Page 34 - ARNM-2-1

Advances in Radiotherapy
& Nuclear Medicine 99m Tc-DOX in multidrug resistance studies

(FCS; Cultilab, Brazil) and antibiotics at 37°C in 5% CO (Invitrogen, Brazil USA), 2 mM l-glutamine, 100 U/mL
2
atmosphere. Cells were passaged twice a week. penicillin, and 100 µg/mL, 1% sodium pyruvate, and 1%
nonessential amino acids (Sigma-Aldrich, US) for 48 h.
2.5. Detection of Pgp expression After reaching confluency, the cells were collected with

For Pgp detection, cells were washed using phosphate- trypsin-EDTA (Invitrogen) and centrifuged at 1200 rpm
buffered saline (PBS) containing 5% FCS and incubated for 10 min. Cells (10 cells/mL) were suspended in
6
at 4°C for 30 min with mouse anti-human P-glycoprotein DMEM without serum. This cell suspension (100 µL,
– FITC antibody (BD Pharmigen, USA) at a dilution of 10 cells per animal) was injected subcutaneously in
5
1:4 (v/v). Negative control cells were incubated at 4°C for C57/Bl6 mice’s left paws, and tumor growth was
30 min, without the antibody, in PBS containing 5% FCS. monitored for 15 days.
Following this, cells were washed and resuspended in PBS
containing 5% FCS. At least 10,000 events were recorded 2.9. Statistical analysis
within the gated region of the flow cytometer FACSCan Student’s t-test was employed to analyze the significance
(Beckton & Dickinson, USA). of the difference in Pgp expression and cell viability.
Differences were considered statistically significant when
2.6. Assessment of Pgp activity
P < 0.05.
For the assessment of Pgp activity, cells were washed using
a medium containing 10% FCS and incubated at 37°C for 3. Results
30 min with Rhodamine 123 (Rho; Sigma-Aldrich, USA), a 3.1. Radiolabeling and stability
Pgp’s fluorescent substrate, at a concentration of 200 ng/mL,
in the presence or absence of 5 µM verapamil (Sigma- The labeling efficiency was maintained at around 90%
Aldrich, USA), a Pgp inhibitor. After incubation, cells were (Table 1). Moreover, the DOX was found to be stably
washed and further incubated at 37°C for 30 min with a labeled by 99m TC, especially in the first 5 h following
medium containing 10% FCS in the presence or absence labeling (Table 2).
of verapamil to allow cells to extrude or not the fluorescent 3.2. Determination of Pgp expression and activity in
substrate. After incubation, the cells were washed once K562 and Lucena 1 cell lines
more and resuspended in PBS containing 5% FCS. At least
10,000 events were recorded within the gated region of the Pgp expression in both K562 and Lucena 1 lines is
flow cytometer FACSCan (Beckton & Dickinson, USA). graphically depicted in Figure 1A. Lucena 1 cell line
exhibited overexpression of Pgp that is not observed in
2.7. Determination of cell viability K562. The transporter activity of the protein (Figure 1B)
K562 or Lucena I cells were seeded onto a 96-well plate was evaluated using a fluorescent substrate, Rho. When
4
at 2 × 10 cells/mL in RPMI 1640 (Sigma-Aldrich, USA) incubated with Rho, the K562 cells demonstrated a
medium containing 10% FCS. Cells were kept for 3 days at significant increase in intracellular fluorescence, surpassing
37°C in a 5% CO atmosphere in the presence of 300 ng/mL the fluorescence intensity showcased by Lucena 1. However,
2
of the chemotherapic DOX or its radiolabeled form 99m Tc- in the presence of verapamil, a Pgp inhibitor, Lucena 1 cells
DOX and/or the Pgp inhibitor verapamil (5 µM). Controls showed a significant increase in intracellular fluorescence,
were cells maintained in the absence of these substances. a magnitude similar to that seen in K562 cells, suggesting
Each sample had three replicates. After a 72-h culture, that Pgp activity accounts for the Rho extrusion observed
cells were incubated with 20 µL of 3-(4,5-dimethylthiazol- in Lucena 1 cells.
2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-
Aldrich, USA), at a concentration of 5 mg/mL, for 3 h at Table 2. Labeling efficiency using Whatman grade 4 paper in
37°C in a 5% CO atmosphere. Following MTT incubation, different periods
2
a 200 µL aliquot of 100% DMSO (Vetec Química Fina, Time (h) Origin (%) Top (%)
Brazil) was added to each culture to dissolve the crystals.
The absorbance was measured at 490 nm using Sunrise 0 90.9 9.1
TM
microplate absorbance reader (Tecan, Switzerland). 1 86.3 13.7
3 83.6 16.1
2.8. In vivo development of Lewis lung carcinoma 5 82.4 17.6
from 3LL cells
24 74.3 25.7
3LL cells were grown in high-glucose DMEM culture Note: Origin (%) and top (%) referring to the Whatman paper number
medium (Vitrocell, Brazil) supplemented with 10% FBS 4 strips.

Volume 2 Issue 1 (2024) 3 https://doi.org/10.36922/arnm.2822