Introduction

While medical technology to extend human lifespan continues to develop, cancer mortality is still high, and the social burden is also increasing [1]. In particular, colorectal cancer has a high diagnosis rate, and although it has a high survival rate in the early stage, its mortality rate is high among all cancers [2]. When colorectal cancer is detected early, the cure rate is high, but it is often detected late because certain symptoms or pain do not appear in the early stages. With continuing research on conquering cancer, first-generation chemical anticancer drugs, second-generation targeted anticancer drugs, and third-generation immune anticancer drugs have been developed, and recently, fourth-generation anticancer drugs are being developed. Existing anticancer drugs have disadvantages such as side effects and resistance. Natural products are compounds produced by living organisms in nature that can be used as food, and it is expected that safe natural products can be used for anticancer therapy to compensate for the shortcomings of existing anticancer drugs.

Turmeric (Curcuma longa L.) is a perennial herbaceous plant belonging to the Zingiberaceae family that is cultivated in India, Indonesia, China, and other Asian countries. C. longa is frequently used as a medicinal herb and a food supplement owing to its health benefits. C. longa is known for many biological activities, among which anticancer activity has been reported, including against glioma cancer [3], cervical cancer [3], prostate cancer [3, 4], oral cancer [4], breast cancer [5, 6], colon cancer [7], and liver cancer [8] in vitro, and liver cancer [9], colon cancer [10], and breast cancer [11] in vivo.

In addition to studies on extracts, many anticancer studies have been conducted on major compounds contained in C. longa. As a representative compound of C. longa, curcumin has been reported to be highly cytotoxic to cancer cells. DNA damage [12], c-jun N-terminal kinase dependent apoptosis [13], and downregulation of E2F4 expression and apoptosis [14] were investigated in the human colon cancer cell line (HCT116) treated with curcumin. Curcumin is easily converted to demethoxycurcumin by demethoxylation from its benzene ring, resulting in a more stable structure [15]. Demethoxycurcumin has also been subjected to cytotoxic studies of cancer cell lines including fibrosarcoma [16], breast cancer [17], prostate cancer [18], lung cancer [19,20,21], bladder cancer [22], cervical cancer [23], oral squamous cell carcinoma [24], osteosarcoma [25], and brain glioblastoma [26]. However, there are no reports on colorectal cancer. We tried to confirm the growth-inhibiting activity of C. longa extract in colorectal cancer cells, as well as whether demethoxycurcumin exhibited the same activity. To support the interpretation of their metabolic mechanisms, metabolites of colorectal cancer cells treated with extract of C. longa and demethoxycurcumin were analyzed using nuclear magnetic resonance (NMR) spectroscopy. Metabolites are final products generated by metabolic processes, and because they reflect reactions that occur in the body, insight into the mechanism of cancer cells can be additionally obtained by applying metabolomics.

Materials and methods

Plant materials

The Curcuma longa used in this study was cultivated in the **do region of the Republic of Korea. A voucher specimen (MPS004295) was deposited at the Herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Republic of Korea. Dried rhizome of C. longa was ground and extracted twice by reflux extraction with 50% aqueous fermented ethanol at 80 °C for 4 h. The extract was filtered through a filter paper (Whatman, Maidstone, UK) and vacuum-concentrated under reduced pressure. The concentrated extract was lyophilized under reduced pressure (100 mTorr) (EYELA, Tokyo, Japan). Demethoxycurcumin was isolated from the C. longa using medium-pressure liquid chromatography (MPLC), NMR techniques and comparison with literature sample (Sigma, CAS # 22608-11-3).

Cell culture

The HCT116 human colorectal cancer cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). HCT116 cells were cultured in DMEM containing 10% FBS with 100 U/mL penicillin and 100 U/mL streptomycin, and cultured at 37 °C in an atmosphere containing 5% CO2.

Cell viability assay

HCT116 cells (2 × 104 cells/well) were seeded in 96-well plates, and cell viability was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). HCT116 cells were cultured with various concentrations of C. longa extract (0, 50, 100, 200, and 300 μg/mL) or demethoxycurcumin (0, 1.5, 3.125, 6.25, 12.5, 25, and 50 μM). Following treatment for 24 h, 10 µL MTT solution (0.5 mg/mL) was added to each well, and cells were incubated at 37 °C for 2 h. The culture medium was subsequently removed and 100 µL dimethyl sulfoxide was added to each well at room temperature while being shaken for 20 min. The optical density of HCT116 cells was measured at 570 nm wavelength on a multiwell plate reader.

Western blot analysis

HCT116 cells (5 × 105 cells/well) were seeded in 6-well plates, and the expression of PARP, caspase-3, caspase-9, Bcl-2, Bax, and p53 protein was detected via Western blot analysis. Following treatment for 24 h with demethoxycurcumin (0, 20, 40, and 60 µM) or doxorubicin (1 µM), HCT116 cells were washed twice with ice-cold PBS and lysed for 30 min on ice in cell-lysis buffer (1 × RIPA; Thermo Fisher Scientific, USA). Protein concentration was determined using a BCA protein assay kit. Protein samples (10–30 µg/well) were resolved using 10% SDS-PAGE. Separated proteins were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes at 70 V for 1.5 h. The membranes were blocked with 5% nonfat milk powder in TBS Tween 20 (TBST) buffer at room temperature for 1 h with shaking. The PVDF was incubated with anti-PARP (1:1000; Cell Signaling Technology, USA), anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, USA), anti-cleaved caspase-9 (1:1000; Cell Signaling Technology, USA), anti-Bcl-2 (1:500; Santa Cruz Biotechnology, USA), anti-Bax (1:500, Santa Cruz Biotechnology, USA), anti-p53 (1:1000, Santa Cruz Biotechnology, USA) and anti-β-actin (1:5000, Santa Cruz Biotechnology, USA) at room temperature for 3 h. Following 3 washes with TBST twice for 30 min, the membranes were incubated with anti-immunoglobulin secondary antibody (1:2000–1:5000, Santa Cruz Biotechnology, USA) at room temperature for 1 h with shaking. The bands were detected using an Enhanced Chemiluminescence Prime Western blotting kit (Thermo Fisher Scientific, USA). Statistical significance was tested using Student’s t-test. P < 0.05 was considered statistically significant.

Sample preparation for NMR analysis

For the metabolic analysis, HCT116 cells were cultivated in the same conditions as the cell viability assay. In order to obtain statistical significance, 10 samples of the control group (CON), 11 samples of the doxorubicin-treated group (DOX), and 10 samples of the C. longa-treated group (CL) were cultivated and analyzed. In addition, 10 samples of CON, 11 samples of DOX, 10 samples of the demethoxycurcumin 20 μM group, 10 samples of the demethoxycurcumin 40 μM group, and 10 samples of the demethoxycurcumin 60 μM group were cultivated and analyzed. Harvested HCT116 cells were washed 3 times with PBS, then an HCT116 cell pellet (approximately 1 × 107 cells) was suspended in 400 μL of pre-chilled methanol, and 325 μL of cold water and 400 μL of cold chloroform were added step-by-step. After centrifugation (4 °C, 4000 rpm, 10 min), aqueous phase and organic phase were separated. The aqueous phase of each sample was collected in a glass vial and the methanol in the sample was eliminated using SpeedVac (EYELA, Tokyo, Japan) for 6 h, and all solvent was lyophilized overnight. Samples were re-dissolved using 560 μL of deuterated 0.2 M sodium phosphate buffer containing 0.2 mM of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP-d4) and transferred to 5 mm NMR tubes.

NMR data acquisition and processing

One-dimensional (1D) 1H-NMR spectroscopy was conducted using a 700 MHz Bruker NMR spectrometer (Bruker Biospin, Germany) equipped with a cryoprobe at the KBSI Ochang Center, Republic of Korea. Pulse sequence for 1D experiment was a 1H nuclear Overhauser effect spectroscopy (NOESY). The parameters were set as 2.00 s of relaxation delay, 50.0 ms of mixing time, 12.2 μs of 90º pulse width, and 128 transients. This resulted in a total experiment time of 7 min 48 s. Two-dimensional (2D) NMR spectroscopy experiments were performed using a 900 MHz Bruker NMR spectrometer equipped with a cryoprobe at the KBSI Ochang Center. 2D 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear single quantum correlation (HSQC) were performed. All acquired data were manually phased and baseline corrected using Topspin v3.6.5 (Bruker Biospin, Germany). Metabolic identification and quantification were performed using Chenomx NMR Suite 8.4 Professional (Chenomx Inc., Edmonton, Canada). Biomarker and correlation analyses of quantified metabolites were conducted by MetaboAnalyst 5.0 (https://www.metaboanalyst.ca). For multivariate statistical analysis, NMR spectra were binned to 0.001 ppm size and normalized to total area using Chenomx NMR Suite 8.4 Professional. The binning results were aligned by the icoshift algorithm of MATLAB (MathWorks, USA). Principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) were performed using SIMCA 15.0.2 (Umetrics, Sweden).

Results and discussion

The cytotoxic effects of C. longa extract and demethoxycurcumin, a single compound in the plant, on the HCT116 cell line were examined by MTT assay. The results show that HCT116 cell growth was dose-dependently inhibited by C. longa extract treatment for 24 h (Fig. 1A) and 48 h (Fig. 1B). The cell viability was less than 40% after treatment with 300 µg/mL of C. longa extract for 24 h, and less than 20% after treatment with 300 µg/mL of extract for 48 h. C. longa extract effectively inhibited the survival of HCT116 cells. Demethoxycurcumin treatment also inhibited the growth of HCT116 cells in a dose-dependent manner (Fig. 1C). C. longa extract majorly contains curcumin, and there have been many reports of curcumin inhibiting the growth of HCT116 cells [12,13,14]. As a result of confirming the cytotoxicity of the single compound contained in C. longa, the IC50 of curcumin was 12 µM (data was not shown), and the IC50 of demethoxycurcumin was 38.5 µM. The reason why C. longa extract effectively inhibited the growth of the HCT116 cell line is expected to be due to the combined action of the activity of curcumin, which is high in turmeric extract, and the activity of demethoxycurcumin. We attempted to determine how C. longa extract and demethoxycurcumin affect metabolic changes associated with HCT116 cell growth inhibition.

Fig. 1
figure 1

Effects of C. longa extract or demethoxycurcumin on cell viability of HCT116 cells. A Cell viability of HCT116 cells treated with C. longa extract (0, 50, 100, 200, and 300 µg/mL) for 24 h and B 48 h, C Cell viability of HCT116 cells treated with demethoxycurcumin (0, 1.5, 3.125, 6.25, 12.5, 25, and 50 µM) for 24 h

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To confirm the metabolic perturbation by C. longa treatment in HCT116 cells, NMR-based metabolic analyses were performed. To obtain the 1D 1H-NMR spectra, a 700 MHz NMR spectrometer was used. To identify metabolites in the 1D spectra, 2D COSY and HSQC-DEPT experiments were performed using a 900 MHz NMR spectrometer. All spectra were analyzed for identification and quantification of metabolites using Chenomx NMR suite software, the open Human Metabolome Database (HMDB), and measured 2D NMR spectra (Additional file 1: Figures S1 and S2). A total of 45 primary metabolites were analyzed in the extract of HCT116 cells (Additional file 1: Table S1). Figure 2 shows a representative NMR spectrum of the HCT116 extract and annotation of identified metabolites.

Fig. 2
figure 2

Representative one-dimensional NMR spectra of the HCT116 cell extract with annotation of major metabolites. 4-HP trans-4-hydroxy-L-proline, Ala alanine, Asn asparagine, Asp aspartate, Glu glutamate, Gly glycine, GPC sn-glycero-3-phosphocholine, GSH glutathione, His histidine, Ile isoleucine, Leu leucine, mI myo-inositol, NAA N-acetylaspartate, PC O-phosphocholine, Phe phenylalanine, Pro proline, Thr threonine, Tyr tyrosine, Val valine

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Quantified metabolites were analyzed by biomarker discovery analysis using receiver operator characteristic (ROC) curves with sensitivity and specificity. The area under the ROC curve (AUC) for each metabolite was calculated when comparing the C. longa extract treated group with the control group and the demethoxycurcumin 60 μM treated group with the control group (Table 1). In the comparison of the C. longa extract treated group with the control group, the AUC value of alanine, arginine, aspartate, betaine, creatine, glutamate, glutathione, glycine, lactate, phenylalanine, threonine, uracil, and valine was 1.00, which makes it an excellent prediction biomarker. In general, AUC values of 0.9–1.0, 0.8–0.9, and 0.7–0.8 are considered excellent, good, and fair predictive biomarkers, respectively [33]. In our results, the contents of choline and glycerophosphocholine formed by the degradation of phosphatidylcholine were the highest in the control group, and were decreased with doxorubicin and demethoxycurcumin treatment. Phosphocholine, which is also part of the CDP-choline pathway, was decreased after treatment with demethoxycurcumin. It is considered that the CDP-choline pathway, which is activated for the survival of cancer cells, is blocked by treatment with demethoxycurcumin (Fig. 7).

Fig. 6
figure 6

Pearson correlation heatmap of metabolites in the HCT116 cell extracts. A Comparison of control group, doxorubicin treatment group and C. longa extract treatment group, B comparison of control group, doxorubicin treatment group and demethoxycurcumin treatment (20, 40, 60 µM) groups

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Fig. 7
figure 7

CDP-choline pathway actively occurring for the survival of cancer cells

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