Yonsei Med J.  2016 Nov;57(6):1312-1323. 10.3349/ymj.2016.57.6.1312.

Knockdown of the M2 Isoform of Pyruvate Kinase (PKM2) with shRNA Enhances the Effect of Docetaxel in Human NSCLC Cell Lines In Vitro

Affiliations
  • 1Department of Oncology, Jinshan Hospital, Medical Center of Fudan University, Shanghai, China. qiaotk@163.com
  • 2Department of Radiotherapy, Donghua Hospital of Sun Yat-sen University, Dongguan, China.

Abstract

PURPOSE
The aim of our study was to explore the relationships between the M2 isoform of pyruvate kinase (PKM2) and the sensitivity of human non-small cell lung cancer (NSCLC) cells to docetaxel in vitro.
MATERIALS AND METHODS
With the method of plasmid transfection, we silenced the expression of PKM2 successfully in A549 and H460 cells. Western blotting and real-time PCR were applied to detect PKM2 expression at protein and gene levels. Cell viability was examined by CCK8 assay. Cell cycle distribution and apoptosis were examined by flow cytometry. P21 and Bax were detected.
RESULTS
Expression of PKM2 mRNA and protein were significantly decreased by shRNA targeting PKM2. Silencing of PKM2 increased docetaxel sensitivity of human NSCLC A549 and H460 cells in a collaborative manner, resulting in strong suppression of cell viability. The results of flow cytometric assays suggested that knockdown of PKM2 or docetaxel treatment, whether used singly or in combination, blocked the cells in the G2/M phase, which is in consistent with the effect of the two on the expression of p21. Cells with PKM2 silencing were more likely to be induced into apoptosis by docetaxel although knockdown of PKM2 alone can't induce apoptosis significantly, which is in consistent with the effect of the two on Bax expression.
CONCLUSION
The results suggest that PKM2 knockdown could serve as a chemosensitizer to docetaxel in non-small lung cancer cells through targeting PKM2, leading to inhibition of cell viability, increase of cell arrest of G2/M phase and apoptosis.

Keyword

The M2 isoform of pyruvate kinase; shRNA; non-small cell lung cancer; chemotherapy; docetaxel; cell cycle; apoptosis

MeSH Terms

Apoptosis/*drug effects
Carcinoma, Non-Small-Cell Lung/drug therapy/genetics
Cell Cycle
Cell Line, Tumor
Cell Proliferation/*drug effects
Humans
Lung Neoplasms/genetics/*metabolism/pathology
MicroRNAs
Protein Isoforms
Pyruvate Kinase/*antagonists & inhibitors/genetics/metabolism
RNA, Small Interfering/genetics
Real-Time Polymerase Chain Reaction
Taxoids/*pharmacology
Transfection
Tumor Cells, Cultured
Up-Regulation/*drug effects
MicroRNAs
Protein Isoforms
RNA, Small Interfering
Taxoids
Pyruvate Kinase

Figure

  • Fig. 1 PKM2 protein expression in A549 and H460 cells. Both A549 cells (A) and H460 cells (B) were untransfected (Control), or transfected by the method of plasmid transfection, including PKM2 shRNA-transfection (PKM2 shRNA) or control shRNA-transfection (Control shRNA). 48 hrs after transfection, Western blot analyses were performed to examine the inhibition effect. The graph depicts mean±SEM for three independent determinations of optical density of the PKM2 Western blot bands. *Compared to control, p<0.05, †Compared to PKM2 shRNA (946), p<0.05. PKM2, the M2 isoform of pyruvate kinase.

  • Fig. 2 PKM2 mRNA expression in A549 and H460 cells. Both A549 cells (A) and H460 cells (B) were untransfected (Control), or transfected by the method of plasmid transfection, including PKM2 shRNA-transfection (PKM2 shRNA) or control shRNA-transfection (Control shRNA). 48 hrs after transfection, RT-PCR were performed to examine the inhibition effect. The graph depicts mean±SEM for three independent determinations of PKM2 mRNA relative to β-actin mRNA value. *Compared to control, p<0.05, †Compared to PKM2 shRNA (946), p<0.05. PKM2, the M2 isoform of pyruvate kinase.

  • Fig. 3 The effect of shRNA-PKM2 and docetaxel or the combined treatment of both on the viability of A549 and H460 cells. 48 hrs after transfection, all A549 and H460 cells, untransfected (Control), PKM2 shRNA-transfected (PKM2 shRNA) or control shRNA-transfected (Control shRNA), were all cultured with different concentrations of docetaxel, ranging from 0 to 25 nM, for up to 72 hrs. (A and B) Cells were incubated with docetaxel at 0 nm for 72 hrs. The cell viability was detected at 24 hrs, 48 hrs and 72 hrs after incubation. (C and D) Cells were incubated with docetaxel at concentrations ranging from 0 nm to 25 nm for 72 hrs. Cell viability was quantified using Cell Counting Kit-8 and expressed as the percentage of the viability of control cells (0 h). Results are presented as mean±SEM of three separate experiments conducted in duplicate. *Compared to control cells with the same dose of docetaxel incubation at the same time point, p<0.05, †Compared to PKM2 shRNA-transfected cells with 24 hrs incubation of 0 nM docetaxel, p<0.05, ‡Compared to PKM2 shRNA-transfected cells with the same dose of docetaxel incubation in the same time point, p<0.05, §Compared to control cells with 72 hrs incubation of 0 nM docetaxel, p<0.05. PKM2, the M2 isoform of pyruvate kinase.

  • Fig. 4 Effects of PKM2 shRNA and docetaxel on cell cycle distribution. 48 hrs after transfection, all A549 and H460 cells, PKM2 shRNA-transfected (PKM2 shRNA), control-shRNA transfected (Control shRNA) or non-transfected (Control), were incubated with docetaxel at concentrations ranging from 0 to 25 nM for up to 48 hrs, and the cell cycle distribution was evaluated using PI staining and flow cytometry analysis. In both A549 and H460 cells, cell cycle distribution was measured at two different time points after incubation, 24 hrs (A and B) and 48 hrs (C and D). Results are presented as mean±SEM of three separate experiments conducted in duplicate. *Compared to control cells, p<0.05, †Compared to control cells with incubation of 0 nM docetaxel, p<0.05. PKM2, the M2 isoform of pyruvate kinase; PI, propidium oidide.

  • Fig. 5 p21 protein expression induced by 0 nM and 20 nM docetaxel in PKM2-shRNA transfected, shRNA-control transfected or non-transfected cells at 48 hrs. 48 hrs after transfection, all A549 (A) and H460 (B) cells, PKM2 shRNA-transfected (PKM2 shRNA), control-shRNA transfected (Control shRNA) or non-transfected (Control), were incubated with 20 nM docetaxel for up to 48 hrs, Western blot analyses were performed to detect the expression of p21. The graph depicts mean±SEM for three independent determinations of optical density of the p21 Western blot bands. *Compared to control cells with incubation of 0 nM docetaxel, p<0.05, †Compared to PKM2 shRNA-transfected cells with incubation of 20 nM docetaxel, p<0.05, ‡Compared to control shRNA-transfected cells with incubation of 0 nM docetaxel, p<0.05. PKM2, the M2 isoform of pyruvate kinase; DOC, docetaxel.

  • Fig. 6 Effects of shRNA PKM2 and docetaxel on apoptosis. 48 hrs after transfection, all A549 and H460 cells, PKM2 shRNA-transfected (PKM2 shRNA), control-shRNA transfected (Control shRNA) or non-transfected (Control), were incubated with docetaxel at concentrations ranging from 0 to 25 nM for up to 48 hrs, and apoptosis was evaluated using Annexin V-FITC/PI staining and flow cytometry analysis. In both A549 and H460 cells, apoptosis was detected at two different time points after incubation, 24 hrs (A and B) and 48 hrs (C and D). Results are presented as mean±SEM of three separate experiments conducted in duplicate. *Compared to control cells, p<0.05, †Compared to control cells with incubation of 0 nM docetaxel, p<0.05. PKM2, the M2 isoform of pyruvate kinase; PI, propidium iodide.

  • Fig. 7 Representative analysis of apoptosis induced by 20 nM docetaxel in PKM2-shRNA transfected, shRNA-control transfected or non-transfected cells at 48 hrs. 48 hrs after transfection, all A549 and H460 cells, PKM2 shRNA-transfected (PKM2 shRNA), control-shRNA transfected (Control shRNA) or non-transfected (Control), were incubated with 20 nM docetaxel for up to 48 hrs, and apoptosis was evaluated using Annexin V-FITC/PI staining and flow cytometry analysis. (A) PKM2-shRNA transfected A549 cells treated with 20 nM DOC. (B) shRNA-control transfected A549 cells treated with 20 nM DOC. (C) Non-transfected A549 cells treated with 20 nM DOC. (D) PKM2-shRNA transfected H460 cells treated with 20 nM DOC. (E) shRNA-control transfected H460 cells treated with 20 nM DOC. (F) Non-transfected H460 cells treated with 20 nM DOC. Bottom right quadrant; cells stained mainly by Annexin V (early apoptotic cells); top right quadrant, cells stained by both PI and Annexin V (late apopototic cells); top left quadrant, cells stained mainly by PI (necrotic cells); bottom left quadrant, cells negative for both Annexin V and PI. PKM2, the M2 isoform of pyruvate kinase; PI, propidium iodide.

  • Fig. 8 Bax protein expression induced by 0 nM and 20 nM docetaxel in PKM2-shRNA transfected, shRNA-control transfected or non-transfected cells at 48 hrs. 48 hrs after transfection, all A549 (A) and H460 (B) cells, PKM2 shRNA-transfected (PKM2 shRNA), control-shRNA transfected (Control shRNA) or non-transfected (Control), were incubated with 20 nM docetaxel for up to 48 hrs, Western blot analyses were performed to detect the expression of Bax. The graph depicts mean±SEM for three independent determinations of optical density of the Bax Western blot bands. *Compared to control cells with incubation of 0 nM docetaxel, p<0.05, †Compared to PKM2 shRNA-transfected cells with incubation of 20 nM docetaxel, p<0.05, ‡Compared to control shRNA-transfected cells with incubation of 0 nM docetaxel, p<0.05. PKM2, the M2 isoform of pyruvate kinase; DOC, docetaxel.


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