Qici Sanling Decoction Suppresses Glutamine Consumption and Bladder Cancer Cell Growth through Inhibiting c-Myc Expression

Traditional Chinese medicine (TCM) is widely used as an alternative therapy for cancer treatment in China. Glutamine catabolism plays an important role in cancer development. Qici Sanling decoction (QCSL) suppresses bladder cancer growth. However, the association between QCSL and glutamine catabolism remains unknown. In this study, different doses of QCSL were applied to T24 cells, followed by the measurements of cell viability and apoptosis using CCK-8 and Annexin V/PI assay, respectively. Furthermore, glutamine consumption was detected using the glutamine assay kit. QCSL was observed to inhibit cell growth and induced cell apoptosis in a dose-dependent manner. Analysis of glutamine consumption revealed that QCSL suppressed glutamine consumption in T24 cells. Furthermore, QCSL decreased the mRNA and protein levels of c-Myc, GLS1, and SLC1A5. All these effects induced by QCSL could be alleviated by c-Myc overexpression, indicating c-Myc was involved in the protective role of QCSL in bladder cancer. In addition, QCSL was found to inhibit tumor growth in the xenograft tumor model. The similar results were obtained in tumor samples that protein levels of c-Myc, GLS1, and SLC1A5 were decreased upon treatment with QCSL. In conclusion, QCSL suppresses glutamine consumption and bladder cancer cell growth through inhibiting c-Myc expression.


Introduction
Bladder cancer (BC) is one of the most common malignancies, which accounts for an estimated 500,000 new cases and 200,000 deaths each year throughout the world [1]. e high recurrence rate is the main cause of death. Cigarette smoking is considered as the main risk of factor, but advanced age, Schistosoma haematobium infection, and male sex also contribute to the development of BC [2]. Recently, the tumor microenvironment (TME) attracts researchers' attentions as playing a key role in tumor development, including BC. e diverse TME observed in tumor indicates that adequate nutrients are needed for growth of tumor cells.
Glutamine, one of the most abundant amino acids in the TME, is of great importance in the cancer cell metabolism and proliferation [3].
To fulfill the rapid proliferation of cancer cells, enhancing the carbon flux through glutaminolysis is observed [4]. Glutaminolysis is the process of glutamine catabolism. First, glutamine is catalyzed by glutaminase (GLS) to generate glutamate. en, glutamate dehydrogenase (GDH) converts glutamate to α-ketoglutarate (α-KG), which is the intermediate of tricarboxylic acid (TCA) cycle [5]. Glutaminase 1 (GLS1) and glutaminase 2 (GLS2) are the two types of GLS. GLS1, frequently upregulated in tumors, is regulated by the transcription factor c-Myc [6]. c-Myc is a member of the Myc family which plays critical roles in numerous cellular processes [7]. c-Myc has been reported to activate the expression of glutaminase, GLS, and SLC1A5 (solute carrier family 1 member 5), resulting in the regulation of glutamine uptake and catabolism [8].

Cell Culture.
Human bladder cancer cell line T24 was obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in the RPMI-1640 medium, supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL), in a 5% CO 2 humidified incubator at 37°C.

Preparation of QCSL and
Lentivirus. QCSL was purchased from E-Fong Pharmaceutical (Guangdong, China) and dissolved with PBS as previously described [9]. Overexpressing lentivirus targeting c-Myc was purchased from GeneChem Company (Shanghai, China).

Cell Apoptosis
Assay. T24 cells were planted into a 6-well plate and cultured overnight in an incubator; then, QCSL were applied to T24 cells. After 48 h incubation, all the cells including the culture medium were collected according to the instruction of the Annexin V/PI cell apoptosis detection kit (BioRad Laboratories). Cells were stained with Annexin V for 5 min in the dark, followed by staining with PI for 2 min. Finally, cell apoptosis was analyzed using flow cytometry.

Glutamine Consumption.
Glutamine concentration of each group was measured using the glutamine assay kit (Abcam) following the manufacturers' instructions. Glutamine consumption was the glutamine concentration in the original medium minus that in the treated medium.

Real-Time PCR.
Total RNA of treated cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). e reverse transcription system (TaKaRa, Dalian, China) and SYBR Green qPCR Mixes (TaKaRa) were used to measure gene expression. e reactions were performed on a 7900HT Sequence Detection System (Applied Biosystems, USA). Relative gene expression level was analyzed using the comparative Ct method (2 −ΔCt ). e primers used are as follows: c-Myc: primer F 5′ ATCCTGTCCGTCCAAGCA 3′, primer R 5′ CGCACAAGAGTTCCGTAG 3′ GLS1: primer F 5′ CTGTGCTCCATTGAAGTG 3′, primer R 5′ TGCCCTGAGAAGTCATAC 3′ SLC1A5: primer F 5′ ATCCATGGGCTCCTGGTACT 3′, primer R 5′ CACGCACTTCATCATCAGCG 3′ GAPDH: primer F 5′ AATCCCATCACCATCTTC 3′, primer R 5′ AGGCTGTTGTCATACTTC 3′ 2.8. Xenograft Tumor Model. BALB/c-nu nude mice (4-6 weeks) were purchased from Shanghai Laboratory Animal Research Center (Shanghai, China). All mice were housed at 20-23°C in a 12 h light/dark cycle with food and tap water supplied ad libitum. Xenograft tumor models were established as previously described [9]. After 2 weeks, 18 mice were randomly divided into three groups (vehicle, 200 mg/ kg QCSL, and 400 mg/kg QCSL). QCSL was applied to the mice in QCSL groups by gavage once a day for 3 weeks, and the mice in the vehicle group was given the same amount of PBS. en, the mice were anesthetized by isoflurane. e tumor samples from each group were fixed in 10% (v/v) formalin for TUNEL examination and frozen for Western blot. e experimental procedures for nude mouse xenograft tumor models and daily care were approved by the Committee on Ethical Use of Animals of Shanghai University of Traditional Chinese Medicine. All experiments were performed in accordance with the national legislation and with the National Institutes of Health Guidelines regarding the care and use of animals for experimental procedures.

TUNEL Assay.
After 48 h fixation in 10% (v/v) formalin, tumor tissues were paraffin embedded and cut into sections (5 mm; Leica RM2125, Germany). Cell apoptosis of tumor tissues were detected using a TUNEL Kit (Roche, Indianapolis, IN, USA) following the manufacturer's instructions. Percentages of apoptotic cells were evaluated in five randomly selected fields.

Statistical
Analysis. Data were presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to compare the differences between 3 or more groups. Differences between two groups were determined by Student's t-test. In addition, P < 0.05 was considered statistically significant.

QCSL Inhibits Cell Viability and Induces Cell Apoptosis in BC Cells.
To explore the function of QCSL in BC, different doses (0, 0.05, 0.1, 0.2, 0.5, 1, and 2 mg/ml) of QCSL were applied to BC cell line, T24 cells. As shown in Figure 1(a), QCSL significantly inhibited cell viability in a dose-dependent manner in T24 cells and 0, 0.2, 0.5, and 1 mg/ml of QCSL were selected for the following experiments. e results indicated that all three concentrations of QCSL markedly increased the cell apoptosis (Figure 1(b)).

QCSL Inhibits GLS1 Expression and Glutamine
Consumption. Given the critical role of glutamine catabolism in cancer cell proliferation, it is rationale to investigate whether and how QCSL regulates glutamine catabolism in BC cell line. To this end, first, glutamine consumption was measured after treatment with QCSL. As shown in Figure 1(c), glutamine consumption was significantly suppressed by QCSL. Next, we hypothesize the decreased glutamine consumption due to reduction of the GLS1 level which is a rate-limiting enzyme of glutamine catabolism. To test, we measured GLS1 mRNA and protein levels and found that GLS1 was downregulated after treatment with QCSL in T24 cells (Figure 1(d)). In addition, the mRNA and protein levels of SLC1A5 were reduced as well implicating decrease of glutamine uptake. To further investigate the mechanism contributing to reductions of GLS1 and SLC1A5, the level of c-Myc was measured and decrease of c-Myc was observed upon QCSL treatment (Figure 1(d)). e results showed that QCSL treatment could increase the cell apoptosis which can be alleviated by c-Myc overexpression (Figure 2(b)). Similarly, overexpression of c-Myc robustly increase the glutamine consumption, which can be rescued by QCSL treatment. Cotreatment with QCSL and c-Myc overexpressing lentivirus exhibited higher glutamine consumption than treatment with QCSL alone but still lower than c-Myc overexpression (Figure 2(c)). Mechanistically, the decrease of GLS1, SLC1A5, induced by QCSL treatment could be rescued by c-Myc overexpressing (Figure 2(d)). Taken together, QCSL increased cell apoptosis in T24 cells by repressing c-Myc expression.

QCSL Suppresses Tumor Growth in the BC Xenograft Mouse Model.
e previous results in Figures 1 and 2 render the rationale to treat BC with QCSL in vivo. To study the tumor suppressing role of QCSL, T24 cells were injected into nude mice to construct the xenograft tumor model. As shown in Figures 3(a) and 3(b), treatment with 200 and 400 mg/kg QCSL markedly inhibited tumor growth. TUNEL results of tumor samples showed that the apoptotic cells were much more in QCSL groups than that in the control group (Figure 3(c)), indicating that QCSL induced cell apoptosis in vivo. Furthermore, the decrease of GLS1, SLC1A5, and c-Myc induced by QCSL was also found in tumor samples (Figure 3(d)).
ese results suggest that QCSL suppresses BC tumor growth by regulating glutamine catabolism.

Discussion
TCM has been wildly used in Asia, particularly in China for thousand years. In the present time, many TCMs were accepted to be an alternative therapy for cancer treatment. However, the major limitation for applications of TCM is the unclear mechanisms due to the complicated components of TCM. Even some of TCMs has been reported to treat bladder cancer [10][11][12], and the role of QCSL in therapy of bladder cancer is still unclear.
Instead of the consumption of glucose, cancer cells have also been shown to favor glutamine as a preferential fuel. e glutaminolysis metabolism plays a vital role in the tumorigenesis and the occurrence of chemoresistance. In our previous study, we reported that QCSL inhibited T24 cells growth by inactivating the Wnt/β-catenin pathway. Of note, QCSL treatment dramatically reduced the level of c-Myc [9]. ese results render the rationale to further study the role of c-Myc in BC.
c-Myc has been reported to active the expression of glutaminase, GLS, and SLC1A5, resulting in the regulation of glutamine uptake and metabolism [8]. To study, we overexpressed c-Myc in T24 cells and observed increase of glutamine consumption upon c-Myc overexpressing (Figure 2). In addition to glutamine consumption, elevation of SLC1A5 indicates increase of glutamine uptake which makes  e collective data from in vitro study render strong rationale for the translational study in the xenograft tumor model. In the study, we observed QSCL inhibited tumor growth in a dose-dependent manner. It is important to determine the appropriate dosage of QSLC to treat BC in balance with toxicity and best therapeutic effects.
is finding provides us a new strategy to treat BC by controlling glutamine catabolism. e results also shed light on the new therapeutic strategy in combination of QSLC with glutamine  Journal of Oncology 7 catabolism inhibitor in the future. However, more studies are required before this application but anyway projects the dawn.

Conclusions
QCSL suppresses glutamine consumption and bladder cancer cell growth through inhibiting c-Myc expression.
Data Availability e data that support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.