MMP-9抑制剂对人口腔鳞状细胞癌细胞系CAL27生物学行为影响机制的研究

doi:10.12439/kqhm.1005-4979.2023.06.002

摘要/Abstract

摘要：

目的：探究基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）抑制剂（BB-94）对人口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞系CAL27恶性生物学行为的影响及可能的作用机制。方法：用含不同浓度（0、0.125、0.25、0.5、1.0 μg/mL）BB-94培养液培养对数生长期CAL27细胞48 h，明胶酶谱实验分析细胞培养上清液中MMP-9的活性；细胞计数试剂盒-8（cell counting kit‐8，CCK-8）实验检测细胞存活率；流式细胞术、伤口愈合实验及Transwell实验检测CAL27细胞凋亡、迁移及侵袭情况；实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）技术检测磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）、蛋白激酶B（protein kinase B， PKB，又称Akt）mRNA表达水平；蛋白质印迹法（Western blotting）检测PI3K、磷酸化蛋白激酶B（phosphorylated protein kinase B，又称p-Akt）、MMP-9、波形蛋白（vimentin，Vim）、神经钙黏素（N-cadherin，N-cad）、上皮钙黏素（E-cadherin，E-cad）表达情况。结果：CAL27细胞MMP-9相对活性、存活率、划痕愈合率、细胞侵袭数、PI3K和Akt mRNA相对表达水平及PI3K、p-Akt、Vim、N-cad、MMP-9蛋白相对表达水平均随BB-94作用浓度增大而降低（P<0.05），CAL27细胞凋亡率、E-cad蛋白相对表达水平均随BB-94作用浓度增大而升高（P<0.05）。结论：MMP-9抑制剂可抑制人口腔鳞状细胞癌细胞系CAL27恶性生物学行为，其作用机制可能与阻止PI3K/Akt信号通路激活及上皮间质转化（epithelial mesenchymal transition，EMT）进展有关。

关键词: 基质金属蛋白酶-9抑制剂, 人口腔鳞状细胞癌, 恶性生物学行为, 磷脂酰肌醇3-激酶, 蛋白激酶B

Abstract:

Objective: To explore the potential of BB-94, a matrix metalloproteinase-9 (MMP-9) inhibitor, in alleviating malignant biological behavior of human oral squamous cell carcinoma (OSCC) cell line CAL27 and its possible mechanism. Methods: CAL27 cells in logarithmic growth phase were cultured in BB-94 culture medium with different concentrations (0, 0.125, 0.25, 0.5, 1.0 μg/mL) for 48 hours; the activity of MMP-9 in cell culture supernatant was analyzed by gelatin zymogram; the cell survival rate was detected by cell counting kit-8 (CCK-8) method; the apoptosis, migration, and invasion of CAL27 cells were detected by flow cytometry, wound healing test and Transwell test; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt); the expressions of PI3K, phosphorylated protein kinase B (p-Akt), MMP-9, vimentin (Vim), N-cadherin (N-cad) and E-cadherin (E-cad) proteins were detected by Western blotting. Results: The relative activity of MMP-9, the survival rate of CAL27 cells, the wound healing rate, the number of cell invasion, the relative expression level of PI3K and Akt mRNA and the relative expression level of PI3K, p-Akt, Vim, N-cad and MMP-9 protein decreased with the increase in the concentration of BB-94 (P<0.05). The apoptosis rate of CAL27 cells and the relative expression level of E-cad protein increased as the concentration of BB-94 went up (P<0.05). Conclusion: These results indicate that MMP-9 inhibitor could inhibit the malignant biological behavior of human OSCC cell line CAL27, and its mechanism may be related to preventing the activation of PI3K/Akt signaling pathway and the progress of epithelial mesenchymal transition (EMT).

Key words: matrix metalloproteinase-9 inhibitor, human oral squamous cell carcinoma, malignant biological behavior, phosphatidylinositol 3-kinase, protein kinase B

中图分类号:

R739.5

王静璇, 刘红悦, 庄志征, 胡燕, 杨英顺. MMP-9抑制剂对人口腔鳞状细胞癌细胞系CAL27生物学行为影响机制的研究[J]. 《口腔颌面外科杂志》, 2023, 33(6): 363-369.

WANG Jingxuan, LIU Hongyue, ZHUANG Zhizheng, HU Yan, YANG Yingshun. Study on the mechanism of MMP-9 inhibitor on biological behavior of human oralsquamous cell carcinoma cell line CAL27[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(6): 363-369.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I6/363

图/表 12

图1 各浓度BB-94作用下的CAL27细胞MMP-9明胶酶谱图中A—E分别代表0、0.125、0.25、0.5、1.0 μg/mL BB-94。

Figure 1 MMP-9 gelatinase spectrum of CAL27 cells at different concentrations of BB-94

表1 CAL27细胞MMP-9相对活性比较($\bar{x} \pm s$)

Table 1 Comparison of MMP-9 relative activity of CAL27 cells ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	MMP-9相对活性
0	1.00±0.02
0.125	0.82±0.04^①
0.25	0.55±0.03^①②
0.5	0.41±0.02^①②③
1.0	0.23±0.02^①②③④
F值	389.068
P值	<0.05

表2 不同浓度BB-94培养下CAL27细胞存活率比较($\bar{x} \pm s$)

Table 2 Comparison of survival rates of cal27 cells cultured with different concentrations of BB-94 ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	细胞存活率/%
0	100
0.125	92.16±3.58^①
0.25	76.74±2.91^①②
0.5	57.44±2.73^①②③
1.0	39.23±2.39^①②③④
F值	271.327
P值	<0.05

图2 流式细胞仪检测各浓度BB-94作用下CAL27细胞的凋亡情况 A—E.各浓度BB-94干预后的细胞凋亡情况，浓度分别为0、0.125、0.25、0.5、1.0 μg/mL。

Figure 2 Apoptosis of CAL27 cells at different concentrations of BB-94 detected by flow cytometry

表3 不同浓度BB-94培养下CAL27细胞凋亡率比较($\bar{x} \pm s$)

Table 3 Comparison of apoptosis rates of CAL27 cells cultured with different concentrations of BB-94 ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	细胞凋亡率/%
0	1.33±0.14
0.125	6.49±0.57^①
0.25	18.24±2.07^①②
0.5	34.33±4.19^①②③
1.0	52.47±5.64^①②③④
F值	122.286
P值	<0.05

图3 各浓度BB-94作用下CAL27细胞迁移情况(×20) A—E. 0 h时各浓度BB-94干预后细胞迁移情况，浓度分别为0、0.125、0.25、0.5、1.0 μg/mL；F—J. 48 h时各浓度BB-94干预后细胞迁移情况，浓度分别为0、0.125、0.25、0.5、1.0 μg/mL。

Figure 3 Migration of CAL27 cells at various concentrations of BB-94 (×20)

表4 不同浓度BB-94培养下CAL27细胞划痕愈合率比较($\bar{x} \pm s$)

Table 4 Comparison of wound healing rates of CAL27 cells cultured with different concentrations of BB-94 ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	划痕愈合率/%
0	89.71±3.96
0.125	73.58±2.84^①
0.25	59.30±4.28^①②
0.5	42.49±3.58^①②③
1.0	26.17±1.85^①②③④
F值	161.009
P值	<0.05

图4 各浓度BB-94作用下CAL27细胞侵袭情况（×200） A—E. 各浓度BB-94干预后细胞侵袭情况，浓度分别为0、0.125、0.25、0.5、1.0 μg/mL。

Figure 4 Invasion of CAL27 cells at various concentrations of BB-94 (×200)

表5 不同浓度BB-94培养下CAL27侵袭细胞数比较($\bar{x} \pm s$)

Table 5 Comparison of CAL27 invasive cells cultured with different concentrations of BB-94 ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	侵袭细胞数/个
0	215.33±8.74
0.125	183.67±8.50^①
0.25	141.67±7.02^①②
0.5	97.33±5.51^①②③
1.0	47.33±3.06^①②③④
F值	283.250
P值	<0.05

表6 CAL27细胞PI3K、Akt mRNA相对表达水平比较($\bar{x} \pm s$)

Table 6 Comparison of relative expression levels of PI3K and Akt mRNA in CAL27 cells ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	PI3K	Akt
0	1.01±0.01	1.01±0.01
0.125	0.92±0.03^①	0.91±0.02^①
0.25	0.79±0.02^①②	0.75±0.04^①②
0.5	0.52±0.03^①②③	0.49±0.02^①②③
1.0	0.39±0.03^①②③④	0.31±0.02^①②③④
F值	325.453	438.000
P值	<0.05	<0.05

图5 各浓度BB-94作用下CAL27细胞相关蛋白Western blotting条带图图中A—E分别代表0、0.125、0.25、0.5、1.0 μg/mL BB-94。

Figure 5 Western blotting banding of CAL27 cell related proteins at various concentrations of BB-94

表7 CAL27细胞PI3K等蛋白相对表达水平比较($\bar{x} \pm s$)

Table 7 Comparison of relative expression levels of PI3K and other proteins in CAL27 cells ($\bar{x} \pm s$)

浓度/ (μg·mL^-1)	PI3K	p-Akt	Vim	N-cad	MMP-9	E-cad
0	0.93±0.03	0.86±0.02	0.93±0.02	0.84±0.03	0.89±0.03	0.16±0.01
0.125	0.87±0.03^①	0.72±0.03^①	0.59±0.03 ^①	0.56±0.02^①	0.74±0.02^①	0.24±0.02^①
0.25	0.53±0.02^①②	0.52±0.02^①②	0.37±0.02 ^①②	0.34±0.02^①②	0.53±0.03^①②	0.31±0.02^①②
0.5	0.38±0.02^①②③	0.32±0.03^①②③	0.23±0.02 ^①②③	0.25±0.02^①②③	0.42±0.04^①②③	0.51±0.02^①②③
1.0	0.10±0.02^①②③④	0.17±0.02^①②③④	0.09±0.01 ^①②③④	0.14±0.01^①②③④	0.36±0.03^①②③④	0.81±0.03^①②③④

参考文献 22

[1]	Di Pardo BJ, Bronson NW, Diggs BS, et al. The global burden of esophageal cancer: A disability-adjusted life-year approach[J]. World J Surg, 2016, 40(2): 395-401. doi: 10.1007/s00268-015-3356-2 pmid: 26630937
[2]	Zeng Q, Tao XA, Huang F, et al. Overexpression of miR-155 promotes the proliferation and invasion of oral squamous carcinoma cells by regulating BCL6/cyclin D2[J]. Int J Mol Med, 2016, 37(5): 1274-1280. doi: 10.3892/ijmm.2016.2529 pmid: 26986233
[3]	Chamoli A, Gosavi AS, Shirwadkar UP, et al. Overview of oral cavity squamous cell carcinoma: Risk factors, mechanisms, and diagnostics[J]. Oral Oncol, 2021, 121: 105451. doi: 10.1016/j.oraloncology.2021.105451 URL
[4]	Du F, Liu H, Lu YY, et al. Epithelial-to-mesenchymal transition: Liaison between cancer metastasis and drug resistance[J]. Crit Rev Oncog, 2017, 22(3-4): 275-282. doi: 10.1615/CritRevOncog.2018024855 pmid: 29604904
[5]	Umashankar K, Jayaraj G, Sherlin HJ, et al. Evaluation of matrix metalloproteinase 9 gene expression in oral squamous cell carcinoma and its clinicopathological correlation[J]. Natl J Maxillofac Surg, 2021, 12(3): 339-343. doi: 10.4103/njms.NJMS_279_20 URL
[6]	Xu GF, Ou L, Liu Y, et al. Upregulated expression of MMP family genes is associated with poor survival in patients with esophageal squamous cell carcinoma via regulation of proliferation and epithelial‑mesenchymal transition[J]. Oncol Rep, 2020, 44(1): 29-42. doi: 10.3892/or.2020.7606 pmid: 32627007
[7]	Wang X, Chen QQ. FERMT1 knockdown inhibits oral squamous cell carcinoma cell epithelial-mesenchymal transition by inactivating the PI3K/AKT signaling pathway[J]. BMC Oral Health, 2021, 21(1): 598. doi: 10.1186/s12903-021-01955-9 pmid: 34814915
[8]	Siqueira AS, Pinto MP, Cruz MC, et al. Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2[J]. Oncotarget, 2016, 7(30): 47904-47917. doi: 10.18632/oncotarget.10062 pmid: 27323814
[9]	Jabłońska-Trypuć A, Matejczyk M, Rosochacki S. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs[J]. J Enzyme Inhib Med Chem, 2016, 31(sup1): 177-183. doi: 10.3109/14756366.2016.1161620 URL
[10]	Buttacavoli M, Di Cara G, Roz E, et al. Integrated multi-omics investigations of metalloproteinases in colon cancer: Focus on MMP2 and MMP9[J]. Int J Mol Sci, 2021, 22(22): 12389. doi: 10.3390/ijms222212389 URL
[11]	Huang CY, Yang JL, Chen JJ, et al. Fluoroquinolones suppress TGF-β and PMA-induced MMP-9 production in cancer cells: Implications in repurposing quinolone antibiotics for cancer treatment[J]. Int J Mol Sci, 2021, 22(21): 11602. doi: 10.3390/ijms222111602 URL
[12]	Patil R, Mahajan A, Pradeep GL, et al. Expression of matrix metalloproteinase-9 in histological grades of oral squamous cell carcinoma: An immunohistochemical study[J]. J Oral Maxillofac Pathol, 2021, 25(2): 239-246. doi: 10.4103/0973-029X.325121 URL
[13]	Smriti K, Ray M, Chatterjee T, et al. Salivary MMP-9 as a biomarker for the diagnosis of oral potentially malignant disorders and oral squamous cell carcinoma[J]. Asian Pac J Cancer Prev, 2020, 21(1): 233-238. doi: 10.31557/APJCP.2020.21.1.233 URL
[14]	Wu ZK, Mulatibieke T, Niu MY, et al. Inhibition of matrix metalloproteinase with BB-94 protects against caerulein-induced pancreatitis via modulating neutrophil and macrophage activation[J]. Gastroenterol Res Pract, 2020, 2020: 8903610.
[15]	Jiang AN, Liu JT, Zhao K, et al. Specific inhibitor of matrix metalloproteinase decreases tumor invasiveness after radiofrequency ablation in liver tumor animal model[J]. Front Oncol, 2020, 10: 561805. doi: 10.3389/fonc.2020.561805 URL
[16]	Yu XD, Shi JS, Wang X, et al. Propofol affects the growth and metastasis of pancreatic cancer via ADAM8[J]. Pharmacol Rep, 2020, 72(2): 418-426. doi: 10.1007/s43440-019-00015-y pmid: 32048249
[17]	Pećina-Šlaus N, Kafka A, Gotovac Jerčić K, et al. Comparable genomic copy number aberrations differ across astrocytoma malignancy grades[J]. Int J Mol Sci, 2019, 20(5): 1251. doi: 10.3390/ijms20051251 URL
[18]	Smith BN, Bhowmick NA. Role of EMT in metastasis and therapy resistance[J]. J Clin Med, 2016, 5(2): 17. doi: 10.3390/jcm5020017 URL
[19]	Yang HL, Thiyagarajan V, Shen PC, et al. Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis[J]. J Exp Clin Cancer Res, 2019, 38(1): 186. doi: 10.1186/s13046-019-1196-x
[20]	Sun L, Zhang J. Icariin inhibits oral squamous cell carcinoma cell proliferation and induces apoptosis via inhibiting the NF-κB and PI3K/AKT pathways[J]. Exp Ther Med, 2021, 22(3): 942. doi: 10.3892/etm.2021.10374 pmid: 34306206
[21]	Liu L, Ye Y, Zhu XM. MMP-9 secreted by tumor associated macrophages promoted gastric cancer metastasis through a PI3K/AKT/Snail pathway[J]. Biomed Pharmacother, 2019, 117: 109096. doi: 10.1016/j.biopha.2019.109096 URL
[22]	Jiang WP, Zhang CX, Zhang XM, et al. CircRNA HIPK₃ promotes the progression of oral squamous cell carcinoma through upregulation of the NUPR1/PI3K/AKT pathway by sponging miR-637[J]. Ann Transl Med, 2021, 9(10): 860. doi: 10.21037/atm URL

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