| Breast cancer is one of the most common malignant diseases with the highest mortality among all the cancers in women worldwide. Although thanks to the recent improvement of modern medicine, the five-year survival rate of breast cancer has increased steadily,currently, it still cannot be cured. In fact, it is still one of the main life-threatening diseases for women. The cancer stem cells(CSCs) are thought to be involved in the relapse and metastasis, which have proved to be the main reasons that directly lead to death.It is well-known that CSCs with the tumor initiation ability are the crucial component of breast cancer, but the number of CSCs is thought to be very low, even though the exact numbers or proportions of CSCs in particular clinical contexts are currently unclear.CSCs have the capacity both to self-renewal and differentiate to tumor cells, so that CSCs are believed to be cancer seeds with high tumorigenicity. In addition, CSCs play a crucial role in the resistance to chemotherapy and radiotherapy, since the conventional radiotherapy and chemotherapy only kill ordinary fast proliferating tumor cells and have little effect on the quiescent CSCs, which eventually causes the recurrence and metastasis of cancers.As reported in the literatures, Alexidine(AD) in certain concentration can promote apoptosis of laryngeal and neck tumor cells. AD is thought to be a potential effective treatment for tumor. Previous studies from the lab of our American collaborators alsofound that AD can specifically inhibit the activity of a tyrosine phosphatase named protein tyrosine phosphatase, mitochondrial 1(PTPMT1), and inhibiting the activity of PTPMT1 can block the embryonic stem cell differentiation. Therefore, we were wondering that whether inhibition of PTPMT1 in breast cancer activity could block breast CSCs differentiation, which in turn, could reduce the malignant tumor into a less malignant chronic disease. In this study, we directly test this hypothesis by using AD to inhibit PTPMT1 activity chronically. Since CSCs have the two characteristics, i.e.,differentiation to and self-renewal, we reason that using two drugs that inhibit both of the characteristic features of CSCs, should be a more effective therapy. Interestingly,Salinomycin(S) has the ability not only to inhibit the growth of tumor, but also the self-renewal of CSCs. In this study, we directly test whether combinational treatment with AD and S is more effective, using in vitro and in vivo models.Our in vitro studies found that AD promote the apoptosis of human breast cancer MCF-7 cells and mouse breast cancer E0771 cells, and also inhibit the proliferation of breast cancer cells. Since EMT is closely associated with the malignancy of CSCs, we evaluated the expression of EMT related markers, i.e., Vimentin, MMP3 and beta-catenin by western-bloting, and found that the low dosage of AD treatment induced the expression of EMT related markers, indicating that the chronic AD treatment increase the level of malignancy of breast cancer cells. These AD induced breast cancer E0771/MCF-7 cells lines were subsequently used in a series of biological behavior experiments, and we found that the colony-formation ability and the ability of migration were significantly higher than those untreated parental control cells. Furthermore, we found that the numbers of CSCs in the AD induced E0771/MCF-7 cells were higher than the control group by ALDEFLUOR. In contrast, the numbers of CSCs were lower in cells received combined treatment of AD and S. This data confirmed our hypothesis.Complimentarily, our in vivo studies found that, AD can induce chronic inhibition of tumor growth, but the tumor growth rebound after AD withdrawal. In vivo tumorigenesis with limited diluted AD induced E0771 cells or control cells found that the tumorigenicity of AD induced cells was higher, which is consistent with our in vitro experiments. Consistently, the percentage of CSCs was also increased by AD treatment.In contrast, we found that when the animal was inoculated with the E0771 cells that received the combinational treatment of AD and S, not only the tumors grew slower but also the tumor size was significantly smaller than the control group. This data supports the idea that the combinational treatment is more effective.In order to further explore the malignant biological behavior of CSCs and understand the underlying molecular mechanisms, we hypothesized that PTPMT1 plays an important role in the process. We found that in the normal breast cell line MCF-10 A the expression of PTPMT1 was significantly higher than that in breast cancer cell.We then collect the clinical breast cancer and adjacent tissue specimens, and we confirmed the expression of PTPMT1 in the adjacent tissue was higher than that in the breast cancer tissue by immunohistochemistry, indicating that absence of PTPMT1 activity promotes the progression of tumors.Overall, these data suggested that combinational treatment of AD and S not only reduced the malignant tumor into a less malignant chronic disease, but also reduce the proportion of CSCs. This study may have direct clinical implications for the treatment of breast cancer. |
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