Digitoflavone inhibits IκBα kinase and enhances apoptosis induced by TNFα through downregulation of expression of nuclear factor κB-regulated gene products in human pancreatic cancer cells.

Tumor necrosis factor-α (TNFα) activates both cell death and cell survival pathways. The activation of survival pathway renders most cancer cells resistant to TNF-induced cytotoxicity. We found that pretreatment with digitoflavone, a plant flavonoid, greatly sensitized TNFα-induced apoptotic cell death in several human pancreatic cancer cells. In search of the molecular basis of the sensitization effect of digitoflavone, digitoflavone was found to inhibit TNFα-induced activation of nuclear transcription factor-kappa B (NF-κB) which is the main survival factor in TNFα signaling. NF-κB suppression occurred through inhibition of IκBα kinase activation, IκBα phosphorylation, IκBα degradation, and NF-κB nuclear translocation. This inhibition correlated with suppression of NF-κB-dependent genes involved in antiapoptosis (mcl-1, bcl-2, bcl-xl, c-iap1, c-iap2, flip, and survivin), proliferation (c-myc, cyclin d1), and angiogenesis (vegf, cox-2, and mmp-9). In addition, digitoflavone can activate JNK through inhibition of NF-κB signaling, provide a continuous blockade of the feed-back inhibitory mechanism by JNK-induced NF-κB activation. This study found a novel function of digitoflavone and enhanced the value of digitoflavone as an anticancer agent

Digitoflavone inhibited TNFα-induced NF-κB transcriptional activity.

<p>PANC-1 cells were cotransfected with NF-κB dependent firefly luciferase reporter construct and constitutively expressing Renilla luciferase construct. The cells were then treated with digitoflavone (40 µM) for different times (1 h, 3 h, 5 h, 7 h). Another group cells (the pretreatment group) were treated with digitoflavone (40 µM) for different times (1 h, 3 h, 5 h, 7 h), followed by TNFα (20 ng/mL) for 2 h. The post-treatment group were treated with TNFα (20 ng/mL) for 2 h, followed by digitoflavone (40 µM) for different times (1 h, 3 h, 5 h, 7 h). Luciferase activity was expressed as fold increased over control after normalized with Renilla luciferase activity. Data are presented as means±s.d. from at least three independent experiments. *<i>P</i><0.05 comparing to TNFα-nontreated control (0 h); ^#<i>P</i><0.05 comparing to the group with TNFa only, and ^△<i>P</i><0.05 comparing to the group TNFα-nontreated Dig-treated control (7 h).</p

Digitoflavone could activate JNK, as well as TNFα, and the activation effect was not weakened when they used together.

<p>Pancreatic cancer cells were incubated with 40 µM digitoflavone for 7 h at 37°C, and then tested for phosphorylated JNK in cytosolic fractions by Western blotting analysis.</p

Digitoflavone inhibited TNFα-induced IKK activation.

<p>A, digitoflavone inhibited TNFα-induced phosphorylation of IKKα/β and IκBα. Pancreatic cancer cells were incubated with 40 µM digitoflavone for 7 h before exposing to TNFα for different times, and then tested for phosphorylated IKKα/β and IκBα in cytosolic fractions by Western blotting analysis. B, digitoflavone had a good binding potency to the ATP binding site of IKK, with Kds of 7.3 µM and 5.2 µM for IKKα and IKKβ respectively.</p

Digitoflavone inhibited expression of antiapoptosis proteins, proliferation proteins, and angiogenesis proteins induced by TNFα.

<p>Pancreatic cancer cells were left untreated or incubated with 40 µM digitoflavone for 7 h and then exposed to TNFα for different times. Whole cell extracts were prepared and analyzed by Western blotting (Fig. 7A–C) or qPCR (Fig. 7D).</p

Pancreatic cancer cells were serum starved for 12α (20 ng/mL), digitoflavone (40 µM), alone or together for 24 h.

<p>Cell apoptosis was determined by Annexin V FITC Apoptosis Kit. *<i>P</i><0.05 comparing to non-treated control; and #<i>P</i><0.05 comparing to the group with TNFα only.</p

Overexpression of p65 prevented digitoflavone-induced JNK activation.

<p>PANC-1 Cells were transfected with pCMV-p65 or empty vector and treated with digitoflavone (40 µM) for 3 h or 7 h. JNK activity was determined by western blotting in whole-cell lysates.</p