Improving the Efficacy of Cisplatin in Colon Cancer HT- 29 Cells via Combination Therapy with Selenium

Research Article

Austin J Pharmacol Ther. 2014; 2 (2). 1013

Improving the Efficacy of Cisplatin in Colon Cancer HT– 29 Cells via Combination Therapy with Selenium

Stockert A1*, Kinder D1, Christ M2, Amend K3, Aulthouse A4

1Department of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University, Ada, OH

2Mount Carmel Health System, Columbus, OH

3Physicians Assistant Program, University of Saint Francis, Fort Wayne, IN

4Department of Biological Sciences and Allied Health, Getty College of Arts and Sciences, Ohio Northern University, Ada, OH

*Corresponding author: : Amy Stockert, Department of Pharmaceutical and Biomedical Sciences, Raabe College of Pharmacy, Ohio Northern University, Ada, OH

Received: February 03, 2014; Accepted: February 10, 2014; Published: February 13, 2014


Cis–platinum is generally not effective against slow growing colon cancer. Glutathione peroxidase is a seleno–enzyme that inactivates reactive oxygen species (ROS). Research suggests that high levels of ROS may alter cisplatinum efficacy. The effect of selenite supplementation during cis–platinum treatment in HT–29 colon cancer cells was explored. Agarose culture allowed cells to grow in 3D, form colonies, as well as allow independent analysis of mitosis, cell viability, and ROS breakdown. Single cells were suspended in agarose and grown 7 days. Cultures were un– or pre– treated with selenite at day 0. On day 4, cultures were treated with cis–platinum alone or in conjunction with selenite. At 7 days, cell viability and mitotic activity were evaluated. ROS breakdown was quantified using an assay of glutathione peroxidase activity. Selenite at low doses did not affect cell viability or mitosis. Cultures treated with the selenite ⁄ cis–platinum combination exhibited higher ROS breakdown and increased cis–platinum efficacy. Where colonies are already formed, cisplatinum alone was not as effective as the cis–platinum ⁄ selenite combination. Additionally, ROS breakdown was increased in cells treated with the cisplatinum ⁄ selenite combination suggesting a link between ROS levels and cisplatinum efficacy.


The debate regarding selenium and cancer has remained unresolved [1,2]. Research has been completed examining the cancer preventative affects of selenium and have suggested a preventative role for selenium while others concluded that there is no preventative effect [3–7]. In addition to these differences, clinical evidence demonstrates that some cancers are more resistant to traditional therapies such as cis–platin (cis–pt), thus necessitating improved therapeutic options either through combination therapy. Slow growing coloncancer cell lines are among those that have greater tendencies to develop resistance and are less susceptible to cis–pt and other metalcoordination therapies [8–10]. Strong evidence suggests that selenium supplementation with chemotherapeutic agents can decrease the nephrotoxicity from these drugs [11–14]. Similarly, selenium seems to play some role in decreasing the development of resistant cell lines [15–18].

Selenium is linked to a decrease in oxidative damage due to its role in the selenoenzyme glutathione peroxidase. Glutathione peroxidase (GPox) is responsible for the breakdown of hydrogen peroxide into water via the oxidation of reduced glutathione. A flavin dependent enzyme, glutathione reductase is responsible for regenerating the reduced form of glutathione using NADPH as an electron donor. It is expected that selenite supplementation would increase the availability of glutathione peroxidase among other selenium dependent enzymes [19,20].

The majority of the studies thus far have focused on how to either increase reactive oxygen species (ROS) selectively in cancer cells or manage ROS in normal cells thereby protecting them from damage [21–24]. Here we present data suggesting that controlling the amount of ROS in cancer cells may improve the efficacy of cis–pt in colon cancer cell line HT–29. We examined the effects of selenite on the colon cancer cell line HT–29 pre–treated with selenite (sodium selenite) and co–treated with selenite (sodium selenite) and cis–pt. Although it has been demonstrated by this study that cis–pt is capable of blocking proliferation when administered at plating, we were able to show that cis–pt is not as effective when given after colonies have had the opportunity to form. Unfortunately, this loss in effectiveness observed once colonies have formed is the situation that is most physiologically significant. Chemotherapeutics would only be administered after cancerous polyps has developed. Our study demonstrates that combination therapy of cis–pt and selenite increases the number of dead cells found in colonies even when administered after colonies have had the chance to form, making cis–pt a potentially viable option for colon cancer patients despite previous studies suggesting it is not an appropriate first line treatment. Although we hypothesized that pre–treatment with selenite would have this effect, and this was not observed in our study to the extent that co–treatment was effective, co–treatment of se with cis–pt is the clinically relevant treatment regimen.

Materials and Methods

Monolayer culture

HT–29 colon adenocarcinoma cell line was purchased from American Tissue Culture Collection (ATCC). Cells were grown with standard monolayer culture techniques at 37° C in a humidified CO2 incubator. The media consisted of Dulbecco’s modified Eagle’s media (DMEM) with 4.5 gm⁄l glucose containing 10% fetal calf serum and 0.1% penicillin–streptomycin. Monolayer culture was used to establish IC50, to give a starting dose for agarose experimentation, and to expand the cell line.

XTT assay for determining IC50 values

Studies to determine the IC50 values of cis–pt in the HT–29 cell lines were conducted using the XTT assay according to manufacturer’s specification (Sigma–Aldrich). Na2SeO4 and cis–pt were purchased from Sigma–Aldrich. All treatment compounds were dissolved in dimethylsulfoxide (DMSO) and mixed with media prior to adding to cell cultures. DMSO concentration in cell culture did not exceed 0.1%. The XTT assay, which measures mitochondrial activity, was used to determine IC50 values for the cis–pt. The assay was conducted according to manufacturer’s directions, and results are reported as percent of control.

Agarose Cell Culture Methods

HT29 cells were grown in monolayer and then suspended inagarose. Details of this agarose method are described in Kinder andAulthouse 2004 [25]. In brief, 10µl of single cell suspension (5x105 cells in 1ml of 0.5% low temperature agarose) were plated on 35 mm tissue culture dishes which were previously coated with high temperature agarose. The cell⁄agarose suspension was allowed to gel and were grown for 7 days. The cultures were fed⁄treated at plating (day 0) and at day 4 with complete media change. Cultures fed DMEM only served as controls and cultures treated with dimethylsulfoxide (DMSO) served as vehicle controls. Final DMSO concentration was 0.1% in all cultures. The cultures were treated with Na2SeO4 or cis–pt (prepared as for monolayer above). The media consisted of Dulbecco’s modified Eagle’s media (DMEM) with 4.5 gm⁄l glucose containing 10% fetal calf serum and 0.1% penicillin–streptomycin.

Rationale for using the agarose cell culture method

The use of the agarose cell culture provides several advantages over monolayer culture. Long term experiments, up to two weeks, can be conducted. Plating of single cells suspended in agarose allows analysis of both cytotoxicity (trypan blue exclusion) and mitotic activity (number and size of cell clusters). In addition, this culture method is amiable to determine enzyme function.

Control (DMEM only) and vehicle control (VC, DMSO) cultures were established for each experiment and for all treatment groups (n=6 for each treatment group). All cultures were analyzed for viability using the trypan blue exclusion assay and for mitotic activity by counting both single cells and cell colonies (clusters of 2 or more cells) using an Olympus IM inverted microscope. Cultures were examined on day 4 and day 7. For analysis on day 7, the cultures were first centered at 4x and then counted at 10x to prevent bias. Approximately 30% of the cell culture was evaluated. The amount of single cells and cell colonies alive and dead, between treatment groups were analyzed using a t–test and controlled for overall error using a modified Bonferroni.

Reactive oxygen species breakdown assa

ROS breakdown was determined using the Total ROS detection kit available from Enzo. Cells were collected from agarose culture by homogenizing the agarose cell mixture in lysis buffer supplied by the assay kit. Correlation from absorbance values to ROS breakdown was determined as specified by the manufacturer.


In order to estimate appropriate cis–pt concentrations for experiments, the IC 50 for cis–pt was determined in the HT–29 cell line. Using the XTT assay in monolayer culture we estimated an IC50 of 70µM. The XTT assay can be used as an indication of cell growth inhibition, but does not answer the question of cytotoxicity. HT–29 cells grown in monolayer were treated with selenite (as Na2SeO4) at increasing concentrations. Selenite is not considered a cytotoxic agent, but has an LD50 in rats of 1.6 mg⁄kg. At 29 µg⁄ml, the number of cells present had dropped to 80% compared to control. The results of the monolayer studies suggest there is a cytostatic or a cytotoxic component to selenite at higher concentrations.

Supplementation with selenite: cell growth inhibition and cell death in agarose

In order to determine the minimum concentration of selenite that was required to observe an effect, but not high enough to contribute to cell death, we completed a dose response in agarose culture. This was a necessary step since values estimated in monolayer are often not representative of those observed in agarose culture. The dose response of the HT–29 cells to selenite was completed by treating cells continuously for 7 days with selenite concentrations of 0.75, 1.5, 3, 6, 11, 23, and 45 µg⁄ml (data not shown). Based on this data we determined that the original concentration of selenite was high enough at all doses tested to result in increased cell death. We repeated the experiment at lower concentrations.

Figure 1 shows the results for the lower dose response with selenite including 0.05, 0.09, 0.18 and 0.33 µg⁄ml. This data allowed us to determine the highest dose tested that did not show significant cell death from selenite alone. Although it was possible that we could have increased the selenite dose beyond 0.33 µg⁄ml and still not have contributed to cell death, our data examining ROS breakdown indicated that a dose of 0.05 µg⁄ml was sufficient to increase selenite enzyme function over control (Figure 2). Based on this data we progressed forward with 0.33 µg⁄ml selenite in the combination experiments, since it was enough to increase GPox activity and still not contribute to cell death.