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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 4  |  Page : 602
 

Significance and expression of p-Akt in oral squamous cell carcinoma


Department of Oral Diagnosis, College of Dentistry, University of Babylon, Hillah, Iraq

Date of Submission17-Jun-2020
Date of Decision27-Oct-2020
Date of Acceptance03-Nov-2020
Date of Web Publication22-Dec-2022

Correspondence Address:
Suha Abdulhussein Hindy
Department of Oral Pathology, College of Dentistry, University of Babylon, Hillah
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.JOMFP_256_20

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   Abstract 


Background: Oral cancer is considered as the sixth-most common malignant neoplasm in the world and the most common type is squamous cell carcinoma (SCC). Akt is a serine/threonine-protein kinase, that acts in the regulation of different signaling downstream pathways, including; proliferation, growth, cell metabolism, angiogenesis and survival. The objective of the present study was to show the expression of p-Akt and its prognostic effect in oral SCC (OSCC).
Materials and Methods: A total of 72 cases of OSCC were included in this study; the expression of the Akt gene was done on 4 μm tumor sections using immunohistochemical staining of Akt antibody.
Results: Histopathological examination showed that: (43) were GIII, (13) cases were GII, and (16) cases were GI. The positivity of immunohistochemical staining were appear as brown stain. Results showed increase expression of p-Akt in most cases of oral SCC and mean expression about (54.86 ± 31.58), and the expression of p-Akt was increased with the progression of the histological grade of the tumor.
Conclusion: High expression of p-Akt and its relation with the progression of tumor grade and invasion of tumor may indicate the relationship between p-Akt expression and aggressiveness and progression of the tumor.


Keywords: Oral cancer, Oral squamous cell carcinoma, Phosphorylated Akt, Immunohistochemistry


How to cite this article:
Hindy SA. Significance and expression of p-Akt in oral squamous cell carcinoma. J Oral Maxillofac Pathol 2022;26:602

How to cite this URL:
Hindy SA. Significance and expression of p-Akt in oral squamous cell carcinoma. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Jan 27];26:602. Available from: https://www.jomfp.in/text.asp?2022/26/4/602/364796





   Introduction Top


Squamous cell carcinoma (SCC) is considered the most common type of oral cancer.[1]

Carcinogenesis is a multistep event involving many factors of inactivation or activation genes and cancer generated by an altered cell cycle, with increasing cell proliferation and block of apoptosis. Oral SCC (OSCC) is an aggressive tumor, categorized by a high rate of early local recurrence and prognosis of affected patients.[2],[3]

Akt is a serine/threonine-protein kinase, and established as Akt kinase, is act in the direction of different signaling downstream pathways included in cell proliferation, growth, cell metabolism, angiogenesis and survival.[4]

Genomic mutations in controllers of Akt signaling pathway have been recorded to cause alteration in oncogene of the normal cell of human[5] and identified in malignant glioma, cancer of endometrium and prostate,[6],[7] nonsmall lung cancer,[8] melanoma,[9] hepatocellular carcinoma,[10] and cancer of the breast.[11] The significance of Akt in human malignancy is mainly concluded from frequent mutations in the enzymes that adjust the activity of phospholipids of the second messenger (PtdIns [3, 4, 5] P3, PtdIns [3,4] P2) and eventually lead to triggering of Akt via membrane employment.[12]

Silencing of Akt1 lead to reduce survival of the cell and cause the arrest of the cell cycle at G2/M phase, and was established to lessen the expression of proteins that regulate the survival and proliferation of cancer cell, such as; Bcl2, cyclooxygenase-2, survivin and cyclin D1. Therefore, the role of Akt1 has been clarified in oral cancer.[13]

Objective

To estimate the expression and the prognostic value of p-Akt in (OSCC).


   Materials and Methods Top


Selection of cases

A retrospective study of 72 specimens for patients diagnosed with OSCC, from formalin-fixed paraffin-embedded blocks. The patients included in this study were selected with primary tumor who had not received any previous treatment.

Superficial layers of oral mucosa that present normally in the studied samples of tumor tissue were used as negative control for immunohistochemistry of examined antibody.

Tissue processing and staining

Immuno histochemical staining

Sequential sections of (4 μm) were cut from formalin-fixed paraffin embedded blocks for each case. The sections mounted on poly-L-lysine coated glass slides and then stained by immunohistochemical technique using rabbit polyclonal Akt antibody.

Antigen retrieval was done by pressure cooking with ethylenediaminetetraacetic acid solution as a buffer solution and then the slides were quenched in 30% hydrogen peroxide and blocking.

After that, incubation of the sections was done with primary antibody (Akt) for 1 h with dilution 1:300. Then, subsequently, apply biotinylated anti-rabbit immunoglobulin and streptavidin conjugated to horseradish peroxidase. Then using 3, 3-diaminobenzidine for the elaboration of dye and using hematoxylin as counter staining.

Immunohistochemical staining results were examined by two observers that appear as brown pigmentation for positive areas. The sections were examined with double-headed Leitz light microscope, using a (×40) objective. The expression level of protein was measured by evaluation the percentage of cells, which were positive for antibody in at least five random high power fields in hot spot areas. The tumor cells containing >(5%) immunoreactive cells (brown pigmentation) were defined as positive. In each case, at least (1000) cells were counted.


   Results Top


Histopathological examination revealed that (43) cases were GIII, (13) cases GII and (16) cases GI.

The basal layer of epithelium showed positive stain in addition to the carcinomatous islands, as shown in [Figure 1], carcinomatous islands showed intracellular positivity with high intensity, which is mainly cytoplasmic as shown in [Figure 2], [Figure 3], [Figure 4].
Figure 1: Expression of p-Akt in the epithelium of basal layer and in carcinomatous islands

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Figure 2: Expression of p-Akt in well-differentiated squamous cell carcinoma

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Figure 3: Expression of p-Akt in moderately differentiated squamous cell carcinoma demonstrate aberrant mitosis

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Figure 4: Cytoplasmic expression of p-Akt in carcinomatous cells

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[Figure 5] shows the invasion of carcinomatous islands in the underlying tissue, and [Figure 6], shows perinural invasion of carcinomatous cells.
Figure 5: Invasion islands of carcinomatous cells in the underlying tissue

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Figure 6: Perineural invasion of carcinomatous cells

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Immunohistochemical expression of p-Akt, in oral squamous cell carcinoma cases according to the grading system (GI, GII and GIII)

The immunohistochemical staining for p-AKT was demonstrated between 10% and 100% expression in sixty-eight cases of OSCC, and their percentage was 94.44%.

The mean expression of p-Akt in all cases of OSCC was (54.86 ± 31.58).

The mean expression of p-Akt in Grade III of OSCC was (72.09 ± 24.05), while the mean expression of p-Akt in Grade II of OSCC was (36.92 ± 14.22), and the mean expression of p-Akt in Grade I of OSCC was (23.125 ± 27.13), as shown in [Table 1].
Table 1: Mean and standard deviation of p-Akt protein expression in different grades of oral squamous carcinoma cases

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According to the grading system of OSCC, results showed that the expression of p-Akt was increased with the advancing grade, which showed increasing in p-Akt expression in moderately differentiated SCC (GII) than well differentiated (GI) but not reach statistically significant level [Table 1].

While it highly increased in poorly differentiated type (GIII) with significant statistically, P < 0.0001, and the difference in expression between moderately and poorly differentiated type highly significant, P < 0.0001 [Table 1].

The difference between the mean expression of p-Akt in both GI and GII in relation to GIII was highly significant, P < 0.0001 [Table 1].


   Discussion Top


To create suitable modalities in the treatment of OSCC, a precise evaluation of some factors affecting the advancement of tumor and its prognosis is important.

Despite the conventional grading system is beneficial for the classification of OSCC and its outcome, but still, the prognosis may be poor even in Grade I or II. Therefore, numerous molecular markers used to predict tumor outcome, treatment modalities and patient prognosis.[1]

Akt is an essential factor in the pathway of signal transduction that stimulated by growth factors or insulin and believed to participate in some cells functions such as proliferation, cell growth, invasion, angiogenesis and apoptosis.[14] Unusual regulation of these procedures may lead to cellular perturbations, which considered hallmarks of malignancy. Many studies showed that recurrent hyperactivation of Akt signaling in a lot of malignancies. Many oncoproteins and tumor suppressor genes cross the signal transduction pathway of Akt and are activated or inactivated.[15]

Stimulation of Akt proteins leads to send signals to other proteins in the cell signaling cycle, which leads to metabolism, growth or division.[1]

The present study revealed that the expression of p-Akt was seen in most cases of OSCC with a percentage 94.44%, but the mean expression increased with the grading of tumor. That mean expression in GII more than GI and in GIII more than GI and GII, and differences between grades showed; significant difference between GI and GIII, GII and GIII, GI and GII together with GIII. The mean expression of p-Akt was higher in GII than GI, but there are no significant differences. These findings support that the aggressiveness of the tumor increased with advancing grade of tumor and higher grade showed higher expression of p-Akt, which indicates the progression of tumor, and affect the prognosis of treatment.

Lim et al., in 2005, revealed that activated Akt serine/threonine kinase (p-Akt) is an important indicator for the prognosis of OSCC[1] which is in agreement with the findings of the present study.

Lamaroon and Krisanaprakornkit in 2009 found overexpression of p-Akt in OSCC, which may be involved in carcinogenesis.[16] The same results were demonstrated in the present study.

Pontes et al., in 2009, found that activation of p-Akt plays a key role in the alteration of a potentially malignant lesion like oral leukoplakia into a malignant lesion from its earlier stages.[17] That means the activation of p-Akt was associated with carcinogenesis and aggressiveness of tumor that was found in this study, which demonstrated overexpression of p-Akt with advancing grading of the neoplasm.

The positivity of p-Akt in the carcinomatous cells aid in the determination of invasion and aggressiveness of tumor, which play a role in the prognosis of tumor. Therefore, the evaluation of p-Akt expression will give an insight into the role of Akt on the behavior of the tumor.

[TAG:2]Conclusion [/TAG:2]

High expression of p-Akt and its relation with progression of tumor grade and invasion of tumor may indicate the relationship between p-Akt expression and aggressiveness and progression of the tumor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Lim J, Kim JH, Paeng JY, Kim MJ, Hong SD, Lee JI, et al. Prognostic value of activated Akt expression in oral squamous cell carcinoma. J Clin Pathol 2005;58:1199-205.  Back to cited text no. 1
    
2.
Malone JP, Stephens JA, Grecula JC, Rhoades CA, Ghaheri BA, Schuller DE. Disease control, survival and functional outcome after multimodal treatment for advanced – Stage tongue base cancer. J Head Neck 2004;26:561-72.  Back to cited text no. 2
    
3.
Plame CE, Gullane PJ, Gilbert RW. Current treatment options in squamous cell carcinoma of the oral cavity. J Surg Oncol Clin N Am 2004;13:47-70.  Back to cited text no. 3
    
4.
Nicholson KM, Anderson AG. The protein kinase B/Akt signaling pathway in human malignancy. J Cell Signal 2002;14:381-95.  Back to cited text no. 4
    
5.
Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the P13K/AKT pathway for cancer drug discovery. J Nat Rev Drug Discov 2005;4:988-1004.  Back to cited text no. 5
    
6.
Ayala G, Thompson T, Yang G, Frolov A, Li R, Scardino P, et al. High levels of phosphorylated form of Akt-1 in prostate cancer and non-neoplastic prostate tissues are strong predictors of biochemical recurrence. J Clin Cancer Res 2004;10:6572-8.  Back to cited text no. 6
    
7.
Majumder PK, Sellers WR. Akt-regulated pathways in prostate cancer. J Oncogene 2005;24:7465-74.  Back to cited text no. 7
    
8.
David O, Jett H, LeBeau H, Dy G, Hughes J, Friedman M, et al. Phospho- Akt overexpression in non- small cell lung cancer confers significant stage – Independent survival disadvantage. J Clin Cancer Res 2004;10:6865-71.  Back to cited text no. 8
    
9.
Dhawan P, Singh AB, Ellis DL, Richmond A. Constitutive activation of Akt/ protein kinase B in melanoma leads to up-regulation of nuclear factor-kB and tumor progression. J Cancer Res 2002;62:7335-42.  Back to cited text no. 9
    
10.
Liu LX, Liu ZH, Jiang HC, Qi SY, Zhang WH, Zhu AL, et al. Overexpression of Akt-1 gene in hepatocellular carcinoma. Chinese J of Cancer Research 2002;14:161-4.  Back to cited text no. 10
    
11.
Chua BT, Gallego-Ortega D, de Molina AR, Ullrich A, Lacal JC, Downward J. Regulation of Akt (ser473) phosphorylation by Choline kinase in breast carcinoma cells. J Mol Cancer 2009;8:131.  Back to cited text no. 11
    
12.
Kumar A, Purohit R. Cancer associated E17K mutation causes rapid conformational drift in Akt1 Pleckstrin Homology (PH) Domain. J PLoS One 2013;8:64364.  Back to cited text no. 12
    
13.
Roy NK, Monisha J, Padmavathi G, Lalhruaitluanga H, Kumar NS, Singh AK, et al. Isoform – Specific Role of Akt in Oral Squamous Cell Carcinoma. J Biomol 2019;9:253.  Back to cited text no. 13
    
14.
Vivanco I, Sawyers CL. The phosphatidylinositol 3- kinase Akt pathway in human cancer. J Nat Rev Cancer 2002;2:489-501.  Back to cited text no. 14
    
15.
Testa JR, Tsichlis PN. AKT signaling in normal and malignant cells. J Oncogene 2005;24:7391-3.  Back to cited text no. 15
    
16.
Lamaroon A, Krisanaprakomkit S. Overexpression and activation of Akt2 protein in oral squamous cell carcinoma. J Oral Oncol 2009;45:175-9.  Back to cited text no. 16
    
17.
Pontes HA, de Aquino Xavier FC, da Silva TS, Fonseca FP, Paiva HB, Pontes FS, et al. Metallothionein and p- Akt proteins in oral dysplasia and in oral squamous carcinoma: An immunohistochemical study. J Oral Pathol Med 2009;38:644-50.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1]



 

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    Abstract
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    Materials and Me...
   Results
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