|Year : 2014 | Volume
| Issue : 3 | Page : 651-657
In vivo preventive effects of insect tea on buccal mucosa cancer in ICR mice
Xin Zhao, Rui Wang, Yu Qian, Guijie Li, Yalin Zhou, Peng Sun
Department of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, China
|Date of Web Publication||14-Oct-2014|
Department of Biological and Chemical Engineering, Chongqing University of Education, Chongqing 400067
Source of Support: None, Conflict of Interest: None
Background: Insect tea is a particular drink or health product in China and it is also used as Chinese medicine now. Its functional effects need to be proved.
Materials and Methods: The ICR mice buccal mucosa cancer model was established by injecting the mice with U14 cells and mice was treated with insect tea. Tumor volumes and lymph node metastasis rates were determined. And the buccal mucosa tissues and cancer cervical lymph node were also checked by histology test, real-time polymerase chain reaction, and western blot assays.
Results: The tumor volumes for the group treated with insect tea mice was smaller than those from the control mice. The sections of buccal mucosa cancer tissue showed that the canceration of insect tea mice was weaker than control mice. Insect tea significantly induced apoptosis in buccal mucosa tissues by upregulating Bax, caspases, and downregulating Bcl-2. Nuclear factor-κB, inducible nitric oxide synthase, and COX-2 gene, was significantly downregulated by insect tea, demonstrating its anti-inflammatory properties. Insect tea also exerted a great anti-metastasis effect on tissues as demonstrated by decreased expression of Matrix metalloproteinases genes and increased expression of tissue inhibitors of matrix metalloproteinases.
Conclusion: The highest concentration of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea showed the best anticancer effects. Based on the results, insect tea showed the strong in vivo buccal mucosa cancer preventive effects.
Keywords: Anticancer, gene expression, insect tea, squamous cell carcinoma cells U14, tissue
|How to cite this article:|
Zhao X, Wang R, Qian Y, Li G, Zhou Y, Sun P. In vivo preventive effects of insect tea on buccal mucosa cancer in ICR mice. J Can Res Ther 2014;10:651-7
FNx01X. Zhao and R. Wang contributed equally to this work.
| > Introduction|| |
Insect tea is the only one of the best natural organic tea, which set the essence of animals and plants in. As early as the Ming Dynasty (1368-1644), there are records about the effect of nutrition and health care.  Local residents put wild rattan and leaves of Kudingcha (Ilex Kuding tea C.J. Tseng), vine tea (Ampelopsis megalophylla Diels et Gilg), dyetree (Platycarya strobilacea Sieb. et Zucc.) and toringo (Malus sieboldii [Regel] Rehd.) together to lure larvae's of Hydrillodes morosa Butler; Nodaria niphona Butler; Aglossa dimidiata Haworth; Herculia glaucinalis L. and Fujimacia bicoloralis leech to eat them, leaving the droppings there. People take residue of these rattan and tea leaves out of droppings, which is highly named as dragon ball. They fry it in oven and the insect droppings tea is ready for drinking. 
Oral squamous cell carcinoma is a type of cancer that usually develops on the squamous or epithelial cells that cover the lips and the oral cavity. The malignant or cancerous cells are usually found on the floor of the mouth or on the surface of the tongue.  The squamous cells carcinoma is the main part of the epidermis of the skin, and this cancer is one of the major forms of skin cancer. Squamous cell carcinoma is the second most common cancer of the skin.  The U14 mouse tumor is a squamous cell carcinoma. It was an ectopically induced carcinoma by treating the uterine cervix with 20-methylcholanthrene.  U14 cells are widely used in studies of tumor invasion, metastasis, recurrence and drug screening. Establishment of a cultured tumor cell line, which can form a tumor in vivo, would be helpful for the study of tumor biology on a cellular and molecular level. 
Buccal mucosa cancer is the most common cancer of the oral cavity.  In the present study, the cancer preventive effect of insect tea was evaluated using a mouse model that the mice were induced by squamous cell carcinoma cells U14 for in vivo evaluation of buccal mucosa cancer researches. The insect tea was shown to enhance anti-cancer activities effect in mice. As a functional food, insect tea was demonstrated as an oral health benefits.
| > Materials and methods|| |
Preparations of insect tea
Insect tea (Guizhou Hongchishui Green Ecological Food Co., Ltd., Zunyi City, China) was purchased in Guizhou, China. The insect tea was stored at -80°C and freeze-dried to produce a powder. A 10-fold volume of boiling water was added to the powdered insect tea and extracted twice. The water extract was evaporated using a rotary evaporator (Eywla, N-1100, Tokyo, Japan), concentrated.
Cancer cell preparation
The squamous cell carcinoma cells U14 was obtained from Chinese Academy of Medical Sciences (Beijing, China) were used for the in vivo experiment. Cancer cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco Co., Grand Island, NY, USA) at 37°C in a humidified atmosphere containing with 5% CO 2 (Forma, model 311 S/N29035, Waltham, MA, USA). The medium was changed 2 or 3 times each week.
In vitro cultured U14 cells (5 × 10 6 /mouse) were injected into the abdominal cavity of 7-week-old female Institute for Cancer Research ( ICR) mice. After 1 week, the carcinoma ascites were collected and diluted in sterile saline for the concentration of 1 × 10 7 /mL.
Induction of buccal mucosa cancer
Female ICR mice (n = 50, 6 weeks old) were purchased from Experimental Animal Center of Chongqing Medical University (Chongqing, China). They were maintained in a temperature-controlled (temperature 25 ± 2°C, relative humidity 50 ± 5%) facility with a 12 h light/dark cycle and had unlimited access to a standard mouse chow diet and water.
To investigate the preventive effect of the insect tea against buccal mucosa cancer induced by injecting squamous cell carcinoma cells U14 into the mice, the animals were divided into five groups with 10 mice each. The experiment design was as follows: Insect tea solutions (A group: 400 mg/kg, B group: 800 mg/kg, C group: 1600 mg/kg) were administered respectively to three groups by gavage, and the three insect tea group's mice were smeared with the insect tea solutions (A group: 100 mg/mL, B group: 200 mg/mL, C group: 400 mg/mL) on the buccal mucosa of mice every 12 h for 14 days. The control and insect tea groups were then inoculated with the cancer cell suspension (1 × 10 7 /mL) of 0.05 mL on the buccal mucosa for each mouse. The insect tea continued to be smeared on the buccal mucosa of the mice every 12 h. The mice were killed 14 days later and their tumor volumes and lymph node metastasis rates were determined. 
Histological grading of buccal mucosa cancer
Buccal mucosa and lymph node tissues were removed and embedded into paraffin for histological analysis with hematoxylin and eosin staining. Buccal mucosa cancer was graded as follows: (I) Well-differentiated carcinoma: Cells look much like the adjacent benign squamous epithelium; (II) Moderately differentiated carcinoma: Cells form large anastomosing areas in which keratin pearls are formed. They are not numerous and the main component consists of cells with pronounced cytonuclear atypia; (III) Poorly differentiated carcinoma: Cells have lost most of their squamous epithelial characteristics and architecture. 
Real time-polymerase chain reaction to measure messenger ribonucleic acid expression
Total ribonucleic acid (RNA) from buccal mucosa tissues were isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. The RNA was digested with RNase-free DNase (Roche, Basel, Switzerland) for 15 min at 37°C and purified using an RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Complementary deoxyribonucleic acid was synthesized from 2 μg of total RNA by incubation at 37°C for l h with avian myeloblastosis virus reverse transcriptase (GE Healthcare, Little Chalfont, UK) with random hexanucleotides according to the manufacturer's instruction. Sequences of primers used to specifically amplify the genes of interest are shown in [Table 1]. Amplification was performed in a thermal cycler (Eppendorf, Hamburg, Germany). The polymerase chain reaction (PCR) products were separated in 1.0% agarose gels and visualized with ethidium bromide staining. 
Data are presented as the mean ± standard deviation differences between the mean values for individual groups were assessed with a one-way analysis of variance with Duncan's multiple range test. Differences were considered significant when P < 0.05. Statistical analysis system (SAS) version 9.1 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.
| > Results|| |
Tumor volumes and lymph node metastasis rates
Buccal mucosa cancer was induced by injecting squamous cell carcinoma cells U14 in mice. After 14 days, the mice in all of the groups showed carcinogenesis. The tumor volumes of buccal mucosa tissues were checked. The tumor volumes for the control and A, B and C insect tea groups were 12.4 mm 3 , 10.3 mm 3 , 7.5 mm 3 , and 5.8 mm 3 , respectively [P < 0.05, [Table 2]]. There were 5 mice in the control group, 4 mice in A group, 3 mice in B group and 2 in C group showing lymph node metastasis. Consequently, the lymph node metastasis rate was 50%, 40%, 30%, and 20%, respectively. These results demonstrated that the insect tea was effective in impeding carcinogenesis, proliferation, and metastasis.
|Table 2: Tumor volumes and lymph node metastasis rates of different concentrations of insect tea smeared on mice|
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Histologic changes in the buccal mucosa in U14 (squamous cell carcinoma cells)-injected mice were examined by hematoxylin and eosin staining. The histologic tissue sections of mice in normal group showed the normal histologic morphology of squamous epithelium tissue. Histopathological evaluation showed indications of buccal mucosa cancer in all groups receiving U14 cells [Figure 1]. The sections from the mice in the control and A group showed that all of the tissue lost its squamous epithelial characteristics and architecture, but the tissues from the A group had some intracellular bridging between normal squamous cells. The histopathology sections indicated that mice of the control and A group developed poorly differentiated carcinoma (Grade III), and the control group experienced more serious carcinogenesis. The tumor cells of B group mice were still in nests, and there were some larger, eosinophilic, polygonal cells that were trying to layer themselves in a squamous-like fashion. However, for the B group, the overall resemblance to normal squamous epithelium is less striking (Grade II). The tissue sections of C group looked only a little different from normal squamous epithelium. The cells look much like the adjacent benign squamous epithelium (Grade I). From these sections, insect tea showed a preventive effect of buccal mucosa cancer. The lymph node sections from the control group exhibited a large area with liquefaction necrosis of the lymph node [Figure 2]. A group reduced this situation. Marked liquefaction necrosis was also identified in the B group, whereas only a small amount of liquefaction necrosis was observed in the C group. These results demonstrate that insect tea is effective in preventing lymph node metastasis.
|Figure 1: Histology of buccal mucosa tissues induced by injecting squamous cell carcinoma cells U14 in mice (H and E, ×100). A group: Mice were administered by gavage with 400 mg/kg and smeared with 100 mg/mL. B group: Mice were administered by gavage with 800 mg/kg and smeared with 200 mg/mL. C group: Mice were administered by gavage with 1600 mg/kg and smeared with 400 mg/mL|
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|Figure 2: Histology of buccal mucosa cancer cervical lymph node metastasis (H and E, ×100)|
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Apoptosis-related gene expression of Bax, Bcl-2 and caspases
To elucidate the mechanisms underlying the in vivo anticancer effects of insect tea expression of Bax, Bcl-2, and caspase-3 and caspase-9 in mice buccal mucosa tissues were measured by real time (RT)-PCR analyze after 48 h incubation with three concentrations of insect tea water solutions. As shown in [Figure 3], expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 showed significant changes in the presence of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea (P < 0.05). These results suggest that the insect tea induced apoptosis in the buccal mucosa cancer tissues through a Bax- and Bcl-2-dependent pathway. The messenger ribonucleic acid (mRNA) expression levels of caspase-3 and caspase-9 were very low in untreated normal mice, but significantly increased after the mice were treated with 600 mg/kg oral gavage and 400 mg/mL smear of insect tea. With the insect tea treatment, mRNA expression of caspase-9 and caspase-3 was gradually elevated with increased concentrations. More specifically, apoptosis induction by the insect tea was related to upregulation of Bax, caspase-3, and caspase-9, and downregulation of Bcl-2 in terms of mRNA expression. The effects of 1600 mg/kg oral gavage and 400 mg/mL smear insect tea were greater compared with those of the lower insect tea solutions.
|Figure 3: Effects of different concentrations of insect tea on the messenger ribonucleic acid expressions of Bax, Bcl-2, caspase-3 and caspase-9 in buccal mucosa tissues of squamous cell carcinoma cells U14 injected mice. Fold ratio = gene expression/glyceraldehyde 3-phosphate dehydrogenase • control numerical value (control fold ratio: 1). a-eMean values with different letters over the bars are significantly different (P < 0.05) according to Duncan's multiple-range test|
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Inflammation-related gene expression of nuclear factor-kB, IkB-α, inducible nitric oxide synthase and COX-2
The anti-cancer actions of insect tea were determined by nuclear factor-kB (NF-kB), IkB-α, inducible nitric oxide synthase (iNOS), and COX-2 gene expressions. As shown in [Figure 4], mRNA expressions of NF-kB and IkB-α were reduced in mice treated with 1600 mg/kg oral gavage and 400 mg/mL smear. Insect tea significantly modulated the expression of genes associated with inflammation. The mRNA expression of NF-kB was decreased while IkB-α mRNA levels were increased. Additionally, mRNA expression of COX-2 and iNOS was gradually decreased in the presence of the insect tea depending on the concentrations (P < 0.05). These findings indicate that insect tea may help preventing cancer in the early stages by increasing anti-inflammatory activities. Overall, the results of this experiment showed that higher concentration of insect tea had a stronger anti-inflammatory effect on buccal mucosa cancer than lower concentration solution.
|Figure 4: Effects of different concentrations of insect tea on the messenger ribonucleic acid expressions of nuclear factor-κB, IκB-α, nitric oxide synthase and COX-2 in buccal mucosa tissues of squamous cell carcinoma cells U14 injected mice. Fold ratio = gene expression/glyceraldehyde 3-phosphate dehydrogenase • control numerical value (control fold ratio: 1). a-eMean values with different letters over the bars are significantly different (P < 0.05) according to Duncan's multiple-range test|
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Metastasis-related matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases gene expression
Real-time polymerase chain reaction analyze was conducted to determine whether the anti-metastatic effect of the insect tea was due to gene regulation of metastatic mediators, specifically matrix metalloproteinases (MMPs) (MMP-2 and MMP-9), and tissue inhibitors of matrix metalloproteinases (TIMPs) (TIMP-1 and TIMP-2), in mice. As shown in [Figure 5], insect tea a significantly decreased mRNA expression of MMP-2 and MMP-9, and increased the expression of TIMP-1 and TIMP-2 (P < 0.05). These changes in TIMP and MMP expression resulting from insect tea treatment could effectively lead to metastatic inhibition in vivo. These results also showed that insect tea had strong anti-metastatic activity.
|Figure 5: Effects of different concentrations of insect tea on the messenger ribonucleic acid expressions of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in buccal mucosa tissues of squamous cell carcinoma cells U14 injected mice. Fold ratio = gene expression/ glyceraldehyde 3-phosphate dehydrogenase • control numerical value (control fold ratio: 1). a-eMean values with different letters over the bars are significantly different (P < 0.05) according to Duncan's multiple-range test|
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| > Discussion|| |
Although insect tea has been used as traditional drink, little scientific data on its effects is available. Insect tea has a large amount of flavonoids, insect hormone, prothrombin, essential amino acids and trace elements, especially the contents of Fe, Zn, Ca and Mg all of which were higher than those in many famous green tea. And insect tea also contained many other nutriments, such as crude protein, crude fiber, fat, polyphenols, caffeine, sugar, and vitamins. ,, Insect tea has been reported to have various functional activities, such as antipyretic effects, hemostatic effect, preventative effects of high blood pressure, hyperlipoidemia, and coronary heart disease.  In this study, we checked the in vivo anti-cancer effects of insect tea.
Histopathology is an important tool in anatomical pathology, since accurate diagnosis of cancer usually requires histopathological examination of samples. And histopathology is an important clinical standard to diagnosis oral cancer.  As buccal mucosa cancer is the most common cancer of the oral cavity .  The cancer preventive effect of insect tea was evaluated by the model of buccal mucosa cancer in mice. Accordingly, insect tea can be expected to contribute to the prevention of buccal mucosa cancer.
Apoptosis is a fundamental cellular event, and understanding its mechanisms of action will help harness this process for use in tumor diagnosis and therapy.  In a healthy cell, the anti-apoptotic protein Bcl-2 is expressed on the outer mitochondrial membrane surface.  Because the Bax and Bcl-2 genes are mainly expressed during apoptosis, we determined that these genes regulate apoptotic activity. Apoptosis results from activation of caspase family members that act as aspartate-specific proteases.  Caspases form a proteolytic network within the cell whereby upstream initiator caspases are activated early in the apoptotic process (caspase-9) and in turn activate other downstream caspases (caspase-3). Cytochrome c and procaspase-9 processing is highly dependent on caspase-3, placing this caspase in a central position as a regulator of essential apoptotic pathways in cancer cells.  Caspase-3 was also reported to play a role as an amplifier of apoptotic signals. 
In addition, anti-cancer mechanisms underlying the effect of insect tea on buccal mucosa cancer involve the induction of apoptosis by increasing the number of apoptotic bodies, regulating the mRNA expression of Bax and Bcl-2, and promoting anti-inflammatory effects by downregulating iNOS and COX-2 gene expression. COX-2 has been suggested to play an important role in colon carcinogenesis, and NOS, along with iNOS, may be a good target for the chemoprevention of colon cancer.  NF-kB is one of the most ubiquitous transcription factors, and regulates the expression of genes required for cellular proliferation, inflammatory responses, and cell adhesion.  NF-kB is present in the cytosol where it is bound to the inhibitory protein, IkB. Following its induction by a variety of agents, NF-kB is released from IkB and translocates to the nucleus where it binds to the kB binding sites in the promoter regions of target genes.  These mechanisms could be involved in the anti-cancer effects of insect tea in mice. Based on the results of the expression patterns of pro-apoptotic genes observed in the present study, it concluded that cancer tissues treated with insect tea underwent apoptosis.
MMPs, a family of zinc-dependent endopeptidases, play a very important role in tumorigenesis and metastasis. MMPs can cleave virtually all extracellular matrix (ECM) substrates. Degradation of the ECM is a key event in tumor progression, invasion, and metastasis.  Among the MMP family members, MMP-2 and MMP-9 are molecules important for cancer invasion, and highly expressed in breast and colon cancer cells.  In fact, inhibition of MMP activity is useful for controlling tumorigenesis and metastasis. TIMPs are naturally occurring inhibitors of MMPs which prevent catalytic activity by binding to activated MMPs, thereby blocking ECM breakdown.  Disturbances in the ratio between MMPs and TIMPs have been observed during tumorigenesis.  Maintaining the balance between MMPs and TIMPs or increasing TIMP activity is useful ways to control tumor metastasis.  Experimental evidence demonstrating the role of MMPs in metastasis has been obtained by in vivo invasion assays. Spontaneous and experimental metastasis to the liver is decreased in mice overexpressing TIMP1, and increased in mice expressing antisense TIMP-1 mRNA.  Ectopic overexpression of TIMP-1 in the brain of transgenic mice also reduces experimental metastasis to the brain.  In particular, MMP-2 and MMP-9 are important for tumor invasion and angiogenesis. Thus, tumor metastasis can be inhibited by blocking MMP synthesis and activity.  Metastasis is defined as the spread of cancer cells from one organ or area to another adjacent organ or location.  It is thought that malignant tumor cells have the capacity to metastasize. Cancer occurs after cells in a tissue are genetically damaged in a progressive manner, resulting in cancer stem cells possessing a malignant phenotype. After the tumor cells come to rest in another site, they penetrate the vessel walls, continue to multiply, and eventually form another tumor.
| > Conclusions|| |
In summary, the in vivo anti-cancer effects of insect tea were checked by injecting squamous cell carcinoma cells U14 into the mice. Results from the present study demonstrated that the in vivo anti-cancer, anti-inflammation and anti-metastatic effects of high concentration of insect tea were stronger than the low concentration one. Overall, insect tea showed potent in vivo anti-cancer activities. The active compounds resulting from insect tea need to be identified and evaluated in future studies.
| > Acknowledgment|| |
This study was supported by Program for Innovation Team Building at Institutions of Higher Education in Chongqing (KJTD201325)
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]