Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

Introduction

Glioblastoma (astrocytoma grade IV) is the most aggressive tumor of the central nervous system. Glioblastoma cells are highly mobile and invasive, easily repopulating the tumor bulk after surgical resection. Additionally, glioblastoma stem cells are considered to be particularly chemoresistant, making a complete therapeutic elimination of these cells almost impossible.[1] After surgery, radiation, and chemotherapy with temozolomide (TMZ), a standard chemotherapeutic agent used to treat glioblastoma, the survival period for patients diagnosed with glioblastoma remains low, between twelve to fifteen months,[2,3] indicating the need for novel and effective reagents that can be applied in combination with existing chemotherapeutics to overcome resistant mechanisms.

Dinaciclib is a small-molecule inhibitor of cyclin-dependent kinases (CDKs). CDKs contribute to tumor cell progression, and dinaciclib has been shown to exert anti-proliferative effects by inhibiting several CDKs including CDK9.[4] In contrast to dinaciclib, isothiocyanates (ITCs) are natural products of the Cruciferae family of plants, such as broccoli or radish, and interfere with tumor cell growth.[5,6] ITCs selectively cause an accumulation of reactive oxygen species (ROS), which in turn provoke apoptosis in tumor cells. Prominently, healthy, non-transformed cells are less susceptible to ROS.[7] Previous studies have reported the synthesis of diisothiocyanate-derived mercapturic acids which were able to kill human adenocarcinoma cells.[8] In this context, we found that the application of diisothiocyanate-derived mercapturic acids (J1, J2, J3, and J4) interfered with cell viability of glioblastoma cells and glioblastoma stem cells.[9] Here, we extend our examination by combining diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ and present that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ supplement their effect to reduce cell viability, including primary glioblastoma cells from a glioblastoma patient.

Results and Discussion

Diisothiocyanate-Derived Mercapturic Acids Combined with Dinaciclib or TMZ Reduce the Metabolic Activity in Glioblastoma Cell Line U87

In a first set of experiments, the EC50 (half-maximal effective concentration) values were precisely determined for diisothiocyanate-derived mercapturic acid compounds (J1, J2, J3, and J4, Figure ), dinaciclib, and TMZ by titrating different concentrations of these compounds to the established glioblastoma cell line U87, sphere-cultured stem cell-enriched glioblastoma cell populations (SCs) that were generated from the tissue of a patient diagnosed with glioblastoma (SC40), and primary differentiated glioblastoma cells (SCs adhesively cultured in the presence of FBS, PC40, Supporting Information S1). The metabolic activity was analyzed by MTT assay and expressed as a dose response curve. Having verified the precise EC50, glioblastoma cell line U87 was incubated with compound J1 (0.17 μM), J2 (0.25 μM), J3 (1.1 μM), J4 (0.65 μM), dinaciclib (7 nM), TMZ (21.5 μM), or a combination of J1, J2, J3, or J4 with dinaciclib or TMZ and cell viability was analyzed. J1, J2, J3, J4, dinaciclib, and TMZ significantly reduced U87 cell viability and cell density (Figures and 3). The combination of J1 with dinaciclib or TMZ significantly decreased further metabolic activity of U87; this was more prominent when using the combination of J1 and dinaciclib. In addition, the combination of J2 or J3 with dinaciclib reduced cell viability but the combination of J2 or J3 with TMZ and J4 with dinaciclib or TMZ did not reach statistical significance.

Figure 1

Scheme of diisothiocyanate-derived mercapturic acids. (a) General method for diisothiocyanate-derived mercapturic acids synthesis. (a)—(1) CS2, Et3N, DMF, 0 °C, 15 min; (2) HBTU, 0 °C, 15 min; (b)—N-acetyl-l-cysteine, NaHCO3, H2O, isopropanol, RT. (b) Chemical structures of diisothiocyanate-derived mercapturic acids: J1, J2, J3, and J4.[8]

Figure 2

U87 cell density was analyzed by light microscopy. U87 cells were treated with EC50 of J1, J2, J3, J4, dinaciclib, or TMZ or a combination of J1, J2, J3, or J4 with dinaciclib or TMZ. DMSO served as a vehicle control. After an incubation time of three days, the medium was removed and cells were treated with the respective components for three additional days. Magnification of microscopic images was 10×.

Figure 3

Metabolic activity in U87 was determined by MTT assay. The metabolic activity in human glioblastoma cell line U87 was determined by MTT assay. U87 cells were cultured with J1, J2, J3, J4, dinaciclib, or TMZ or J1, J2, J3, or J4 in combination with dinaciclib or TMZ. Cells were incubated for three days, the medium was removed, the cells were treated with the respective components for three additional days, and cell viability was measured. The DMSO sample served as a vehicle control. The MTT assay was performed in triplicate and with three independent experiments (n = 3). TMZ, temozolomide; EC50, drug concentration yielding half-maximal response. Only significance between the components is shown. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****).

Diisothiocyanate-Derived Mercapturic Acids Combined with Dinaciclib or TMZ Reduce the Metabolic Activity in Primary Glioblastoma Cells

In order to test the components on patient-derived glioblastoma stem cells and primary glioblastoma cells, SC40 were treated with J1 (2.6 μM), J2 (1.8 μM), J3 (2.4 μM), J4 (1.95 μM), dinaciclib (9 nM), or TMZ (14.01 μM) and PC40 incubated with J1 (0.2 μM), J2 (0.2 μM), J3 (0.78 μM), J4 (0.5 μM), dinaciclib (8 nM), TMZ (15.76 μM), or a combination of J1, J2, J3, or J4 with dinaciclib or TMZ. SC40 and PC40 cells were susceptible to diisothiocyanate-derived mercapturic acids, dinaciclib, and TMZ (Figures and 5). The combination of J3 or J4 with dinaciclib or TMZ decreased SC40 cell viability significantly (Figure ). In the case of PC 40, the combination of J2 or J4 with dinaciclib or TMZ significantly decreased cell viability (Figure ). Primary astrocyte-enriched cultures, which was used as control cells, treated with J1, J2, J3, or J4 did not provoke any changes in metabolic activity; only the combination of J3 with TMZ reduced the cell viability in primary astrocytes (Figure ). Next, TMZ-resistant glioblastoma T98G cells were incubated with compounds J1 (0.17 μM), J2 (0.25 μM), J4 (0.65 μM), and TMZ (50 μM). We observed that the diisothiocyanate-derived mercapturic acid could not sensitize T98 for TMZ, but a higher amount of these components (excess) had a strong effect on cell viability (Supporting Information S2).

Figure 4

Determination of cell viability of SC40. Sphere-cultured stem cell-enriched glioblastoma cell population from patient 40 (SC40) was incubated with J1, J2, J3, J4, dinaciclib, or TMZ or J1, J2, J3, or J4 in combination with dinaciclib or TMZ, after an incubation time of three days the medium was removed and SC40 cells were treated with the respective components (three days). The metabolic activity was determined by using an MTT assay. Three independent experiments were performed (n = 3).

Figure 5

Determination of cell viability of PC40. SC40-derived primary differentiated cells (PC40) were co-cultured with J1, J2, J3, J4, dinaciclib, or TMZ or J1, J2, J3, or J4 in combination with dinaciclib or TMZ for three days. Afterward, the medium was removed and PC40 cells were treated with the components for three days. The cell viability was measured by using an MTT assay. Three independent experiments were performed (n = 3).

Figure 6

Metabolic activity in primary astrocyte-enriched cultures. For three days, primary astrocyte-enriched cultures were treated with J1, J2, J4, or TMZ or J1, J2, or J4 in combination with TMZ. Then, the medium was removed, cells were treated with the respective components for three additional days, and cell viability was measured. The DMSO sample served as a vehicle control. The MTT assay was performed in triplicate; the summary of three independent experiments (n = 3) is shown.

While the combination of J2 with TMZ showed an antagonistic outcome, J1, J2, J3, or J4 with dinaciclib or J1, J3, or J4 with TMZ have an additive effect, when analyzing the U87 data by Bliss analysis (Table ). Moreover, SC40 and PC40 showed additive and synergistic results (comparable to published data treating PC with imipridones, a class of small molecules used for anti-seizure medication, in combination with 2-deoxyglucose, showed a synergistic inhibitory effect to PC[10]), whereby additional synergy between combinations of J3 with TMZ, J3 with dinaciclib, and J4 with TMZ were found for SC40. Similar results were obtained from PC40, where synergistic effects were due to the combination of J3 with dinaciclib, J4 with TMZ, and J4 with dinaciclib.

Table 1

Recently, we demonstrated that J1, J2, J3, and J4[8] were selectively cytotoxic to glioblastoma cells.[9] However, SC40 treated with J1, J2, J3, or J4 (at EC50) together with TMZ (3.09 μM), a concentration found in the brain of patients after being therapeutically treated with TMZ,[11] had neither a stabilizing nor a sensitizing effect on SC40. One reason might be that glioblastoma cells were treated with component J1, J2, J3, or J4 in combination with TMZ for a limited time (three days). This latter aspect is of particular importance, as these glioblastoma stem cells proliferate slowly[12] and the current model of how TMZ affects cells postulates several rounds of cell cycle progression and futile repair attempts.[13] Here, we increased the time of treatment up to seven days. Indeed, J1, J2, J3, or J4 combined with dinaciclib or TMZ showed additive or even synergistic results on the reduction of cell viability depending on the source of glioblastoma cells, in contrast to when compounds were used separately.

The current standard treatment for glioblastoma patients encompasses a combination of radiotherapy and chemotherapy after surgery; however, most clinical trials of diverse molecular targeted therapies for glioblastoma have not revealed a significant survival advantage.[14] Thus, novel chemotherapy strategies are needed in combination with multi-targeted drugs and immunotherapy in addition to surgery and radiotherapy in order to overcome tumor resistance.[14,15] The most commonly used chemotherapeutic drug, TMZ, methylates DNA leading to double-stranded DNA breaks and eventually to apoptosis.[16] In general, dinaciclib limits glioma cell growth by inhibiting CDKs, promotes degradation of Mcl-1 by the proteasome and enhances ABT-737-mediated cell death of glioma cell lines.[17,18] ITC can selectively trigger the accumulation of ROS, leading to apoptosis of transformed cells.[7]

Conclusions

Combinatorics, which provokes double-stranded DNA breaks (TMZ) or the CDKs inhibitor (dinaciclib) with ROS-induced components (ITC or diisothiocyanate-derived mercapturic acids), might be advantageous in targeting different pathways for therapeutic treatment of glioblastoma.

Methods

Diisothiocyanate-Derived Mercapturic Acids

Diisothiocyanate-derived mercapturic acids were generated as described.[8] Briefly, J3 and J4 diisothiocyanates were synthesized from diamine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) by using carbon disulfide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and triethylamine (Avantor Performance Materials Poland S.A., Gliwice, Poland) for generation in situ ditiocarbamate salt and followed by an addition of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (Iris Biotech GmbH, Marktredwitz, Germany) as a desulfurization agent. The diisothiocyanates for J1 and J2 were purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). To obtain diisothiocyanate-derived mercapturic acid, diisothiocyanate (dissolved in isopropanol) was added to an aqueous solution of N-acetyl-l-cysteine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and sodium hydrogen carbonate (Avantor Performance Materials Poland S.A.). The product was purified by HPLC (Discovery BIO Wide Pore C8; 10 μm, 25 cm × 21.2 mm).

Cells

The human glioblastoma cell line U87-MG (U87, American Type Culture Collection, ATCC, Manassas, VA, USA) was grown in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Inc.) with 10% FBS and 1% penicillin (120 mg/mL) combined with streptomycin (120 mg/mL) at 37 °C (5% CO2 atmosphere).

Sphere-cultured stem cell-enriched glioblastoma cell populations (SCs) were generated from the tissue of a patient diagnosed with astrocytoma grade IV (glioblastoma, patient number 40, 57 years, female, SC40),[12] which was minced, washed in PBS, and incubated with TrypLE Express (Gibco; Thermo Fisher Scientific, Inc.). Afterward, cells were filtered, cultured at 37 °C (5% CO2 atmosphere) in DMEM/Ham’s F-12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing l-glutamine, 0.01% (v/v) epidermal growth factor (EGF; Biomol GmbH, Hamburg, Germany), 0.04% (v/v) fibroblast growth factor (FGF; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), 1% (v/v) B27 (Gibco; Thermo Fisher Scientific, Inc.), 2% fungizone (Gibco; Thermo Fisher Scientific, Inc.), and 1% penicillin (120 mg/mL)/streptomycin (120 mg/mL; Thermo Fisher Scientific, Inc.),[12] and defined as sphere-cultured stem cell-enriched glioblastoma cell populations according to the patient number from which they were derived (SC40), expressing stem cell and differentiation markers.[19] PC40 cells are adherent glioblastoma cells which were generated from SC40.[12] SC40 were kept at 37 °C (5% CO2 atmosphere) in DMEM supplemented with 10% FBS plus 2 mM glutamine, 1% penicillin (120 mg/mL)/streptomycin (120 mg/mL; Thermo Fisher Scientific, Inc.) since SCs differentiated into PCs when FBS was added.

Primary astrocyte-enriched cultures, which are patient-derived non-tumor cell culture isolated and grown from surgical specimen after patient’s consent was obtained as described previously,[20] were treated according to the protocol for human astrocytes (Thermo Fisher Scientic Inc.). Cells were maintained in Dulbecco’s Modified Eagle Medium with high glucose (DMEM+ GlutaMax; Gibco, Thermo Fisher Scientic Inc.), and supplemented with 10% fetal calf serum (Gibco), 1% N-2 (Gibco), 0.01% human EGF (Biomol), and 1% penicillin/streptomycin (Gibco). Cell culture plates were coated with Geltrex matrix (reduced growth factor basement membrane matrix) (Gibco) for 1 h prior to cell seeding. Plates were then washed with DPBS + Ca/Mg (Gibco), and cells were seeded at a cell density of 1 × 104 cells/cm2. After overnight incubation, cells were treated with J1, J2, J3 J4, TMZ, dimethyl sulfoxide (DMSO), or the combination. Following an incubation time of 3 days, the media were removed and cells were again treated with the respective components. The metabolic activity (MTT test) was assessed after 3 additional days.

The use of SCs and primary astrocyte-enriched cultures for experiments was approved by the local ethics committee of Ulm University, Germany, number: 162/10.

Determination of Cellular Metabolic Activity and Cell Viability by Using MTT Test

Adherent glioblastoma cells (U87, PC40, or T98G from the ATCC) were placed in 96-well flat-bottomed tissue culture plates at 1.5 × 104 cells/mL in 100 μL DMEM containing 10% FBS and 1% penicillin/streptomycin. SC40 were seeded in 96-well flat-bottomed tissue culture plates at 1.5 × 104 cells/mL in 90 μl DMEM/Ham’s F-12 containing 0.01% EGF, 0.04% FGF, 1% B27, 2% fungizone, and 1% penicillin/streptomycin. After an incubation time of one day, the medium was removed. Different concentrations of J1, J2, J3, J4, dinaciclib (ChemieTek, Indianapolis, IN, USA), TMZ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), or the solvent and vehicle control DMSO (Serva Electrophoresis GmbH, Heidelberg, Germany) were prepared in DMEM containing 1.5% FBS and 1% penicillin/streptomycin and added to U87 and PC40 cells (final volume, 100 μL). In the case of SC40, the medium was not changed and J1, J2, J3, J4, dinaciclib, TMZ, or DMSO were added directly (final volume, 100 μL) and cells were cultured for three days. The medium was removed, and J1, J2, J3, J4, dinaciclib, TMZ, or DMSO was added and incubated for an additional three days (in total, cells were cultured for seven days). The medium was removed, and U87 and PC40 cells were incubated with 100 μL MTT working solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), diluted 1:5 in RPMI 1640 medium without l-glutamine and phenol red. For SC40, the SC40 plate was centrifuged for 390g for 5 min at room temperature. The supernatant was then discarded, the cells re-suspended in 100 μL MTT working solution, and incubated for an additional 3 h at 37 °C. Formazan crystals were solubilized with 100 μL isopropyl alcohol, and cell viability was tested by optical density at 550 nm using a microplate spectrometer (Tecan Spectra Classic, Tecan Group Ltd., Männedorf, Switzerland).

Optical Microscopy

Images were taken using a PrimoVert microscope combined with an AxioCam ICc1 camera (Zeiss AG, Oberkochen, Germany) and documented.

Statistical Analysis

Data are presented as the mean ± standard error of the mean, and the statistical analysis was assessed by one-way ANOVA with Bonferroni correction, which was considered to be significant when P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), or P < 0.0001 (****) by using Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). In addition, EC50 values were calculated (Prism 6, GraphPad Software, Inc., La Jolla, CA, USA).

The expected response to the combination treatment was calculated as fractional response to drug A (Fa) + fractional response to drug B (Fb) – (Fa × Fb). Bliss (Excel) analysis was conducted to detect synergistic (ratio of the actual total response and the expected total response > 1.1), additive (this quotient equaled 0.9 to 1.1), or antagonistic effects (quotient < 0.9).