Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism

Abstract The purpose of the present study was to evaluate the anti‐cancer property of Lobetyolin on colorectal cancer and explore its potential mechanism. Lobetyolin was incubated with HCT‐116 cells in the absence or presence of ASCT2 inhibitor Benser or p53 inhibitor Pifithrin‐α. The levels of glutamine, glutamic acid, α‐ketoglutarate, ATP and GSH were determined to measure the glutamine metabolism. Annexin V‐FITC/PI staining and TUNEL assay were applied to estimate the apoptotic condition. The levels of ASCT2 were examined by RT‐qPCR, Western blot and immunofluorescence staining. The expressions of cleaved‐caspase‐3, caspase‐3, cleaved‐caspase‐7, caspase‐7, cleaved‐PARP, PARP, p53, p21, bax and survivin were detected using Western blot analysis. As a result, the treatment with Lobetyolin effectively induced apoptosis and glutamine metabolism in HCT‐116 cells through ASCT2 signalling. The inhibition of ASCT2 reduced the glutamine‐related biomarkers and augmented the apoptotic process. We further found that the effect of Lobetyolin on HCT‐116 was related to the expressions of p21 and bax, and transportation of p53 to nucleus. The inhibition of p53 by Pifithrin‐α promoted the inhibitory effect of Lobetyolin on ASCT2‐mediated apoptosis. Lobetyolin also exerted anti‐cancer property in nude mice. In conclusion, the present work suggested that Lobetyolin could induce the apoptosis via the inhibition of ASCT2‐mediated glutamine metabolism, which was possibly governed by p53.


| INTRODUC TI ON
As one of the most common malignant cancer types, colon cancer remains the second leading cause of cancer-related deaths worldwide. Colorectal tumour is highly ranked in the morbidity and even accounts for the 8.5% mortality. Colon cancer deeply influences the life quality and causes heavy economic burden on the patients and society. 1 During the past few decades, great advance has been achieved on the exploration of therapeutic target and compound.
Unfortunately, most drugs are not satisfied due to their resistance, side effect and limited efficacy in clinical. Thus, it is urgent to develop the new candidate compound for the intervention of colorectal carcinogenesis.
The cell metabolism which provides energy and substance for the proliferation of tumour has been recognized as the key feature of carcinogenesis. Growing evidence elicited that the augmented requirement of carbon and nitrogen source conduced to the reliance of cancer cell on glutamine. Glutamine serves as a crucial metabolic substrate in cancer cell and participates in tumour development and proliferation. Alanine-serine-cysteine transporter 2 (ASCT2), the membrane-associated protein, is a broad specificity bidirectional transport of glutamine. 2 As the mediator of glutamine uptake and metabolism, ASCT2 is involved in the aetiology of multiple cancers, such as breast cancer, prostate cancer and melanoma. It was reported that the inhibition of ASCT2 repressed reduced cancer cell growth and proliferation in LS174T cell line. 3 Thus, it was assumed that ASCT2 might be a candidate target for the intervention of colon cancer, whereas the underlying role of ASCT2 in the pathogenesis of colorectal cancer remains not fully understood.
Codonopsis pilosula (Franch.) Nannf., the famous traditional Chinese medicine, has been used to enhance the immune system, suppress blood pressure, attenuate gastrointestinal function, improve appetite and treat gastric ulcer. 4 Its bioactive compound, Lobetyolin, is the critical ingredient of polyacetylenes in C pilosula.
Growing evidence indicated that the Lobetyolin exerted anti-inflammatory, anti-oxidative 5,6 and xanthine oxidase inhibiting properties. 7 It was noteworthy that Chinese medicine Bu-Fei decoction, containing the constituent of Lobetyolin, was proved to relieve epithelial-mesenchymal transition of non-small-cell lung cancer. 8 Moreover, the steamed Codonopsis lanceolate, which consisted of Lobetyolin, was also confirmed to show beneficial effect on H22 tumour-bearing mice. 9 Nevertheless, there has been limited report focused on the anti-cancer effect of Lobetyolin in colon cancer. Thus, the present study was carried out to investigate the pharmacological effect of Lobetyolin on colon cancer and explore its underlying mechanism.

| Reagents
The tested compounds were provided by Dongdong Sun (Nanjing University of Chinese Medicine). For the treatment, the compounds were dissolved in culture medium and DMSO [with DMSO less than 0.1%(v/v)]. HCT116 colorectal cancer cells were supplied from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 culture medium was provided by Biological Industries (Beit-Haemek). TUNEL apoptotic commercial kit was produced by Beyotime.

| Cell culture
The HCT-116 and NCM460 cells were cultured in RPMI 1640 medium complemented with 10% foetal bovine serum (Thermo) and 1% penicillin-streptomycin (Biological Industries) at 37°C with 5% CO 2 in a humidified incubator. The culture medium was refreshed every 1-2 days.

| The determination of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH
The concentrations of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH were measured in accordance with the procedure of manufacturer's instructions.

| Annexin V-FITC/PI staining
The apoptosis was evaluated by Annexin V-FITC/PE staining. HCT-116 cells were washed using cold PBS for two times and resuspended in 500 μL binding buffer. Afterwards, the cells were exposed to 1 μL V-FITC/PE solution in dark environment at 25°C for 10 minutes. The percentage of apoptotic cell was analysed in a FACS flow cytometer (Becton Dickinson).

| TUNEL assay
The apoptotic situation of HCT-116 cells was estimated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The cells were seeded on the chamber slides and treated with Lobetyolin for 24 hours. After washing with PBS, the TUNEL staining was carried out in accordance with the manufacturer's procedure. Anti-evaporation membranes were applied to maintain the humidification. The anti-fluorescence quenching solution was used prior to the visualization under fluorescence microscope. The experiment was conducted in triplicate. were removed and weighed. The tumours were applied for mRNA and Western blot analyses.

| RT-qPCR
2 mL 2.5 × 10 5 /mL HCT-116 cells were seeded on the six-well plate for 24 hours. Then, the culture medium was removed and the compound was added to the cells. 24 hours later, the cells were harvested for the RT-qPCR assay. Total cellular RNA was isolated using TRIzol reagent.
1 μg total RNA was reverse transcribed to make cDNA using SYBR Premix Ex Taq™. The reaction mixer consisted of supermix, total RNA and DNase-free water. RNA was transcribed using M-MuLV Reverse Transcriptase (Invitrogen). The primers were Synthesized by Sangon Biotech, and the sequences were listed as Table 1. Fluor in the dark for 1 hour and exposed to 150 μL DAPI. After sealing with anti-fluorescence quenching solution, the samples were observed by fluorescence microscope.

| Statistical analysis
All the results were illustrated in mean ± SD. One-way analysis of variance with Student's t test was carried out using GraphPad 6.0. P < .05 was considered as significant. To examine the mechanism of Lobetyolin on apoptotic process, the protein expressions of cleaved-caspase-3, caspase-3, cleavedcaspase-7, caspase-7, cleaved-PARP and PARP were examined.

| The effect of Lobetyolin on HCT-116 cells
As illustrated in Figure  Lobetyolin was capable of mediating glutamine metabolism.

| The effect of Lobetyolin on the transportation of p53 to nucleus and the expressions of p21 and bax
Next, we investigated the effect of Lobetyolin on p53 protein transportation to the nucleus. As revealed in Figure 6A, the expression of p53 were down-regulated in nucleus and up-regulated in cytoplasmic (both P < .01) caused by the treatment with Lobetyolin (10, 20, 40 μmol/L). Figure 6B also revealed that the nucleoprotein expression of p53 was improved compared with that of control group. The obtained data confirmed that Lobetyolin administration promoted the translocation of p53 from cytoplasm to nucleus.
Moreover, the mRNA of apoptosis-related protein p53 and cell cycle-related protein p21 were detected by RT-qPCR. As shown in Figure 6C, the augmented mRNA levels of Bax (P < .01) and p21 (P < .05) were observed owing to the stimulation with Lobetyolin

| The role of p53 in Lobetyolinmediated apoptosis
The above results indicated that Lobetyolin inhibited glutamine metabolism to induce apoptosis by suppressing ASCT2. To further explore the role of p53 during this process, Pifithrin-α, the selective p53 inhibitor was applied. As illustrated in Figure 7A,

| The effect of Lobetyolin on tumour growth in vivo
In order to further evaluate the antitumour effect of Lobetyolin on colon cancer in vivo, the nude (nu/nu) mice were transplanted with HCT-116 cells. As summarized in Figure 9, the Lobetyolin (10, 20, 40 mg/kg) treatment obviously inhibited the tumour volume compared with that in control group. The ASCT2 mRNA level in Lobetyolin (10, 20, 40 mg/kg) groups was remarkably decreased compared with that in control group. Besides, the administration of Lobetyolin (10, 20, 40 mg/kg) markedly down-regulated the ASCT2 protein expression compared with that in control group. The in vivo results suggested that Lobetyolin exerted anti-cancer activity in nude mice, which was possibly associated with the suppression of glutamine metabolism.

| D ISCUSS I ON
Increasing evidence has emerged indicating that C pilosula possesses antitumour property. Codonopsis pilosula component inhibited the cancer cell proliferation and migration. 10 It was proposed that C pilosula restrained hepatocellular carcinoma via GDF15 and HMOX1. 11 Lobetyolin also presented cytotoxic activity against lung cancer. 12 Although there was limited literature focused on the pharmacological effect of Lobetyolin on colon cancer, we assumed that Lobetyolin might function as a therapeutic candidate for colorectal tumour.
Glutamine, the critical precursor of nucleotides and proteins, is generally considered as an essential mediator for cellular metabolism. which induces the apoptotic process. 15 Cleaved-PARP, cleavedcaspase-3 and cleaved-caspase-7 were applied for diagnosing the apoptosis in colorectal cancer. 16 Survivin, the member belongs to the apoptosis inhibitor protein family, was reported to be highly related to caspase-3/7 pathway and participate in the modulation of colon cancer. 17 Our data proved that Lobetyolin evidently induced apoptosis by promoting the expressions of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP and PARP.
Nonetheless, the blockade of ASCT2 and p53 notably inhibited the apoptotic progression.
A variety of metabolic approaches have been developed as pivotal targets for anti-cancer drugs. It is widely acknowledged that the metabolism of specific nutrients including glucose and glutamine are required for the energy generation of cancer cell. Previous investigators illuminated that the prevention of glutamine uptake could induce the apoptosis of glutamine addicted tumours. 18 ASCT2 drives glutamine transport through nutrient transporters in diverse cancers. 19 The silence of ASCT2 repressed intracellular glutamine accumulation and contributed to apoptotic cell death in human breast cancer. 20 The experimental data revealed that the inhibition of ASCT2 by Benser successfully reduced the levels of glutamine metabolism indices including Glutamine, glutamic acid, α-ketoglutarate, ATP and GSH. The combination of Lobetyolin and Benser slightly promoted apoptosis-related protein expressions more than Lobetyolin-treated alone group, which suggested that the ASCT2 was involved in the regulation of Lobetyolinmediated HCT-116 cells.
p53, the key tumour suppressor gene, has been clarified to regulate cellular stress. p53 plays a critical role in mediating DNA repairing, cell cycle progression and apoptosis to prevent cancer development.
It was elicited that p53 regulates energy metabolism and ATP synthesis, which further elevated glutathione (GSH) level in cancer cells. 21 Nikkuni et al 22 demonstrated that ASCT2 was highly related to the p53 expression in laryngeal squamous cell carcinoma. ASCT2 functioned as the significant prognostic and preventive factor in tongue cancer through p53. 23 It was proposed that glutaminase was a p53 target gene for tumour suppression. 13 The expressions of p21 and p53 were altered in glutamine-deficient hepatoma cells. 24 To explore the underlying p53 In conclusion, the present study demonstrated that Lobetyolin exhibited anti-cancer effect on colon cancer HCT-116 cells through the apoptosis regulated by ASCT2-modulated glutamine metabolism, which was governed by p53. Further investigations are warranted before the clinical application of Lobetyolin.

ACK N OWLED G EM ENTS
This work was financially supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support findings of the study are available from the corresponding author upon request.