In Vitro Effects of Selective COX and LOX Inhibitors and Their Combinations with Antineoplastic Drugs in the Mouse Melanoma Cell Line B16F10.

Ines Da-Costa-Rocha,Jose M Prieto

doi:10.3390/ijms22126498

Abstract

The constitutive expression or overactivation of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes results in aberrant metabolism of arachidonic acid and poor prognosis in melanoma. Our aim is to compare the in vitro effects of selective COX-1 (acetylsalicylic acid), COX-2 (meloxicam), 5-LOX (MK-886 and AA-861), 12-LOX (baicalein) and 15-LOX (PD-146176) inhibition in terms of proliferation (SRB assay), mitochondrial viability (MTT assay), caspase 3-7 activity (chemiluminescent assay), 2D antimigratory (scratch assay) and synthesis of eicosanoids (EIA) in the B16F10 cell line (single treatments). We also explore their combinatorial pharmacological space with dacarbazine and temozolomide (median effect method). Overall, our results with single treatments show a superior cytotoxic efficacy of selective LOX inhibitors over selective COX inhibitors against B16F10 cells. PD-146176 caused the strongest antiproliferation effect which was accompanied by cell cycle arrest in G1 phase and an >50-fold increase in caspases 3/7 activity. When the selected inhibitors are combined with the antineoplastic drugs, only meloxicam provides clear synergy, with LOX inhibitors mostly antagonizing. These apparent contradictions between single and combination treatments, together with some paradoxical effects observed in the biosynthesis of eicosanoids after FLAP inhibition in short term incubations, warrant further mechanistical in vitro and in vivo scrutiny.

Highlights

Despite the latest advances in adjuvant combination therapeutic strategies most patients with metastatic melanoma still have poor prognosis [1,2]
Our aim is to compare the effects of a panel of selective COX-1, COX-2, 5-LOX (MK-886 and arachidonic acid (AA)-861), 12-LOX and 15-LOX (PD146176) inhibitors and their in vitro synergies with two classic antineoplastic drugs used in melanoma treatment: dacarbazine and temozolomide (Figure 1) in the same cell line and conditions
Tamoxifen was included in our study as a positive control as it is considered a reference apoptosis-inducing agent in human melanoma cell line (A375) (10 μM, 24 h) [29], but it did not have any major effect on the activation of caspase 3/7 in B16F10 under the same conditions (Figure 3B)

Summary

Introduction

Despite the latest advances in adjuvant combination therapeutic strategies most patients with metastatic melanoma still have poor prognosis [1,2]. It is important to continue to study newer strategies to increase melanoma patients’ survival. The idea of using COX and LOX inhibitors together with other chemotherapy agents is a promising research field in oncology. Synergetic effects of COX inhibitors with several anti-cancer agents have attracted more attention perhaps due to the clinical availability of a variety of NSAIDs vs the experimental nature of the selective LOX inhibitors. Clinical data on combinatorial therapies involving NSAID and other medication such as anti-PD-1 therapy or sorafenib [11,12] are not compelling, despite preclinical studies suggesting a potentially synergistic relationship

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