The Expression and Prognosis of Caspase-9 in Hepatocellular Carcinoma

Objective: We aim to investigate the expression of Caspase-9 and its prognostic significance in hepatocellular carcinoma (HCC). Methods: The expression of Caspase-9 in 108 specimens of HBV-related HCC, 30 cases of cirrhosis tissue and 20 cases of normal liver tissue was examined by immunohistochemical staining. And we explored the relationship of Caspase-9 with clinicopathological parameters, 5-year overall survival (OS) and disease-free survival (DFS) in HCC patients. Results: Caspase-9 expression in HCC was significantly higher than that in normal liver tissue (χ = 4.349, P = 0.037). The expression of Caspase-9 was not different both between normal liver tissue and cirrhosis, cirrhosis and HCC (χ = 0.347, P = 0.556; χ = 2.713, P = 0.100). Caspase-9 expression in HCC was correlated with Edmondson grade and satellite nodule (P = 0.001 and P = 0.005, respectively). Survival analysis showed that Caspase-9 expression was associated with OS and DFS (P = 0.001 and P = 0.006, respectively). Conclusion: The expression of Caspase-9 correlated with the carcinogenesis and progression of HCC. Assessment the expression of Caspase-9 may predict the recurrence and survival of HCC patients.


Introduction
Hepatocellular carcinoma is one of the common malignant tumors threatening the health of humans [1].
Apoptosis plays an important role in the pathogenesis and progression of tumors [2]. The activation of cysteinyl aspartate specific proteinase (Caspase) is the core step to regulate apoptosis [3]. There are two pathways of apoptosis [4]. One is the extrinsic pathway triggered by the death receptor and the ligand system. The other one is known as intrinsic pathway which is mediated by mitochondria and cytochrome c (Cyto-C). Cyto-C is released by mitochondria. It can combine Apaf-1 and ATP to activate Caspase-9. Caspase-9 can then invoke the activation of the downstream effector Caspase-3 and Caspase-7 to induce apoptosis. Caspase-9 is the foremost initiator in the process of intrinsic apoptosis [5,6]. The abnormal expression of Caspase-9 and its association with pathological parameters have been reported in many tumors, such as breast carcinoma [7] [13,14].
However, the association of Caspase-9 expression and the prognosis of HCC patients has not been reported. In the present study, we investigated the expression of Caspase-9 and its association with clinical parameters and prognosis in HCC.

Patients and samples
We selected 108 HCC specimens (11 women  None of these patients has undergone any pre-operative therapy. The study was approved by the local ethics committee.
After deparaffinization and rehydration, antigen retrieval was conducted by boiling the slides for 2 min in a pressure cooker filled with 10 mM citric buffer (PH=6.0, Maxin-bio, Fuzhou, China). After cooling at room temperature, the slide were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Subsequently, the slides were incubated in rabbit anti-Caspase-9 polyclonal antibody (Boster, Wuhan, China) at a dilution of 1:150 at 37.0°C for 60 min. The slides were then incubated in HRP-Ploymer anti-Rabbit antibody (Maxin-bio, Fuzhou, China, KIT-5005) for 10 min at 37.0°C. The slides were then dyed with diaminobezidine tetrahydrochloride (DAB, Maxin-bio, Fuzhou, China). At last, the slides were counterstained with hematoxylin. Between each step, we used PBS to wash the slides for 3 times. We chose PBS to replace the primary antibody as a negative control.
Immunohistochemical assessment was performed by two investigators respectively without any knowledge of the clinicopathological details of the patients. For each slide, we chose five fields at X400 magnification randomly. We used a scoring system based on staining intensity and percentage of positive part. The staining intensity was scored as "0" (no staining), "1" (weakly stained), "2"(moderately stained) and "3" (strongly stained). The percentage of positive tumor cells was scored as "0" (< 5%), "1" (5 -25%), "2" (26 -50%), "3" (51 -75%) and "4" (> 75%). The final score was calculated by multiplying the staining intensity score by the score of the percentage positive part. Caspase-9 expression was defined: negative when score less than 8 or positive when score equal or more than 8. Kaplan-Meier survival analysis was used to analyze the 5-year OS and DFS. The log-rank test was performed to compare the differences. P<0.05 was considered statistically significant.

Expression of Caspase-9 and prognosis
All of the 108 HCC patients were followed up for 60 months after the operation. The survival analysis revealed that the expression of Caspase-9 was associated with OS and DFS (P = 0.001 and P = 0.006, respectively) ( Table 3). And patients with positive Caspase-9 expression had favorable OS and DFS than those with negative Caspase-9 expression ( Figure 2).

Discussion
The carcinogenesis of tumors is closely related to the imbalance of cell proliferation and cell death [15].
Apoptosis is one of the mechanism of cell death by which unwanted, senescent and damaged cells can be eliminated to achieve the homeostasis of organs.