Pancreatic cancer is a malignancy with a particularly poor prognosis (1). Pancreatic ductal adenocarcinoma (PDAC) accounts for 85% of all cases of pancreatic cancer, and is the most frequently observed type of malignancy derived primarily from pancreatic cells (2). It is equally common in men and women, and mainly involves the head of the pancreas (2). In the majority of cases, the tumor already infiltrates the adjacent structures at the time of diagnosis, including the common bile duct, duodenum and ampulla (1). Lesions are typically irreversible, and the 5-year survival rate is 1–2% (1). The low percentage of survival in pancreatic cancer cases results principally from the weak response of cancer cells to treatment, which is mainly due to their high level of resistance to apoptotic stimuli (1).

Apoptosis, also known as programmed cell death, is involved in the physiological maintenance of normal cell homeostasis, as well as having a role in pathological lesions that accompany the course of various diseases (3,4). The caspase activation mechanism is a major signaling pathway leading to apoptosis (1). Caspases constitute a group of cysteine proteases that occur in an inactive and an activated form (4). They become activated via the action of proteases, including their mutual activation. Caspases may be divided into effectors (caspase-3, −6 and −7) and initiators (caspase-8 and −9) (4). Two mechanisms that lead to the activation of the caspase initiators have been proposed: i) A mechanism by which the inactive form of caspase-8 is activated upon binding of its death receptor to a protein containing a death domain, and whereby the activated protein directly stimulates effector caspases such as caspase-3 (1,3,4); ii) and an alternative B-cell lymphoma 2 (Bcl-2) family-dependent mechanism that involves the cytosol and is associated with the permeabilization of the mitochondrial outer membrane and subsequent release of cytochrome c, apoptotic protease activating factor 1 and additional apoptotic agents. This leads to the formation of apoptosomes that activate caspase-9, which subsequently triggers a cascade of effector caspases (caspase-3 and −7) that induce the process of apoptosis (5). Disorders in these signaling pathways are associated with cancer cell immortality (5).

Therefore, the aim of the present study was the immunohistochemical assessment of the expression of caspase-8 and the inactive and cleaved (active) forms of caspase-3 in the tissues of patients exhibiting PDAC, and the subsequent analysis of the association of caspase expression in these patients with specific clinicopathological parameters.

Normal pancreatic cells, identical to other body cells, undergo the physiological process of controlled cell death, known as apoptosis (6). This mechanism conditions normal cell function and enables maintenance of homeostasis (7). The process of apoptosis involves the Bcl-2 family-dependent signaling pathway and the caspase-regulated signaling pathway (7). It has been previously demonstrated that the action of these proteins is disrupted in pancreatic cells affected by various pathologies (6–8).

In normal pancreatic tissue, positive expression of caspase-8 has been identified in the cytoplasm of glandular cells, single islets of Langerhans and ductal epithelial cells (8). The results of the present study are consistent with the observations reported by Virkajärvi et al (9), who described the expression of caspase-8 in all types of structural cells in the pancreas, as well as strong expression of caspase-8 in inflamed areas surrounding the islets of Langerhans. Analysis of caspase-8 expression in cancer cells confirmed its presence in 74% of patients exhibiting pancreatic adenocarcinoma, and strong expression was observed in a small percentage of cases (9). In the present study, ~93% of cases exhibiting PDAC demonstrated positive expression of caspase-8. Similar results have been reported in other gastrointestinal tumors, including gastric or colorectal cancer. Sträter et al (10) reported moderate or strong caspase-8 expression in the cancer cells of a small number of colon tumors. Shintani et al (11) observed immunopositive cells for caspase-8 in ~50% of gastric and colorectal adenocarcinoma cases. Previous studies have indicated that this may be due to disturbances in apoptosis induction within the Fas/Fas ligand system, or alternative death ligands, including tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand, which may cause dysfunction in initiator caspases such as caspase-8 and inhibition of apoptosis during its initial stage (12,13).

The present study additionally focused on the expression of caspase-3 in its precursor and activated forms in normal pancreatic and cancer cells. A positive pro-caspase-3 color reaction was identified by immunohistochemistry in the cytoplasm of normal and cancerous cells in the majority of cases analyzed. Thus, it was speculated that PDAC cells may retain the potential to produce the precursor form of caspase-3. To the best of our knowledge, the present study is the first to assess the expression of the two forms of caspase-3 separately. Virkajärvi et al (9) evaluated the expression of the precursor and active forms of caspase-3 using one antibody. Expression of caspase-3 was observed in 80% of patients exhibiting pancreatic adenocarcinoma. However, no correlation was identified between apoptotic index and caspase-3 expression, which may indicate the expression of the inactive form of caspase-3 in the cancer cells of these patients (9). Satoh et al (14) observed high expression levels of caspase-3 in PDAC cells in the majority of cases evaluated, and suggested that the precursor form of caspase-3 may have contributed to the color observed upon immunohistochemical staining (14). In that study, caspase-3 expression was assessed in the cytoplasm and nuclei of PDAC cells, and a correlation was observed between cytoplasmic expression of caspase-3 and malignant tumor stage. In addition, an association was observed between nuclear reaction and benign tumor phenotype (14).

The present study particularly focused on the expression of activated caspase-3. More frequent expression of cleaved caspase-3 was observed in normal pancreatic cells, compared with pancreatic duct cell tumors. In the present study, a statistically significant correlation was identified between caspase-8 and active caspase-3 expression in the tumor mass. A decrease in the expression levels of caspase-8 or −3 caused a reduction in the expression levels of the other, which confirmed the involvement of these two proteins in the common apoptotic signaling pathway of PDAC cells. Meggiato et al (15) noted an increased percentage of positive expression of cleaved caspase-3 in the cytoplasm of tumor cells, compared with the cytoplasm of epithelial cells that lined the pancreatic ducts. It was suggested that the strong expression of active caspase-3 in pancreatic tumor cells, as opposed to normal ductal cells, indicated stimulation of the apoptosis-inducing mechanisms (15). Similarly, Luo et al (16) reported an increase in the expression of cleaved caspase-3 in PDAC tissue (43.9%), compared with normal tissue (3.5%). Furthermore, statistically significant correlations were noted between caspase-3 expression and high tumor grade, tumor location in the head of the pancreas, cyclin D1 gene amplification and presence of polysomy of chromosome 11 (16). A detailed analysis of these patients additionally revealed an association between positive expression of active caspase-3 and shorter survival time in patients exhibiting PDAC, and the expression of active caspase-3 was identified as an independent risk factor (16). In contrast to PDAC, high expression levels of cleaved caspase-3 have been associated with positive prognosis in patients diagnosed with colorectal cancer (17). The previously mentioned reports are not consistent with the results of the present study, which may be due to the small size of the current patient cohort. However, reduced expression of effector cleaved caspase-3 may be associated with a lack of adequate agents to trigger the activation of the protein, including caspase-8, or may result from disorders in the Bcl-2 family-dependent signaling pathway of apoptosis, which conditions the levels of cytochrome c present in the cytosol (18). In contrast with normal cells, pancreatic cancer cells demonstrate a reduced ability to activate caspases via cytochrome c (18). It has been suggested that cancer cells may require increased levels of cytochrome c in order to activate the apoptotic signaling pathway of caspases (1). The reduced capacity of caspase-3 stimulation in PDAC cells may induce their resistance to apoptosis, as well as determining their longevity and invasiveness (18). In consequence, PDAC belongs to a group of tumors characterized by short survival times and poor prognoses (18).

Despite the small size of the patient cohort in the current study, the findings of the present study suggest that pancreatic cancer may involve disturbances to the initiation of cancer cell apoptosis via a decrease in the expression of caspase-8, which may cause dysregulation in the activation of effector caspase-3. Therefore, assessment of the expression of caspase-8 and cleaved caspase-3 in the tissues of patients exhibiting PDAC may aid the determination of the degree of apoptotic activity in pancreatic cancer cells. This may be utilized in the future to develop targeted therapies for the treatment of PDA using activators of the proteins investigated in the present study.