Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contact Us Login 
An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists

  Table of Contents    
Year : 2020  |  Volume : 24  |  Issue : 2  |  Page : 400-401

The efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide-induced squamous cell carcinoma of the tongue in rats

1 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Başkent University, İstanbul, Turkey
2 Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Medipol University, İstanbul, Turkey
3 Department of Pathology, Faculty of Medicine, Başkent University, İstanbul, Turkey
4 Pathology Laboratory, Council of Forensic Medicine, Ankara Group Chairmanship, İstanbul, Turkey
5 Animal Research Center, Başkent University, İstanbul, Turkey

Date of Submission16-Apr-2018
Date of Acceptance11-May-2020
Date of Web Publication09-Sep-2020

Correspondence Address:
Secil Cubuk
Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Baskent University, 11 Sokak No: 26 Bahçelievler, Ankara 06490
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jomfp.JOMFP_88_18

Rights and Permissions



Aims: The aim of this study to investigate the efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinogenesis of the tongue in rats.
Subjects and Methods: The sample was composed of 27 male Sprague Dawley rats that received 50 ppm 4NQO for 20 weeks in drinking water. Group 1 (n = 9) was treated with 50 mg/kg/day propranolol for 20 weeks, Group 2 (n = 9), after carcinogenesis inducement for 20 weeks, received propranolol (50 mg/kg/day) for 2 weeks and Group 3 (n = 9) received no treatment. At the end of the experimental stage, the tongue specimens were evaluated under a light microscope and categorized as low- or high-risk lesions according to a binary system.
Statistical Analysis Used: The statistical comparison was performed with a likelihood ratio test.
Results: Histopathological analysis revealed the risk of malignant transformation rates as 33.3% in Group 1, 55.5% in Group 2 and 77.8% in Group 3; however, the difference between the groups was not statistically significant (P > 0.05).
Conclusion: The results of the study suggest that propranolol has a tendency to preventive effect against carcinogenesis.

Keywords: 4-nitroquinoline 1-oxide, carcinogenesis, chemoprevention, propranolol

How to cite this article:
Cubuk S, Uckan S, Ozdemir H, Taslica ZF, Bacanli D. The efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide-induced squamous cell carcinoma of the tongue in rats. J Oral Maxillofac Pathol 2020;24:400-1

How to cite this URL:
Cubuk S, Uckan S, Ozdemir H, Taslica ZF, Bacanli D. The efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide-induced squamous cell carcinoma of the tongue in rats. J Oral Maxillofac Pathol [serial online] 2020 [cited 2021 Aug 1];24:400-1. Available from: https://www.jomfp.in/text.asp?2020/24/2/400/294650

   Introduction Top

Squamous cell carcinoma (SCC) is the most common type of oral carcinoma and is the eighth most common human malignancy. Although many studies have been conducted on treatment and chemopreventive techniques for SCC of the oral cavity, the 5-year survival rates for patients have been reported as approximately 56%.[1],[2]

Carcinogenesis is a multistep process in which accumulated genetic changes are induced by carcinogenic agents.[3] 4-nitroquinoline 1-oxide (4NQO) is widely used as a carcinogen for the development of oral SCC via damaging DNA similar to other carcinogens present in tobacco.[3] 4NQO can be applied to rats in drinking water or topically, and precancerous or cancerous lesions can be produced in this model. One of the advantages of this cancer model is that it simulates the development of human SCC within premalignant white oral lesions; thus, the developmental stages of carcinogenesis can be studied in this model.[4] The growth and metastasis of solid tumors depends on angiogenesis; therefore, suppression of tumor vascularization is one of the main targets for therapy.

Propranolol is a nonselective β-blocker drug widely used for the treatment of problematic infantile hemangiomas.[5],[6],[7],[8],[9] The mechanism of therapeutic effect of propranolol on hemangiomas is not known precisely, but may be due to vasoconstriction, inhibition of angiogenesis and induction of apoptosis in capillary epithelial cells.[5],[10] Based on the agent's antiangiogenic effect, it has been reported that propranolol may inhibit tumor progression, metastasis and the secretion of angiogenic cytokines in several types of cancers.[11] Propranolol reduces head and neck SCC cell line viability, induces apoptosis and inhibits production of the proangiogenic protein vascular endothelial growth factor.[12] In addition, prostate carcinoma, lung carcinoma and growth of neuroblastoma in mice models were reduced by administration of propranolol.[13],[14],[15],[16],[17]

We hypothesized that since oral tissues and oral SCC are rich in vascularization, propranolol, an antiangiogenic agent, might have an inhibitory and/or therapeutic effect on the carcinogenesis stages of oral SCC. Therefore, the aim of this study was to investigate the efficiency of propranolol on occurrence and development of 4NQO-induced squamous cell carcinogenesis of the tongue in rats.

   Subjects and Methods Top

Animals and experimental design

All experimental protocols involving animals conformed to procedures describing in the Guiding Principles for the Use of Laboratory Animals. The study was approved by the Animal Committee of Baskent University of Ankara, Turkey (number D-DA 13/04). The power analysis revealed that 9 animals per group were needed to detect clinically meaningful differences between the groups at a power of 90% and at 0.05 significance level. A total of 27 male Sprague Dawley rats weighing approximately 350 g were obtained from Baskent University (Ankara, Turkey) and maintained under controlled temperature (24°C ± 2°C) and 12 h: 12 h light/dark period conditions and with free access to water and food.

The rats were distributed into three groups (n = 9 per group). All groups received 50 ppm 4NQO (Sigma Aldrich, St Louis, USA) for 20 weeks in drinking water. Group 1 was treated with 50 mg/kg/day propranolol by oral gavage for 20 weeks. Group 2 was treated with propranolol for 2 weeks after carcinogenesis inducement for 20 weeks, and Group 3 (the control group) received no treatment. The experimental periods were established to evaluate the effect of propranolol on all stages of carcinogenesis (from the initiation phase to 20 weeks) and after 20 weeks of the carcinogenesis process. The rats were euthanized with high-dose ketamine at the end of the experimental stage. The tongues were longitudinally cut into halves for histopathological examinations. The tissues were fixed in 10% buffered formalin, embedded in paraffin blocks and stained with hematoxylin and eosin.

Histopathological analysis

Histopathological evaluation was performed under a light microscope. The tongue sections were evaluated as normal, hyperplasia/hyperkeratinization, dysplasia or carcinoma. The scoring was based on the architectural and cytological changes described in the World Health Organization classification, and precancerous lesions were classified as low- or high-risk lesions according to the binary system [Table 1].[18] If there were at least four architectural changes and five cytological changes, the lesion was considered as high-risk lesion for malignant transformation. If there were fewer than four architectural changes or five cytological changes, the lesion was considered as low-risk lesion for malignant transformation.
Table 1: Architectural and cytological changes described in the World Health Organization

Click here to view

Statistical analysis

Data analysis was performed using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL, USA). Nominal data were analyzed with the likelihood ratio test. P < 0.05 was considered statistically significant.

   Results Top

Clinical view of the lesions

All lesions were localized at the posterior dorsal tongue, and most were characterized as white lesions with a straight or papillary/nodular surface [Table 2]. There was 1 tumor occurrence in each group. The diameter of the mass was 0.6 cm, 0.9 cm and 1.8 cm in Groups 1, 2 and 3, respectively [Figure 1], [Figure 2], [Figure 3].
Figure 1: Tumor sample from Group 1. The maximum width of the tumor surface is measured as 0.6 cm

Click here to view
Figure 2: Tumor sample from Group 2. The maximum width of the tumor surface is measured as 1.2 cm

Click here to view
Figure 3: Tumor sample from Group 3. The maximum width of the tumor surface is measured as 1.8 cm

Click here to view

Histopathological evaluation

The histopathological analysis showed that the SCC had spread into the submucosa and underlying muscle layer, forming small islands with typical keratin pearl formation in the tumor samples from Group 2 and Group 3. In contrast, the malignant transformation in the tumor sample from Group 1 was limited to the epithelial layers, and a histopathological diagnosis of carcinoma in situ was determined for this sample [Figure 4].
Figure 4: Histopathologic views of the tumor sample from Group 3. The invasive keratin pearls and tumor islands were seen (H&E, 10 × 10)

Click here to view

Decreased risk of malignant transformation in Group 1 (33.3%) compared with Group 2 (55.5%) and 3 (77.8%) was detected based on the binary system of oral precancerous lesions. There were slight differences between the three groups regarding the high-risk category (Group 1, 11.1%; Groups 2 and 3, 22.2%). The rates of low risk were 22.2% in Group 1, 33.3% in Group 2 and 55.5% in Group 3. The difference between the groups was not statistically significant (P > 0.05).

   Discussion Top

Although many data have been published about treatment approaches to and diagnosis methods for oral SCC, the survival rates of patients have not improved significantly during the last 40–50 years.[19] Consequently, investigations related to chemoprevention that can extend the latent period of carcinogenesis or prevent the development of carcinoma have received attention.

The 4NQO cancer model is one of the most frequently used methods in these types of studies. One of the advantages of the 4NQO model has been reported that it can be used for studying the early stages of oral cancer progression.[19] Although there are differences in the dose and application periods of 4NQO, general histopathological alterations are associated with carcinogenesis such as progression from early hyperplasia to dysplasia and then to SCC occur within 28 weeks.[20],[21]

The binary system introduces to evaluate the prediction of malignant potential of oral premalignant lesions using quantitative morphological characteristics scoring system.[18] The binary system was used for classification of the lesions which occurred during carcinogenesis stages in this study. According to our experience, this technique is also feasible for animal studies considering that sample size is generally limited in comparison with clinical studies.

Ever since suppression of hemangiomas after the administration of propranolol was shown, investigators have been concerned about propranolol's ability to reduce the development of a malignancy. Several clinical studies were reported that propranolol might impact tumor development and decrease the cancer-specific mortality in breast cancer.[1] Pasquier et al. showed that propranolol can potentiate the antiangiogenic effects and antitumor efficacy of chemotherapy in breast cancer treatment.[11] Severalin vivo studies have demonstrated that propranolol can reduce metastasis of PC-3 prostate cancer cells and avoid tumor growth in ovarian carcinoma.[14],[16] Wolter et al. demonstrated that propranolol reduced head and neck SCC cell line viability, induced apoptosis and inhibited production of the proangiogenic protein. The authors also recommended propranolol as a useful adjuvant in head and neck SCC that can aid in reversing chemotherapy and radiation resistance.[12]

In our knowledge, this study was the firstin vivo study that evaluated the prevention and curative effect of propranolol on oral SCC. At the end of the experimental stage, the tumor diameter was three times that of the control group compared with Group 1 (1.8 cm vs. 0.6 cm) and the risk of malignant transformation of precancerous lesion was almost two times for the control group compared with Group 1 (78% vs. 34%).

   Conclusion Top

These results suggest that propranolol has potential to reduce the development of SCC in tongue. This is important for the determination of beneficial drug effects on patients who are treated for cardiac disease with propranolol. Furthermore, this study will inspire the investigators for future studies on curative effect of propranolol on SCC.

Financial support and sponsorship

Başkent University Research Fund.

Conflicts of interest

There are no conflicts of interest.

   References Top

Clark JI, Eisner RM, Hofmeister C, Norton J, Thomas S, Choudhury A, et al. Phase I adjuvant radiation with docetaxel in high-risk head and neck cancer. Am J Clin Oncol 2009;32:396-400.  Back to cited text no. 1
Zhou H, Tang Y, Liang X, Yang X, Yang J, Zhu G, et al. RNAi targeting urokinase-type plasminogen activator receptor inhibits metastasis and progression of oral squamous cell carcinoma in vivo. Int J Cancer 2009;125:453-62.  Back to cited text no. 2
Kanojia D, Vaidya MM. 4-nitroquinoline-1-oxide induced experimental oral carcinogenesis. Oral Oncol 2006;42:655-67.  Back to cited text no. 3
Dayan D, Hirshberg A, Kaplan I, Rotem N, Bodner L. Experimental tongue cancer in desalivated rats. Oral Oncol 1997;33:105-9.  Back to cited text no. 4
Leaute-Labreze C, de la Rooque ED, Hubiche T. Propranolol for severe hemangiomas of infancy. N Engl J Med 2010;358:2649-51.  Back to cited text no. 5
Truong MT, Chang KW, Berk DR, Heerema-McKenney A, Bruckner AL. Propranolol for the treatment of a life-threatening subglottic and mediastinal infantile hemangioma. J Pediatr 2010;156:335-8.  Back to cited text no. 6
Jacobson D, Nayan S, Macnay R. Intrathyroidal hemangioma – A rare congenital anomaly: Case presentation and literature review. Int J Pediatr Otorhinolaryngol 2014;78:1779-83.  Back to cited text no. 7
Greene AK, Rogers GF, Mulliken JB. Management of parotid hemangioma in 100 children. Plast Reconstr Surg 2004;113:53-60.  Back to cited text no. 8
Peridis S, Pilgrim G, Athanasopoulos I, Parpounas K. A meta-analysis on the effectiveness of propranolol for the treatment of infantile airway haemangiomas. Int J Pediatr Otorhinolaryngol 2011;75:455-60.  Back to cited text no. 9
Lamy S, Lachambre MP, Lord-Dufour S, Béliveau R. Propranolol suppresses angiogenesis in vitro: Inhibition of proliferation, migration, and differentiation of endothelial cells. Vascul Pharmacol 2010;53:200-8.  Back to cited text no. 10
Pasquier E, Ciccolini J, Carre M, Giacometti S, Fanciullino R, Pouchy C, et al. Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: Implication in breast cancer treatment. Oncotarget 2011;2:797-809.  Back to cited text no. 11
Wolter NE, Wolter JK, Enepekides DJ, Irwin MS. Propranolol as a novel adjunctive treatment for head and neck squamous cell carcinoma. J Otolaryngol Head Neck Surg 2012;41:334-44.  Back to cited text no. 12
Schuller HM, Cole B. Regulation of cell proliferation by beta-adrenergic receptors in a human lung adenocarcinoma cell line. Carcinogenesis 1989;10:1753-5.  Back to cited text no. 13
Palm D, Lang K, Niggermann B. The norepinephrine-driven metastasis development of PC-3 human prostate cancer cells in BALB/c nude mice is inhibited by β-blokers. Int J Cancer 2006;118:2744-9.  Back to cited text no. 14
Barron TI, Connolly RM, Sharp L, Bennett K, Visvanathan K. Beta blockers and breast cancer mortality: A population- based study. J Clin Oncol 2011;29:2635-44.  Back to cited text no. 15
Lee JW, Shahzad MM, Lin YG, Armaiz-Pena G, Mangala LS, Han HD, et al. Surgical stress promotes tumor growth in ovarian carcinoma. Clin Cancer Res 2009;15:2695-702.  Back to cited text no. 16
Xu T, Xiao X, Zheng S, Zheng J, Zhu H, Ji Y, et al. Antiangiogenic effect of propranolol on the growth of the neuroblastoma xenografts in nude mice. J Pediatr Surg 2013;48:2460-5.  Back to cited text no. 17
Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol 2006;42:987-93.  Back to cited text no. 18
Ribeiro DA, Fávero-Salvadori DM, da Silva RN, Darros BR, Alencar-Marques ME. Genomic instability in non-neoplastic oral mucosa cells can predict risk during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Oral Oncol 2004;40:910-5.  Back to cited text no. 19
Tamura I, Sakaki T, Chaqour B. Correlation of P-cadherin and β-catenin expression and phosphorylation with carcinogenesis in rat tongue cancer induced with 4-nitroquinoline-1-oxide. Oral Oncol 2003;39:506-14.  Back to cited text no. 20
Sato K, Okazaki Y, Tonogi M, Tanaka Y, Yamane GY. Expression of beta-catenin in rat oral epithelial dysplasia induced by 4-nitroquinoline 1-oxide. Oral Oncol 2002;38:772-8.  Back to cited text no. 21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


Print this article  Email this article


    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
  Related articles
    Article in PDF (1,505 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  

   Subjects and Methods
    Article Figures
    Article Tables

 Article Access Statistics
    PDF Downloaded54    
    Comments [Add]    

Recommend this journal

Journal of Oral and Maxillofacial Pathology | Published by Wolters Kluwer - Medknow
Online since 15th Aug, 2007