Biochemical Pharmacology br Calcein AM
Biochemical Pharmacology 159 (2019) 52–63
2.9. Calcein AM exclusion assay
For calcein AM exclusion assays, MDA-MB-231 shRNA-ACSL4, MDA-MB-231 mock (in the presence or absence of 1 µM triacsin C), MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty vector Tirapazamine (2 × 106) were incubated in DMEM with calcein AM (0.5 μM) and protected from light for 30 min at 37 °C. Cells were then washed and incubated in calcein AM-free medium for 30 min to assess eﬄux. Cells were har-vested after trypsin treatment, washed twice and resuspended in PBS containing 2% v/v fetal bovine serum. The amount of intracellular calcein AM was immediately measured by FACSCalibur II flow cyto-metry (Becton Dickinson Biosciences) using excitation with argon laser at 488 nm and detected on an FL2 channel (excitation 488 nm, emission 530 nm).
2.10. Statistical analysis
Data were analyzed using GraphPad InStat Software 3.10 (La Jolla, CA, USA). Statistical significance was determined through analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison tests.
3.1. ACSL4 modulation of cell survival upon treatment with chemotherapeutic drugs
We used a pharmacological approach to elucidate ACSL4 impact on cell resistance to treatment with chemotherapeutic agents. We ex-amined the inhibitory eﬀect on cell survival of three diﬀerent che-motherapeutic drugs – i.e. cisplatin, doxorubicin and paclitaxel – in MCF-7 Tet-Oﬀ/ACSL4 and MCF-7 Tet-Oﬀ empty vector cells. Cisplatin treatment revealed significantly higher inhibitory eﬀects on MCF-7 Tet-Oﬀ empty vector than MCF-7 Tet-Oﬀ/ACSL4 cells between 1 µM and 5 µM. In turn, doxorubicin treatment produced significant diﬀerences in inhibition between 0.025 µM and 0.1 µM, while paclitaxel rendered significant diﬀerences between 0.1 µM and 0.5 µM (Fig. 1A, B and C, respectively). Most importantly, the downregulation of ACSL4 expres-sion through doxycycline treatment produced a recovery in cell survival rates for all three drugs, with MCF-7 Tet-Oﬀ/ACSL4 cell resistance re-sembling control values.
Further supporting evidence of ACSL4 participation in cell re-sistance to chemotherapeutic treatment was obtained using the highly aggressive triple negative MDA-MB-231 breast cancer cells, which constitutively overexpress ACSL4, and disrupting ACSL4 endogenous expression using short hairpin RNA (shRNA), which markedly decreases ACSL4 protein expression, as previously described . Once again, cisplatin showed significantly higher inhibitory eﬀects on MDA-MB-231 shRNA-ACSL4 cell survival than on MDA-MB-231 mock cells between 1 µM and 5 µM, doxorubicin produced significantly diﬀerent eﬀects between 0.025 µM and 0.05 µM, and paclitaxel rendered significant diﬀerences between 0.1 µM and 0.2 µM (Fig. 1D, E and F, respectively).
In order to validate a potential clinical significance of ACSL4 in-volvement in cell resistance to drugs, we used a combined pharmaco-logical approach in order to establish the eﬃcacy of ACSL4 activity inhibition in combination with chemotherapeutic agents. To this end, we analyzed MDA-MB-231 wild type cell proliferation using sub-max-imal concentrations of cisplatin, doxorubicin and paclitaxel in combi-nation with triacsin C, a well-known inhibitor of ACSL4 activity [20,33,34]. Results show that neither single chemotherapeutic drug treatment nor single triacsin C treatment aﬀected cell proliferation at any of the concentrations used. However, these inhibitor concentrations combined with cisplatin, doxorubicin or paclitaxel (Fig. 2A, B and C, respectively) rendered a significant and concentration-dependent re-duction in MDA-MB-231 cell survival, showing remarkable synergistic eﬀects.
3.2. ACSL4 involvement in compound eﬄux
Given that compound eﬄux is one of the mechanism associated to tumoral cell resistance and that doxorubicin is widely used to measure the capacity of cell eﬄux systems [35–37], flow cytometry analyses were carried out in doxorubicin-treated cells. Interestingly, MCF-7 Tet-
Oﬀ empty vector, MDA-MB-231 shRNA-ACSL4 and MDA-MB-231 mock cells treated with triacsin C showed a right shift in the peak of fluor-escence intensity as compared to MCF-7 Tet-Oﬀ/ACSL4 and untreated MDA-MB-231 mock cells, which indicates an increase in doxorubicin accumulation when ACSL4 expression decreases (Fig. 3A and B). In-deed, MDA-MB-231 shRNA-ACSL4 and MDA-MB-231 mock cells treated
with triacsin C rendered comparable right shifts (Fig. 3B).
To corroborate these results and further establish transporter par-ticipation, assays were conducted on Hoechst eﬄux from cells. Results showed a significantly lower Hoechst 33342 integrated density in MCF-