Dimethyl sulfoxide inactivates the anticancer effect of cisplatin against human myelogenous leukemia cell lines in in vitro assays.

OBJECTIVES: To investigate the effect of DMSO on cisplatin induced cytotoxicity (invitro) against K562 (Human mylogenous leukemia) cell line and to study the cisplatin-DMSO adduct formation using UV-spectrophotometer.
MATERIALS AND METHODS: Effect of DMSO on the cytotoxicity of cisplatin was studied in K562 (Chronic mylogenous leukemia) cell line by MTT assay. Cisplatin-DMSO adduct formation was studied by continuously monitoring the increase in absorption peaks for 30 minutes using UV-spectrophotometer.
RESULTS: 0.1-0.3% DMSO markedly reduced the cytotoxic activity of cisplatin in K562 cells. Cisplatin-DMSO adduct formation was detected using UV-spectrophotometer. Continuous increase in UV absorbance between 250nm-290nm was observed when cisplatin (0.5mg/ml) and DMSO (10%) were mixed.
CONCLUSION: Present study revealed that DMSO inactivates the cytotoxicity of cisplatin. Cisplatin-DMSO mixture showed increased absorbance at 250-290nm. Therefore, using DMSO in invitro assays might result in misinterpretation of actual efficacy of drugs.

Introduction

Dimethyl sulfoxide (DMSO) is widely used to dissolve hydrophobic drugs in both in vitro and in vivo studies. There are other known functions of DMSO. For example, it is used to dissolve water insoluble formazan crystals formed inside cells during performing cell viability assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, it is used as cryoprotectant for preserving cell cultures and it has anti-inflammatory and reactive oxygen species scavenging properties.[12] Most of the in vitro and in vivo studies, which had used DMSO as vehicle to dissolve drugs had not checked the effect of DMSO on the drug itself. A study showed that many platinum- based drugs can form adduct with DMSO. The synergetic effect of other hydrophobic anticancer drugs with cisplatin is also widely studied by dissolving them in DMSO. In many such in vitro studies adduct formation of DMSO with cisplatin was not taken into concern.[34] A recent report showed that about 11–34% of the total laboratory studies on cisplatin had utilized DMSO to dissolve cisplatin. Dissolving cisplatin in DMSO has removed cisplatin from DNA.[5] This study evaluated the effect of DMSO on cisplatin-induced cytotoxicity and has detected cisplatin-DMSO complex using ultraviolet (UV)-spectrophotometric measurement. Therefore, our study showed that using DMSO for drug studies in cell cultures might cause a misinterpretation of actual efficacy of the drugs.

Cisplatin has been widely used to treat various types of cancers because of its broad spectrum of activity.[6] Cisplatin is a platinum-based metal complex, which binds to DNA and forms intrastrand crosslinks between adjacent guanines as Pt (NH3)2(2+) ions are chelated to the N7 atoms.[7] Previous studies show that cisplatin is cytotoxic to various cancer cell lines. Cisplatin induces apoptosis and activates various signal transduction pathways including mitochondrial pathways.[8] Cytotoxicity (PC12 and L1210) and neurotoxicity (mouse embryonic rat dorsal root ganglion neurons) induced by cisplatin was significantly reduced during combination treatment with DMSO. Cisplatin formed an adduct with DMSO, and the resulting product showed less ability to bind with DNA.[9] Recently, it was reported that co-treatment of cisplatin with DMSO exacerbates the cisplatin-induced sensory hair death in zebrafish (model system for studying the sensory hair loss in human ears during chemotherapy).[10] A previous study shows that treatment with DMSO has induced a delayed appearance of cell differentiation characteristics. The ability to reduce nitroblue tetrazolium dye and engulfment of latex particle by differentiated human myeloblastic leukemia cells delayed for 48 h compared to the 12-O-tetradecanoylphorbol-13-acetate treated cells.[11] These studies clearly show that treatment of DMSO has affected the normal activities of cells (at noncytotoxic concentrations) and it also directly reduced the efficacy of platinum-based drugs by forming complexes. Therefore, use of DMSO in cell culture and drug discovery studies has to be cautioned to avoid misinterpretation of actual efficacy of drugs and cellular activities.

Materials and Methods

Cisplatin (1 mg/mL) (Cisteen) Miracalus Pharma Pvt. Ltd, Mumbai, India was obtained as a gift from Amala Cancer Hospital and Research Centre. MTT was obtained from, Himedia, India. DMSO (suitable for UV-spectroscopy) was obtained from SRL, India. K562 (human myeloblastic leukemia) was maintained in Roswell Park Memorial Institute media with 10% fetal bovine serum and antibiotics. Cells were incubated at 37°C under 5% carbon dioxide environment.

Results and Discussion

Cell Viability Assay

Approximately 105 cells/mL were seeded to each well and allowed to incubate overnight. The cells were co-treated with varying concentration of DMSO (0.1–0.3%) and fixed concentration of cisplatin (5 μg/mL) and incubated for 48 h. At the end of incubation, supernatant media was removed, and MTT (5 μg/mL) was added to each well and incubated further for 4 h. The supernatant was removed and the water-insoluble formazan crystals formed inside cells were dissolved in DMSO. The optical density was read at 570 nm.[12]

Cisplatin, when treated alone, was found to be cytotoxic against K562 cell lines. IC50 value of cisplatin in K562 cell line was 7.53 ± 2.1 μg/mL. When cells were treated with 5 μg/mL of cisplatin only 34.76% of viability was recorded. Treatment of 0.1% DMSO along with 5 μg/mL of cisplatin markedly reduced the cytotoxic activity of cisplatin, and the percentage of viability of cells increased to 77.276%. This increased viability may be due to inactivation of cisplatin by DMSO. The increase in the concentration of DMSO from 0.1% to 0.3% along with cisplatin proportionally reduced the percentage of dead cells, indicating the reduction in cisplatin-induced cytotoxicity [Figure 1].

Figure 1

Co-treatment of cisplatin (5 μg/mL) and 0.1–0.3% dimethyl sulfoxide (DMSO) reduced the cisplatin induced cytotoxicity. Increase in the concentration of DMSO proportionally reduced the cytotoxicity of cisplatin

Ultraviolet Detection of Cisplatin-Dimethyl Sulfoxide Complex

In order to detect the cisplatin-DMSO adduct formation using UV-spectrophotometer three different solvent mixtures, A (Cisplatin-0.5 mg/mL and water) B (Cisplatin-0.5 mg/mL, DMSO-10% and water) and C (DMSO-10% and water) were prepared. Each sample was incubated at room temperature for 15 min and scanned for UV-absorption peaks ranging from 199 nm to 600 nm in Shimadzu-UV-spectrophotometer. Double distilled sterile water used to dissolve cisplatin was also scanned under UV-spectrophotometer to rule out the effect of water, if any. The continuous monitoring of cisplatin-DMSO adduct formation was studied by preparing the mixture B and then continuously scanning (between 199 nm and 600 nm) the mixture using UV-spectrophotometer at every 5 min interval for 30 min.

In order to observe the formation of cisplatin-DMSO adduct by UV-spectroscopy the concentration of cisplatin and DMSO was proportionally increased to 100 times. 0.5 mg/mL of cisplatin along with 10% DMSO showed a characteristic increase in absorption between 250 nm and 290 nm. 10% DMSO or cisplatin alone didn’t show absorbance at this region. Therefore the characteristic increase in absorption appeared at this region is due to the formation of cisplatin-DMSO adducts [Figure 2a]. Cisplatin-DMSO adduct formation was continuously observed under UV-spectrophotometer for 30 min and gradual increase in the UV-absorption was observed between 250 nm and 290 nm, this clearly demonstrate the rapid increase in adduct formation with time [Figure 2b].

Figure 2

(a) Cisplatin (0.5 mg/mL) and 10% dimethyl sulfoxide (DMSO) showed increased absorbance between 250 nm and 299 nm. Cisplatin (0.5 mg/mL) or 10% DMSO alone did not showed any characteristic increase in absorbance between 250 nm and 299 nm. Water used in the experiment also had no effect on cisplatin. (b) Continuous monitoring of cisplatin-DMSO adduct formation at every 5 min intervals for 30 min using ultraviolet spectrophotometer showed steady increase in the absorption between 250 nm and 299 nm

Efficacy of cisplatin against the proliferation of leukemic cells (K562) was reduced when the cells were co-treated with cisplatin and DMSO therefore, UV-spectrum analysis was done using the mixture of cisplatin and DMSO. Cisplatin-DMSO adduct formation was clearly detected using UV-spectrophotometer. Steady increase in the UV-absorption peaks between 250 nm and 290 nm was observed, indicating the rapid formation of the cisplatin-DMSO adduct. Results from the present study indicate that adduct formation between cisplatin and DMSO might be the reason for the reduction of cytotoxicity of cisplatin.

Based on the above results we do not recommend the use of DMSO along with cisplatin as DMSO might interfere with the antitumor effect of the cisplatin. Similar results were previously reported when DMSO was used along with cisplatin.[8] Therefore, before using DMSO as vehicle for dissolving any platinum-based drugs or hydrophobic molecules it is necessary to study the effect of DMSO on the same drugs using UV-spectrometer or mass spectrometer. Thus studying the effect of DMSO on platinum-based drugs or any hydrophobic molecules can possibly avoid the misinterpretation of the actual efficacy of the drug in cell culture studies.