Xuejuan Li, Weihua Lou1, Fei Ma, Xiaoyi Cha, Changan Pang, Qinghua Ma and Fei Ma*

Department of Otorhinolaryngology, The Seventh People’s Hospital of Zhengzhou, 1Department of Otorhinolaryngology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450000, China

*Corresponding Author:
Fei Ma
Department of Otorhinolaryngology, The Seventh People’s Hospital of Zhengzhou, Zhengzhou, Henan Province 450000, China
E-mail: mafei846@126.com
Date of Received 10 February 2023
Date of Revision 05 August 2023
Date of Acceptance 22 January 2024
Indian J Pharm Sci 2024;86(1):308-313  

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DOI: 10.36468/pharmaceutical-sciences.1279

Abstract

Isoliquiritin has anti-tumor effects in a variety of cancers. circ_0023028 has been confirmed to enhance laryngeal squamous cell carcinoma progression. However, whether isoliquiritin affects laryngeal squamous cell carcinoma progression by regulating circ_0023028 is unknown. TU177 cells were divided into 8 groups; control group, isoliquiritin 25 μmol/l group, isoliquiritin 50 μmol/l group, isoliquiritin 100 μmol/l group, si-NC group, si-circ_0023028 group, isoliquiritin+plasmid cloning deoxyribonucleic acid group and isoliquiritin+plasmid cloning deoxyribonucleic acid-circ_0023028 group. Cell functions were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, colony formation assay, flow cytometry, transwell assay and wound healing assay. circ_0023028 expression was examined by quantitative real-time polymerase chain reaction, and cleaved caspase-3 level was evaluated using Western blot. circ_0023028 expression was higher in laryngeal squamous cell carcinoma tissue. Isoliquiritin increased cell inhibition rate, apoptosis rate and cleaved caspase-3 level, while reduced scratch healing rate, colony cell number, migrated cell number, and circ_0023028 expression in TU177 cells. Silencing of circ_0023028 enhanced cell inhibition rate, apoptosis rate and cleaved caspase-3 level, while suppressed scratch healing rate, colony cell number and migrated cell number in TU177 cells. Besides, overexpression of circ_0023028 reversed the effect of isoliquiritin on laryngeal squamous cell carcinoma cell proliferation, apoptosis and migration. Isoliquiritin inhibited laryngeal squamous cell carcinoma cell growth and migration via reducing circ_0023028 expression.

Keywords

Isoliquiritin, circ_0023028, laryngeal squamous cell carcinoma, caspase-3, metastasis

Laryngeal Squamous Cell Carcinoma (LSCC) is one of the common malignant tumors of the head and neck worldwide, in which malignant metastasis and recurrence are the main reasons for LSCC patient’s poor survival[1,2]. Patients with mid to late-stage LSCC are treated with manual excision of the diseased tissue in combination with chemotherapy or radiotherapy[3,4]. But with the prolongation of treatment, the appearance of drug resistance limits the effectiveness of LSCC treatment in some patients[5,6]. Therefore, it is necessary to clarify molecular mechanism affecting LSCC process to provide new way for LSCC treatment.

Isoliquiritin (ISL) is a phenolic flavonoid compound extracted from within the traditional Chinese medicine Glycyrrhiza glabra[7]. ISL has been shown to have significant anti-tumor effects[8,9]. Recent findings revealed that ISL could induce lung cancer cell apoptosis[10]. Also, ISL promoted ferroptosis to improve the doxorubicin resistance in breast cancer[11]. In this, we found that ISL had an inhibition on LSCC cell apoptosis. However, ISL roles in LSCC progression have not been revealed.

Circular Ribonuclic Acid (circRNA), a special non-coding RNA, can be acted as target for cancer treatment[12]. It was reported that circ_0023028 was upregulated in laryngeal cancer tissues, and it’s silencing restrained cancer malignant progression[13]. Besides, circ_0023028 knockdown was confirmed to inhibit LSCC proliferation and metastasis[14]. Therefore, circ_0023028 has the potential to be a molecular target of LSCC treatment. In this, we found that ISL reduced circ_0023028 expression. However, whether ISL regulates circ_0023028 expression to exert anti-tumor effect in LSCC is unclear.

Here, we aimed to reveal ISL roles in LSCC progression and explore its underlying molecular mechanisms. Based on the above, we proposed the following hypothesis; ISL inhibited LSCC progression by suppressing circ_0023028 expression.

Materials and Methods

Sample collection:

The study was approved by the Seventh People’s Hospital of Zhengzhou. All participants signed an informed consent form. LSCC tissues and paracancerous tissues of 73 cases were taken from 73 individuals diagnosed with LSCC (age 35 y-72 y, 44 males and 29 females) in the Seventh People’s Hospital of Zhengzhou.

Cell culture and grouping:

TU177 cells (Biovector, Beijing, China) were grown in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10 % Fetal Bovine Serum (FBS). Cells were inoculated in 24-well plates and treated with different concentrations of ISL (Meilune, Dalian, China), and labelled as; ISL 25 μmol/l group, ISL 50 μmol/l group, and ISL 100 μmol/l group. Normal cultured cells were used as control group. TU177 cells were transfected with si-NC/si-circ_0023028 or plasmid cloning Deoxyribonucleic Acid (pcDNA)/pcDNA-circ_0023028, and then treated with 100 μmol/l ISL, and labelled as; si-NC group, si-circ_0023028 group, ISL+pcDNA group and ISL+pcDNA-circ_0023028 group.

Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR):

Total RNAs were extracted by TRIzol reagent (Invitrogen, Carlsbad, California, United States of America (USA)). Extracted RNA was reverse-transcribed into complementary DNA (cDNA) and then used for PCR amplification. circ_0023028 expression was analyzed by 2−ΔΔCt method.

Cell proliferation assay:

3-[4,5-Dimethylthiazol-2-yl]-2,5 Diphenyl Tetrazolium Bromide (MTT) assay: TU177 cells in 96-well plates were cultured for 48 h. Cells were treated with MTT reagent (Beyotime, Shanghai, China) and then incubated with Dimethyl Sulfoxide (DMSO) reagent. After that, cell inhibition rate was evaluated using a microplate reader.

Colony formation assay: TU177 cells in 6-well plates were cultured for 14 d. After fixed using 4 % paraformaldehyde and stained with crystal violet, colony cell numbers were counted under the microscope.

Flow cytometry:

TU177 cells were incubated with Annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI) solution (Beyotime). Apoptosis rate was analyzed under flow cytometry.

Western blot:

Protein was extracted, separated and transferred to Polyvinylidene Difluoride (PVDF) membranes. Membrane was incubated with anti-cleaved caspase-3 (ab2302, 1:1000), anti-Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (ab9485, 1:2500) and secondary antibody (ab205718, 1:50000). Afterwards, membrane was incubated with Beyo Electrochemiluminescence (ECL) plus solution (Beyotime) to detect protein signals.

Transwell assay:

TU177 cells were inoculated in the upper of transwell chamber, and complete medium was filled into lower chamber. After 24 h, cells were fixed and stained, and then migrated cell numbers were counted under a microscope.

Wound healing assay:

TU177 cells were inoculated in 24-well plates, and cell layer was created a wound using a 10 μl pipette. After 24 h, cell wound area was photographed to count the scratch healing rate.

Statistical analysis:

Data were presented as mean±Standard Deviation (SD) using Graph Pad Prism 7.0 software. Results were compared using Student’s t-test or Analysis of Variance (ANOVA). p<0.05 was considered as significant.

Results and Discussion

As shown in Table 1, circ_0023028 expression was remarkably increased in LSCC tissues. The inhibition rate, apoptosis rate and cleaved caspase-3 level were significantly increased, while the colony cell numbers were markedly decreased by ISL in a dose-depend manner (fig. 1 and Table 2). The scratch healing rate and migrated cell numbers of TU177 cells were reduced by ISL in a dose-dependent manner (Table 3). Circ_0023028 expression was gradually decreased in TU77 cells with the increasing of ISL treatment concentrations (Table 4).

Group n circ_0023028
Paracancerous tissues 73 0.78±0.14
LSCC tissues 73 2.40±0.28*
t 44.214
p 0.000

Table 1: circ_0023028 expression In LSCC tissues.

cleaved

Fig. 1: Effect of ISL on apoptosis and cleaved caspase-3 level in TU177 cells, (A): Flow cytometry plot and (B): Cleaved caspase-3 protein expression.

Group Percentage Cloned cell numbers Cleaved caspase-3
Inhibition Apoptosis
Control 0.00±0.00 6.67±0.36 134.89±6.94 0.15±0.02
25 μmol/l ISL 18.56±1.01* 11.90±0.78* 101.11±4.43* 0.32±0.02*
50 μmol/l ISL 32.18±2.28*# 16.60±1.05*# 76.00±2.54*# 0.52±0.04*#
100 μmol/l ISL 46.46±4.59*#△ 21.53±1.11*#△ 50.22±2.78*#△ 0.74±0.06*#△
F 517.438 474.428 573.258 389.350
p 0.000 0.000 0.000 0.000

Table 2: Effect of ISL on TU177 cell proliferation and apoptosis.

Group Scratch healing rate Migrated cell numbers
Control 64.48±2.54 180.89±11.37
25 μmol/l ISL 52.24±2.06* 153.33±5.96*
50 μmol/l ISL 40.76±1.99*# 111.00±6.83*#
100 μmol/l ISL 30.63±1.47*#△ 62.33±2.00*#△
F 456.645 447.565
p 0.000 0.000

Table 3: Effect of ISL on migration of TU177 cells.

Group circ_0023028
Control 1.00±0.00
25 μmol/l ISL 0.74±0.05*
50 μmol/l ISL 0.44±0.04*#
100 μmol/l ISL 0.22±0.02*#△
F 912.000
p 0.000

Table 4: Effect of ISL on circ_0023028 expression in TU177 cells.

Circ_0023028 expression was reduced by the transfection of si-circ_0023028. Circ_0023028 silencing increased the inhibition rate, apoptosis rate, and cleaved caspase-3 level, whereas decreased the scratch healing rate, colony cell number, and migrated cell number in TU177 cells (fig. 2 and Table 5).

knockdown

Fig. 2: Effect of circ_0023028 knockdown on apoptosis and cleaved caspase-3 expression in ISL-treated TU177 cells, (A): Flow cytometry plot and (B): Cleaved caspase-3 protein expression.

Group circ_0023028 Percentage Numbers Cleaved caspase-3
Inhibition Apoptosis Scratch healing Colony cell Migrated cell
si-NC 1.00±0.00 0.00±0.00 6.69±0.43 64.57±2.61 133.22±8.66 179.78±10.95 0.17±0.02
si-circ_0023028 0.27±0.03* 54.08±2.45* 23.39±1.51* 24.61±1.60* 43.00±2.00* 52.78±1.81* 0.83±0.05*
t 73.000 66.220 31.910 39.159 30.452 34.329 36.768
p 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Table 5: Effect of circ_0023028 knockdown on proliferation, apoptosis and migration in ISL-treated TU177 cells.

Circ_0023028 expression was elevated by circ_0023028 overexpression vector. The inhibition rate, apoptosis rate, and cleaved caspase-3 level were enhanced, while the scratch healing rate, colony cell number and migrated cell number were suppressed in ISL+pcDNA-circ_0023028 group (fig. 3 and Table 6).

expression

Fig. 3: Effect of circ_0023028 overexpression on apoptosis and cleaved caspase-3 expression in ISL-treated TU177 cells, (A): Flow cytometry plot and (B): Cleaved caspase-3 protein expression.

Group circ_0023028 Percentage Cells number Cleaved caspase-3
Inhibition Apoptosis Scratch healing Cloned migrated
ISL+pcDNA 1.00±0.00 46.92±3.97 21.67±1.28 29.98±1.99 52.00±1.63 60.44±3.13 0.74±0.05
ISL+pcDNA-circ_0023028 3.44±0.11* 20.18±1.04* 13.49±0.93* 47.07±2.42* 94.33±2.91* 136.22±6.25* 0.35±0.03*
t 66.545 19.547 15.510 16.364 38.073 32.524 20.065
p 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Table 6: Effect of circ_0023028 overexpression on proliferation, apoptosis and migration in ISL-treated TU177 cells.

Glycyrrhiza glabra, a leguminous plant with a long history of medicinal use, and is rich in chemical components such as triterpenoids, flavonoids, alkaloids and polysaccharides[15]. Modern pharmacological experiments show that it has immune regulation, hypoglycemic and anti-tumor effects[16,17]. ISL is extracted from Glycyrrhiza glabra[18]. Previous studies had shown that ISL promoted gastric cancer cell apoptosis in dose-dependent manner[19]. Also, ISL decreased cell viability and increased apoptosis in breast cancer[11]. The above findings confirmed that ISL could resistant to tumorigenesis. Here, we found that ISL treatment promoted apoptosis, while suppressed proliferation and migration, suggesting that ISL inhibited cell growth and migration to hinder LSCC malignant progression.

With the discovery and reporting of more and more circRNAs, much attention has been paid to exploring the biological functions of circRNAs[20,21]. CircRNAs, vary widely in their expression abundance, are usually tissue-specific and disease-specific[22]. Wang et al.[23] found that circRNA_100290 downregulation inhibited LSCC cell growth and metastasis. Circ-CCND1 facilitated LSCC cell proliferation by regulating miR-646 expression[24]. Previous study had revealed that circ_0023028 contributed to LSCC cell proliferation and migration. Similar to this reports, our study confirmed the high circ_0023028 expression in LSCC tissues. Circ_0023028 knockdown enhanced apoptosis, while suppressed proliferation and migration in LSCC cells, verifying that circ_0023028 might promote LSCC progression. Also, circ_0023028 expression was reduced in ISL-treated LSCC cells, and its overexpression reversed ISL-mediated the inhibition on LSCC cell growth and migration, showing that ISL affected LSCC progression via reducing circ_0023028 expression.

In summary, ISL inhibited LSCC cell growth and migration by inhibiting circ_0023028 expression. The results of this study provided new ideas for the clinical management of LSCC.

Conflict of interests:

The authors declared no conflict of interests.

References