P13.09 Inconsistent effect of temozolomide exposure on cell viability in glioblastoma cell line models - a systematic review

BACKGROUND The alkylating agent temozolomide is part of standard care for patients with glioblastoma. Potential novel therapeutic agents are often first evaluated against temozolomide in glioblastoma cell line models. Despite the importance of this step in compound development, there is no standard concentration or exposure duration of temozolomide in laboratory research, and consistency in the effect of temozolomide on glioblastoma cell lines has not been assessed. This systematic review aimed to summarise the concentration and exposure duration of temozolomide and its effect on cell viability in studies using glioblastoma cell lines. MATERIAL AND METHODS We searched Medline and Embase Jan 1994 - Feb 2021 for studies that used at least one glioblastoma cell line and reported a measure of cell viability associated with temozolomide exposure. Studies were excluded if they used modified cell lines or did not report a cell viability measure associated with temozolomide as monotherapy. One reviewer screened all records and two reviewers assessed potentially eligible studies for inclusion. The main data items included the cell lines used, the concentration and exposure duration to temozolomide, and cell viability measures. We summarised findings using descriptive statistics. RESULTS Of 1,533 potentially eligible studies we included 213 studies reporting 209 different cell lines. The most common cell lines were U87, U251 and T98G, used in 61%, 41%, and 27% of studies, respectively. Twenty-five (12%) studies used patient-derived cell lines. The concentration of temozolomide used ranged from 0 to 8000μM. The temozolomide exposure duration ranged from <24 hours to >96 hours, with 29% studies using 72 hours. The most common cell viability measure was half maximal inhibitory concentration (IC50), which was reported in 183 (86%) studies. The median IC50 in 32 studies using the U87 cell line was 180μM (interquartile range [IQR]: 52–254μM) at 48-hour temozolomide exposure and 202μM (IQR 52–518μM) at 72-hour exposure. The median IC50 in 31 studies using U251 cell line was 84μM (IQR: 34–324μM) at 48-hour exposure and 102μM (IQR: 35–358μM) at 72-hour exposure. CONCLUSION Experimental setup of temozolomide and its effect on cell viability vary widely between studies using similar glioblastoma cell lines. This inconsistency of response to temozolomide questions reproducibility and the translational value of study findings.

Lepidium graminifolium L.: Glucosinolate Profile and Antiproliferative Potential of Volatile Isolates

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active.

SN38 loaded nanostructured lipid carriers (NLCs); preparation and in vitro evaluations against glioblastoma

SN38 is the active metabolite of irinotecan with 1000-fold greater cytotoxicity compared to the parent drug. Despite the potential, its application as a drug is still seriously limited due to its stability concerns and low solubility in acceptable pharmaceutical solvents. To address these drawbacks here nanostructured lipid carrier (NLC) containing SN38 was prepared and its cytotoxicity against U87MG glioblastoma cell line was investigated. The formulations were prepared using hot ultrasonication and solvent evaporation/emulsification methods. NLCs with a mean size of 140 nm and particle size distribution (PDI) of 0.25 were obtained. The average loading efficiency was 9.5% and its entrapment efficiency was 81%. In order to obtain an accurate determination of released amount of SN38 a novel medium and extraction method was designed, which lead to an appropriate in vitro release profile of the drug from the prepared NLCs. The MTT test results revealed the significant higher cytotoxicity of NLCs on U87MG human glioblastoma cell line compared with the free drug. The confocal microscopy images confirmed the proper penetration of the nanostructures into the cells within the first 4 h. Consequently, the results indicated promising potentials of the prepared NLCs as a novel treatment for glioblastoma.

Enhanced Anti-Tumor Activity in Mice with Temozolomide-Resistant Human Glioblastoma Cell Line-Derived Xenograft Using SN-38-Incorporated Polymeric Microparticle

Glioblastoma multiforme (GBM) has remained one of the most lethal and challenging cancers to treat. Previous studies have shown encouraging results when irinotecan was used in combination with temozolomide (TMZ) for treating GBM. However, irinotecan has a narrow therapeutic index: a slight dose increase in irinotecan can induce toxicities that outweigh its therapeutic benefits. SN-38 is the active metabolite of irinotecan that accounts for both its anti-tumor efficacy and toxicity. In our previous paper, we showed that SN-38 embedded into 50:50 biodegradable poly[(d,l)-lactide-co-glycolide] (PLGA) microparticles (SMPs) provides an efficient delivery and sustained release of SN-38 from SMPs in the brain tissues of rats. These properties of SMPs give them potential for therapeutic application due to their high efficacy and low toxicity. In this study, we tested the anti-tumor activity of SMP-based interstitial chemotherapy combined with TMZ using TMZ-resistant human glioblastoma cell line-derived xenograft models. Our data suggest that treatment in which SMPs are combined with TMZ reduces tumor growth and extends survival in mice bearing xenograft tumors derived from both TMZ-resistant and TMZ-sensitive human glioblastoma cell lines. Our findings demonstrate that combining SMPs with TMZ may have potential as a promising strategy for the treatment of GBM.