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

Multidrug resistance (MDR) constitutes the major Based on these aforementioned studies and drawing
obstacle to successful cancer treatment. Multidrug- from our experience in labeling different types of cells
resistant cell lines have been used as models to study MDR and molecules, such as leukocytes, stem cells, thymine,
in vitro and in vivo. We selected for this study a cell line anti-CD3, and anti-TNF-α, for the diagnostic purposes of
derived from K562, a chronic myeloid leukemia cell. The cancers and other diseases, 23-39 we successfully labeled DOX
4
resistant cell line, Lucena 1, has been characterized and with 99m Tc. Previously, we conducted a pilot study in this
40
features the overexpression of P-glycoprotein (Pgp), an regard involving women with breast cancer. The current
ATP-dependent transporter capable of extruding substrates study aims to assess whether 99m Tc-DOX can be taken up
from the cell, including a range of chemotherapeutic drugs, by susceptible and multidrug-resistant cell lines. We used
therefore being less affected by the cytotoxic effects of the human chronic myeloid leukemia K562 cells and the resistant
drugs. 5-13 Encoded by the MDR1 gene, Pgp is a 170 kDa counterpart Lucena1 cells to evaluate Pgp detection and
protein found in the cytoplasm, serving as a storage pool activity for this purpose. For in vivo evaluation, we applied
to maintain a steady-state level of membrane Pgp. Of note, 99m Tc-DOX in mice with Lewis lung carcinoma (3LL lineage).
the transport activity of these cells can be inhibited by the 2. Materials and methods
chemosensitizers, such as verapamil, trifluoperazine, and
cyclosporine. 14-18 2.1. Radiolabeling of DOX

DOX is actively extruded from cancer cells DOX (Eurofarma, Brazil) was labeled based on the direct
−
overexpressing Pgp. Thus, high cumulative doses of DOX reduction of 1 mCi (37 mBq) of 99m TcO freshly eluted
4
99m
99
are required in cancer chemotherapy to achieve a sufficient from a Mo/ Tc generator (IPEN/CNEN) with stannous
therapeutic effect. However, higher doses lead to dose- chloride (SnCl ; Sigma-Aldrich, USA). This labeling
2
dependent side effects, such as cumulative cardiotoxicity, process is under patent registration. In the preparation
nephrotoxicity, and extravasation, which compromise of the radiopharmaceutical, all reagents were used under
its clinical applications. Moreover, intracellular DOX sterile conditions.
accumulation is a complex process, which includes cellular 2.2. Chromatography
uptake, retention, relocalization, and efflux from the cell.
At any given time, the drug accumulation in cells is the Chromatography was performed using methanol,
difference between the quantity of drug uptake and efflux. isopropyl alcohol, and distilled water (3:2:5) as the mobile
Thus, it is required to design the next generation of DOX phase. Thin-layer chromatography (TLC) was used to
derivatives to overcome drug resistance, improve their characterize the labeled DOX using Whatman grade 4
99m
retention in the cell, and enhance the sustained therapeutic paper. The TLC was performed using 2 µL of Tc-DOX.
effect. 19 The radioactivity of the strips was counted in Gamma
Counter (Wizard 2, PerkinElmer), as presented in Table 1.
Many studies have been performed to assess the efficacy
of DOX therapies via novel research strategies. Despite 2.3. Stability evaluation
the potential benefits of the currently available liposomal The stability of Tc-DOX was evaluated in saline solution.
99m
DOX delivery systems, relevant problems still persist. For The percentage of free 99m TcO and the radioactivity
-
4
example, cardiotoxicity arising after administration is still of 99mTc-DOX were measured at different periods
evident, although at a reduced frequency. Doxil/Caelyx use (immediately, 1, 3, 5, and 24 h).
is commonly associated with the development of palmar-
plantar erythema (PPE), which is believed to reflect the 2.4. Cell line culture
gradual accumulation and nonspecific release of DOX The human chronic myeloid leukemia K562 cells and
in the extremities. PPE manifests as a painful swelling of the resistant counterpart Lucena1 cells (4) were cultured
the hands and feet that ultimately requires a reduction in in 25 cm cell culture flasks, at the final concentration of
3
the administered dose, therefore resulting in less effective 2 × 10 cells/mL, in 5 mL of RPMI-1640 medium (Sigma-
4
treatment. 20-22 Aldrich, USA) supplemented with 10% fetal calf serum
Table 1. Efficiency of DOX labeling compared with control carried out with sodium pertechnetate (Na 99m TcO4-)

Whatman paper number 4 Bottom (%) Top (%) Total (%)
99m Tc-DOX 90.9 63.0 100
Na TcO - 0.7 99.3 100
99m
4
Note: Bottom (%) and top (%) referring to the Whatman paper strips.
Volume 2 Issue 1 (2024) 2 https://doi.org/10.36922/arnm.2822