Yi Qi Jie Du Formula and Salinomycin Combination Treatment Mediates Nasopharyngeal Carcinoma Stem Cell Proliferation,Migration and Apoptosis via CD44/Ras Signaling Pathway

HE Ln,ZHOU Fng-Ling,ZOU Pn,WANG Xin-Wen,JIANG Yi-Ln*,HE Ying-Chun*,LIAO Dun-Fng,CAO De-Ling

a.Graduate School,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China

b.Hunan Provincial Key Laboratory for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Traditional Chinese Medicine,Changsha,Hunan 410208,China

c.Diseases Prevention and Treatment with Traditional Chinese Medicine and Visual Function Protection Engineering and Technological Research Center,Changsha,Hunan 410208,China

d.Department of Medical Microbiology,Immunology &Cell Biology,Simmons Cancer Institute,Southern Illinois University School of Medicine,Springfield,IL 62794,USA

ABSTRACT Objective To assess the effects of Yi Qi Jie Du Formula (YQJDF)combined with salinomycin (SAL) on nasopharyngeal carcinoma stem cells (NPC-SCs) and investigate the underlying molecular mechanisms.Methods Cell counting methods,the CCK-8 assay,transwell migration assay and JC-1 staining,were used to observe the effects of the combination on the proliferation,migration and apoptosis of NPC stem cells,respectively.Western blot was used to detect the levels of protein in NPC-SCs.Results YQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPCSC apoptosis.Mechanistically,YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins,including cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9.Moreover,YQJDF combined with SAL synergistically decreased the levels of CD44,p-c-Src,Ras,p-PKCδ,p-MEK,p-c-Raf,p-ERK1/2 and p-AKT proteins.Conclusions The combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis,which may be closely related to the downregulation of the CD44/Ras signaling pathway.

Keywords Yi Qi Jie Du Formula (YQJDF)Salinomycin Nasopharyngeal carcinoma stem cells(NPC-SCs)Proliferation Apoptosis CD44/Ras signaling pathway

1 Introduction

Nasopharyngeal carcinoma (NPC) is one of the most common head and neck malignancies that affects southern China.Owing to the serious reactions associated with radiotherapy and chemotherapy,there is an urgent need to identify new safe and effective treatment options.Chinese medicine antitumor drugs may represent a safer and more effective treatment option than conventional chemotherapy.Cancer stem cells (CSCs) are a type of tumor cell with stem cell characteristics,unlimited proliferation and self-renewal ability.Moreover,NPC stem cells (NPCSCs) are considered one of the main causes of NPC formation and recurrence.Thus,targeting NPC-SCs may represent a novel therapeutic approach to NPC treatment.

The occurrence of NPC involves changes in a series of tumor regulatory factors.Antitumor drugs often play a role via the inhibition of the proliferation and induction of the apoptosis of cancer cells[1].A variety of cytokines,growth factors,inflammatory factors and other extracellular stimulatory signals can be transmitted to the cytoplasm and nucleus through relevant cell signal transduction pathways,particularly via the activation of the Ras oncogene and its associated signal transduction pathway,which leads to the excessive proliferation and progression of tumor cells[2-4].

Yi Qi Jie Du Formula (YQJDF) is an empirical recipe that has been researched and applied by our group during the past 20 years and has demonstrated a therapeutic effect for the clinical treatment of NPC[5-7].YQJDF consists of Astragali Radix (Huang Qi,黃芪),Coptidis Rhizoma (Huang Lian,黃連),Codonopsis Radix (Dang Shen,黨參),Trichosanthis Radix(Tian Hua Fen,天花粉),Hedyotis Diffusa (Bai Hua She She Cao,白花蛇舌草),Poria (Fu Ling,茯苓) and Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草).In previous studies,YQJDF has been shown to inhibit proliferation and induce cancer cell death via the modulation of nuclear transcription factors,regulation of the G0/G1 phase of the cell cycle,and induction of the expression of apoptosis-related genes[8-15].Recently,we observed that YQJDF inhibits the proliferation of NPC-SCs[16].

As a highly effective and broad-spectrum antitumor drug,salinomycin (SAL) can inhibit stem cell proliferation,as well as reduce stem cell marker expression,spheroid-forming ability and invasiveness[17-22].Additionally,SAL has been shown to inhibit NPC CSCs via the upregulation of miRNA-200C[23].As an area of interest in the study of drug combinations that can be used for the treatment of tumors,the combination of SAL and chemotherapeutic drugs can effectively eliminate cancer cells and CSCs[24,25].In our previous study[26],we determined that YQJDF combined with SAL affects the proliferation,apoptosis and migration of NPC cells;however,the precise effect of YQJDF treatment combined with SAL on NPC-SCs proliferation,apoptosis and migration remains unknown.

In this study,we aimed to elucidate the effects of YQJDF combined with SAL on the biological behavior of NPC-SCs and the related mechanisms.

2 Materials and Methods

2.1 Cell culture

The NPC cell lines,CNE2 and 5-8F,were purchased from Sun Yat-sen University (Guangzhou,China).NPC cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco,Grand Island,NY,USA),2 mmol/L L-glutamine and 100 U/mL penicillin-streptomycin.NPC-SCs were cultured in a serum-free culture solution,which was prepared by the addition of B27 (1:50,Gibco,Grand Island,NY,USA),20 ng/mL epidermal growth factor(Gibco),20 ng/mL basic fibroblast growth factor(Gibco),and 0.04% bovine serum albumin (Sigma-Aldrich LLC.,St.Louis,MO,USA) to RPMI 1640.

2.2 Preparation of aqueous extract of YQJDF

YQJDF consists of seven Chinese herbs:Astragali Radix (Huang Qi,黃芪) 30 g,Coptidis Rhizoma(Huang Lian,黃連) 10 g,Codonopsis Radix (Dang Shen,黨參) 10 g,Trichosanthis Radix (Tian Hua Fen,天花粉) 10 g,Hedyotis Diffusa (Bai Hua She She Cao,白花蛇舌草) 10 g,Poria (Fu Ling,茯苓) 10 g and Glycyrrhizae Radix Et Rhizoma (Gan Cao,甘草) 6 g,after some modifications based on clinical practice experience.YQJDF herbs were purchased from the Pharmacy Department of the First Hospital of Hunan University of Chinese Medicine.

An aqueous extract of the identified YQJDF medicinal material was prepared as previously described[16,27].The constituents of the YQJDF were mixed and soaked overnight in 10 times the volume of distilled water and boiled at 100 °C for 30 min.The drug solution was then separated for further use and the sediment residue was mixed with three times the volume of distilled water and boiled at 100 °C for 20 min.All drug solutions were refluxed for 4 h in a Soxhlet extractor,cooled in a vacuum,and dried to a powder.Then,a 10 mg/mL solution of YQJDF was prepared by dissolving the powder in a stem cell culture solution,filtering to remove bacteria,and adjusting to a pH of 7.2 for future experiments.

2.3 CSCs cell culture in serum-free suspension

NPC cells were routinely cultured in an incubator at 5% CO2and 37 °C under saturated humidity.NPCSCs were enriched in a serum-free culture system.Briefly,the NPC cells concentration was adjusted to 1×105cells/mL with serum-free medium and 5×105cells/mL were collected in a 25 cm2low adhesion culture bottle.The culture bottle was placed upright and shaken several times a day to maintain the cells in suspension and 1 mL of fresh serum-free culture medium was added every two days.After seven days of culture,the first generation of tumor microspheres was collected and passaged.Specific experimental procedures were performed following those reported previously[28,29].

2.4 Cell proliferation assay

Cell proliferation was detected using a cell counting method.According to the preliminary experimental results.The concentration gradients of the water extract with YQJDF were 0.5 mg/mL,1 mg/mL and 2 mg/mL (prepared with 10 mg/mL solution of YQJDF).The SAL concentration gradients were 10 μmol/L,20 μmol/L,40 μmol/L in CNE2-SCs and 10 μmol/L,20 μmol/L,30 μmol/L in 5-8F-SCs,respectively.Cells were inoculated into a 12-well plate (1.5×105cells/well) and treated with the drugcontaining culture solution for 72 h.The cells were counted using a cell counter and the cell proliferation rate was calculated.These experiments were repeated three times.According to the results of CCK-8,concentration of YQJDF was 1 mg/mL.The concentration of SAL used 40 μmol/L in CNE2-SCs and 30 μmol/L in 5-8F-SCs for subsequent expermients.

After the initial determination of the proper drug concentration,the cells were divided into four groups:solvent group (vehicle),YQJDF group,SAL group and YQJDF combined with SAL group (YQJDF + SAL).The cell counting kit-8 (CCK-8) method was used to detect the cell proliferation activity:1.5×104cells/well were seeded into a 96-well plate and treated with the indicated drugs for 48 h.Then,10 μL of CCK-8 solution was added to each well and the cells were incubated for 4 h.There were six duplicate wells for each group of cells.A microplate reader was used to measure the absorbance at 570 nm.The experiment was repeated three times to obtain an average.

2.5 Measurement of cell migration ability

Transwell inserts (8 μm pore) were placed in each well of a 24-well plate.Then,500 μL medium/well was added to the upper Transwell insert and 750 μL of complete medium was added to the well (under chamber).The well plate was preheated in a 37 °C incubator.The culture medium in the upper chamber was aspirated and 500 μL of NPC-SC suspension (4×104cells) was added and cultured in an incubator.After adherence to the upper chamber,the cell culture solutions were replaced with a drugcontaining culture medium for 20 h.Any nonmigrated cells in the upper chamber were gently wiped with wet cotton swabs.Then,10% methanol was added for 5 min to fix the cells.The cells were then air-dried and washed with water.Then,the samples were stained with crystal purple solution for 5 min and washed with distilled water.The samples were dried after washing,pictures were obtained,and the number of cells was counted.The experiment was repeated three times to calculate the average number of migratory cells.

2.6 Apoptosis assay

The cells were grouped as described in 2.4.According to the results of CCK-8,concentration of YQJDF was 1 mg/mL.The concentration of SAL used 40 μmol/L in CNE2-SCs and 30 μmol/L in 5-8F-SCs for subsequent experiments.After 48 h of treatment,the drug-treated cells were stained using a mitochondrial membrane potential assay kit (JC-1 staining).JC-1 reagent was equilibrated to room temperature and diluted 100 times with culture medium.Then,JC-1 was added to the cultured cells at a 1:10 ratio,which were incubated in a 37 °C incubator for 20 min and photographed under a fluorescence microscope.Three fields were randomly selected for each group and the number of apoptotic and normal cells was counted.The apoptotic rate was calculated and averaged.The experiments were repeated three times.

2.7 Western blot analysis

The expression levels of proteins were detected by western blot.The cells were washed three times with cold phosphate-buffered saline and lysed on ice in complete lysis-M buffer (Roche,Indianapolis,IN,USA) containing a protease inhibitor cocktail for 15 min.Lysates were centrifuged at 1 000×g at 4 °C for 10 min and the supernatant was collected.Protein separation,membrane blotting,and antibody probing were conducted as previously studies described[30,31].Finally,the band density was quantified using densitometry (LI-COR,USA).The antibodies and dilutions were cleaved caspase-3,cleaved caspase-7,cleaved caspase-8,cleaved caspase-9,CD44,p-protein kinase B (p-AKT),p-protein kinase C-δ (p-PKCδ),RAS,p-extracellular signal-regulated protein kinase 1/2 (p-ERK1/2),p-mitogen-activated protein kinase (p-MEK),p-c-Raf and p-c-Src,all from Cell Signaling Technology,Inc.

2.8 Statistical analysis

SPSS 22.0 was used for all statistical analyses.Data were expressed as mean ± SD.The data followed a normal distribution.Comparisons among groups,including a single-factor analysis of variance,and multiple comparisons between the groups were performed (the least significant difference method was adopted in the case of the variance uniform and Tamhane’s T2 test was used when the variance was not uniform).For data that did not follow a normal distribution,a rank-sum test was used.P<0.05 was considered statistically significant (two-sided).

3 Results

3.1 Synergistic antiproliferation effects of YQJDF combined with Sal on NPC-SCs

The effects of different concentrations of YQJDF or SAL on CNE2-SCs and 5-8F-SCs proliferation were detected and analyzed using a cell counting assay(Figure1A and 1B).The cell proliferation rates in the drug-treated groups were significantly lower than those in the solvent control group (vehicle) (P<0.01)in a concentration-dependent manner.

Based on the inhibitory effects that occurred when the drug was used alone,in the combination drug group (YQJDF + SAL),YQJDF 1 mg/mL and SAL 40 μmol/L (for CNE2-SCs) or 30 μmol/L (for 5-8FSCs),were used to detect cell proliferation using CCK-8.The proliferation rates in the drug combination groups were significantly lower than that in the solvent control group.There was a significant difference in the proliferation rate among treatment groups (P<0.01).The cell proliferation rate in the drug combination group was significantly lower than that when each drug was used alone (P<0.01).The interaction effect of YQJDF and SAL was statistically significant (for CNE2-SCs:F=15.299,P=0.002;for 5-8F-SCs:F=50.854,P=0.002).Thus,the combined application of the two drugs had a synergistic effect on the inhibition of NPC-SCs proliferation (Figure1C and 1D).

3.2 Synergistic inhibitory effects on NPC-SCs migration of YQJDF combined with SAL

To detect the antitumor effects of YQJDF combined with SAL,we assessed cellular migration in YQJDF and/or SAL-treated cells.There was a significant difference in the level of cell migration among the groups (P<0.01,Figure2).The cell migration rates in the drug-treated groups were significantly lower than those in the solvent group (P<0.01).The rate of cell migration in the drug combination group was significantly lower than that in the two single drug groups (P<0.01).The interaction effects of YQJDF and SAL were statistically significant (P<0.05).Thus,the combined application of the two drugs had a synergistic effect on the inhibition of NPC-SCs migration.

3.3 Synergistic inductive effects on NPC-SCs apoptosis of YQJDF combined with SAL

We observed the apoptosis-inducing effects of YQJDF combined with SAL on NPC-SCs.There were significant differences in the apoptosis rate among the groups (P<0.01,Figure3).The apoptosis rates in the drug-treated groups were significantly higher than those in the solvent control group (P<0.01).The apoptosis rate in the drug combination group was significantly higher than that in the two single drug groups (P<0.01) and the interaction effect of YQJDF and SAL was statistically significant (P<0.05).Therefore,the combined application of the two drugs had a synergistic inductive effects on NPC-SCs apoptosis.

3.4 Synergistic effects of YQJDF combined with SAL on key proteins in the apoptosis signaling pathway in NPC-SCs

To clarify the mechanism by which YQJDF + SAL induces NPC-SC apoptosis,the expression levels of key proteins in the apoptotic signaling pathway were detected in CNE2-SCs and 5-8F-SCs following drug treatment for 24 h.The levels of cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9 in the drug-treated groups were significantly higher than those in the solvent group (P<0.01,Figure4).The expression level of each protein in the drug combination group was significantly higher than that in the single drug groups and the interaction effects of YQJDF and SAL were statistically significant (P<0.05).Thus,the combined application of YQJDF and SAL induced a synergistic increase in the levels of cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9 in NPC-SCs.These data suggest that the effects of the combination on the level of cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9 protein activation is consistent with its synergistic effects on apoptosis.However,the expression of cleaved Caspase-8 was almost unchanged in all groups,which indicated that the drug may exert its proapoptotic effects primarily through the mitochondrial pathway.

3.5 Synergistic effects on CD44 and key proteins in the Ras signaling pathway in NPC-SCs of YQJDF combined with SAL

We next determined whether the effects of YQJDF combined with SAL on the proliferation,migration and apoptosis of NPC-SCs are related to changes in the levels of CD44 and key proteins in the Ras signaling pathway and downstream.The levels of CD44 and key proteins in the Ras signaling pathway and downstream in CNE2-SCs and 5-8F-SCs treated for 48 h were detected by western blot.

The levels of CD44,Ras,p-c-Raf,p-MEK,p-PKCδ,p-AKT and p-c-Src protein in the drug-treated groups were significantly lower than those in the solvent group in CNE2-SCs (P<0.01;Figure5A,5B and 5C).The levels of p-ERK1/2 in the YQJDF and YQJDF + SAL groups were significantly reduced (P<0.01);however,there was no change in the level of p-ERK1/2 in the SAL group (P>0.05) compared with that in the control group.YQJDF combined with SAL exhibited an interaction effect regarding the inhibition of CD44,p-c-Raf,p-MEK,p-ERK1/2,p-PKCδ,p-AKT and p-c-Src levels (P<0.05);however,there was no interaction effect regarding Ras protein expression (P>0.05).

The levels of CD44,Ras,p-c-Raf,p-MEK,p-ERK1/2,p-PKCδ,p-AKT and p-c-Src in the drugtreated groups were significantly lower than those in the solvent group in 5-8F-SCs (P<0.01).The levels of these proteins in the drug combination group were significantly lower than those in the YQJDF and SAL groups (P<0.01).YQJDF combined with SAL exhibited an interaction effect regarding the inhibition of CD44,Ras,p-c-Raf,p-MEK,p-ERK1/2,p-PKCδ,p-AKT and p-c-Src.Therefore,the combined application of YQJDF and SAL can synergistically reduce the level of CD44 and key proteins in the Ras signaling pathway and downstream in 5-8F-SCs (Figure5D,5E and 5F).

4 Discussion

Traditional Chinese medicine (TCM) therapy is safe,effective,widely used and gaining worldwide attention[32]for the treatment of tumors owing to its extensive efficacy and low toxicity[33,34].SAL can specifically target CSCs.Our results showed that YQJDF or SAL alone or in combination could inhibit the proliferation and migration of NPC-SCs,as well as induce their apoptosis.The combination of YQJDF and SAL has synergistic antitumor effects.

Apoptosis,an important homeostatic mechanism in multicellular organisms[35],is related to the regulation of various cellular factors.It is an important means by which the cell maintains the stability of the internal environment.The core regulatory molecules of apoptosis are in the caspase family of cysteine proteases.The caspase cascade,which promotes the occurrence of apoptosis,contains multiple members.According to its role in apoptosis,caspases can be divided into apoptosis promoters and execution factors.Caspase-8 and Caspase-9 are involved in the initiation of apoptosis,whereas Caspase-3 and Caspase-7 are apoptosis executing factors that normally exist in the form of zymogen[36].When activated,cleaved Caspase marks the cell for apoptosis and the cell enters an irreversible phase[37,38].The apoptotic pathway is divided into the death receptor pathway(exogenous pathway) and mitochondrial pathway(endogenous pathway).Caspase-9 is a marker protein in the mitochondrial apoptotic pathway and Caspase-8 is a protein marker in the exogenous apoptotic pathway,whereas Caspase-3 is a common downstream gene in both pathways and plays a vital role in the process of apoptosis.Inducing the apoptosis of tumor cells may be an effective method to treat tumors[39-44].In this study,the expression of apoptosisrelated proteins,cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9,increased after SAL,YQJDF,or YQJDF + SAL treatment,especially in the drug combination group (YQJDF + SAL).Although both drugs can induce apoptosis,the combined application of the drugs exhibited a synergistic effect;the level of cleaved Caspase-8 expression was not significantly increased,whereas the level of cleaved Caspase-9 expression was significantly increased.The findings indicated that the drug induced the apoptosis of NPC-SCs via the mitochondrial pathway.

The Ras/Raf/MEK/ERK signaling pathway is a widely activated mitogen-activated protein kinase(MAPK) pathway that can transmit extracellular signals from the cell surface to the interior of the nucleus,thereby mediating the expression of intracellular specific proteins and participating in the regulation of cell proliferation,differentiation,apoptosis,autophagy,and other functions[45,46].The MAPK signaling pathway is a promising therapeutic target in cancer[47,48].Studies have shown that Chinese herbal medicines and their extracts have certain effects on MAPK signal transduction pathways[13,14].For example,curcumin upregulates the level of p53 protein expression,an upstream factor of FOXO3a,through ERK1/2,thereby inducing apoptosis and inhibiting NPC cell proliferation[49].Aqueous extracts of YQJDF can inhibit proliferation and induce the apoptosis of NPC cells via the downregulation of the MAPK/ERK1/2 signaling pathway[13,14].However,as the current research on the MAPK signaling pathway is insufficient and its mechanism of action remains poorly understood,further studies are required.The continuous elucidation of the mechanism of the MAPK signaling pathway will provide theoretical guidance and novel research directions for the treatment of NPC with Chinese herbal medicine.With such advances,the prospect of using Chinese herbal medicine to treat NPC will become clearer.

In this study,the expression of CD44,an NPC-SC marker,was significantly downregulated after being treated with SAL or YQJDF alone,or YQJDF + SAL.The levels of Ras protein and its downstream p-PKCδ,p-MEK,p-ERK1/2,p-c-Raf,p-c-Src and p-AKT proteins were also significantly decreased.Furthermore,the levels of CD44,p-PKCδ,p-MEK,p-ERK1/2,p-c-Raf,p-c-Src and p-AKT protein were significantly lower in the drug combination group than in the SAL or YQJDF alone groups,which indicated that SAL and YQJDF exhibited a synergistic effect.The combined group had more pronounced effects on proliferation,apoptosis and regulation of protein expression levels.This result suggests that the combined application of YQJDF with SAL may reduce CD44 expression,and subsequently,downregulate Ras as well as other key downstream proteins,such as p-PKCδ,p-MEK,p-ERK1/2,p-c-Raf and p-AKT,in the Ras pathway via p-c-Src,which results in the downregulation of apoptosis proteins (e.g.,cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9).This inhibits NPC-SC proliferation and induces cell apoptosis via the mitochondrial pathway.

5 Conclusions

Both YQJDF or SAL alone and in combination can inhibit the proliferation and migration of NPC-SCs as well as induce apoptosis.Such effects may be exerted by the downregulation the expression levels of CD44 expression level and the subsequent downregulation the expression levels of key proteins in the Ras signaling pathway via p-c-Src.

Acknowledgements

We thank for the funding support from the National Natural Science Foundation of China (No.81874408,No.81973914 and No.81573721),the Natural Science Foundation of Hunan Province,China (No.2019JJ40216) and Study Fundation of Hunan Provincial Education Department (No.19B439).

Competing Interests

The authors declare no conflict of interest.