Latexin inhibits the proliferation of CD133+ miapaca-2 pancreatic cancer stem-like cells

Abstract

Background: An increasing number of evidence suggests that pancreatic cancer contains cancer stem cells (CSCs), which may be relevant to the resistance of chemotherapy. Latexin (Lxn) is a negative regulator of stem cell proliferation and we investigate the effects of Lxn on CD133+ pancreatic cancer stem-like cells.

Methods: CD133+ miapaca-2 cells, a human pancreatic carcinoma cell line, were isolated and sorted by magnetic activated cell sorting and flow cytometry. The capacity for self-renewal, proliferation, and tumorigenicity of CD133+ miapaca-2 cells was determined by the floating spheres test and tumor xenograft assays. Protein and mRNA expression of Lxn in CD133+ and CD133- miapaca-2 cells were detected by Western blotting and qRT-PCR, respectively. After CD133+ miapaca-2 cells were treated with Lxn in serum-free medium (SFM), cell proliferation was assayed with a Cell Counting Kit 8 (CCK-8) and apoptosis was analyzed by flow cytometry. The protein and mRNA expression levels of Bcl-2, bax, and c-myc were also analyzed.

Results: We successfully isolated CD133+ miapaca-2 cells that exhibited the capacity for self-renewal in SFM, a proliferation potential in DMEM supplemented with FBS, and high tumorigenicity in nude mice. Lxn protein and mRNA expression levels in CD133+ miapaca-2 cells were significantly lower than those in CD133- cells. Lxn-treated CD133+ miapaca-2 cells exhibited increased apoptosis and low proliferation activity, down-regulation of Bcl-2 and c-myc expression, and up-regulation of Bax expression in a dose-dependent manner.

Conclusions: Lxn induces apoptosis and inhibits the proliferation of CD133+ miapaca-2 cells. These changes are associated with down-regulation of Bcl-2 and c-myc and up-regulation of Bax.

Figure 1

The percentage of CD133+ cells in miapaca-2 and sorted CD133+ cells determined by flow cytometry using CD133/2 (293C3)-PE antibodies. The mouse IgG2b-PE antibody was used as an isotype. (A) Miapaca-2 mouse IgG2b-PE (isotype), (B) Miapaca-2 CD133-PE, (C) Miapaca-2 CD133+ cells mouse IgG2b-PE (isotype), (D) Miapaca-2 CD133+ cells CD133-PE.

Figure 2

Proliferation ability of the CD133+ and CD133- cells in serum-free medium. CD133- cells showed slow growth and good adherence; and almost all cells died within 2 weeks (A). CD133+ cells formed floating spheres (B). When the floating spheres were cultured in DMEM supplemented with 10% FBS, the cells were adherent and proliferated similarly to unsorted miapaca-2 cells (C). The expression of CD133 mRNA in CD133+ cells was dramatically higher than unsorted miapaca-2 cells and re-adherence CD133+ cells (D). ^a P < 0.05.

Figure 3

Differences of tumor size and tumor weight between CD133 + 、 CD133- and unsorted miapaca-2 cell groups. (A) Tumor size of CD133+ cell groups were greater than that of CD133- cell groups and unsorted miapaca-2 cell groups by naked eye. (B) Tumor weight of CD133+ cell groups were more than that of CD133- cell groups and unsorted miapaca-2 cell groups. ^a P < 0.05, ^b P < 0.05.

Figure 4

Lxn expression in CD133+ and CD133- cells. (A) The Lxn protein expression level was decreased in CD133+ cells compared to CD133- cells. (B) The Lxn mRNA expression level in CD133+ cells was significantly lower than that in CD133- cells. ^a P < 0.05.

Figure 5

Cell death analysis in CD133+ and CD133- cells. (A-D) Apoptosis of CD133+ miapaca-2 cells treated with 0 ng/μL (A), 10 ng/μL (B), 20 ng/μL (C) and 40 ng/μL (D) of Lxn. The lower left indicates live cells (Annexin V-FITC negative/PI negative); the lower right shows early apoptotic cells (Annexin V-FITC positive/PI negative). The upper left shows damaged cells (Annexin V-FITC negative/PI positive), while the upper right demonstrates necrotic cells and late apoptotic cells (Annexin V-FITC positive/PI positive). The number represents the percentage of early apoptotic cells, necrotic cells, and late apoptotic cells in each condition (right quadrant). (E) The percentage of apoptotic cells, necrotic cells, and late apoptotic CD133+ miapaca-2 cells treated with 0 ng/μL, 5 ng/μL, 10 ng/μL, 20 ng/μL and 40 ng/μL of Lxn. ^a P < 0.05, ^b P < 0.05, ^c P < 0.05, ^d P < 0.05. FITC, fluorescein iodothiocyanate; PI, propidium bromide.

Figure 6

Cytotoxic effects of Lxn treatment in CD133+ miapaca-2 cells treated with Lxn at various concentrations of Lxn. The inhibitory ratio was calculated according to the following formula: Inhibitory ratio = (OD450 nm value of control group - OD450 nm value of Lxn treatment group)/(OD450 nm value of control group - OD450 nm value of blank group). ^a P < 0.05, ^b P < 0.05, ^c P < 0.05, ^d P < 0.05.

Figure 7

The expression level of Bcl-2、c-myc and bax protein after treatment with Lxn in CD133+ cells. (A) The expression level of Bcl-2、c-myc and bax protein by Western blot analysis. (B) The relative expression level of Bcl-2、bax、bcl-2/bax and c-myc protein compared to the controls. ^a P < 0.05, ^b P < 0.05, ^c P < 0.05, ^d P < 0.05.

Figure 8

The mRNA expression of Bcl-2 (A) and bax (B). The ratio of Bcl-2/bax (C) and c-myc (D). The relative amount of target gene mRNA was normalized to β-actin and compared with the controls. ^a P < 0.05, ^b P < 0.05, ^c P < 0.05, ^d P < 0.05.