Anoikis as a novel mode of shikonin derivatives anticancer action on C6 glioma cells

Abstract

Background: Shikonins are naturally occurring naphthoquinones found in the roots of several genera of the Boraginaceae family, widely known for their antiinflammatory, antioxidant, antimicrobial, and anticancer properties. This study aimed to investigate the antitumor potential of six shikonins isolated from the roots of Onosma visianii against highly aggressive rat glioma cell line C6 and to explore the mechanisms involved. Material and Methods: Cell viability was estimated by MTT and CV assays. Cell death, proliferation rate, and caspase activity were assessed using flow cytometric analysis of annexin V-FITC/propidium iodide, CFSE, and ApoStat staining, respectively. Fluorescent microscopy of propidium iodide-stained cells was employed for the detection of nuclear morphology. To evaluate the viability of detached cells, an acidic phosphatase assay was used. The cells’ property to adhere was assessed by cell adhesion assay while western blot was engaged to measure the expression of relevant proteins responsible for the observed phenomenon. Results and Conclusions: All examined shikonins dose-dependently decreased the viability of C6 cells, with compounds 5 and 6 being the most potent ones. Compound 5 had a more profound effect on the proliferation rate of C6 cells than compound 6, resulting in almost 70% of inhibition of cell division. Additionally, compound 5, but not compound 6 generated a significant number of early and late apoptotic cells in treated cultures as detected by flow cytometry. In collision with this, typical morphological signs of apoptotic cells were not observed, and fluorescent microscopy revealed only the presence of enlarged nuclei. This paradox was resolved by the discovery of massive detached cell presence, indicating that glioma cells underwent anoikis, a cell attachment-dependent programmed cell death, in response to treatment with both agents. Decreased ability of C6 cells to adhere to the extracellular matrix and compromised integrin signaling was further confirmed by adhesion assay and western blot, respectively. Interestingly, while compound 5 triggered caspase-mediated anoikis, compound 6 realized anoikis in a caspase-independent manner and under sustained ERK1/2 activation, indicating the deviation from standard proanoikis signaling. This study represents the first proof of shikonin derivatives’ strong anticancer potential realized through the induction of anoikis of highly proliferative and invasive malignant glioma cells.