Cordycepin,a metabolite of Cordyceps militaris,inhibits xenograft tumor growth of tongue squamous cell carcinoma in nude mice

ZHENG Qingwei ,SHAO Yidan ,ZHENG Wanting ,ZOU Yingxue

1Key Laboratory of Infection and Immunity of Anhui Higher Education Institutes,Bengbu Medical College,Bengbu 233030,China;2Academy of Medical Engineering and Translational Medicine,Tianjin University,Tianjin 300072,China;3Children's Hospital of Tianjin University,Tianjin 30074,China

Abstract: Objective To evaluate the inhibitory effect of cordycepin on oral cancer xenograft in nude mice and explore the underlying mechanisms.Methods Sixteen BALB/c mice bearing subcutaneous human tongue squamous cell carcinoma (TSCC)TCA-8113 cell xenografts were randomized into model group and cordycepin treatment group for daily treatment with saline and cordycepin for 4 weeks.After the treatment,the tumor xenografts were dissected and weighed to assess the tumor inhibition rate.Histological changes in the heart,spleen,liver,kidney,and lung of the mice were evaluated with HE staining,and tumor cell apoptosis was examined using TUNEL staining;The expressions of Bax,Bcl-2,GRP78,CHOP,and caspase-12 in the xenografts were detected using RT-qPCR and Western blotting.Results Cordycepin treatment resulted in a tumor inhibition rate of 56.09%in the nude mouse models,induced obvious changes in tumor cell morphology and significantly enhanced apoptotic death of the tumor cells without causing pathological changes in the vital organs.Cordycepin treatment also significantly reduced Bcl-2 expression (P＜0.05)and increased Bax,GRP78,CHOP,and caspase-12 expressions at both the RNA and protein levels in the tumor tissues.Conclusion Cordycepin treatment can induce apoptotic death of TCA-8113 cell xenografts in nude mice via the endogenous mitochondrial pathway and endoplasmic reticulum stress pathways.

Keywords:cordycepin;edible fungi;Cordyceps militaris;oral cancer;cell apoptosis;xenograft

INTRODUCTION

Cordyceps militarisis an entomopathogenic fungi used in traditional Chinese medicine (TCM),and similar to its high-valued counterpartCordyceps sinensis,it possesses high medicinal values while serving also as a nutritious food material［1］.As one of the most important metabolites ofCordyceps militaris［2-3］,cordycepin(3'-deoxyadenosine)has been shown to have a wide range of biological activities with immunomodulatory,anti-inflammatory,anti-aging,and anti-cancer effects［4-12］.Studies have shown promising antitumor effects of cordycepin in different cancer types［13-18］,but its inhibitory effect on tongue squamous cell carcinoma (TSCC) remains to be characterized.

TSCC is one of the most prevalent head and neck tumors in China［19-21］.Due to the sensitive location of these tumors,chemotherapy and radiotherapy often cause severe side effects in patients with TSCC,and it is therefore urgent to develop a novel therapeutic strategy with better safety and efficacy profiles is therefore urgent to improve the patients' quality of life.We previously demonstrated that cordycepin was capable of inhibiting proliferation,promoting apoptosis and inducing AMPK/mTOR pathway-mediated autophagy in tongue cancer TCA-8113 cellsin vitro［22］.Whether cordycepin produces the same inhibitory effect against TSCC xenografts in nude mice has so far remained unclear,and we therefore conducted this study to evaluate the therapeutic efficacy of cordycepin in a nude mouse model bearing subcutaneous TCA-8113 cell xenografts.

METHODS

Reagents and cell culture

Cordycepin was obtained from Dalian Meilun Biotechnology Co.Ltd.Fetal bovine serum (FBS) and RPMI 1640 were the products of GIBCO.TUNEL kit was supplied by Roche (USA).The PCR primers were synthesized by Shanghai Shenggong.The antibodies specific to Bcl-2,GRP78,CHOP,and caspase-12 were purchased from Proteintech.Human TSCC TCA-8113 cells were obtained from Shanghai Meixuan Biotechnology Co.,Ltd and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS).The cells in logarithmic growth were used in subsequent experiments.

Animals,modeling and treatment

The protocol of this animal experiment was approved by the Ethics Committee of Bengbu Medical College([2021]No.023) and carried out in line with the Institutional Laboratory Animal Care and Use Committee Guideline.Sixteen specific pathogen-free male BALB/c nude mice(6-8 weeks old,body weight 20±2 g) were provided by Shanghai Slake Laboratory Animals Co.,Ltd.[Certificate No.scxk (Shanghai) 2017-0005].The mice were allowed to acclimate to laboratory conditions for one week prior to experiment with free access to normal chow and water.

A total of 5×106TCA-8113 cells were injected subcutaneously into the flank of 2 nude mice.Ten days after tumor cell inoculation,tumor formation in the mice was observed.When the tumors grew to about 10 mm,the mice were euthanized and the tumors were dissected and cut into small tissue blocks of about 1 mm3under sterile conditions.The tumors were then inoculated subcutaneously in the right armpit of 16 mice using a No.16 lumbar puncture needle［23］.The tumor-bearing mouse models were divided into model group and treatment group(n=8)for daily treatment with saline and 10 mg/kg cordycepin via oral gavage for 8 weeks,respectively.The tumor size was monitored using a vernier caliper every other day,and body weight of the nude mice was measured using an electronic balance.On the second day after the last treatment,the mice were euthanized via cervical dislocation and the tumor xenografts,along with the heart,liver,spleen,kidney,and lung,were dissected and weighed.The tumor volume was calculated using the formula:V=1/2ab2,wherearepresents the major diameter andbrepresents the minor diameter.The tumor inhibition rate was calculated using following formula: Tumor inhibition rate (%)=[Mean tumor mass (Control)-Mean tumor mass (cordycepin group)]/Mean tumor mass(Control)×100%.

Histological examination of the tumor

The dissected tumors and normal tissue and the vital organs were fixed with 4% paraformaldehyde for hematoxylin and eosin (HE) staining and TUNEL staining.The fixed tissues and organs were embedded in paraffin,cut into 4-μm slices,and stained with HE for observation under a Nikon NI-U microscope.

Apoptosis analysis

The paraffin-embedded tumor tissue was cut into 5-μm slices,deparaffinized,and rehydrated for TUNEL staining following the manufacturer's instructions of the kit.After staining,the slices were washed with tap water for 15 min,re-dyed,sealed,and observed under an optical microscope.The apoptotic index (AI) was calculated as the number of apoptotic cells/total cell number×100%.

Real-time quantitative PCR(RT-qPCR)

The primers used for RT-qPCR were synthesized by Sangon Biotech Co.,Ltd.(Shanghai,China),and the primer sequences are listed in Tab.1.The total RNA was extracted from the tumor samples,and a reverse transcriptase kit (Takara) was used for cDNA synthesis.Using the SYBR Green dye and a 7300 instrument(ABI),amplification was initiated with predenaturation at 95 ℃for 1 min,followed by 40 thermal cycles of 95 ℃for 10 s,60 ℃for 30 s,and 72 ℃for 30 s.GAPDH was used for normalization,and the relative gene expression was calculated with the 2-△△CTmethod.

Tab.1 Sequences of the primers used in RT-qPCR

Western blotting

The total protein was extracted from tumor tissue homogenate and quantified using BCA kit(Solarbio).The proteins separated with SDS-PAGE were transferred onto nitrocellulose membranes,and after blocking with 5%skimmed milk for 1 h,the membranes were incubated overnight with the primary antibodies against caspase-3,caspase-9,Bcl-2 or Bax (Proteintech,26593-1-AP,1:1000) at 4 ℃.After washing for 3 times with TBST,the protein blots were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,BBI) at room temperature for 2 h.An enhanced chemiluminescence kit (ECL,Beyotime) was used for protein visualization.Image Pro Plus 6.0 software was used to quantify the protein levels in the samples with GAPDH (BBI,D110016) as the internal control.

Statistical analysis

The statistical data are presented asMean±SDand analyzed using SPSS 17.0 software.One-way ANOVA was used for comparison between the two groups,and aPvalue less than 0.05 was considered to indicate a statistically significant difference.

RESULTS

Cordycepin inhibits TCA8113 tumor growth in nude mice

After the 8-week-long treatment,the tumor mass in the mouse models increased by 1.23±0.12 g in the model group and by 0.54±0.05 g in cordycepin treatment group.Cordycepin treatmentresulted in a tumor inhibition rate of 56.09%.We noted that both the tumor volume and body weight of the mice increased rapidly over time in the model group,whereas cordycepin treatment significantly slowed down tumor growth in the mice(P＜0.05;Fig.1).

Fig.1 Effect of cordycepin treatment on body weight(A) and tumor growth (B) in nude mice.*P＜0.05,**P＜0.01,***P＜0.001 vs model group.

Effect of cordycepin on histology of tumor tissues and vital organs

We examined the heart,liver,spleen,kidney,lung and tumor tissues of the nude mice with HE staining and TUNEL staining,and we observed no abnormalities in the vital organs in terms of cellular morphology or pathological changes.Histological examination of the tumor tissues in the model group showed that the round or oval tumor cells were large in size and closely arranged,with patchy or diffuse distribution;the cells contained rich cytoplasm with enlarged and hyperchromatic nuclei,and the mitotic phase was frequently seen.In cordycepin treatment group,the tumor cells showed decreased cell size,widened intercellular gap,decreased nucleopyknosis,and reduced nucleoplasm ratio;nuclear division in the tumor cells was scarce,and large necrotic areas were seen in the tumor tissue.In addition,the tumors in the treatment group also exhibited a reduction in the number of visible blood vessels(Fig.2).

Fig.2 Effect of cordycepin treatment on tumor cell morphology in the tumor-bearing nude mice (HE staining,original magnification: ×200).A: Model group.B:Cordycepin-treated group.

Cordycepin induces TCA-8113 tumor cell apoptosis in vivo

We analyzed cell apoptosis in the tumor tissues using TUNEL staining.The results showed that very few tumor cells were TUNEL-positive in the model group,while the tumor tissues in cordycepin treatment group showed a significantly higher apoptosis rate(P＜0.01;Fig.3).

Fig.3 Effect of cordycepin on TCA8113 tumor cell apoptosis in the tumor-bearing nude mice(TUNEL staining,×200).A:Model group;B:Treatment group.**P＜0.01.

Effect of cordycepin on endoplasmic reticulum (ER) stress pathway

We further explore the changes in the ER stress pathway in the tumor tissues of the mice.Compared with that in the model group,the tumor tissues in cordycepin treatment group showed a significant reduction in Bcl-2 mRNA expression (P＜0.05) and significantly increased expressions of Bax,GRP78,CHOP,and caspase-12 (P＜0.05;Fig.4).Consistently,the protein expression level of Bcl-2 was decreased and those of GRP78,CHOP,and caspase-12 increased significantly in the tumor tissues of cordycepin-treated mice (Fig.5).These results suggest that cordycepin induces TCA-8113 tumor cell apoptosis possibly by regulating the ER stress pathway in the tumor-bearing nude mouse models.

Fig.4 Effect of cordycepin on gene expressions in the xenografts in the tumor-bearing nude mice detected by RT-qPCR.**P＜0.01 vs model group.

Fig.5 Effect of cordycepin on protein expressions in the xenografts in the tumor-bearing nude mice detected by Western blotting.A: Model group;B:Cordycepin treatment group.**P＜0.01 vs model group.

DISCUSSION

Monitoring changes in tumor weight and tumor growth inhibition is a direct means for assessing anti-tumor activity of a specific compound of interest［24］.In this study we observed that cordycepin treatment significantly reduced both tumor weight and tumor growth rate in nude mouse models with a tumor inhibition rate exceeding 50%,demonstrating a strong ability of cordycepin to suppress TSCC tumor growth in nude mice.

We previously demonstrated that cordycepin treatment induced apoptotic death of TCA-8113 cellsin vitrovia the endogenous pathway.As such,we furtherevaluated the activation of this apoptotic pathway in the tumor tissues in nude mice,with Bcl-2 and Bax as the key pathway components of interest.We found that in the tumor-bearing nude mouse model,cordycepin treatment increased Bax expression and reduced Bcl-2 expression to result in a decrease of Bcl-2/Bax ratio in the tumor tissue,suggesting that cordycepin also induces apoptotic death of TCA-8113 cellsin vivovia the endogenous mitochondrial pathway.

The ER stress pathways serve as a protective mechanism to maintain homeostasis in eukaryotic cells,and prolonged ER stress can cause apoptotic cell death［25-30］.GRP78,as a marker of ER stress,binds to unfolded proteins and drives the activation of CHOP and caspase pathway,which ultimately results in apoptotic cell death［31］.CHOP is also capable of further suppressing Bcl-2 expression to enhance cell apoptosis［32］.We show in this study that cordycepin treatment significantly increased intratumoral GPR78,CHOP,and caspase-12 expressions at both the mRNA and protein levels,suggesting that cordycepin induces apoptotic cell death at least in partviathe ER stress pathway in tumor cells.

Taken together,our results demonstrate that cordycepin is capable of inducing apoptotic death of TCA-8113 tumor cells in nude mice via the mitochondrial and ER stress pathways.How other signaling molecules such as PARK,ATF6,and IRE-1 contribute to this effect remains to be clarified.Given the well-documented interactions between the mitochondria and the ER［33］,further study is warranted to determine the contribution of these proteins to cordycepin-induced tumor cell apoptosis.