Contributed equally
Hypoxia is an important factor in radioresistance of laryngeal carcinoma. Glucose transporter-1 (GLUT-1) is an important hypoxic marker in malignant tumors, including laryngeal carcinoma. Apigenin is a natural phytoestrogen flavonoid that has potential anticancer effects. Various studies have reported that the effects of apigenin on lowering GLUT-1 expression were involved in downregulation of the PI3K/Akt pathway. Thus, apigenin may improve the radiosensitivity of laryngeal carcinoma by suppressing the expression of GLUT-1 via the PI3K/Akt pathway. The effect of GLUT-1 and PI3K/Akt pathway-related factor expressions by apigenin or antisense oligonucleotides (AS-ODNs) on the radiosensitivity of laryngeal carcinoma
Laryngeal carcinoma, the second most common head and neck type of cancer, accounts for 2% of all malignant tumors worldwide (
Resistance to head and neck cancer radiotherapy occurs for three main reasons: intrinsic radiation resistance, tumor cell proliferation and hypoxia. It has been previously reported that hypoxia plays an important role in the resistance of tumors to radiotherapy (
Apigenin is a natural phytoestrogen flavonoid present in a wide range of fruits, vegetables (particularly celery), beans and tea. Moreover,
The aim of the present study was to examine the expression of GLUT-1 and PI3K/Akt pathway-related factors in tumors in nude mice. Specifically, we investigated whether GLUT-1 expression was decreased by inhibiting the PI3K/Akt pathway and whether apigenin enhanced the radiosensitivity of laryngeal carcinoma. In addition, we assessed whether apigenin plays a role in the effects of GLUT-1 AS-ODNs on enhancing the radiosensitivity of laryngeal carcinoma.
Apigenin was purchased from Selleckchem (Houston, TX, USA). AS-ODNs GLUT-1 was prepared as reported previously (
The laryngeal Hep-2 carcinoma cell line was purchased from the Cell Research Institute of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at the Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in an atmosphere containing 5% CO2. Cells in the logarithmic growth phase were used in the experiments.
This experiment was conducted in accordance with the institutional guidelines of the First Affiliated Hospital, College of Medicine, Zhejiang University and with appropriate institutional certification. Four-week-old male athymic nude mouse with a BALB/c background weighing 18±2 g (n=30) were raised under specific pathogen-free (SPF) conditions at the Surgical Laboratory Animal Center of the First Affiliated Hospital, College of Medicine, Zhejiang University. Under sterile conditions, a 2×106/ml Hep-2 cell suspension was injected subcutaneously into the right forelimb of each nude mouse at a volume of 0.2 ml. The inoculation site of each nude mouse was observed daily after inoculation. After 1 week, a grain-sized induration may develop at the visible inoculation site, which confirmed that the xenograft model was established successfully.
Two separate experiments were performed: one using 15 and a second using 18 tumor-bearing mice.
When the tumors reached a volume of ~100 mm3, 15 tumor-bearing mice were divided randomly into five groups of three mice. The tumors were then treated as follows: 50
When the tumors reached a volume of ~100 mm3, 18 tumor-bearing mice were divided randomly into six groups of three mice: A negative control, apigenin alone, GLUT-1 AS-ODNs alone, 10 Gy X-ray irradiation alone, apigenin and GLUT-1 AS-ODNs and the combination of apigenin, GLUT-1 AS-ODNs and 10 Gy X-ray irradiation. In the apigenin-only group, the mice were injected intraperitoneally with 100
The mental state, food intake and activity of all the mice were monitored daily. The tumor growth conditions (tumor formation rate, size, weight and inhibition ratio) were assessed after vaccination, drug and radiological intervention. The tumor volume (V) was calculated by measuring the i) long and ii) short tumor diameter after tumor formation using the equation V=1/2(ab2). The tumor formation rate was calculated as the number of mice whose tumor volume was >5 mm diameter/the number of the mice in the experimental group × 100%. Animal surgery was performed under general anesthesia, using 50 mg/kg ip injection of pentobarbital sodium. The inhibition ratio (IR) was defined as 1-(tumor weight of test group/control group).
Total RNA was extracted from cells using TRIzol according to the manufacturer's instructions. The concentration of total RNA was measured using ultraviolet spectrophotometry, with an optical density (OD) 260/280 ratio of 1.8–2.1 being considered acceptable. First-strand cDNA was synthesized in a 20-
GLUT-1, p-Akt and PI3K protein levels were analyzed using a BAC protein quantification kit. Briefly, 80
Paraffin-embedded sections of xenograft tumor tissues were dewaxed and hydrated. TUNEL staining of tumor sections was then performed according to the manufacturer's instructions (Roche). Staining was visualized under an optical microscope, and cells with brown or brown-yellow nuclei were interpreted as positive and observations were confirmed using the morphological features of apoptotic cells. Specifically, unstained cells became smaller, the membrane appeared to be foaming, apoptotic bodies formed in the later stages and adherent cells became shrunken, round and shed. In addition, stained cells exhibited chromatin condensation, marginalization, nuclear membrane cracking and the chromatin was divided into block/apoptotic bodies. The sections were observed at a magnification of ×400 and the percentage of apoptotic cells in 100 cells/field was counted and used to calculate the mean apoptosis index (AI).
Subcutaneous xenograft tumors were visible ~1 week after inoculation with Hep-2 cells (
In the first experiment, tumor size in the 100
In the second experiment, tumors were significantly smaller in the GLUT-1 AS-ODNs group compared to the control group 17 days after treatment (P<0.05;
In the first experiment, the weight of the tumors harvested from the mice in each group is shown in
The weight of the tumors harvested from the mice in each group from the second experiment is shown in
In the second experiment, the rates of tumor growth inhibition in the apigenin, GLUT-1 AS-ODNs, 10 Gy, apigenin plus GLUT-1 AS-ODNs and apigenin plus GLUT-1 AS-ODNs plus 10 Gy groups were −2.05, 37.33, 7.02, 4.79 and 27.16%, respectively.
The apoptotic indices were significantly higher in the apigenin, GLUT-1 AS-ODNs, 10 Gy, apigenin plus GLUT-1 AS-ODNs and apigenin plus GLUT-1 AS-ODNs plus 10 Gy groups compared to the control (P<0.05;
In the first experiment,
In the second experiment,
In the first experiment, GLUT-1 expression was significantly reduced in the 100
In the second experiment, the expression of GLUT-1, p-Akt, and PI3K was significantly decreased in the apigenin group, GLUT-1 AS-ODNs group and apigenin plus GLUT-1 AS-ODNs group compared to the control group (P<0.05;
Radioresistance is one of the major obstacles in the treatment of laryngeal carcinoma, however, the mechanism behind the radioresistance of laryngeal carcinoma remains unclear. We demonstrated previously that GLUT-1 AS-ODNs inhibit glucose uptake and proliferation in Hep-2 cells by inhibiting the expression of GLUT-1 (
In the second experiment in the present study, the results revealed that tumor size and weight did not decrease significantly in the 10 Gy group compared to the control group. However, GLUT-1 mRNA and protein were significantly higher in the 10 Gy group compared to the control group, which was consistent with previous studies.
As determined above, the mechanism behind the radioresistance of laryngeal carcinoma is unclear and likely caused by an interplay of multiple factors. With the development of molecular biology-based techniques, the factors that regulate GLUT-1 have been gradually clarified. Specifically, several signaling pathways regulate GLUT-1, including the PI3K/Akt pathway. Melstrom
The second experiment in the present study revealed that GLUT-1 protein expression was higher in the 10 Gy group compared to the control group (P<0.05;
Apigenin is a natural phytoestrogen flavonoid that exerts biological effects including anti-oxidative, anti-inflammatory, antiviral, immune-adjusting, anti-mutation and anticancer activities (
In the first experiment of the present study before X-ray radiation (the 10 Gy group was equivalent to the control group), 100
However, in the second experiment, apigenin reduced tumor size compared to the control group, although not significantly (P>0.05), which may have been due to the different apigenin dosing intervals used in the two experiments. GLUT-1 AS-ODNs reduced tumor size significantly compared to the control group 17 days after treatment (P<0.05). In addition, GLUT-1 AS-ODNs, apigenin plus GLUT-1 AS-ODNs and apigenin plus GLUT-1 AS-ODNs plus 10 Gy reduced tumor size significantly compared to the control 24 days after treatment (P<0.05), suggesting that these treatments inhibited xenograft tumor growth. In addition, apigenin, GLUT-1 AS-ODNs, 10 Gy X-ray radiation, apigenin plus GLUT-1 AS-ODNs and apigenin plus GLUT-1 AS-ODNs plus 10 Gy X-ray radiation all increased tumor cell apoptosis, as detected using TUNEL staining. Apigenin plus GLUT-1 AS-ODNs also enhanced the effect of apigenin or GLUT-1 AS-ODNs alone on tumor cell apoptosis significantly. Apigenin plus GLUT-1 AS-ODNs significantly enhanced the effects of X-ray radiation on tumor cell apoptosis. Collectively, these results suggest that apigenin and GLUT-1 AS-ODNs inhibited laryngeal carcinoma growth. The combination of apigenin and GLUT-1 AS-ODNs enhanced the effects of apigenin or GLUT-1 AS-ODNs alone. In addition, apigenin and apigenin plus GLUT-1 AS-ODNs improved the radiosensitivity of laryngeal carcinoma.
We investigated the molecular mechanisms by which apigenin and GLUT-1 AS-ODNs enhanced the radiosensitivity and suppressed xenograft tumor growth. The expression of
In conclusion, the overexpression of GLUT-1 and increased activation of the PI3K/Akt signaling pathway may be involved in the radioresistance of laryngeal carcinoma
antisense oligonucleotides
glucose transporter-1
The present study was supported by the Traditional Chinese Medicine Scientific Research Project of Zhejiang Province, China (grant no. 2013ZA075), Health Department of Zhejiang Province, China (grant no. 2015116850), and the National Natural Science Foundation of China (grant nos. 81172562 and 81372903).
Establishment of the nude mouse model of laryngeal carcinoma. (A) Subcutaneous xenograft tumors were visible 1 week after inoculation with Hep-2 cells. (B) Tumor volume reached ~100 mm3 after 2 weeks.
The effects of apigenin, GLUT-1 AS-ODNs or the combined treatment plus radiation on xenograft tumor growth
Tumors harvested from the mice in each group. Tumors were resected from the mice in each group of the (A) first experiment and (B) second experiment.
The effects of apigenin and GLUT-1 AS-ODNs on xenograft apoptosis (the second experiment), observed an under optical microscope (magnification, ×400), and the percentage of apoptotic cells in 100 cells per field was counted and used to calculate the mean apoptosis index (AI). AI, apoptotic index.
Effects of apigenin and GLUT-1 AS-ODNs on GLUT-1, Akt and PI3K mRNA. (A) First experiment: *P<0.05, indicated that there were significant differences in the tumor mRNA compared to the 10 Gy group. +P<0.05, indicated that there were significant differences in the tumor mRNA between apigenin 100
Frst experiment. (A) Western blot analysis shows the expression of GLUT-1, p-Akt and PI3K protein in xenograft tumors. (B) *P<0.05, indicated that there were significant differences in the tumor protein compared to the 10 Gy group.
Second experiment. Western blot analysis shows the expression of GLUT-1, p-Akt and PI3K protein in xenograft tumors. (A) *P<0.05, indicated that there were significant differences in the tumor protein compared to the control group. (B) *P<0.05, indicated that there were significant differences in the tumor protein compared to the control group. #P<0.05, indicated that there were significant differences in the tumor protein between the apigenin+anti-GLUT-1 group and the apigenin group.
Tumor weight of each group in the first experiment.
Group | Tumor weight (g) |
---|---|
Apigenin 50 |
0.6070±0.0195 |
Apigenin 100 |
0.5260±0.0285 |
10 Gy | 0.6770±0.0609 |
Apigenin 50 |
0.4937±0.2430 |
Apigenin 100 |
0.4820±0.1421 |
Data are presented as a mean value ± standard deviation.
P<0.05, indicated that there were significant differences in tumor weight between the apigenin 100 and 50
P<0.05, indicated that there were significant differences in tumor weight between the apigenin 100
Tumor weight, inhibitory rate and apoptotic index of each group in the second experiment.
Group | Tumor weight (g) | Inhibitory rate (%) | AI (%) |
---|---|---|---|
Control | 0.9733±0.1358 | 1.50±1.77 | |
Apigenin | 0.9933±0.2108 | −2.05 | 23.65±6.41 |
Anti-GLUT-1 | 0.6100±0.0346 |
37.33 | 6.63±3.13 |
Apigenin+anti-GLUT-1 | 0.9267±0.6888 | 4.79 | 34.71±7.48 |
10 Gy | 0.9050±0.1598 | 7.02 | 15.26±3.43 |
Apigenin+anti-GLUT-1+10 Gy | 0.7090±0.2383 | 27.16 | 53.28±5.81 |
Data are presented as a mean value ± standard deviation.
P<0.05, indicated that there were significant differences in the tumor weight compared to the control group.
P<0.05, indicated that there were significant differences in the tumor apoptotic index compared to the control group.
P<0.05, indicated that the combination of apigenin and GLUT-1 AS-ODNs significantly enhanced the effects of apigenin or GLUT-1 AS-ODNs alone on tumor cell apoptosis.
P<0.05, indicated that the combination of apigenin and GLUT-1 AS-ODNs significantly enhanced the effects of X-ray irradiation on tumor cell apoptosis. AI, apoptotic index.