Use of a colorimetric assay to evaluate the proliferation of canine mammary tumor cells exposed to propofol

Abstract

Drugs applied on human cancer cells can influence the rate of cell proliferation. The present study investigates the use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) colorimetric assay to evaluate canine tumor cell proliferation after exposure to the injectable anesthetic, propofol.

Primary (CIPp) and metastatic (CIPm) canine tubular adenocarcinoma cell lines were incubated with cell culture medium (control) or propofol (1, 5, and 10 μg/mL). The MTT assays were performed after 6 and 12 hours of exposure. Measurements of absorbance were obtained for each condition with a spectrophotometer and compared with controls using a 3-way analysis of variance (P < 0.05).

An increased cell proliferation rate was observed in CIPp exposed to 5 and 10 μg/mL of propofol for 6 hours and 1, 5, and 10 μg/mL for 12 hours. No significant changes were observed in CIPm after 6 hours of exposure. All propofol concentrations decreased the cell proliferation rate in CIPm after 12 hours of exposure.

The MTT assays showed that exposure of CIPp to propofol for 6 and 12 hours increased cell proliferation. A decrease in the CIPm proliferation rate was observed when propofol exposure lasted for 12 hours. Further studies are warranted to better understand the role of propofol on cancer cell proliferation.

Introduction

Cancer develops as a result of the continual unregulated proliferation of cells. Therefore, the study of cancer cell proliferation has been regarded as a key component when evaluating tumor initiation and metastasis (1).

Recently, it has been suggested that anesthetic drug choice during primary tumor removal may affect cell proliferation, consequently affecting the development of cancer and the spread of metastatic cells (2,3). However, contradictory results have been found in studies in which the effects of propofol, an intravenous (IV) anesthetic agent that facilitates inhibitory neurotransmission mediated by gamma-aminobutyric acid, were investigated on human tumor cell proliferation. For instance, propofol induced the proliferation and invasion of gallbladder cancer cells (4) and neuroblastoma cells (5) but inhibited the proliferation and invasion of osteosarcoma cells (6). Interestingly, some studies suggested that propofol inhibits breast cancer cell proliferation, supporting the hypothesis that propofol decreases the incidence of cancer recurrence and prolongs survival time in women affected with breast cancer (7,8).

The possibility of using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay for measuring proliferation of cells has long been studied (9–11). The assay is broadly used to measure in vitro cytotoxic effects of drugs on cells and has the advantages of being easy to handle and not requiring radioactivity (12). To the authors’ knowledge, no other studies have been designed to evaluate the effects of propofol on canine tumor cell proliferation. The aim of the present study, therefore, was to evaluate the effects of a clinically available formulation of propofol on the proliferation of 2 canine mammary tumor cell lines using an MTT assay. We hypothesized that the exposure of canine mammary tumor cells to propofol would significantly decrease the cell proliferation rate.

Materials and methods

Cell culture

Primary canine tubular adenocarcinoma (CIPp) and meta-static canine tubular adenocarcinoma (CIPm) cell lines originating from primary and metastatic lesions on the same individual (13) were grown in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum (Fetal Bovine Serum; Sigma-Aldrich, St. Louis, Missouri, USA), 100 μg/mL penicillin (Penicillin-Streptomycin; Sigma-Aldrich), 100 μg/mL streptomycin (Penicillin-Streptomycin; Sigma-Aldrich), and 1.5 mg/mL amphotericin B (Amphotericin B; Sigma-Aldrich) at 37°C in humidified atmospheric air with 5% carbon dioxide. Cells were grown as monolayers in 75 cm² standard tissue culture plasticware (TC Flask T75; Standard, Sarstedt, Dublin, Ireland) and media were changed every 3 d. For the experiments, cells were harvested from 70% confluent cultures by trypsinization and counted with an automated cell counter (Automated Cell Counter TC20; Bio-Rad, Milan, Italy).

In order to determine the optimal experimental conditions, the time-dependent exponential cell growth curve was evaluated at 4, 6, and 12 h for each cell line. Cells were seeded in normal medium in a range from 1000 to 10 000 cells per well. The experiment was conducted in triplicate in a 96-well plate (Eppendorf Cell Culture Plate; Eppendorf S.r.l., Milan, Italy) and a proliferation index was assessed with the MTT colorimetric assay. Based on the obtained time-dependent exponential cell growth curve, it was established that 3000 cells per well provided the optimal experimental conditions for testing cell proliferation during 6 and 12 h of treatment exposure (e.g., no signs of cell death due to over-confluence). Therefore, 3000 cells per well were seeded in a 96-well cell culture plate and incubated with 100 μL of cell culture medium for 12 h to allow cell adhesion.

Drug exposure

Treatments were performed with a clinically available propofol formulation (10 mg/mL propofol) containing a lipid-based emulsion (LE) (100 mg/mL soybean oil, 22.5 mg/mL glycerol, and 12 mg/mL egg lecithin) and a sodium hydroxide adjuvant to adjust the pH (Vetofol; Esteve SpA, Milan, Italy). The LE propofol was diluted with cell medium to 1, 5, or 10 μg/mL (P1, P2, and P3 treatments, respectively) before incubating the cells. These concentrations correlated with reported blood levels of propofol in greyhounds (14) after IV administration of clinically relevant doses [4 mg/kg body weight (BW) followed by infusions of 0.2 to 0.4 mg/kg BW per minute]. Medium containing LE propofol was then added to the cell cultures for 6 and 12 h. Cells grown in culture medium alone were used as controls. The experiment was performed 6 times for each condition.

Proliferation assay

An MTT colorimetric assay for cell survival and proliferation was conducted on both cell lines according to Tada et al (9). This method measures the conversion of MTT into purple-colored formazan crystals that are induced by redox activity of living cells. A decrease of cellular redox activity indicates a reduction in cell viability (12). After completion of the different exposure times, the solutions containing propofol treatments were removed. Thereafter, 20 μL of MTT diluted in phosphate-buffered saline at a concentration of 5 mg/mL and a pH of 7.5 was added to each well and incubated for 4 h at 37°C. After the first incubation period, 0.1 mL of 10% sodium dodecyl sulfate (Sodium Dodecyl Sulfate; Sigma-Aldrich) diluted in a solution of 0.01 M HCl was added to each well in order to dissolve the formazan crystals and incubated overnight. Subsequently, measurements were performed with a spectrophotometer (Microplate Model 680; Bio-Rad) on an enzyme-linked immunosorbent assay (ELISA) plate reader at 590 nm. An absolute value of absorbance was obtained. Absorbance values lower than those of controls indicated a reduction in cell proliferation rate, while values higher than those of controls indicated an increase in cell proliferation rate (12).

Statistical analysis

The mean absorbance value of the controls was compared with the mean absorbance values from the treatments using a 3-way analysis of variance (ANOVA) (time, cell line, concentration of LE propofol). All statistical analyses were performed using an open-source statistical software package (R-studio, version 3.2.0; Boston, Massachusetts, USA; www.r-project.org). A value of P < 0.05 was considered statistically significant. Data are presented as mean ± standard error (SE) and ranges for the percentage increases or decreases of the cell proliferation rates.

Results

No differences were found in CIPp between the mean absorbance value of the P1 treatment group and that of the control cells. Meanwhile, treatment groups P2 and P3 showed statistically significant increases in cell proliferation rates (18% and 27%, respectively) compared to CIPp control cells after 6 h of exposure (Figure 1A). No significant differences in cell proliferation at the same incubation time were observed between control CIPm cells and any LE propofol-treated CIPm cells (Figure 1B). After 12 h of exposure, all LE propofol concentrations induced a significant increase in cell proliferation in CIPp cells (P1 = 105%, P2 = 114%, P3 = 123%) (Figure 2A) and a significant decrease in cell proliferation in CIPm cells (P1 = −22%, P2 = −14%, P3 = −22%) (Figure 2B).

Figure 1.

Changes in absorbance values between primary (CIPp; A) and metastatic (CIPm; B) canine mammary tubular adenocarcinoma cells receiving different concentrations (P1 = 1 μg/mL; P2 = 5 μg/mL; P3 = 10 μg/mL) of a lipid-based propofol emulsion when compared to control cells (C) after 6 h of drug exposure. Statistical significance is represented by *** P < 0.001, ** P < 0.01, and * P < 0.05.

Figure 2.

Changes in absorbance values between primary (CIPp; A) and metastatic (CIPm; B) canine mammary tubular adenocarcinoma cells receiving different concentrations (P1 = 1 μg/mL; P2 = 5 μg/mL; P3 = 10 μg/mL) of a lipid-based propofol emulsion when compared to control cells (C) after 12 h of drug exposure. Statistical significance is represented by *** P < 0.001, ** P < 0.01, and * P < 0.05.

Discussion

In the present study, LE propofol modified the cell proliferation rate of cultured CIP cells in a cell line-dependent way. Lipid-based propofol emulsion treatments increased and decreased cell proliferation in CIPp and CIPm cells, respectively, compared with the control group. Time effects (i.e., 6 h versus 12 h of exposure) were also observed, mainly in CIPm.

The results of previous reports on the effects of propofol on cancer cell growth have been controversial since, for instance, propofol induced proliferation in gallbladder cancer cells (4) and neuroblastoma cells (5) but decreased proliferation in osteosarcoma cells (6). Interestingly, some studies suggested that propofol inhibited breast cancer cell proliferation supporting the hypothesis that propofol decreases the incidence of cancer recurrence and prolongs survival time in women affected with breast cancer (7,8).

In the case of breast cancer cells, propofol inhibited cell growth when used at 5 and 10 μg/mL on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cell lines (7). Other investigators also showed that propofol conjugates applied to ER-negative MDA-MB-231 breast cancer cells significantly inhibited cell growth (8), although these results were only seen when higher concentrations of propofol conjugates were applied (i.e., approximately 20 μg/mL). In the current study, a reduction of absorbance was observed in CIPm cell lines treated with LE propofol for 12 h at concentrations between 1 and 10 μg/mL, which are considered to be within the blood levels obtained from dogs anesthetized with propofol (14).

Paradoxically, in the current study, an increase in tumor cell proliferation was observed when applying LE propofol on CIPp at almost all concentrations and at both studied time points. In a recent study, breast cancer cells from the line MDA-MB-231 treated with propofol (2 to 10 μg/mL) for 1, 4, and 12 h also showed an increase in proliferation rate in a dose- and time-dependent manner (15), disagreeing with results from Deegan et al (7) and Siddiqui et al (8). The authors postulated that the increased proliferation was partially associated with the inhibition of the expression of p53, a gene involved in the suppression of cell mutation (15). This observation becomes extremely interesting when considering that human and canine mammary tumors have in common not only similar malignancy rates, disease courses, and prognoses (16) but also some molecular attributes such as changes in the expression of steroid receptors (17) and mutations of the suppressor gene p53 (18).

It is not clear why LE propofol induced different effects in CIPp and CIPm cell lines in the present study. A clear cell-type dependency has not been previously reported. For instance, in a study by Ecimovic et al (19), propofol did not reduce breast cancer cell proliferation in either ER-positive or ER-negative cell lines with any of the tested concentrations (1 to 10 μg/mL) or exposure times (6, 12, 24, and 36 h), showing a lack of effect as well as a lack of dependency on dose, time, and cell type. In a previous study, a decrease in the neuroepithelial gene 1 expression was observed in CIPm exposed to LE propofol (20). The study also showed a dual effect on the neuroepithelial gene 1 expression in CIPp exposed to LE propofol, with increases of gene expression at 6 and 48 h and decreases at 12 and 24 h after exposure. Even though the aim of both trials was to observe possible propofol anti-cancer activity, the laboratory techniques involved were different, with one study evaluating cell number (i.e., proliferation) and the other one evaluating the expression of a gene related to cell migration. Indeed, the ability to proliferate and the ability to migrate do not have to be the same in a particular cell evolutionary state or cell type and could be extremely different in primary and metastatic cells from the same tumor type (21). Unfortunately, the current study did not evaluate the molecular features of the cultured cells and therefore, could not determine why CIPp and CIPm cell lines showed different effects regarding proliferation when exposed to LE propofol.

Different assays can be chosen to study cell proliferation. Therefore, diverse results among research projects can be the consequence of different methodologies. The MTT assay used herein is based on the measurement of absorbance, which is associated with changes in cell number (9). The actual reason why the cell number varies (e.g., increase or decrease in proliferation, cell death, or apoptosis) cannot be determined with this technique. Other reports, for instance, use techniques that combine the evaluation of cell growth using the water-soluble tetrazolium-1 assay and the investigation of cellular viability and apoptosis using a Vybrant apoptosis assay kit (8).

Exposure times used in the present study were chosen based on what has been reported in vitro in human medicine, to facilitate comprehensible comparisons between studies (22).

A clinically available formulation of propofol was chosen for this study. This formulation is widely used in veterinary anesthesia and contains soybean oil, glycerol, and egg lecithin as adjuvants. In a previous study on prostate cancer cell proliferation, the effects of an intralipid-containing propofol formulation similar to the one reported here, were compared with those of a propofol-free 10% LE, using an MTT assay (23). The authors did not report differences between the control group and the group treated with the propofolfree 10% LE. Unfortunately, there are no reports that evaluate the blood or plasma concentrations of the components of the LE obtained after LE propofol administration to dogs. For this reason, it was unclear what concentration of the LE should have been used to check cancer cell proliferation in vitro. Therefore, the use of a sham group treated with propofol-free LE was disregarded in the present study.

A deep understanding of the molecular mechanisms underlying tumor spread is definitely needed. However, the variable environment in a clinical setting makes the direct translation of information from in vitro to in vivo conditions difficult. In fact, the few retrospective clinical studies available in human medicine show an advantage of propofol over inhalant anesthetics when anesthetizing cancer patients for surgical cancer removal (24,25), while data from in vitro studies are still not completely in agreement.

In conclusion, in the present study, MTT assay tests showed that higher but still clinically relevant concentrations of LE propofol and longer exposure times resulted in an increase in cell proliferation when applied to CIPp cells and a decrease in cell proliferation when applied to CIPm cells. Further studies are warranted to determine the role of propofol in CIP cell proliferation and recurrence.