Researchersmade a big impact on developing a large number of matching cells to use inlaboratory experiments. There are different cell lines or types, one of thecell line is called HeLa cells which are human cervical cancer cells found atthe top of the vagina and the entrance to the uterus. This was the first humancancer cell to be cultured constantly for experiments and it was refined in1951 from Henrietta Lacks.
HeLa cells develop quicklyif the correct nutrients and disorder and space is used, reason being HeLacells are cancer cells, which can multiply and develop rapid in a muchuncontrolled way and competed to normal cells. HeLa cells developed harmful dueto infection with human papilloma virus 18 (HPV18) as HeLa cells can spreadaround the body and interrupt the normal activity of the cells to make cells convertcancerous.Innatural cells, the Hayflick limitphenomenon is the number of times a normal human cell population will divideuntil cell division stops. Which means cells can only divide by mitosis aspecific number of times as the telomeres at the end of the chromosomes reducewith each division. This doesn’t relate to several types of cancer cells asthey produce an enzyme called telomerase, which extends the telomeres whenchromosomes are copied and permits the cells to reproduce constantly. The aim of this experiment is totest the extracted DNA samples to see whether Arsenic Trioxide (ATO) can affectHPV18 E6 DNA levels of treated HeLa cells. Method 8tubes were prepared for this experiment. 7 tubes were labelled 1-6appropriately and the remaining 1 tube was labelled as NTC.
we pipetteddifferent reagents required for PCR reactions into the PCR tubes which isstated in the following table.Afterpipetted the reagents, closed the PCR tubes and placed it into the thermocycler(PCR machine). The following table is showing the thermocycler which wasprogrammed.PCRtook approximately 2 hours to complete. Then tubes were collected and storedunder frozen at -20ºC, then we analyzed PCR productusing gel electrophoresis We used prepared 1% agarose gel containing8 sample wells.Electrophoresis buffer gel waspoured in to the tank till the gel was full covered.
Then we loaded our samples(10µL of PCR reactions) into the wells including the 5µLof the ladder.Samples were loaded into the wellsby using pipettes then tank was covered with the lid and the power cables wereconnected to the power supply (90 Volts).Started the timer for 30 minutesafter incubation of 30 minutes gel was removed with the tray from theelectrophoresis tank and placed it on a piece of tissue paper then we proceededusing UV/Dark Reader transilluminator to visualize and analyze our gel. Results The results obtain are shown in thefigure below and explained in the table- Theladder is used to adjust electrophoresis gelsso that samples of unknown DNA thathave been initiatedinto the gelcan be measured. HPV18 HeLa and GAPDH – HeLa was untreated which is a template but HPV18 HeLa with 5 µM of ATO was effective andis seen with a band where it is not as bright as DNA ladder because ATO brokedown its DNA. When ATO concentration increased to 5µM ATO was used fortreatment this was too toxic, therefore all the cell has been destroyed by ATOso this band is invisible.
GAPDH is one of the key enzymesinvolved in glycolysis, also constitutively expressed in almost all tissues inhigh amounts. Therefore, GAPDH is widely used as a loading control for proteinnormalization and also deregulated in various cancer cells. Discussion Arsenic Trioxide (ATO) is a cancermedication which delays the growth of the cancer cells as well as spreadingaround the body. This drug is used to treat acute leukemia, promyelocytic orAPL. Generalreports have establish that the anti-proliferative accomplishments of ATO aremediated by several ways of apoptosis. ATO have been used to treat many cancersincluding acute leukemia, promyelocytic orAPL.
The molecular bases are not yet stated in solid tumor cells, particularly cervical cancercells carrying HPV genome.Therelation between HPV and cervical cancer is definite then again the treatmentoptions are limited. The efficient toxicity of ATO has prohibited its extensiveuse in medical uses, mainly at the concentrations higher than 5?M as we haveused ATO at low dosage of 2?M which precisely target HPV infected cervicalcancer cells. Where most of the cells were destroyed when we increased theconcentration of ATO to 5?M due to higher drug toxicity, regardless of HPVinfection.
Our results showed that HPV18 HeLa (Band4) with 5µMconcentration of ATO was effective where it is not visible compare to the DNAladder, because all the cell has been destroyed by ATO due to high concentration.HPV18 HeLa (Band 2) was used astemplate to compare untreated ATO with treated ATO which is HPV18 HeLa ATO with concentrationof 2µM (Band 3), where I can see that it has effected the cells and its lessvisible than untreated HPV HeLa from the results it is showing that the treatmenton the cancer was as successful as I was expected. GAPDH (Band 8) was untreatedas it was a template also. GAPDH is used as loading control for protein to normalizeand deregulate in several cancer cells. As GAPHD is an enzyme which involves inglycolysis constitutively expressing in majority tissues in high amounts. Band 4,5, and 6 are DNA extra used as template, this was used as an internal control. Conclusion From my results I can conclude that ATOhas affected HPV18 E6 DNA levels as there is a strong evidence that ATOrestrain proliferation and induced apoptosis to the HeLa cells showing directeffects on cervical cancer.
As ATO considerablydecreased cell in comparison withcontrol cells in a time and does dependent method. At the highest concentration(5µM),ATO inhibited the progression prospective of the cell proliferation whichcaused a rapid decline in the number of visible cells after 2 days, there wasno visible cells remained (HPV18 HeLa ATO 5 µM band 4), at the lowestconcentration (2µM) cell proliferation was initially increased as we comparedwith control, and had small effect (HPV18 HeLa ATO 2 µM band 3). High concentrationand low concentration had no effect on the HPV18 HeLa Untreated band 2 andGAPDH bands.