(M1430-02-08) Transcriptomic Analysis Reveals that Nymphaea odorata Extracts and Fractions Upregulate the Apoptosis, Autophagy, and Unfolded Protein Canonical Pathways in MCF-7 Cells

Purpose: Nymphaea odorata Aiton (Nymphaeaceae; NO; American white water-lily) is a perennial aquatic plant native to USA and Canada and used in traditional medicine for the management of cancer. In this work, we investigated the effects of an extract (MeNO), fractions and one purified compound (methyl gallate) from MeNO in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line MCF-7 and the ER negative line SKBr3. RNA-seq was also performed for whole transcriptome analysis in MCF-7 cell to determine biological networks and canonical pathways involved in its mechanism of action.

Methods: MCF-7 and SKBr3 cells were cultured and maintained as we have previously described.¹ Cell viability and cytotoxicity were determined using CellTiter-Glo® 2.0 assay at concentrations from 1 to 100 μg/mL. Caspase activity and apoptosis were determined using Caspase-Glo® 3/7, Caspase-Glo® 8, and ApoTox-Glo™ triplex assays, and qPCR. Autophagy was measured using a Cyto-ID Autophagy Detection Kit. To determine potential mechanisms of action, RNA-seq was performed. Total RNA was isolated from MCF-7 cells treated with MG, and RNA-seq libraries were prepared using a Universal Plus mRNASeq kit. Sequencing was carried out using a NovaSeq 6000. General quality-control metrics were measured using FastQC and raw reads were aligned to human reference genome hg38 using STAR and BWA MEM. ENSEMBL genes were quantified using FeatureCounts, and differential expression statistics were computed using EdgeR. Specific gene expression was validated using qPCR.

Results: The MeNO extract inhibited the growth of MCF-7 cells with an IC50 of 20.6 µg/ml and SKBr3 cells with an IC₅₀ of 35.5 µg/ml.  Thus, the extract was more cytotoxic to estrogen receptor positive human breast adenocarcinoma cells. Flow cytometry data clearly indicated that, MeNO induced apoptosis in MCF-7 cells. The active fraction of the extract contained the known compound, methyl gallate (MG), and had an IC50 of 8.6 µg/ml. Transcriptomic analysis showed that of 26,048 genes analyzed, the expression of 10,634 genes was significantly altered (5643 up-regulated and 4991 down-regulated, FDR < 0.05). Ingenuity Pathway analysis revealed the involvement of 67 canonical pathways, with the top upregulated pathways included: apoptosis, autophagy, myc-mediated apoptosis, p53 and the unfolded protein response pathway. These data suggest that MeNO has antiproliferative effects in breast cancer cells and one of the active constituents is MG, that has multiple mechanisms of action in MCF-7 breast cancer cells.

Conclusion: Nymphaea odorata, a medicinal and food plant used traditionally for the treatment of cancer inhibits the growth of breast cancer cells but appears more effective in ER+ cell lines. Whole transcriptomic analysis suggests that the active compound MG induces apoptosis, autophagy and the unfolded protein response in MCF-7 cells indicating multiple mechanisms of action.

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Acknowledgements: This work was funded in the Schlumberger Foundation Fellowship award to TOL, and a Raman Post-Doctoral Fellowship by University Grants Commission, Govt. of India to NAR. PK and MMC were supported partly by the University of Illinois at Chicago (UIC) Center for Clinical and Translational Science (CCTS) by Grant #UL1TR002003 from the National Center for Advancing translational Sciences, National Institutes of Health. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the funding agencies. The funding agency had no role in the design of the study and collection, analysis, or in interpretation of data and in writing the manuscript.

Figure 1. NOM extract upregulates Myc-mediated apoptosis signaling in MCF-7 cells