Liensinine perchlorate inhibits colorectal cancer tumorigenesis by inducing mitochondrial dysfunction and apoptosis

Yang Wang , Yang-Jia Li , Xiao-Hui Huang , Can-Can Zheng , Xing-Feng Yin , Bin Li * and Qing-Yu He *
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. E-mail:;; Fax: +86-20-85227039; Fax: +86-20-85224372; Tel: +86-20-85227039 Tel: +86-20-85224372

Received 8th June 2018 , Accepted 18th August 2018

First published on 21st August 2018

Scope: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. Methods and results: Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. Conclusion: This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.

1. Introduction

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths among both men and women worldwide with poor overall survival.1 The incidence rate of CRC in those aged under 50 years increased by 22% from 2000 to 2013.1 Currently, chemo- and radiotherapies remain the chief strategies for cancer therapy.2 However, the main obstacle to an effective treatment is the failure of the initial chemotherapy to eradicate a sufficient number of tumor cells to prevent tumor recurrence.3 Secondly, another reason for the failure of conventional chemotherapy is that conventional chemotherapy drugs have serious toxic effects on the human body. For example, many chemotherapeutics target proliferating cells, including both cancer cell and somatic cells.4–6 Therefore, food source phytocompounds with a potential anti-tumor effect may be given priority to be adopted due to their stability and safety for the clinical application.7 Screening the novel chemotherapeutics from a food source compound library is a promising strategy to guarantee their safety and effectiveness.

Using a natural product library consisting of 429 food source compounds, we identified liensinine, an active constituent of Nelumbo nucifera Gaertn, as a novel anticancer agent. Nelumbo nucifera, also called sacred lotus, is a daily food in Asia and is used as a diuretic, vasodilator, anti-bacterial, and anti-helminthic and in the treatment of strangury, vomiting, and nervous exhaustion.8 Different parts of N. nucifera, including the roots, rhizomes, flowers, leaves, fruits and seeds, have been used as various medicated diets to prevent cardiovascular disease and cancer for centuries and have shown tremendous potential. Liensinine, an isoquinoline alkaloid extracted from the seed embryo of Nelumbo nucifera Gaertn, has a wide range of biological activities, including treatment of arrhythmias, hypertension, pulmonary fibrosis9 and cancer.10 Liensinine was found to inhibit late-stage autophagy/mitophagy through blocking autophagosome–lysosome fusion, and suppress mitochondrial fission and the growth of breast cancer cells.11 It was reported that liensinine could block the binding of E2F1 at the transcription factor binding site of the FGFR2 promoter region and inhibit the FGFR2 gene expression.12 However, the effects of liensinine on colorectal cancer have rarely been reported, and the underlying mechanisms are largely unknown.

Researchers became interested in investigating which proteins or signaling pathways can be regulated by liensinine, which warrants a proteome level study. As a major advance in the field of quantitative proteomics, data-independent acquisition (DIA) mass spectrometry (MS) is considered to be a promising method to improve the comprehensiveness and reproducibility of large-scale discovery proteomics. The basic theory of DIA-MS is to combine the reproducibility of selected reaction monitoring with the breadth of the data-independent acquisition (DIA) method by simultaneously fragmenting all precursors whose mass-to-charge ratios (m/z) fall into one of a small number of wide windows that traverse the entire m/z range. DIA-MS is allowed to measure low-abundance proteins in biological samples with high reproducibility,13 and our previous study had applied DIA-MS in profiling protein alterations in ESCC cells treated with propafenone, an FDA-approved drug, and revealed its action mechanisms.4 In the present study, our in vitro and in vivo experiments demonstrated a strong anticancer effect of liensinine on CRC. Using DIA-MS and a series of experimental confirmations, we found that liensinine may induce apoptosis and suppress the growth of CRC cells via the Jun N-terminal kinase (JNK)-mitochondrial dysfunction pathway.

2. Materials and methods

2.1 Cell lines and drugs

The human CRC cell lines HT29 and DLD-1 (ATCC, Rockville, MD, USA) were cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies) at 37 °C in 5% CO2. The human immortalized colonic epithelial cell line NCM460 was obtained from IN CELL (San Antonio, TX, USA) and cultured according to the recommendation of the manufacturer. The natural product compound library and liensinine perchlorate were purchased from TargetMol (Boston, MA, USA).

2.2 Cell viability assay

Cell viability was measured by the WST-1 assay (Beyotime, Jiangsu, China) as described previously.14 In brief, a total of 5000 cells in 100 μl were seeded into 96-well plates and treated with various concentrations of liensinine (0, 5, 10, 20 μM) for 24 h and 48 h, respectively. WST-1 was added and incubated at 37 °C for 1 h, and then the absorbance was measured using an automated microplate spectrophotometer (BioTek Instruments, Vermont, USA) at a wavelength of 450 nm.

2.3 Colony formation assay

CRC cells were seeded into 6-well plates at a density of 2000 cells per well and cultured for 14 days. After washing twice with PBS, the cells were fixed with methanol for 15 min and stained with 1% crystal violet for 20 min at room temperature. The number of colonies was counted, and all statistical measurements were obtained from three independent experiments.15

2.4 Annexin V-FITC/propidium iodide (PI) staining assay

Cell apoptosis was determined using an Annexin V-FITC/PI Apoptosis Detection kit (KeyGen, Jiangsu, China).16 Cells were suspended in binding buffer, and stained with annexin V-FITC and PI for 15 min at room temperature in the dark. Apoptosis was assessed and analyzed using a C6 flow cytometer (BD Biosciences, San Diego, CA, USA).

2.5 Measurement of ROS and mitochondrial transmembrane potential

The intracellular ROS level in HT29 and DLD-1 cells was determined by measuring the cell permeable 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Beyotime) as previously described.16 The JC-1 assay (Beyotime) was used for the measurement of the mitochondrial transmembrane potential. Cells were treated with the indicated concentrations of liensinine for 48 h, and stained with JC-1 according to the manufacturer's instructions.

2.6 Western blot analysis

Whole-cell lysates were prepared in lysis buffer (Cell Signaling Technology, Beverly, MA, USA).17 A BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration. The samples were loaded onto a sodium dodecyl sulfate (SDS)-PAGE gel and subsequently electrotransferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk for 1 h, the membrane was incubated with primary antibodies for 2 h at room temperature and washed three times for 10 min per wash with 1× Tris-buffered saline with Tween (TBST). Next, the membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The reaction was visualized using Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and detected by exposure to an autoradiographic film.18 The antibodies used included caspase-3, cleaved caspase-3, phospho-CDK1 (p-CDK1), cyclin A2, JNK, phospho-JNK (p-JNK), Bax, Bcl-2, Bcl-xL, PAPR, cleaved PARP (Cell Signaling Technology) and actin (from Santa Cruz Biotechnology, Santa Cruz, CA, USA).

2.7 Flow cytometric cell cycle analysis

The cells were fixed with 70% alcohol for 1 h at −20 °C and stained with PI staining buffer (PBS containing 33 μg ml−1 PI, 0.13 mg ml−1 RNase A, 10 mM EDTA, 0.5% Triton X-100) for 10 min at room temperature. A cell cycle analysis was performed using a BD Accuri C6 Analyzer (BD Biosciences).

2.8 Mass spectrometry and bioinformatics analyses

Whole-cell lysates were homogenized in RIPA lysis buffer. After further trypsin digestion through the method of filter-aided sample preparation (FASP), the peptide samples were vacuum-freeze-dried and resuspended in anhydrous acetonitrile solution for further desalination using a MonoTIPTM C18 Pipette Tip (GL Sciences, Tokyo, Japan). Next, the peptides were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). The raw data were searched using Proteome Discoverer (Thermo Fisher Scientific) and Spectronaut (Omicsolution Co., Ltd, Shanghai, China) software. An FDR of 1% was set to identify proteins. The differentially expressed proteins were analyzed by Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City, CA, USA) as described previously.4,19,20

2.9 Tumor xenograft experiments

Female BALB/c nude mice aged 6–8 weeks were maintained under standard conditions. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Jinan University and approved by the Animal Ethics Committee of Jinan University. HT29 cells in equal volumes of PBS and Matrigel were subcutaneously implanted into the flanks of mice to establish tumor xenografts.21 When the tumor xenografts reached ∼5 mm in diameter, the mice were randomly divided into treatment and control groups (6 mice per group). The treatment group received oral gavage of liensinine at a dose of 30 mg kg−1 every other day for 15 days, whereas the control group received the vehicle only. The body weight of the mice was monitored every two days during the experiments to evaluate overall health. Tumor size was measured every two days, and the tumor volume was calculated using the following equation: V = (length × width2)/2. At the end of the study, tumors, lungs, liver, and kidneys were collected for further analysis. Immunohistochemical analysis of the proliferative index was performed using antibodies against Ki-67 (Dako, Mississauga, ON, Canada).22

2.10 Statistical analysis

All in vitro experiments were performed in triplicate, and the data were expressed as the means ± SD. The GraphPad Prism software (San Diego, CA, USA) was used to calculate statistically significant differences using a Student's t-test method. All statistical tests were two-sided, and P < 0.05 was considered to be statistically significant.

3. Results

3.1 A high-throughput screening of food source compounds with anticancer bioactivity in CRC cells

In order to identify candidate therapeutic agents for CRC, as shown in Fig. 1A, we firstly selected 52 food-source compounds, which have rarely been reported for their bioactivity, in particular, the anticancer effects, from a natural product library containing 429 food source compounds. To screen the compounds with a strong anticancer effect on CRC cells, HT29 cells were treated with 52 food source compounds (10 μM) individually for 48 h, and the inhibitory effect for each compound is shown in Fig. 1B. We found that liensinine, an isoquinoline alkaloid extracted from the seed embryo of Nelumbo nucifera Gaertn (Fig. 1C), with its chemical structure shown in Fig. 1D, had the strongest inhibitory effect (89.99 ± 0.52%) among the candidate agents. These data suggest that liensinine could be a potential food-source chemotherapeutics for CRC treatment and warrants further investigation.
image file: c8fo01137k-f1.tif
Fig. 1 Food-source library screening identified liensinine as a potential anticancer agent for treating CRC. (A) Flow chart for food-source library screening. A total of 52 compounds were selected from a food-source natural product library (429 compounds) according to a literature study, and subjected to cell viability screening. (B) HT29 cells were individually treated with the 52 compounds at a concentration of 10 μM for 48 h. Liensinine showed a significant anticancer effect in three independent experiments. (C) Liensinine is an isoquinoline alkaloid extracted from the seed embryo of Nelumbo nucifera Gaertn. (D) Chemical structure of liensinine.

3.2 Liensinine suppresses CRC cell proliferation

To evaluate the inhibitory effect of liensinine on cancer cell proliferation, both CRC cell lines HT29 and DLD-1 were exposed to increasing concentrations (up to 20 μM) of liensinine for 24 h and 48 h, and the results showed that liensinine markedly inhibited cell viability and cell proliferation with increasing concentration in DLD-1 and HT29 cells (Fig. 2A), with the IC50 of 11.265 ± 0.83 μM and 14.507 ± 0.834 μM for 24 h, 5.65 ± 0.57 μM and 6.16 ± 0.267 μM for 48 h, respectively. Note that the cytotoxicity of liensinine was not observed in the normal colorectal epithelial cells NCM460 (ESI Fig. S1). In addition, we determined the effect of liensinine on colony-formation in CRC cells. As shown in Fig. 2B, the number of colonies formed by CRC cells treated with liensinine was significantly decreased in a dose-dependent manner. Collectively, these results suggest that liensinine exhibited a strong anticancer effect in CRC.
image file: c8fo01137k-f2.tif
Fig. 2 Liensinine inhibits CRC cell proliferation. (A) DLD-1 and HT29 cells were incubated with various concentrations (up to 20 μM) of liensinine for 24 and 48 h, respectively, and cell viability was determined by the WST-1 assay. (B) Liensinine inhibited the colony formation ability of DLD-1 and HT29 cells. Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control cells.

3.3 Liensinine induces apoptosis in CRC cells

Next, we investigated the effect of liensinine on apoptosis in CRC cells. Both DLD-1 and HT29 cells were treated with the indicated concentrations of liensinine (up to 20 μM) for 48 h, respectively, and the percentages of apoptotic cells were determined by the Annexin V-FITC/PI double staining assay using flow cytometry. As shown in Fig. 3A and B, liensinine induced apoptosis in DLD-1 and HT29 cells in a dose-dependent manner, with the fold change of apoptotic cell increased over 13.5 and 4.3 times in DLD-1 and HT29 cells, respectively. The effect of liensinine on apoptosis was also evidenced by the increased expression levels of cleaved caspase-3 and cleaved PARP in liensinine-treated CRC cells (Fig. 3C). Taken together, these results indicated that liensinine can induce apoptosis in CRC cells.
image file: c8fo01137k-f3.tif
Fig. 3 Liensinine induces apoptosis in CRC cells. (A) DLD-1 and HT29 cells were treated with different concentrations (up to 20 μM) of liensinine for 48 h; the apoptotic cells were detected by an Annexin V-FITC/PI double staining assay. (B) The fold change of apoptotic cells including early apoptosis and late apoptosis upon liensinine treatment was statistically presented. (C) Western blot analysis of cleaved caspase-3, caspase-3, cleaved PARP and PARP expressions in the DLD-1 and HT29 cells treated with indicated concentrations of liensinine for 48 h. Bars, SD; *, P < 0.05; ***, P < 0.001 compared with control cells.

3.4 Liensinine induces G2/M arrest of CRC cells in a dose-dependent manner

We further studied the effect of liensinine on the cell cycle in CRC cells. Both DLD-1 and HT29 cells were incubated with various concentrations of liensinine (up to 20 μM) for 48 h, respectively, and the distribution of cell cycle was determined by a PI staining assay using flow cytometry. As shown in Fig. 4A and B, liensinine induced G2/M arrest in DLD-1 and HT29 cells in a dose-dependent manner, with the percentage of G2/M cells increased from 22.98% to 40.51% and from 25.27% to 39.77% in DLD-1 and HT29 cells, respectively. The effect of liensinine on G2/M arrest was also evidenced by the increased expression levels of p-CDK1 and CyclinA223 in liensinine-treated CRC cells (Fig. 4C). These results indicated that liensinine can induce G2/M arrest in CRC cells.
image file: c8fo01137k-f4.tif
Fig. 4 Liensinine causes G2/M arrest in CRC cells. (A) DLD-1 and HT29 cells were treated with liensinine (0, 5, 10 and 20 μM) for 48 h, and the distribution of cell cycle was determined by flow cytometry. (B) The percentages of cells in G1, S and G2/M phases were statistically presented. (C) Expression levels of p-CDK1, CDK1 and CyclinA2 were detected by western blot analysis. Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control cells.

3.5 DIA-based quantitative proteomics suggests the involvement of JNK-mitochondrial dysfunction signaling in the anticancer effect of liensinine in CRC

To elucidate the proteomics alterations and explore the potential action mechanism of liensinine in CRC cells, we used DIA-MS, which has been justified as a good tool for protein quantitation,4,24 especially for low abundance proteins to identify the liensinine-regulated proteins, and the experimental flow chart was shown in Fig. 5A. Here, a total of 3309 proteins were identified and quantified from triplicate samples of the cells treated with 10 μM liensinine for 48 h and DMSO control (Fig. 5B). Meanwhile, a total of 376 differentially expressed proteins (ESI Table 1) was identified through the power law global error model (PLGEM) algorithm,25,26 a statistical test for analyzing protein abundance, with a slope of 0.809 and an adjusted r2 of 0.987 (Pearson r = 0.75) (Fig. 5C). The residuals distributed evenly and were independent from the rank of mean abundances (Fig. 5D and E). The quantile–quantile (Q–Q) plot presented that the data had a fitted normal distribution of the residual standard deviations between the modeled and the actual values (Fig. 5F). A total of 376 differentially expressed proteins with 273 up-regulated proteins and 103 down-regulated proteins (FC ≥ 1.5, p-value ≤0.05, ESI Table 1) were generated according to PLGEM model (Fig. 5G). We further investigated the molecular mechanism involved in the anticancer effect of liensinine in CRC cells, IPA software was performed to characterize the canonical pathways in which the differentially expressed proteins participated, and the signaling associated with mitochondrial dysfunction ranks first with a p-value of 8.35 × 10−17 (Fig. 5H). Network analysis showed that JNK signaling plays a hub role in mediating liensinine-induced apoptosis (Fig. 5I). These data suggested JNK-mitochondrial dysfunction may play a critical role in anticancer effects of liensinine.
image file: c8fo01137k-f5.tif
Fig. 5 DIA-based quantitative proteomics and bioinformatics analyses identified liensinine-regulated proteins and pathways. (A) Experimental flow chart of the identification of liensinine-regulated proteins. (B) Venn diagram showing the number of overlapped proteins in three biological replicates. (C–F) The overlapped proteins were subjected to a PLGEM model. (C) PLGEM fitting of the abundance of liensinine-regulated proteins. (D) Histogram of residuals of identified proteins. (E) Residual distribution along with the rank of mean abundances. (F) Q–Q plot of the residual versus standard normal. (G–I) According to the PLGEM model, differentially expressed proteins with fold change ≥1.5 and P-value ≤0.05 were subjected to IPA analysis (G), and the top canonical pathways were listed (H). Note that a cluster of liensinine-regulated proteins pointed to JNK signaling (I).

3.6 Liensinine activates JNK pathway to induce mitochondrial damage

Based on the DIA-MS data, we evaluated the effect of liensinine on mitochondria function. The integrity of mitochondrial membrane permeability was determined with the fluorescent dye JC-1, which fluoresces a red color in healthy mitochondria but a green color when mitochondria are damaged. HT29 and DLD-1 cells were treated with increasing concentrations of liensinine for 48 h and stained with JC-1. A dose-dependent decrease of the red/green fluorescence ratio was observed (Fig. 6A and B), suggesting that liensinine may induce apoptosis through decreasing the mitochondrial membrane potential. Moreover, we observed a significant increase of intracellular ROS in both CRC cell lines (Fig. 6C). Our western blot results demonstrated that expression levels of Bax and p-JNK were remarkably increased upon liensinine treatment, whereas Bcl-2 and Bcl-xL expression were decreased (Fig. 6D). Taken together, these results demonstrated that liensinine may induce apoptosis through the JNK-mitochondrial dysfunction pathway.
image file: c8fo01137k-f6.tif
Fig. 6 Liensinine activates JNK signaling and causes mitochondrial dysfunction in CRC cells. (A) The mitochondrial membrane potential levels of the DLD-1 and HT29 cells treated with the indicated concentrations of liensinine (up to 20 μM) were evaluated by a JC-1 assay. (B) Percentages of cells with low mitochondrial membrane potential were quantified. (C) DLD-1 and HT29 were treated with different concentrations of liensinine for 48 h, and the ROS level was determined by a DCFH-DA assay. (D) Western blot analysis of the expressions of Bax, Bcl-2, Bcl-xL, p-JNK and JNK in the DLD-1 and HT29 cells treated with various concentration of liensinine. Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with control cells.

3.7 Liensinine suppresses tumor growth in vivo

Given that liensinine displays a significant anticancer effect in CRC cells, we next evaluated the therapeutic potential of liensinine using a mouse model. The nude mice bearing established subcutaneous tumor xenografts were orally administrated with 30 mg kg−1 liensinine, and its effect on tumor growth was monitored. The results showed that the tumor burden was markedly inhibited by liensinine treatment with a decrease of 63% (Fig. 7A and B). We also determined the Ki-67 proliferation index in the tumor xenografts, and the results suggested that liensinine inhibited tumor growth through decreased cell proliferation (mean index decreased from 43.0 ± 9.5% in the vehicle group to 11.7 ± 4.5% in the treatment group, Fig. 7C). As shown in Fig. 7D, western blot analysis indicated that expressions of p-JNK and Bax were upregulated in the tumor xenografts treated with liensinine (Fig. 7D). Furthermore, no significant difference between the treatment and control groups was observed in terms of body weight (Fig. 7E). Histological examination of vital organs, including the lungs, livers and kidneys, did not reveal any overt changes in morphology (Fig. 7F), suggesting that liensinine treatment had no toxic effects on animals.
image file: c8fo01137k-f7.tif
Fig. 7 Liensinine suppresses tumor growth in vivo. Nude mice bearing HT29-derived xenografts were orally administered either liensinine (30 mg kg−1) or vehicle every other day (n = 6 per group). (A) Images of tumors. (B) Tumor curves showed that liensinine exerted a significant inhibitory effect on the growth of tumor xenografts. (C) Immunohistochemical analysis of the Ki-67 proliferation index in tumor xenografts treated with liensinine and vehicle. (D) Comparison of the expression levels of Bax, p-JNK and JNK between the tumors from mice treated with liensinine or vehicle by western blot analysis. (E) Body weight of nude mice during the experimental period. (F) Hematoxylin and eosin (H&E) staining of the lungs, livers and kidneys collected from the mice of the treatment and control groups. Bars, SD; **, P < 0.01 compared with control group.

4. Discussion

Increasing evidence suggested that some natural compounds, such as curcumin and resveratrol, achieve remarkable clinical outcome in cancer therapy.27–29 In order to screen the food-source natural products with significant anticancer effect on CRC cells, we selected 52 compounds by literature research from a natural product library containing 429 food-source compounds to investigate their bioactivity in CRC cells. This study demonstrated for the first time that liensinine perchlorate, a natural chemical compound extracted from seed embryo of Nelumbo nucifera Gaertn, significantly induced apoptosis and suppressed tumor growth through causing mitochondrial dysfunction and activation of JNK signaling in colorectal cancer.

As a bioactive compound of the Nelumbo nucifera, liensinine is generally recognized as safe for use in daily food,30 moreover, accumulating evidence showed that liensinine and its analogue exhibits potential anticancer agents.11,31 Liensinine could increase CHOP and Bcl-2-dependent apoptosis in renal NRK-52E cells.32 The analogue of liensinine, isoliensinine and neferine, were found to increase the production of reactive oxygen species (ROS) and induce apoptosis in breast cancer cells.31 In this study, a systematic investigation on the action mechanisms of liensinine in cancer was performed. Our results from quantitative proteomics and bioinformatics analysis showed that many liensinine-regulated proteins in CRC cells were enriched in mitochondrial dysfunction and oxidative phosphorylation (Fig. 5), suggesting that the mitochondrial related pathway may significantly contribute to the biological function of liensinine. Our in vitro and in vivo data indicated that liensinine suppressed colorectal tumorigenesis by inducing cell apoptosis via the JNK-mitochondrial dysfunction signaling pathway in colorectal cancer.

JNK, known as stress activated protein kinases, can be activated by various stress stimulations.33,34 It has been well documented that JNK plays a critical role in activating apoptotic signaling by up-regulating pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro-apoptotic proteins through phosphorylation.35,36 Moreover, JNK is a critical regulator of cell cycle progression, which is linked to tumorigenesis and carcinogenesis.37 Therefore, JNK could be considered as a therapeutic target in cancer treatment, and some potential agents activating this pathway have been developed.38 Our results showing that liensinine treatment significantly induced phosphorylation of JNK and triggered G2/M phase arrest in vitro and in vivo supported the potential implication of liensinine in the prevention and treatment of colorectal cancer.

Colorectal cancer is one of the most common malignant diseases and remains the leading cause of cancer-related deaths currently. A limited treatment efficiency and obvious side effects are the major obstacles in cancer therapy; therefore, the development of novel chemotherapeutics is urgently needed.39 A food-source compound is a valuable source for new drug discovery. In this study, we identified liensinine as a potential anticancer natural product from a 429 food-source compound library. Our in vitro and in vivo studies provide solid evidence that liensinine exerted a strong inhibitory effect on CRC tumorigenesis. Mechanically, liensinine can induce cell apoptosis via JNK-mitochondrial dysfunction signaling pathway. Therefore, our findings suggest that liensinine may serve as a potential anticancer strategy for treating CRC.


CRCColorectal cancer
DIAData-independent acquisition
MSMass spectrometry
PIPropidium iodide
FASPFilter-aided sample preparation
JNKJun N-terminal kinases
CDK1Cyclin-dependent kinases 1
PLGEMPower law global error model
IPAIngenuity pathway analysis
H&E stainingHematoxylin and eosin staining

Conflicts of interest

The authors declare no conflict of interest.


This work was supported by the National Key Research and Development Program of China (2017YFA0505100), the National Natural Science Foundation of China (31770888, 31570828, and 81773085), and Guangdong Natural Science Research Grant (S2013030013315 and 2016A030313838).


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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8fo01137k
These authors contributed equally to this work.

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