Nimodipine Used with Vincristine: Protects Schwann Cells and Neuronal Cells from Vincristine-Induced Cell Death but Increases Tumor Cell Susceptibility

Abstract

The chemotherapeutic agent vincristine is commonly used for a variety of hematologic cancers, as well as solid tumors of the head and neck, bronchial carcinoma, as part of the procarbazine, lomustine and vincristine (PCV) regimen, for glioma. Damage to nerve tissue (neuropathy) is often dose-limiting and restricts treatment. Nimodipine is a calcium antagonist that has also shown neuroprotective properties in preliminary studies. In this approach here, we investigated the effects of the combination of vincristine and nimodipine on three cancer cell lines (A549, SAS and LN229) and neuronal cells (RN33B, SW10). Fluorescence microscopy, lactate dehydrogenase (LDH) assays and Western blot analyses were used. Nimodipine was able to enhance the cell death effects of vincristine in all tumor cells, while neuronal cells were protected and showed less cell death. There was an opposite change in the protein levels of Ak strain transforming/protein kinase B (AKT) in tumor cells (down) and neuronal cells (up), with simultaneous increased protein levels of cyclic adenosine monophosphate response element-binding protein (CREB) in all cell lines. In the future, this approach may improve tumor response to chemotherapy and reduce unwanted side effects such as neuropathy.

Keywords: Schwann cells; glioblastoma; neuronal cells; neuropathy; neuroprotection; nimodipine; non-small lung cancer; squamous tongue cancer; vincristine.

Figure 1

Microscopic images of RN33B (a) and SW10 (b) cells with 100× magnification. Top row shows control. Middle row shows cells after 24 h treatment with VCR. Bottom row shows cells after 24 h pretreatment of cells with NIM and subsequent addition of VCR. Brightfield (first column) and fluorescence microscopic images with NucBlue^™ staining (blue, middle column) and CellROX^™ (green, right column). Scale bar = 100 µm.

Figure 2

Microscopic image of A549 (a), SAS (b) and LN229 (c) cells with 100× magnification. Top row shows control. Middle row shows cells after 24 h treatment with VCR. Bottom row shows cells after 24 h pretreatment of cells with NIM and subsequent addition of VCR. Brightfield (left column) and fluorescence microscopic images with NucBlue™ (Hoechst 33342) staining (blue, middle column) and CellROX^™ (green, right column). Scale bar = 200 µm.

Figure 3

Cell viability of RN33B (a) and SW10 (b) cells measured after 24 h and 48 h by LDH assay. The viability was calculated using a sample treated with Triton X100 (cell death = 100%). The vehicle (ethanol) and the NIM-pretreated samples are compared. The diagrams show the mean values and SDs of three independent biological replicates. Tukey’s multiple comparisons test was performed for statistical analysis. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Figure 4

Cell viability of A549 (a), SAS (b) and LN229 (c) cells measured after 24 h and 48 h by LDH assay. The viability was calculated using a sample treated with Triton X (cell death = 100%). The data for the samples without stress are shown on the left and the samples with VCR treatment on the right. The vehicle (ethanol) and the NIM-pretreated samples are compared. The diagrams show the mean values and SDs of three independent biological replicates. Tukey’s multiple comparisons test was performed for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

Detection of transcription factors in neuronal cells RN33B (a) and SW10 (b) treated with VCR alone as well as with co-application of NIM. After the transfer of the proteins separated by SDS-PAGE onto nitrocellulose membranes, phosphorylation and total protein levels of the cell signaling components were determined by specific antibodies. The GAPDH protein level was used as a loading control. The Western blot shown is representative of the results from three independent biological replicates.

Figure 6

Quantification of pAKT and pCREB. Quantification of the bands in RN33B (a) and SW10 (b) was normalized to the GAPDH control. The mean values including standard deviation are shown. Two independent biological replicates were analyzed. Student’s t-test was performed for statistical analysis. * p < 0.05; ** p < 0.01; ns: not significant.

Figure 7

Detection of transcription factors in cancer cell lines A549 (a), SAS (b) and LN229 (c) treated with VCR alone as well as with co-application of NIM. After the transfer of the proteins separated by SDS-PAGE onto nitrocellulose membranes, phosphorylation and total protein levels of the cell signaling components were determined by specific antibodies. The GAPDH protein level was used as a loading control. The Western blot shown is representative of the results from three independent biological replicates.

Figure 8

Quantification of pAKT and pCREB in A549 (a), SAS (b) and LN229 (c). Quantification of the bands normalized to the GAPDH control. The combination treatment was also normalized to the monotherapy. The mean values including deviation are shown. Two independent biological replicates were analyzed. Student’s t-test was performed for statistical analysis. * p < 0.05; ** p < 0.01; ns: not significant.

Figure 9

Overview of the main results.

Figure 10

Overview of treatment scheme and experimental set-up (Created with BioRender^®, Agreement number: WR273JJ9DL).

Figure 11

Observed effects of the combination of VCR and NIM. Improved treatment response of tumor cells, which could lead to longer progression free survival (PFS) and overall survival (OS). Simultaneously, we demonstrated protection against chemotherapy-associated cell death of Schwann and neuronal cells, which could lead to a reduction in adverse effects (Created with BioRender^®, Agreement number: N273JM90A).