5-Fluorouracil (5-FU) and its prodrug TF have been demonstrated to possess potent anti-tumor activity in vitro. However, their severe side effects, including myelotoxicity, gastrointestinal toxicity (diarrhea and stomatitis) and central nervous system (CNS) disturbance, mean that it is impossible for these drugs to achieve sufficient clinical benefits. TS-1 was designed with the simultaneous aim of enhancing the efficacy of 5-FU while reducing the associated adverse reaction. Specifically, TS-1 includes both CDHP, an inhibitor of 5-FU degradation, to maintain an effective plasma concentration of FT, and Oxo to reduce the level of gastrointestinal toxicity. By reducing gastrointestinal toxicity, which causes great discomfort to patients, it is possible to use TS-1 for an extended period of time. Thus, TS-1 has become the most frequently-used anti-cancer drug for the treatment of cancer patients in Japan .
TS-1 is administered orally for 4 weeks, followed by a 2-week rest. This treatment protocol can be repeated if no serious side effects are apparent. Nonetheless, TS-1 has been reported to cause a variety of adverse reactions, such as anemia, leukopenia, neutropenia, diarrhea, thrombocytopenia, stomatitis, anorexia and proteinuria . In particular, TS-1 suppression of bone marrow is a very serious problem. When a decrease in WBC count is detected in TS-1-treated patients, TS-1 therapy must be immediately discontinued. If the development of TS-1-induced leukopenia can be inhibited, however, the patients can continue to receive therapy. Additionally, the dosage of anticancer agent might even be increased to improve therapeutic gain. This would be a great benefit to patients with gastroenterological cancer.
Our study aimed to solve this problem through three individual steps. First, we investigated whether JTT has a preventive effect on bone marrow suppression induced by TS-1. Second, we attempted to identify candidate biomarkers that can detect myelosuppression before the onset of leukopenia. If we use appropriate precautionary measures such as the strict control of the treatment protocol of TS-1 and/or the prophylactic administration of hematopoietic reagents (e.g. G-CSF), the discontinuation of TS-1 therapy may be avoided or at least delayed. Third, we investigated whether the candidate biomarker could predict whether the patient is a “JTT-responder”.
JTT has been reported to improve the general condition of cancer patients receiving chemotherapy and/or radiation therapy . In mice, it has been suggested that JTT improves the decline in bone marrow function induced by anticancer therapy or radiotherapy and that the effect is mediated, at least partly, by enhancing the proliferation of hematopoietic stem cells. This effect has been demonstrated as an increase in colony-forming units in spleen (CFU-S) and/or granulocyte-macrophage colony-forming cells (CFU-GM) in cisplatin-, mitomycin C-treated or irradiated animals [18, 20], and is mediated by mitogenic activity of oleic acid and linolenic acid contained in JTT. The same mechanism may be involved in the inhibition of TS-1-induced leukopenia/bone marrow suppression demonstrated in the present study; however, further extensive studies are necessary to clarify this point.
It has been long supposed that there are distinct groups of responders and non-responders to each Kampo medicine. Certain Kampo medicines have been reported to produce a dramatic therapeutic effect in “responder” patients, but there are always a number of “non-responder” patients. Many clinical trials using Kampo medicines, including more than 10 multicenter, placebo-controlled, double-blind studies, have demonstrated significant beneficial effects of Kampo medicines [23, 24]. However, these trials also suggest that it is important to distinguish responders from non-responders at an early stage of therapy to achieve the anticipated therapeutic outcome. Thus, in order to maximize the beneficial effects of Kampo medicines in modern medical practice it is crucially important to identify a suitable biomarker to distinguish a “responder” from a “non-responder”.
Although a huge number of research papers have been devoted to identifying biomarkers, few biomarker(s) have been validated using diagnostic criteria. Currently, proteomic technology is one of the most effective methods for identifying biomarkers. SELDI is an MS-based proteomic technique that has been used in the discovery of disease-related biomarkers derived from biological fluids. We have previously reported several biomarker candidates for Kampo medicines using SELDI [15, 16, 25]. Among them, haptoglobin alpha 1 chain may be used to predict the efficacy of the Kampo medicine keishibukuryogan in the treatment of rheumatoid arthritis. The strategy used in the present study employed SELDI to find a predictive marker for TS-1-induced leukopenia. Specifically, we focused on protein peaks whose intensity changed significantly prior to a decrease in WBC count. To generate a shortlist of candidate proteins, we focused on peaks that changed upon treatment with TS-1 but were normalized by co-treatment with JTT. Using this approach, we identified three biomarker candidates, which all increased 3 days after administration of TS-1 although coadministration of JTT reduced these changes. Interestingly, the TS-1-induced up-regulation of these biomarker candidates was only transient, because they showed no significant difference on days 5 and 7 as compared with the negative control. Our results suggest that these candidates may be induced in the early phase of TS-1-mediated myelosuppression. As such, these candidate biomarkers may play an important role in the development of a serious adverse effect of TS-1.
We successfully identified one (m/z 4223.1) of the three candidate biomarkers as the C-terminal fragment of albumin using a combination of SDS-PAGE and LC-MS/MS analysis. It is implausible that this albumin fragment directly mediates the side effects associated with administration of TS-1, such as leukocytopenia and bone marrow suppression. Therefore, we believe that the albumin fragment may be derived from early molecular events during the onset of bone marrow suppression. Indeed, a decrease in albumin is associated with myelosuppression induced by several chemotherapeutic reagents, including TS-1 [26–30]. Recent studies have suggested that the plasminogen fibrinolytic pathway is required for hematopoietic regeneration [31, 32]. Thus, dynamic alteration of the protease-protease inhibitor network might occur in myelosuppression. We intend to investigate whether JTT directly inhibits the protease responsible for producing the albumin fragment of m/z 4223.1 in a future study.
Albumin fragments of various length and amino acid sequence have been identified as biomarker candidates for other diseases [33–35]. Those results suggest that the fragments are generated from circulating albumin by specific or nonspecific proteases activated in various disease states. Albumin is the most abundant protein in blood and has a diverse range of functions, including maintenance of intravascular volume and colloid osmotic pressure, binding and transport of various molecules (including hormones, lipid and drugs), antioxidant and anti-inflammatory actions, and exertion of a stabilizing effect on the endothelium [36–39]. Thus, albumin may act as a “buffer” against stress from disease and/or exposure to certain drugs. Various mediators (e.g., inflammatory, oxidative, fibrolytic, chemotactic), including proteases liberated from injured or disabled organs, might be “buffered” or “neutralized” by inactivation upon binding to, reduction with, and digestion of, circulating albumin. It must be noted that fragments of various major serum proteins, such as haptoglobins , transferrin , fibronectin and apolipoproteins , have also been identified as serum biomarkers in various diseases. Abundant serum proteins might represent a reservoir of “scapegoats” for activated proteases. Identification of the protease responsible for generation of the present peptide will contribute to elucidating the mechanisms underlying both the development of bone marrow suppression by TS-1 and the improvement by JTT.
It should be noted, however, that there are species differences in the amino acid sequence between human and murine albumin. The peptide fragment found in this study is supposed to be generated from the cleavage of mouse albumin at lysine 569, which corresponds to lysine 569 of human albumin. The overall identity and similarity of the amino acid sequence of mouse and human albumin (consisting of 609 and 608 AA, respectively) are high (72.3 and 92.9%, respectively), and those of the sequence of the anterior segment of the cleavage sites (541–569) are also high (86.2 and 93.1%, respectively). It therefore is possible that a similar fragment may be generated in human. On the other hand, the identity and similarity of the posterior sequence (570–609) are lower (55 and 82.5%, respectively). Furthermore, recognition of the cleavage site by proteases has varying degrees of stringency depending on each protease. Therefore validation studies in human patients, as well as clarification of the protease responsible for generation of the fragment, are necessary. We plan to execute these studies in the near future in our laboratory.