Enhanced In Vitro Antimitotic Effect of Colchicine Exhibited by Anti-Hypercholesterolemic Drugs, Statins, Using Allium cepa Root Tip Assay Coruña, C.J., Llena, A.S., Libres, M.J. Bayugan National Comprehensive High School

Abstract Cancer is defined as the uncontrollable growth of malignant cells. At least 8 million people die each year from cancer, an estimated 13% of all global deaths. One major obstacle in chemotherapy is multidrug resistance in many anti-cancer drugs like Doxurubocin. Recent clinical studies have shown that patients, who took the cholesterol-lowering drugs, statins, had less incidents of cancer. This observation suggests that statins may have cancer-preventive properties. This study was conducted to evaluate the potential effects of Statins on the anti-mitotic and chemo preventive actions of Colchicine using Allium cepa assay. Allium cepa roots were equally treated with the 8 treatment groups namely, Negative Control (Natural Saline Solution), Rusovastatin, Atorvastatin, Simvastatin, Rusovastatin+Colchicine, Atovastatin+Colchicine, Simvastatin+Colchicine, and Positive Control (Colchicine). Three slides per concentrations were prepared and then proceed to microscopic viewing. Allium cepa roots microscopic investigation at 400× and 630× magnification reveals that Rusovastatin+Colchicine treatment group had the least percentages of abnormalities per 100 cell counts, such as in Mitotic Index (0.15%), Chromosome Aberration (2.33%), and Micronucleus in Interphase (3.00 %) and Overall Abnormalities (5.48%). Both Atorvastatin and Simvastatin, Atorvastatin+Colchicine and Simvastatin+colchicine, and Rusovastatin and Colchicine exerted antimitotic and chemo preventive activities, which were not significantly different from each other in all parameters. From the data gathered, we can conclude that statins have potentiating effects on the antimitotic activity of colchicine. Combined Rusovastatin and Colchicne exhibited the highest antimitotic activity of Allium cepa. Keywords: Statins, Colchicine, Anti-hypercholesterolemic, Cancer, Micronucleus in Interphase, Mitotic Index, Chromosome Aberration

Introduction Cancer is characterized by the wild development of dangerous cells. No less than 8 million individuals pass on every year from malignancy, an estimated 13% of all global death (Ngelangel & Wang, 2002). Regardless of this, low disease aversion awareness perseveres. Tumor is thought to be bought on

by the communication between heredity vulnerability and ecological poisons. One noteworthy hindrance in chemotherapy is the building up of the resistance on the commonly used cancer agents on some cancer cell lines. (Irvine, 2011). However, late clinical studies have demonstrated that patients who took the

cholesterol-bringing down medications, Statins, had less occurrence of growth malignancy (Villanueva, 2015). This perception proposes that Statin drugs may have malignancy preventive properties. Chemotherapeutic medications forms the main stray of cancer treatment and agent that will disrupt the mitotic spindle assemble so called antimitotic drugs. The antimitotic property of the medicine can be for starters screened using Allium cepa root meristematic cells which have been utilized widely as a part of the screening of medication with antimitotic movements (Baldwin, 2012). Meristematic locale is like the tumor division in people which can be utilized for preparatory screening of medication with anticancer action. However, just a couple concentrates on have investigated this determinant connection of tumor and the potential parts of antihypercholesterolemic medications like statins as far as restraining the seriousness of such disease cases. It is imperative to specify that cholesterol and its subordinates in high fixations exceedingly potentiate cell separation and expansion, growth movement, and improvement of general metastatic potential. The theory of this anticipates is that Statins can potentiate the antimitotic action of colchicine to onion (Allium cepa) effectively creating root tips. Hereafter, the venture proposes to explore the capability of the Statins, to be specific, Atorvastatin, Rosuvastatin, and Simvastatin to improve the antimitotic impact of Colchicine. The general target of this study was to expand growth aversion by surpassing the restrictions of routine treatments for malignancy and taking a gander at new treatment mixes that have a few focal points over customary strategies as well as more available to everybody. There is dependably a high probability that the quest for the perfect medication blend may

decrease the quick shooting up of growth cases around the world. It takes after that this examination is basic as a practical basis to determine this significant wellbeing issue.

Materials and Methods Collection of Drugs Colchicine was purchased from a local drug store and pure Statins namely Atorvastatin, Simvastatin and Rosuvastatin was kindly provided by a medical doctor for combination and experimentation only.

Preparation of Treatment Groups Eight treatment groups (test solutions) were used as the experimental new drug combinations for screening enhanced antimitotic effect on Allium cepa test. 3 set up for each statin (40mg) was mixed in 40 ml distilled water. Then 3 set up for each statin (20mg) that was mixed in 20mg colchicine and mixed in 40 ml distilled water. Each treatment group was mixed thoroughly until no visible powder content can be seen under a magnifying glass. Allium cepa Assay Pre-Treatment The Allium cepa bulbs were grown on tap water at room temperature for 2–3 days. When the roots reach 2–4 cm in length, the bulbs were then subjected to different treatments. A set of Allium cepa was placed in Colchicine treated solution as positive control while for the negative control, a set of Allium cepa was grown in Natural Saline Solution. The solutions were changed daily. After 48 hours, root tips from each bulb were harvested and fixed in Carnoy’s fixative (1:3 acetic acid: alcohol) for 24 h. Slides Preparation

Allium cepa root tips were washed a few times with distilled water. These were hydrolyzed with 1 N HCl at 60– 70 °C for 5 min. After hydrolysis, the roots were washed once again. About 1–2 mm of the root tips was cut and placed on the slide. A small amount of aceto-orcein was dropped on the root tip and left for 2 min. The root tip was squashed with metal rod and another small drop of aceto-orcein was added and left for another 2 min. The cover slip was carefully lowered on to avoid air bubbles and the sides of the slides were sealed with clear fingernail polish.

Mitotic index = (Number of cells in mitosis) / (Total number of cells) Statistical Analysis The data was statistically analyzed using the Excel program and the SPSS software version 16. Amounts on diagram were stated based on means ± the standard error. Difference between the control sample and the treatment sample were determined by Student’s t-test and P values less than 0.1 was considered significant. Disposal

Observation of Specimens The slides was observed under the light microscope at 400× and 630× magnification. An Olympus light microscope with digital camera was used in order to get the clear image of the chromosome aberrations. Photomicrographs were made and minimum of 100 cells per slide was analyzed. The mitotic index, micronucleus in interphase and chromosome aberrations in mitotic phases, was determined by the examining a minimum of 100 cells per slide. The experiment were replicated three times with three roots for each replicate, therefore, nine slides was prepared for each treatment group. The mitotic index was obtained as follows: Results and Discussions

Results Figure 1 shows the overall number of abnormalities observed on Allium cepa root tips after the treatment, with the data being plotted on the graph. Three trials were conducted for each treatment group. The One – Way Analysis of Variance (ANOVA) in Appendix A1 revealed that there was a significant difference among the means of the different treatments /solutions, since the p value of 0.000 did not fall at the level of

All the used materials and unused materials with broken seals were disposed of to prevent contamination. The organic compounds are flammable so Researchers placed the materials in an organic waste container as well as the smelly compound such as the treated roots. The containers were sealed after disposal. Glassware’s with smelly compound were rinsed with acetone. Rubber and paper items were placed in a garbage can. Entire disposal was supervised by the closely monitoring laboratory assistant, Mrs. Cherry Mar T. Tiempo, to ensure meticulous maintenance of aseptic technique.

significance (0.1). Furthermore, the Duncan’s Multiple Range Test (DMRT) in Appendix B1 also showed that the treatment means were significantly different between the negative control (Normal Saline Solution) and the Rusovastatin+Colchicine concentration. The mean for the positive control and Rusovastatin+Colchicine concentration were significantly different with each other. This proves that the Rusovastatin+Colchicine concentration was the most effective test solutions in terms of antimitotic and chemopreventive activity

from overall observed abnormalities on Allium cepa root tips. The means for Atorvastatin and Simvastatin were not significantly different with each other. In the meantime, the means for Atorvastatin+Colchicine and Simvastatin+Colchicine were not significantly different with each other.

Figure 2. Bar graph showing percentages of Mitotic Index on Allium cepa root tips after being treated with various treatment group

Figure. 1 Bar graph showing percentages of overall abnormalities on Allium cepa root tips after being treated with different treatment groups.

Figure 2 shows the overall number of mitotic index observed on Allium cepa root tips after the treatment, with the data being plotted on the graph. One – Way Analysis of Variance (ANOVA) in Appendix A2 revealed that there was a significant difference among the means of the different treatments /solutions, since the p value of 0.000 did not fall at the level of significance (0.1). Furthermore the Duncan’s Multiple Range Test (DMRT) in Appendix B2 shows that the treatment means were significantly different between the negative control (NSS) and Rusovastati+Colchicine concentration. The means for Atorvastatin and Simvastatin were not significantly different with each other. Also the means for Atorvastatin+Colchicine and Simvastatin+Colchicine were not significantly different with each other. At the same time, the means for Rusovastatin and positive control (Colchicine) were not significantly different with each other

In the meantime, Figure 3 shows the overall number of Chromosome Aberration observed on Allium cepa root tips after the treatment, with the data being plotted on the graph. One – Way Analysis of Variance (ANOVA) in Appendix A3 revealed that there was a significant difference among the means of the different treatments /solutions, since the p value of 0.000 did not fall at the level of significance (0.1). Furthermore the Duncan’s Multiple Range Test (DMRT) in Appendix B3 shows that the treatment means were significantly different between the negative control (NSS) and Rusovastatin+Colchicine concentration. The means for Atorvastatin and Simvastatin were not significantly different with each other. Also the means for Atorvastatin+Colchicine and Simvastatin+Colchicine were not significantly different with each other. At the same time, the means for Rusovastatin and positive control (Colchicine) were not significantly different with each other.

Discussions Figure 3. Bar graph showing percentages of Chromosome Aberration found on Allium cepa root tips after being treated with various treatment group Furthermore, figure 4 shows the overall number of Micronucleus in Interphase observed on Allium cepa root tips after the treatment, with the data being plotted on the graph. One – Way Analysis of Variance (ANOVA) in Appendix A4 revealed that there was a significant difference among the means of the different treatments /solutions, since the p value of 0.000 did not fall at the level of significance (0.1). Furthermore the Duncan’s Multiple Range Test (DMRT) in Appendix B4 shows that the treatment means were significantly different between the negative control (NSS) and Rusovastatin+Colchicine concentration. The means for Atorvastatin and Simvastatin were not significantly different with each other. Also the means for Atorvastatin+Colchicine and Simvastatin+Colchicine were not significantly different with each other. At the same time, the means for Rusovastatin and positive control (Colchicine) were not significantly different with each other. Figure 4. Bar graph showing percentages of Micronucleus in Interphase found on Allium cepa root tips after being treated with various treatment groups

The overall gathered data, showed that as the root tip soaked in the negative control (Natural Saline Solution) concentration, revealed that the number of Over All Abnormalities counted per 100 cells, yields the most counts. The Atorvastatin concentration and Simvastatin concentration are having almost the same number of Over All Abnormalities found, that yields the second most number of anomaly lower than the negative control. The Atorvastatian+Colchicine concentration and Simvastatin+Colchicine are also having the same number of Over All Abnormalities found that yields the third highest number of anomaly lower than the negative control, Rosuvastatin concentration and Simvastatin concventration. The number of anomaly found in positive control (Colchicine) is much higher than the Rosuvastatin+Colchicine concentration but lower than the Rosuvastatin concentration and other concentration stated above. This proves that the Rosuvastatin+Colchicine concentration yields the lowest number of overall abnormalities found. As the lower the number of anomaly, the effective the medicine. Analysis of Data The obtained data on the antimitotic and chemo preventive activities of all the treatment groups confirmed that the positive control and the various concentration of statins, and the combination of statin and

colchicine exerted varying biological activities. Consistently, the Rusovastatin+Colchicine concentration exerted the most potent antimitotic and chemo preventive activity at significant levels of differences with the positive control on overall number abnormalities, mitotic index, chromosome aberration, and micronucleus in interphase. The antimitotic and chemo preventive activity of the Rusovastatin+Colchicine concentration was not significantly different from the positive control. The other concentrations showed less antimitotic and chemo preventive activity, which were significantly different from the negative control (NSS). Conclusion This study showed that the Statins (Rosuvastatin, Atorvastatin and Simvastatin) concentrations has potent antimitotic and chemopreventive activity on Allium cepa root tips abnormalities in vivo based on the results gathered. The Rosuvastatin concentration potentiated the antimitotic and chemopreventive action of Colchicine in Allium cepa model. Rosuvastatin concentration showed a lowest number of abnormalities than the other Statin concentration (Atorvastatin alone and Simvastatin alone). The Rosuvastatin+Colchicine concentration exerted the highest antimitotic and chemopreventive actions, which was much higher than the positive controlColchicine alone in any parameter. Rosuvastatin+Colchicine concentrations consistently expressed the best disrupting agent on Allium cepa root tips, as evidenced by the least number of abnormalities per criterion. Root tips from the Rosuvastatin+Colchicine group showed only 0.15% Mitotic Index on observed cells per 100 counts, 2.33% for Chromosome

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