The Effect of Glucose and Salt Concentrations on HepG2 Human Liver

Cancer Cells

Abstract

Hyperglycemia, the excess of glucose and a symptom of diabetes, and

hypernatremia, the excess of sodium in the body and a symptom of hypertension both

have a relation to cancer, mostly causing or helping cause it. However, while not

everything is known about this, millions or billions suffer from diabetes, hypertension,

and/or cancer, which is even more reason to perform this experiment. In the

experiment, HepG2 cancer cells were cultured in media in three groups: high glucose,

high salt, and the control; the cells’ proliferation was found with an MTT assay after 5

days. It was predicted that if high glucose media was used to culture the cells, the cell

proliferation would be greatest. This was supported, with all data being significant

(measured with a t-test), and the high salt group having the lowest proliferation. The

standard deviation was also low. Other journal articles supported the data, except for

one article that stated that sodium increased tumor growth. (Radišaukas et al., 2016)

Introduction

A. The excess of glucose, or hyperglycemia, is one of the key factors of diabetes.

According to an article by the CDC, diabetes rates are on the rise, with 9.4 percent of

Americans suffering in 2017. On top of this, diabetes is the 7 th most deadly disease in

the U.S. (CDC, 2017). However, the average American eats over 70 grams of added

sugar a day, while the AHA recommends 25, 38, and 12-25 grams for women, men and

children respectively. (UCSF). In the blood stream, there should only be about 1 g/L of
glucose, although many have well over 2 g/L, which signifies diabetes. (Mayo Clinic,

2018). Increased sugar intake is linked with diabetes, a major health issue in the USA.

(Chang & Yang, 2016).

Diabetes is also linked to hypertension, or high blood pressure. High blood

pressure has many symptoms, one being kidney problems. Kidney problems can cause

increased blood sodium levels, with anything above 3.3 g/L in the blood stream being

hypernatremia, the excess of sodium. (Mayo Clinic, 2018). (Mayo Clinic, 2018). The

recommended sodium intake is a maximum of 2,300 mg a day, but the average

American adult consumes over 3,400 mg a day, leading to increased sodium levels,

hypertension, heart disease, and stroke. (CDC)

Finally, there is liver cancer. Liver cancer is a mutation of the cells in and from

the liver, causing cells to rapidly reproduce. (Mayo Clinic, 2019). It is the fifth-most

common cancer and second-most deadly on Earth. While there are many treatments for

early stages, most cancers are diagnosed at late stages, where options are more limited

and ineffective, with first-line therapies only increasing survival at about three months.

Unlike other cancers, liver cancer has had a rise in mortalities, at 43% from 2000-2016.

(Reghupaty & Sarkar, 2019). Limiting alcohol, maintaining diet and exercise, and getting

vaccinated against Hepatitis B are all ways to prevent the cancer. Hypernatremia and

hyperglycemia both may be linked to cancer the way they are to hypertension and

diabetes, but parts of this remains to be seen. (Mayo Clinic, 2019)

B. While the above is quite important, their link is just as important, if not more. For

example, hyperglycemia can increase several factors that are a part of cancer, including

increase in proliferation and migration, as well as increase the resistance to death and
treatments like chemotherapy. Not only this, but hyperglycemia is also associated with

the cause of many cancers, particularly in the breast, liver, bladder, and other organs.

(Li et al., 2019). The major reasons as to why an increase in sugar causes greater

tumor growth is that, when cancer cells perform cellular respiration, they need a greater

amount of glucose to supply the cells, and since glucose is more readily available,

cancer cells can grow faster, causing more proliferation. In general, increased sugar

intake leads to creation and growth of tumors and cancer cells. (Chang & Yang, 2016).

While hyperglycemia does have a link to cancer, so does hypertension, and

hypernatremia, although indirectly. One issue is that hypertension is a side effect of

many anti-cancer drugs, leading to hypernatremia. Generally, many antihypertension

drugs are used alongside anti-cancer drugs to prevent hypertension, especially in the

elderly. (Małysko et al., 2018). Hypertension is closely linked with other forms of cancer,

including a form of kidney cancer, and is also a factor in metabolic syndrome, which is

when the metabolic cycle of a cell is disturbed. (Radišaukas et al., 2016). Because of

these two theories, it is visible that hypertension could be both a cause and effect of

cancer, which simply strengthens both the link and the problem of hypertension and

cancer.

C. Hyperglycemia, hypertension, and their link to cancer are quite important and

alarming, but if treated, it could save millions with diabetes and hypertension from the

claws of cancer, particularly liver cancer. Such an experiment could boost the

experimenter’s role in liver cancer and start paving the road towards research and

recognition in this field, as the researcher had always heard and sometimes seen his

father do cancer research, causing him to want to do the same. The experimenter had
always enjoyed learning about biology, being surrounded by biology textbooks, and

scientific papers, allowing for his heightened interest in cancer research.

D. For the experiment, the independent variable was the glucose/salt content used

to culture liver cancer cells, or HCC cells, and the dependent variable was the amount

of cancer cells living at the end. This research project was meant to investigate the

effects of diabetes and hypertension on cancer cells as to grasp a better idea of how

these are related. The control was a medium containing 6.4 g/L of sodium chloride and

1 g/L of glucose, 6.4 g/L of sodium chloride used in Dulbecco’s Modified Eagle Medium,

a medium to culture cells, and 1 g/L of glucose is the general desired amount of glucose

in and around cells. (Thermo Fisher Scientific). (Mayo Clinic, 2018). It was hypothesized

that if higher concentrations of sugar were used, there would be the greatest amount of

cancer cells surviving. This is due to, as said above, higher glucose levels supply

cancer cells with greater levels of sugar. (Chang & Yang, 2016).

Literature Cited (Introduction)

Castro, M. Regina, editor. "Diabetes." Mayo Clinic, Mayo Foundation for Medical

Education and Research, 8 Aug. 2018, www.mayoclinic.org/diseases-

conditions/diabetes/diagnosis-treatment/drc-20371451.

Chang, Shu-Chun, and Wei-Chung Vivian Yang. "Hyperglycemia,

Tumorigenesis, and Chronic Inflammation." Critical Reviews in

Oncology/Hematology, vol. 108, Dec. 2016, pp. 146-53. Science Direct, doi:

10.1016/j.critrevonc.2016.11.003. Accessed 19 Oct. 2019.

"11965 - DMEM, High Glucose." Thermo Fisher Scientific,

 www.thermofisher.com/us/en/home/technical-resources/media-

formulation.8.html. Accessed 3 Nov. 2019.

"Get the Facts: Sodium and the Dietary Guidelines." Centers for Disease Control and

Prevention, U.S. Department of Health and Human Services,

www.cdc.gov/salt/pdfs/sodium_dietary_guidelines.pdf. Accessed 20 Oct. 2019.

"High Blood Pressure Fact Sheet." Centers for Disease Control and Prevention, U.S.

Department of Health and Human Services,

www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_bloodpressure.htm. Accessed

20 Oct. 2019.

"How Much Is Too Much?" Sugar Science, U of California San Francisco,

sugarscience.ucsf.edu/the-growing-concern-of-

overconsumption.html#.XayvOhKjIU. Accessed 20 Oct. 2019.

Li, Wenjie, et al. "Effects of Hyperglycemia on the Progression of Tumor Diseases."

PubMed. PubMed, www.ncbi.nlm.nih.gov/pubmed/31337431. Accessed 19 Oct.

2019. Originally published in Journal of Experimental and Clinical Cancer

Research, vol. 38, 23 June 2019, p. 327.

Małyszko, Jolanta, et al. "Hypertension in malignancy–an underappreciated problem."

Oncotarget [Online], 9.29 (2018): 20855-20871. Web. 20 Oct. 2019

"New CDC Report: More than 100 Million Americans Have Diabetes or Prediabetes."

Centers for Disease Control and Prevention, U.S. Department of Health and

Human Services, 18 July 2017, www.cdc.gov/media/releases/2017/p0718-

diabetes-report.html.

Picco, Michael F., editor. "Liver Cancer." Mayo Clinic, edited by Sandhya Pruthi, Mayo
 Foundation for Medical Education and Research, 4 May 2019,

www.mayoclinic.org/diseases-conditions/liver-cancer/symptoms-causes/syc-

20353659.

Radišaukas, Ričardas, et al. "Hypertension, Serum Lipids, and Cancer Risk: A Review

of Epidemiological Evidence." Medicina, vol. 52, no. 2, 2016, pp. 89-98.

ScienceDirect, DOI:10.1016/j.medici.2016.03.002. Accessed 2 Nov. 2019.

Regupathy, Saranya Chidambaranath, and Devanand Sarkar. "Current Status of Gene

Therapy in Hepatocellular Carcinoma." Cancers, vol. 11, no. 9, p. 1265. MDPI,

doi:10.3390/cancers11091265. Accessed 21 Oct. 2019.

Sheps, Sheldon G., editor. "High Blood Pressure (Hypertension)." Mayo Clinic, edited

by Sandhya Pruthi, Mayo Foundation for Medical Education and Research, 12

May 2018, www.mayoclinic.org/diseases-conditions/high-blood-

pressure/symptoms-causes/syc-20373410.

---, editor. "Hyponatremia." Mayo Clinic, edited by Sandhya Pruthi, Mayo Foundation for

Medical Education and Research, 8 May 2018, www.mayoclinic.org/diseases-

conditions/hyponatremia/symptoms-causes/syc-20373711.

Methods and Materials

HepG2 is a line of human HCC cells. To begin, HepG2 cells from a tissue culture

dish were trypsinized using 1 mL of 1X trypsin and a pipet to detach the cells from the

bottom of the dish. After the cells were trypsinized, the cells were suspended in 9 mL of

Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS).

The DMEM containing the cells was removed from the dish and added to a 15 mL

centrifuge tube. A hemocytometer, a specially designed slide used to count cells, was
used to measure the number of the cells per mL. The hemocytometer is divided into 9

sections, each with a volume of 0.1 mm^3, or 100 nL. The values were only taken by

the 4 corner sections, average, and then multiplied by 10^5 to count them at full scale.

The hemocytometer was in the biosafety cabinet. There were 16 wells/trials per group,

in each well there were 5000 cells in 100 µL of media for three groups each, being

control, high sugar, and high salt, each in 96 well plates.  Therefore, 16 times 5000 cells

were needed, which equals 80000 cells in 1600 µL. However, to account for loss,

enough cells and media for 20 wells were used, so 100000 cells in 2000 mL. After the

cells were counted, the 100000 cells for each level of the IV were taken in 3 centrifuge

tubes each. The independent variable was the concentration of sodium and glucose in

the media when cultured: the control had 6.4 g/L of salt and 1 g/L of glucose, the high

glucose had 4.5 g/L of glucose instead, and the high salt had 10 g/L of salt. The

centrifuge tubes were centrifuged at 1500 rpm for 5 minutes at room temperature. The

centrifuge is a device that spins around tubes called centrifuge tubes that hold cells with

media. This was to spread out the cells more evenly throughout the media. The

centrifuge was in the biosafety cabinet. The media was then removed, the cells being

resuspended after with 2000 µL of media suitable for each group. Next, the cells were

plated with 100 µL of media per well in a 96 well plate. Note that, while 100000 cells

were meant to be counted, only 80000 of those cells would go in the wells. After that,

the cells were cultured in their respective media (done for 5 days, at 37 degrees

Celsius, 5% CO2 in the atmosphere, and 100% relative humidity). Next, an MTT assay

was performed.

Cell proliferation is measured using an MTT assay. Yellow MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) was converted to purple

formazan in the mitochondria of cancer cells. The absorbance of this colored solution

can be measured by using a multiplate reader at a wavelength of 600 nm. The

conversion can only occur when the mitochondria of a cell is active, so it can define how

many cells are alive and active, so it can be used as a measure of cell proliferation, the

variable being tested for, or the cell proliferation in different glucose and salt

concentrations. One mL of a 5 mg/mL MTT solution was diluted in 9 mL of DMEM.

Media was removed from each well, and then 100 µL of the diluted MTT solution is

added to each of the 16 wells. The plate was stored for 4 hours at 37°C. Because the

formazan eventually solidifies into crystals, 100 µL of a solubilization solution (prepared

by adding 42 µL hydrochloric acid to 50 mL 10% sodium dodecyl sulfate) was added to

each of the 16 wells in order to dissolve the formazan. The 96-well plate was wrapped

in plastic wrap and incubated for 24 hours at 37°C. Absorbance of each well was read

at 600 nm using a Promega Glomax multiplate reader. This multiplate reader uses light

at 600 nm to scan for the absorbance of the light in formazan. This was in a biosafety

cabinet at MCV (see Appendix C). This procedure was used for each level of the

independent variable, and was performed with goggles, gloves, a lab coat, and a

biosafety cabinet. The absorbance values were averaged for each group, with a bar

graph being made.

Results

As said above, the absorbance values were averaged each group, the units

being absorbance units at 600 nm. Even before the plates were put in the multiplate

reader, it was still somewhat visible on which cancer cells fared better than others
based on the amount of purple. The more purple, the more cells alive. In this

experiment, the high glucose section had the most purple. Of the values, the highest

was in the high glucose group, as expected, with the absorbance values at 1.6873 Abs

units, with the control being at 1.2638 Abs units and the high sodium being at the

lowest, at 0.447 Abs units. Note that the more Abs units (absorbance units) at 600 nm,

the more formazan, and therefore the more cells alive.

The medians for high glucose, control, and high salt were at 1.7093, 1.331, and

0.4361 Abs units respectively. Because all values were different, there was no mode.

The standard deviation for the control was highest, at about 0.2078 Abs units, the

standard deviation was 0.1072 and 0.0996 Abs units for high glucose and high salt

respectively. For the T-test, the P value for the control to high glucose was 6.2227 ×

10^-9, and the P value for the control to high sodium was 3.69001 × 10^-11.
 Graphs (Results)

The Effect of Glucose and Salt Concentrations on HepG2

Mean of AbsoAbsorbance Units at 600 nm

Human Liver Cancer Cells

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Control (6.4 g/L salt, 1 g/L High Glucose (4.5 g/L High Salt (10 g/L salt)
glucose) glucose)

Concentrations of Glucose and Salt

The bars at the top of each bar show the standard deviation for each group. This graph,

as shown, shows the mean.

Tables and Charts

Control High Glucose High Sodium

Mean (AU at 600 nm) 1.2638 Abs 1.6878 Abs 0.4470 Abs

units units units

Median (AU at 600 nm) 1.331 Abs units 1.7093 Abs 0.4361 Abs

units units
Standard Deviation for Mean 0.2078 0.1072 0.0996
 The top most row contains levels of the Independent Variable, the concentrations. All

values are in Abs units at 600 nm.

Discussion & Conclusion

The main purpose of the experiment was to find a link between hypertension and

diabetes to liver cancer. Hypertension, diabetes, and cancer have numerous people

across the world affected, and this experiment would help those people.

It was found that the high glucose group caused cell proliferation to be the

highest, with about 1.69 Abs units at 600 nm in the MTT assay, supporting the

hypothesis. The group with the lowest cell proliferation was the high sodium group, with

proliferation being at about 0.45 Abs units at 600 nm. The control was in the middle, at

1.26 Abs units at 600 nm. The standard deviation varied as well, with the values being

about 0.21, 0.11, and 0.01 for the control, high glucose, and high sodium respectively.

The hypothesis, stating that if higher concentrations of sugar were used, there

would be the greatest amount of cancer cells surviving, was supported, as the high

glucose group’s proliferation values were the highest. As shown below, increased

amounts of glucose did increase proliferation, shown in the data. The standard deviation

for the high glucose group was 0.11, so the data points stayed close together. For the t-

test, the p value was 6.22 x 10^-9, so the data points were extremely significant.

Without comparing to other data, this article would have been baseless. For the

high sodium/salt group, similarities were found. According to an article in Frontiers in

Immunology, high salt conditions on mice inhibited tumor growth. (Ralf et al., 2019). As

stated in the introduction, sodium is a factor is kidney cancer growth and metabolic
syndrome, a disturbance in cell proliferation. (Radišaukas et al., 2016). The latter did

not support the data because of it stating that salt increases cancer cell growth.

As for the high glucose group, there was more information to compare to. As

stated in the introduction, increased glucose causes greater cell proliferation, as this

provides enough glucose to tumors. (Chang & Yang, 2016). Glucose, again, increases

proliferation, but also increases migration of cells and resistance to cancer cell death

and treatments. (Li et al., 2019). These two journal articles reinforced the theory that

increased glucose does increase cancer proliferation, and therefore the data.

There were many reasons why the data turned out how it did. A reason why the

high sodium group had the lowest cell proliferation is osmosis. In osmosis, water

molecules moved to where there is a greater concentration of a solute to balance that

concentration. The greater salt concentration was outside the cells, causing water to

exit the cells. It may seem that the same would have gone for high glucose, but cells

likely took in the glucose as it was used to produce ATP. For these reasons, the high

salt and high glucose groups had the lowest and highest proliferation, respectively.

There were many possible improvements to the experiment performed. To start,

there could have been more groups tested in the experiment. With this, lower than

normal concentrations should have been tested as well. Greater documentation could

also have been used. Using solutes other than salt and glucose could have improved

study on the topic. As for further study, testing on mice and/or humans would have

brought other factors like the immune system, giving more insight.

Literature Cited (Abstract, Methods & Materials, Discussion and Conclusion)

"MTT Proliferation Assay." Provost and Wallert Research, U of San Diego,

home.sandiego.edu/~josephprovost/MTT%20Proliferation%20Assay

%20Protocol.pdf. Accessed 6 Nov. 2019.

Chang, Shu-Chun, and Wei-Chung Vivian Yang. "Hyperglycemia, Tumorigenesis, and

Chronic Inflammation." Critical Reviews in Oncology/Hematology, vol. 108, Dec.

2016, pp. 146-53. Science Direct, doi: 10.1016/j.critrevonc.2016.11.003.

Accessed 19 Oct. 2019.

Li, Wenjie, et al. "Effects of Hyperglycemia on the Progression of Tumor Diseases."

PubMed. PubMed, www.ncbi.nlm.nih.gov/pubmed/31337431. Accessed 19 Oct.

2019. Originally published in Journal of Experimental and Clinical Cancer

Research, vol. 38, 23 June 2019, p. 327.

Radišaukas, Ričardas, et al. "Hypertension, Serum Lipids, and Cancer Risk: A Review

of Epidemiological Evidence." Medicina, vol. 52, no. 2, 2016, pp. 89-98.

ScienceDirect, doi: 10.1016/j.medici.2016.03.002. Accessed 2 Nov. 2019.

Willebrand, Ralf et al. "High Salt Inhibits Tumor Growth by Enhancing Anti-tumor

Immunity." Frontiers in Immunology vol. 10 1141. 4 Jun. 2019,

doi:10.3389/fimmu.2019.01141

Appendix A
 The Effect of Glucose and Salt Concentrations of HepG2 Human
Liver Cancer Cells

Median of Absorbance Units at 600 nm

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Control (6.4 g/L salt, 1 g/L High Glucose (4.5 g/L High Salt (10 g/L salt)
glucose) glucose)

Concentrations of Glucose and Salt

This graph shows similar data to the graph above, with the mean of absorbance units,

but this graph contains the median instead.

Appendix B

Title: The Effect of Glucose and Salt Concentrations on HepG2 Human Liver Cancer
Cells
Hypothesis: If a high glucose concentration is used, there will be greater cell
proliferation.

IV: Glucose and Salt Concentrations in Cancer Cell Media

Levels of IV Control High Glucose High Sodium (10 g/L

(6.4 g/L of NaCl, 1 g/L (4.5 g/L NaCl)
glucose) glucose)

Number of 16 16 16
Trials

DV: Cell proliferation based on amount of formazan after MTT assay five days after
cells left in incubator and cultured, measured in Abs units at 600 nm

Constants: number of trials, multiplate reader, hemocytometer, temperature (37

oC), relative humidity (100%), CO2 concentration (5%), type of dish used, time to
culture, amount of media (200 µL per trial), number of starting cells (5000 cells per
trial)

The above is the experimental design diagram for the experiment.

Appendix C

The above is the main workspace in the biosafety cabinet.

Appendix D
The above is a shot of the biosafety cabinet. The off-white box with light blue all the way

at the right of the image is the incubator, where the cells were cultured.

Appendix E

This is a shot of the Promega Glomax multiplate reader, used to calculate the results.