Enhancement of 5-Flurouracil Uptake by Insulin in Human Colonic Cancer

Hamid Naji Obeid,Najat A. R. Hasan, Adeeb A. Al-Zubaidy
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Keywords : Insulin,5-Flurouracil Uptake ,SW-480 cell line
Medical Journal of Babylon  11:2 , 2014 doi:1812-156X-11-2
Published :16 July 2014

Abstract

SW-480 cell line was cultivated on two microtiter-plates of 48 wells, by using the routine culture methods and all what needed from culture media and reagents. One microtiter-plate of SW-480 cell line was exposed to 0.5 IU/L of neutral insulin for 10 hours of incubation, followed by serial double dilutions of 5FU starting from 80 µg/mL to 1.25 µg/mL of 5FU for 12 hours incubation. The second cultured plate was exposed to same 5FU serial dilution but without proceeding with insulin. Gas chromatographic analysis was performed to determine the residual 5FU concentration in the culture medium of SW-480 cell line wells in both cultured plates. There was significant differences (P<0.05) in the residual 5FU between them. In conclusion: insulin increases the uptake of 5FU into human colonic cancer SW-480 cell line significantly.

Introduction

Colorectal cancer (CRC) is a significant health problem; it is the most common malignancy of the gastrointestinal tract ]1[. CRC accounts for 10–15% of all cancers ]2 [, and it is the second leading cause of cancer-related deaths ]3[. It is among the top three causes of cancer death in both men and women in the Western world ]4[. There are currently eight chemotherapeutic agents licensed for use in treatment of CRC in the US and Europe, one of them is 5FU ]5[. It is an antimetabolite of the pyrimidine analogue type ]6[ which has been frequently used clinically in patients with various cancers ]7[. It causes DNA damage, during S phase due to the misincorporation of fluoro-deoxyuridine triphosphate (FdUTP) into DNA]8[. The activity of 5FU is markedly limited by its rapid degradation into 5FUH2 via the action of the cytosolic enzyme DPD, the first enzyme in the catabolic cycle of 5FU. It has been demonstrated that this enzyme deactivates more than 85% of the injected dose of 5FU ]9[. The overall response rate for advanced CRC to 5FU alone is still only 10–15% ]10[.However, nowadays 5FU resistance during the course of treatment has become common, which is an important cause of failure for cancer therapy ]11[. To increase the activity of 5FU, various researchers have proposed utilization of biochemical modulators ]12[ enhancement of 5FU with insulin or insulin potentiation therapy (IPT) was developed by the Mexican medical doctor "Donato Perez Garcia" in the 1930s ]13[. IPT utilizes insulin to deliberately induce a hypoglycemic state to starve the cancer cells. However, during the state of hypoglycemia, the starving cancer cells are given low dose chemo agents or natural anti-cancer substances immediately followed by infusion of dextrose sugar. The low dose chemo agents are therefore devoured by and readily absorbed into the cancer cells ]14[. Insulin as a metabolic accelerant, may be able to increase the metabolism of cancer cells and stimulate them into a cell cycle stage in which they are more sensitive to anticancer drugs ]15 [. In addition, insulin promotes the uptake of many nutrition materials by facilitated diffusion like the entry of glucose into many tissues ]16[.The present study demonstrated the enhancement of intracellular uptake of 5FU by insulin.

Materials and methods

1- Cytotoxicity assays
          According to Freshney, (1994) cytotoxicity assays were applied on human colonic cancer SW-480 cell line using MEM culture medium. When the growth in the  flask became as monolayer before it reach the exponential phase, the cell monolayer were harvested and re-suspended with a culture medium in a concentration of 5x 105  cell / ml and seeded into two microtiter plate. When the cell growth reach 80%, the wells were exposed to 0.5 IU/L of insulin and incubated for 10 h. ]17[.Then the culture media was changed by fresh one containing 5FU at two-fold serial dilutions (80, 40, 20, 10, 5, 2.5, 1.25 µg/ml). The plates were incubated at 37°C for 12 hours.
2- Gas chromatography analysis
         A volume of one ml of cell culture medium was collected from five replicate of each concentration were mixed and centrifuged at 10000 rpm for 10 min ]18[. One cubic milliliter of the supernatant was lyophilized and kept overnight in the oven at 50°C for further dryness. The samples were re-suspended with 1mL methanol and analyzed by gas chromatography (Shemadzo 2010- GC) to determine the concentration of 5FU in the culture medium. The injection volume: 1µl, the injector temperature: 280°C, the detector temperature: 300°C and the column oven temperature: 250°C ]19[. Results are presented as mean ± standard deviation (SD). A differences was considered to be significant at P<0.05 level and is represented by (*).



Results

Figure (1 and 2) shows the standard curve of 5FU detected by Shimadzu GC monitored by florid ion current detector. 5FU standard solutions consisting 100, 50, 25, 12.5, 6.25µg/ml. Figure (3) appears the gas chromatograph of the residual 5FU concentrations in the culture medium of SW-480 cell line wells after treatment with different concentrations of 5FU at 12h incubation. Whereas, Figure (4) appears the gas chromatograph results of the residual 5FU concentrations in the culture medium of SW-480 cell line wells after treatment with different concentrations of 5FU pretreated with 0.5U/L insulin at 12h incubation. Table (1) appears the residual 5FU concentration in the culture medium of SW-480 cell line wells after 5FU treatment with and without insulin groups. There was significant differences (P<0.05) in the residual 5FU between both treated groups.

Discussions

The significant differences in both treated groups indicates that insulin increase the uptake of 5FU into the cells so increase its absorption and consumption. Ke et al., (2007) proposed that the detection of 5FU concentration in the culture medium can indirectly analyze the total consumption of 5FU by tumor cells and avoid the effect of drug metabolism and drug efflux via drug pumps at the cell membranes]18 [. Insulin stimulates Na+, K+ -ATPase activity, and translocation to the plasma membrane via phosphorylation of the alpha-subunits of insulin receptors by the extracellular signal-regulated kinase ]20[. Previous studies demonstrated that the uptake of 5FU is apparently Na+-dependent. The carrier-mediated transport of 5FU may require some other factors that are yet to be identified ]21[. In conclusion, GC analysis confirmed that insulin increased the uptake of 5FU in human colonic cancer SW-480 cell line.

Conclusions

N/A

References

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