1.
Introduction
All around the world, breast cancer
represents the most widely diffuse type of cancer in women. It is also reported
as the second most frequent cause of cancer related death, usually as a result
of metastatic spread [1]. Surgical removal of
the primary tumor is considered to be the most effective treatment for patients
diagnosed with malignant breast cancer [2].
However, recent studies suggest that some anesthetic techniques may facilitate
or impede cancer spread via different mechanisms [3].
In particular, the intravenous anesthetic agent propofol may have anti-cancer
properties by promoting apoptosis in some cancer cell lines [4], initiating the activation of T-helper cells and
promoting the differentiation of T-helper 1 cells [5] among some described mechanisms.
In this context, interest is
raising towards the effects of drugs on the expression of genes that are
associated with tumor cell migration like the Neuroepithelial Cell Transforming
(NET) 1 gene. The NET1 gene, a RhoA specific Guanine Nucleotide Exchange Factor
(GEF), has a fundamental role in the organization of actin filaments in the
cytoskeleton and its overexpression may increase the ability of breast
adenocarcinoma cells to migrate and invade [6]. In vitro
studies have shown that NET1 gene expression in human tumor cells can be
influenced by drugs used in the perioperative period. In particular, in a study
from Ecimovic and colleagues (2014), it was shown that cells cultured under
propofol exposure significantly decreased NET1 gene expression reducing cells
migration, a phenomenon, the latter, which was reversed after NET1 gene
silencing [7].
In
recent years, canine tumors have been postulated as translational models of
naturally occurring cancer in people [8]. Based
on the fact that dogs have shorter life spam and consequently that canine
tumors require shorter time to metastasize, clinical canine patients affected
with mammary tumors could be adopted as a natural model for the study of
mammary tumor progression in a faster fashion. However, to the author’s
knowledge, there are no studies evaluating the influence of anesthetics on the
expression of NET1 gene in canine mammary tumor cells. Based on this, the aim
of the present study is to evaluate NET1 gene expression and the effects of two
concentrations of a clinically available Propofol formulation on such
expression in primary (CIPp) and metastatic (CIPm) canine mammary tumor cell
lines.
2.
Methods
2.1.
Cell
Culture
Primary canine tubular
adenocarcinoma’s cells (CIPp) and metastatic canine tubular adenocarcinoma’s
cells (CIPm) derived from the same patient were used for this study [9]. Both cell lines were cultivated in Roswell Park Memorial Institute (RPMI)
medium supplemented with 10% fetal bovine serum, 100 μg ml-1 penicillin, 100 μg
ml-1 streptomycin, 1.5 mg ml-1 amphotericin B and incubated for 24 hours at
37°C in a humidified atmosphere with
5% CO2 (Sigma-Aldrich, Italy).
2.2.
Drug
Exposure
Cells were seeded in triplicates
onto p6 culture plates (Eppendorf, Italy) and treated with 1 and 10 µg ml-1 (P1
and P10 treatments, respectively) of Vetofol®
(Esteve SpA, Italy) a clinically available propofol formulation, for 6, 12, 24
and 48 hours. Cells cultured without anesthetics were used as baseline.
2.3.
Total RNA
Extraction
Total RNA was isolated using the
Trizol Reagent (Thermo Fisher Scientific, Italy) according to the
manufacturer`s instruction. In short, after removing the growth medium and
washing the cells with PBS, Trizol Reagent was added to 80% confluent cell
cultures. After a 5-minute incubation period, cell lysates were transferred
into a 1.5 ml microfuge tube and 200 μl of
chloroform were added. Thereafter, the samples were incubated at room
temperature (25°C) for 15 minutes
followed by centrifugation at 13,000 × g at 4°C
for other 15 minutes. The upper aqueous layer containing RNA was transferred
into a 1.5 ml tube. Ice-cold isopropanol (0.5 ml) was added to the aqueous
phase, the tube was shaken and left to stand on ice for 10 minutes before it
was centrifuged at 13,000 × g at 4°C
for 10 minutes. The supernatant was removed and the pellet was washed with 1 ml
75% ethanol. After centrifugation at 7,500 × g for 5 minutes, ethanol was
removed and the pellet was allowed to air-dry for 5 minutes. Pellets were
re-suspended in 50 μl of nuclease-free water and
incubated at 60°C for 15 minutes. RNA
was stored at -80°C.
2.4.
Complementary
DNA (cDNA) synthesis
Total RNA was quantified using the
Experion Electrophoresis System (Bio-Rad, Italy). cDNA
was synthesized from 1 µg of total RNA using a
quantiscript reverse transcriptase test (QuantiTect Reverse Transcription kit;
Qiagen, Italy) as follows: 1 µg of total RNA was
subjected to DNAse treatment using 2 μl of gDNA Wipeout Buffer in a total reaction volume of 14 μl. Samples were incubated at 42°C for 2 minutes and chilled on ice for 10
minutes. Then 1 μl of Quantiscript Reverse
Transcriptase, 4 μl of Quantiscript RT Buffer 5X
and 1 μl of RT Primer mix were added. The
samples were subsequently incubated for 15 minutes at 42°C following 3 minutes at 95°C to inactivate Quantiscript Reverse
Transcriptase.
2.5.
Quantitative
PCR expression by real time PCR
To determine the relative amounts
of specific NET1 gene transcript, 1 μl of cDNA
was used for quantitative PCR using the IQ SYBR Green Super mix (Bio-Rad,
Italy) and the IQ5 detection system (Bio-Rad, Italy). The sequences of primers
used for PCR were: canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
GenBank entry: AB038240.1) forward 5′-GGCACAGTCAAGGCTGAGAAC-3′, canine GAPDH reverse 5′-CCAGCATCACCCCATTTGAT-3′, canine NET1 (GenBank entry: XM_54427.5) forward 5’-CATCAAGAGGACGATCCGGG-3’,
canine NET1 reverse 5’-ATTGCTTGGCTCCTCTTGCT-3’. The reaction conditions were as follows: reverse
transcription: 95°C for 3 minutes (1
cycle) followed by denaturation at 95°C
for 30 seconds and annealing at 60°C
for 30 seconds (35 cycles). Data analysis using the delta cycle threshold (ΔΔCt) method was performed using an optical system
software (IQ5, Bio-Rad, Italy). The GAPDH expression levels were used to
normalize NET1 expression. The level of gene expression was calculated using a
relative quantification assay corresponding to the comparative Ct method: the
amount of target, normalized to the endogenous housekeeping gene (GAPDH) and relative to the calibrator
(control sample), was then transformed by 2ΔΔCt
(fold increase), where ΔΔCt = ΔCt (sample)-ΔCt
(control) and ΔCt is the Ct of the target gene
subtracted from the Ct of the housekeeping gene. To perform statistical
analysis, ΔCts were compared using a 3-way ANOVA
test (cell, treatment, time) or a student t-test as relevant. Significance was
set at p < 0.05.
2.6.
Results
In the present study expression of
NET1 gene was detected in both primary and metastatic cell lines cultured
without Propofol in the sole culture medium with a statistically significant
higher expression in CIPp compared to CIPm (median ΔCt
5.82 and 6.48 respectively; p = 0.000375). Mean ΔCts
values over time for CIPp and CIPm are presented in (Table
1 and Table 2), respectively.
After 6 hours of exposure, CIPp
cells treated with both concentrations of Vetofol® showed significantly higher
NET1 gene expression than controls (Figure 1).
In CIPm, a statistically
significant difference was found between controls and the higher concentration
of Vetofol®, the latter showing lower
gene expression (Figure 2).
After 12 hours of exposure, NET1
mRNA levels were significantly decreased with both concentrations of Vetofol® in CIPp and the lower concentration of Vetofol® in CIPm compared to controls.
After 24 hours of exposure, no
differences were found in CIPm, while in CIPp both concentrations of Vetofol®
induced a significant reduction in NET1 gene expression compared to controls.
After 48 hours of exposure, no
differences were found between controls and treatments in CIPm. Conversely, in
CIPp the higher concentration of Vetofol®
induced a significant increase in gene expression.
3.
Discussion
This is the first study focused on
the biological effect of a clinically available propofol formulation on NET1
gene expression in canine mammary cell lines. Both, P1 and P10 induced a
decreased NET1 gene expression in CIPp after 12 and 24 hours of exposure while
only the higher concentration (i.e. P10) caused a reduction of NET1 gene
expression in CIPm after 6 and 12 hours of exposure. Paradoxically, an
increased NET1 gene expression was observed in CIPp with both Vetofol® concentrations and with the higher Vetofol® concentration after 6 and 48 hours of
exposure, respectively. To the authors’ knowledge, this is the first time that
such a divergent effect has been described for a clinically available propofol
formulation in a primary canine tumor cell line. Unfortunately, the reasons why
these divergent effects were observed cannot be explained with the current
study.
As previously described, the NET1
gene is critical for Transforming Growth Factor (TGF) 1-induced cytoskeletal
reorganization, N-cadherin expression, and RhoA activation [10]. As a RhoA specific GEF it plays an important
role in the Epithelial-Mesenchymal Transition (EMT), enabling tumor cells to
invade and migrate [11]. Silencing of
this gene has been associated with abrogation of the inhibitory effect of
propofol on cancer cells migrating ability [7].
Therefore, a reduction in NET1 gene expression may be interpreted as a
potentially anti-metastatic effect. On the other hand, an increase of NET1 gene
expression could be interpreted as an increase in the ability of the tumor
cells to cause metastasis [12]. Consistently,
the co-expression of NET1 gene and α6β4 integrin in the primary tumors of node-positive
patients with invasive breast carcinoma was associated with decreased distant
metastasis-free survival [6].
Cell lines originating from the
same individual, but with different malignant potentials, were chosen to
compare the effects of propofol on primary and metastatic cells. NET1 mRNA was
detectable in both CIPp and CIPm; however, NET1 gene showed a higher expression
in CIPp compared to CIPm. These results may suggest that CIPp have a higher
invasiveness potential than CIPm. Indeed, it has been speculated that intrinsic
mechanisms promoting cell invasion and migration would be enhanced in CIPp
since this cell line is responsible for the dissemination of the tumor to
remote locations within the body [9]. On the
other hand, mechanisms responsible for cell migration may be attenuated in CIPm
in order for these cells to be able to adhere to each other and grow,
establishing metastatic lesions in distant organs.
The
doses of Propofol reported in the present study were chosen because they
reflect the clinically achieved plasma concentrations obtained during Propofol
anesthesia and sedation in dogs [13]. Interestingly,
a generic chemical form of Propofol at concentrations like the ones reported
here (i.e. between 1 and 10 µg ml-1)
reduced NET1 gene expression by 49-79%
in MDA-MB-231 cells, an estrogen-receptor-negative human breast
adenocarcinoma cell line and by 42-88% in MCF7 cells, an estrogen and
progesterone-receptor-positive human breast adenocarcinoma cell line [7].
Different to the present study in which the NET1 expression was reduced on CIPm
only with the high concentration of Vetofol®, the authors reported a lack of dose-effect
in MDA-MB-231 cells and MCF7 cells.
However, this is comparable to the results obtained from CIPp in which both
doses of Vetofol® influenced
NET1 gene expression and a dose-effect could not be demonstrated.
In the
present study different results were found at different time-points; however,
the observed time-effects did not follow a linear pattern. Interestingly,
with the shortest exposure time (i.e. 6 hours), an increase of NET1 mRNA levels
in CIPp was observed. This finding was surprising and seems to speak for an
enhancement of malignancy in primary tumor cells when exposed to Vetofol® for that period of time. However, this effect
was reversed after 12 and 24 hours of exposure, where Vetofol® induced a decrease in gene expression in CIPp.
Thereafter, a significant increase in NET1 gene expression was observed once
again in that cell line. This observation could suggest that there is a time-dependent
response in the expression of NET1 gene of CIPp when exposed to Vetofol®. Some sort of time dependency was already
noticed after observing that NET1 expression tended to return to the baseline
in MDA-MB-231 cells and MCF7 cells after 4 hours of incubation with
lysophosphatidic acid [7]. On the
other hand, the only significant changes observed in CIPm were the reduction in
NET1 gene expression. Unfortunately, the present study does not
provide enough elements for a comprehensive explanation of the phenomenon.
It is unclear how Propofol
modulates the expression of NET1 gene. In a previous study it was suggested
that Propofol modulates NET1 by changing the cellular microenvironment, rather
than by a specific receptor pathway [7]. Other
studies underline the relationship between TGF-β,
microRNAs and NET1 expression as combined targets for Propofol anti-metastatic
activity [14-17], with, for instance, TGF-β increasing NET1 and mediating stress fiber formation
in human keratinocytes [16].
The potential anticancer properties
of Propofol have been previously reported in several studies. For instance,
Propofol induces cancer cell apoptosis in human promyelocytic leukemia HL-60 [18] and hepatic cancer cells HepG2 [19]. In pancreatic cancer cells (i.e. MIA-PaCa-2)
Propofol promoted apoptosis in a dose-dependent manner (20). Propofol inhibited
invasion, angiogenesis and induced apoptosis of human esophageal squamous Cell
Carcinoma (i.e. EC-1) cells in vitro through regulation of S100A4 expression [21]. In addition, propofol suppressed the
epithelial-mesenchymal transition and consequently kidney fibrosis through TGF-β/Smad 3 signaling and regulating miR-155 levels [18] and decreased cancer cell invasion via nuclear NF-kb pathway inhibition and
subsequent reduction of matrix
metalloproteinase 2 and matrix
metallopeptidase 9 levels in human
MDA-MB-231 cells [19]. Mammoto and colleagues showed that sub-anesthetic
propofol infusions for 4 weeks effectively inhibited pulmonary metastasis in
mice inoculated with murine osteosarcoma cells and suggested its possible
anti-invasive action in vivo [22].
Paradoxically, propofol has also
been associated with mechanisms that may promote cancer. Propofol induced
proliferation and promoted invasion of gallbladder cancer cells through
activation of Nrf2, in a dose- and time- dependent manner [23]. In another study, human breast cancer cells
(i.e. MDA-MB-468) migrated in a higher proportion and at a faster velocity than
controls in a dose-dependent manner when exposed to propofols [24].
There were some limitations to the
current study. The gene expression of NET1 was investigated in canine mammary
tumor cells without performing biological tests. Although it can be stated that
Vetofol® effectively influenced the
expression of a gene closely related with increased cancer cells malignancy,
the conclusions cannot be extended to the effects on cellular behavior. In
particular, it would be interesting to verify in the future whether the expression
of NET1 gene is correlated with increased migration potential in canine mammary
tumor cells and if the decreased NET1 gene expression caused by propofol
effectively decreases (or increases) cell migration. In addition, it would be
expected that silencing NET1 gene in the presence of propofol would return the
migration parameters to those obtained at baseline. Finally, the use of a
clinically available propofol formulation cannot ensure that the changes
observed in the present study are solely related to the active compound,
propofol. In addition to propofol, the emulsion contains soybean oil (100
mg/mL), glycerol (22.5 mg/mL), egg lecithin (12 mg/mL) and disodium edetate
(0.005%), with sodium hydroxide to adjust pH. The role of the adjuvants in the expression
of NET1 gene would need further clarification.
4.
Conclusions
The reported propofol formulation
effectively modified canine mammary cancer cells NET1 gene expression. Both the
concentrations examined in the present study induced a decrease in gene
expression in the most treatment time-points, although increases in gene
expression were also observed. Further studies, including biological tests and
gene silencing are warranted to better understand this phenomenon.
5.
Acknowledgements
The authors thank Claus Vogel,
Institute of Animal Breeding and Genetics, Veterinary University Vienna,
Vienna, Austria, for the support with data analysis and Kohei Saeki, Laboratory
of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Japan, for providing the cell lines.
research article 
