Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

Abstract

Mammalian target of rapamycin (mTOR) and the microtubules are shown to be potential targets for treating hepatocellular carcinoma (HCC). PI3K/Akt/mTOR activation is associated with resistance to microtubule inhibitors. Here, we evaluated the antitumor activity by cotargeting of the mTOR (using allosteric mTOR inhibitor everolimus) and the microtubules (using novel microtubule-stabilizing agent patupilone) in HCC models. In vitro studies showed that either targeting mTOR signaling with everolimus or targeting microtubules with patupilone was able to suppress HCC cell growth in a dose-dependent manner. Cotargeting of the mTOR (by everolimus) and the microtubules (by patupilone, at low nM) resulted in enhanced growth inhibition in HCC cells (achieving maximal growth inhibition of 60-87%), demonstrating potent antitumor activity of this combination. In vivo studies showed that everolimus treatment alone for two weeks was able to inhibit the growth of Hep3B xenografts. Strikingly, the everolimus/patupilone combination induced a more significant antitumor activity. Mechanistic study demonstrated that this enhanced antitumor effect was accompanied by marked cell apoptosis induction and antiangiogenic activity, which were more significant than single-agent treatments. Our findings demonstrated that the everolimus/patupilone combination, which had potent antitumor activity, was a potential therapeutic strategy for HCC.

Figures

Figure 1

Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 105) were treated with 0.1 μM everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Figure 2

Patupilone inhibited proliferation in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by patupilone. The effect of patupilone on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC50 values of patupilone in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM.

Figure 3

Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 104) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 105) were treated with everolimus (0.1 μM) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Figure 4

Potent antitumor effects of the everolimus/patupilone combination in in vivo models of HCC. Hep3B cells (3 × 106 cells) were inoculated into nude mice by subcutaneous injection. Drug treatments were started on day 20 after inoculation. Mice received administration of drugs for two weeks (black arrow: patupilone i.p. injection; red arrow: everolimus orally given). (a) Treatment of mice with everolimus, patupilone, or combination suppressed tumor growth in established xenografts of Hep3B. Tumor growth was monitored twice weekly. Arrows indicated time of drug administration. The in vivo antitumor activity of everolimus/patupilone combination was more significant than either agent alone (n = 10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group). (b) Tumor weight of Hep3B xenografts in each group. The tumor weight of everolimus/patupilone combination group was significantly reduced (n = 10 per group, **P < 0.01 versus vehicle group). (c) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment in Hep3B xenograft. Tumor xenografts were harvested, fixed, and stained for pi-mTOR and pi-S6 by immunohistochemistry. Representative images (400x magnification) were shown. Quantitation of pi-mTOR and pi-S6 staining using immunohistochemistry scoring was shown in (B) (n = 10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group).

Figure 5

Everolimus/patupilone combination induced cell apoptosis and exerted antiangiogenic effect in HCC models. Tumor xenografts were harvested, fixed, and stained for cleaved PARP and CD34 by immunohistochemistry. Representative images (400x magnification) were shown. Quantitation of cleaved PARP and CD34 (microvessel density, MVD) staining was shown in (b) (n = 10 per group, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle group).