Abstract
Background/Aim: Plexiform neurofibromas (PNFs) are benign tumors composed mainly of tumorous Schwann cells and non-tumorous fibroblasts. This study examined the possible enhancing effect of vitamin D on the efficacy of drugs used for the treatment of PNF in vitro. Materials and Methods: Paired Schwann cells and fibroblasts were cultured from 10 PNFs and treated with imatinib and nilotinib in the absence and presence of calcipotriol, an analogue of the active metabolite of vitamin D. IC50 values for cell proliferation were calculated. Results: Calcipotriol reduced the IC50 of the two drugs in both tumorous Schwann cells and non-tumorous fibroblasts by 40 to 45%. Conclusion: Calcipotriol enhanced the efficacy of imatinib and nilotinib on PNF-derived cells in vitro, though rather non-specifically. Nevertheless, sustaining vitamin D at 100-200 nM, the physiological range, may be beneficial for reducing the dose of drugs without scarifying efficacy.
Plexiform neurofibromas (PNFs) are benign tumors of peripheral nerve sheaths which are frequent in neurofibromatosis type 1 (NF1), a tumor suppressor gene syndrome (1). Despite of their benign nature, these congenital tumors can grow to a large size and cause various complications such as pain, severe disfigurement, and neurological impairment (2-4). In addition, PNFs have a high risk of malignant transformation into malignant peripheral nerve sheath tumors (3). PNFs are composed mainly of Schwann cells and fibroblasts; the former have been proven to be the tumor cells, since they carry the somatic NF1 inactivating genetic alterations (5). Due to their infiltrating and invading nature, total resection of PNFs is generally not possible without damaging functions and organs (3, 4). Systemic drug-therapies that suppress the growth or/and reduce the size of the tumors are therefore highly desirable.
Imatinib mesylate (Gleevec/Glivec) is a receptor tyrosine kinase inhibitor primarily developed for chronic myeloid leukemia and gastrointestinal stromal tumors (6). Our previous studies found the expression of PDGFR-α and PDGFR-β in PNFs and PNF-derived primary Schwann cells. Indeed, imatinib suppressed the proliferation of PNF-derived Schwann cells in vitro and induced regression of PNF xenografts in vivo (7). A previous study reported >20% decrease in tumor volume in 26% of NF1 patients treated with imatinib (8). Nilotinib is the second-generation tyrosine kinase inhibitor, which targets Breakpoint Cluster Region of Abelson Murine Leukemia-gene (BCR-ABL) more selectively and has a 30-fold higher activity (6). Our recent study revealed that nilotinib is also more potent for treating PNF cells in vitro and xenografts in vivo (9).
Vitamin D is well known for its role in regulating calcium homeostasis and bone metabolism (10). In addition, its possible anticancer effects have been postulated (11, 12). Studies also suggested the potential enhancing effect of vitamin D on chemotherapies for cancer patients (13, 14). The desirable serum concentration of 25-OH-vitamin-D3 (calcidiol), a precursor of the active form of vitamin D, is around 75 nM (30 ng/ml), while more than 95% of the population have >50 nM (20 ng/ml) (15). Our previous studies revealed significantly lower calcidiol level in NF1 patients with a mean of 35 nM (14 ng/ml) (16). Since many NF1 patients have a reduced vitamin D intake and can develop osteoporosis in an early phase of life, many of these patients are substituted with vitamin D. In addition, calcidiol level is inversely correlated with cutaneous neurofibromas in NF1 patients (16). A previous study also reported the effect of vitamin D3 in improving the pigmentation of café au lait spots and in suppressing the development of neurofibromas (17).
Like pharmaceuticals in general, drugs and candidate drugs for PNF including imatinib and nilotinib also have various toxic effects. It is therefore essential to keep the dose as low as possible while not sacrificing their therapeutic efficacy. This study aimed to examine the possible enhancing effect of calcipotriol on the efficacy of imatinib and nilotinib in PNF-derived cells in vitro. To examine the specificity of the drugs and the effect of calcipotriol, all treatments were carried out in paired Schwann cells and fibroblasts cultured from each of a total of 10 unrelated PNFs.
Materials and Methods
PNFs were obtained from surgically resected tissues, after ensuring sufficient specimen for pathological diagnosis. All specimens were anonymized. According to local regulations, anonymized application of surgical specimens does not require an approval from the local ethical review board. Patients were informed about the study and have given written consent.
From each PNF, Schwann cells and fibroblasts were separately cultured and enriched as previously described (9). Purity of Schwann cells was examined by immunofluorescence staining for S100 and only cultures consisting of more than 60% Schwann cells were used for drug treatment, which was carried out in 3-5 passage cells.
For drug treatment, 103 cells were seeded into each well of 96-well plates with 100 μl medium. Each drug at each concentration was tested in 4 replicates. On the next day, half of the medium was changed to imatinib- or nilotinib-containing one, to reach the final concentrations of 0, 5, 10, 15, 20, 25 μM for the former and 0, 2, 4, 6, 8, 10 μM for the latter. The treatment was carried out with 4 doses of calcipotriol (LEO Pharma A/S, Ballerup, Denmark): 0, 10, 100 and 1000 nM, and continued for 10 days. Half of the media containing drugs and calcipotriol were refreshed every day. At the end of the treatment, proliferation of the cells was measured using a BrdU cell proliferation Elisa kit (Roche, Mannheim, Germany). Inhibition was calculated as [max value - value]/(max value), whereas max value is the mean value of respective controls at the drug concentration of 0. IC50 is the drug concentration for 50% inhibition.
Statistical analysis. Differences between Schwann cells and fibroblast cells, and between treatment with and without calcipotriol were examined using Student’s t-test. Data were presented as mean±standard deviation. Statistical significance was set at p<0.05. Statistical analysis was performed using SPSS software (SPSS version 17.0 software: SPSS, Chicago, IL, USA).
Ethics approval and consent to participate. Administrative permissions were acquired by our team to access the data used in our research. The study protocol was approved by the Hamburg University ethics committee (No: REC 0296/4/2017). Accordingly, all samples were coded with numbers and all personal identification of the patients were removed. All parents or guardians of participants provided written informed consent for using their tumor, which otherwise would have been discarded as waste.
Results
From each of the 10 PNFs, paired primary cultures of Schwann cells and fibroblasts were successfully obtained (Figure 1).
Cells derived from a plexiform neurofibroma (PNF) cultured under conditions for enriching Schwann cells (A) and fibroblasts (B). (C) The majority of cells are S100-positive (green) Schwann cells. (D) Some CD-90 (red) positive fibroblasts were also present. Nuclei were stained in blue with DAPI.
Efficacy of the drugs on Schwann cells and fibroblasts. Efficacy of imatinib on Schwann cells and fibroblasts was comparable: IC50 value ranges were 9.5-13.0 μM and 9.0-11.2 μM, respectively. Also, within each pair of Schwann cells and fibroblasts from a PNF, no significant difference in IC50 values was observed (Figure 2A).
IC50 of imatinib and nilotinib for the proliferation of paired Schwann cells and fibroblasts. *p<0.05, **p<0.01, ***p<0.001.
In contrast, nilotinib was slightly more potent in Schwann cells than fibroblasts with an IC50 range of 3.7-7.9 for the former compared to 5.5-9.5 μM for the latter. Also, in each pair, the IC50 was generally higher for fibroblasts than Schwann cells (Figure 2B).
Effect of calcipotriol alone on PNF cells. Calcipotriol alone exhibited a weak dose-dependent inhibitory effect on the proliferation of both Schwann cells and fibroblasts (Figure 3). The maximal inhibition at 1000 nM ranged from 11.1% to 50.4% for the total of 20 cultures.
Mild inhibitory effect of calcipotriol alone on the proliferation of paired Schwann cells and fibroblasts from four plexiform neurofibromas. In all cases, proliferation of the both types of cells was dose-dependently inhibited. Broken lines: Tumorous schwann cells; solid lines: non-tumorous fibroblasts.
Influence of calcipotriol on the efficacy of imatinib and nilotinib. Adding calcipotriol to imatinib- or nilotinib-treatment led to dose-dependent enhancement of the efficacy of both drugs. The enhancing effect of calcipotriol was more prominent at lower doses of imatinib and nilotinib, whereas it nearly vanished at the highest doses (Figure 4).
An example of the inhibitory effect of imatinib and nilotinib combined with or without 10, 100 and 1,000 nM calcipotriol on paired Schwann cells and fibroblasts.
Generally, the IC50s of imatinib and nilotinib were lowered in the presence of calcipotriol (Figure 5). At 100 nM of calcipotriol, the IC50 of imatinib and nilotinib in both cell types was decreased by 40 to 45%. However, this enhancing effect of calcipotriol on the efficacy of imatinib and nilotinib was similar in Schwann cells and fibroblasts, indicating that it is rather non-specific.
Enhancing effect of calcipotriol on the efficacy of imatinib (A) and nilotinib (B). IC50 values for the 10 pairs of cultures are summarized as mean±standard deviation. Solid lines: Schwann cells; Dashed lines: fibroblasts.
Discussion
In this study, we carried out drug treatments on 10 paired cultures of Schwann cells and fibroblasts, each derived from the same PNF. We first confirmed the previous findings that nilotinib is more potent than imatinib and more specific for Schwann cells (9).
We further found an inhibitory effect of calcipotriol on the proliferation of PNF-derived cells. However, this effect was similar on Schwann cells and fibroblasts, suggesting that it is rather non-specific. Furthermore, under combined treatment, calcipotriol substantially enhanced the efficacy of imatinib and nilotinib. However, this enhancing effect was again similar for Schwann cells and fibroblasts, indicating that it is also non-specific.
In the present study, Schwann cells and fibroblasts were cultured separately, under different conditions, since Schwann cells require special additives and surface coating. The two types of cells also had different growth rates in culture. These differences prohibit a direct comparison the drug effect and the effect of the combined drugs between these cell types. A more desirable experimental setting for future studies would be culturing and treating all cells from a PNF in a mixed culture under identical conditions, and measuring the effect of the drugs on each cell type separately. Determining the genetic features of tumor cells, as in the case of PNF cells with a somatic NF1 mutation or allele loss, provides a strategy to enable such study (18).
Physiological range of serum calcidiol is 50-100 nM (19), while possible vitamin D-related toxicity such as hypercalcemia may only occur with serum calcidiol concentrations above 300 nM (20, 21). Therefore, elevating serum calcidiol to 100-200 nM by means of vitamin D substitution may provide a safe and feasible strategy for enhancing the efficacy of imatinib and nilotinib. Especially considering that NF1 patients have generally lower vitamin D levels, substitution is more attractive.
Conclusion
Keeping vitamin D at the high end of the physiological range may allow dose reduction of imatinib and nilotinib and thus, alleviate related toxicities. This strategy may also apply to other drugs and other tumors. In addition, the potential effects of co-medication should be considered when assessing pharmacologically-induced tumor reductions, especially in the treatment of NF1-associated PNF.
Footnotes
Authors’ Contributions
YZ: conceived the study, supervised the experiments and drafted the manuscript. LK: performed the data collection and drafted the manuscript. REF: designed the experiments and drafted the manuscript. MY: data evaluation, medical writing and editorial assistance in preparing this manuscript for publication. RS: performed data collection. MG: analyzed the data and revised the manuscript. All Authors have read and approved the manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests in relation to this study.
- Received May 5, 2021.
- Revision received May 17, 2021.
- Accepted May 25, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.