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, 15 (2), 1789-1798

Piperlongumine Inhibits Cancer Stem Cell Properties and Regulates Multiple Malignant Phenotypes in Oral Cancer

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Piperlongumine Inhibits Cancer Stem Cell Properties and Regulates Multiple Malignant Phenotypes in Oral Cancer

Yin-Ju Chen et al. Oncol Lett.

Abstract

Piperlongumine (PL), a natural product of Piper longum, inhibits multiple malignant phenotypes. Therefore, the present study examined whether PL suppresses cancer stemness in oral cancer. The cellular effects of PL were determined by examining alterations in tumor sphere formation, cell migration, invasion, proliferation ability, chemosensitivity and radiosensitivity. Reverse transcription-quantitative polymerase chain reaction analysis and western blotting were performed in order to determine molecular expression levels. The present study revealed that PL inhibited cancer stem cell-forming ability and suppressed the expression of the stemness-related transcription factors SRY-Box 2, POU class 5 homeobox 1, and Nanog homeobox. However, it increased the expression of the differentiation marker cytokeratin 18. PL also suppressed cell migration and invasion, resulting in the elimination of the epithelial-mesenchymal transition. Furthermore, PL increased chemo- and radiosensitivity and suppressed tumor growth in vitro and in vivo. The results of the present study suggested that PL inhibits malignant phenotypes via the suppression of cancer stemness in oral cancer. Thus, PL may serve as an effective therapeutic agent for oral cancer.

Keywords: cancer stem cells; chemosensitivity; epithelial-mesenchymal transition; oral cancer; piperlongumine; radiosensitivity.

Figures

Figure 1.
Figure 1.
PL suppressed cancer stem cell properties including tumor sphere-forming ability. (A) Representative image of the number (magnification, ×40) and (B) size of tumor spheres (magnification, ×400) formed by SAS and CGHNC8 cells following treatment with 0.0 (0.1% DMSO), 2.5 and 5.0 µM PL. The bar graphs present the number and diameter of the tumorspheres (scale bar, 100 µm). *P<0.05 vs. the untreated control group. PL, piperlongumine.
Figure 2.
Figure 2.
PL reduced the expression of the cancer stem cell markers SOX2, Oct-4, and NANOG but increased the expression of the differentiation marker CK18. SAS and CGHNC8 cells were treated with 5.0 µM PL for 48 h; subsequently, the cells were harvested and analyzed. (A) mRNA expression levels of SOX2, NANOG, and Oct-4 and CK18 were measured using reverse transcription-quantitative polymerase chain reaction. (B) Cellular protein levels were determined using western blotting (control cells, 0.1% DMSO). Experiments were performed in triplicate. PL, piperlongumine; SOX2, SRY-box 2; Oct-4, POU class 5 homeobox 1; NANOG, NANOG homeobox; CK18, cytokeratin 18.
Figure 3.
Figure 3.
PL suppressed cell migration and invasion, and regulated the expression of EMT-associated markers. (A) PL suppressed cell migration, revealed using a wound healing assay. (B) PL suppressed cell invasion ability, revealed using a Matrigel invasion assay. (C) Western blotting was used to examine the expression of EMT-associated molecules. *P<0.05 vs. the untreated control group. PL, piperlongumine; EMT, epithelial-mesenchymal transition; E-, epithelial; N-, neural; Snail1, Snail family transcriptional repressor 1; Slug, Snail family transcriptional repressor 2.
Figure 4.
Figure 4.
PL increased the radio- and chemosensitivity levels of oral cancer cells. (A) Clonogenic survival assays were used to determine the effect of PL on the radiosensitivity of SAS and CGHNC8 cells. Cells were treated with either PL alone (0.625 µM), radiation alone (2 Gy) or a combination of the two treatments. (B) PL increased chemosensitivity, as determined by the cytotoxic assay. Cells were treated with 0.03 µM 5-FU alone, 2.50 µM PL alone, or a combination of the two treatments for 48 h. Cell survival was examined using an MTS assay. The data of the cytotoxic assay were compared with the data of control cells. Experiments were performed in triplicate. *P<0.05 vs. the ctrl or RT/5-FU treated groups, #P<0.05 vs. the RT/5-FU or RT+PL/5-FU+PL treated groups, with comparisons indicated by lines. PL, piperlongumine; Ctrl, control; RT, radiotherapy; 5-FU, 5-fluorouracil.
Figure 5.
Figure 5.
PL suppressed cell growth. (A) PL suppressed tumor growth in vitro, as determined by the cell number enumeration. (B) PL suppressed colony formation, demonstrated using a colony formation assay. (C) PL reduced tumor growth in xenografted tumors. SAS cells were subcutaneously injected into BALB/c nude mice. After 7 days, PL was injected intraperitoneally. The tumor size was measured weekly following injection. (D) Once the mice were sacrificed, the tumors were removed and weighed. (E) The grafted tumors were confirmed by hematoxylin and eosin staining. The results are presented as the mean ± the standard deviation. *P<0.05 vs. the untreated control group. PL, piperlongumine; Ctrl, control.

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