Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells

Abstract

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Keywords: Chenopodium quinoa wild; patient-derived glioblastoma cell lines; quinoin; ribosome-inactivating proteins; temozolomide.

Figure 1

IC₅₀ values estimation. IC₅₀ values of U87Mg cells and two primary glioblastoma cell lines NULU and ZAR after 24, 48, and 72 h of incubation with quinoin using concentrations of 0.01, 0.1, 1.0, 2.5, and 5.0 μM. The control was assumed as part of the dose–response curve, considering it as a very low concentration (10⁻¹¹ µM). Data were processed using GraphPad Prism and data are reported as Mean ± SD.

Figure 2

Growth curve and MTT assay of the U87Mg glioblastoma continuous cell line and primary cell lines. (A) On the left, the graphs of the growth curves of the continuous glioblastoma cell line U87Mg and of two primary cell lines obtained from the patient’s biopsy (NULU and ZAR) are shown. Quinoin was administered at various doses of 2.5 and 5.0 nM daily, at various time intervals (1, 2, 3 days). (B) On the right, the graphs of the cell viability assessed by MTT assay. U87Mg and patient-derived glioblastoma cell lines NULU and ZAR treated daily with quinoin 2.5 and 5.0 nM, at various time intervals (1, 2, 3 days). Data shown are representative of three separate experiments and values are presented as Mean ± SEM. Statistical analysis was performed by one-way ANOVA. According to GraphPad Prism, * p-value 0.01 to 0.05 (significant), ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (extremely significant), **** p-value < 0.0001 (extremely significant).

Figure 3

Morphological change of quinoin-treated U87Mg. The glioblastoma continuous cell line was exposed to 0.01, 0.1, 1.0, 2.5, and 5.0 μM quinoin for 72 h. Cells were imaged with an Evos FL microscope at 20× magnification.

Figure 4

Western blot analysis of quinoin-treated primary cell lines for 24 h. (A) Quinoin induced a significant time-independent reduction of Cyclin D1 and (B) activation of apoptosis by a decrease of procaspase 3 when administered at a concentration of 250 nM in the NULU cell line. (C) Western blot analysis of the expression of Cyclin D1, (D) procaspase, and autophagic markers p62 (E) and LC3B (F) after treatment of the ZAR cell line with different concentrations of quinoin for 24 h. Densitometric analysis of protein levels represent the means ± SEM of three individual determinations. Data were normalized to the housekeeping gene actin and are expressed as a fold change over control-treated cells. * Unpaired t-test. According to GraphPad Prism, * p-value 0.01 to 0.05 (significant), ** p-value 0.001 to 0.01 (very significant), *** p-value 0.0001 to 0.001 (extremely significant).

Figure 5

Quinoin and oxidative stress. Effect of primary cell line NULU’s pre-treatment with the ROS scavenger NAC (3.0 mM) and evaluation of the cell viability under different concentrations of quinoin (0.01, 0.1, 1, and 2.5 µM) at 24 h from treatment. Data analyzed with the unpaired t-test revealed no significance.

Figure 6

Combined treatment of quinoin and TMZ on primary glioblastoma cell lines. (A) IC₅₀ values of TMZ of two primary glioblastoma cell lines, NULU and ZAR, after 24 h of incubation with TMZ. Data were processed using GraphPad Prism and data are reported as Mean ± SD. (B) Combined treatment in the presence of quinoin 2.5 nM and TMZ 1.0 μM for 24 h on NULU and ZAR primary cell lines. Data shown are representative of three separate experiments and values are presented as Mean ± SEM. Statistical analysis was performed by one-way ANOVA. According to GraphPad Prism, *** p-value 0.0001 to 0.001 (extremely significant), **** p-value < 0.0001 (extremely significant) significance vs. control cells; # p-value 0.01 to 0.05 (significant) significance of TMZ 1 μM vs. quinoin 2.5 nM plus TMZ 1.0 μM; ## p-value 0.001 to 0.01 (very significant) significance of quinoin 2.5 nM vs. quinoin 2.5 nM plus TMZ 1 μM; ### p-value 0.0001 to 0.001 (extremely significant) significance of TMZ 1 μM vs. quinoin 2.5 nM plus TMZ 1.0 μM.