Caspase 3/8/9 Apoptotic Pathway Deregulation Analysis in Laryngeal Squamous Cell Carcinoma

Abstract

Objectives

Among the molecules that are implicated in the regulation of apoptosis (programmed cell death), caspases are most significant acting as strong enhancers. The aim of the current study was to co-analyze the caspases 3, 8 and 9 at their protein expression level in a series of laryngeal squamous cell carcinomas (LSCCs).

Materials and methods

Tissue specimens derived from 50 LSCC cases were selected and co-analyzed for determining the caspase 3/8/9 expression. A combination of immunohistochemistry and digital image analysis assays was implemented.

Results

According to the analysis of the immunostained slides, females showed higher median expression levels of caspase 3/9 and lower caspase 8 compared to males. However, none of these differences reached statistical significance. Furthermore, median caspase 3 expression levels decreased from Grade 1 to Grade 3, and similar gradual changes were noted for other markers. However, none of these differences reached statistical significance, indicating that the distributions of the biomarkers did not differ significantly by grade. Interestingly, the median caspase 3 levels strongly decreased with stage and this difference was significant (p = 0.033). No significant differences were found across stages for caspase 8/9 and also regarding the anatomical region of the examined tumors.

Conclusion

Deregulation of caspase 3/8/9 molecules that regulate a crucial cataract of reactions in the apoptotic pathway is a relatively frequent event in LSCCs. Progressive loss of their expression is correlated with an aggressive phenotype in the corresponding malignancies, especially referring to caspase 3. Enhancing caspase 3/8/9 expression – that induces apoptotic rates in malignant tumors including LSCC – is a significant goal and challenge for developing novel targeted therapeutic strategies.

INTRODUCTION

Equilibrium between cell proliferation and death is a critical, necessary and sufficient condition ensuring the homeostasis, stability and normal functionality of the tissues, even from the embryogenesis (1). Concerning the development and progression of malignancies, abnormal cell immortalization – as a result of crucial chromosome and gene imbalances – is correlated with aggressive phenotypes and increased resistance to chemo-targeted therapeutic regimens (2, 3). Genetically programmed cell death is referred as apoptosis. In this phenomenon, intrinsic and extrinsic pathways are involved. Receptors and ligands that binds to them – including the tumor necrosis (TNF) and FAS molecules – mediate the apoptotic signal transduction from the extracellular to intracellular environment, whereas mitochondrial depended molecules – such as BCL family proteins – and caspases act directly in the cytoplasm (4, 5). Biochemically, caspases are cysteine-aspartic proteases and their enzymatic activity affect apoptosis, tissue differentiation and also inflammation pathways (6, 7). Concerning their involvement in apoptosis, they are characterized as initiators (caspases 2/8/9/10) and executioners (caspases 3/6/7). More specifically, after its recruitment and activation by cytochrome C/AFAP-1 complex, caspase 9 induces the caspase 3/7 pathway (8). Additionally, FAS-FAS ligand complex enhances caspase 3/8 protein expression and activity (9). In the current research study, we analyzed and estimated the caspase 3/8/9 protein co-expression levels in a series of laryngeal squamous cell carcinomas (LSCCs).

MATERIALS AND METHODS

Patients and specimens

A pool of 50 formalin-fixed paraffin-embedded archival LSCC bioptic material extracted by extensive or regional surgical operations was obtained for the purposes of the research. The Department of Pathology of "Hippokratteion" Hospital, Athens, Greece, and the corresponding Ethics Committee of National and Kapodistrian University of Athens consented to the use of the previous referred bioptic material for the present study (Reference ID research protocol: 59936/26-06-24), according to the World Medical Association Declaration of Helsinki guidelines (2008, updated 2014) dovetailing with the GDPR EU/2016 guidelines. Concerning the inclusion criteria for the study group, we selected the cases based on a normal stratification regarding the most advanced grade and stage, and also a normal representation of the anatomical regions (glottis, transglottic, supraglottic). The corresponding tissue sections were fixed in a 10% concentration neutral formalin buffer. Initially, the slides were stained by hematoxylin and eosin (H&E) and screened by two independent pathologists for determining the histological diagnosis including the grading and staging of the examined LSCC based on the World Health Organization (WHO) pathology guidelines (10).

Immunohistochemistry assay (IHC)

For the immunohistochemistry assay (IHC) procedure, ready-to-use anti-caspase 3 (rabbit polyclonal, Abcam, UK; concentration 0.002 mg/ML), anti-caspase 8 (mouse monoclonal, AMB14C1, Abcam, UK; concentration 0,5 mg/ML) and anti-caspase 9 (rabbit monoclonal, E23, Abcam, UK; concentration 1.301 mg/ML) were applied in the corresponding cases. The IHC procedure was performed on 4 μm tissue sections for the proteins of the study. Initially, the slides were deparaffinized in xylene solution and then rehydrated by graded ethanol stages. In the next step, the slides were immunostained according to the EN Vision+ (Dako, Glostrup, Denmark) assay using an automated IHC staining system (BOND – MAX, Leica Biosystems, Deer Park, IL, USA). Following the peroxidase blocking, the sections were incubated with the primary antibody for 35 min at room temperature and then incubated with horseradish peroxidise-labelled polymer-HRP LP for 30 min. Additionally, the antigen-antibody reaction was determined by the use of 3-3, diaminobenzidine tetrahydrochloride (DAB) as a chromogen substrate. In the next step, the slides were slightly counterstained with hematoxylin for 30 seconds, dehydrated and finally mounted. Breast carcinoma tissues that express normally the examined markers were considered positive controls. For negative controls, the primary antibodies were omitted in three experimental slides. Diffuse cytoplasmic predominantly and sub-membranous staining pattern was considered acceptable for caspase 3/8/9 protein expression (Figure 1a, b, c).

Digital image analysis (DIA) assay

Caspase 3/8/9 protein expression levels were objectively calculated by measuring the staining intensity values (densitometry estimation) in the immunostained malignant tissue sections. We implemented a DIA-based protocol using a semi-automated computerized system with the Image Pro Plus, Media Cybernetics, Rockville, MD, USA digital image analysis software in five optical fields at magnification of 100x in every immunostained slide. A broad range of continuous grey scale values (0-255) at the RedGreenBlue (RGB) colored spectrum was available for discriminating and detecting different protein expression levels (Figure 2). According to the DIA algorithms, staining intensity values that progressively decrease to 0 represent a progressive over expression of the marker. In contrast, values that increase to 255 represent a progressive loss of its staining intensity.

Statistical analysis

Statistical analysis was based on the statistical package SPSS vr 21.00 (IBM Corporation, Somers, NY, USA). A one-sample Kolmogorov–Smirnov (K–S) test was conducted to examine the normality of the distributions for caspases 3, 8 and 9. The Mann-Whitney U and Kruskal-Wallis tests were conducted to determine whether there was a significant difference regarding caspases' expression levels and the examined clinicopathological characteristics. Spearman's correlation analysis was also performed. Statistical significance was set at p<0.05.

RESULTS

The study sample comprised 50 patients [46 men (92.0%) and four women (8.0%)] diagnosed with laryngeal carcinoma. Participants' ages ranged from 45 to 94 years (M = 65.92, SD = 11.46). Tumors were distributed across supraglottic (n = 24, 48.0%), glottic (n = 10, 20.0%) and transglottic (n = 16, 32.0%) anatomical regions. Regarding histopathological grading, 10.0% (n = 5) of tumors were Grade 1, 52.0% (n = 26) Grade 2 and 38.0% (n = 19) Grade 3. According to clinical stage, 6.0% (n = 3) of patients were classified as stage II, 50.0% (n = 25) stage III and 44.0% (n = 22) stage IV. With respect to tumor characteristics, ulceration was observed in 46.0% (n = 23) of cases and absent in 54.0% (n = 27). During follow-up, 40.0% (n = 20) of participants experienced relapse, whereas 60.0% (n = 30) did not. The mean maximum tumor diameter was 2.53 cm (SD = 2.00, range 0.3–8.0 cm).

Concerning the sex parameter, median values for caspase 3/8/9 were compared between female and male patients (Table 2). Women showed higher median expression levels of caspase 3/9 and lower caspase 8 compared to men. However, none of these differences reached statistical significance. Furthermore, median caspase 3 expression levels decreased from Grade 1 to Grade 3, and similar gradual changes were noted for other markers. However, none of these differences reached statistical significance, indicating that the distributions of biomarkers did not differ significantly by grade. Interestingly, the median caspase 3 levels strongly decreased with stage and this difference was significant (p = 0.033). No significant differences were found across stages for caspase 8/9. Additionally, no statistically significant differences among the three anatomical groups (glottic/transglottic/supraglottic) for any of the biomarkers were assessed. All the results and statistical correlations are described in Tables 1-4.

FIGURE 1.

Caspase different expression levels: a) caspase 3 low expression; b) caspase 8 strong expression; and c) caspase 9 loss of expression. Note the diffuse cytoplasmic pattern of their expression (original magnification 100x, DAB chromogen, light to dark brown staining)

FIGURE 2.

Digital image analysis (DIA) procedure for calculating caspase protein expression staining intensity levels. Microscopic snapshot digitized images are the substrate for the detection of different protein expression levels visualized as reddish and green areas. Values range from 0 to 255 in a RGB-based continuous grey scale spectrum. According to the DIA algorithms, immunostaining intensity values decreasing to 0 represent a progressive over expression of the marker, whereas values increasing to 255 show a progressive loss of its staining intensity (caspase 3 expression, original magnification 100x, DAB chromogen, light to dark brown staining).

TABLE 1.

Expression levels and Mann–Whitney U tests for caspases vs sex

TABLE 2.

Expression levels and Kruskal–Wallis tests vs grade

TABLE 3.

Expression levels and Kruskal–Wallis tests vs stage

TABLE 4.

Expression levels and Kruskal–Wallis tests vs anatomical regions

DISCUSSION

Deregulation of caspases' mediated apoptotic pathway is detected in a variety of solid malignancies, including LSCC. In the current research study, we explored the impact of caspase 3/8/9 protein expression on the clinic-pathological features of a LSCC series. Concerning the limitations of the present study, the number of examined patients (n=50), imbalance between the genders' number (men/women) and also the lack of stage I cases were the most obvious, but not critical. We observed a progressive loss of expression in significant subgroups of the enrolled patients, but the most statistical significance was succeeded correlating the caspase 3 overall expression with the stage of the tumors. In fact, the loss of marker's expression has negatively affected the biological behavior of the examined malignancies (advanced stage). Additionally, although not statistically significant, progressive loss of caspase 3/8/9 was obvious in low grade and glottic-based as anatomical region tumors. Focused on the role of pro- and active caspase 3, another study group implementing IHC in LSCC tissues revealed loss of its expression influencing grade, especially in malignancies derived from the glottis region (11). Interestingly, another study group concluded that Bcl-2/Bak complex, and not the caspase 3/8, was critical for the positive human papillomavirus (HPV) depended LSCC cases (12). Regarding the molecular substrate of caspase 3 deregulation, a significant genetic analysis in a series of solid and non-solid malignancies including LSCC reported a subgroup of them that harbored specific missense and silent mutations leading to its loss of functionality and normal expression (13). In conjunction to mutations, there are robust data regarding the role of specific single nucleotide polymorphisms (SNPs) in CASP3 and CASP9 genes in the inherited cases of head and neck squamous cell carcinomas (HNSCC), including LSCCs. A study group concluded that CASP3 c.-1191A>G and CASP9 c.-1339A>G SNPs were implicated in as significant risk factors in the development and progression of these malignancies (14). Furthermore, specific micro-RNA markers (miRs) seem to modify the caspase 3 expression levels and activity. MiR-199-a-5p, miR-4500, miR-101, miR-4497, miR-384, miR-15a, miR-340, miR-145 and miR-24 up or downregulate caspase functionality in these malignancies and are challenging molecules for specific chemo-targeted therapies (15-23).

Concerning caspase 8 and 9 in LSCCs, there is strong evidence that their loss of expression negatively affects chemosensitization of cisplatin in the corresponding patients, especially in HPV positive cases (24-26). In order to enhance caspase 3/8/9 expression and activity, many studies have explored the impact of a variety of agents on LSCC cell series. In one of them, the researchers reported elevated protein rates of them leading to apoptosis acceleration under the influence of a recombinant adenovirus encoding H-ras ribozyme (27). Similarly, a plant steroid named diosgenin has been analyzed for its role in LSCC and also in specific high-malignancy tumors such as laryngosarcoma (28). Two study groups showed that the agent positively affected the p53/caspase-mediated apoptotic cataract of reactions. Furthermore, deglycosylated bleomycin, a glycopeptide antibiotic, induces apoptosis by a different pathway compared to conventional bleomycin caspase-mediated one (29, 30). Additionally, oridonin, an active factor of Rabdosia rubescens plant, induces apoptosis by a caspase 9 independent pathway (31). Interestingly, another plant agent, epigallocatechin-3-gallate, a polyphenol detected in green tea, has been found to increase caspase 3 activity (32). In conjunction, a proteasome inhibitor, bortezomib, and also a plant polysaccharide from Boschniakia rossica is also implicated in caspase 3 protein enhancement (33, 34). Concerning the diagnosis and monitoring of LSCCs, there are strong data that specific imaging modalities, including laryngeal ultrasonography combined with computerized tomography and endoscopy, provide a complete diagnostic algorithm for the management of the corresponding patients (35, 36).

CONCLUSIONS

Deregulation of the proliferation/apoptotic cell death equilibrium is a significant abnormality that characterizes malignant transformation of normal tissues. Concerning LSCC, caspase 3/8/9 loss of expression seem to correlate with aggressive phenotypes (advanced stage, progressive tissue dedifferentiation). Novel agents that target the caspase-mediated apoptotic pathway by enhancing their expression and activity seems to be a very challenging field in molecular oncology due to its impact on preventing cancerous enlargement and expansion in LSCCs and also generally in HNSCCs (37).

Conflicts of interest

None declared.

Financial support

None declared.