Study of As2O3 regulating proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1

Abstract Objective Our study aims to investigate the effect of arsenic trioxide (As2O3) on proliferation and apoptosis of Tca8113 tongue squamous carcinoma cells. Methods Cell proliferation and the expression of Id-1 mRNA in Tca8113 cells after treatment with different concentrations of As2O3 were detected by MTT and qRT-PCR, respectively. The expression of Id-1, cell proliferation and apoptosis in Id-1 silencing Tca8113 cells were detected by qRT-PCR, Western blot, MTT and flow cytometry, respectively. The pcDNA 3.1-Id-1 overexpression vector was transfected into Tca8113 cells combination with 3 μmol/L As2O3. The detection of cell proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in transfected Tca8113 cells were performed by MTT, flow cytometry and Western blot assay, respectively. Results As2O3 of different concentration could inhibit the proliferation of Tca8113 cells and IC50 value was 3.004 ± 0.2379 μmol/L. The expression of Id-1 mRNA was down-regulated in Tca8113 cells treated with 3 μmol/L As2O3 for 48 h. The results of qRT-PCR, Western blot, MTT and flow cytometry indicated that the expression level of Id-1 and cell proliferation ability were decreased while the apoptosis rate was increased in Tca8113 cells after transfection of Id-1 siRNA. Overexpression of Id-1 could attenuate the inhibition or promotion of As2O3 on proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in Tca8113 cells. Conclusion As2O3 could regulate the proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1.


Introduction
The overall incidence of malignant tumors is on the rise, seriously endangering the health of human. There were 4.29 million new cancer cases and 2.81 million deaths in China only in 2015. Among them, the number of oral cancer cases and deaths were 48,100 and 22,100, respectively [1]. Tongue squamous cell carcinoma accounts for 43.4% of oral cancer. It is the most common oral cancer, with high lymph node metastasis rate and strong invasiveness [2]. The incidence of tongue squamous cell carcinoma is related to age, sex and race. Smoking, alcohol abuse and poor oral hygiene can promote the occurrence of tongue squamous cell carcinoma. According to the survey, the incidence of tongue squamous cell carcinoma tends to be younger [3,4]. Surgical treatment, supplemented by radiotherapy, chemotherapy and other multidisciplinary treatment models are frequently used for the treatment of tongue squamous cell carcinoma. However, the tongue is special and slight carelessness seriously affects the patient's speaking, swallowing ability and other functions and postoperative recurrence and metastasis limit the therapeutic effect. The 5-year survival rate of young patients with tongue squamous cell carcinoma is only 42% [5]. Arsenic trioxide (As 2 O 3 ) is one of the main components of traditional Chinese medicine arsenic, which has been widely used in the clinical treatment of acute promyelocytic leukemia [6]. With continuous research, As 2 O 3 has a certain effect on the treatment of a variety of solid tumors [7]. In this paper, Tca8113 cells were used as the research object to observe the effect of As 2 O 3 on Id-1 expression, cell proliferation and apoptosis.

Cell culture and grouping
Tca8113 cells were cultured in RPMI 1640 medium containing 10% FBS at 37 C, 5% CO 2 incubator. The cells in the logarithmic growth phase were collected, seeded in a 24-well plate at 1 Â 10 5 cells/well. After incubating for 24 h, fresh medium containing As 2 O 3 with different concentration (0.25, 0.50, 1.00, 2.00, 4.00, 8.00, 16.00, 32.00 lmol/L) was added. The cells were cultured for another 48 h and labeled as 0.25 lmol/L group, 0.50 lmol/L group, 1.00 lmol/L group, 2.00 lmol/L group, 4.00 lmol/L group, 8.00 lmol/L group, and 16.00 lmol/L group and 32.00 lmol/L group. When Tca8113 cells were grown to a confluency of about 45%, the cells were transfected with siRNA and Id-1 siRNA and continuously cultured for 24 h according to the instructions of LipofectamineTM2000 transfection reagent. The cells were divided into si-con group and si-Id-1 group, respectively.
After transfecting the cells with pcDNA 3.1 empty vector and pcDNA 3.1-Id-1 overexpression vector for 24 h, the culture medium was replaced by fresh medium containing 3 lmol/L As 2 O 3 for 48 h, The cells were divided into As 2 O 3 þCtrl group and As 2 O 3 þId-1 group.
MTT assay was used to detect cell proliferation Cells were cultured for 48 h with the control group, 0.25 lmol/L group, 0.50 lmol/L group, 1.00 lmol/L group, 2.00 lmol/L group, 4.00 lmol/L group, 8.00 lmol/L group, 16.00 lmol/L group, and 32.00 lmol/Group, respectively. When the cells were cultured for 24 h, 48 h, and 72 h, 20 lL MTT (5 mg/mL) was added and incubation was continued for 4 h. The superfluous medium was discarded and 150 lL of dimethyl sulfoxide (DMSO) was added to react for 10 min. The absorbance at 490 nm was measured by a microplate reader. Cell proliferation rate (%) ¼ (experiment group OD value/control group OD value) Â 100%.

qRT-PCR
Cells from the control group, As 2 O 3 group, si-con group and si-Id-1 group were collected, fully lysed, and total RNA was extracted by Trizol method to detect RNA purity and concentration. The RNA was reverse transcribed into cDNA using the TaKaRa reverse transcription kit, and the reaction system was prepared according to the TaKaRa fluorescence quantification kit. The b-actin was used as an internal reference and was placed on an ABI-7300 real-time PCR instrument for amplification. The samples were repeated 3 times, and the experimental results were analyzed using the F ¼ 2 ÀDDCt method. Id-1 primers: upstream: 5 0 -CTCCAACTGAAGGTCCCTGATGTAG-3 0 , downstream: 5 0 -ACGACATGAACGGCTGTTACTC AC-3 0 ; b-actin primer: upstream:

Western blot
Cells in control group, As 2 O 3 group, si-con group, si-Id-1 group, As 2 O 3 þCtrl group and As 2 O 3 þId-1 group were ultrasonically pulverized, then RIPA lysate containing protease inhibitor was added and the cells were lysed on ice for 5 min, The supernatant was collected by centrifugation at 12,000 g for 15 min at 4 C. The protein samples were electrophoresed on SDS-PAGE, transferred to PVDF membrane, and blocked with 5% skim milk powder for 2 h. Rabbit anti-human Bax polyclonal antibody (1:5000), rabbit anti-human Bcl-2 polyclonal antibody (1:1000), rabbit anti-human Caspase-3 polyclonal antibody (1:500), rabbit anti-human Id-1 polyclonal antibody (1:1000) and rabbit anti-human beta-actin polyclonal antibody (1:1000) were added respectively and incubated overnight at 4 C. After rinsing with TBST for 3 times, HRP labeled secondary antibody was added for 2 h at room temperature. The samples were rinsed with TBST for 3 times, 10 min each time, ECL kit was developed by light, and each sample was repeated for three times.

Flow cytometry for apoptosis
The cells of the control group, As 2 O 3 group, si-con group, si-Id-1 group, As 2 O 3 þCtrl group and As 2 O 3 þId-1 group were digested by trypsinase, washed with PBS for 3 times and collected. 5 lL Annexin V-FITC and 10 lL PI were added to each group according to the instructions of Annexin V-FITC/PI apoptosis detection kit, gently mixed, and incubated at room temperature for 15 min in the dark, and apoptosis was detected by flow cytometry.

Statistical analysis
The experimental data were expressed as x ± s, and analyzed by SPSS 22.0 (SPSS, Inc., Chicago, IL,USA). The data were compared between the two groups by t-test. The data of multiple groups were compared by one-way analysis of variance. p < .05 was considered statistically significant.

As 2 O 3 inhibits Tca8113 cell proliferation
Tca8113 cells were treated with As 2 O 3 of different concentration and cell proliferation was detected. The results (Figure 1) showed that As 2 O 3 could inhibit Tca8113 cell proliferation. IC50 ¼ 3.004 ± 0.2379 lmol/L and 3 lmol/L was used for subsequent experiments.

As 2 O 3 inhibits Id-1 expression in Tca8113 cells
Tca8113 cells were treated with 3 mol/l As 2 O 3 for 48 h, and Id-1 expression was detected by qRT-PCR. As shown in Figure  2, the expression level of Id-1 mRNA in group As 2 O 3 was significantly lower than that in the control group (p < .05).

Detection of Id-1 gene silencing efficiency in Tca8113 cells
As displayed in Figure 3, the expression of Id-1 mRNA and protein in Tca8113 cells transfected with Id-1 siRNA was significantly lower than that in si-con group (p < .05). The difference was statistically significant.

Silencing Id-1 inhibits proliferation and induces apoptosis in Tca8113 cells
As can be seen in Figure 4, after Id-1 siRNA transfection, the cell proliferation ability was significantly decreased (p < .05), and the number of apoptotic cells was significantly increased in Tca8113 cells, (p < .05), and the difference was statistically significant.
Overexpression of Id-1 attenuates the effect of As 2 O 3 on proliferation and apoptosis of Tca8113 cells As shown in Figure 5, the intervention of 3 lmol/L As 2 O 3 in Tca8113 cells inhibited cell proliferation and induced apoptosis (p < .05). However, the cell proliferation ability was restored (p < .05) and the apoptotic rate was reduced (p < .05) in Tca8113 cells transfected with Id-1 siRNA.

Id-1 ectopic expression partially restored the effect of As 2 O 3 on the expression of apoptosis protein in Tca8113 cells
As can be seen in Figure 6, As 2 O 3 could inhibit the expression of Caspase-3 and Bax protein in Tca8113 cells (p < .05) and up-regulate the expression of Bcl-2 protein (p < .05). The expression of Caspase-3, Bax and Bcl-2 in Tca8113 cells were restored to a certain extent after Id-1 siRNA transfection.

Discussion
As 2 O 3 is powdery, tasteless, soluble in acid, alkali, water, ethanol, etc. Once entering in the human body, it will show extremely strong toxicity and cause blood vessel and visceral damage, and seriously will lead to circulatory failure or even death. As 2 O 3 was firstly used as a drug in the clinical treatment of tuberculosis and it has been confirmed by continuous research that As 2 O 3 has an inhibitory effect on the growth and metastasis of various tumor cells. Clinical evidence indicates that [8] the use of As 2 O 3 in the treatment of multiple myeloma patients is of significant efficacy. It is reported that As 2 O 3 could inhibit the growth and development of glioma cells [9]. After treatment with As 2 O 3 , the survival rate of xenotransplantation model mice has been improved. In addition, As 2 O 3 could regulate the biological processes such as apoptosis and proliferation of tumor cells. For example, Xia et al. [10] found that As 2 O 3 could induce apoptosis of breast cancer cells and inhibit cell growth by down-regulating the expression of Bcl-2 and nuclear factorkappa B (NF-jB). Gao et al. [11] found that As 2 O 3 could regulate the cell cycle and stagnate the pancreatic cancer cell cycle in the G2/M phase, thereby effectively inhibiting cell proliferation and promoting apoptosis by regulating p53 expression; Moreover, Nakamura et al. [12] analyzed the role of As 2 O 3 in the pathogenesis of osteosarcoma and found that As 2 O 3 could induce apoptosis by causing DNA damage and up-regulating the activity of caspase-3. However, there is a limited study of As 2 O 3 on the pathogenesis of tongue squamous cell carcinoma. In this study, As 2 O 3 was able to effectively inhibit the proliferation of Tca8113 cells by using As 2 O 3 with different concentration to interfere with Tca8113 cells and detect cell proliferation. The IC50 was 3.004 ± 0.2379 lmol/L.
The transcription factor that regulates the activity of basic helix-loop-helix in the 11 groups of an inhibitor of DNA binding and/or differentiation (Id) was first cloned in 1990 [13].
Mammals contain four Id factors, namely Id1, Id2, Id3, Id4, which are involved in the regulation of cell growth, differentiation, maturation, apoptosis, etc., and are abnormally expressed in tumors such as melanoma and pancreatic cancer [14,15]. A number of studies [16,17] have shown that the abnormal expression of Id-1 can be detected in prostate, ovarian and cervical cancer tissues, and its expression level is  related to the tumor stage, disease grade, lymph node metastasis and survival rate of patients. Li et al. [18] found that ectopic expression of Id-1 could regulate the cell cycle, promote cell proliferation and metastasis, while down-regulation of Id-1 expression had the opposite effect. Lai et al. [19] used RNAi technology to knock down Id-1 expression in colorectal cancer cells. It was found that the cell proliferation ability decreased, the number of migration and invasion cells decreased significantly, and the expression of matrix metalloproteinase-2 decreased. It suggested that Id-1 could regulate cell proliferation and metastasis by regulating downstream target genes. Caspase-3, Bcl-2 and Bax proteins are key proteins in apoptosis, and BH1 and BH2 domains in Bcl-2 and Bax proteins can bind to form dimers, and Bcl-2/Bax homodimers are the main switch of apoptosis, when the expression of Bax in cells increases, the Bcl-2/Bax homologous dimer dissociates, promotes the release of cytochrome C, and then activates Caspase-3, initiates the caspase cascade  reaction, promotes cell apoptosis. Evidence [20] shows that Id-1 can inhibit Bax expression and up-regulate Bcl-2 expression in breast cancer cells, up-regulate Bcl-2 expression and reduce cell apoptosis. In this study, 3 lmol/L As 2 O 3 treatment could inhibit the expression of Id-1, Caspase-3 and Bax in Tca8113 cells and promote the expression of Bcl-2. After silencing Id-1 by RNAi technology, the cell proliferation ability decreased and the number of apoptotic cells increased. Overexpression of Id-1 could attenuate the inhibition of As 2 O 3 on the proliferation and apoptosis of Tca8113 cells to some extent.
In summary, As 2 O 3 can effectively inhibit the proliferation of Tca8113 cells and induce apoptosis, accomplished by regulating the expression of apoptosis-related proteins and down-regulating Id-1 expression. The study is expected to provide an experimental basis for the clinical application of As 2 O 3 in the treatment of tongue squamous cell carcinoma.

Disclosure statement
No potential conflict of interest was reported by the authors.