The targeting of phosphoinositide-3 kinase attenuates pulmonary metastatic tumor growth following laparotomy

Abstract

Objective: We aimed to characterize a potential role for phosphatidylinositol 3-kinase (PI3k) in leading to accelerated postoperative metastatic tumor growth.

Background: PI3k enhances tumor cell survival in part by phosphorylating Akt and reducing apoptosis. Postoperatively, apoptosis is reduced within local recurrences and distant metastases. This reduction is associated with the phenomenon of accelerated postoperative tumor growth.

Methods: Balb/c mice underwent a tail vein injection of 1x10 metastatic murine mammary adenocarcinoma 4T1 cells. Animals were divided into the following treatment groups (n=10/group): group A, controls; group B, DMSO intraperitoneally (IP) daily from days 14 to 21; group C, IP LY294002 daily from days 14 to 21; group D, laparotomy only; group E, laparotomy followed by IP DMSO for 7 days; and group F, laparotomy followed by LY294002 IP for 7 days. All laparotomies were performed on day 14. Animals were killed at day 28. Metastatic tumor burden was assessed using the lung/body weight ratio and a histologic metastatic index. Mitotic counts and apoptotic indices were established using a combination of hematoxylin and eosin histology and TUNEL immunohistochemistry. A parallel survival study was performed, and PI3k activity was assessed using western blots for phospho-Akt.

Results: Laparotomy was associated with increased systemic tumor burden (P=0.001). Postoperatively, LY294002 significantly attenuated metastatic tumor growth (P<0.001). Effective PI3k inhibition was confirmed by demonstrating a reduced Akt phosphorylation. Moreover, PI3k inhibition led to reduced proliferation, increased apoptosis (P<0.001), and enhanced postoperative survival (P<0.001).

Conclusions: Targeting PI3k with postoperative LY294002 significantly attenuates the acceleration in postoperative metastatic tumor growth seen following laparotomy.

FIGURE 1. Bar charts summarizing pulmonary metastatic burden as indicated by the lung/body weight ratio (A) and the histologic metastatic index (D). Group A were control animals that received no treatment, group B received DMSO (35%) intraperitoneally (IP) daily from days 14 to 21, group C received IP LY294002 (100 μg/day in 35% DMSO) daily from days 14 to 21 (Sigma), group D underwent laparotomy only, group E underwent laparotomy and received IP DMSO (35%) postoperatively for 7 days, and group F underwent laparotomy and received LY294002 (100 μg/day) IP for 7 days postoperatively (n = 10 animals per group). B, Photograph of lungs in a control Balb/c mouse (ie, no laparotomy) killed 4 weeks following the onset of the study. Normal lung parenchyma is evident surrounding pulmonary metastases. C, Photograph of lungs excised from a Balb/c mouse that underwent laparotomy and was killed 2 weeks following the latter. No normal parenchyma is evident.

FIGURE 2. Bar charts summarizing the apoptotic (A), mitotic (D), and mitotic/apoptotic (E) indices for groups A to F (see Fig. 1 for description of groups). B, Photomicrograph (original magnification ×40) demonstrating TUNEL-positive (and hence apoptotic) cells in a metastasis present in the lungs shown in Figure 1B (ie, no laparotomy). C, Photomicrograph (original magnification ×40) demonstrating reduced numbers of TUNEL-positive cells in a metastasis present in the lungs shown in Figure 1C (ie, following laparotomy).

FIGURE 3. A, Line chart summarizing survival in group A (following laparotomy alone), in group B (laparotomy plus DMSO), and group C (laparotomy plus LY294002) (†P < 0.001, ‡P < 0.001, ¥=P < 0.001). B, Photograph demonstrating whole-cell phosphorylated Akt levels in control animals that did not undergo laparotomy, in animals that underwent laparotomy, and in animals that underwent laparotomy and received LY294002.