In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474

Abstract

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton-Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O₂^•-) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

Keywords: Herceptin®; Kadcyla® (T-DM-1); Trastuzumab (Tz); antibody drug conjugate (ADC); epidermal growth factor receptor (EGFR); human epidermal growth factor receptor 2 (Her/2); monoclonal antibody (mab); reduced glutathione (GSH); selenium (Se); superoxide (O2•−).

Figure 1

Original morphology of Se-Trastuzumab treated JIMT-1, Trastuzumab resistant and BT-474, Trastuzumab sensitive cell lines. Doubling cell division times for JIMT-1 and BT-474 cells were approximately 30 and 100 h, respectively.

Figure 2

(a) Se-BHR: selenium conjugating Bolton–Reagent; (b) chemical structure of Se-BHR; (c) control and selenium labeled Tz following exhaustive dialysis, pH 7.4.

Figure 3

(a) O₂^•− CL generated by treatments over time. Figure (b) is the sum total of O₂^•− CL generated by each of the treatments over 5 min. For the measurement of superoxide generation, 330 μL of Se-Tz was added to 500 μL of CL cocktail and similar steps were followed for the native Tz (330 μL) and Se-BHR (50 μL) in PBS. Mean CL generation by Se-BHR (20 mg/mL), Tz (protein concentration 3 mg/mL) and Se-Tz (protein concentration 3 mg/mL) was 2.53 ± 1.68, 0.05 ± 0.036 and 21.5 ± 5.17 CLU (** p < 0.001), respectively.

Figure 4

20X Microphotographs of Trypan Blue Stained Cells after Treatments. (a) JIMT-1 cells photographed at the concentrations and times indicated; (b) BT-474 cells photographed at the concentrations and times indicated. Selenite was used as a redox toxic positive control at a concentration of 10 µgSe/well. Selenite at concentrations less than 10 µgSe/well had no visible effect on cancer cell lines under these experimental conditions.

Figure 5

Superoxide Generation by Dihydroethidium (DHE) detection after selenium treatments. (a,b) Photographs of superoxide staining of JIMT-1 & BT-474 cells after Se treatments at magnifications of 4X= 1000 µm; 20X= 100 µm; (c,d) Quantitative measurement of superoxide generation of JIMT-1 & BT-474 cells after Se treatments. After acclimatization for 24 h, 50 units/well of SOD and 100 units/well of catalase were added to all wells in media. All cells, control, selenite, Tz and Se-Tz were appropriately treated and then DHE was added to the cells at a final concentration of 10 μM in media. Selenite was used as a redox toxic positive control at a concentration of 10 µgSe/well. Selenite at concentrations less than 10 µgSe/well had no visible effect on cancer cell lines under these experimental conditions. The fluorescence was measured using a microtiter plate reader with an excitation at 520 nm and emission at 610 nm.

Figure 6

Photographic hydrogen peroxide detection by DCFH-DA after selenium treatments at magnifications of 1000–1010 µm. (a) Hydrogen peroxide staining of JIMT-1 cells after Se treatments; (b) hydrogen peroxide staining of BT-474 cells after Se treatments. After acclimatization for 24 h, 50 units/well of SOD and 100 units/well of catalase were added to all wells in media. All cells, control, selenite, Tz and Se-Tz were appropriately treated and then DCFH-DA was added to the cells at a final concentration of 30 μM in media. Fluorescence intensity was read using a fluorescence microplate reader with an excitation at 475 nm and emission at 525 nm. Selenite was used as a redox toxic positive control at a concentration of 10 µgSe/well. Selenite at concentrations less than 10 µgSe/well had no visible effect on cancer cell lines under these experimental conditions.

Figure 7

Caspase-3 activity from cell lines after selenium treatments. (a) JIMT-1 cells after 24 h of Trastuzumab and selenium treatments; (b) BT-474 cells after 72 h of Trastuzumab and selenium treatments. Cells were treated with Se-Tz at the concentration of 4.8 µgSe/well and Tz cells were treated with an equal mab protein concentration. Selenite was used as a redox toxic positive control at a concentration of 10 µgSe/well. Selenite at concentrations less than 10 µgSe/well had no visible effect on cancer cell lines under these experimental conditions.

Figure 8

Cells were treated with Se-Tz at the concentrations of 2.4 and 4.8 µgSe/well and Tz cells were treated with an equal mab protein concentration. Selenite was used as a redox toxic positive control at a concentration of 10 µgSe/well with significant decrease in % cell viability. MTT cell viability assay demonstrated significant decrease in % cell viability in Se-Tz treated JIMT-1 cells (a) and BT-474 cells (b) in a time and dose dependent manner compared to control cells. Se-Tz at 2.4 µgSe/well concentration up-regulated % cell viability of BT-474 cells (b). Figure 8 (c,d) represent the time dependent decrease in % cell viability of JIMT-1 and BT-474 cells, respectively, of Se-Tz treated cells at the concentration of 4.8 µgSe/well and selenite at 10 µgSe/well compared to control cells. Tz cells were treated with an equal mab protein concentration. Selenite at concentrations less than 10 µgSe/well had no visible effect on both the cancer cell lines under these experimental conditions.

Figure 9

(a) IC₅₀ calculations for Se-Trastuzumab treated JIMT-1 cells from Figure 8d and (b) IC₅₀ calculations for Se-Trastuzumab treated BT-474 cells from Figure 8e. (c) The IC₅₀ is calculated here for Se-Tz after treating the two cancer cell lines from low to high concentrations using the Graph Pad Prism software from 9a and 9b.

Figure 10

Morphology of control and selenium treated JIMT-1 and BT-474 cells by scanning electron microscopy. Representative control and Se treated cancer cells at 72 h were fixed in 10% buffered formalin on glass cover slips and allowed to air-dry for 4–5 h. SEM images of both the cancer cell lines were taken using a Hitachi Model S-4700 FE-SEM at magnifications of 5–20 µm after 96 h of selenium treatment.