Effects of combining rapamycin and resveratrol on apoptosis and growth of TSC2-deficient xenograft tumors

Abstract

Lymphangioleiomyomatosis (LAM) is a rare neoplastic metastatic disease affecting women of childbearing age. LAM is caused by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) as a consequence of tuberous sclerosis complex (TSC) 1/2 inactivation. Clinically, LAM results in cystic lung destruction. mTORC1 inhibition using rapamycin analogs (rapalogs) is partially effective in reducing disease progression and improving lung function. However, cessation of treatment results in continued progression of the disease. In the present study, we investigated the effectiveness of the combination of rapamycin treatment with resveratrol, an autophagy inhibitor, in the TSC2-null xenograft tumor model. We determined that this combination inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase PI3K/Akt/mTORC1 signaling and activates apoptosis. Therefore, the combination of rapamycin and resveratrol may be an effective clinical strategy for treatment of LAM and other diseases with mTORC1 hyperactivation.

Keywords: LAM; TSC2; mTORC1; rapamycin; resveratrol.

Figure 1.

Combination of rapamycin (Rapa) and resveratrol (Rsv) inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt and mechanistic target of rapamycin (mTOR) signaling pathways in lymphangioleiomyomatosis (LAM) and Eker rat uterine leiomyoma-derived TSC2-deficient (ELT3) cells. (A) LAM cells were treated with 20 nM rapamycin and/or 100 μM resveratrol for 24 hours. Cells were lysed, and indicated proteins were detected by immunoblot. (B) LAM cells were treated as described in (A). (C and D) ELT3 cells were treated as described in (A). Each experiment was performed at least three times to ensure reproducibility. PARP, poly(ADP-ribose) polymerase-1.

Figure 2.

The combination of rapamycin and resveratrol reduces cell migration of LAM and ELT3 cells. (A) LAM cells grown to confluent monolayer were scratched and treated with 20 nM rapamycin and/or 100 μM resveratrol for 17 hours. Representative images at the 0- and 17-hour time points are shown. (B) ELT3 cells were treated as described in (A). (C) Measurement and quantification of LAM cell migration was performed using EVOL FL Auto and Excel software. Statistical analysis was performed using two-tailed Student’s t test. (D) Measurement and quantification of the ELT3 cell migration was performed as described in (C). Scale bars, 400 μm. *P < 0.05; **P < 0.01. C, control; R, rapamycin.

Figure 3.

The combination of rapamycin and resveratrol prevents cell migration. Cell migration/Boyden chamber assay was performed as described in Materials and Methods. (A) Representative images of ELT3 cells stained with 4′,6-diamidino-2-phenylindole after a 6-hour migration assay. (B) Histogram representing the number of cells migrated relative to untreated control. Scale bars, 400 μm. **P < 0.01.

Figure 4.

Combination treatment with rapamycin and resveratrol reduces tumor size and inhibits growth. Female CB17-scid mice were inoculated with ELT3-luciferase cells subcutaneously. Mice were treated with vehicle, rapamycin, resveratrol, or a combination of rapamycin and resveratrol for 4 weeks. (A) Body weight was measured every week. (B) Tumor volume was measured weekly using a digital caliper. The y axis indicates the relative fold growth of tumor size versus the baseline measurement before drug treatment. (C and D) Bioluminescent intensity in xenograft tumors was recorded and quantified weekly. The y axis indicates the relative tumor growth versus the baseline quantification before drug treatment. *P < 0.05; **P < 0.01. Vehicle- and resveratrol-treated tumors progressed faster than the tumors in rapamycin or rapamycin–resveratrol groups, and ulcers developed in the skin of tumor areas; thus, those animals had to be killed at Week 3.

Figure 5.

The combination of rapamycin and resveratrol inhibits mechanistic target of rapamycin complex 1/Akt signaling in xenograft tumors. (A) Tumors were lysed as described in Materials and Methods, and indicated proteins were detected by immunoblot. Numbers represent different mice within the treatment group. (B) Quantification of immunoblots was performed using Image Studio 4.0 program and Excel. *P < 0.05; **P < 0.01.

Figure 6.

The combination of rapamycin and resveratrol reduces proliferation in xenograft tumors. (A) Quantification of Ki617-positive cells was performed as described in Materials and Methods. *P < 0.05. After immunohistochemistry, representative images for control (B), rapamycin (C), resveratrol (D), or the combination of rapamycin and resveratrol (E) were created using an EVOS Fl Auto microscope (original magnification: ×40). Arrows indicate Ki67-positive cells. Scale bars, 100 μm.

Figure 7.

The combination of rapamycin and resveratrol induces apoptosis in xenograft tumors. (A) Tumors were lysed as described in Materials and Methods, and caspase-3 and actin were detected by immunoblot. (B) Quantification of caspase-3 was performed using Image Studio 4.0 program and Excel. *P < 0.05; **P < 0.01. (C) Quantification of TUNEL-positive cells was performed as described in Materials and Methods. *P < 0.05. After immunohistochemistry, representative images for control (D), rapamycin (E), resveratrol (F), or the combination of rapamycin and resveratrol (G) were created using an EVOS Fl Auto microscope (original magnification: ×40). Arrows indicate TUNEL-positive cells. Scale bars, 100 μm. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.