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. 2010 Jun;101(6):1447-53.
doi: 10.1111/j.1349-7006.2010.01562.x. Epub 2010 Mar 15.

Silencing of pkm2 increases the efficacy of docetaxel in human lung cancer xenografts in mice

Affiliations

Silencing of pkm2 increases the efficacy of docetaxel in human lung cancer xenografts in mice

Hua-shan Shi et al. Cancer Sci. 2010 Jun.

Erratum in

  • Cancer Sci. 2010 Jun;101(6):1575

Abstract

Tumor aerobic glycolysis, or the Warburg effect, plays important roles in tumor survival, growth, and metastasis. Pyruvate kinase isoenzyme M2 (PKM2) is a key enzyme that regulates aerobic glycolysis in tumor cells. Recent research has shown that PKM2 can be used as a tumor marker for diagnosis and, in particular, as a potential target for cancer therapy. We investigated the effects of combining shRNA targeting PKM2 and docetaxel on human A549 lung carcinoma cells both in vivo and in vitro. We observed that the shRNA can significantly downregulate the expression level of PKM2. The decrease of PKM2 resulted in a decrease in ATP synthesis, which caused intracellular accumulation of docetaxel. Furthermore, the combination of pshRNA-pkm2 and docetaxel inhibited tumor growth and promoted more cancer cell apoptosis both in vivo and in vitro. Our findings suggest that targeting tumor glycolysis can increase the efficacy of chemotherapy. In particular, the targeting of PKM2 could, to some extent, be a new way of reversing chemotherapy resistance to cancer therapy.

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Figures

Figure 1
Figure 1
Plasmid expressing shRNA targeting pyruvate kinase isoenzyme M2 (pshRNA‐pkm2) downregulated the expression of PKM2 in human A549 lung carcinoma cells. (A) pshRNA‐pkm2 (PKM2) and pshRNA‐Con (Control) were transfected for 48 h, then Western blot analysis was used to detect PKM2 protein. pshRNA‐pkm2 effectively diminished PKM2 protein levels compared to pshRNA‐Con and blank groups. (B) The level of PKM2 in the pshRNA‐pkm2 group was 19.7 ± 3.5% that of the pshRNA‐Con group and 21.2 ± 4.1% of the blank group. β‐actin was used to normalize for any differences in protein loading between lanes. Bars, SD; columns, mean.
Figure 2
Figure 2
(A) Detection of intracellular chemotherapeutics in human pulmonary adenocarcinoma A549, human hepatocellular liver carcinoma HepG2, and murine Lewis lung carcinoma LL/2 cell lines. Data showed that treatment with plasmid expressing shRNA targeting pyruvate kinase isoenzyme M2 (pshRNA‐pkm2) + docetaxel (PKM2 + DOX) increased the content of docetaxel in A549 and HepG2 cells compared to other treatment groups (both P < 0.05), but there was no obvious difference in LL/2 cells. (B) Detection of ATP in A549 cells. After transfection with pshRNA‐pkm2 and pshRNA‐Con (CONTROL) for 24 h, 2 μg/mL docetaxel was added. The ATP content was detected after a further 24 h. Data shows the ATP content of 106 cells. The pshRNA‐pkm2 group showed a significant decrease compared with control groups (P = 0.0136). Bars, SD; columns, mean. NS, normal saline.
Figure 3
Figure 3
Flow cytometry analysis and MTT assay of human A549 lung carcinoma cells in vitro. (A) To measure the effects on cell proliferation, cells were transfected with plasmid expressing shRNA targeting pyruvate kinase isoenzyme M2 (pshRNA‐pkm2) (PKM2) and pshRNA‐Con (CONTROL) for 24 h, then 2 μg/mL docetaxel (DOX) was added. After 24 h, viable cells were determined by MTT assay. Data are presented as the absorbance of each group. The pshRNA‐pkm2 + docetaxel (PKM2 + DOX) group showed a significant decrease compared with control groups (P = 0.0057), PKM2 alone (P = 0.0012), or DOX alone (P = 0.0127). (B) A549 cells were treated as before, then apoptosis was determined by flow cytometry. Data are expressed as percentages. The PKM2 + DOX group showed a significant decrease in apoptosis compared with control groups (P = 0.0011), PKM2 alone (P = 0.0008), or DOX alone (P = 0.0015). Bars, SD; columns, mean. NS, normal saline.
Figure 4
Figure 4
Detection of expression levels of pyruvate kinase isoenzyme M2 (PKM2) in vivo in human A549 lung cancer cells implanted into female nude mice. Staining of PKM2 in cancer cells was clearly positive in the group treated with normal saline (NS). Weak cancer cell staining was observed in the group treated with pshRNA‐pkm2. Magnification, ×400.
Figure 5
Figure 5
In vivo effects of plasmid expressing shRNA targeting pyruvate kinase isoenzyme M2 (pshRNA‐pkm2) (PKM2) combined with docetaxel (DOX). (A) Antitumor effects. Tumor‐bearing mice were treated with NS (♦), PKM2 alone (▮), DOX alone (), or PKM2 + DOX (*). There was a significant difference in tumor volume (P = 0.0049) between NS treated mice and other groups. There was also a difference between the PKM2 + DOX group and the PKM2 (P = 0.0347) and DOX (P = 0.0296) groups. Bars, SD; points, mean (n = 5). (B) Inhibition of proliferation activity in A549 lung cancer cells. Proliferated tumor cells were detected by an antibody to Ki67 and the positive cell densities were quantified by counting the number of cells per high power field (×400). (a) Normal saline (NS) group, (b) PKM2 group, (c) DOX group, (d) PKM2 + DOX group. (e) Ki67 positive cells in each group. The PKM2 + DOX group showed a significant decrease compared with the control group (P = 0.0063). Bars, SD; columns, mean (five high power fields/slide).
Figure 6
Figure 6
Detection of apoptotic A549 lung cancer cells using TUNEL analysis. The percentage of apoptosis was determined by counting the number of apoptotic cells and dividing by the total number of cells in the field (five high power fields/slide). (A) Normal saline group; (B) plasmid expressing shRNA targeting pyruvate kinase isoenzyme M2 (pshRNA‐pkm2) (PKM2) group; (C) docetaxel (DOX) group; (D) PKM2 + DOX group. (E) Percent apoptosis in each group. The combined treatment with PKM2 + DOX resulted in significantly increased apoptosis compared to that of other groups (P < 0.05). Bars, SD; columns, mean.

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