In this study, CY produced myelosuppression manifested as leukopenia (Fig. ). It also significantly reduced the blood supply and proliferating cell number in both the gastric and duodenal mucosae. Subcutaneous administration of AP at the dose of 5 mg/kg daily significantly promoted the recovery rate of immune system in mice in a 14-day treatment (Fig. ). It also significantly increased the number of blood vessel and PCNA-positive cell in both the gastric (Fig. A and B respectively) and duodenal tissues (Fig. A and B respectively). Dose-dependent effects were observed in general. Western blot analysis implicated the reduction by CY and normalization by AP of blood vessel count was VEGF dependent in gastric tissue (Fig. A). On the other hand, the decrease in proliferating cell number in gastric mucosa by CY administration was found to be c-Myc and ODC-dependent (Fig B and C respectively).
CY is the non-cytostatic drug that acts non-specifically on both tumor cells and normal healthy cells with high proliferating capacity like immune cells and GI tissues. It exerts its cytotoxic effect via transfer of its alkyl groups to DNA, leading to cell cycle arrest and apoptosis. The major site of alkylation within the DNA is the N7 position of guanine. Alkylation of guanine results in depurination by excision of guanine residues, causing DNA strand breakage through scission of the sugar-phosphate backbone 3
. Patients under chemotherapeutic regimen are often subject to leukopenia which greatly increases the chance of opportunistic infections. As a result drug-free period is routinely introduced during regimen to prevent any or even fetal infections. In this model, we showed that 7 days were needed for the CY-treated mice to restore their immunity to normal level as indicated by WBC count. Such a long drug-free period is indeed undesirable because it allows the restoration of tumor tissue into active proliferating stage 2
, as vascular endothelial cells can proliferate again and tumor will be nourished by supply of nutrients and oxygen. However, in mice treated with AP 5mg/kg, it was observed that a 5-day drug free period was enough to fully restore their normal immune response (Fig. ). The present findings suggest that AP has immunostimulatory effect which can accelerate the recovery from leukopenia induced by CY and thereby shortens the drug-free period to allow a more frequent administration of anticancer drug e.g. CY so as to increase the efficacy of chemotherapy. In previous studies, AP has been shown to activate polyclonal B cells 20
, induce interferon 21
and also activate helper T cells 22
. Lymphocyte proliferation assays, e.g. mitogen-mediated lymphocyte proliferation test and mixed lymphocyte culture also proved that AP could increase the rate of lymphocyte proliferation in vitro 23
. Oral administration of You-Gui-Wan, a classical prescription of TCM containing AP, was shown to protect mice against hydrocoticoid-induced inhibition of IFN-γ, IL-2, IL-4 and IL-10 transcription 24
. In addition, vitamin B12, folinic acid and biotin identified in AP may also contribute to stimulated hematopoiesis 25
. All of these results are consistent with the present findings that AP is a tonic to hematopoietic system.
Concerning angiogenesis, it is believed that one of the anti-tumor mechanisms of CY is through the suppression on endothelial cell growth in tumor bed 26
. In addition, the down-regulation of VEGF by CY has been shown to be due to p53 activation 27
. This would decrease the blood supply to cancer cells so as to reduce nutrients and oxygen to support the growth and differentiation of tumor. However, it would also adversely affect the repairing capacity and the defensive mechanism of the GI mucosae that have been damaged during chemotherapy. In this regard, AP was shown to be beneficial to cancer patients because it normalized blood vessel number, which could probably supply more nutrients and oxygen to gastric and duodenal mucosae. This also promoted the defensive mechanism and also the repairing capacity of the GI system which has been damaged by CY administration. However, it should be noted that AP might also have a similar effect on the vascular endothelial cells in tumor bed, of which the proliferation and differentiation would be enhanced with a good supply of blood flow. Whether or not AP could affect blood vessels in tumors remains unknown. In this regard, further studies are needed to clarify this phenomenon.
CY exerts its cytotoxicity by cross-linking DNA strands and activation of p53-dependent growth arrest and apoptosis 28
. It was therefore not surprising that CY administration resulted in a decrease in the proliferating cell number in both the gastric and duodenal tissues. Indeed the decrease in cell proliferation in gastric tissue was supported by the down-regulation of c-Myc and ODC protein in the Western Blot analysis (Fig. B and C). It has been reported that p53 activation suppresses the transcription of c-Myc, an immediate early gene related to cell proliferation in which mitogenic stimulation leads to increased expression and drives the cell cycle from G0
to G1 27
. Furthermore, c-Myc is known to induce the transcriptional activity of ODC gene 29
, which is involved in polyamine synthesis. It is likely that the cell cycle-arresting action of CY in the stomach in this study was due to the activation of p53 and therefore leading to the suppression of the c-Myc/ODC pathway. All these findings could explain the mechanism of CY on repression of cell proliferation in the gastroduodenal mucosae. In general, dose-dependent effect on the reversal action of AP on this suppressive effect could be observed. Although our previous study has shown that ODC was involved in AP-induced normal gastric epithelial cell proliferation 10
, the increased proliferating rate in this study was independent of the c-Myc, ODC or EGF as indicated by the corresponding protein levels in Western Blot analysis. However this could be partly contributed to the effect of AP on angiogenesis that increased the blood supply to tissues for growth and repairment.
To conclude, the above findings not only provide a fundamental insight into the mechanism of CY-induced systemic cytotoxicity, particularly in the gastrointestinal system, but also propose a role for polysaccharides from Angelica sinensis as a cytoprotective agent to spare the hemopoietic and gastrointestinal toxicities of CY. Whether or not the present study can be translated into practical benefits, warrants further investigation.