Identification of Gedunin from a Phytochemical Depository as a Novel Multidrug Resistance-Bypassing Tubulin Inhibitor of Cancer Cells

Abstract

The chemotherapy of tumors is frequently limited by the development of resistance and severe side effects. Phytochemicals may offer promising candidates to meet the urgent requirement for new anticancer drugs. We screened 69 phytochemicals, and focused on gedunin to analyze its molecular modes of action. Pearson test-base correlation analyses of the log₁₀IC₅₀ values of 55 tumor cell lines of the National Cancer Institute (NCI), USA, for gedunin with those of 91 standard anticancer agents revealed statistically significant relationships to all 10 tested microtubule inhibitors. Thus, we hypothesized that gedunin may be a novel microtubule inhibitor. Confocal microscopy, cell cycle measurements, and molecular docking in silico substantiated our assumption. Agglomerative cluster analyses and the heat map generation of proteomic data revealed a subset of 40 out of 3171 proteins, the expression of which significantly correlated with sensitivity or resistance for the NCI cell line panel to gedunin. This indicates the complexity of gedunin's activity against cancer cells, underscoring the value of network pharmacological techniques for the investigation of the molecular modes of drug action. Finally, we correlated the transcriptome-wide mRNA expression of known drug resistance mechanism (ABC transporter, oncogenes, tumor suppressors) log₁₀IC₅₀ values for gedunin. We did not find significant correlations, indicating that gedunin's anticancer activity might not be hampered by classical drug resistance mechanisms. In conclusion, gedunin is a novel microtubule-inhibiting drug candidate which is not involved in multidrug resistance mechanisms such as other clinically established mitotic spindle poisons.

Keywords: cancer; microtubuli; natural product; network pharmacology; targeted chemotherapy.

Figure 1

Screening of 69 phytochemicals in drug-sensitive parental CCRF-CEM and multidrug-resistant CEM/ADR5000 cells using the resazurin assay. A fixed concentration of 10 µM of each concentration was used. Shown here are the mean values ± SD of three independent experiments.

Figure 2

Dose-response curves of 14 selected phytochemicals in drug-sensitive parental CCRF-CEM and multidrug-resistant CEM/ADR5000 cells using the resazurin assay. The IC₅₀ values were determined from the dose-response curves, and the degrees of resistance were obtained by dividing the IC₅₀ of CEM/ADR5000 cells by the corresponding IC₅₀ value of CCRF-CEM cells. Shown here are the mean values ± SD of three independent experiments.

Figure 3

Molecular docking of seven selected phytochemicals to a human homology model of P-glycoprotein in the open conformation. (A) Extracellular domain, transmembrane domain, intracellular domain with ATP-binding sites, and drug-binding site of P-glycoprotein. (B) Correlation of lowest binding energies (LBE, kcal/mol) and predicted inhibition constants (pKi, µM) of seven selected phytochemicals. (C) Binding orientation of the selected phytochemicals at the drug-binding site. (D) Interaction of the selected phytochemicals with amino acid residues at the drug binding site. The established anticancer drugs paclitaxel and vincristine were chosen as control drugs, as they are well-known to be transported by P-glycoprotein.

Figure 4

Cytotoxicity of eight selected phytochemicals to the NCI panel of the NCI consisting of cell lines derived from different tumor origins (leukemia; melanoma; brain tumors; carcinoma of the prostate, colon, breast, lung, kidney, or ovary). Shown are the mean log₁₀IC₅₀ values for each tumor type and each compound.

Figure 5

Cross-resistance profiling of gedunin to standard anticancer drugs. (A) Chemical structure of gedunin. (B) Percentage of standard anticancer drugs from different pharmacological classes that significantly correlated to the responsiveness of NCI tumor cell lines to gedunin (with p < 0.05 and r > 0.3 as cutoff values). Oncobiograms of (C) tubulin-inhibiting drugs or (D) geldanamycin derivatives correlating to the response of NCI tumor cell lines to gedunin.

Figure 6

Disruption of the microtubule network in gedunin-treated U2OS cells. Micrographs of fixed U2OS cells were taken after 24 h of treatment with DMSO, various gedunin concentrations, vincristine, and paclitaxel. The microtubules were imaged at 40× magnification using an AF7000 widefield fluorescence microscope (scale bars = 10 µm). The images were merged with DAPI (blue) to represent the nucleus.

Figure 7

Molecular docking of gedunin to human α- and β-microtubules using AutoDock4.2.6. Gedunin was preferentially bound to the Vinca alkaloid binding site but not to the taxane or colchicine binding sites.

Figure 8

Heat map and agglomrative cluster analyses of the protein expression in the response of 55 tumor cell lines to gedunin (log₁₀IC₅₀). The proteins are labeled as numbers at the top of the heatmap. The proteins assigned to each number are given in Supplementary Table S2. The cell lines, their tumor origins, and their sensitivity/resistance to gedunin are shown at the right side of the heat map. Cell lines with individual log₁₀IC₅₀ values smaller than the median value of all of the 55 cell lines tested (−5.125 M) were defined as being sensitive, while all of the others with log₁₀IC₅₀ values above the median were defined as being resistant to gedunin. The cluster analysis (Ward method) separated the proteins into five clusters (clusters A-E) and the cell lines also into five clusters (clusters 1–5). The cell lines were clustered according to their degrees of relatedness to each other on the basis of their protein expression included in the analysis. Color code: red, 0–25% quartile; orange, 26–50% quartile; grey, median value; light green, 50–75% quartile; dark green, 76–100% quartile.

Figure 9

Canonical signaling pathways were predicted by using the proteomic data of 40 proteins in a panel of 55 NCI tumor cell lines and IPA™.

Figure 10

Network analysis using the proteomic data of 40 proteins in a panel of 55 NCT tumor lines and IPA™.