Experimental Study Of Nimesulide Treatment Enhances Photodynamic Therapy-mediated Lethal Effect On Human Cholangiocarcinoma Cell Line QBC939 In Vitro

Cholangiocarcinoma is a common neoplasm in the biliary tract. Due to lack of specific symptoms, it is very difficult to diagnose patients with cholangiocarcinoma in the early stage and resect the tumor radically. Along with its sensitivity to chemotherapy and radiotherapy, the prognosis is still very poor. Recent years, the incidence rate and the mortality rate of cholangiocarcinoma are increasing markedly, so it is regarded as crucial to find new effective therapeutic approaches of them.Photodynamic therapy (PDT) is a minimally invasive therapeutic modality approved for treatment of cancer; it involves the systemic administration of a tumor-localizing photosensitizer followed by focal light activation with laser of specific wavelength. This procedure results in the photochemical generation of cytotoxic reactive oxygen species within the target, leading to cell death. As part of ongoing studies on PDT, we confirmed that PDT has a significant lethal effect on human cholangioncarcinoma cells, however, recurrences can occur. Therefore, we should further explore the mechanism of PDT treatment of cancer, and find new ways to improve PDT efficacy.Cyclooxygenase (COX) is rate-limiting enzymes in the metabolism of arachidonic acid (AA) to prostaglandins (PGs); it has two isoforms, COX-1 and COX-2, they have different biological function. COX-1 is expressed constitutively in many human tissues. In contrast, COX-2 is a type of inducible enzyme. Under normal condition, it is not detected, but can be induced by various kinds of cytokine such as tumor necrosis factor (TNF), epidermal growth factor (EGF), interleukin-1 (IL-1) quickly and continuously. COX-2 has been found high expression in colonic, hepatic carcinoma, cholangiocarcinoma and other tumors and closely associated with occurrence and development of them in recent years, while the selective COX-2 inhibitors can significantly inhibit the proliferation of tumor.Research has proved that PDT stimulates the release of prostaglandin E₂ (PGE₂), COX-2 consistently overexpressed after PDT, and these phenomena can be blocked by COX-2 inhibitors. Selective COX-2 inhibitors may increase the therapeutic effect of PDT. In the current study, the effect of selective COX-2 inhibitor nimesulide combined with hematoporphrphyrin derivative (HpD) as photosensitizer of PDT on cultured human cholangiocarcinoma cell line QBC939 in vitro is investigated, we evaluated the lethal effect and possible mechanisms of the combination of nimesulide and PDT on human cholangiocarcinoma, in order to explore new methods of prevention and treatment of cholangiocarcinoma.Objective To observe the lethal effect of COX-2 inhibitor nimesulide enhanced HpD-PDT on human cholangiocarcinoma cell line QBC939 in vitro, study the effect of photodynamic therapy and the combination of nimesulide and photodynamic therapy on caspase-3 activity in human cholangiocarcinoma cells and explore possible mechanism of HpD-PDT and the combination lethal effect on human cholangiocarcinoma cells.Methods The cells were divided into four groups: blank control group, simple HpD-PDT group, simple nimesulide group and combined group (HpD-PDT first, nimesulide after). The cholangiocarcinoma cell line QBC939 cultured in vitro was treated with different concentrations of HpD-PDT and nimesulide, and then MTT assay was applied to measure the OD₅₇₀ value. The lethal effects of the combination of PDT and nimesulide on human cholangiocarcinoma were observed, and then growth curve was described according above ideal parameters, and a correlation analysis of lethal effects between HpD concentration and nimesulide concentration was made. The morphous of cells was observed under the light microscope after treatment at different time. The caspase-3 activity of different time after treatment was detected by spectrophotometry.Results (1) The growth inhibition of QBC939 cell line induced by HpD-PDT, nimesulide or HpD-PDT+nimesulide was enhanced in a time-dependent and dose-dependent manner. (2) Compares to HpD-PDT or nimesulide alone, nimesulide enhanced HpD-PDT by increasing apoptosis obviously. (3) The combination can reduce the dose both HpD and nimesulide as the same inhibition rate of QBC939. (4) When the QBC939 was treated with HpD-PDT, the activity of caspase-3 increased early in a dose-dependent, negative correlation with time. (5) Compares to HpD-PDT or nimesulide alone, the activity of caspase-3 increased even more significantly, the activation time for longer.Conclusions (1) HpD-PDT can activate caspase-3 early; its effect was dose dependent. The results indicate that the activation of caspase-3 may be one of the mechanisms of HpD-PDT lethal effect on human cholangiocarcinoma cells; the effect of a negative correlation with time can also explain that HpD-PDT on human cholangiocarcinoma cells lethal effects of limited and not persistent. (2) HpD-PDT and nimesulide in combination, the cell lethal effect is more apparent, the use of fewer drugs, caspase-3 activation higher, longer activation time. These indicate that nimesulide can enhance the HpD-PDT on human cholangiocarcinoma cells lethal effect, the effect may be depent on that HpD-PDT and nimesulide activated caspase-3 sequentially, and strengthen the activity of caspase-3, lengthen the caspase-3 activation tmie, and the activation of caspase-3 may be the common pathway.