9-Deazaadenosine directly binds PYCR1 and inhibits cancer cell proliferation through disruption of NAD+ metabolism

Highlights

• •9-DAA effectively inhibited colon cancer proliferation and metastasis.
• •9-DAA directly bound to PYCR1.
• •PYCR1 was upregulated in colon cancer under hypoxic conditions and in 3D spheroids.
• •The inhibitory effects of 9-DAA were NAD+-dependent.

Abstract

Cancer cells exhibit abnormal proliferation and dysregulated cell cycle checkpoints. Therefore, discovering cell cycle inhibitors is a promising strategy for cancer treatment. The pyrroline-5-carboxylate reductase 1 (PYCR1) is a key enzyme that controls proline metabolism by regulating the conversion of pyrroline-5-carboxylate to proline. PYCR1 is highly expressed in various cancers, including colon cancer. In this study, we discovered a novel role of 9-deazaadenosine (9-DAA) as a cell cycle inhibitor and demonstrated that this compound directly binds to PYCR1. Our results suggest that the inhibitory effects of 9-DAA are due to nicotinamide adenine dinucleotide. We further demonstrated that PYCR1 was elevated under hypoxia and in 3D spheroids in colon cancer and that 9-DAA effectively inhibited cancer progression under cancer-mimicking conditions and in vivo.

Graphical abstract

Introduction

Colorectal cancer is one of the most common cancers worldwide [1,2]. In recent years, colon cancer, including lung, prostate, and breast cancers, has been considered the main cause of cancer-related deaths [3]. Various genetic and environmental risk factors, including smoking, alcohol intake, obesity, and a high-fat diet, play important roles in the development of colorectal cancer [1,4,5]. Colorectal cancer is caused by various genetic mutations, including adenomatous polyposis coli (APC), AKT, TP53, and K-RAS mutations; p53 mutations are responsible for 50 % of colorectal cancers [1,6]. Additionally, these mutations promote cancer progression and metastasis, making it difficult to treat [7,8].

The cell cycle is a well-controlled process of cell division [9]. Thus, various proteins carefully regulate the cell cycle, and checkpoints can tightly control the cell cycle in response to cellular damage, such as DNA damage or cell death [[10], [11], [12]]. Contrastingly, genetic mutations in cancer impair cell cycle checkpoints and sustain cell division [13]. Thus, cell cycle inhibitors are considered therapeutic drugs for cancer treatment [14]. Furthermore, various cell cycle inhibitors that inhibit cyclin-dependent kinases (CDK) 4 and 6 have been approved by the Food and Drug Administration for anticancer treatment, and others are being tested in clinical trials as novel anticancer drugs [15,16].

Pyrroline-5-carboxylate reductase 1 (PYCR1) is a mitochondrial enzyme that catalyzes the conversion of pyrroline-5-carboxylate (P5C) into proline [17]. This conversion process uses the reducing power of NADH to produce NAD+. Proline metabolism controls proline synthesis and catabolism, which occur in the cytoplasm and mitochondria [18]. PYCR3, which has 45 % similarity to PYCR1, is the most abundantly expressed in the cytoplasm and regulates proline biosynthesis through the conversion of P5C to proline in an NADPH-dependent manner [18]. PYCR1 and PYCR2, which share 85 % similarity, are expressed in the mitochondria. Both these enzymes control the conversion of P5C to proline and use NADH as a cofactor, unlike PYCR3 [17,18]. Proline is used in protein synthesis and supports cancer cell proliferation [17]. Additionally, NAD+ or NADP+, which are byproducts of PYCR, are used in other metabolic pathways, such as the tricarboxylic acid (TCA) cycle and pentose phosphate pathway, respectively [19,20]. Moreover, some studies have reported that PYCR1 is overexpressed in various malignant tumors, including non-small cell lung cancer, breast cancer, and colon cancer; consequently, it is considered an oncogene [[21], [22], [23]]. Therefore, proline metabolism and related enzymes are potential targets for cancer therapy [21].

Adenosine and adenosine analogs have long been used as drugs [24,25]. Generally, adenosine analogs are used as antiviral or anticancer drugs because they inhibit DNA and RNA synthesis [26]. Here, we identified the cellular target protein of 9-deazaadenosine (9-DAA), a known anticancer agent [27], and a newly suggested mode of action. Unlike the traditional role, we suggested a detailed mechanism of action of 9-DAA as a cell cycle and metastasis inhibitor and as a direct binding reagent of PYCR1 in colon cancer. This compound arrests the cell cycle, does not cause cell death, and plays a p53-independent role. These effects were associated with the direct binding of 9-DAA to PYCR1, and dysregulation of PYCR1 inhibited cell cycle progression and suppressed cancer metastasis. Furthermore, the inhibitory effects of 9-DAA on cancer cells decreased tumor volume in xenograft models. Thus, we suggest that 9-DAA may be useful for the treatment of PYCR1-overexpressed cancers.

Materials and methods

Reagents

9-DAA was provided by J&C SCIENCES Co. in Korea. The detailed synthetic methods are described in the Supplementary Information.

HCT 116 (CCL-247), SW480 (CCL-228), and SW620 (CCL-227) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The phosphatase inhibitor cocktail 3 (P0044) and 5-ethynyl-2′-deoxyuridine (#900584) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Lipofectamine™ 3000 transfection reagent (L3000001), penicillin/streptomycin buffer (100 U/100 μg/mL; #15140122), RIPA buffer (#89900), Click-iT™ Plus Alexa Fluor™ 647 picolyl azide toolkit (C10643), SuperSignal West Pico Chemiluminescent Substrate (#34080), SuperSignal West Femto maximum sensitivity substrate (#34095), and M-PER buffer (#78501) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The protease inhibitor cocktail (#5056489001) and X-tremeGENE™ siRNA Transfection Reagent (#4476093001) were purchased from Roche (Basel, Switzerland). Fetal bovine serum (FBS; S001–07) and Dulbecco’s modified Eagle’s medium (DMEM; LM 001–05) were purchased from Welgene (Gyeongsangbuk-do, Korea). Nitrocellulose membranes (0.2 μm; 162–0112) were purchased from Bio-Rad (Hercules, CA, USA). The Human Topoisomerase ICE assay kit (TG1020) was purchased from TopoGEN (Buena Vista, CO, USA). CellTiter-Glo® Luminescent assay kit (G7570) was purchased from Promega (Madison, WI, USA).

The primary antibodies were purchased from the following resources: CDK4 (#12790), Cyclin D1 (#2978), p27 (#3686), PYCR1 (#37635), p-AMPK (#2535), AMPK (#2532), p-p38 (#4511), p38 (#9212), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signalling Technology; β-actin (sc-47778), p21 (sc-756), and p53 (sc-126) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

Animal studies

Animal studies were performed following the ARRIVE guidelines [28]. All the experiments were approved by the KRIBB Institutional Animal Care and Use Committee (KRIBB-AEC-23338). Five-week-old male BALC/c-nude mice were purchased from NARA Biotech (Seoul, Korea). The mice were acclimated to the animal facility for 1 week. The mice were maintained under a 12:12 h light/dark cycle in constant-temperature-controlled environments.

Xenograft studies in mice

For the xenograft studies, one million HCT 116 cells were subcutaneously injected into the hind flanks of mice. When the tumor volume reached approximately 50 mm³, the mice were randomly divided into two groups. Each mouse received intraperitoneal injections of dimethyl sulfoxide (DMSO) or 9-DAA (0.5 mg/kg) once every 2 days for a total of five times according to the experimental scheme in Fig. 8A. The 9-DAA dose was determined based on a previous study [27]. The tumor sizes were measured every 2 days. The volumes were calculated using the following equation:

$$\text{Tumor}\text{volume}=\text{lengh}\times \text{widt}{\mathrm{h}}^2/2$$

Fig. 8.

9-DAA has inhibitory effects in xenograft mouse models. (A) Scheme of xenograft mouse experiments. (B) Weight of mouse was measured once every three days after treatment with 9-DAA (0.5 mg kg⁻¹). (mean ± SD; DMSO = 5; 9-DAA = 5). (C) The tumor volume was measured once every two days after treatment with 9-DAA (0.5 mg/kg). Data were analyzed using two-way ANOVA followed by Šídák‘s post hoc test. (mean ± SD, n = 5; ** p < 0.01 compared to the DMSO control). (D) The images of isolated tumors. (E) Tumor weight was measured two weeks after first treatment with 9-DAA (0.5 mg/kg). Data were analyzed using student’s T-test. (mean ± SD, n = 5; * p < 0.05 compared to the DMSO control).

The body weights of the mice were measured every 3 days. Fourteen days after the first injection of the test compounds, the mice were sacrificed and their weights were measured.

Cell lines and cell culture

HCT 116 (CCL-247), p53-null HCT 116, SW480 (CCL-228), and SW620 (CCL-227) cells were cultured in DMEM supplemented with 10 % FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin, cultured at 37 °C under 5 % CO₂ conditions in a humidified atmosphere. The cell line was revived every 3 months. The p53-null HCT 116 cell line used in this study was established and used in our earlier study [29].

Cell viability assay

Cell viability and proliferation were measured using the Cyto X colorimetric assay (WST; water-soluble tetrazolium salt method) (0793, Daeil Lab Service, Seoul, Korea). For the cytotoxicity assays, HCT 116 cells were seeded in 96-well cell culture plates (7 × 10³ cells/well) overnight and treated with 9-DAA at the indicated concentrations. After a 24 h incubation, the WST reagent solution and DMEM mixture were added, and cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader (SpectraMax 190, Molecular Devices, San Jose, CA, USA). The cell viability was normalized to that of the DMSO control group.

For proliferation analysis, the cells were seeded in 96-well cell culture plates (3 × 10³ cells/well) overnight and treated with 9-DAA at the indicated concentrations. After the indicated periods, the WST reagent solution and DMEM mixture were added, and cells were incubated at 37 °C for 2 h.

LDH release assay

LDH release was measured according to the manufacturer’s instructions. Briefly, HCT 116 cells were incubated with the indicated concentrations of 9-DAA for 24 h. After 24 h, only the cell culture medium was used for further analyses. The cell culture medium was transferred to a new empty plate. A mixture of the catalyst and dye solution was added to each well. After incubation for 1 h, absorbance was measured using a microplate reader (SpectraMax 190, Molecular Devices, San Jose, CA, USA) at 490 nm. LDH release was normalized to that of the DMSO control group.

Colony formation assay

HCT 116 cells were seeded overnight in 6-well cell culture plates (1.2 × 10³ cells/well). Cells were treated with the indicated concentrations of 9-DAA. The medium was replaced every 2 days. After 7 days of incubation, the cells were fixed with 4 % paraformaldehyde for 15 min and stained with 0.2 % crystal violet.

Topoisomerase in vivo complex of enzyme (ICE) assay

HCT 116 cells were incubated with indicated concentrations of 9-DAA for 24 h. For positive control, cells were incubated with etoposide (50 μM) for 30 min. The topoisomerase ICE assay was performed according to the manufacturer’s instructions. Briefly, cells were lysed using a lysis buffer containing a protease inhibitor cocktail. The isolated DNA-protein complex was loaded in each dot blot slot, and subsequently, topoisomerase Ⅱα was analyzed using immunoblot analysis.

Cell cycle analysis

Cells were seeded in 6-well cell culture plates (2.2 × 10⁵ cells/well) overnight. The cells were treated with the indicated concentrations of 9-DAA for 24 h. For p53-null cell line experiments, both p53-wild type (WT) and p53-null HCT 116 cells were incubated with DMSO control or 100 nM 9-DAA for 24 h. After incubation, cells were harvested using trypsin/ETDA solution, and subsequently fixed with cold 70 % ethanol for 30 min at 4 °C. Fixed cells were washed with PBS and stained with phosphate buffer saline (PBS) containing propidium iodide (PI) and RNase A for 30 min at 37 °C in the dark. Stained cells were analyzed using a flow cytometer (BD Biosciences). The results were analyzed using the FlowJo v11 software (BD Biosciences).

Transfections

HCT 116 cells were transiently transfected with the EGPF-C3 and EGFP-PYCR1 plasmids using Lipofectamine 3000 reagent.

For small interfering RNAs analysis, cells were transfected with siPYCR1 #1 (5′-GUGGAAUAGUGGAGGCCUU-3′), siPYCR1 #2 (5′-GUGGAGGCCUUCAACUGAU-3′), or scrambled siRNA control using X-treme GENE transfection reagent. All the siRNAs were purchased from Bioneer (Daejeon, Korea).

Western blot analysis

For immunoblot analysis, cells were lysed with ice-cold RIPA buffer containing a protease inhibitor cocktail and phosphatase inhibitor cocktail 3. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (0.2 μm). After 1 h of blocking with 5 % skim milk in TBS-T buffer, the membranes were incubated with the indicated primary antibodies at 4 °C overnight. Following a 2 h incubation with HRP-conjugated secondary anti-mouse or anti-rabbit antibodies, protein bands were visualized using SuperSignal West Pico chemiluminescent substrate or SuperSignal West Femto maximum sensitivity substrate. Band intensities were determined using ImageJ software (NIH, Bethesda, MD, USA).

EdU staining

Cells were seeded in 6-well plates (2.2 × 10⁵ cells/well) for 24 h and then treated with the indicated compounds in each condition. After 24 h of incubation, the cells were incubated with EdU (10 μm) and subsequently fixed with 4 % paraformaldehyde. The incorporated EdU was stained using the Click-iT toolkit, according to the manufacturer’s instructions. Briefly, the cells were permeabilized with 0.5 % PBS-T for 20 min and then incubated with a mixture of Click-iT for 30 min. The stained cells were measured using a flow cytometer, and the results were analyzed using the FlowJo v11 software.

For image analysis, EdU-stained cells were counterstained with DAPI for 10 min. Fluorescence images were captured using a laser scanning confocal microscope (LSM 700, ZEISS, Oberkochen, Germany).

Thermal shift assay

Recombinant human His-PYCR1 was added to 10X TNC buffer. Next, DMSO or 10 mM 9-DAA was added to PYCR1. The mixture was then incubated at room temperature for 1 h. After 1 h, each group was separated and heated at the indicated temperature for 5 min, then cooled for 10 min at 25 °C. Heated samples were centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was separated using SDS-PAGE and subsequently analyzed by immunoblotting. Band intensities were determined using ImageJ software (NIH, Bethesda, MD, USA).

Surface plasmon resonance (SPR) analysis

Recombinant human His-PYCR1 was used for the SPR analysis to determine the binding constant between a single protein and a small molecule. SPR analysis was performed on an SR7500DC instrument (Reichert) by WOOJUNGBIO, Inc. (Suwon, Korea) to determine the direct binding of 9-DAA to PYCR1. All binding reactions were performed in PBS containing 2 % DMSO as the running buffer. Kinetic affinity was monitored at a flow rate of 30 μL/min for 3 min of association, followed by 10 min of dissociation at the same flow rate. The sensor chip surface was regenerated using a 2 mM NaOH solution. All binding experiments were analyzed using Scubbers2 (Scrubber).

3D spheroid formation assay

The spheroid formation assay was performed according to a previous study [30]. HCT 116 cells were seeded into 96-well cell culture plates (5 × 10³ cells/well). After incubation for 2 days, the spheroids were treated with DMSO or the indicated concentrations of 9-DAA, and the medium was replaced every 2 days. The volume of the spheroids was measured daily after treatment with 9-DAA.

Annexin V-PI staining

HCT 116 cells were seeded in 6-well plates (2.2 × 10⁵ cells/well) overnight and then incubated with the indicated concentration of 9-DAA for 24 h. Cells were harvested using trypsin/EDTA solution and subsequently incubated in Annexin V binding buffer (400 µL of 10 mM HEPES/NaOH, pH 7.5 containing 2.5 mM CaCl₂ and 0.14 M NaCl) containing 0.5 µg/mL APC-Annexin V and 2 µg/mL PI for 15 min at 25 °C in the dark. The stained cells were measured using a flow cytometer, and the results were analyzed using the FlowJo v11 software.

Intracellular reactive oxygen species (ROS) measurement

HCT 116 cells were seeded in 6-well plates (2.2 × 10⁵ cells/well) overnight and then incubated with the indicated concentration of 9-DAA for 24 h. The cells were stained with CM-H₂DCFDA (10 mM) for 30 min at 37 °C. The stained cells were harvested, and fluorescence was determined using a flow cytometer. The results were analyzed using the FlowJo v11 software.

Intracellular ATP measurement

ATP levels were measured using the CellTiter-Glo® Luminescent assay kit, following the manufacturer’s instructions. Briefly, HCT 116 cells were seeded into 96-well plates (7 × 10³ cells/well) and incubated overnight. The cells were then treated with the indicated concentrations of 9-DAA for 24 h. After 24 h, a mixture of the substrate and buffer was directly added to each well. Following the 20-min incubation to stabilize the luminescent signal, luminescence was measured using a multilabel microplate (Victor™ X2, PerkinElmer). To eliminate the influence of cell number, the luminescence values were normalized using the cell viability ratio. The final ATP levels were normalized to those of the DMSO control group.

Molecular docking simulations

Molecular docking was performed as described previously. The structures of PYCR1 (5UAU) and PYCR2 (6LHM) were obtained from the Protein Data Bank. Noncovalent docking was conducted using AutoDock Vina software. The crystal structures of each protein and 9-DAA were prepared using standard AutoDock tools. The starting coordinates of each protein and 9-DAA were docked using AutoDock Vina software. The protein–ligand interactions were analyzed using Discovery Studio Visualizer v17.2 and LigPlot v2.2.8.

Statistical analysis

All data are expressed as the mean ± standard deviation of three or more independent experiments. Statistical analyses were conducted using the GraphPad Prism software (GraphPad 9.5.1, La Jolla, CA, USA). The treatments were considered statistically significant at * p < 0.05 and ** p < 0.01, compared to the DMSO control group.

Results

9-DAA suppresses cell proliferation and is not associated with DNA intercalation

To evaluate the cytotoxicity of 9-DAA (Fig. 1A), HCT116 cells were incubated with various concentrations of 9-DAA for different periods. As shown in Fig. 1B and 1C, 9-DAA reduced cell viability in a dose- and time-dependent manner. However, LDH release from cells was not affected by 9-DAA treatment (Fig. 1D). These results indicated that 9-DAA did not induce cell death but inhibited cell proliferation. To further validate apoptosis at high concentrations, the cells were treated with high concentrations of 9-DAA and analyzed using flow cytometry. 9-DAA did not induce apoptosis, even at concentrations up to 500 nM (Fig. S1A). Therefore, we evaluated the inhibitory effects of 9-DAA on cancer cell proliferation and found that it effectively inhibited cancer cell proliferation in a dose-dependent manner (Fig. 1E). In addition, 9-DAA suppressed colony formation of HCT116 cells (Fig. 1F).

Fig. 1.

9-DAA inhibits cell proliferation. (A) Chemical structure of 9-DAA. (B and C) HCT 116 cells were treated with indicated concentration of 9-DAA for 24 hours. (B) Cell viability was determined the using Cyto X colorimetric kit. Cell viability was normalized to DMSO control. Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. (mean ± SD, n = 3; * p < 0.05 and ** p < 0.01 compared to the DMSO control). (C) LDH release was measured using an LDH detection kit. The cell culture medium was transferred to a new plate, and a catalyst mixture was added to each well to measure the activity of LDH released into the medium. (mean ± SD, n = 3). (D) HCT 116 cells were treated with 100 nM of 9-DAA for indicated periods. Cell viability was determined the using Cyto X colorimetric kit. Cell viability was normalized to DMSO control. Data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the DMSO control). (E) HCT 116 cells were treated with the indicated concentration of 9-DAA for indicated time periods. Cell viability was determined the using Cyto X colorimetric kit. (mean ± SD, n = 3). (F) HCT 116 cells were treated with different concentration of 9-DAA for 7 days. Media containing 9-DAA was exchanged every 2 days. After 7 days incubation, cells were fixed with 4 % PFA and then stained using crystal violet.

Various nucleoside analogs have recently been used as anticancer agents owing to their ability to inhibit DNA synthesis [31,32]. As 9-DAA is a nucleoside analog, validating its effects as a DNA-damaging agent is scientifically justified. We performed a topoisomerase ICE assay to evaluate the potential of 9-DAA as a DNA-damaging agent. Etoposide, a representative topoisomerase inhibitor, inhibited topoisomerase Ⅱα. However, 9-DAA did not exhibit such activity (Fig. S1B) nor induce ROS (Fig. S1C), which are phenotypes of DNA damage that contribute to cell proliferation arrest. Collectively, our results indicate that 9-DAA inhibits cancer cell proliferation and that this effect is not associated with DNA damage or cellular oxidative stress.

9-DAA induces G0/G1 phase cell cycle arrest in colon cancer cells

As mentioned above, 9-DAA effectively inhibited cell proliferation but did not induce apoptosis. These results suggest that 9-DAA acts as a cell cycle inhibitor. To evaluate cell cycle arrest induced by 9-DAA, DNA was stained with PI and analyzed using flow cytometry. 9-DAA caused cancer cell cycle arrest in the G0/G1 phase in a concentration-dependent manner (Fig. 2A and B). To further evaluate the inhibition of cell proliferation, the cells were incubated with EdU and stained using the Click-iT toolkit. Flow cytometry data indicated that DNA synthesis during cell division was suppressed by 9-DAA (Fig. 2C), and fluorescence imaging revealed a dose-dependent reduction in EdU-positive cells (Fig. 2D). These results indicated that 9-DAA arrested cancer cell proliferation in the G0/G1 phase.

Fig. 2.

9-DAA induces G1 phase arrest. (A) HCT 116 cells were treated with indicated concentration of 9-DAA for 24 hours. After 24 hours, cells were fixed and then cellular DNA contents were stained by using PI. DNA contents were measured using flow cytometry. (B) The graph showed proportion of phase of cell cycle in (A). (C and D) HCT 116 cells were treated with the indicated concentration of 9-DAA for 24 hours. Two hours prior to harvesting, cells were incubated with EdU. After incubation, cells were fixed, and EdU was stained by using Click-iT™ EdU imaging kit. (C) The EdU-positive cells were analyzed using flow cytometry. (D) Cells were counterstained with DAPI, and the stained cell was captured by fluorescence microscopy. (Scale bar = 20 μm).

Cell cycle is regulated by various proteins, including members of the CDK family [33]. Thus, we hypothesized that 9-DAA affects genes involved in cell cycle regulation. As expected, 9-DAA significantly decreased the expression of CDK4 and Cyclin D1, regulatory genes involved in cell division, in a dose- and time-dependent manner (Fig. S2A and S2B). The expression levels of p27 and p21, tumor suppressor genes, and regulators of CDK4 and Cyclin D1 were elevated by 9-DAA in a dose- and time-dependent manner (Fig. S2C and S2D). These results demonstrated that 9-DAA suppresses cell division by regulating tumor suppressor genes.

9-DAA strongly inhibited the cell cycle in HCT116 cells, suggesting that it may exert inhibitory effects on colon cancer cells. To validate the effects of 9-DAA in various colon cancer cell lines, SW480 and SW620 cells were treated with 9-DAA and cell cycle progression was analyzed. In cell proliferation analysis, the proliferation rates of SW480 and SW620 cells were significantly suppressed by 9-DAA treatment (Fig. S3A). Therefore, to evaluate the effects of 9-DAA on the cell cycle, the DNA of both cell lines was stained with PI and analyzed using flow cytometry. As shown in Fig. S3B, 9-DAA inhibited the cell cycle progression of SW480 and SW620 cells in the G0/G1 phase. Newly synthesized DNA was assessed during cell division using an EdU incorporation assay. As expected, 9-DAA considerably reduced the proportion of EdU-positive cells in both cell lines (Fig. S3C). The effects of 9-DAA as a cell cycle inhibitor were consistently observed in colon cancer cell lines. These results suggest that 9-DAA is a potential agent for the treatment of colon cancer.

9-DAA induces cell cycle arrest through a p53-dependent and independent manner

TP53 is a well-known tumor suppressor gene widely associated with cancer cell proliferation [34]. Additionally, p53 suppresses cell division by activating p21, inhibiting CDK4 and Cyclin D1, which are cell cycle regulators [35]. Thus, we hypothesized that the inhibition of cell proliferation induced by 9-DAA may be affected by p53 signalling. When both p53-WT and p53-null HCT116 cells were treated with 9-DAA, the viability and proliferation of the p53-null cells were inhibited (Fig. 3A and B). However, 9-DAA exhibited a slightly lower inhibition rate in p53-null cells than in p53-WT cells. To further evaluate cell cycle inhibition in p53-null cells, both WT and null cells were analyzed using flow cytometry and EdU staining. Flow cytometric analysis showed that 9-DAA induced G0/G1 arrest in p53-null cells; however, this effect was lower than that observed in p53-WT cells (Fig. 3C and D). Moreover, EdU staining revealed that 9-DAA treatment decreased the EdU-positive ratio (Fig. 3E and F). The protein levels of the tumor suppressor genes p21 and p27 were also elevated by 9-DAA treatment (Fig. S4). Therefore, 9-DAA effectively inhibited the proliferation of p53-null cells, although the 9-DAA-induced cell cycle arrest was only slightly mitigated. Consequently, these results suggest that 9-DAA exerts anticancer effects in a cell cycle-independent manner and may be effective against drug-resistant cancers with p53 mutations.

Fig. 3.

9-DAA arrests cancer cell proliferation both of p53 dependency and independency. (A) p53 WT or p53 null HCT 116 cells were treated with the indicated concentration of 9-DAA for 24 hours. Cell viability was determined the using Cyto X colorimetric kit. Data were analyzed using two-way ANOVA followed by Dunnett's post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the DMSO control). (B) p53 null HCT 116 cells were treated with indicated concentration of 9-DAA for indicated time. Cell viability was determined the using Cyto X colorimetric kit. (mean ± SD, n = 3). (C) p53 WT or p53 null HCT 116 cells were treated with DMSO control or 9-DAA for 24 hours. After 24 hours, DNA was stained by using PI and then DNA contents were measured using flow cytometry. (D) The graph showed proportion of phase of cell cycle in (C). (E and F) p53 WT or p53 null HCT 116 cells were treated with DMSO control or 9-DAA for 24 hours. Two hours prior to harvesting, cells were incubated with EdU. After incubation, cells were fixed, and EdU was stained by using Click-iT™ EdU imaging kit. (E) The EdU-positive cells were analyzed using flow cytometry. (F) Cells were counterstained with DAPI, and the stained cell was captured by fluorescence microscopy. (Scale bar = 20 μm).

PYCR1 is a direct target of 9-DAA

9-DAA inhibited cancer cell proliferation by regulating the expression of tumor suppressor genes (Figs. 2 and S2). However, the exact targets of 9-DAA remain unknown. To identify the cellular target of 9-DAA, we conducted a cellular thermal shift assay and validated the target proteins using LC/MS analysis. As shown in Fig. 4A, a distinct protein band was validated and identified as PYCR1. To evaluate the direct binding of 9-DAA to PYCR1, PYCR1 protein was isolated and a SPR assay with 9-DAA was performed. The results showed that 9-DAA binds directly to PYCR1 (Fig. 4B). The association rate (k_a) and dissociation binding constant (k_d) were 2.65 M⁻¹ s⁻¹ and 2.092 × 10⁻⁵ s⁻¹, respectively. The actual binding equilibrium dissociation constant (K_d) of 7.89 μM was obtained as the ratio of binding rates (K_d = k_d/k_a). To further validate binding by assessing changes in thermal stability, the purified His-tagged PYCR1 protein was heated in the absence or presence of 9-DAA. The thermal shift assay data showed an increase in PYCR1 in the 9-DAA-treated group (Fig. 4C). These findings demonstrate that 9-DAA directly binds to PYCR1.

Fig. 4.

PYCR1 is direct target of 9-DAA (A) HCT 116 cell lysates were treated with DMSO control or 10 μM of 9-DAA for 1 hours and then heated for 5 min at indicated temperature. The protein aggregates were separated by centrifugation. The eluted proteins were separated by SDS-PAGE and stained by silver staining. The enhanced protein targets (red quadrangle) of 9-DAA were identified using LC−MS/MS. (B) SPR analysis of the binding kinetics between the indicated concentrations 9-DAA and His-PYCR1. (C) His-PYCR1 proteins were treated with DMSO (control) or 10 μM of 9-DAA for 1 hours. The mixture was divided and then heated for 5 min at indicated concentrations. The protein aggregates were separated by centrifugation. The soluble proteins were separated by SDS-PAGE and analyzed by immunoblot analysis. (D) Binding interaction images of PYCR1 and 9-DAA based on molecular docking results. (E) Detailed view of interacting residues between 9-DAA and PYCR1. (F) Binding affinity profiles showing predicted free energies of 9-DAA binding to PYCR1 and PYCR2.

To evaluate the binding site between 9-DAA and PYCR1, we performed molecular docking analysis. The docking model revealed that 9-DAA bound to the predicted site and interacted with the amino acid residues (Fig. 4D and E). Furthermore, the binding-affinity data showed that the binding free energy of 9-DAA to PYCR1 was approximately 1 kcal/mol lower than that to PYCR2 (Fig. 4F). A difference of 1 kcal/mol in binding energy corresponds to an approximately 5-fold increase in binding affinity. These findings demonstrate that 9-DAA preferentially binds to PYCR1 over its isoform, PYCR2.

PYCR1 regulates cancer cell proliferation, and chronic inhibition of PYCR1 causes apoptosis

We demonstrated that 9-DAA effectively inhibited cell proliferation and elevated the expression of the tumor suppressor genes p27 and p21 (Figs. 2 and S2). Moreover, PYCR1 was validated as a direct target of 9-DAA by thermal shift and SPR assays (Fig. 4). PYCR1 is a well-established oncogene that promotes cancer proliferation [36]. Furthermore, TCGA analysis showed that PYCR1 expression was significantly elevated in clinical colon cancer samples (colon and rectum adenocarcinomas) (Fig. 5A). These observations suggest that targeting PYCR1 is a promising strategy for PYCR1-overexpressing cancer treatment, particularly colon cancer. Therefore, we hypothesized that alteration of PYCR1 protein may regulate cancer cell proliferation. To evaluate the effects of PYCR1 on cell proliferation, HCT 116 cells were transfected with siRNAs targeting PYCR1 or scrambled RNA (scRNA). Cell proliferation was significantly reduced in the siRNA of PYCR1-transfected groups (Fig. 5B). To further validate the effects of PYCR1 on cell division, cells were incubated with EdU and stained. Fig. 5C and D show that a reduction in PYCR1 levels effectively inhibited the EdU-positive cell population. Additionally, the tumor suppressor genes p27 and p21 were elevated by PYCR1 knockdown (Fig. 5E and F), similar to that observed in 9-DAA-treated cells.

Fig. 5.

Reduction of PYCR1 arrests cell proliferation. (A) The graph showed expression levels of PYCR1 in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). The graph was generated using GEPIA2 platform based on TCGA and GTEx datasets. The expression levels were compared between tumor and normal tissues. (** p < 0.01 compared to the normal tissues). (B) HCT 116 cells were transfected with scRNA or indicated siRNA using X-tremeGENE™ siRNA transfection reagent. After indicated time, cell viability was determined the using Cyto X colorimetric kit. Cell viability was normalized to scRNA control. Data were analyzed using two-way ANOVA followed by Dunnett's post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the scRNA control). (C) HCT 116 cells were transfected with scRNA or indicated siRNA. After 48 hours transfection, two hours prior to harvesting, cells were incubated with EdU. After incubation, cells were fixed, and EdU was stained by using Click-iT™ EdU imaging kit. Cells were counterstained with DAPI, and the stained cell was captured by fluorescence microscopy. (Scale bar = 20 μm). (D) The graph showed population of EdU-positive cells in (C). Data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the scRNA control). (E) HCT 116 cells were transfected with scRNA or indicated siRNA. After 48 hours transfection, protein levels of p21 were analyzed by immunoblotting. β-Actin was used as a loading control. (E) The graphs showed quantitative analysis of protein levels in (F). Data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. (mean ± SD, n = 3; * p < 0.05 and ** p < 0.01 compared to the scRNA control).

To evaluate the effects of elevated PYCR1 levels on cell proliferation, we induced the ectopic expression of GFP-PYCR1 in HCT 116 cells and measured cell proliferation. The proliferation of HCT 116 correlated with PYCR1 expression, and PYCR1 mitigated the inhibition of proliferation induced by 9-DAA (Fig. S5A). Moreover, EdU staining showed that the ectopic expression of PYCR1 alleviated the decrease in EdU-positive cells induced by 9-DAA (Fig. S5B and C). As shown in Fig. S5D and E, the protein levels of tumor suppressor genes that were decreased by 9-DAA treatment were reversed by PYCR1 expression. Our results indicate that PYCR1 affects cell proliferation and that 9-DAA-induced cell cycle arrest is associated with PYCR1.

Our findings showed that 9-DAA treatment and reduced PYCR1 levels effectively inhibited cell proliferation. Thus, we hypothesized that chronic exposure to 9-DAA induces cell death. To validate the cellular apoptosis, we determined the levels of apoptotic marker proteins after chronic exposure to 9-DAA or siRNA transfection. HCT 116 cells were treated with 9-DAA for 3 days. Protein levels were analyzed by immunoblotting. As shown in Fig. S6A and B, chronic exposure to 9-DAA significantly elevated the levels of apoptotic marker proteins such as cleaved PARP in a dose-dependent manner. Similar to 9-DAA treatment, PYCR1 downregulation increased cleaved PARP levels (Fig. S6C and D). Furthermore, flow cytometry data showed that a reduction in PYCR1 protein increased early and late apoptotic cell populations (Fig. S6E). Thus, our data demonstrate that inhibition of PYCR1 effectively inhibits cell proliferation and that chronic inhibition of PYCR1 induces cell apoptosis.

9-DAA and PYCR1 regulation control cancer metastasis

PYCR1 levels are associated with cancer cell metastasis, and inhibitors of PYCR1 increase E-cadherin expression, which is associated with the epithelial-to-mesenchymal transition during cancer progression [37,38]. Therefore, we hypothesized that 9-DAA regulates cancer cell metastasis by directly binding to PYCR1. The Transwell invasion assay showed a significant decrease in the number of invaded cells with increasing 9-DAA concentrations (Fig. S7A and B). Furthermore, E-cadherin, a marker protein of epithelial structures, was considerably upregulated after 9-DAA treatment (Fig. S7C and D).

To validate the effects of PYCR1 protein levels on colon cancer cells, PYCR1 cells were transfected with siRNA or GFP-PYCR1. A decrease in PYCR1 levels via siRNA inhibited cell invasion (Fig. S7E). By contrast, overexpression of PYCR1 increased invasiveness, and PYCR1 mitigated the inhibitory effects of 9-DAA on colon cancer cell metastasis (Fig. S7F).

9-DAA inhibitory effects are NAD+-dependent and associated with the AMPK–p38 signalling pathway

PYCR1 uses NADH as a cofactor for the conversion of P5C to proline and produces NAD+ as a byproduct of the reaction. NAD+ metabolism is widely associated with cell survival and proliferation via regulation of DNA repair, energy production, and epigenetics [19]. Therefore, we hypothesized that the inhibition of cell proliferation induced by 9-DAA may depend on NAD+. To evaluate the NAD+ dependence, cells were treated with 9-DAA in the presence or absence of NAD+. The cell viability and proliferation rates, which were reduced by 9-DAA treatment, were significantly rescued by NAD+-dependent cell division (Fig. 6A and B). The cells were treated with DMSO or 9-DAA in the presence or absence of NAD+ for 7 days. Colony formation by HCT 116 cells was restored by NAD+ co-treatment (Fig. 6C). To further validate the dependence of the cell division rate on NAD+, we performed EdU staining in the presence or absence of NAD+. The population of EdU-positive cells increased in cells co-treated with 9-DAA and NAD+ compared to that in cells treated with 9-DAA alone (Fig. 6D). At the protein level, p21, a tumor suppressor gene upregulated by 9-DAA, was notably diminished in the 9-DAA and NAD+ co-treated groups (Fig. 6E and F).

Fig. 6.

Inhibition of cell proliferation induced by 9-DAA is dependent on NAD+ (A) HCT116 cells were treated with DMSO control or 9-DAA in the absence or presence of NAD+ for 24 hours. Cell viability was determined the using Cyto X colorimetric kit. Cell viability was normalized to DMSO control. Data were analyzed using two-way ANOVA followed by Šídák‘s post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the DMSO control). (B) HCT 116 cells were treated with indicated concentration of 9-DAA in the presence or absence of NAD+ (50 μM) for indicated periods. Cell viability was determined the using Cyto X colorimetric kit. Data were analyzed using two-way ANOVA followed by Tukey’s post hoc test. (mean ± SD, n = 3; ** p < 0.01 compared to the DMSO control; ## p < 0.01 compared to the group treated with 9-DAA only). (C) HCT116 cells were treated with DMSO control or 9-DAA in the absence or presence of NAD+ for 7 days. Media containing 9-DAA was exchanged every 2 days. After 7 days incubation, cells were fixed with 4 % PFA and then stained using crystal violet. (D) HCT116 cells were treated with DMSO control or 9-deazainosine in the absence or presence of NAD+ for 24 hours. Two hours prior to harvesting, cells were incubated with EdU. After incubation, cells were fixed, and EdU was stained by using Click-iT™ EdU imaging kit. Cells were counterstained with DAPI, and the stained cell was captured by fluorescence microscopy. (Scale bar = 20 μm). (E) HCT116 cells were treated with DMSO control or 9-deazainosine in the absence or presence of NAD+ for 24 hours. Protein levels of p21 were analyzed by immunoblotting. β-Actin was used as a loading control. (F) The graphs showed quantitative analysis of protein levels in (E). Data were analyzed using two-way ANOVA followed by Tukey's post hoc test. (mean ± SD, n = 3; * p < 0.05 compared to the DMSO control; # p < 0.05 compared to the group transfected with GFP only).

NAD+ is an important cofactor for energy production through glycolysis, β-oxidation, and the TCA cycle [19]. Thus, an imbalance in NAD+ affects energy balance, and AMP-activated protein kinase (AMPK) activation restores NAD+ metabolism [39]. Because we previously demonstrated that 9-DAA-induced cell cycle arrest was alleviated by treatment with NAD+ (Fig. 6), we hypothesized that 9-DAA might affect intracellular ATP levels, which could subsequently activate the AMPK and p38 signalling pathways. The ATP levels were measured after treatment with 9-DAA to test this hypothesis. The result showed that 9-DAA significantly reduced intracellular ATP levels in a dose-dependent manner (Fig. S8A). Additionally, the levels of phosphorylated AMPK and p38 were determined by immunoblotting. Phosphorylated AMPK and p-p38, which are downstream molecules of AMPK, were significantly activated by 9-DAA in a dose-dependent manner (Fig. S8B and C). Interestingly, a reduction in PYCR1 levels via siRNA also increased the phosphorylation of AMPK and p38, similar to the results of 9-DAA treatment (Fig. S8D and E). Therefore, our results demonstrated that the inhibition of cell division induced by 9-DAA is linked to NAD+ and involves disrupting ATP homeostasis and activating the AMPK–p38 signalling pathway.

PYCR1 is upregulated in hypoxic and 3D spheroid models, and 9-DAA has anticancer effects in both models

PYCR1 enzyme activity was upregulated under hypoxic conditions [40]. Another study indicated that PYCR1 promotes cancer cell stemness and is upregulated in spheroid cancer cells [41]. Thus, we hypothesized that 9-DAA is effective against cancer cells in 3D spheroid models under hypoxic conditions. Before evaluating the anticancer effects of 9-DAA, we determined the levels of PYCR1 in hypoxic and 3D spheroid models. Interestingly, the protein level of PYCR1 was upregulated in both instances (Fig. 7). Therefore, we treated cancer cells under hypoxic conditions using a 3D spheroid model with 9-DAA. As shown in Fig. S9A and B, 9-DAA successfully inhibited cell proliferation under hypoxic conditions, and the reduction of PYCR1 by siRNA suppressed cell proliferation under these conditions. In the 3D spheroid models, 9-DAA effectively suppressed spheroid growth dose-dependently (Fig. S9C and D). These results indicated that 9-DAA is effective in other cancer model systems.

Fig. 7.

PYCR1 levels is upregulated under hypoxia condition and 3D spheroid cancer cell. (A) Representative images of HCT 116 cells under normoxia or hypoxia condition and 3D spheroid. (B) Protein levels of each group were analyzed by immunoblotting. β-Actin was used as a loading control. (C) The graphs showed quantitative analysis of protein levels in (B). Data were analyzed using one-way ANOVA followed by Dunnett's post huc test. (mean ± SD, n = 3; * p < 0.05 and ** p < 0.01 compared to the Normoxia control).

9-DAA exhibits anticancer effects in an HCT 116 xenograft mouse model

Having successfully demonstrated the effects of 9-DAA as a cell-cycle inhibitor, we administered it to a xenograft mouse model to determine its effects in vivo. HCT 116 cells were subcutaneously injected into the flanks of nude mice, and then the mice were injected with 9-DAA when the tumor volume reached 50 mm³ according to the experimental scheme (Fig. 7A). When the weights of the mice were measured once every 3 days after 9-DAA injection, no significant changes were observed (Fig. 7B). The tumor volume was measured once every 2 days after treatment with 9-DAA. This volume was significantly reduced by the 9-DAA injections (Fig. 7C). Additionally, 14 days after the first injection, the isolated tumor volumes decreased considerably with 9-DAA treatment in the xenograft mice. These results demonstrate that 9-DAA exerts anticancer effects in a xenograft mouse model. These in vivo findings corroborate the anticancer effects observed in the 2D and 3D cell culture models.

Discussion

Cancer cells have an abnormal metabolic system due to excessive cell proliferation [42]. Therefore, nonessential amino acids are important in cancer progression [43]. In particular, proline supports cancer cell progression by aiding protein biosynthesis, collagen production, controlling redox reactions, and regulating epigenetic modifications [[44], [45], [46]]. Therefore, proline metabolism and related enzymes are considered effective therapeutic targets for cancer treatment [36]. PYCR1 is the final enzyme involved in proline metabolism and converts P5C to proline. Additionally, regulating PYCR1 levels affects various cellular signalling pathways involved in cell growth, inflammation, and metastasis [17]. Multiple studies have reported that PYCR1 dysregulation suppresses cancer cell progression and metastasis [37,38,[47], [48], [49]]. Herein, we demonstrate that PYCR1 is a direct target of 9-DAA, with the latter effectively inhibiting cancer cell proliferation and, in turn, suppressing cancer metastasis.

Some researchers have suggested that proline can rescue cancer cell progression inhibited by the disruption of proline biosynthesis via dysregulation of P5C synthase [50]. However, other studies indicated that direct administration of proline does not affect the growth of PYCR1-deficient cells [40]. The current study’s findings suggest that the energy imbalance caused by dysregulation of the NAD+/NADH ratio, which is a byproduct of PYCR1, may play an important role in cancer cell progression. The inhibitory effects of 9-DAA on colon cancer progression were reversed by ectopic NAD+ support (Fig. 6). These data supported the hypothesis that the inhibitory effects on colon cancer proliferation induced by 9-DAA were dependent on NAD+. Additionally, 9-DAA inhibited cellular ATP levels, and both PYCR1-knockdown and 9-DAA-treated cells showed significant activation of AMPK and subsequent activation of the p38 pathway. These data suggest that energy imbalance may stimulate AMPK and p38 and that this pathway is one of the causes of cell division arrest.

A previous study indicated that although the enzymatic activity of PYCR1 is upregulated under hypoxia, it does not alter the protein levels [40]. Another study showed that PYCR1 levels increased in sphere cultures of TNBC cells; however, downregulation of PYCR1 inhibited sphere growth [41]. Interestingly, in our study, 3D spheroid culture of colon cancer cells caused an increase in PYCR1 expression, and this shift was observed under oxygen-limiting conditions, in contrast to a previous study. Consequently, knockdown of PYCR1 or 9-DAA effectively inhibits cell growth under hypoxia or in 3D spheroid culture, which mimics the solid cancer environment [[51], [52], [53]]. Our findings suggest that PYCR1 is an effective target for cancer treatment, particularly in colon cancer, as PYCR1 expression is upregulated under both oxygen-limiting conditions and in 3D spheroids of colon cancer. Thus, 9-DAA may be effective for treating solid cancer-mimicking conditions.

PYCR1 inhibitors are reported to be proline-based analogs, such as pargyline, which is an MAO inhibitor based on the benzylamine structure [54,55]. In this study, we report a novel role of 9-DAA as a direct target of PYCR1. In addition, 9-DAA effectively inhibited colon cancer cell division by regulating the expression of cancer suppressor genes, including p27 and p21. Additionally, our data show that PYCR1 is a potent target for colon cancer treatment under hypoxia or in 3D spheroids, with 9-DAA showing inhibitory effects under both conditions. However, in this study, we did not exclude the direct effects of our compound on RNA polymerase inhibition, because 9-DAA is an adenosine analog that could act as an inhibitor of RNA polymerase. Nevertheless, we suggested a potential role for 9-DAA as an anticancer agent and demonstrated that it directly binds to PYCR1. Thus, improving its pharmacological properties and minimizing its potential side effects, such as those associated with RNA polymerase inhibitors, is necessary. Based on our molecular docking analysis and binding affinity to PYCR1, further preclinical studies should focus on structure–activity relationship analysis and the design of 9-DAA derivatives to improve their safety and drug-like properties.

Glossary

• •AMPK — AMP-activated protein kinase
• •APC — adenomatous polyposis coli
• •CDK — cyclin-dependent kinases
• •DMEM — Dulbecco’s modified Eagle’s medium
• •DMSO — dimethyl sulfoxide
• •FBS — fetal bovine serum
• •ICE — in vivo complex of enzyme
• •9-DAA — 9-deazaadenosine
• •PBS — phosphate buffer saline
• •P5C — pyrroline-5-carboxylate
• •PI — propidium iodide
• •PYCR1 — pyrroline-5-carboxylate reductase 1
• •ROS — reactive oxygen species
• •SPR — surface plasmon resonance
• •SDS-PAGE — sodium dodecyl sulfate–polyacrylamide gel electrophoresis
• •TCA — tricarboxylic acid
• •WT — wild type
• •WST — water-soluble tetrazolium salt method

CRediT authorship contribution statement

Jongtae Roh: Writing – original draft, Formal analysis, Data curation, Conceptualization. Inho Ahn: Resources, Formal analysis. Jong-Ryoo Choi: Resources, Formal analysis. Sung-Kyun Ko: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request.