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, 32 (6), 585-92

Honokiol-mediated Inhibition of PI3K/mTOR Pathway: A Potential Strategy to Overcome Immunoresistance in Glioma, Breast, and Prostate Carcinoma Without Impacting T Cell Function

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Honokiol-mediated Inhibition of PI3K/mTOR Pathway: A Potential Strategy to Overcome Immunoresistance in Glioma, Breast, and Prostate Carcinoma Without Impacting T Cell Function

Courtney Crane et al. J Immunother.

Abstract

Inhibition of the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is an appealing method for decreasing the immunoresistance and augmenting T cell-mediated immunotherapy. A major impediment to this strategy is the impact of conventional PI3K/mTOR pathway inhibitors on T cell function. In particular, rapamycin, is a well-known immunosuppressant that can decrease the activity of the PI3K/mTOR pathway in tumor cells, but also has a profound inhibitory effect on T cells. Here we show that Honokiol, a natural dietary product isolated from an extract of seed cones from Magnolia grandiflora, can decrease PI3K/mTOR pathway-mediated immunoresistance of glioma, breast and prostate cancer cell lines, without affecting critical proinflammatory T cell functions. Specifically, we show that at doses sufficient to down-regulate levels of phospho-S6 and the negative immune regulator B7-H1 in tumor cells, Honokiol does not significantly impair T cell proliferation or proinflammatory cytokine production. In contrast to classic inhibitors, including LY294002, wortmannin, AKT inhibitor III and rapamycin, Honokiol specifically decreases the PI3K/mTOR pathway activity in tumor cells, but not in freshly stimulated T cells. Collectively, our data define a unique application for Honokiol and provide the impetus to more fully elucidate the mechanism by which T cells are resistant to the effects of this particular inhibitor. Honokiol is clinically available for human testing and may serve to augment T cell-mediated cancer immunotherapy.

Conflict of interest statement

Financial Disclosure: JA has filed for international patent rights for Honokiol. Emory University has filed for US rights. All other authors have declared there are no conflicts of interest in regards to this work.

Figures

Figure 1
Figure 1. Honokiol inhibits PI3K/mTOR pathway activation, B7-H1 protein expression and T cell apoptosis in PTEN deficient glioma cells
(a) PTEN deficient glioma cell lines U87 and U251 were treated for 72 hours with PI3K inhibitors LY294002 (100µM) (Ly294), Wortmannin (100µM) (WORT), AKTIII inhibitor (50µM) (AKTIII), Honokiol (20µM) (HNK) or Rapamycin (100µM) (Rapa) and whole cell lysates were analyzed by western blot as described in Materials and Methods. (b) CD3+ lymphocytes (T cells) were stimulated with anti-CD3/anti-CD28 (3/28) or left unstimulated (NS) and cocultured with inhibitor treated U87 or U251 cell lines for 4 hours. Cocultures were analyzed for T cell apoptosis using CD45/AnnexinV/7-AAD staining. Bars represent the average of triplicate samples from a representative experiment. Error bars represent +/− SD.
Figure 2
Figure 2. Honokiol inhibits PI(3) kinase/mTOR pathway activation, B7-H1 protein expression and T cell apoptosis in PTEN deficient breast and prostate cells
(a) PTEN deficient tumor cell lines BT549 (breast cancer) or PC-3 (prostate cancer) were treated for 72 hours with PI3K inhibitors described in Figure 1 and whole cell lysates were analyzed by western blot as described in Materials and Methods. (b) T cells were stimulated with anti-CD3/anti-CD28 (3/28) or left unstimulated (NS) and cocultured with inhibitor treated BT549 or PC-3 cell lines for 4 hours. Cocultures were analyzed for T cell apoptosis using CD45/AnnexinV/7-AAD staining. Bars represent the average of triplicate samples from a representative experiment. Error bars represent +/− SD.
Figure 3
Figure 3. Honokiol treatment does not reduce T cell apoptosis by increased toxicity to tumor cells
PTEN deficient U87 and U251 tumor cell lines were treated with the following inhibitors for 72 hours : LY294002 (100µM) (Ly294), Wortmannin (100µM) (WORT), AKTIII inhibitor (50µM) (AKTIII), Honokiol (20µM) (HNK) or Rapamycin (100µM) (Rapa). Tumor cells were subsequently analyzed for apoptosis by flow cytometry using Annexin/ 7-AAD staining. Bars represent the average of triplicate samples from a representative experiment. Error bars represent +/− SD.
Figure 4
Figure 4. Honokiol treated T cells proliferate in response to stimulation
(a) Representative flow cytometry of T cells stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with CD3/CD28 in the presence of inhibitors. 72 hours later, cells were analyzed for dilution of CFSE. (b) Average percentage of proliferating T cells following 72 hour CD3/CD28 stimulation. Percentages were determined using the gates drawn on the raw data shown in (a). Bars represent the average of triplicate samples from a representative experiment. Error bars represent +/− SD. (c) CFSE dilution by CD3+ T cells 72 hours after CD3/CD28 stimulation in the presence of increasing doses of Honokiol.
Figure 5
Figure 5. Honokiol treated CD8 + T cells produce pro-inflammatory cytokines in response to stimulation
(a) Average percentage of CD8+ T cells expressing CD25 on the cell surface. T cells were stimulated for 72 hours as described in Materials and Methods and analyzed for surface expression of CD4 (x-axis) and CD25 (y-axis) by flow cytometry (b–c) T cells were stimulated for 72 hours and analyzed for CD8 (x-axis) expression and intracellular accumulation of (b) IFNγ (y-axis) or (c) IL-17 (y-axis). Bar graphs are representative of the average expression of triplicate samples. Percentages were determined using the gates drawn on raw flow cytometry data.
Figure 6
Figure 6. Honokiol treatment, but not AKT III inhibitor treatment allows activation of PI3K/TOR pathway in T cells
(a) T cells or (b) U87 glioma cells were stimulated for 72 hours in the presence of Honokiol (20µM) or AKT III inhibitor (50µM) and analyzed for S6 phosphorylation by western blot. Alpha tubulin was used as a loading control. Densitometry data provided a quantitative measure of protein expression levels.

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