Myelosuppression is one of the common side effects of chemotherapy, characterized by depletion of cells within the bone marrow[40, 41]. In general, myelosuppression is primarily attributable to the direct cytotoxicity to bone marrow cells, inhibition of bone marrow precursor or progenitor cell proliferation, the reduction in HSC reserves and impairment in HSC self-renewal. Notably, because of the reduction of HM cellularity in varying degrees, the damaged-hematopoietic microenvironment may result in diminished or delayed hematopoiesis function, immune-related disorders, as well as long-term damage to the bone marrow recovery[42, 43]. It has been shown that chemotherapeutic treatment damage the hematopoietic microenvironment in vitro and vivo[5, 44, 6, 7, 9, 10]. As chemotherapy disrupts the steady-state function of hematopoietic and stromal cell, disruptions over time may cause severe bone marrow toxicity and the failure of cancer treatment. To ensure this does not occur, finding appropriate agents to promote the recovery process following discontinuation of chemotherapy and to lessen the bone marrow damage has a profound significance.
Since it was first synthesized in 1957, 5-FU has remained one of the most widely used chemotherapeutic agents with broad-spectrum activity against many solid tumors[45]. 5-FU exerts its anticancer effects through inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA, leading to cytotoxicity and cell death[12]. Recent studies have indicated that 5-FU suppressed the proliferation of HSCs and induced the myelosuppression of mice by down-regulating PI3K-AKT signaling pathway[14, 46]. However, the definite mechanism for 5-FU caused myelosuppression remains unclear. Focused on bone marrow stromal cells, we provided the evidence that 5-FU inhibited stromal cell growth and induced apoptosis, which was related to downregulation of Wnt/β-catenin signaling, also up-regulation of FoxO1 concomitant with an increase of cellular oxidative stress. Furthermore, the current work revealed that anti-oxidative property and role in Wnt signaling regulation might be the key mechanisms of ASP to prevent against 5-FU-induced stromal damage.
Stem cells display the defining capacity to self-renew, and their fate are primarily dictated by extrinsic, short-range signals, which typically emanated from the stem cell niche[47]. The non-hematopoietic cells in hematopoietic microenvironment have a functional role in regulating hematopoiesis and the signaling pathways that regulate HM may be necessary for the development of functional niches that regulate hematopoietic stem cells and their progenitors[48, 49]. The Wnt signaling pathway exerts a variety of effects on target cell developmental processes, including cell proliferation, apoptosis, and differentiation. The canonical Wnt pathway affects cellular functions by accumulating of β-catenin in the cytoplasm and eventually translocating into the nucleus. Within the nucleus, β-catenin binds to T cell factor (TCF) family/lymphoid enhancer factor (LEF) and regulates cell proliferation through Wnt downstream target genes[16, 50, 51]. It was reported that Wnt/β-catenin signaling regulates HSCs function in dosage-dependent manner[52–55]. Various degrees of activation of the pathway may cause different outcomes, leading to either enhanced repopulation capacity or exhaustion of the HSCs. A mild increase in Wnt signaling enhanced HSC function[18, 56]. However, a high Wnt level in HSCs eventually leads to stem cell exhaustion and impairment of reconstitution in irradiated recipients[57–59]. Most importantly, Wnt signaling regulates HSC reconstruction in a stromal-dependent manner. It was found that when hematopoietic cells were co-cultured with BMSCs supplemented with Wnt3a conditioned medium, the cellularity of Lin−Sca-1+c-kit+ hematopoietic stem cells were increased, and the hematopoietic transplantation and reconstruction capability were enhanced[60, 20]. Hence, in the current study we focused on the Wnt signaling regulation on BMSCs following chemotherapy. It was found that 5-FU induced a decrease in cytoplasmic expression of total β-catenin, p-GSK-3β, and CyclinD1, meanwhile weakened nuclear expression of β-catenin, LEF-1, and C-myc proteins, causing HS-5 cells proliferation inhibition. The results herein are in line with the other data related to the relationship between canonical Wnt signaling and cell proliferation, which has confirmed that Wnt/β-catenin signaling positively stimulates cell growth via cell cycle regulation[61, 62].
Reactive oxygen species (ROS) are free radicals and active metabolites of oxygen containing unpaired electrons, which take a significant role in cell signal transduction and regulation[63]. Chemical agents, as well as irradiation can cause a persistent ROS production. This accumulation of ROS may lead to excessive oxidative stress and DNA damage such as DSBs (double-strand breaks), which is considered to be the main potential mechanisms causing cellular damage[64, 65]. Previous study in our group has demonstrated that 5-FU weakened the antioxidant capacity of HS-5 cells and caused high sensitivity of cells to ROS, thus HS-5 cells underwent DSB which eventually resulted in either apoptosis or senescence[15]. Oxidative stress is also related to cell cycle arrest. DSBs initiate DNA damage response through sequential stimulation of ATM, Chk2, and p53[66]. Activation of p53 and its downstream p21 may induce the cell cycle arrest. Meanwhile ROS can activate p38 MAPK pathway[67]. Activation of p53 and p38 pathways can converge at p16 and augment of p16 expression may also lead to permanent cell cycle arrest[68, 69]. Interestingly, it is reported that β-catenin may be critical for antagonizing oxidative stress. Exposing β-catenin knock- down mice to chemotherapeutic agent or radiation caused a decreased expression of the hydrogen peroxide (H2O2) detoxifying enzyme catalase and led to the accumulation of ROS and superoxide (O2¯) free radicals in cells and an inability to repair DNA damage[70]. On the opposite, effector molecules generated from oxidative DNA damage may also down-regulate the Wnt pathway by inhibiting transcriptional activity or participating in post-translational modifications to enhance ubiquitination degradation[71]. These evidences above hint that Wnt signaling also closely corelated with oxidative stress. Therefore, in the current study, increased oxidative stress may be one of the reasons for down-regulation of wnt signaling induced by 5-FU treatment. Whereas decrease in β-catenin protein accompanying reduction of antioxidase SOD and CAT induced by 5-FU treatment may be another mechanism of cell proliferation inhibition.
Forkhead box O (FOXO) family are transcription factors, which promote cell survival by regulating the cell cycle, apoptosis and the response to oxidative stress[23]. The accumulation of ROS may interrupt 14-3-3 combine to FoxO via JNK (c-Jun N terminal kinase), permit FoxO entrance into nucleus, and induce its transcriptional activation[72, 73]. FoxO can be phosphorylated by phosphatidylinositol 3-kinase-Akt pathway[74, 75]. It is of note that FoxO-mediated transcription requires binding of β-catenin. FoxOs can compete with TCF/LEF by directly binding β-catenin, thereby inhibit Wnt/β-catenin downstream signaling[76, 77, 24, 78]. It was demonstrated herein, compared with the control group FoxO1 expression in 5-FU treated HS-5 cells rose dramatically concurrent with decreased p-FoxO1 expression. The reason for up-regulation of FoxO1 may be related to 5-FU triggered oxidative stress, whereas FoxO1 up-regulation may be another reason for 5-FU induced decrease in Wnt signaling[27, 26, 79]. FoxO transcription factor family regulate the proteins that are crucial for the apoptosis, as well as the proteins involved in proliferative status of a cell. FoxO factors may regulate antiapoptotic and proapoptotic proteins at multiple levels, finally trigger activation of the effector caspases. Bim promotes apoptosis by inhibition of antiapoptotic Bcl-2 family members or through direct activation of Bax-like molecules. FoxO factors may regulate Bim protein expression to cause cell death due to cytokine deprivation. FoxO factors may also repress transcription of Bcl-XL through the induction of the transcriptional repressor[80–82]. Caspase-3 is an important effector protease, when it is cleaved, it acts as the final executor during apoptosis. In the current study, it was found that in 5-FU treated HS-5 cells, FoxO1 targeted apoptosis-related proteins to cause increase in Bim, Bax, and caspase-3 whereas decrease in Bcl-2. FoxO1 targeted apoptosis to disturb the dynamic balance of the cellularity of HS-5 cells, which may be one of reasons for cell growth inhibition. Moreover, the cyclin kinase inhibitor p27Kip1, a downstream target of FoxO1, acting as a potent inhibitor of cyclin/cdk complexes in the S-phase of cell cycle progression was also tested[83–86]. It was found herein that 5-FU increased the expression of p27kip1. In addition, 5-FU simultaneously reduced the expression of Cyclin D1. It is of note that transcriptional repression of D-type cyclins is vital to the FoxO-induced cell-cycle arrest, which is evidenced by transcriptional profiling and mRNA analysis. D-type cyclins are required for phosphorylation and inactivation of the retinoblastoma tumor suppressor protein (pRb), an essential determinant of cell-cycle progression in G1[87, 88]. To sum up, 5-FU-induced HS-5 cell growth inhibition is probably associated with FoxO1 targeted apoptosis or cell cycle arrest.
The traditional Chinese medicine Angelica sinensis which is commonly used to enrich blood, promote blood circulation[39]. The active constituents of Angelica sinensis include polysaccharides, organic acid sand phthalides, among which Angelica sinensis polysaccharides (ASP) are regarded as the main biological activity ingredient responsible for pharmacological effects with multi-target property[89]. ASP have attracted more and more attention to its beneficial effects, such as hematopoietic effects[90], immunologic enhancement[91], anti-tumor activity[92, 93], and anti-radiation damage[94]. The antioxidant properties of ASP suppress the production of ROS and protected the endothelial progenitor cells, hepatocytes, myocardial cell and nerve cells from oxidative damage[95, 96, 40]. Moreover, evidences demonstrated that ASP promote cell proliferation, including in total spleen cells, macrophages[91], and gastric epithelial cells[97]. Our previous studies suggested that ASP reduced oxidative stress and oxidative DNA damage, boosted direct cell-cell contact between stromal cells and hematopoietic cells through Cx43 junctions, regulated cytokines, growth factors and chemokines such as CXCL12, SCF, GM-CSF, RANTES and thus provided a homeostatic microenvironment for hematopoietic stem/progenitor cells to regenerate following chemotherapeutic myelosuppression. In the present study, it was further demonstrated that ASP protected HS-5 cells from 5-FU-induced proliferation inhibition and ameliorated cellular oxidative stress via the mechanism of up-regulation of Wnt/β-catenin signaling. Most importantly, it was first evidenced herein that ASP balanced the relationship between FoxO-mediated transcription and Wnt signaling in BMSCs under oxidative stress, which might be promising for clinical therapeutic use of ASP to myelosuppression.