Team:JSNU-China/Description
Background
Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality all over the world, with 14 million new cases and over 8 million deaths per year. It is also the second cause of death and is responsible for quarter of the death cases among developing countries.
People have tried many ways to keep health or prevent cancer, such as eating some healthy diet, keeping active, avoiding certain infections and so on. Anthocyanin works as a natural colorant in food with health-promoting properties, which could protect our hearts and boost our cancer defense, and during the process of preventing and curing cancer, doctors always encourage patients to use anthocyanin-like foods and drugs. However, anthocyanin cannot kill cancer cells and prevent more from growing in our bodies effectively.
About anthocyanin
Anthocyanin is a type of natural flavonoid and antioxidant with powerful anti-cancer properties. Anthocyanin will be able to protect against cancer indirectly through the prevention of DNA damage. Anthocyanin can also make cancer cells not be successfully diffused, thereby protecting the healthy cells not being eroded by cancerous ones.
About KLF4
Kruppel-like-factor 4（KLF4）is a zinc-finger transcriptional factor that regulate gene expression. KLF4’s expression level is different in gastric cancer, lung cancer, bladder cancer ,breast cancer and oral squamous cell.
Experiment
1.We constructed GFP-plasmid and transfected it into SUM52 cells. After 72h, we could detect green fluorescent protein in almost 90% of SUM52 cells.
Fig1.Transfect GFP-plasmid into SUM52 with liposome
2.We set up one SUM52-GFP stable cell line and treated it with different concentrations of anthocyanin. The results of fluorescence microscopy and cell counting kit-8(CCK-8) suggested anthocyanin can kill SUM52 cells.
Fig2.SUM52-GFP treated with different concentrations of anthocyanin(4x)
Fig3.CCK-8 kit to test the cell viability of SUM52-GFP cells
3.We treated normal human gastric epithelial cell line- GES1 and gastric cancer cell line-HGC27 with different concentrations of anthocyanin and tested the cells' viability with CCK-8 kit.The result of CCK-8 showed that anthocyanin had the effect of killing HGC27 cell and had a little effect on GES1 cell lines. We also found normal cells can't endure high concentration of anthocyanin for a long time.
Fig4.CCK-8 kit to test the cell viability of cell lines
(a)GES1 cell (b)HGC27
These experiments showed anthocyanin was a kind of powerful anti-cancer substance. There are many opportunities for humanity to intake anthocyanin for a long time in the course of their entire life. But no research has data to prove the cancer incidence is different between inadequate or excessive intake anthocyanin. Why humanity can't prevent and destroy cancers with it? Maybe, we need push it. We try to find a gene which is special for cancer cell. Is there a hidden traitor in cancer cells? KLF4 is a transcription factor acting as both activator and repressor.
4.We tested KLF4’s expression in various cancer cells by q-PCR and western blot. The results showed that KLF4’s expression is different in a bunch of cancer cells cultured in our lab, mostly downregulation compared with normal human gastric epithelial cell line- GES1.
Fig5. KLF4’s expression in different cancer cell lines
(a)q-PCR (b)Western Blot
5.We constructed KLF4 over-expression and 5 mutant plasmids.
1)Design 6 templates with specific restriction sites for cloning
Fig6. The schematic diagram of five mutants
2)
3)Cloning of "master" into vector
Fig7.BBa_K2000006
After 3 weeks, we selected one KLF4 over-expression cell line from HEK293T cell with G418. WB’s results make sure KLF4 over-expressed in our HEK293-KLF4 cell.
Fig8.KLF4 over expressed in HEK293T-KLF4 cell.
6.Then, we treated HEK293T-KLF4 and HEK293T cells with different concentrations of anthocyanin. After 3 days, HEK293T-KLF4 cells all dead while HEK293T cells dead after 6 days with anthocyanin treatment. The results of CCK-8 kit and light microscopy are consistent which showed upregulation of KLF4 will make HEK293T cell more sensitive to anthocyanin.
Fig9.HEK293T cells treated with different concentrations of anthocyanin
(a)Light microscope showed HEK293T cells dead after 6 days with anthocyanin treatment.
(b)CCK-8 also detected HEK293T cells dead with anthocyanin treatment.
Fig10.HEK293T-KLF4 cells treated with different concentrations of anthocyanin
(a)Light microscope showed HEK293T-KLF4 cells dead after 3 days with anthocyanin treatment.
(b)CCK-8 also detected HEK293T cells dead with anthocyanin treatment.
Our results are so funny, just as the Chinese old saying goes, A mix of Jacks and Jills makes a tough job a breeze. KLF4 maybe is Ms. anthocyanin's Mr. right. Upregulation of KLF4's expression maybe help increase cancer cells' sensitivity to anthocyanin. Another truth is , cancer cells will tolerate it after a long time of taking in anthocyanin. KLF4 downregulated in some cancer cells to help its cancer stem cell to escape from immune system, including lost sensibility to anti-cancer drug. That's why we can't use anthocyanin in clinical cancer therapy. Maybe, our work throw a little light in this dilemma, we can find a plant inside the cancer cells to cooperate with anthocyanin and destroy cancer cell.
In the future, we'll do more jobs about the molecular mechanism of anthocyanin and KLF4. We also finished constructing 5 mutants to explore the relation of KLF4 and anthocyanin.
References:
[1] Zhang ZF, Lu J, Zheng YL, et al. Purple sweet potato color attenuates hepatic insulin resistance via blocking oxidative stress and endoplasmic reticulum stress in high-fat-diet-treated mice.J Nutr Biochem. 2013,Jun 24(6):1008-18.
[2] Lu J, Wu DM, Zheng YL et al. Purple Sweet Potato Color Alleviates D‐galactose‐induced Brain Aging in Old Mice by Promoting Survival of Neurons via PI3K Pathway and Inhibiting Cytochrome C‐mediated Apoptosis . Brain Pathol. 2010 May;20(3):598-612.
[3] Capra hircus Kruppel-like factor 4 (KLF4) mRNA, complete cds.https://www.ncbi.nlm.nih.gov/nucleotide/1021691550?report=genbank&log$=nuclalign&blast_rank=10&RID=Z74WM35U015
Future Work
Compared with traditional chemotherapy, combining eating foods rich in anthocyanin with gene therapy has many advantages, such as no harm and lower cost in treatment. Since everybody's genes are different, we can treat patients one-to-one in gene therapy. In this way, we will get a more efficient result and we don't need to take medicine any more. We can take in anthocyanin from grapes, cherries, strawberries and some food that we can purchase easily.
Protocal
1.Molecular Cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.
In the molecular cloning experiment, the KLF4 to be cloned is combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into E. coli bacteria. This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. This is single cell then can be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule.
2.Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions, outside their natural environment, an essential aspect of cloning technology. We can get a lot of cells or metabolites through cell culture.
Cell recovery
1. We put cryopreserved tubes in water bath pot (37℃) and shake it until cells dissolved. We can complete the thawing in 1-2 minutes.
2. Move cells into the centrifuge tube (5 ml).
3. Centrifugation for 5 minutes, 1000r/min.
4. After centrifugation, we discard the supernatant and move the precipitation to a petri dish.
Cell culture
1．Add 55ml serum and 6ml Penicillin-Streptomycin Solution to the medium.
2．Add the medium to the petri dish.
Cell collection
1.Digest cells with trypsin, then stop it with cell culture medium.
2.Remove the medium, then the cells sedimentated.
3.The cells are moved to a centrifuge tube and centrifuged for 5 minutes.
4.Discard the supernatant, and wash cells with PBS.
5.Centrifugation for 5 minutes, 1000r/min.
6.Discard the supernatant and cryopreservate the cells at -80℃.
3.Western blot
Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for klf4, and visualized by using secondary antibodies detection reagents.
4.Cell Counting Kit-8
Cell Counting Kit-8 is based on WST-8 (chemical name: 2- (2-methoxy-4-nitro-phenyl) -3- (4-nitro-phenyl) -5- (2, 4-sulfophenyl) -2H- tetrazolium monosodium salt) which is widely used in drug screening, cell proliferation assay, cytotoxicity assay, tumor susceptibility testing and detection of biological activity and so on.
CCK-8 was produced by TransGen Biotech.
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