Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells

Abstract

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.

Figure 1. BME treatment increases cytotoxic activity of NK3.3 cells on HNSCC cells

(A) NK3.3 cells were treated with BME for 24 hr. Cell number and viability was measured by Trypan blue dye exclusion method. Results shown are an average of three independent experiments. Cell viability at 0 hr time point was arbitrarily set to100%. (B) Cal27 or JHU-29 cells were co-cultured with different T:E ratios of control or BME treated NK cells, and cytotoxicity was measured. Data are represented as mean ± SD.* p<0.05. (C) Representative microscopic images (10X) showing untreated HNSCC cells (control), HNSCC cells exposed to untreated NK (NK) or BME treated NK cells (NKBME) with T:E ratio 1:10. Photographs were taken after 24 hr of co-culturing BME treated NK cells with target cells.

Figure 2. BME treatment increases granzyme B expression in NK 3.3 cells

(A) Western blot analysis of granzyme B expression in control or BME treated NK3.3 cells when co-cultured with Cal27 and JHU-29 cells. (B) PhosphoSTAT3/STAT3 expression in control or BME treated NK3.3 cells co-cultured with Cal27 was examined by Western blot using specific antibody. Similarly, lysates from NK cell were untreated or BME treated were analyzed for phosphoSTAT3/STAT3 expression. Blots were re-probed with an antibody to actin for comparison of protein loading in each lane. Densitometric analyses of was performed using Image J software. Data are represented as mean ± SD. *, p<0.05, **, p<0.01

Figure 3. BME treatment increases CD107a/LAMP1 expression on NK 3.3 cells

(A) FACS analysis of the cell surface expression of CD107a/LAMP-1 on NK cells. Dot plot analysis of the surface expression of CD107a on CD56 positive NK3.3 cells and BME exposed NK3.3 cells with or without co-culture with HNSCC cells. (B) Western blot data showing CD107a/LAMP1 expression in NK3.3 cells. Blots were re-probed with an antibody to actin for comparison of protein loading in each lane. Densitometric analyses were performed using Image J software. Data are represented as mean ± SD. * p<0.05

Figure 4. BME treatment increases CD16 and NKp30 surface expression on NK3.3 cells

(A) FACS analysis showing the populations of CD16⁺ NK3.3 cells exposed with BME or left untreated when co-cultured with Cal27 or JHU-29 cells. The right panel showed the quantitation of the CD16 as % of NK cell population (B) FACS analysis showing expression of NKp30 on NK3.3 and BME treated NK3.3 cells co-cultured with Cal27 and JHU-29 cells. Data are represented as mean ± SD. * p<0.05 (C) Overlay of histograms showing expression of CD314/NKG2D, NKp46, and CD161 by FACS analysis on NK3.3 cells exposed with BME as described above.