IN VITRO STUDY OF THE ANTICANCER POTENTIAL OF GINGER-DERIVED EXOSOME-LIKE NANOVESICLES ISOLATED FROM ZINGIBER OFFICINALE VAR. AMARUM BY MODULATING OF MACROPHAGE ACTIVITY

• Main Author: Firdaus Aryantha, Hasna
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/79139
• Institution: Institut Teknologi Bandung

Cancer is currently one of the most urgent public health challenges, both globally and nationally. In Indonesia, this issue is illustrated by a death toll of 234.511 lives per 2018, placing cancer as the second highest cause of death due to non-communicable diseases. Various challenges in conventional cancer therapy—such as multi-drug resistance (MDR) and pathological side effects—contribute in exacerbating the problem of cancer and its treatment, especially in low to middle income countries, such as Indonesia. Therefore, the development of alternative cancer therapeutics remain a proliferative area of research, including those derived from natural sources. Ginger-derived exosome-like nanovesicles (GDEN), nanovesicles isolated from the ginger rhizome, has been spotlighted as a promising drug candidate in nanomedicine. Several studies have demonstrated its potential as an antiinflammatory agent with high biocompatibility and low immunogenicity. The antiinflammatory characteristics possessed by GDENs have inspired the question on whether GDENs pose anticancer properties through modulation of inflammatory signaling. This study serves to characterize the potential of GDENs as immunomodulatory anticancer agents through modulating macrophage activity in vitro, by determining the effects of conditioned media (CM) isolated from RAW 264.7 murine macrophage cells induced with GDENs on the viability and apoptotic activity of HeLa cervical cancer cells. The proposed hypothesis presents that GDENs pose the potential as nontoxic anticancer immunomodulator agents, and that the conditioned media derived from GDEN-induced macrophages (CMGM) are capable of reducing cellular viability and inducing apoptosis in HeLa cells. GDENs were isolated from the ginger rhizome by serial filtration, centrifugation, and polyethylene glycol MW 6000 (PEG6000) precipitation. Characterization of the size distribution and morphology of GDENs were conducted using a particle size analyzer (PSA) and transmission electron microscopy (TEM). Quantification of GDEN concentration was conducted by the estimation of protein concentration with the bicinchoninic acid (BCA) assay. Internalization of GDENs into RAW 264.7 macrophages was evaluated by fluorescent labelling of GDENs and cell nuclei and observed by confocal fluorescent microscopy. The toxicity of GDENs towards RAW 264.7 cells were evaluated using the MTT assay in a range of GDEN concentrations: 0, 5, 10, 20, 50, 100, dan 200 ?g/mL; in 2 treatment durations: 24 and 48 hours. To collect CMGM, RAW 264.7 cell cultures were induced with 10 ?g/mL and 20 ?g/mL GDEN—proven nontoxic concentrations—and incubated for 48 hours in serum-free media. The effect of CMGM treatment towards HeLa cell viability was evaluated with the MTT assay in a range of concentrations: 25%, 50%, 75% dan 100% v/v; in 2 treatment durations: 24 and 48 hours. The effect of CMGM treatment in inducing HeLa cell apoptosis was evaluated by DAPI staining and visualized by fluorescent microscopy. Apoptosis was evaluated qualitatively by morphological observation of stained nuclei, and semi-quantitatively by the morphometric assessment of the nuclear area factor (NAF). Statistical analysis was conducted by the Kruskal-Wallis and two-way aligned rank transform ANOVA (ART-ANOVA) with the Dunn post-hoc test. The results show that the GDEN isolate had a size range consistent with previous literature with moderate homogeneity. Toxicity evaluation demonstrate that GDENs were relatively nontoxic towards RAW 264.7 cells, with a cell viability value above 50% up to treatment with 200 ?g/mL GDENs for 48 hours. Evaluation of HeLa cell viability after CMGM treatment show that CMGM 10 ?g/mL and 20 ?g/mL significantly reduced HeLa cell viability compared to the group treated with CMGM 0?g/mL and serum-free media as a negative control (p ? 0.05) in 50% v/v to 100% v/v CMGM concentrations and both treatment durations. DAPI staining revealed that HeLa cells treated with 100% v/v CMGM 10 ?g/mL and 20 ?g/mL showed nuclear morphology consistent with apoptosis. This finding was fortified by a significantly lower NAF value shown in HeLa cells treated with CMGM 10 ?g/mL and 20 ?g/mL as compared to the cells treated with CMGM 0 ?g/mL and serum-free media after 24 hours of treatment (p ? 0.05). Taken together, these results show that GDENs are capable of modulating macrophage activity into suppressing cancer growth. Further studies, particularly in vivo, would provide further insight to fortify findings in this preliminary study.