Abstract
Background/Aim: Pyra–Metho–Carnil (PMC) has been identified as a novel candidate compound for treating numerous malignancies; however, its mechanism of action remains unknown. In this study, we conducted RNA-sequencing (RNA-seq) analyses to elucidate the mechanism of PMC against human colorectal cancer cells harboring mutant KRAS (mtKRAS). Materials and Methods: RNA-seq analyses of the HKe3-wild-type KRAS and HKe3-mtKRAS spheroids treated with DMSO or PMC for 6 days were performed. Results: RNA-seq data suggested that PMC treatment suppresses the aerobic glycolysis pathway in HKe3-mtKRAS spheroids through the down-regulation of the HIF1 pathway. Indeed, treatment with PMC markedly suppresses the absorption of glucose by spheroids and the secretion of lactate from them. Conclusion: PMC suppresses growth of cancer spheroid through down-regulation of cancer-specific glucose metabolism.
KRAS is a key molecule in the aberrant proliferation and malignant progression of cancer cells (1, 2).
Activated KRAS or KRAS-related tumor-promoting pathways are important targets for cancer therapeutics. Inhibitors of KRAS with specific mutations such as G12C (sotorasib) have been developed; however, 10% of oncogenic KRAS mutations are attributable to the G12C mutation (3-5). In addition, the efficacy of direct KRAS inhibitors and inhibitors of downstream KRAS signals targeting BRAF and MEK is temporal because cells often acquire drug resistance (6-8). Remarkably, conventional cancer treatments exhibited considerable toxicity when administered as therapy (9). To overcome these problems, we developed two isogenic colorectal cancer-derived HKe3 cell lines, whose endogenous allele of mutant KRAS (mtKRAS) G13D was disrupted and re-expressed either wild-type KRAS (wtKRAS; normal cell model) or mtKRAS G13D (cancer cell model) (10, 11). Further, we conducted a screening process on these cells to find natural compounds that selectively and effectively eliminate cancer spheroids. We identified a compound, Pyra–Metho–Carnil (PMC), which exhibits a specific antitumor activity for HKe3-mtKRAS spheroids (12). Furthermore, PMC demonstrates growth-restricting properties against cancer spheroids with various genetic mutations, regardless of tissue type (12), suggesting that PMC can target common features of cancer spheroids. However, the exact mechanisms of how PMC exerts its effects remain uncertain. In this study, we used RNA-sequencing (RNA-seq) analyses on cancer spheroids treated with PMC for 6 days and revealed the mechanisms underlying the PMC-induced tumor growth inhibition.
Materials and Methods
Compounds. PMC (IUPAC Name: 1-{3-[(3,5-dimethylpyrazol-1-yl)methyl]-4-methoxyphenyl}-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole) was synthesized by Namiki Shoji Co., Ltd. (Tokyo, Japan) as described previously (12).
Cell culture. HKe3-wtKRAS and HKe3-mtKRAS cultures were established and maintained as previously described (10, 11).
3D floating cell culture. Cells were seeded in 96-well plates with round bottoms and ultralow attachment surfaces (product number 7007; Corning Inc., Corning, NY, USA) and treated with PMC on day 0 as described previously (10, 12).
RNA-seq. RNA-seq libraries were prepared using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (E6310, NEB, Ipswich, MA, USA) and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420, NEB) per the manufacturer’s protocols, with 700 ng of total RNA as the starting material and eight polymerase chain reaction cycles for library amplification. The RNA-seq libraries were sequenced on a HiSeq X platform (Illumina, San Diego, CA, USA) in the paired-end mode (150 bp ×2) at the GENEWIZ NGS Laboratory (South Plainfield, NJ, USA). Sequence reads in the FASTQ format were aligned to the hg19 human reference genome using hisat2-2.1.0. To conduct an exon-wise expression analysis, we compiled a nonredundant list of 316,759 exons in a bed file format from the gencode.v27lift37.gtf file obtained from the GENCODE website (https://www.gencodegenes.org). Bam files generated by hisat2 were subjected to the coverage command of bedtools to count mapped read numbers for each of the 316,759 exons. The count data of eight samples were combined and normalized by the total read count numbers and used to search for exons showing different expression levels. Data were visualized using the Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA).
RNA-seq analysis. To identify genes that were upregulated in HKe3-mtKRAS spheroids and repressed by PMC treatment, the genes whose expression levels were 1.5 times higher in HKe3-mtKRAS spheroids compared to HKe3-wtKRAS spheroids and 0.66 times lower in PMC-treated HKe3-mtKRAS spheroids compared to dimethyl sulfoxide (DMSO)-treated HKe3-mtKRAS spheroids (p<0.05) were selected. Furthermore, genes whose expression levels were significantly changed by PMC in the normal cell model (HKe3-wtKRAS spheroids) and whose fragments per kilobase of exon per million reads mapped values were <50 in HKe3-mtKRAS samples were excluded.
Glucose uptake. HKe3-wtKRAS spheroids (4×103 cell/well) and HKe3-mtKRAS spheroids (1×103 cell/well) were cultured using 10% FBS DMEM (high glucose) in 96-well ultralow attachment plate for 7 days. Spheroids were treated with PMC (30 μM) in 400 μl of no-glucose DMEM (Thermo Fisher Scientific, Waltham, MA, USA), without the FBS medium. Cells were incubated at 37°C with 5% CO2 (20% O2) for 1 h. The medium (200 μl) was removed without disturbing the cells. A 200-μl aliquot of 400-μM 2-NBDLG {2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose} (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) resolved in DMEM without glucose was gently added to each well, and spheroids were incubated at 37°C with 5% CO2 for 1 h. Spheroids were washed using PBS and dispersed using accutase solution (Merck, Darmstadt, Germany) for 10 min at 37°C. Flow cytometry was performed using FACSVerse™ (BD Biosciences, Fraklin Lakes, NJ, USA) and analyzed using FlowJo™ (BD Biosciences).
Lactate assay. We performed 3D floating culture using HKe3-wtKRAS or HKe3-mtKRAS cells. A 20-μl aliquot of the culture supernatant was used for the Lactate Assay Kit-WST (Dojindo, Kumamoto, Japan) per the manufacturer’s protocol.
Antibodies. Anti-HIF1α (D1S7W), anti-HIF1β (D28F3), anti-PDK1(#3062), anti-GLUT1(D3J3A), and anti-Hexokinase II (#2106) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Western blotting. Cells were lysed in RIPA buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and subjected to immunoblotting as described previously (13).
Results
Expression analysis of molecules repressed by treatment with PMC in spheroid cultures. To gain a more comprehensive understanding of how PMC suppresses tumor growth, we conducted RNA-seq on RNA extracted from HKe3-mtKRAS spheroids and HKe3-wtKRAS spheroids, treated with either PMC or DMSO as a control for 6 days (Figure 1A). Using RNA-seq data, we identified 81 genes that may underlie PMC-induced growth inhibition (Table I). As their expression levels were specifically affected by PMC treatment, these genes potentially include molecules targeted by PMC in HKe3-mtKRAS spheroids.
Transcriptional down-regulation of genes involved in glycolysis and HIF1-mediated responses in HKe3-mtKRAS spheroids upon PMC treatment. (A) Experimental scheme of cell preparation for RNA-sequencing. HKe3-mtKRAS and HKe3-mtKRAS cells were seeded into 96-well plates with round bottoms and ultralow attachment surfaces, and treated with or without PMC (30 μM) for 6 days. Spheroids were collected from 96 wells and subjected to RNA-sequencing on day 6. Gene expression levels were analyzed using data from four independent experiments. (B) Gene ontology enrichment analysis using the Metascape software for genes whose expression is specifically down-regulated by PMC in HKe3-mtKRAS spheroids. (C) Summary of the enrichment analysis performed using Metascape for transcription factor targets of genes specifically down-regulated by PMC in HKe3-mtKRAS spheroids. Enriched terms are shown as bar graphs and color-coded by p-values. PMC, Pyra–Metho–Carnil.
Genes up-regulated in HKe3-mtKRAS spheroids and repressed by PMC treatment.
To identify the biological processes affected by PMC in cancer spheroids, we performed an ontology analysis on genes identified in our RNA-seq data. Ontology analysis revealed that biological processes such as glycolysis, HIF1 signaling, and protein hydroxylation were remarkably enriched among the genes that were down-regulated in PMC-treated HKe3-mtKRAS spheroids (Figure 1B). Furthermore, the in-silico analysis of transcription factor targets revealed an abundance of pathways controlled by HIF1 (Figure 1C). These data suggest that treatment of HKe3-mtKRAS spheroids with PMC reduces the transcriptional level of factors in the glycolysis pathways regulated by HIF1.
To further substantiate these results, we assessed the changes at the protein level of some HIF1-regulated genes by immunoblotting using spheroid cell lysates (Figure 2A). As expected, PMC reduced the expression of glycolysis-associated proteins (PDK1, GLUT1, and HK 2). Given that cancer cells consume high energy and prefer to use glycolysis for ATP production, these data suggest that PMC exerts its antitumor effects through the inhibition of cancer-specific glucose metabolism known as the Warburg effect in cancer spheroids. Interestingly, HIF1 protein levels were found decreased in PMC-treated spheroids on day 6 (Figure 2B). In HKe3-mtKRAS spheroids, mRNAs of glycolysis-related genes (PDK1, GLUT1, HK2, and LDHA) were decreased by PMC; however, the RNA-seq analysis did not reveal a decrease in the HIF1A and B mRNAs (Figure 2C), suggesting that PMC can degrade HIF1 proteins through mechanisms that are currently unknown.
PMC suppresses the expression of the hypoxia adaptation marker, HIF1, and its downstream factors in HKe3-mtKRAS spheroids. (A, B) Immunoblotting analysis of PDK1, GLUT1, HK2, HIF1α, and HIF1β in HKe3-mtKRAS spheroids treated with DMSO (Control) or PMC (30 μM) for 6 days. Actin was used as the loading control. (C) Visual representation of RNA-seq results (screenshot of the Integrative Genomics Viewer) of HKe3-mtKRAS and HKe3-wtKRAS spheroids treated with DMSO or PMC (30 μM) for 6 days for genomic loci containing the HIF1α, HIF1β, PDK1, GLUT1, HK2, or LDHA genes. The MYC gene was used as an unaffected control. The bottom rows represent the exon-intron structures of the genes. Values in the Y-axes of the panels represent the ranges of mapped reads. PMC, Pyra–Metho–Carnil; DMSO, dimethyl sulfoxide; PDK1, pyruvate dehydrogenase kinase 1; GLUT1, glucose transporter 1; HK2, hexokinase 2; HIF1, hypoxia inducible factor 1; LDHA, lactate dehydrogenase A.
PMC suppresses glucose uptake and lactate production. GLUT1 is a representative glucose transporter involved in the uptake of glucose. We performed a glucose uptake assay in PMC-treated spheroids (Figure 3). Under control treatment with DMSO, glucose levels of HKe3-mtKRAS spheroids were higher than those of HKe3-wtKRAS spheroids. PMC treatment led to reduction of glucose levels of HKe3-mtKRAS spheroids to levels similar to those of HKe3-wtKRAS spheroids (~50% reduction compared to HKe3-mtKRAS spheroids treated with DMSO). Lactate levels were also decreased by PMC treatment in the culture supernatant of HKe3-mtKRAS spheroids (Figure 4). These data confirm that cellular glucose metabolism was actually suppressed by PMC treatment on day 6.
PMC suppresses glucose uptake in HKe3-mtKRAS spheroids. Fluorescence images (A) or flow cytometry analyses (B and C) of HKe3-wtKRAS or HKe3-mtKRAS spheroids treated with DMSO (Control) or PMC (30 μM) for 1 h on day 7, using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG) to visualize glucose uptake levels. (A) Representative images of spheroids are shown. Blue, nuclear DNA; green, 2-NBDLG. Scale bar, 100 μm. (B) Distribution of the 2-NBDLG signal intensity in cells dispersed from spheroids. (C) Median values of the 2-NBDLG signal normalized to the signal intensity of unstained control samples. The data represent the mean±SD of three independent experiments. PMC, Pyra–Metho–Carnil; DMSO, dimethyl sulfoxide.
PMC suppresses lactate production. Relative lactate levels of the culture supernatant of HKe3-wtKRAS or HKe3-mtKRAS spheroids treated with DMSO (control) or PMC (30 μM) for 3 or 6 days. The data represent the mean±SD of three independent experiments. *p<0.05: **p<0.01 (Student’s t-test). PMC, Pyra–Metho–Carnil; DMSO, dimethyl sulfoxide.
Discussion
Together, RNA-seq and immunoblotting analyses revealed that PMC treatment down-regulates the expression of a group of glycolysis-associated proteins (Table I; Figure 1 and Figure 2). Among the proteins down-regulated by PMC, GLUT1 promotes glucose uptake into the cell, HK2 phosphorylates glucose to produce glucose-6-phosphate in the first step of glycolysis, and PGK1 catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate to generate ATP molecules (14-16). In contrast, PDK1 inactivates pyruvate dehydrogenase, thereby inhibiting the influx of glucose-derived acetyl-CoA into the tricarboxylic acid (TCA) cycle, and LDHA promotes the production of lactate from pyruvate supplied by glycolysis (14-16) (Figure 5). These proteins are regulated by HIF1, as suggested by the ontology analysis (Figure 1), which plays a central role in the adaptation to hypoxic conditions (15, 17). Cancer cells also promote the production of lactate even in aerobic conditions. This aerobic glycolysis and TCA cycle inhibition in cancer cells are known as the Warburg effect (14, 18). While HIF1 signaling modulates the key processes required for the Warburg effect, aerobic glycolysis contributes to accumulation of HIF1 proteins in cancer cells, suggesting a complex association between HIF1 signaling and the Warburg effect (14, 15, 19).
A model for the effect of PMC to suppress spheroid growth through the negative regulation of HIF1 signaling pathways. The cancer spheroid environment stimulates HIF1 signaling pathways to up-regulate the expression of glycolysis-associated proteins such as GLUT1, PDK1, HK2, PGK1, and LDHA. GLUT1 promotes glucose uptake into cells. PDK1 inhibits the tricarboxylic acid (TCA) cycle in mitochondria. HK2, PGK1, and LDHA promote energy metabolism through anaerobic glycolysis and lactate production. These glycolysis-associated proteins contribute to tumor growth through aerobic glycolysis, a phenomenon known as the Warburg effect. PMC may inhibit cancer spheroid growth by suppressing cancer-specific and HIF1-regulated glucose metabolism. PMC, Pyra–Metho–Carnil; HIF1, hypoxia inducible factor 1; GLUT1, glucose transporter 1; PDK1, pyruvate dehydrogenase kinase 1; HK2, hexokinase 2; PGK1, phosphoglycerate kinase 1; LDHA, lactate dehydrogenase A.
Metabolic reprogramming is among the hallmarks of cancer, and the reprogramming of metabolic pathways in mutant KRAS cells contributes to active protein synthesis and cell proliferation (16, 20, 21). The regulation of glycolysis for energy production in cancer cells has become a focus of research in therapies that selectively target cancer cells (14). The findings of this study suggest that PMC exerts its anticancer effects through inhibition of HIF1-mediated pathways (Figure 5). We also hypothesized that the uptake of glucose into spheroid cells and cellular levels of lactate were reduced by PMC (Figure 3 and Figure 4), suggesting that PMC (and its derivatives) can pave the way for the development of anticancer therapies targeting the Warburg effect (which is a typical feature of tumors). In this study, RNA-seq of 3D spheroids of HKe3-wtKRAS and HKe3-mtKRAS cells treated with PMC was performed on day 6. Further analyses at earlier time points after the addition of PMC would reveal the precise mechanisms leading to down-regulation of HIF1.
Acknowledgements
The Authors thank Yuriko Isoyama and Yumiko Hirose for technical assistance. This work was supported by Grant-in-Aid for Scientific Research (KAKENHI, Grant Number 15K06847, 18K07215, 21K07161, and 22K07221) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and the Fukuoka Foundation for Sound Health Cancer Research Fund.
Footnotes
Authors’ Contributions
K.Y., Kensuke N., T.M., and T.T. performed experiments, analyzed the data, and wrote the first manuscript draft. S.I., Kazuhiko N., R.Y., and M.S. participated in study design, data collection, and analysis. T.O. and S.S. conceived the idea, designed the study, interpreted the data, provided important intellectual content, and obtained final approval for manuscript submission.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received May 13, 2023.
- Revision received June 12, 2023.
- Accepted June 19, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
