As evidence on bone marrow HM has accumulated over the past few years, it has become widely acknowledged that MSCs affect a great number of different cell types besides hematopoietic parenchymal cells, including leukemia cells [11–13]. With this close relationship between MSCs and leukemia cells, it may be that the influence of MSCs is what ultimately determines the prognosis of leukemia.
In general, MSCs in the HM have been considered to be nurse-like cells that exert a form of protective modulation. Leukemic MSCs can reportedly inhibit the chemotherapeutic-induced apoptosis of Jurkat cells and HL-60 cells. Moreover, they can interfere with the cell cycle of Jurkat cells at the G0-G1 phase [14, 15]. They can also negatively regulate cancer immunotherapy involving NK cells and inhibit cytotoxic T cells by secreting cytokines [16, 17]. Thus, there appear to be multiple roles of MSCs in proliferation, differentiation, and survival of leukemia cells [18–20] as well as normal immune cells. In the present study, the role of leukemic MSCs on K562 cells was explored under normal nutritional conditions or under serum starvation. We noticed a marked increase in K562 cell apoptosis after serum starvation for 24 hours. However, a marked decrease in apoptosis was observed when these starved cells were cocultured with MSCs, supporting the protective role of leukemic MSCs against apoptosis. This inhibition existed both in contact coculture and in separated coculture, and was induced even by supernatant culture medium from MSCs. Thus, our data support that cytokines, adherent reactions and gap junctions participated in inhibiting leukemic cell proliferation.
When K562 cells were cocultured with normal MSCs, they also showed cell cycle blockade. These K562 cells also showed drug-resistance to daunorubicin (DNR), which is consistent with their increased G0-G1 phase and reduced S phase. The reasons for this drug resistance may also relate to the upregulation of antiapoptotic gene expression and the cytokines secreted by MSCs. Our data also showed a similar cell cycle blockade of K562 cells resulting from coculture with leukemic MSCs obtained from 4 patients, although these nurse cells may have undergone malignant transformation in vivo or over their long time in culture. Inhibition of cell growth is a primary method of treating leukemia; however, the blockade of the cell cycle may prevent the efficacy of chemotherapeutic agents, which mainly target the proliferative phase of tumor cells. When most tumor cells are blocked at the quiescent phase, they may evade the killing powers of chemotherapeutics and may ultimately form micro residual disease (MRD). We hypothesize that leukemic MSCs may provide a niche for tumor stem cells, in which K562 cells back up the proliferation and self-renewal potential. These tumor cells may then be the source of relapse.
Constitutive activation of Akt, one downstream target of PI3K, is also believed to promote proliferation and increase cell survival, leading to cancer progression. The PI3K-Akt signal pathway is involved in the antiapoptotic activity of tumor cells and culminates in the phosphorylation of the BCL-2 family member, Bad, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates Bad both in vitro and in vivo, and blocks Bad-induced cell death . The PI3K-Akt-Bad pathway may represent a form of general antiapoptotic machinery, although there is insufficient evidence to support this hypothesis at present.
We determined the expression levels of Akt, p-Akt, Bad, p-Bad proteins in K562 cells after inoculation with MSCs. Under the condition of K562 cells alone, there was a basal expression of p-Akt, and p-Bad, which might have been related to the bcr/abl fusion protein-activated PI3K-Akt signal pathway. In addition, the expression of p-Akt and p-Bad was increased after coculture with leukemic MSCs. The addition of the specific inhibitor LY294002, which competes with PI3K for ATP binding sites , resulted in a dramatic decrease in levels of both phosphorylated proteins, while no obvious difference in Akt and Bad expression was observed among the three groups. Hence, we showed that the PI3K-Akt pathway was activated after coculture with MSCs. The pro-apoptotic molecule, Bad, was then phosphorylated and exerted inhibitory effects on starvation-induced apoptosis.
Taken together, serum deprivation appears to mimic the effects of an adverse HM for leukemia cells. MSCs of leukemia patients can retard the cell cycles of K562 cells, inhibiting their proliferation and reducing their apoptosis. Consequently, MSCs protect leukemia cells against adverse conditions like serum deprivation and ultimately sustain their viability. The activation of the PI3K-Akt-Bad signaling pathway seems to be involved in the protective machinery. Therefore, approaches that block the activation of this signaling pathway may in turn remove this shielding and consequently may prove to be of benefit in the effective treatment of leukemia.