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. 2022 Aug;73(1):e12808.
doi: 10.1111/jpi.12808. Epub 2022 Jun 5.

Melatonin and andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling

Daniil Sokolov  1 , Neha Sharda  1 , Banabihari Giri  2 , Md Sazzad Hassan  3   4 , Damandeep Singh  1 , Agnieszka Tarasiewicz  1 , Charity Lohr  1 , Urs von Holzen  3   4   5   6 , Tibor Kristian  7   8 , Jaylyn Waddell  1 , Russel J Reiter  9 , Hafiz Ahmed  10 , Aditi Banerjee  1
Affiliations

Melatonin and andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling

Daniil Sokolov et al. J Pineal Res. 2022 Aug.

Abstract

β-catenin signaling, and angiogenesis are associated with colospheroid (CSC), development. CSCs, spheroids derived from colon cancer cells, are responsible for metastasis, drug resistance, and disease recurrence. Whether dysregulating β-catenin and inhibiting angiogenesis reduce CSC growth is unknown. In this study, the molecular mechanism of CSC growth inhibition was evaluated using a novel combination of melatonin (MLT) and andrographolide (AGP). These drugs have anticarcinogenic, antioxidant, and antimetastatic properties. CSCs were obtained from two metastatic colon cancer cell lines (HT29 and HCT-15). The viability and stemness were monitored (FDA propidium iodide staining and immunoblot for CD44, CD133, Nanog, Sox2, and Oct4). The drug combination synergistically diminished stemness via increased reactive oxygen species (ROS) levels, reduced mitochondrial membrane potential and ATP level. MLT + AGP induced cell death by inhibiting β-catenin expression and its downregulatory signals, Cyclin D1, c-Myc. MLT + AGP treated cells exhibited translocation of phospho-β-catenin to the nucleus and dephosphorylated-β-catenin. Downregulation of β-catenin activation and its transcription factors (TCF4 and LEF1) and GTP binding/G-protein related activity were found in the dual therapy. Angiogenic inhibition is consistent with downregulation of VEGF messenger RNA transcripts (VEGF189), phosphorylated VEGF receptor protein expression, matrigel invasion, and capillary tube inhibition. In vivo, the intravenous injection of MLT + AGP slowed HT29 metastatic colon cancer. Histopathology indicated significant reduction in microvascular density and tumor index. Immunohistochemistry for caspase 7, and β-catenin found increased apoptosis and downregulation of β-catenin signals. The mechanism(s) of decreased colospheroids growth were the inhibition of the Wnt/β-catenin pathway. Our results provide a rationale for using MLT in combination with AGP for the inhibition of CRCs.

Keywords: Wnt/β-catenin signals; andrographolide; angiogenesis; colospheroids; melatonin; xenograft.

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Figures

Figure 1.
Figure 1.. Impact of MLT alone or in combination on colospheroids.
A. HT29 and HCT-15 cells were used for CSCs chronological development from HT29 and HCT-15 mCRC cells, HT29-s (upper lane) and HCT-15-s (lower lane). Mature and dividing CSCs were found at day 5 and day 3, respectively. Mature CSCs were treated with or without the IC50 dose, MLT (0.18 mM) and AGP (9.3 μM) at 48 h. Untreated group maintained their spheroid formation (B-upper HT29-s and lower HCT-15-s). Membrane disintegration and reduced size were observed in the treated group in comparison with the untreated or single treatment groups using phase contrast microscope. Magnification: 10X and scale bar 100 micron. C-D. Fluorescence microscopy images showing the viability of HCT-15-s and HT29-s cultured in vitro as indicated, phase contrast image, FDA stain, PI stain, overlay of FDA and PI stain. E. Apoptotic features were identified by condensation and fragmentation in MLT+AGP treated colospheroids using DAPI staining. Images were captured using inverted fluorescence microscope.
Figure. 2.
Figure. 2.. Combination of MLT and AGP reduces colospheroids stemness.
A-D. Characterization of stemness. CSCs lysates were analyzed by immunoblot for CD44, Nanog, CD133, and GAPDH expression. Left panel is a representative photograph from an experiment that was repeated three times. HT29-s (A) and HCT-15-s (C). Right panel (B-D) represents quantitative estimations of protein levels determined by densitometry measurements of immunoblots from three independent experiments after normalization with GAPDH (* P<0.05, ***P<0.001). E-F. Treatment of spheroids with or without AGP (9.3 μM) and MLT (0.18 mM) for 48 h suppresses CSC-s stemness. G-I. Quantitative measurements (G, CD44; H, Nanog; I, CD, 133; **P<0.01, ***P<0.001).
Figure. 3.
Figure. 3.. Co-treatment of MLT and AGP suppresses β-catenin signaling.
A. Immunoblots from treated or untreated CSCs extracts were used for monitoring β-catenin signaling protein expression. GAPDH was used as a loading control. Quantification of B. Phospho EGFR, C. Phospho β-catenin, D. Sox2, E. Oct4. Statistical significance was determined by one way-ANOVA followed by the Bonderroni test. (*P<0.05, **P<0.01, ***P<0.001). F. HT29-s were grown on poly-L-Lysine coated coverslips. Phospho- (left panel)-and total β-catenin (right panel) protein expression were evaluated by confocal microscopy. Nuclei were stained using DAPI. Fluorescence intensity was determined and compared with untreated CSCs (G-H). I. Quantification. H. Analysis of phospho- and total β-catenin distribution. Equal number spheroids equivalents were electrophoresed and assayed by immunoblot. H3 and GAPDH levels served as the nuclear and cytoplasmic loading control, respectively. I. Representative quantification for phospho/total β-catenin using densitometry (**P<0.01).
Figure. 4.
Figure. 4.. Impact of MLT and AGP on β-catenin and Rho-activity in HT29-s.
The impact of MLT and AGP on luciferase reporter activity. Spheroids were treated with or without AGP/MLT as previously described. The results are presented as ratio of firefly luminescence to Renilla luminescence. The y-axis represents the luciferase activity of treated colospheroids relative to untreated spheroids (**P< 0.01, ***P<0.001). B. A pull-down assay was used to detect RhoA activity as described. Quantification of relative phospho-Rho kinase level/total Rho kinase by densitometry measurements of immunoblots from three independent experiments. (**P<0.01, ***P<0.001).
Figure 5.
Figure 5.. MLT AGP inhibits β-catenin/TCF signaling.
mRNA derived from treated or untreated HT29s were monitored for the β-catenin/Tcf signal associated gene expression by qRT-PCR. A. lgr5, tcf4, and lef1 (Left, middle and right). Bar graphs show quantitative results normalized to GAPDH mRNA levels. Results are from three independent experiments. B. Immunoblot analysis of treated or untreated HT29-s extracts as indicated and C. quantification. LGR5, TCF4, LEF1, Axin-1. D-E. HCT-116 cells were transfected with plasmid for overexpression of β-catenin and then treated with or without AGP/MLT. Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of phospho- β-catenin, GSK3β, and total β-catenin, GSK3β, Cyclin D1, c-Myc. F-G. HCT-116 parental cell lysates were monitored as indicated and quantified. All statistical significance were determined using one-way ANOVA followed by Bonderroni test (*P<0.05, **P< 0.01, ***P<0.001).
Figure 6.
Figure 6.. MLT and AGP synergistically downregulates angiogenic signaling in colospheroids.
Treated/untreated HT29-s lysates were subjected for protein expression as indicated. Phospho protein was normalized with total protein B-D. All statistically significant differences were determined using one-way ANOVA followed Bonferroni test (*P<0.05, **P< 0.01, ***P<0.001). D, F-G. MLT+AGP suppressed angiogenic branching and total capillary tube length. E. Matrigel invasion inhibition of HT29-s after treatment as indicated. H. The invaded cells were quantified by counting using an optical microscope at 100X magnification and averaged after counting five fields per membrane (**P< 0.01, ***P<0.001).
Figure 7.
Figure 7.. Tumor growth inhibition by MLT and AGP in HT29 cell-derived subcutaneous xenografts.
HT29 tumor-bearing nude mice were treated with MLT (40 mg/kg, 5 times a week) and AGP (20 mg/kg, 5 times a week) alone and in combination for 2 weeks. A. Tumor size was measured twice a week using calipers and the data were plotted. RTV changes over a period of 2 weeks after injection of drugs were shown. B. RTV changes after 2 weeks of drug treatments were plotted and compared. C. Mean tumor weight was calculated from final day tumor weights and were plotted. D. Mouse body weight was measured twice a week during the 2-week therapy period and were plotted. Data are representative of mean values ± standard deviations from 8 mice per group. E. H&E-stained tumor sections identifying the microvascular density in treated and untreated tumor tissue. Arrows indicate microvessels. F. H&E staining identifying the mitotic index (arrowheads). The right-side representative histograms are for the microvessels area and percentage of mitotic index from ten representative areas of each tumor. Statistical significance was determined using one way-ANOVA followed Bonferroni test (**P<0.01, ***P<0.001). G. Immunohistochemistry for Ki67 expression in HT29 CRC tumor sections with or without MLT, and AGP (200X). The histogram on the right panel represents the average percentage of Ki67 expression.
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