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6α-Acetoxyanopterine: A Novel Structure Class of Mitotic Inhibitor Disrupting Microtubule Dynamics in Prostate Cancer Cells Claire Levrier1,2, Martin C. Sadowski1, Anja Rockstroh1, Brian Gabrielli3, Maria Kavallaris4,5, Melanie Lehman1,6, Rohan A. Davis2, and Colleen C. Nelson*,1 1 Australian Prostate Cancer Research Centre−Queensland, School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Princess Alexandra Hospital, Translational Research Institute, Brisbane, QLD 4102, Australia 2 Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia 3 The University of Queensland Diamantina Institute, Translational Research Institute; Brisbane, QLD 4102, Australia 4 Tumour Biology and Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, NSW, Australia 5 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Australian Centre for NanoMedicine, UNSW Australia, NSW, 2052 6 Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada Supplementary Information Supplementary Figures S1−S9 and figures legends Supplementary Tables S1−S6 Supplementary Videos S1−S5 and legends 1 Supplementary Figure S1. Quantitative image analysis of PHH3-positive cells and αtubulin intensity using CellProfiler software. Example (LNCaP cells treated with 2.5 nM of 6-AA for 24 h) of the automated image segmentation and object quantification performed with CellProfiler software. First, nuclei were identified based on DAPI staining with a mean fluorescence intensity threshold that captured all nuclei (relaxed and condensed chromatin, top left panel). Next, PHH3-positive nuclei were identified based on PHH3 staining (top right panel). To quantify PHH3-positive cells, all nuclei were scored for PHH3-positive (red) and -negative staining (blue, bottom left panel). Mitotic cells displayed strong nuclear PHH3 staining, condensed chromatin and a round cell morphology. Cell outlines were identified based on α-tubulin staining (bottom left panel) and the α-tubulin staining mean intensity per cell was correlated with α-tubulin polymer mass per cell. 2 Supplementary Figure S2. 6-AA induces a time- and concentration-dependent growth arrest and inhibits cell migration. A, following treatment of HeLa cells with various doses of 6-AA, proliferation was analyzed for 72 h using real-time cell imaging (IncuCyte), showing that 6-AA caused a concentrationdependent growth inhibition. DMSO (vehicle) and vinblastine (Vinb) were used as controls (n=3, mean±SD). 3 B, 6-AA inhibited PC-3 cell migration. Wound closure was measured by live-cell imaging (IncuCyte) after 16h of treatment with vehicle control (DMSO), 6-AA, vinblastine (Vinb) or the positive control cytochalasin D (Cytoch) (left panel, n=2, mean±SD). Cytochalasin D is an actin inhibitor commonly used as positive control in migration assays. Representative images of treatments with DMSO, 6-AA and cytochalasin D are shown (right panel). C, phase contrast and GFP-H2B images acquired on an Olympus IX81 microscope showed that 6-AA- and vinblastine-treated HeLa GFP-H2B cells presented the classical features of mitotic arrest (round cells with condensed chromatin) and apoptosis (membrane blebbing, DNA ultra-condensation and fragmentation). Scale bar=50 µm. D, FACS analysis revealed that 6-AA (5 nM) arrests LNCaP cells in the G2-M phase of the cell cycle and induces cell death in a time-dependent manner. DMSO and vinblastine (Vinb) were used as controls (n=2, mean±SD). Two-ways ANOVA with Sidak’s multiple comparisons test was used (ns=non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). 4 Supplementary Figure S3. DNA microarray analysis of 6-AA-treated LNCaP cells. A, heatmap depicting the fold changes of all 228 reference genes differentially expressed in LNCaP cells treated for 24 h with either 6-AA (10 nM) or vinblastine (Vinb, 3.25 nM), showing that the directionality of change was the same for both compounds. Red indicates up-regulation and blue down-regulation. The darker the shade of color, the higher the fold-change of expression. B, GeneOntology analysis (GOrilla) revealed “microtubule binding” as the top enriched molecular function within the set of differentially expressed genes after 6-AA and vinblastine treatments. 5 6 7 Supplementary Figure S4. 6-AA is a reversible inhibitor of mitosis. A, immunofluorescence microscopy coupled with automated image analysis (CellProfiler) was used to quantify PHH3-positive (mitotic) LNCaP cells (~3,000 cells/treatment) after the indicated treatment conditions (n=2, mean±SD). 6-AA (1.25-10 nM) and vinblastine (10 nM) induced a significant increase in PHH3-positive cells when treated for after 8 h (orange bars). Longer treatment (24 h, purple bars) further increased the proportion of PHH3-positive cells. Removal of 6-AA and vinblastine after 8 h of treatment followed by 16 h of recovery decreased the number of PHH3-positive cells to levels seen in vehicle control (DMSO) irrespective of the inhibitor dose. Two-ways ANOVA with Sidak’s multiple comparisons test was used (ns=nonsignificant, ** P < 0.01, **** P < 0.0001; orange label=statistical comparison to DMSO 8 h). B, proliferation of LNCaP cells after different treatment modalities (continuous, 8 h and 24 h before inhibitor removal) with the indicated concentrations of 6-AA (left panels) and vinblastine (right panels) was monitored by real-time imaging (IncuCyte) for 72 h (n=2, mean±SD). Removal of 6-AA and vinblastine after 8 h and 24 h of treatment enabled LNCaP cells to visibly increase cell confluence at all inhibitor concentrations tested. Arrow=washout. C, LNCaP cells were subjected to the same treatment modalities as described in B, and cell viability was measured after 72 h (alamarBlue, n=2, mean±SD). Intermittent treatment with 6AA (8 h and 24 h) significantly less reduced cell viability compared to continuous treatment (72 h). A shorter intermitted treatment period resulted in higher cell viability (8h vs 24h), suggesting that the reversibility of the effect of 6-AA has a time-dependent component. Twoways ANOVA with Sidak’s multiple comparisons test was used (ns=non-significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001). D, cell cycle analysis (FACS) of LNCaP cells treated with DMSO, 6-AA (2.5 nM) continuously for 24 h or with 6-AA (2.5 nM) for 8 h plus 16h recovery demonstrated that the 6-AA-induced accumulation of cells in G2-M is reversible and that removal of 6-AA restored a cell cycle distribution which was similar to control (DMSO). Representative DNA histograms of two independent experiments are shown. E, immunofluorescence microscopy coupled with automated image analysis (CellProfiler) was used to quantify PHH3-positive (mitotic) PC-3 cells (~3,000 cells/treatment) after the indicated modalities of transient and continuous treatments (n=2, mean±SD). Intermittent 6-AA treatment for 8 h resulted in significant lower levels of PHH3-positive PC-3 cells when compared to continuous treatment for 24 h at all concentrations tested. A similar, reversible 8 effect on the accumulation of PHH3-positive cells was observed with vinblastine (20 nM). Two-ways ANOVA with Sidak’s multiple comparisons test was used (ns=non-significant, * P < 0.05, *** P < 0.001). F, real-time imaging of cell confluence (IncuCyte) revealed that washout of 6-AA and vinblastine after 8 h allowed HeLa cells to resume proliferation, while transient treatment with 6-AA for 24 h visibly reduced cell proliferation (n=2, mean±SD). Arrow=washout. Supplementary Figure S5. 6-AA induces mitotic spindle defects in PC-3 cells. PC-3 cells treated with 6-AA, vinblastine, nocodazole (Noc), or vehicle control (DMSO) for 24 h were subjected to immunofluorescence microscopy of α-tubulin (green) and PHH3 (red) with a DeltaVision Elite microscope (60×). Representative images of cells in metaphase with misaligned chromosomes and abnormal spindle pole organization (6-AA, vinblastine and nocodazole treatments) are shown. Scale bar=10 µm. 9 Supplementary Figure S6. All anopterine analogs share the same mechanism of action. A, chemical structures of tested C20 diterpenoid alkaloids, which included 6αacetoxyanopterine (6-AA, 1), 4'-hydroxy-6α-acetoxyanopterine (2), 4'-hydroxyanopterine (3), 10 and 11α-benzoylanopterine (4), anopterine (5), 7β-hydroxyanopterine (6), 7β,4'- dihydroxyanopterine (7), and 7β-hydroxy-11α-benzoylanopterine (8). B, LNCaP cells treated with 6-AA (1, 5 nM), 2 (30 nM), 3 (1000 nM), 4 (400 nM), 5 (100 nM), 6 (300 nM), 7 (400 nM), 8 (10 nM), vinblastine (Vinb, 10 nM), or vehicle control (DMSO) for 24 h were subjected to immunofluorescence microscopy of α-tubulin (green) and DNA (blue) with an INCell 2200 automated imaging system (40×). Representative images of cells in metaphase with misaligned chromosomes and abnormal spindle pole organization are shown. Scale bar=10 µm. C, anopterine (5), like vinblastine (Vinb), inhibited tubulin polymerization in a cell-free system, while paclitaxel (Paclit) stimulated polymerization. A representative experiment (n=2) is shown. 11 Supplementary Figure S7. 6-AA does not affect polo-like kinase-1 and Aurora A kinase activities. A, LNCaP cells treated with 6-AA, the Aurora A kinase inhibitor MNL8237, the polo-like kinase 1 inhibitor BI2536, or vehicle control (DMSO) for 24 h were subjected to immunofluorescence microscopy of Aurora A (green) and DNA (blue) with a DeltaVision Elite microscope (60×). MNL8237 inhibited localization of Aurora A kinase at the spindle poles, which was not affected by 6-AA or BI2536. Representative images are shown. Scale bar=10 µm. B, LNCaP cells treated with 6-AA, the polo-like kinase 1 inhibitor BI2536, or vehicle control (DMSO) for 24 h were subjected to immunofluorescence microscopy of α-tubulin (green) and PHH3 (red) with a DeltaVision Elite microscope (60×). BI2536 inhibited spindle pole organization, uniformly generating cells with monopolar spindles and expanded chromosome 12 configurations, while 6-AA generated heterogeneous spindle pole abnormalities (monopolar, bipolar and multipolar) with visibly contracted chromosome alignments. Representative images are shown. Scale bar=10 µm. Supplementary Figure S8. The microtubule-depolymerizing action of 6-AA in LNCaP cells is reversible. LNCaP cells were treated with high doses of 6-AA, vinblastine or colchicine for 2 h, then washed and left to recover for 4 h. After washout of 6-AA and vinblastine, microtubules repolymerized, forming microtubule networks. Washout of the irreversible inhibitor colchicine failed to restore microtubule networks. Microtubules were visualized by immunofluorescence microscopy of α-tubulin with an INCell 2200 automated imaging system (40×). Scale bar=5 µm. 13 Supplementary Figure S9. 6-AA reduce the length of the polymerizing portion of microtubules in HeLa-EB1-GFP cells. HeLa-EB1-GFP cells were treated with 6-AA or vinblastine for 2 h, and EB1-GFP was imaged by spinning disk microscopy. EB1 comets were tracked and analyzed using Imaris software. The average displacement length of EB1 tracks is represented (10 cells/treatment), demonstrating that 6-AA caused a concentration-dependent decrease of the length of the polymerizing portion of microtubules. 14 Supplementary Table S1. Primer sequences of primers used quantitative Real Time-PCR (qRT-PCR) Primer Forward sequence Reverse sequence RPL32 5’-GCACCAGTCAGACCGATATG-3’ 5’-ACTGGGCAGCATGTGCTTTG-3’ CCNB1 5’-AGAGCCATCCTAATTGACTG-3’ 5’-CAACCAGCTGCAGCATCTTC-3’ CDC25B 5’-AGCGAAACCCCAAAGAGTCAGGT-3’ 5’-AGCAGCCTCACCGGCATAGACT-3’ PLK1 5’-TGCCCTACCTACGGACCTGG-3’ 5’-AAGTTGATCTGCACGCTGCC-3’ HMMR 5’-CTACCTTGCCTGCTTCAGCT-3’ 5’-AGCTGAAGCAGGCAAGGTAG-3’ PTTG1 5’-GAAAAGCTGTTTCAGCTGGGC-3’ 5’-CAGAATGCTTGAAGGAGACTGC-3’ CDKN3 5’-GGAAGAGCTTACAACCTGCC-3’ 5’-ACAGGTATAGTAGGAGACAAGC-3’ Supplementary Table S2: Cytotoxic activities of 6-AA against a human malignant cell lines. Compound IC50 ± SD (nM) LNCaP HeLa PC3 6-AA 3.1 ± 1.8 3.2 ± 1.1 5.1 ± 0.2 Vinb 3.2 ± 0.1 11.6 ± 4.2 2.5 ± 0.4 IC50 values of 6-AA and vinblastine (Vinb) in the indicated cell lines were calculated based on cell viability (alamarBlue) after 72 h of treatment (n=3, mean±SD). 15 Supplementary Table S3: List of selected mitosis-related genes upregulated by 6-AA (10 nM) as identified in the microarray. Gene symbole CDC20 AURKA CENPA CCNB1 HMMR KIF18A CKS2 KIF20A CCNB2 NUF2 TPX2 CCNA2 NEK2 CDC25B BUB1B CDCA8 KIF4A PLK1 BIRC5 BUB1 NDC80 CDKN3 KIF2C SPC25 CDK1 KIF23 AURKB KIFC1 PLK4 BORA NCAPH SMC4 CDK5RAP2 INCENP CENPK CNTRL CKS1B PCNT PTTG1 Gene description cell division cycle 20 aurora kinase A centromere protein A cyclin B1 hyaluronan-mediated motility receptor kinesin family member 18A CDC28 protein kinase regulatory subunit 2 kinesin family member 20A cyclin B2 NDC80 kinetochore complex component TPX2, microtubule-associated cyclin A2 NIMA (never in mitosis gene a)-related kinase 2 cell division cycle 25 budding uninhibited by benzimidazoles 1 homolog beta cell division cycle associated 8 kinesin family member 4A polo-like kinase 1 baculoviral IAP repeat containing 5 (survivin) budding uninhibited by benzimidazoles 1 NDC80 kinetochore complex component cyclin-dependent kinase inhibitor 3 kinesin family member 2C NDC80 kinetochore complex component cyclin-dependent kinase 1 kinesin family member 23 aurora kinase B kinesin family member C1 polo-like kinase 4 bora, aurora kinase A activator non-SMC condensin I complex, subunit H structural maintenance of chromosomes 4 CDK5 regulatory subunit associated protein 2 inner centromere protein centromere protein K Centriolin CDC28 protein kinase regulatory subunit 1B Pericentrin pituitary tumor-transforming 1 Fold change 2.80 2.43 2.42 2.40 2.40 2.37 2.31 2.23 2.21 2.21 2.21 2.20 2.14 2.05 2.03 2.03 2.03 2.03 2.01 2.01 2.01 1.95 1.94 1.91 1.80 1.79 1.77 1.77 1.75 1.72 1.71 1.69 1.66 1.66 1.60 1.60 1.57 1.51 1.48 For genes with multiple probes, the ‘differentially expressed reference probe with the highest fold change’ was used to represent the gene for functional analysis. 16 Supplementary Table S4: Cytotoxicity of 6-AA, 2-methoxyoestradiol, vincristine and doxorubicin against resistant cell lines. Compound CEM CEM/2ME2-14.4R CEM/2ME2-28.8-R IC50 (nM) IC50 (nM) RF IC50 (nM) RF 2ME2 765.9 ± 177.4 14,310.0 ± 4,851.4 18.7 23,410.0 ± 13,392.3 30.6 6-AA 2.6 ± 1.0 5.9 ± 0.7 2.3 4.5 ± 0.4 1.7 CEM Vinc CEM/VCR-R IC50 (nM) IC50 (nM) RF 0.5 ± 0.3 6,168.0 ± 1,359.2 12,336.0 6-AA 2.6 ± 1.0 117.7 ± 50.7 45.3 Dox 99.4 ± 34.9 24,75.3 ± 222.0 24.9 Cytotoxicity (IC50) of 6-AA, vincristine (Vinc), 2-methoxyestradiol (2ME2), and doxorubicin (Dox) was measured (72 h, alamarBlue) in indicated cell lines (upper panel, n=3, mean±SD). The resistance factor (RF) expresses the quotient IC50 (drug-resistant cell line)/IC50 (CEM). Supplementary Table S5: Cytotoxicity of 6-AA, vincristine, and doxorubicin in combination with verapamil against the multi-drug resistant CEM/VCR-R cell line. CEM CEM/VCR-R Compound IC50 (nM) IC50 (nM) RF Verap >10,000 >10,000 - Vinc + verap 1.3 ± 0.4 82.7 ± 42.2 63.6 6-AA + verap 1.6 ± 0.3 3.4 ± 0.4 2.1 Dox + verap 146.8 ± 54.7 163.9 ± 63.0 1.1 Cytotoxicity (IC50 ± SD) of indicated compounds in combination with verapamil (Verap) was measured in indicated cell lines (72 h, alamarBlue, left panel, n=3). The resistance factor (RF) expresses the quotient IC50 (CEM/VCR-R + verapamil)/IC50 (CEM + verapamil). Supplementary Table S6: Statistical data for Figure 1D Compound Sub G0-G1 G0-G1 S G2-M 6-AA (1.25 nM) ns ns ns ns 6-AA (2.5 nM) ** ** ns **** 6-AA (5 nM) ** *** ns **** Vinb (10 nM) ns *** ns **** For each phase of the cell cycle, one-way ANOVA with Dunnet's multiple comparisons test was used (comparison to DMSO, ns=non-significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001). 17 Supplementary Videos Supplementary Videos S1. Time-lapse microscopy of HeLa-H2B-GFP cells treated with DMSO, vinblastine (20 nM) or 6-AA (0.62 nM). HeLa-H2B-GFP cells were imaged using an Olympus IX81 (1 image/5 min for 24 h). Top panels: brightfield, bottom panels: H2B-GFP. DMSO-treated cells normally progressed through mitosis, which was delayed with vinblastine (20 nM) and low dose of 6-AA (0.62 nM). Both compounds induced asymmetric division. In addition, vinblastine caused cell death. Scale bar=20 µm. Supplementary Videos S2. Time-lapse microscopy of HeLa-H2B-GFP cells treated with 6-AA (2.5, 5 and 10 nM). HeLa-H2B-GFP cells were imaged using an Olympus IX81 (1 image/5 min for 24 h). Top panels: brightfield, bottom panels: GFP channel. 6-AA increased in a concentration-dependent manner mitotic duration, leading to cell death or asymmetric division. Scale bar=20 µm. Supplementary Videos S3. Interphase microtubule dynamics in HeLa-EB1-GFP treated with DMSO. EB1-GFP was imaged by spinning disk microscopy (1 image/s for 1 min). EB1 comets were clearly discernible and followed regular trajectories. Scale bar=10 µm. Supplementary Videos S4. Interphase microtubule dynamics in HeLa-EB1-GFP treated with 6-AA (2.5 nM). EB1-GFP was imaged by spinning disk microscopy (1 image/s for 1 min). EB1 comets followed irregular and short trajectories. Scale bar=10 µm. Supplementary Videos S5. Interphase microtubule dynamics in HeLa-EB1-GFP treated with vinblastine (20 nM). EB1-GFP was imaged by spinning disk microscopy (2 images/s for 2 min). EB1 comets appeared faint and followed irregular and short trajectories. Scale bar=10 µm. 18