Gallbladder carcinoma (GBC) is a gastrointestinal and often fatal malignancy, ranked fifth overall and the third most common biliary tract malignancy [1,2]. Correct diagnosis at a time which would enable a cure for GBC remains problematic due to its asymptomatic nature during the initial stages [3,4]. In general, it is one of the most aggressive biliary cancers with a very short survival duration [5]. The higher incidence of GBC in females has been attributed in part to hormonal factors, including estrogen and progesterone receptor expression and their effects on GBC prognosis [6,7]. Many other reports have also referred to the effects of hormone receptor status in GBC [8-14]. More recently, a significant correlation between hormone receptor polymorphism and gallbladder (GB) carcinogenesis has been reported [15]. Androgen receptor (AR) CAG repeats have been directly correlated with gallstone formation and GB carcinogenesis [16]. These studies have shown that hormone receptors are important for GB carcinogenesis, with whole GB resection being the preferred curative treatment [17]. Adjuvant chemotherapy and molecular-targeted therapy are potential therapeutic options for advanced-stage cancer. In GBC, due to its typically late diagnosis and presentation, chemotherapy is administered before surgical removal of the GB tumor. It is therefore vital to develop new drugs for GBC, with a view to combating the cancer at the molecular level.

There are 14 caspases in mammals [18,19]. The initiator caspases (e.g., caspases 8, 9, and 10) are activated by apoptotic stimulation, which activates further effector caspases [20]. Additionally, many studies have shown that active caspase-3 is needed to induce apoptosis in response to chemotherapeutic treatments using regimens such as taxanes, 5-fluorouracil, and doxorubicin [21-24]. Caspase-3 is synthesized as a 32-kDa proenzyme, which is cleaved into 12- and 17-kDa subunits. Two 12-kDa and two 17-kDa subunits are reassociated to form the functionally active caspase-3 enzyme [25]. After activation, the effector caspases, which include caspase-3, -6, and -7, initiate the cleavage of many key cellular proteins, including poly (ADP-ribose) polymerase, inhibitors of caspase-activated DNase, gelsolin, 4-GDI, α- and β-fodrin, and epidermal growth factor receptors [19,26]. These cellular proteins cause blebbing of the membrane, condensation of chromatin and DNA fragmentation. Based on the central role played by caspase-3 in apoptosis, we assessed the expression levels of procaspase-3 and activated caspase-3 in advanced GB tumor tissue samples to determine the apoptotic status of estrogen receptor (ER)-, AR-, and HER/neu-positive GBC tissues.

Chemoresistance is a principal cause of treatment failure in many cancers. To date, our understanding or resistance to cancer drugs remains limited. In order to identify and study hormone receptor expression and apoptosis rates, we have assayed procaspase-3 and active caspase-3 proteins in GB carcinoma. We show here that a triple negative hormone receptor GBC demonstrates less active apoptosis, whereas a triple positive hormone receptor expressing GBC demonstrates more active apoptosis. This indicates that expressing GBC hormone receptors may be more sensitive to hormone-dependent chemotherapeutic agents. As hormone receptors play a role in cancer initiation, it is important to study the status of apoptosis in hormones involved in GBC. Endocrine therapy using tamoxifen, a selective ER modulator, and aromatase inhibitors, which ablate peripheral estrogen synthesis, have been shown to substantially improve disease-free survival [28]. We first assessed procaspase-3 and active caspase-3 expression in triple negative and triple positive GBC so as to identify a correlation between hormone receptor expression and the rate of apoptosis. This information is clinically important because caspase-3 activation is necessary for initiation of apoptosis and regulation of processes such as membrane blebbing and internucleosomal DNA fragmentation. Caspase-3 expression in the MCF-7 human breast cancer cell line (caspase-3 deficient) revives the apoptotic response [28-30]. Caspase-3 is also involved in breast cancer apoptosis when cells are exposed to anthracyclines [23,31,32] and cisplatin [33-36]. Apoptosis failure is a crucial step in the initiation and progression of cancer. High apoptotic rates have been reported in invasive cancers compare to the paired normal rates [37-40]. We found no significant difference between procaspase-3 and active caspase-3 expression in triple negative GBC. However, we found a significantly higher active caspase-3 concentration in triple positive GBC, which exhibits high apoptotic activity. Our results show that triple negative GBC is more resistant to apoptosis than triple positive GBC. This information can be used clinically to select chemotherapeutic drugs in a hormone-dependent or independent manner. For therapeutic chemotherapy, triple positive GBC is more sensitive to apoptosis-inducing agents than is triple negative GBC.

To investigate the effects of hormones in more detail, we selected ER-, AR-, and HER/neu-positive GBC and assayed procaspase-3 and active caspase-3 by Western blotting. ER-positive GBC showed significant expression of active caspase-3, indicating that the ER-positive cases were more susceptible to apoptosis and had a high rate of internal apoptosis. Supplementary induction of apoptosis by chemotherapeutic agents may thus accelerate its effect. HER/neu-positive GBC also exhibited high active caspase-3 expression. However, the AR-positive GBC results were interesting, in terms of the increased levels of active procaspase-3 and less active caspase-3 expression, compared to ER- and HER/neu-positive GBC. Overall, the level of caspase-3 protein was lowest among other hormone-responsive GBC, with AR being the least sensitive to apoptosis.

In conclusion, our findings reflect the status of apoptosis in GBC, and indicated that ER- and HER/neu-positive GBC tumors are highly sensitive to apoptosis and may respond better to chemotherapeutic drugs such as tamoxifen, whereas AR-positive GBC is more resistant to apoptosis and hence requires more aggressive treatment than ER- and HER/neu-positive GBC.

1. HER and estrogen receptor (ER) positive gallbladder tumors have more sensitivity for apoptosis compare to androgen receptor (AR) positive gallbladder tumors.

2. HER and ER positive gallbladder tumor may give better chemotherapy response in comparison to AR positive gallbladder tumor with apoptotic inducing drugs.

3. AR positive gallbladder cancer may require more aggressive treatment compare to HER and ER positive gallbladder cancer.