Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death

Abstract

Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing.Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment.

Keywords: Citrus limon L.; TRAIL-mediated cell death; cancer; exosome-like nanovesicles.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there is no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported. A patent on the use of Citrus limon L.-derived nanovesicles as antineoplastic agents has been filed (RM2015A000162).

Figures

Figure 1. Nanovesicles characterization and uptake by target cells

A. Citrus nanovesicles were collected after 30% sucrose gradient ultracentrifugation and analyzed at transmission electron microscope. The scale bar indicates 200 nm. B. Nanovesicles size distribution was determined by DLS analysis. C. The Venn diagram shows a comparison between the Citrus nanovesicle data set and protein ExoCarta data set. The overlapping area contains about 60% of proteins identified in Citrus nanovesicles. Most of these common proteins belong to functional groups highly associated with exosomes. D. Analysis at confocal microscopy of A549 (left panel) or LAMA84 cells (right panel) treated, for 3 and 6 hours, with 20 μg/ml of Citrus nanovesicles, compared with untreated cells (Ctrl). Cells were stained with Actin Green 488 (green), nuclear counterstaining was performed using Hoechst (blue), nanovesicles were labeled with PKH26 (red).

Figure 2. Citrus nanovesicles inhibit the growth of tumor cell lines

A. Cell growth was measured by MTT assay after 24, 48, 72 h of treatment with 5 or 20 μg/ml of nanovesicles. The values were plotted as absorbance. Each point represents the mean ± SD of three independent experiments. B. Cancer cell survival was assessed by colony formation assay in methylcellulose. Cells were plated in methylcellulose in presence or not of 5 and 20 μg/ml of Citrus nanovesicles. The values were plotted as fold change compared to control cells (untreated cells). Each point in the histogram represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (Ctrl) (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). Pictures are representative of observed colonies.

Figure 3. Citrus nanovesicles affect the balance between pro- and anti-apoptotic molecules

A. Real-time PCR analysis was performed on A549, SW480 and LAMA84 cell lines treated for 24 or 48 hours with 5 or 20 μg/ml of nanovesicles to evaluate mRNA levels of the pro-apoptotic and anti-apoptotic genes. The values were plotted as fold change compared to control (untreated cells). Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (Ctrl) (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). B. Western blot analysis was performed on cells treated for 48 h with 20 μg/ml of Citrus nanovesicles. Protein levels of the pro-apoptotic (BAX) and anti-apoptotic (BCL-xL) were evaluated. Blots were stripped and subsequently re-probed with an antibody against β-actin to ensure equal loading. Histograms represent densitometry analysis of protein levels in treated cells (Nanoves) versus untreated cells (Ctrl). Each point represents the mean ± SD of three independent experiments.

Figure 4. Citrus nanovesicles induce TRAIL-mediated cell death

A. Real-time PCR analysis was performed on A549, SW480 and LAMA84 cell lines treated for 24 or 48 hours with 5 or 20 μg/ml of Citrus nanovesicles to evaluate mRNA levels of Trail and Dr5. The values were plotted as fold change compared to control (untreated cells). Each point represents the mean ± SD of three independent experiments. B. ELISA was performed to determine TRAIL concentration in the conditioned medium of A549, SW480 and LAMA84 cell lines treated for 24 or 48 hours with 5 or 20 μg/ml of Citrus nanovesicles. The values are expressed in ng/ml. Each point represents the mean ± SD of three independent experiments. Asterisks indicate statistically significant values in comparison to control (Ctrl). C. Cell growth was measured by MTT assay after 48 h of treatment with 20 μg/ml of nanovesicles in presence or not of 5 or 20 ng/ml of neutralizing anti TRAIL antibodies. The values were plotted as % of growth vs control (untreated cells). Each point represents the mean ± SD of three independent experiments. D. Cell death was detected by Annexin V staining after 48 h of treatment with 20 μg/ml of Citrus nanovesicles in presence or not of 20 ng/ml of neutralizing anti TRAIL antibodies. Figure shows representative overlay histogram from untreated cells (solid line), cells treated with nanovesicles (dashed line) or with nanovesicles and neutralizing anti TRAIL antibodies (dotted line). Histogram reported % of Annexin V positive cells in samples treated compared to untreated (Ctrl). Asterisks indicate statistically significant differences (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Figure 5. Citrus nanovesicles inhibit in vivo tumor growth

A. LAMA84 cells were injected subcutaneously in NOD/SCID mice as described. After palpable tumor formation, mice were treated as described in Materials and Methods. Comparison of the median tumor weight was used as an index of the antitumor efficacy of Citrus nanovesicles. Asterisks indicate statistically significant values in comparison to control (Ctrl) (***p ≤ 0.001) B. mRNA levels of pro-, anti-apoptotic genes, Trail and Dr5 were evaluated in samples from mice xenografts. The values were plotted as fold change compared to xenograft control. Each point represents the mean ± SD for three independent experiments. Asterisks indicate statistically significant values in comparison to control (Ctrl) (**p ≤ 0.01; ***p ≤ 0.001). C. Representative images of confocal fluorescence microscopy show TRAIL (green) immunolabeling in paraffin sections from xenografts. Nuclear counterstaining was performed using Hoescht (blue). Arrows indicate TRAIL positive cells. D. Multiplex cytokine evaluation of VEGF-A, IL6 and IL8 in the serum of mice treated or not with nanovesicles. The values are expressed in pg/ml (upper panel). mRNA levels of Vegf-A and Vegf-A receptor were evaluated in samples from mice xenografts (lower panel). The values were plotted as fold change compared to control. Each point represents the mean ± SD for three independent experiments. Asterisks indicate statistically significant values in comparison to control (Ctrl) (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Figure 6. In vivo Citrus nanovesicles biodistribution

A. Representative in vitro fluorescence images of DiR-labeled Citrus nanovesicles dilutions from 50 to 5 μg of nanovesicles in 150 μl of PBS; the quantification of fluorescence signal was calculated in the entire area of each well through the use of ROIs. Data are expressed as average radiance efficiency ([p/s/cm2/steradian]/[μW/cm2]). B. NOD/SCID mice bearing CML xenograft tumors in the right flank were injected intraperitoneally with PBS, Free-DiR, 50 μg Nanovesicles-DiR in a volume of 150 μl PBS. Mice were imaged at 15 min, 1 h and 24 h post injection. A scale of the radiance efficiency is presented to the right of each live mouse image. C. Organs and tumors were excised and imaged after 24 h. A scale of the radiance efficiency is presented to the right. Histogram represents ex vivo quantification of tumor fluorescence.