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PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines - PubMed

  • ️Thu Jan 01 2015

PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines

Alba Ginés et al. PLoS One. 2015.

Abstract

Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of PKM2 silencing on OXA cytotoxicity in HT29, SW480 and HCT116 cells.

Dose-response curves for HT29, SW480 and HCT116 cell lines after PKM2 gene silencing and OXA treatment at 0–140 μM and 0–32 μM for 24 hours. Curves represent the average values from at least three independent experiments. Cell proliferation was measured by MTT assay. Vertical bars in the graphics represent ± SD. Insets show PKM2 inmunoblotting after siRNA-directed inhibition. Specific IC50 values for oxaliplatin in all conditions are displayed in the table. IC50 values for Mock conditions (without transfection) were very similar to those of siNTC and are not shown. *p-values are result of comparison to the siNTC condition.

Fig 2
Fig 2. Effect of PKM2 silencing according to p53 in HT29 and HCT116 cell lines.

Bars represent the IC50 ± SD (average values from at least three independent experiments) for oxaliplatin for each condition. Insets show PKM2 and p53 inmunoblotting after PKM2 gene silencing. Specific IC50 values for oxaliplatin in all conditions are displayed in the table. IC50 values for Mock conditions (without transfection) were very similar to those of siNTC and are not shown. *p-values are result of comparison to the siNTC condition. * P- value < 0.05. ** P-value < 0.01; *** P-value < 0.001

Fig 3
Fig 3. PKM2 silencing alters OXA response in HT29 cells but not apoptosis activation.

After siRNA transfection cells were treated with 15 μM OXA for 24 h period, observed by optical microscopy at 0, 24 and 48 h after the end of drug exposure (0 h refers to cells treated for 24h; 24 h refers to cells treated for 24 h and left to recover for additional 24 h) (A) and quantified by trypan blue staining (B). Apoptosis activation after 24 and 48 hours of 10 μM OXA exposure was determined by FITC-Annexin V/ PI double staining (C) and measured as a ratio between percentages of apoptotic treated (T) and non-treated (NT) cells (D). Vertical bars in the graphics represent ± SD. *P-value < 0.05. NT: non-treated cells. Optical microscopy: objective 10x magnification.

Fig 4
Fig 4. Cell cycle distribution in HT29 and HCT116 cells after PKM2 gene silencing and treatment with OXA.

Both cell lines were transfected and/or exposed to 10 μM OXA for 8, 24, 48 and 72 h. After propidium iodide staining, the proportion of cells in cell cycle phases G1, S and G2/M was measured by flow cytometry and quantified by Flowjo v9.2 software. Results are representative of at least three independent experiments. P-values ≤ 0.05 are represented as a *.

Fig 5
Fig 5. PKM2 subcellular localization in response to OXA in colorectal cancer cell lines.

Immunoflourescence staining of PKM2 (red) demonstrates nuclear accumulation in HCT116 and HT29 cells after treatment with OXA in a time and dose-dependent manner but not in resistant HTOXAR3 cells. Nuclei were stained in blue. NT: Non-treated cells. Objective lens: 40x immersion oil. Scale bar: 10 μm.

Fig 6
Fig 6. Changes in cell death genes expression patterns after PKM2 gene silencing and/or OXA treatment.

A. 3-D plot showing fold changes in expression patterns after treatment with 10 μM OXA in HT29, siPKM2-HT29 and HTOXAR3 cells. B. Heat map showing up- and down-regulated genes after OXA treatment according to three different cell death pathways.

Fig 7
Fig 7. BMF is involved in oxaliplatin response and cell death at the transcriptional level A.

Changes in BMF gene expression between OXA treated (T) and non-treated (NT) HT29, siPKM2- HT29 and HTOXAR3 cells. Vertical bars in the graphics represent means obtained from at least 3 independent experiments ± SD. B. Percentage of dead cells after treatment with oxaliplatin and/or BMF gene knockdown. Bars represent means obtained from at least 3 independent experiments ± SD. The Little graph shows percentage (mean ± SD) of BMF expression inhibition after siRNA transfections. * P- value < 0.05. ** P-value < 0.01; *** P-value < 0.001

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