Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib

Abstract

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib-shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.DEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, b,b-dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.DEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DK-MG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCc1, but also together with erlotinib synergistically inhibited DEGFR phosphorylation in U87MG.DEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.Glioblastoma (GBM) is the most common primary brain tumor in adults and one of the deadliest human cancers with a median survival rate of 12 months despite aggressive therapies (i.e., urgical resection, radiotherapy and chemotherapy) due to their highly invasive and neurologically destructive nature.

Natural Compounds As Modulators of Non-apoptotic Cell Death in Cancer Cells

“…deoxyshikonin, isobutyrylshikonin, acetylshikonin, β, β-dimethylacrylshikonin and acetylalkannin) showed synergistic cytotoxicity toward human glioblastoma U87MG ΔEGFR cells (expressing kinase defective mutant of epidermal growth factor receptor, EGFR), as well as in EGFR-expressing cell lines, glioblastoma BS153 cells (wild type EGFR amplified), epidermoid skin cancer A431 cells (wild type EGFR) and glioblastoma DK-MG cells (mutated EGFR), as described elsewhere [285]. Shikonin inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42 MAPK and phospholipase C (PLC)-γ1, but also synergistically (along with erlotinib) inhibited ΔEGFR phosphorylation in U87MG ΔEGFR cells [285]. Shikonin exerted potent cytotoxic effects on human multiple myeloma bortezomib-resistant KMS11/BTZ cells, as described in [286].…”

“…Shikonin could circumvent drug resistance, displayed by tumor cells and mediated by P-glycoprotein, BCL-2 and BCL-xL, by induction of necroptosis [289-293]. Naturally-occurring shikonin analogues (deoxyshikonin, acetylshikonin, isobutyrylshikonin, beta, beta-dimethylacrylshikonin, isovalerylshikonin, alpha-methyl-n-butylshikonin) were found to induce necroptosis in resistant tumor cells overexpressing MDR1 and BCRP1, as indicated in [285-292]. Shikonin induced a cell death in human breast cancer MCF-7 cells distinct from apoptosis, and that the cell death was prevented by NEC-1, as described in [292-295].…”

Shikonin induces mitochondria-mediated apoptosis and enhances chemotherapeutic sensitivity of gastric cancer through reactive oxygen species

“…SHK also has a cytotoxic effect on MDR cell lines and enhances chemotherapeutic sensitivity. It enhances cisplatin-induced colon cancer cell apoptosis, synergistically kills glioblastoma cells in combination with erlotinib and induces cell cycle arrest in triple negative breast cells14151617. Furthermore, SHK has anti-tumour effects by inducing receptor-interacting protein 1 (RIPK1)-dependent necroptosis1819.…”

Anti-tumor effects of shikonin derivatives on human medullary thyroid carcinoma cells

“…The anti-cancer effects have been shown to be linked to fundamental cellular processes such as cell cycle progression, apoptosis (19, 20) and mitochondrial function (27, 28). Shikonins further affect main anti-apoptotic and pro-proliferative pathways such as MAPK and AKT signaling cascades (29, 30), but the effects on MTC cells with respect to migration and invasion as well as in vivo tumor growth have not yet been investigated. IC 50 values on TT cells were found to be in the micromolar range with shikonin exhibiting the lowest IC 50 of 1.1 µM.…”

“…In glioblastoma cells U87MG, various shikonin derivatives have been reported to have cytotoxic properties with IC 50 values ranging from 3.61 µM to 51.97 µM (30). In combination with the tyrosine kinase inhibitor erlotinib, however, synergistic effects of e.g.…”