Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells

Abstract

Nonstop proliferation and vigorous neovascularization are two prominent characteristics of cancer. Antiangiogenic therapy has emerged as an important modality in treatment of solid tumors. Our previous work demonstrated that microparticles derived from apoptotic T-lymphocytes (LMPs) not only reduced the viabilities of high-proliferating cells, but also exhibited potent antiangiogenic effects through inhibition of the vascular endothelial growth factor (VEGF)/VEGF receptor 2 signalling pathway. In the present study, we extended these studies to explore the anticancer potential of LMPs using a murine model of Lewis lung carcinoma (LLC). Results show that intratumoral injection of LMPs (2.5 mg/kg) decreased tumor size by more than 50% relative to control. Tumor microvessel density and VEGF-A levels were also markedly reduced upon LMPs treatment. To elucidate the underlying mechanisms of LMPs-mediated antitumor activity, LLC cells were utilized in in vitro experiments. LMPs suppressed VEGF-A protein levels in LLC cells and led to inhibition of LLC cell viability and proliferation. In addition, knockdown of the low-density lipoprotein receptor (LDLR) expression reduced the uptake of LMPs into LLC cells and attenuated the inhibitory effects of LMPs on cell growth and VEGF-A expression. Our findings demonstrate that LMPs exert antiangiogenic and proapoptotic effects that lead to inhibition of lung carcinoma by reducing VEGF-A levels and LDLR mediates the anti-VEGF effect of LMPs through translocating LMPs into LLC cells. These results suggest that LMPs are promising antiangiogenic therapeutic agent and represent a new therapeutic strategy for treating lung carcinomas.

Figure 1

Inhibitory effects of LMPs on tumor growth in LLC-bearing mice. (A and B) C57BL/6 female mice were sacrificed after 10 days s.c. inoculation with 0.5 × 10⁶ LLC cells in PBS or 50 µg LMPs. Primary tumors were collected and weighed. Representative images (A) and quantifications (B) are presented. (C) After s.c. inoculation with 0.5 × 10⁶ LLC cells for 7 days, C57BL/6 mice were intratumorly injected with 50 µL PBS (control) or 2.5 mg/kg LMPs every 2 days for 4 consecutive injections. Tumors were collected on day 14 and weighed. Representative images of the tumors are shown in (C) and quantification of tumor weight in (D). In each case, values are depicted as mean ± SEM of 8 (B) or 5 (D) mice per group. *p < 0.05, ***p < 0.001 vs. control.

Figure 2

Effect of LMPs on LLC tumor angiogenesis in vivo. (A) Representative images of microvessels in the tumors developed from the mice received LLC and LLC + LMPs injection subcutaneously, stained with TRITC-lectin. (B) Microvessel density was quantified in three different fields (400x). (C) VEGF-A levels were measured via ELISA from fresh frozen LLC tumor lysates and normalized to protein concentrations. Values are presented as percentage of control (set to 100%). *p < 0.05, **p < 0.01 vs. control.

Figure 3

Effects of LMPs on LLC cell viability, proliferation and apoptosis. LLC cell viability (A) and proliferation (B) was measured after 24-h incubation with indicated concentrations of LMPs. (C) Apoptosis was determined by flow cytometry after LMPs treatment (24 h) and expressed as the percentage of apoptotic cells relative to the total number of cells per condition. (D) Representative image of DNA fragmentation assay performed in LLCs with or without 20 ug/mL LMPs. Values are depicted as means ± SEM of 3–5 independent experiments performed in duplicate (C) or triplicate (A and B). The control group was set to 100%. ***p < 0.001 vs. control.

Figure 4

Effect of LMPs on VEGF-A expression and VEGF-induced cell migration. (A) After LMPs treatment (20 µg/mL, 24 h), VEGF-A levels in LLC culture medium and cell lysates were measured by ELISA and normalized to protein concentrations. The values in the control group were set to equal 100%. Values are means ± SEM of 3–5 individual experiments, each performed in triplicate. *p < 0.05, **p < 0.01 vs. control. (B) Representative images of VEGF-induced LLC cell migration in the presence or absence of LMPs (10 µg/mL). Photographs were taken 36 h and 72 h after LMPs treatment. Black arrows indicate direction of cell migration. Images (4x) are from three independent experiments performed in duplicate. (C) Cell migration at 72 h was quantified by MTT assay and is presented as the relative cell migration rate compared to VEGF alone. *p < 0.05.

Figure 5

Influence of LDLR on the uptake of LMPs into LLC cells. (A) Representative fluorescent images of DiI-LMPs uptake into LLC cells. Photos were taken after LLC cells were incubated with 20 µg/mL DiI-LMPs at different time points (10x and 40x magnification in upper and lower parts, respectively). (B) Uptake of DiI-LMPs into LLC cells was evaluated by spectrofluorometry and presented as mean fluorescent intensity (MFI). (C) Efficiency of LDLR downregulation via siRNA in LLC cells. LDLR protein expression was determined by western blot in LLC cells transfected with or without siRNA-LDLR and (D) presented as percentage of control (scramble siRNA). (E and F) Incubation of DiI-LMPs with LLC cells transfected with siRNA-LDLR for 24 h. Fluorescence intensity of DiI-LMPs was assessed in intact cells (E) and in membrane or cytosolic fractions (F) and expressed as a percentage of control (scramble siRNA). *p < 0.05, *p < 0.01 vs. control.

Figure 6

Interfering LMPs effect by LDLR. (A) LLC cells were transfected with siRNA-LDLR subsequent to treatment with LMPs (20 µg/mL). VEGF-A protein expression was determined by western blot analysis and normalized to β-actin. Values are presented relative to control (scramble siRNA without LMPs treatment). (B) Data are presented as means ± SEM. ^#p < 0.05 vs. scramble siRNA + LMPs, ***p < 0.001 vs. scramble siRNA. (C) Cell viability was assessed in LLC cells transfected with siRNA-LDLR and treated with 20 µg/mL LMPs for 24 h. Data are presented as percentage of control (scramble siRNA). ^#p < 0.05 vs. control + LMPs. (D) Cell viability was assessed in LLC cells preincubated with 15 µg/mL LDLR antibody followed by LMPs treatment (20 µg/mL, 24 h). Data are presented as percentage of control. ^#p < 0.05 vs. LMPs, ***p < 0.001 vs. CTL.