Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jun 3;115(22):4455-63.
doi: 10.1182/blood-2009-10-251082. Epub 2010 Mar 18.

Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas

Aadra P Bhatt  1 , Prasanna M Bhende, Sang-Hoon Sin, Debasmita Roy, Dirk P Dittmer, Blossom Damania
Affiliations

Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas

Aadra P Bhatt et al. Blood. .

Abstract

Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcoma-associated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. Our data have broad applicability for the treatment of cytokine-dependent tumors with PI3K/mTOR dual inhibitors.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schematic representation of cellular pathways leading to cell survival or cell death. The roles PI3K, Akt, and AMPK play in signaling pathways important for cell survival or cell death are shown. The influence of rosiglitazone, miltefosine, perifosine, and NVP-BEZ235 on these pathways is also shown. Phosphorylation of proteins is indicated by “+p.”
Figure 2
Figure 2
Inhibition of PEL cell proliferation induced by ciglitazone and rosiglitazone as measured by MTS assay. Shown in each panel (y-axis) is the absorbance at 490 nm in vehicle (DMSO) without drug (♦) or presence of 100μM ciglitazone (A-D, ■) or 150μM (E-F, ■) or 200μM rosiglitazone (E-F, ●) versus time in hours after drug treatment (x-axis). Each data point is the average of triplicate or quadruplicate measurements. Error bars represent the SD and in most cases are smaller than the symbol.
Figure 3
Figure 3
Effect of glitazone treatment on cellular signaling pathways and tumors in mice. (A) Immunoblot analysis of protein extracts from the indicated cell lines exposed to DMSO (−) or 200μM rosiglitazone for 72 hours, or 100μM ciglitazone for 96 hours was performed to visualize the expression of phosphorylated AMPK (Ser172), mTOR (Ser 2448), S6K (T421/S424), or S6 (S235/236) proteins along with total S6 and β-actin as loading controls. (B) Decrease in the average tumor size in mice treated with rosiglitazone compared with those treated with methylcellulose (vehicle). Volumes of tumors in mice treated with methylcellulose as vehicle (n = 5), 30 mg/kg rosiglitazone (n = 5), or 60 mg/kg rosiglitazone (n = 5) are plotted on the y-axis versus time in days after inoculation on the x-axis. Error bars represent the SD for each group of animals. (C) Increase in phospho-AMPK (pAMPK; T172) and decrease in phospho-S6K (pS6K; T421/S424) in mouse xenograft tumors on treatment with rosiglitazone. Immunohistochemistry of mouse xenograft tumors using antibodies specific for pAMPK and pS6K is shown. No staining was observed in the absence of a specific primary antibody (no antibody). Original magnification ×400.
Figure 4
Figure 4
Effects of alkylphospholipids on PEL. (A) Inhibition of PEL cell proliferation induced by miltefosine (right panel) and perifosine (left panel) as measured by MTS assay. Shown in each panel is the absorbance at 490 nm (y-axis) in the absence of drug (gray circles) or increasing, indicated doses of either miltefosine or perifosine, ranging from 10μM to 50μM. Treatment time is represented on the x-axis. Each data point is the average of triplicate or quadruplicate measurements. Error bars represent the SD and, in most cases, are smaller than the symbol. (B) Immunoblot analysis of extracts harvested from indicated PEL cell lines treated with DMSO (vehicle), the indicated drug, or untreated cells (−). Membranes were probed with antibodies raised specifically against the phosphorylated forms of FOXO1, GSK3β, mTOR, S6K, or S6 proteins. Membranes were probed with anti-actin antibody, as a loading control. (C) Tumor progression is delayed in miltefosine-treated mice and significantly delayed in perifosine-treated mice. Mice were treated with 50 mg/kg miltefosine (n = 5), perifosine (n = 5), or vehicle (n = 5) by intraperitoneal injection and followed for 20 days after formation of palpable tumors. Error bars represent the SD for each group of animals. (D) Tumors excised from miltefosine- and perifosine-treated mice display decreased phosphorylation of the ribosomal S6 protein, compared with vehicle controls. Staining is not observed in sections not incubated with primary antibody. Original magnification ×400.
Figure 5
Figure 5
Low doses of NVP-BEZ235 are sufficient to inhibit proliferation of PEL cells in vitro, by inhibiting the downstream targets of PI3K/mTOR pathway. (A) Inhibition of proliferation of the indicated PEL cell lines on treatment with NVP-BEZ235 as measured by the MTS assay. Shown in each panel is the absorbance at 490 nm (y-axis) in the absence of drug (gray diamond) or increasing, indicated doses of NVP-BEZ235. Treatment time is represented on the x-axis. Each data point is the average of triplicate or quadruplicate measurements. Error bars represent the SD and, in most cases, are smaller than the symbol. (B) The IC50 curve for NVP-BEZ235 showing an IC50 value of 5.68 ± 1.76nM for BC-1 PEL cells. (C) Immunoblot analysis of extracts harvested from PEL cell line treated with DMSO (vehicle) or increasing doses of NVP-BEZ235. Membranes were probed with antibodies raised specifically against the phosphorylated forms of Akt (Ser473), mTOR, S6K, pS6, FOXO1, and GSK3β. Membranes were also probed with anti-actin antibody, as a loading control.
Figure 6
Figure 6
Treatment with NVP-BEZ235 delays tumor progression in vivo in a xenograft model of PEL. (A) Tumor progression is significantly delayed (P < .001) in mice treated with 40 mg/kg NVP-BEZ235 administered by oral gavage. Mice were treated 5 times per week with NVP-BEZ235 (n = 7) or vehicle (n = 6) after the development of palpable tumors. Mice were followed for 20 days, at which point vehicle-treated mice were killed. NVP-BEZ235–treated mice had significantly smaller tumors. Error bars represent the SD for each group of animals. (B) Immunohistochemical analyses reveal decreased phosphorylation of ribosomal S6 protein and Akt (Ser473) in NVP-BEZ235–treated mice. No staining was observed in sections that had not been incubated with specific antibodies. (C) PI3K/Akt inhibition induces apoptosis in PEL. A total of 1 × 106 BC-1 PEL cells were treated with 50μM miltefosine, 50μM perifosine, or 20nM NVP-BEZ235, and the appropriate vehicle controls. Cells were harvested and lysed 12 hours later. Equivalent micrograms of cell lysate for all samples were incubated with a fluorogenic caspase-3 substrate (DEVD-AFC). Caspase-3 cleavage of the fluorescent DEVD substrate was measured on a fluorometer. The percentage of caspase-3 activity in BC-1 cells after incubation with perifosine, miltefosine, or NVP-BEZ235 was calculated compared with vehicle-treated cells. Percentage increase in caspase-3 activity of drug-treated cells compared with the respective vehicle control-treated cells is shown on the y-axis, and the specific inhibitor is shown on the x-axis.
Figure 7
Figure 7
Profile of cellular cytokine levels after inhibition of PI3K/Akt/mTOR pathway members. IL-6 (A), IL-10 (B), IP-10 (C), HGF (D), MIG (E), and VEGF (F) levels on treatment with indicated compounds or with vehicle control. Changes in cytokine levels are represented as percentage change from vehicle-treated levels, which were set to 100%. Error bars represent SD. The PI3K/mTOR dual inhibitor NVP-BEZ235 dramatically reduces levels of the indicated cytokines secreted into the growth medium. P < .001 by Tukey post-hoc test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995;378(6559):785–789. - PubMed
    1. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997;91(2):231–241. - PubMed
    1. del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997;278(5338):687–689. - PubMed
    1. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998;282(5392):1318–1321. - PubMed
    1. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129(7):1261–1274. - PMC - PubMed

Publication types

MeSH terms