate.
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Glucosinolates; plant secondary metabolites are known to be very effective against cancer, but some reports suggest that they are also toxic if used as primary food source. Does any researcher has any published material on toxicity limits of these compounds?
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Hi, Saeed,
Ref.:  Das, S., Srinibas, Tyagi, A. K., and Kaur, H. (2000). Cancer modulation by glucosinolates: A review. Curr. Sci. 79: 1665–1671. 
According to Das S et al and Lavecchia T et al, overdose of glucosinolates can cause thyroid dysfunction causing hypertrophy and goiter. 
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Can anyone elaborate how the drug resistance mechanism of cancer is consequence of multipathway connectivity. 
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Cancer resistance, at least for  the case of kinase inhibiting drugs, may have one of many potential origins as detailed by Wagle et al. ( J. Clinical Oncol 29(22), 2011) and others. These may be roughly summarized as follows:
(1) Secondary mutation of the target kinase. Ex: BCR-ABL is resistant to Dasatinib because of a secondary mutation different from the one that resisted the effect of Gleevec (imatinib) and for which Dasatinib was shown to be effective.
(2) Downstream mutation, as is the case for Vemurafenib inhibition of BRAF shown to be accompanied with a RAS-ERK MAPK pathway compensation, driven by a MEKC121S mutation ( MEK is downstream of BRAF). On the other hand the inhibition of MEK itself, by Selumetinib, is accompanied by a different resistant mutation : MEKP1242 .
(3) Upstream activation/amplification. Cancer cells that are resistant to MEK inhibition are found to have amplified BRAFV600E mutants (Corcoran et el. Sci Signal 3(149), 1010) . An example of resistance associated with upstream activation is the exclusive N-RAS mutation or the upregulation of PDGFRβ associated with Vemurafenib inihibition of BRAFV600E in melanomas (Nazarian et al. , Nature 468(7326), 2010)
(4) Activation of an alternate pathway (bypass) – Example: implicated MET oncogene amplification in the activation of PI3K/AKT for the case of lung cancer treatment through EGFR inhibition by gefitinib and eroltinib (Turke, et al. Cancer Cell 17(1), 2010)
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I need liophilized Trastuzumab for NMR structure characterization.
Does anyone know if some company sells the liophilIzed product, because many vendors sell only the reconstituted powder in physiological medium for injection in cancer patients.
Thank you!
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we get Trastuzumab for free, from weekly residues of therapy from the pharmacy of IST, istituto tumori Genova.
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What is the possible anti tumor activity of beta glucan which founds in certain mushrooms causing death of tumor cells?
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Good evening Ali,
I our lab we are carrying out a similar study. We found good anti cancerous activity of the glucans isolated from mushrooms.
During our analysis we found that they majorly elevated the immune response and lead to increased production of ROS which may be a cause of cell death
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The components of the drug are unknown, and we want to test the toxicity of this drug to the tumor cells.
But what is the dose we should add to the cells? Should we choose concentrations of this mixture as our index?
We will complete the component analyses later. Thank you.
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Hello Qingyun Fu,
I would suggest that, after you ave extracted the components of the plant using the solvent of your choice, you chose concentrations of the extract relative to the concentration of cells, but keep varying the concentration of extract from high to low, while keeping the concentration of cells constant. you can chose a factor by which to vary the concentration of your extract eg. 2 etc so that you can be able to determine the toxicity of the extract on cells when concentration is doubled. for example, you could use a ration of 5:200 i.e extract to cells and keep increasing the amount of extract e.g 10:200, 20:200, 40:200, 80:200, 160:200. this way, you will be able to estimate the possible toxic and safe dose levels that you can try and determine more accurately in the next batch of tests.
you have to avoid using very high concentration of the extract to very small number of cells as such results may not have any meaningful interpretations or extrapolation to possible in-vivo effects.
Remember also to read more studies conducted in the same aspect to get more ideas.
I hope this will give you some Idea on what to do.
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My molecule is small and I tried docking with breast cancer protein and it show good results and the binding energy is -2.01 . Tell me can it possibly act as a breast cancer drug...? What next step I can do further...?
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First of all I would like to congratulate you to get a molecule with good docking score. And I would like to add that molecular modelling studies does not guarantee efficient results. It just predicts the results. However there are instances wherein molecules designed using computational studies have come out as potential drug candidates. Therefore, there is a scope for drug discovery using molecular modelling.
The next thing you could do is, synthesize that molecule and evaluate it for its cytotoxicity especially against breast cancer cell lines such as MCF7 or MDA-MB 231 etc. If it is found active then I suggest you to screen it for its effect on estrogen receptor to further confirm its activity against breast cancer.
All the very best. Hope this molecule becomes a potential compound for the treatment of breast cancer.
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We have a sugar-PEG-DOX drug carrier which is water soluble and forms micelle structure whose size is around 60 nm. DOX is bound to PEG block via an enzymaticly cleavable linkage. Our problem is since the polymer forms a micelle structure in aqua system, the enzyme, which will break the bound and leads to releasing of the DOX is not efficient because of limited enzyme can reach the core part of the micelle. Which means the interaction between the enzyme and the linkage is less. My question is how I can increase enzymatic cleavage in our carrier system. I'm open to any suggestion.
Thanks.
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Depending in the pH, and th pI of the enzyme you can add (ou to ,1 mole fraction) of a surfactant with a charge opposite to that of the enzyme. This addition will: a) increase the head group distance and b) the enzyme affinity for the micelle. Several charged lysolipids with + or - charges are available.
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CoCl2 does not induce hypoxia, but it will block degradation of HIF-1a and thereby induce HIF-1 target genes. However, DFX and DMOG are better as chemical inducers of HIF-1a. If you require hypoxia and not just the induction of HIF-1, then you will need to arrange access to a hypoxia chamber.
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What are the chemical compounds of mushrooms that mostly can be used as anti-tumor agents? Especially tropical forest mushrooms from south east Asia.
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You may find anwers to your question in the enclosed articles. Regards. Pr. Sylvie Rapior
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I have different extracts of a medicinal plant. This plant have been known for its medicinal potential in traditional medicinal systems. So, I want to evaluate the biological potential of this plant, but I don't have an in vivo setup for evaluating the above mentioned activities. Is there any in vitro procedure for these activities that would allow me to evaluate this in lab?
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Dear Ajay,
I am sending you three published papers as an attached file on Cyclooxygenase inhibitory effect of medicinal plants by using Cayman assay kits.
SALMAN AHMED
Ph.D Fellow
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When we want to screen or test the effect of a natural product in cancer cells, we have to know how safe is it. Why do some articles mention antiproliferative and others cytotoxicity assays for this screening or testing?
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Proliferation literally means rapid growth of any stuff and in case of cells it is cell proliferation which is nothing but cancer. In my view antiproliferative activity is the ability of a compound to stop the growth of cells. This means that not allowing the cells to multiply rapidly. While cytotoxicity refers to causing harm to cells thereby killing them. Let me explain with an example.
Assume 100 cells are incubated in a plate and after a certain time the cells become 200. After the addition of a drug, if the number of cells in the plate are between 100 and 200 then this refers to growth inhibition which is a measure of antiproliferative activity. On the other hand if the number of cells in the plate are less than 100 then this is referred as cytotoxicity.
Cells incubated = 100
Cells after incubation without drug = 200
Cells after incubation with drug = 100-199 (antiproliferative activity)
Cells after incubation with drug < 100 (cytotoxicity)
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Is there any difference between usual PCR and qRT-PCR
I am got few primer sequences from literature, how could I check complementary sequence with my target gene, for both forward and reverse primers?
What is the minimum and maximum bp (product size) for real time PCR?
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Hi Sathish,
Let me try to answer your queries one by one....let me begin with your query no 2
Yes, ofcourse there exists some significant difference between primers of usual PCR and qRT-PCR . The most important them is the size of the primer and the target sequence for which it is designed.
Now in answering of your query no 4..
The recommended minimum and maximum product size of qRT-PCR is usually between 100 bp to 200 bp and not more than that.
Now coming to your third query,
U can check the complementarity of your primers by using any of the online available tools, they are free to use and very user friendly, at the same time dependable too.
Now coming to your first query..
It is not that easy to discuss with you how you should go for designing ideak qRT-PCR primer over the researchgate platform. But you can always take the help of numerous online available databases and tools. For example..
Hope these may help you with your task.
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We prepare stable bio capped metal and metal oxide nanoparticles using medicinal plants.
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Dear Dr.Mohammad,
I am happy to get a response from you. Can you please mention your terms and conditions for our fruitful collaboration?
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Can any structural features be identified for the binding affinity and what could be the reason for that?
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in general groove binders will prefer AT sequences (distamycin A, netropsin, DAPI), due to steric obstruction of the GC minor groove by the guanine amino group, model intercalators (ethidium bromide, daunomycin, for example) will show no preference for either. Therefore GC binding small molecules are more rare and usually are synthesized for the job, from naturaly ocurring, mithramycin springs to mind. check the ref bellow for the molecular recognition if you want to dig deeper, there is plenty of refs on small mols targeting specific sequences.
Dervan, Bioorganic & Medicinal Chemistry 9 (2001) 2215–2235
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In a recent review I noticed that one of the main handicap of these natural products derived from sharks was that they had not been tested significantly in clinical studies with humans. Can anybody in summary update this knowledge, especially about the results in recent clinical trials with these products?
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Aeterna-Zentaris got great results with their purified shark cartilage extract called AE-941 in preclinical model of glioblastoma and in phase II for lung and kidney cancer and psoriasis as well. One mistake with the phase 3 for kidney cancer, was the recruitment of patients with advanced disease, presenting metastatic sites and resistant to immunotherapy. I don't know if they realized that the mechanisms of action involved 1) prevention of metastasis (via TIMP like protein) and 2) inflammation of the tumor vessel (via expression of cytokines and leukocyte adhesion molecules).
They also started a phase 3 in lung cancer but could not recruit after the publication of the kidney trial. I studied its anti-angiogenic efficacy in glioblastoma during my PhD. The effect was striking in mice, but tumor bleeding was causing intracranial edema, which afraid the clinicians.
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Please explain the details. I need help urgently.
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The half maximal inhibitory concentration (IC50) value determination :
Anticancer Research 32 (2012) 5271-5278; M. Stojak et al.
IC50 is determined usually 48 h after the tested agent application
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I require the caspases for western blotting.
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Cell signal, 9661S for Cleaved Caspase 3 in our lab.
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An anti-cancer compound of herbal origin when in crude plant extracts show substantially good cytotoxic efficacy against cancer cell lines, along with higher IC50 in normal cells (cell lines, mouse bone marrow cells, etc.); but when administered solo, they intend to display higher cytotoxicity to normal cells than previous and at times a lesser IC50 than cancer cells. Well evidenced fact in the contemporary publications.
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The fact that Rajaram explained about various components of a crude formulation triggering more than one pathway is legitimate. And also what Heidi mentioned about the noble prize question of synergistic activity (LOL!) is quite true. Its vast .....very vast and complex to draw down the complete web of action in a cell. like as Heidi you meant that a drug no matter how good it is if not able to penetrate the cell membrane loses its overall efficacy; though with the help of a fellow compound (in the formulation) which may help it get through by increasing its solubility or, manipulating the pH, etc. It is only going to help when we pen down these fragmented thoughts of ours together on a single sheet.
Now again take the example of the traditional medicinal practices like "Ayurveda" etc., where the practitioners (that time) never provided one single plant or derivative as a medicine but they rather made complex formulations involving dozens of plants, which probably complied with their beliefs of theory of Synergism which all four of us mentioned. I adore and wonder how hundreds of years back these guys were sure what herb works where n how, without a western blot PVDF or a Real time!
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I would like to test drug or toxicity with MDA-MB-231 and MCF 7 cells line. Could you tell me which chemical can I use?
Briefly, cancer cells were trapped, incubated, spread on top of microelectrode. I would like to see the response of cells on reagents. I use impedance method so that will be better if reagents cause the changing of cell morphology.
THANK YOU SO MUCH FOR YOUR ANSWERS.
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Epirubicin, Doxorubicin, Docetaxel, Paclitaxel, Cisplatin (Carboplatin is a prodrug), Cyclophosphamid
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I've found that one of my drugs (peptide) causing shrinkage of nucleus and microtubule destabilization. I have also found that the drug is causing apoptosis of cell via p53 and p21 pathway etc. The live cell imaging shows shrinkage of nucleus more than 1/2 w.r.t control cells (without treatment). I want to know whether the effect is common bcz of cells are dying, hence cell cytoplasm to nuclear ration getting decreased or any other reasons...
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You'll have to check
1.PLA2 activation
2.Check similar nuclear shrinkage occurs or not, when you put the peptide+zVAD-fmk
3.Check the levels of HIF1a changes with or without peptide
Then you'll be able to say as you wished for.
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I was going through some article of antibiotic discovery, where they prepared solution of compounds in µg/ml. Two compounds show activity at 25 µg/ml and they concluded that these two compounds are euipotent. But both these compounds have a different molecular weight. Because of that they shows different µM concentration, one shows 0.04µM and other 0.08µM. From this I've concluded that the first compound is more potent than that of second.
I think it is better to look at molar concentration instate of concentration in µg/ml.
Please give your opinion and share your experience. I am bit confused in these two concentration term.
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Generally concentrations are prepared in µg/ml or µM whichever found convenient is taken, for experiments I used micro or nano molar concentrations and the final potency is measure in µM/nM only compared to some reference compound.
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I have already performed MTT assay, Trypan blue exclusion and DNA fragmentation.
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ROS is the Reactive Oxygen Species which results in the cytotoxicity of cells and JC-1 is the Dye used as Mitochondrial Membrane Potential Probe for apoptosis studies.You can use these two parameters to study the effect of your drug on cells.
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It also prevents receptor shedding in general, like HER2 HER3, and decreases beta catenin levels, increases JUN and TNF mRNA levels. And it works in cell culture to either prevent proliferation or cause apoptosis( mechanism unknown as to how it decreases cell numbers).
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Increasing TNFa may be a problem. There is quite a large literature that suggests TNFa activates endothelial cells and up regulates adhesion molecules such as E-selectin, ICAM-1 etc. This can affect a tumor cells ability to invade into the circulation and to exit the circulation at a potential metastatic site. This could increase the metastatic potential of the tumor. These studies have for the most part been carried out in pre-clinical models (mice).
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I was testing the cell killing effects of some synthetic compounds. I found two different kinds of cell death phenomenon. One is that after 48-72 hours incubation with compound A, most of the cells became round and then were released from the bottom of culture dish. This phenomenon is very similar to that caused by Doxorubicin. Another is that after the same time incubated with compound B, the cells were still attached on the bottom of the dish, but all the attached cells are dead (tested by MTT assay and trypan blue staining). In the second type, the cell membrane has somewhat been damaged. Based on this, I think compound A and B may through different mechanisms to kill the cells. I wonder how I should design a further experiment to get more information about this? Have any suggestions?
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No matter what else you have observed with these compounds if they are dead according to the MTT/trypan assay you still need to determine whether it is due to necrosis or apoptosis.
I disagree with the above suggestion for a cell cycle assay first, this will not give you any indication of the type of cell death.
The annexin-V/propidium iodide assay will allow you to distinguisg between necrosis and apoptosis. However you must do either a a time course experiment or a dose-response experiment. I would do a time course experiment where the cells are exposed to the compounds for different time periods, harvesed, stained with AnnexinV and PI then measured using flow cytometry (you don't need a cell sorter so doing worry about FACS).
If the cell death is due to apoptosis you will see the appearance of AnnexinV only stained cells prior to the double stained Annexin/PI cells. If you only see double stained cells then it is likely to be necrosis. Apoptosis can be confirmed using either a caspase 3 specific FLICA reagent or antibody.
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What are the important steps in designing peptide-based drugs? We have anti-cancer peptides that display anticancer activity against a wide range of cancer cell lines. We also checked they are not cytotoxic to normal cells. How should we proceed further?
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If you are currently limited to in vitro assays I would recommend doing human and murine plasma stability, sometimes there is a problem with peptides and plasma peptidases. Plasma protein binding (PPB) could be also of use to evaluate free fraction of your pepide. Depending on peptide nature and your capablilies I would suggest to run BBCEC assay to see if it gets to the brain. Based on that you can leverage your studies of neurotoxicity.
If you can do in vivo - then you can do efficacy (xenografts, singenic, transgenic). Then PK in 3 doses, single and multiple doses, bioavailability, and single and multiple tolerated doses. Based on that you can assess therapetic index and design your tox program.
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Cancer therapy can be initially successful with the elimination of the primary tumor causing remission for a number of years and then return with therapeutic resistance. Does resistance usually involve residual disease after treatment? What is the common basis for relapse 3–5 years after treatment? What are the current thoughts to reduce relapse?
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Over decades it appeared to be very logic for patients and doctors to treat some cancers as aggressive as possible, i.e. removing as much tissue and related lymph nodes surgically and use all aggressive additional treatments possible to guarantee the best survival rate. This may still be the best for many cancers, but, as far as I understood, such highly aggressive therapies may not help in all cases, especially where there are already distant metastases or micrometastases. In such cases a too aggressive treatment may reduce the patients life quality without providing a benefit in life extension. Therefore, it will help to distinguish subsets of patient groups and offer a more individualized therapy, perhaps, depending on many other factors than the histological diagnosis, for example, some genetic and other markers which still may be investigated. And it also turned out that in some cases a patient could benefit from a more aggressive therapy, where a very restricted tumor was removed and further treatment did not appear necessary, but might have been beneficial due to early and unexpectable metastasis (when the basal membrane of the surrounding tissue was intact and no tumor cells appeared to break through - btw this supports the Cancer stem cell theory).
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Structural biology can inform drug discovery and optimization and is frequently used by drug companies as one of their core technologies. Can you suggest specific examples where structures helped provide successful therapies?
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What about GLEEVEC/imatinib to inhibit Abl kinase for the treatment of CML leukemia?
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Etoposide is derived from podophyllotoxins. So what are the changes that can be made in the development of the drug?
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Dear Iyalla
etoposide is derived from 4'-demethyl-9-epi-podophyllotoxin 9-O-beta-D-glucoside by making an 4,6-O-acetal with acetaldehyde at the glucose. The crucial thing is the 9-epi-configuration of the podophyllotoxin and the 4'-O-demethylation at the 3,4,5-trimethoxyphenyl ring of podophyllotoxin. These two manipulations change the antineoplastic characteristics from mitotic poision (podophyllotoxin is a tubulin inhibitor like colchicine) to an eukaryotic topoisomerase II inhibitor (etoposide and teniposide). So for anti-cancer effects you have to decide what antineoplastic effect you want to create. If you want a colchicine-like mitotic poison, you have to start from podophyllotoxin. If you want to create a new human topoisomerase II inhibitor, you have to modify 4'-demethyl-9-epi-podophyllotoxin, eventually as 9-O-beta-D-glucopyranoside.
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Are there specific therapeutic advantages or disadvantages to killing cancer cells by apoptosis versus necrosis or autophagy and how important are microRNAs in regulating these processes?
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Find the answer and we will all be grateful! (and you may publish a well-cited article). In the meantime, we know that in the absence of an immune system, it does not matter much which way they die. See "Chemotherapy induces tumor clearance independent of apoptosis", Jennifer L. Guerriero,1 Dara Ditsworth,2 Yongjun Fan,3 Fangping Zhao,2 Howard C. Crawford,4 and Wei-Xing Zong, Cancer Res. 2008 December 1; 68(23): 9595–9600. http://cancerres.aacrjournals.org/content/68/23/9595.long
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As we learn more from the cancer genome project and therapeutic strategies that provide long-term benefits, what types of targets might allow us to do better? Many successful cancer therapies directly or indirectly damage DNA, and many target replication in some way. Whereas kinase and other inhibitors can show strong initial impacts, resistance seems to be a greater problem with some types of inhibitors. Should we be aiming for drugs that rapidly kill cancer kills but may cause resistance by strong selective pressure or those that weaken cancer cells but better avoid resistance.
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I think you're totally right. It seems logical to treat cancer with those drugs which cancer cells have limit potential to induce resistance against them. But the major problem as you somehow indicated, is genome instability of these cells. Any drug category has its own pathway to function and cancer cell can switch to some alternative pathways to lower the drug effect. I personally think that the best way to combat the cancer cells is to use the body's own potential. Some kind of treatment which trigger the immune responses and potentiate this system will work more efficiently I believe. But the great results will achieve when there is enough time to work for immune responses. In this regard some prophylactic strategy like using cancer vaccine may work properly.
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APE repairing enzyme, a druggable target.
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Yes, it is. I'd suggest you to have a look to: http://www.ncbi.nlm.nih.gov/pubmed/23658942
However, I think it isn't on clinic yet.
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I tried to detect the level of NO production in cartilage explant using Griss reaction which was obtained from promega. The conditioned media (100-50 ul) were assayed by this means but the result didn't work (The color after assay didn't shift to purple. it was still yellow) I don't know whether I must treat something before assay. Can anyone suggest me.
PS: the cartilage explant was treated by IL-1beta and the conditioned media was collected at DAY5.
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My first question is do you have a positive control? Can you spike NO2 into the CM and does it turn purple? What are your detection limits? Based on the literature, how much NO would you expect to get, and could you detect it based on your standard curve amounts?
Also, there is the question of whether your il1 beta is working properly. Can you, for example, do some other assay, like perhaps for aggrecanase with a FRET substrate, or MMP assays perhaps? Or maybe even do an ELISA for TNF. These are just some suggestions.
Also, do you need to let the CM sit for a few days after collection?
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We often dilute antibodies in either blocking buffer or PBS (a buffer which provides physiological conditions and pH) for antibody dilution. But I am doing conjugation of commercially available antibodies with the quantum dots which I am preparing. But when I redisperse the conjugated particles in PBS after conjugation, they precipitate. My particles are stable only in water or MES buffer (its pH is 6.0). I am not sure whether my antibodies will be functional if I dilute them in water.
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Changing pH may also effect on antibody stability and binding affinity! Anyway, my suggestion is firstly you should find a suitable condition to dilute your conjugation (even in water) and then go ahead for staining with a positive control (un-conjugated antibody in same condition, as water). Make sure you get good staining in positive control -> your staining condition is ok -> if not, change other protocol like changing the pH as other people suggested above. Anyway, your positive control is important!
However, I think diluting antibody in water for just several hours staining would be ok ^^ Anyway, it needs to be answered with experiments and controls.
Good luck ^^
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I'm making a presentation on the cancer drug resistance mechanisms in major drugs. I want to include major/representative cancer drugs. I'm thinking about including MAPK inhibitors and TKI inhibitors. What other representative/major drugs would you recommend that I include?
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You may also include cisplatin, we have recently theorized how cisplatin resistance develops and these general mechanisms surely apply to most, if not all, anticancer drugs (see Galluzzi et al, Oncogene 2012)
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I have tried three different concentrations for doxorubicin, 10uM, 50 uM, 100 uM for 2 hrs. The effect attained was similar at all three concentrations, but there was a problem while conducting an image acquisition. All the analogs were stained well with hoechst at 1ug/ml except in doxorubicin treated wells. Somehow its masking the intensity level of hoechst even at 5 ug/ml.
Should I lower the doxorubicin concentration or is there any other alternative?
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which cells you are incubating? and what is the aim of different concentrations? do you want to reach IC50?
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How to identify the correct dose for each one?
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Hi,
1. One chemotherapeutic with one herbal drug you are going to use means this is combination study.
2. First you have to do cytotoxicity assay on colon, ovarian, lung cancer cell line and calculate IC50 drug alone.
3. Based on IC50 you have to do combination study. dose selection will be 1:1 2:2, 3:3, 4:4, 5:5 ratio for combination of both drugs. also you have to do drug alone treatment.