Nicardipine sensitizes temozolomide by inhibiting autophagy and promoting cell apoptosis in glioma stem cells

Susceptibility of GSCs to combining administering of TMZ and nicardipine

SU4 and SU5 cells derived from surgical specimen of glioblastoma patients were applied for cytotoxicity experiments. GSCs (both SU4 and SU5) had a strong ability to form tumor-sphere like cell clusters, and expressed GSCs markers, including CD133, nestin and CD44, consistent with the GSCs stemness characteristics (Figure 1A). To evaluate the cytotoxic effect of TMZ and nicardipine on GSCs, both SU4 and Su5 cells were incubated at the indicated concentrations of TMZ or nicardipine for 48 h. Cell viability was detected by CCK-8 assay, which showed that GSCs were highly resistant to TMZ at the regular concentration, decrease in cell viability appeared at TMZ concentration higher than 400 μM (Figure 1B). Inhibitory effect of TMZ on GSCs at lower concentration (100μm) was poor even after longer time point (96 h) (Supplementary Figure 1). This was related to the innate tolerance of GSCs to chemotherapy [21, 22]. Nicardipine showed no obvious cytotoxic effects against GSCs at the low concentration (<40 μM) (Figure 1B). In order to avoid confusion caused by the direct inhibition effect of high concentration of nicardipine on GSCs, we selected low concentration nicardipine (20 μM) as chemosensitizer. Combined with the preliminary experimental data results and the previous studies [23, 24], 400 μM TMZ and 20 μM nicardipine were selected to explore the sensitization effect of nicardipine on TMZ against GSCs.

Figure 1. GSCs characteristic analysis and cell survival assay evaluating GSCs sensitivity to TMZ and nicardipine. Under the phase contrast microscope, GSCs showed spherical growth (Scale bar 100μm); CD133, Nestin, and CD44 were positively expressed in GSCs, determined by immunofluorescence staining (Scale bar 50μm) (A). Cell survival assay by CCK-8 after administering TMZ ranging from 0 to 1600 μM, nicardipine (0 to 80 μM), or both for 48 h (B). ** p < 0.01, *** p < 0.001.

Nicardipine can enhance TMZ cytotoxic effects on GSCs

CCK-8 assay showed that 400 μM TMZ in combination with 20 μM nicardipine obviously reduced the viability of both SU4 and SU5 GSCs (Figure 2A, 2D). Flow cytometry was carried out to determine whether apoptosis of GSCs was induced by combining administration of TMZ and nicardipine. The apoptosis ratio induced by TMZ plus nicardipine was markedly higher than that of TMZ alone (Figure 2B, 2C, 2E, 2F). Several proteins involved in apoptosis-related signaling pathway were detected by Western blot to further verify initiation of apoptosis induced by TMZ in combination with nicardipine, which showed combination of TMZ and nicardipine promoted Bax accumulation obviously (Figure 2G–2I). In addition, mitochondrial proteins of GSCs were extracted to analyze Bax expression in mitochondrion, compared to TMZ group, combination of TMZ and nicardipine can upregulate mitochondrial Bax expression both in SU4 and SU5 cells, confirming activation of mitochondrial apoptosis induced by TMZ plus nicardipine (Figure 2G, 2J, 2K). Taken together, these data suggest that combination of TMZ with nicardipine can inhibit GSCs proliferation via promoting mitochondrial apoptosis.

Figure 2. Combining dosing of nicardipine and TMZ decreased cell viability and promoted apoptosis of GSCs. SU4 cell viability was assessed by CCK-8 assay under exposure to TMZ (400 μM), nicardipine (20 μM) or both for 48 h (A). SU4 cells were treated with TMZ (400 μM), nicardipine (20 μM) or both for 48 h, then were stained with PI and Annexin V-FITC for apoptosis analysis (B, C). SU5 cell viability was assessed by CCK-8 assay under treatment with TMZ (400 μM), nicardipine (20 μM) or both for 48 h (D). SU5 cells were treated with TMZ (400 μM), nicardipine (20 μM) or both for 48 h, then were stained with PI and Annexin V-FITC for apoptosis analysis (E, F). Western blot analysis of Bcl-2, Bax, mitochondrial Bax and COX IV in GSCs was performed after treatment with TMZ, nicardipine or both for 48 h (G–K). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Combination of nicardipine and TMZ can inhibit GSCs autophagy via upregulating mTOR

To analyze the effect of TMZ in combination with nicardipine on autophagy of GSCs, the expression of p62 and LC3 in SU4 and SU5 cells were measured. Western blot data showed upregulation of p62 and LC3 expression both in nicardipine group and TMZ plus nicardipine group, p62 and LC3 expression in TMZ plus nicardipine group even higher (p<0.05) (Figure 3A–3C, 3E, 3F), suggesting that nicardipine can inhibit both autophagosome transport and autophagy in GSCs [25]. For further verifying this result, LC3 was transfected into GSCs with lentivirus vector to detect autophagic flux via mCherry/GFP dual-color tracing techniques, in which GFP can be quenched in the acidic environment of lysosome, while mCherry emitting red fluorescence can exist longer. The yellow fluorescence formed by fusion of GFP and mCherry represented autophagosome, and the red fluorescence implied the combination of autophagosome and lysosome to form autolysosome. Aggregation of yellow fluorescence in GSCs can be observed treated with TMZ plus nicardipine, which was significantly higher than GSCs treated with TMZ alone, or nicardipine alone, indicating that transportation of autophagy bodies and binding of lysosomes in GSCs decreased obviously, and inhibition of autophagy existed (Figure 3H).

Figure 3. Autophagy-related proteins expression in GSCs after treating with TMZ, nicardipine, or both. Western blot analysis of p62, LC3 II and mTOR in SU4 and SU5 cells treated with TMZ (400 μM), nicardipine (20 μM), or both, respectively for 48 h (A). Statistical analysis of p62, LC3 II and mTOR protein levels in SU4 and SU5 cells (B–G). The mCherry-GFP-LC3 distribution in GSCs cultured with TMZ, nicardipine or both after 48 h was analyzed by confocal microscopy. Scale bar 100 μM (H). * p <0.05, ** p <0.01, *** p <0.001.

Chloroquine (CQ), as an inhibitor of late autophagy, was applied to verify the inhibitory role of nicardipine on GSCs autophagy. The combination of TMZ (400 μM) and CQ (10 μM) increased in p62 and LC3-II expression levels in both SU4 and SU5 cells (Figure 4), which accorded with the effect of nicardipine on GSCs. Previous studies reported that mTOR played a key role in autophagy regulation and can inhibit autophagy through multiple molecular regulatory steps [26], then the protein levels of mTOR and phosphorylated-mTOR (p-mTOR) were analyzed in GSCs treated with TMZ and nicardipine, which suggested that the p-mTOR/mTOR ratio exhibited significant increase in GSCs treated with TMZ plus nicardipine (Figure 3A, 3D, 3G), which implied that combining application of nicardipine and TMZ can inhibit GSCs autophagy by upregulating mTOR expression.

Figure 4. Detection of autophagy level of GSCs treated with TMZ, CQ, or both. SU4 and SU5 cells were exposed to TMZ (400 μM), CQ (10 μM), or both, respectively for 48 h. Then p62 and LC3 II expression levels were analyzed by Western blotting (A). Statistical analysis of p62 and LC3 II expression in SU4 and SU5 cells (B–E). *p < 0.05, ***p<0.001.

Nicardipine can sensitize TMZ against GSCs by promoting cellular apoptosis via suppressing autophagy

Combination of TMZ and nicardipine increased p-mTOR, p62 and LC3-II expression levels of GSCs (Figure 3). Rapamycin, as a selective mTOR inhibitor activating autophagy, can reverse upregulation effect of p-mTOR and mTOR expression in GSCs induced by combined application of TMZ plus nicardipine (Figures 5D, 2G). Rapamycin-treated GSCs showed decline of p62 expression and accumulation of LC3-II (Figure 5A–5C, 5E, 5F), indicating that rapamycin can promote GSCs autophagy and reverse autophagy inhibition of GSCs induced by combined application of nicardipine with TMZ. Flow cytometry was carried out for further exploring the interaction between apoptosis induced by combined treatment and autophagy, which revealed that rapamycin weakened GSCs apoptosis induced by combined treatment (Figure 6A–6D). Western blot analysis on apoptotic-related proteins suggested that rapamycin can reduce Bax/Bcl-2 ratio in GSCs treated with TMZ and nicardipine (Figure 6E–6G). Collectively, these findings indicated that rapamycin can decrease apoptotic efficiency in GSCs treated with TMZ and nicardipine via promoting GSCs autophagy.

Figure 5. Rapamycin promoted GSCs autophagy by activating mTOR. The protein levels of p62, LC3 II and mTOR in GSCs were measured by Western blotting after exposure to TMZ (400 μM) plus nicardipine (20 μM) for 48 h, or in GSCs pretreated with rapamycin (50 nM) for 12 h (A). Statistical analysis of p62, LC3 II and mTOR expression in SU4 and SU5 cells (B–G). *p < 0.05, **p<0.01.

Figure 6. Rapamycin inhibited nicardipine-induced sensitization of TMZ against GSCs. SU4 and SU5 cells were treated with TMZ (400 μM), nicardipine (20 μM), TMZ (400 μM) + nicardipine (20 μM), and TMZ (400 μM) + nicardipine (20 μM) +rapamycin (RPA) (50 nM), respectively, for 48 h. Then cells were stained with PI and Annexin V-FITC for apoptosis analysis (A–D). Western blot analysis of Bcl-2 and Bax in GSCs was performed after treating with TMZ (400 μM), nicardipine (20 μM), TMZ (400 μM) + nicardipine (20 μM), and TMZ (400 μM) + nicardipine (20 μM) +RPA (50 nM), respectively for 48 h (E–G). The survival analysis of GSCs bearing mice after chemotherapy, Kaplan-Meier curve indicated combined treatment with TMZ and nicardipine had positive survival benefit (H). * p < 0.05, ** p < 0.01, ***p<0.001.

Nicardipine can sensitize GSCs inhibition effect of TMZ in vivo

Sensitization effect of nicardipine on temozolomide against GSCs in vivo was also evaluated. There was no obvious drug-related side effect during combined treatment in the tumor bearing mice. The OS of tumor bearing mice was recorded. The median survival of the normal saline control group was 29 days, the temozolomide group was 38 days, and the combined treatment group was 45 days. Kaplan Meier survival curve analysis showed that the median survival of the combined treatment group was longer than that of the other two groups (p<0.05) (Figure 6H).

Cell culture and materials

The primary cultured GSCs SU4 and SU5 were derived from fresh surgical specimen of human GBM tissues using a method described previously with informed consent [37]. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University. Cell lines were maintained in Dulbecco’s modified Eagle’s medium/F12 (Gibco, Grand Island, NY, USA) supplemented with B-27 (Gibco, Grand Island, NY, USA), 20 ng/mL recombinant human epidermal growth factor (EGF; Gibco, Grand Island, NY, USA), 20 ng/mL basic fibroblast growth factor (bFGF; Gibco, Grand Island, NY, USA) and antibiotics (100 U/mL penicillin and 100 mg/L streptomycin (HyClone, South Logan, UT, USA)).

TMZ, Nicardipine, chloroquine (CQ), rapamycin, laminine and poly-L-ornithine solutions were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dimethyl sulfoxide was the product of MP Biomedicals, LLC (Santa Ana, CA, USA). Antibodies against β-actin, Bcl-2, Bax, p62, LC3B, AKT, p-AKT, mTOR and p-mTOR, were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary biotinylated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Neurosphere formation and immunofluorescence staining

GSCs were seeded in 96-well plates (2×10³ cells per well) and supplemented with 50 μL culture medium every 3 days. Then neurospheres formation was observed under a microscope (Nikon, Tokyo, Japan) after 10-14 days of incubation. Immunofluorescence staining of CD133, CD44, Nestin was carried out as described [38].

mCherry- GFP- LC3 lentivirus transfection

mCherry- GFP -LC3 lentivirus was applied to transfect glioma stem cells SU4 and SU5 to detect autophagy flux, following the manufacturer’s instructions. Then the GSCs were treated with TMZ (400μM), nicardipine (20μM), or both, respectively, for 48 h, and intracellular fluorescence was observed by confocal microscopy, yellow fluorescence indicated the presence of autophagosomes, and red fluorescence suggested the existence of autolysosomes.

Cell survival assay

GSCs were cultured in a 96-well plate (2×10³ cells per well) and incubated at 37° C for 12 h, then were treated with different dosage of TMZ, the final concentration of TMZ applied was as following, 0, 50μM, 100μM, 200μM, 400μM, 800μM, 1600μM. Nicardipine was also administered at a series of final concentration gradient, 0, 5μM, 10μM, 20μM, 40μM, 60μM, 80μM. Cells were treated with TMZ, nicardipine, and both of the above mentioned drugs, respectively. CCK-8 solution (Dojindo, Kumamoto, Japan) was supplemented into each well, incubated for 2 h at 37° C on a shaker. Subsequently, the absorbance values were measured at 490 nm on a microplate reader EL×800 (BioTek, Winooski, Vermont, USA). The viability of untreated cells was set as 100%. The experiment was repeated three times.

Cell apoptosis assay

GSCs were seeded in 6-well plate (2×10⁵ cells per well) and treated with TMZ (400μM), nicardipine (20μM) or both, with or without rapamycin (50 nM), incubated for 48 h. A total of 1×10⁵ GSCs were resuspended in the binding buffer and stained with 5 μL of Annexin V-fluorescein isothiocyanate (FITC) and 5 μL of propidium iodide (PI; BD Pharmingen, San Diego, CA, USA) for 30 min, then were detected by flow cytometry (Guava EasyCyte 6HT-2L, Merck Millipore, Darmstadt, Germany). The early apoptotic cells located in the lower right quadrant were calculated as the percentage of apoptotic cells. The experiment was repeated three times.

Western blot

Cells were cultured in 6-well plate and treated with TMZ (400μM), Nic (20μM)/CQ (10μM) or both, with or without rapamycin (50 nM) for 48 h, then cells were digested with accutase and washed thoroughly with PBS for three times, followed by lysing cells in RIPA buffer (Beyotime, Shanghai, China) supplemented with 1% protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). After centrifugation at 12000 g for 2 min at 4° C, protein concentration was determined with a standard BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blot was performed according to the standard protocol. The mitochondrial protein was extracted with a mitochondrial isolation kit (Beyotime Biotechnology; Jiangsu, China), according to the manufacturer’s instructions. Primary antibodies against Bcl-2 (#2872), Bax (#2772), p62 (#8025), LC3 (#3868), mTOR (#2983), p-mTOR (#5536), AKT (#4691) and p-AKT (#4060) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-β-actin (#4970) was used as normal loading control while rabbit anti-CoxIV (#4850) was applied as mitochondrial loading control. An ECL plus kit (Thermo Fisher Scientific, Waltham, MA, USA) and a ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA) were applied to detect the chemiluminescence signals. Each experiment was repeated three times.

Evaluation of nicardipine sensitizing TMZ against GSCs in orthotopic GSCs model

10 μl cell suspension containing 10⁶ GSCs was implanted into the right caudate nucleus of 4-week-old BALB / C nude mice with stereotactic techniques (nude mice were obtained from the Nanjing Institute of Model Animals). After 7 days, the mice were randomly divided into three groups. TMZ group (n=5): intragastric administration of TMZ (50 mg / kg, once a day for 5 days). TMZ + nicardipine group (n=5): TMZ was administration according to the above method, and nicardipine (1.5mg/kg) was injected intraperitoneally slowly on the same day. Control group (n=5): intraperitoneal injection of equal volume of normal saline. The overall survival (OS) time of mice was recorded and analyzed by Kaplan-Meier survival curves.

Statistical analysis

All data was collected and analyzed with GraphPad Prism 7 (GraphPad Software), and expressed as mean ± SD values. Student’s t-test (two tailed) or one-way ANOVA was used for statistical analysis. p value < 0.05 was considered to reflect a statistically significant difference.

Ethics approval and consent to participate

Procedures performed in the study were in accordance with the ethical standards of the institutional research committee.

Statement of human and animal rights

The study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University.

Availability of data and materials

The datasets are available from the corresponding author on reasonable request.