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Knockdown of secretory phospholipase A2 IIa reduces lung cancer growth in vitro and in vivo  Jessica A. Yu, MD, David Mauchley, MD, Howard Li, MD, Xianzhong.

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Presentation on theme: "Knockdown of secretory phospholipase A2 IIa reduces lung cancer growth in vitro and in vivo  Jessica A. Yu, MD, David Mauchley, MD, Howard Li, MD, Xianzhong."— Presentation transcript:

1 Knockdown of secretory phospholipase A2 IIa reduces lung cancer growth in vitro and in vivo 
Jessica A. Yu, MD, David Mauchley, MD, Howard Li, MD, Xianzhong Meng, MD, PhD, Raphael A. Nemenoff, PhD, David A. Fullerton, MD, Michael J. Weyant, MD  The Journal of Thoracic and Cardiovascular Surgery  Volume 144, Issue 5, Pages (November 2012) DOI: /j.jtcvs Copyright © Terms and Conditions

2 Figure 1 Short-hairpin RNA (shRNA) sequence S2 obtains most robust knockdown of secretory phospholipase A2 (sPLA2) group IIa messenger RNA and protein. A549 and H358 cells were stably transduced with green fluorescent protein (GFP)-tagged lentiviral vector particles, (A) to determine the optimal conditions for transduction, before transducing cells with vector particles containing nontargeted shRNA as a control, or shRNA targeting sPLA2 IIa. Growth selection for transduced cells was with puromycin. Four distinct shRNA sequences (S1 to S4) targeting group IIa sPLA2 were used to create 4 different knockdown cell lines. B, Knockdown was confirmed by messenger RNA and (C) protein analysis. The most robust knockdown in protein was with sequence S2. RT-PCR, Reverse-transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

3 Figure 2 Secretory phospholipase A2 group-IIa (sPLA2 IIa) knockdown is stable over several cell passages with corresponding persistent decrease in growth rate of lung cancer cells. Top panels, Quantitative reverse-transcription polymerase chain reaction confirms decreased messenger RNA (mRNA) over several cell passages 1 through 7. Bottom panels, Analysis of proliferation using the same cell passages 1 through 7 shows a stable reduction in the growth rate of both A549 and H358 knockdown cells. Controls for the experiments are wild-type cells and vector controls (NT). Data are presented as mean ± standard error of the mean; n = 3-5 performed in triplicate. ∗P < .05. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

4 Figure 3 Secretory phospholipase A2 group IIa (sPLA2 IIa) knockdown decreases nuclear factor-κB (NF-κB) activity. Tumor necrosis factor-α (TNF-α) induces NF-κB phosphorylation in A549 cells, vector control cells (ANT) and knockdown cells (AS2), though significantly less in the knockdown cells. Representative western blot of a single experiment is shown with a graph of densitometric analysis of cumulative experiments below (mean ± standard error of the mean; n = 5 per group). Pharmacologic sPLA2 IIa inhibition in lung cancer cells results in a similar decrease in NF-κB phosphorylation (data not shown). ∗P < .05. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

5 Figure 4 Secretory phospholipase A2 group IIa knockdown decreases lung cancer cell growth in vivo. A549 and knockdown cells were injected into the left flanks for nude mice and tumor size was serially measured until postinjection day 23. A, Representative photograph of xenograft tumors in situ demonstrating increased A549 tumor size compared with the knockdown tumors. B, Volume of the tumors over time shows significant growth retardation in the knockdown cells (AS2) compared with A549 cells (mean ± standard error of the mean; n = 5) per group. C, Representative histology from knockdown and wild-type tumors exhibit large areas of necrosis representative of fast growth. Wild-type tumors demonstrate increased Ki-67 staining in the compared with more peripheral and less concentrated staining in the knockdown tumors. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

6 Figure 5 Secretory phospholipase A2 group IIa (sPLA2 IIa) messenger RNA (mRNA) remains decreased in knockdown tumors compared with wild-type tumors. Reverse transcriptase-polymerase chain reaction of tumor homogenates shows that wild-type tumors had more than double the amount of sPLA2 IIa mRNA compared with knockdown tumors. Average of ΔΔCt for all 5 knockdown tumors was compared with the average of ΔΔCt for all 5 wild-type tumors. Results presented as mean ± standard error of the mean; n = 3 performed in triplicate. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © Terms and Conditions

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