Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

Saharut Wongkaewkhiaw; Amaraporn Wongrakpanich; Sucheewin Krobthong; Witchuda Saengsawang; Arthit Chairoungdua; Nittaya Boonmuen

doi:10.1371/journal.pone.0266044

. 2022 Apr 4;17(4):e0266044. doi: 10.1371/journal.pone.0266044

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

Saharut Wongkaewkhiaw ¹, Amaraporn Wongrakpanich ², Sucheewin Krobthong ³, Witchuda Saengsawang ^1,^3,⁴, Arthit Chairoungdua ^1,^4,^5,⁶, Nittaya Boonmuen ^1,^*

Editor: Lay-Hong Chuah⁷

PMCID: PMC8979466 PMID: 35377896

Abstract

Colorectal cancer is the leading cause of cancer-related deaths worldwide, warranting the urgent need for a new treatment option. Plant-derived nanovesicles containing bioactive compounds represent new therapeutic avenues due to their unique characteristics as natural nanocarriers for bioactive molecules with therapeutic effects. Recent evidence has revealed potential anticancer activity of bioactive compounds from Boesenbergia rotunda (L.) Mansf. (fingerroot). However, the effect and the underlying mechanisms of fingerroot-derived nanovesicles (FDNVs) against colorectal cancer are still unknown. We isolated the nanovesicles from fingerroot and demonstrated their anticancer activity against two colorectal cancer cell lines, HT-29 and HCT116. The IC₅₀ values were 63.9 ± 2.4, 57.8 ± 4.1, 47.8 ± 7.6 μg/ml for HT-29 cells and 57.7 ± 6.6, 47.2 ± 5.2, 34 ± 2.9 μg/ml for HCT116 cells at 24, 48, and 72 h, respectively. Interestingly, FDNVs were not toxic to a normal colon epithelial cell line, CCD 841 CoN. FDNVs exhibited selective uptake by the colorectal cancer cell lines but not the normal colon epithelial cell line. Moreover, dose- and time-dependent FDNV-induced apoptosis was only observed in the colorectal cancer cell lines. In addition, reactive oxygen species levels were substantially increased in colorectal cancer cells, but total glutathione decreased after treatment with FDNVs. Our results show that FDNVs exhibited selective anticancer activity in colorectal cancer cell lines via the disruption of intracellular redox homeostasis and induction of apoptosis, suggesting the utility of FDNVs as a novel intervention for colorectal cancer patients.

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer-related death and the fourth most frequent malignant tumor worldwide [1]. Several chemotherapeutic drugs are available for CRC; however, the systemic toxicity to normal cells limits their therapeutic efficacy. These harmful side effects to healthy tissues can be fatal. Therefore, the development of new anticancer agents with fewer toxic side effects is strongly needed [2]. For several decades, traditional medicines from plant extracts and natural compounds have been utilized in cancer treatment [3–6]. Boesenbergia rotunda (L.) Mansf., or fingerroot, an herb in the Zingiberaceae family, is a widely found ginger plant in Southeast Asia [7]. Pinostrobin, pinocembrin, and panduratin A are three pharmaceutical bioactive flavonoids isolated from fingerroot [4]. Both extracts and isolated compounds of fingerroot have been found to have anticancer properties in various cancer cells [8, 9]. For example, the fingerroot crude extracts can suppress the growth of nasopharyngeal carcinoma cells (HK1) [8], human promyelocytic cancer cells (HL-60) [10], and human colorectal adenocarcinoma cells (HT-29) [11]. In addition, several isolated compounds from fingerroot have also been reported to have anticancer activities against various cancer cell lines, including human prostate adenocarcinoma (PC3) [12], human lung adenocarcinoma (A549) [9], and human breast cancer (MCF-7) [13]. However, similar to other anticancer compounds, fingerroot extracts and compounds have non-specific cytotoxic effects on non-cancerous cells, thereby limiting their clinical applications [9, 11, 12]. Thus, the efficacy of fingerroot as an anticancer agent is still in question.

In recent years, increasing evidence has shown health benefits of plant-derived nanovesicles (PDNVs). PDNVs are nano-sized, membrane-bound vesicles [14] which contain several biomolecules, including proteins, lipids, mRNAs, and microRNA [15]. Several studies have elucidated the role of PDNVs in intercellular communications through the transferring their components to target recipient cells [16]. An in vivo study found that PDNVs can deliver cargo to distant organs via blood circulation and regulate organ function [17]. Furthermore, PDNVs are stable under the acidic conditions of the digestive tract [18]. For example, curcumin encapsulated in PDNVs is four times more stable than free curcumin, leading to efficient intestinal cell absorption [17]. Additionally, oral intake of ginger-derived nanovesicles can help maintain intestinal homeostasis in mice [19]. Moreover, PDNVs have several unique benefits, including lower toxicity, non-immunogenicity, effective target cell uptake, and the ability for large-scale preparation [20, 21]. Thus, PDNVs are emerging as an important factor for therapeutics and targeted drug delivery [21].

Although the anticancer activity of crude extracts and isolated compounds of fingerroot in CRC have been extensively reported, the effect of fingerroot-derived nanovesicles (FDNVs) in CRC is still unknown. Therefore, in the present study, we focused on developing a novel biotherapeutic from fingerroot that selectively targets cancer cells. Specifically, we isolated FDNVs and characterized their properties and therapeutic potential in CRC.

Materials and methods

Isolation and purification of fingerroot-derived nanovesicles (FDNVs) and fingerroot extract

Fingerroot was obtained from the Nakhon Pathom province, Thailand. As previously described, isolation of PDNVs was performed with some modifications [22]. The fingerroot was washed 5 times and blended using a clean blender without adding other liquid for homogenization. The blended juice was passed through a cheesecloth to exclude non-homogenized residues and centrifuged twice for 1 h at 10,000×g at 4°C. Next, the supernatant was centrifuged again at 50,000×g at 4°C for 1 h followed by filtration with a 1.2 μm filter (Acrodisc^®, Port Washington, NY, USA). The filtrate was further centrifuged at 100,000×g using a fixed-angle rotor 50.2T-Optima L100-XP (Beckman Coulter, Brea, CA, USA) at 4°C for 1.5 h. Next, FDNV pellets were re-suspended in phosphate-buffered saline (PBS), pH 7.4 before filtration with a 0.45 μm filter (Acrodisc^®). The FDNVs were then purified using qEV original size exclusion chromatography (SEC) column (Izon Science, Christchurch, New Zealand) according to the manufacturer’s protocol. Thirty fractions were collected.

The fingerroot extract was isolated from Boesenbergia rotunda (L.) Mansf. as previously described [23] and kindly provided by Patoomratana Tuchinda, Excellence Center for Drug Discovery (ECDD) and Department of Chemistry, Faculty of Science, Mahidol University.

Determinations of protein concentration and size distribution of FDNVs

Total protein concentration was measured by Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. The size and number of vesicles of fractions 7–9 were determined by nanoparticle tracking analysis (NTA) (Malvern Panalytical, Worcestershire, UK). In brief, 2.5 μl sample was diluted with 1 ml PBS, pH 7.4 to obtain optimal signal count per frame according to the manufacturer’s recommendations (30–50 reads/frame). Samples were injected under constant flow conditions at 25°C, and 3 × 60 s videos were captured. Data were analyzed using NTA 3.4 Build 3.4.003 (Malvern Panalytical).

Zeta potential measurements

To evaluate FDNVs stability, zeta potential analysis was performed as previously described [24]. Briefly, 50 μl FDNVs were diluted in 1 ml sterile distilled water and applied to a Malvern Zetasizer Nano-ZS ZEN3600 (Malvern Panalytical). Zeta potential measurements were carried out using standard settings (viscosity = 0.89, dielectric constant = 80, temperature = 25°C). The data were analyzed by the Zetasizer software version 7.11 (Malvern Panalytical).

Transmission Electron Microscopy (TEM)

Morphology of FDNVs was examined using the negative staining methods [22, 25]. Briefly, drops of FDNVs were deposited onto the surface of a carbon grid and stained with 1% uranyl acetate for 1 min. Images were observed by JEM-1400 TEM (JEOL, Tokyo, Japan) at 100,000X and 300,000X.

Metabolomic profiling of FDNVs

The metabolites were extracted using the previous protocol with minor modifications [26]. Briefly, FDNVs samples were mixed with methanol and incubated at -20°C for 48 h. Then, the solution was centrifuged at 15,000·g for 30 min at 4°C, cleaned using Sep-Pak^® C18 Cartridges (Water^™, Milford, MA, USA), and vacuum evaporated using a Rotavapor^® R-300 (BUCHI, Flawil, Switzerland). The sample was reconstituted in methanol and diluted with 1% formic acid/water at a 1:10 ratio (v/v). Liquid chromatography-mass spectrometry analysis was performed using a Q-Exactive Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher Scientific) coupled to UltiMate 3000 HPLC (Thermo Fisher Scientific). The sample (5 μl) was separated using a Hypersil GOLD^™ C18 (Thermo Fisher Scientific) at 28°C (flow rate of 0.3 ml/min). The total time for each analysis was 35 min. MS was operated in positive mode. A spray voltage of 4.0 kV in both positive, sheath gas and the auxiliary gas flow rate was set at 48 and 11 arbitrary units, respectively. The capillary temperature was 350°C. The MS analysis alternated between MS full scans and data-dependent MS/MS scans with dynamic exclusion. LC-MS for full MS: scan range, 90–900 m/z; resolution 120,000; AGC target 3e⁶; max IT 60 ms and LC-MS for full MS/MS: resolution 30,000; AGC target 1e⁵; max IT 200 ms.

Next, the total ion chromatograms of all the samples were extracted. The acquired raw MS files were processed with Compound Discoverer 3.1 (Thermo Fisher Scientific). The retention time (RT) and mass-to-charge ratio (m/z) of different injections were conducted according to the retention time deviation of 0.5 min and the mass deviation of 10 ppm. Then, the peak extraction was performed according to the set information and adduct information: mass deviation = 5 ppm, signal strength deviation = 30%, and signal-to-noise ratio = 2. The target m/z ions were then integrated to predict the molecular formula and compared against mzCloud and ChemSpider online databases to identify and confirm the compounds. Finally, the classes of plant metabolites in FDNVs were classified according to their chemical structure as previously described [27].

Cell culture

Colorectal cancer (HT-29 and HCT116) and normal human colon epithelial (CCD 841 CoN) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HT-29 cells were cultured with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12). HCT116 cells were cultured with DMEM low glucose. The medium was supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Thermo Fisher Scientific). CCD 841 CoN cells were grown in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and 1% antibiotic-antimycotic. Cells were maintained in a humidified incubator with 95% O₂ and 5% CO₂ atmosphere at 37°C. The cells were sub-cultured using 0.05% Trypsin-EDTA (Thermo Fisher Scientific).

Cytotoxicity assay

Cells were plated at a density of 6.0×10³ cells/well in Costar^® 96-well plates (Corning Inc., Corning, NY) and grown overnight. Cells were incubated with 3.13 to 100 μg/ml of FDNVs or fingerroot extracts for 24, 48, and 72 h at 37 °C in a humidified 5% CO₂ incubator. Untreated cells were used as a negative control. Cell viability was determined by MTT assay (Sigma-Aldrich, St. Louis, MO). Briefly, cells were incubated with 0.5 mg/ml MTT solution at 37°C in a humidified 5% CO₂ incubator for 4 h. The medium was then removed before adding 100% DMSO (Sigma-Aldrich). The absorbance was measured at optical density 570 nm using Multiskan^™ GO Microplate Spectrophotometer (Thermo Fisher Scientific).

Apoptosis assay

Cells were seeded at a density of 1×10⁵ cells/well in 24-well plates. After 24 h, cells were treated with FDNVs at concentrations of 25, 50, and 100 μg/ml for 48 h at 37 °C in a 5% CO₂ atmosphere. Cells treated with 5% DMSO (Sigma-Aldrich) were used as a positive control. The untreated group was treated equally PBS volume to the treated group. After treatments, cells were washed, detached by trypsin-EDTA, and stained with FITC/Annexin V and propidium iodide (PI) using Annexin V-FITC Apoptosis Detection Kit (BioLegend Way, San Diego, CA, USA) according to the manufacturer’s instructions. The stained cells were analyzed using a BD FACSCanto^™ flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed by Kaluza Analysis Software version 2.2.1 (Beckman Coulter).

Quantitative real-time PCR

The expressions of apoptosis-associated genes in FDNVs-treated cells were investigated by quantitative real-time PCR. Briefly, cells (2×10⁵ cells/well) were seeded in a 12-well plate and treated with 6.25–25 μg/ml FDNVs for 24 h. Total RNAs were extracted using TRIzol^™ reagent (Invitrogen^™, Waltham, MA, USA) according to the manufacturer’s protocol. cDNA synthesis was conducted using iScript^™ Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). Targeted gene expressions were determined using iTaq^™ Universal SYBR^® Green Supermix (Bio-Rad) with specific primers. The expression was normalized to the constitutive expression of GADPH and was calculated using the comparative 2^-ΔΔCT method [28]. The result is expressed as fold change from three independent experiments carried out in triplicate. Oligonucleotides for the specific primers are as follows: Bax sense strand, 5’-AAGAAGCTGAGCGAGTGT-3’ and antisense strand 5’-GGAGGAAGTCCAATGTC-3’ [29]; Bcl-2 sense strand, 5’-CTTCTCCCGCCGCTAC-3’ and antisense strand 5’-CTGGGGCCGTACAGTTC-3’ [29]; Caspase-3 sense strand, 5’-TGCCGTGGTACAGAAC-3’ and antisense strand 5’-GACTCAAATTCTGTTGCC-3’ [29]; Caspase-9 sense strand, 5’-CCAGAGATTCGCAAACCA-3’ and antisense strand 5’-CCTGACAGCCGTGAGAG-3’ [29]; and GAPDH sense strand, 5’-ATGGGGAAGGTGAAGGTCG-3’ and antisense strand 5’-GGGTCATTGATGGCAACAATAT-3’ [30].

Cellular uptake of FDNVs

FDNVs (12.5 and 25 μg/ml) were stained with PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Cells were seeded at a density of 5×10⁴ cells/well on coverslips in 24-well plates and cultured overnight. Cells were then incubated with PKH67-labeled FDNVs for 24 h at 37 °C in a 5% CO₂ atmosphere. Non-treated cells were used as a negative control. After incubation, cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Next, nuclei and actin filaments were stained for 30 min with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen^™) and Alexa Fluor^® 647 Phalloidin (Invitrogen^™), respectively. Cells were mounted and imaged using an FV1000 confocal laser scanning microscope (Olympus Corporation, Shinjuku, Japan). The fluorescent intensity was quantified using ImageJ software version 1.48h3 (National Institutes of Health; NIH, Bethesda, MD, USA). The mean fluorescence intensity was normalized by the cell number (50,000 cells).

Inhibition of the cellular uptake of FDNVs

For pinocytosis, cells (5×10⁴ cells/well) were pretreated with pinocytosis inhibitors including 1 μg/ml amiloride (macropinocytosis) (Sigma-Aldrich), 5 μg/ml chlorpromazine (clathrin-mediated endocytosis) (Sigma-Aldrich), and 0.25 μg/ml filipin (caveolae-mediated endocytosis) (Sigma-Aldrich) for 1 h [16]. The medium was then removed, and cells were further incubated with 50 μg/ml PKH67-labeled FDNVs in the presence of these inhibitors for 3 h. For phagocytosis, cells were preincubated with 0.005 μg/ml of cytochalasin D (Tocris Bioscience, Bristol, United Kingdom) for 1 h. The medium was then removed and further incubated with FDNVs for 2 h in the presence of this inhibitor. After incubation, cells were fixed, stained with DAPI, and observed using an FV1000 confocal laser scanning microscope (Olympus).

Measurements of intracellular reactive oxygen species (ROS) levels

Cells were seeded at a density of 1×10⁴ cells/well in CellCarrier-96 Ultra Microplates (PerkinElmer, Waltham, MA, USA) and incubated overnight. FDNVs were added to the cells at concentrations of 12.5, 25, and 50 μg/ml. After 6 h, cells were washed with Dulbecco’s Phosphate Buffered Saline (D-PBS) (Sigma-Aldrich) and incubated with 10 μM CM-H₂DCFDA (Thermo Fisher Scientific) at 37 °C for 30 min in the dark. Cells incubated with 200 μM H₂O₂ (Merck, Darmstadt, Germany) for 3 h were used as positive controls. Cells were washed with D-PBS and the fluorescence signals were measured using an EnVision^® multimode plate reader (PerkinElmer). The level of intracellular ROS was expressed as a ratio to untreated cells.

Measurements of intracellular glutathione (GSH) levels

Cells were seeded at a density of 2×10⁵ cells/well in 12-well plates and incubated overnight. Cells were then incubated with FDNVs at concentrations of 12.5, 25, and 50 μg/ml for 6 h at 37 °C in a 5% CO₂ atmosphere. Cells were then deproteinized with 5% 5-sulfosalicylic acid (Sigma-Aldrich) and centrifuged at 10,000×g at 4°C for 15 min. Total glutathione levels in the supernatant were measured using a Glutathione Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.

Statistical analysis

Data are presented as means ± standard deviation (SD). Statistically significant differences were analyzed by one-way ANOVA and Tukey’s multiple comparisons test using GraphPad Prizm software (version 9.0). P-value <0.05 was considered statistically significant.

Results

Isolation, characterization, and metabolite profiling of FDNVs

FDNVs were isolated from fingerroot juice using differential centrifugation, followed by IZON’s qEV size exclusion chromatography (SEC) column. Thirty fractions (total volume of 500 μl) were collected from the qEV column, and protein concentrations were determined. We detected a substantial concentration of proteins in fractions 7, 8, and 9 (S1 Fig), with the highest protein concentration observed in fraction 8. Fractions 7–9 contained a high concentration of nanovesicles with high purity. Therefore, the particle number and size distribution of fractions 7, 8, and 9 were determined by nanoparticle tracking analysis. As shown in Fig 1A and Table 1, the range of FDNVs sizes was similar in these 3 fractions, the maximum of which is less than 500 nm. The modal sizes of fractions 7–9 were 78.4 ± 7.8, 70 ± 6.3, and 71.1 ± 1.4, respectively, whereas average particle size of fractions 7–9 were 102.1 ± 4.3, 100.2 ± 10.1, and 106.7 ± 2.4 nm, respectively. Moreover, fraction 8 contained the highest number of particles (1.5×10¹¹ ± 7.3×10⁹ particles/ml) compared to the other fractions (Fig 1B). FDNV morphology was examined by transmission electron microscopy (TEM). The FDNVs were round-shaped membrane-bound vesicles less than 100 nm in size (Fig 1C). Consistent with the NTA result, TEM revealed a greater number of particles in fraction 8 than fractions 7 and 9. In addition, all FDNVs fractions showed negative zeta potential (Fig 1D). Fraction 8 showed the highest negative zeta potential value at -26.9 ± 6.1 mV, whereas fractions 7 and 9 were -17.4 ± 3.7 mV and -10.6 ± 5.6 mV, respectively. This result indicates that fraction 8 showed the highest mutual repulsion and no tendency toward aggregated states. Taken together, the characteristics of our isolated FDNVs are compatible with the previous reports on nanovesicles from edible plants [22, 31]. Therefore, fraction 8 was selected for subsequent experiments.

Fig 1 — (A) The size distribution and (B) particle concentration of fractions 7, 8, and 9 were analyzed by NTA. (C) FDNV morphology was observed under TEM at 100,000X (scale bar = 200 nm) and 300,000X (scale bar = 100 nm). (D) The zeta potential of isolated FDNVs was measured using a Zetasizer. (E) Distribution of tentatively identified metabolites in FDNVs using LC-MS/MS. Data are represented as means ± SD of three independent experiments in duplicate; ns = not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Table 1. Size of FDNVs in fractions 7, 8 and 9 using nanoparticle tracking analysis (n = 3).

Fraction	Min size (nm)	Max size (nm)	Modal size (nm)	Mean size (nm)
F7	33.5 ± 2.7	428.5 ± 25.3	78.4 ± 7.8	102.1 ± 4.3
F8	34 ± 1.1	483.5 ± 15.4	70 ± 6.3	100.2 ± 10.1
F9	33.2 ± 9.2	419 ± 8.8	71.1 ± 1.4	106.7 ± 2.4

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Next, we investigated the metabolite profiling of FDNVs. As shown in S1 Table, we identified 58 putative metabolites in FDNVs. The distribution of FDNVs metabolites is shown in Fig 1E. Alkaloids were the most common FDNVs metabolites subtype (53%; 31/58), followed by phenolics (21%; 12/58), lipids (14%; 8/58), and organic compounds (12%; 7/58). Importantly, the phenolic compounds naringenin chalcone, pinostrobin, and pinocembrin were found in FDNVs. These phenolic compounds have been found as promising bioactive compounds in fingerroot juice [4].

Cytotoxicity of FDNVs and fingerroot extract on colorectal cancer cells

We next investigated the cytotoxic effect of FDNVs against two colorectal cancer cell lines, HT-29 and HCT116. FDNVs exhibited dose- and time-dependent cytotoxic effects against both CRC cell lines (Fig 2A and 2B and Table 2). At 25 μg/ml, FDNVs caused cytotoxicity on HT-29 and HCT116 cells after 24 h of incubation. The IC₅₀ values of FDNVs against HT-29 cells were 63.9 ± 2.4, 57.8 ± 4.1, and 47.8 ± 7.6 μg/ml at 24, 48, and 72 h, respectively. The IC₅₀ values of FDNVs against HCT-116 cells were 57.7 ± 6.6, 47.2 ± 5.2, and 34 ± 2.9 μg/ml at 24, 48, and 72 h, respectively. Interestingly, FDNVs had no cytotoxic effects toward normal human colon epithelial cells (CCD 841 CoN) (Fig 2C). In addition, we compared the cytotoxic selectivity between fingerroot extract and its nanovesicles. In contrast to the selective cytotoxic effect of FDNVs, fingerroot extract exhibited dose- and time-dependent effects against both cancer cells and normal human colon epithelial cells (Fig 2 and Table 2). Cytotoxicity of fingerroot extract was significantly observed at 25 μg/ml after 24 h of treatment toward all tested cells (P < 0.001). Additionally, there was no difference between the IC₅₀ values of the fingerroot extract against all tested cells. These results indicate the selective cytotoxic effect of FDNVs on colorectal cancer cells with relatively low cytotoxicity toward normal colon cells.

Fig 2 — Cell viabilities of (A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were determined using MTT assay after treatment with FDNVs and fingerroot extract for 24, 48, and 72 h. Data are represented as means ± SD of three independent experiments in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

Table 2. IC₅₀ values of FDNVs and fingerroot extract against colorectal cancer cells.

Cell line	IC₅₀ (μg/ml)
	FDNVs			Fingerroot extract
	24 h	48 h	72 h	24 h	48 h	72 h
HT-29	63.9 ± 2.4	57.8 ± 4.1	47.8 ± 7.6	59.1 ± 0.5	46.3 ± 3.5	34.9 ± 2.9
HCT116	57.7 ± 6.6	47.2 ± 5.2	34 ± 2.9	57.7 ± 3.2	40.8 ± 3.3	30.8 ± 1.7
CCD 841 CoN	N/A	N/A	N/A	57.6 ± 2.9	44.3 ± 0.3	29.6 ± 3.8

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N/A = Not applicable.

Cellular uptake of FDNVs

To investigate the differential cytotoxic effect of FDNVs on CRC and colon epithelial cells, we next examined the uptake of FDNVs into cancer cells and normal colon epithelial cells (Fig 3). Cells were incubated with 12.5 μg/ml FDNVs labeled with PKH67 (green) for 24 h. The intracellular green fluorescence signals of PKH67-labeled FDNVs were detected in both CRC cell lines (Fig 3A and 3B). Moreover, intracellular fluorescence was positively correlated with FDNV concentration in CRC cells, as we observed increased fluorescence after incubation with at a higher concentration of FDNVs (25 μg/ml). In contrast, we detected significantly lower fluorescence intensity of PKH67-labeled FDNVs in CCD 841 CoN cells (Fig 3C) compared with HT-29 (P < 0.001) and HCT116 (P < 0.001) cells (Fig 3A, 3B and 3D). There were no green fluorescence signals in vehicle control-treated cells. The quantification of fluorescence intensity per 50,000 cells is shown in Fig 3D. These findings may partially explain the selective cytotoxic effect of FDNVs on colorectal cancer cells.

Fig 3 — (A) HT-29, (B) HCT116, and (C) CCD 841 CoN cells were incubated with PKH67-labeled FDNVs (green) for 24 h. Cells were also stained with DAPI (blue) and Phalloidin (red) to label the nucleus and actin filaments, respectively. (D) The green fluorescence intensity of FDNVs was determined using ImageJ software. The values are shown as means ± SD of three independent experiments in duplicate. ***P < 0.001 (one-way ANOVA). Scale bar = 10 μm.

Since the selective cytotoxic effects of FDNVs likely involve cellular uptake, we investigated the uptake mechanism of FDNVs in colorectal cells. Generally, there are two major endocytosis pathways, including phagocytosis and pinocytosis. Pinocytosis divides into three subcategories: micropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis [16]. Thus, we incubated cells with FDNVs and uptake inhibitors to block cellular uptake. As shown in Fig 4, the uptake of FDNVs in HT-29 and HCT116 were markedly inhibited by filipin (Fig 4A) and cytochalasin D (Fig 4B), which are inhibitors of caveolae-mediated endocytosis and phagocytosis, respectively (P > 0.001). Conversely, treatment with amiloride, an inhibitor of micropinocytosis, and chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not affect the uptake of FDNVs in both cancer cell lines (Fig 4A). These data suggest that the internalization of FDNVs in colorectal cancer cells is partly via caveolae-mediated endocytosis and phagocytosis pathways.

Fig 4 — (A) HT-29 and HCT116 cells were pretreated with chlorpromazine, amiloride, and filipin for 1 h and then incubated with PKH67-label FDNVs (green) for an additional 3 h in the presence of the inhibitors. (B) HT-29 and HCT116 cells were pretreated with cytochalasin D for 1 h and then incubated with PKH67-label FDNVs for an additional 2 h in the presence of the inhibitor. Cells were then fixed and stained with DAPI (blue). The green fluorescence intensity of FDNVs was determined by ImageJ software. Bar graphs show the uptake efficiency of FDNVs in HT-29 and HCT116 cells. The values are presented as means ± SD of three independent experiments in duplicate. ***P < 0.001 (one-way ANOVA). Scale bar = 10 μm.

FDNVs induce colorectal cancer cells apoptosis

The effect of FDNVs on apoptosis induction in CRC cells was further investigated by FITC/Annexin V staining and flow cytometry (Fig 5). As shown in Fig 5A and 5B, treatment with FDNVs markedly induced apoptosis in HT-29 and HCT116 cells in a dose-dependent manner. Treatments with FDNVs at 25, 50, and 100 μg/ml significantly caused early apoptosis in HT-29 cells, up to 6.4 ± 1.2% (P < 0.05), 12.2 ± 2.4% (P < 0.001), and 18.1 ± 2.4% (P < 0.001), respectively, compared to untreated control (2.9 ± 1.1%) (Fig 5A). In addition, late apoptotic population increased in these cells after treatment with FDNVs at 50 and 100 μg/ml compared with control. Similarly, treatment with FDNVs at 50 and 100 μg/ml significantly induced early apoptosis in HCT116 cells, up to 11.3 ± 5.6% (P < 0.05) and 19.8 ± 3.1% (P < 0.01), respectively, compared to untreated cells (1.4 ± 0.7%). Late apoptotic population also increased in HCT116 cells treated with 50 and 100 μg/ml FDNVs (Fig 5B). However, a significant increase in necrotic cell death was found only in the HT-29 cells after treatment with a higher dose of 100 μg/ml FDNVs (P < 0.05) (Fig 5A). In addition, the percentage of viable cells decreased in both CRC cell lines after treatment with FDNVs (Fig 5A and 5B). These results indicate that the cytotoxic effect of FDNVs in CRC cells are mediated through apoptosis induction. To further elucidate the mechanism of the differential cytotoxic effects of FDNVs on CRC and human colon epithelial cells, we examined FDNV-induced apoptosis in normal human colon epithelial cells, CCD 841 CoN (Fig 5C). There was no significant induction of early apoptosis in all tested concentrations of FDNVs, as compared to untreated control. Statistically significant induction of apoptosis was only found in the presence of 5% DMSO in human colon epithelial cells (32.2 ± 5.1%, P < 0.001). More than 90% of cells remained viable even at a high concentration of FDNVs, indicating that FDNVs exhibited low cytotoxicity against normal colon cells. In contrast, treatment with 5% DMSO resulted in a significant reduction of cell viability (P < 0.001) relative to control. Late apoptosis and necrosis were not significantly different in all tested conditions. These results demonstrated that FDNVs displayed selective induction of apoptosis-mediated cell death in cancerous, but not normal, cells.

To further confirm the underlying mechanism of apoptosis induction of FDNVs, the effect of FDNVs treatment on the expression of apoptosis-related genes was examined by quantitative RT-PCR analysis (Fig 6). Treatment with FDNVs at 12.5 and 25 μg/ml markedly increased the expression of caspase-3 and caspase-9 in HT-29 and HCT116 cells (Fig 6A and 6B). An increase in the expression of Bax, a pro-apoptotic gene, was also observed after treatment with 25 μg/ml FDNVs in both CRC cell lines. In contrast, the expression of Bcl-2, an anti-apoptotic gene, was decreased in both CRC cell lines after treatment with FDNVs at 25 μg/ml. These results indicate that FDNVs-mediated apoptosis induction in cancer cells is associated with the up-regulation of caspase and pro-apoptotic genes and the suppression of an anti-apoptotic gene.

Fig 6 — (A) HT-29 and (B) HCT116 cells were treated with FDNVs for 24 h. The expression of target genes was determined using quantitative RT-PCR. The relative quantitation of each gene was normalized to the constitutive expression of GADPH. The results are mean ± SD of three independent experiments in duplicate and presented as fold change. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with untreated cells (one-way ANOVA).

FDNVs increased ROS generation but decreased glutathione levels in colorectal cancer cells

We next investigated whether FDNVs-induced apoptosis was mediated by reactive oxygen species (ROS) (Fig 7). Treatment with FDNVs at 12.5, 25, and 50 μg/ml significantly increased ROS levels in HT-29 cells, up to 1.2 ± 0.18 (P < 0.05), 1.3 ± 0.04 (P < 0.01), and 1.4 ± 0.09 (P < 0.001), respectively, compared to untreated control (Fig 7A). A similar result was observed in HCT116 (Fig 7B). Relative to control-treated cells, ROS levels were significantly increased up to 1.5 ± 0.22% (P < 0.01), 1.7 ± 0.14% (P < 0.001), and 1.8 ± 0.21% (P < 0.001) in HCT116 cells treated with FDNVs at 12.5, 25, and 50 μg/ml, respectively. Similarly, treatment with H₂O₂, a positive control, significantly increased ROS concentrations up to 1.3 ± 0.13 (P < 0.01) in HT-29 cells and 1.4 ± 0.09 (P < 0.05) in HCT116 cells. Additionally, treatment with FDNVs did not affect the induction of intracellular ROS in CCD 841 CoN (Fig 7C).

To examine the disruption of redox balance, we determined the level of glutathione (GSH) in FDNVs-treated cells (Fig 7). Treatments of HT-29 cells with FDNVs at 25 and 50 μg/ml significantly decreased GSH levels to 65.5 ± 3.2 nmoles/ml (P < 0.05) and 34.3 ± 7.2 nmoles/ml (P < 0.001), respectively, which was significantly lower than control-treated cells (73.8 ± 3.1 nmoles/ml) (Fig 7A). Similarly, after treatment with FDNVs at 12.5, 25, and 50 μg/ml, GSH levels in HCT116 cells were significantly reduced to 66.4 ± 3.9 nmoles/ml (P < 0.01), 60.8 ± 1.3 nmoles/ml (P < 0.001), and 47.8 ± 2.7 nmoles/ml (P < 0.001), respectively. This was significantly lower than untreated cells (80.8 ± 3.9 nmoles/ml) (Fig 7B). Treatment with H₂O₂, as a positive control, reduced GSH levels in both CRC cell lines (Fig 7A and 7B). In contrast to CRC cells, in CCD 841 CoN cells, only H₂O₂ causes significantly reduced levels of GHS (P < 0.01, Fig 7C). These data suggest that FDNVs showed selective cytotoxicity towards cancer cells through increased ROS production. Moreover, FDNVs-induced apoptosis in CRC cell lines is possibly due to disruption of the redox balance leading to apoptotic cell death; thus, FDNVs have significant potential to be developed as selective anticancer drugs.

Discussion

In the present study, we provided the first isolation and characterization of nanovesicles from fingerroot. More importantly, we demonstrated promising selective anticancer effects of these nanovesicles against CRC cells. As such, fingerroot-derived nanovesicles (FDNVs) exerted their anticancer activity by stimulating apoptotic mechanisms mediated through ROS production. Furthermore, FDNVs did not cause toxicity to normal human colon epithelial cells. These findings highlight an alternative approach in using nanovesicles from natural sources in cancer therapy.

Fingerroot possess several pharmacological activities, including antiviral [23], anti-inflammatory [32], and potential anticancer [8, 11] effects. Currently, the nanovesicles extracted from plants exhibit excellent potential for therapeutic applications against various diseases [33]. With the recent increased interest in the therapeutic potential of plant-derived nanovesicles (PDNVs), several groups have attempted to isolate and develop plants as natural green nano-factories to investigate their biomedical utility [21, 22]. However, the isolation, characterization, and biological activity of nanovesicles isolated from fingerroot or FDNVs have not been reported. In this study, we established a protocol to isolate nanovesicles from fingerroot. The standard protocol for PDNV isolation and characterization has only recently been fully developed [14]. Differential centrifugation is widely used for nanovesicles isolation. Consequently, size-, density- and immunoaffinity-based techniques have been applied to purify and reduce non-vesicular extracellular materials [34]. Here, we used the differential centrifugation method followed by a qEV size exclusion chromatography (SEC) column to isolate and purify the FDNVs. We found that the isolated FDNVs from our protocol exhibited characteristics of nanovesicles similar to those isolated from other edible plants [22, 31]. The FDNVs were approximately 100 nm in diameter, which was similar to the report of nanovesicles derived from ginger [16]. Although the previous study showed that nanovesicle isolation using the immunoaffinity-based technique (ExoQuick plus) provided the highest particle concentration, the qEV column and sucrose density-gradient separation methods contain less protein contamination than the immunoaffinity-based technique [34, 35]. Therefore, the established protocol for isolation FDNVs in this study may help isolate nanovesicles from other types of plants.

Plant secondary metabolites play a crucial role in the pharmacological actions of medicinal plants [27]. Our FDNVs contained alkaloids, phenolics, lipids, and organic compounds, which illustrates the diversity of phytochemical constituents in FDNVs. There have been reports of anticancer potentials of phenolic compounds via ROS-mediated apoptosis, such as naringenin chalcone [36], pinostrobin [37], and pinocembrin [38]. In addition, valerenic acid (lipid) and darymid A (alkaloid) have also been found to possess anticancer activity [39, 40]. Perhaps these secondary metabolites may serve as medicinal agents that underlie the therapeutic action of FDNVs, which can improve our understanding of how FDNVs exhibit biological activities. Current chemotherapeutic drugs for CRC have off-target effects that cause toxicity to both cancer cells and their normal counterparts [9, 11, 12]. Therefore, finding anticancer agents with a high level of specificity may help reduce side effects for CRC patients and improve their quality of life [2]. Previously, extracellular vesicles from plant-sap have been revealed to have selective cytotoxic effects on tumor cells rather than normal cells [16]. Herein, we demonstrated that FDNVs exhibit anticancer activity against two CRC cell lines (HT-29 and HCT116); however, FDNVs have reduced cytotoxicity toward normal colon epithelial cells (CCD 841 CoN). On the contrary, the fingerroot extract exhibited a cytotoxic effect against both CRC and normal colon cells. This is consistent with other studies, which have found anticancer activity of fingerroot extract against several cancer cell lines [8, 10, 11]. However, fingerroot extract induced cytotoxicity on non-cancerous cells, such as non-transformed human skin fibroblast cells (SF 3169) [11], normal hepatic cells (WRL68) [12], and normal colon epithelial cells (CCD 841 CoN) [9]. Importantly, our results illustrate a tremendous potential of FDNVs to selectively target CRC cells relative to the parental fingerroot extract.

Our study also found that both types of CRC cells were more susceptible to FDNV uptake than normal colon cells. FDNVs were taken up and preferentially localized in the cytoplasm of cancer cells. For example, ginger-derived nano-lipids loaded with doxorubicin were mainly internalized via the phagocytosis pathway into CRC cancer cells that were significantly inhibited by cytochalasin D [41]. Moreover, the internalization of plant sap-derived extracellular vesicles in breast cancer cells was mediated by phagocytosis and caveolae-mediated endocytosis [16]. Thus, our findings are consistent with other reports that the internalization of FDNVs into CRC cells is likely due to phagocytosis and caveolae-mediated endocytosis. Caveolin-1, the principal structural component of caveolae, is involved in caveolae-mediated endocytosis [42]. Although caveolin-1 function in cancer is controversial, overexpression of caveolin-1 has been reported in colon cancer [43]. Therefore, the caveolae-mediated endocytosis may be more effective in CRC, resulting in larger amounts of FDNVs internalization than in normal colon epithelial cells. In addition, the internalization of garlic-derived nanovesicles is mediated by interaction with the CD98 heavy chain (CD98hc) in liver cancer cells (HepG2) [44]. Expression levels of CD98hc protein were higher in CRC tissues than in matched normal tissues [45]. Therefore, upregulation of CD98hc might support the uptake of FDNVs in CRC cells. Taken together, these specific properties may help cancer to gain nanovesicles uptake inside the cells and explain the greater toxicity of FDNVs toward CRC cells. However, additional experiments are required to understand the FDNVs uptake mechanism in cancer cells.

Our study showed that FDNVs drove apoptosis cell death in CRC cell cultures. The well-known apoptotic mechanism is initiated by the induction of the intrinsic pathway via the targeting and activation of caspase-9 in response to the release of cytochrome c, consequently leading to the activation of effector caspases (-3, -6, and -7) [46]. We detected an increased expression of pro-apoptotic genes, including Caspase-3, Caspase-9, and Bax, as well as a decrease in the expression of the anti-apoptotic gene Bcl-2, in CRC cells after FDNVs treatment. These findings indicate that the anticancer effect of FDNVs was partially mediated through activation of the intrinsic pathway, leading to the execution of apoptosis.

The induction of intracellular ROS reportedly contributes to apoptosis in cancer cells [47, 48]. Several studies have found that medicinal plants can cause excessive production of ROS, leading to irreversible damage to DNA, lipids, and proteins, ultimately leading to the induction of apoptosis [49]. The secondary metabolites present in plants, such as flavonoids, fatty acids, and proteins have been shown to induce ROS generation, i.e. pinostrobin [37], linoleic acid [50], and phospholipase D [51], respectively. Our study showed that ROS levels were elevated in HT-29 and HCT116 cells after FDNVs treatment. Indeed, previous studies have reported ROS induction in response to fingerroot compounds. Boesenbergin A, a chalcone from fingerroot induced oxidative stress-mediate apoptosis in lung adenocarcinoma cells (A549) [9]. Pinostrobin, a flavanone in Fingerroot, exhibited anti-proliferation effects and induced apoptosis in cancer stem-like cells through a ROS-dependent mechanism [37]. Additionally, nanovesicles from several plants, including lemon and ginseng, have also reported ROS-mediated apoptosis [21, 31]. Hence, FDNVs may contain bioactive compounds that play a key role in ROS generation, leading to the induction of apoptosis in CRC cells. Besides the metabolites, plant-derived microRNAs have also been reported to exhibit anticancer effect [52]. Therefore, the miRNA profile in FDNVs needs further investigation to more directly address the molecular mechanism of FDNVs-mediated anticancer properties. Interference of cellular detoxification by reducing GSH was associated with ROS-mediated cytotoxicity. [47, 53]. We, therefore, determined the cellular level of GSH. Indeed, we found significant reductions of GSH in all treated CRC cells, which supports our hypothesis on the disruption of redox balance in FDNVs-treated cells by reducing GSH to neutralize ROS. On the other hand, the ROS and GSH levels were not significantly altered in normal colon cells after treatment with FDNVs. These findings suggest that FDNVs promoted apoptosis through the production of intracellular ROS and the GSH system’s dissipation, resulting in cytotoxicity selectively towards CRC cells.

Recent interest in the study of PDNVs is partly due to their various biological properties, which can have selective targeting, leading to novel opportunities for clinical applications in various diseases [14]. Additional advantages of PDNVs include large-scale production, possessing high biocompatibility, and stability under gastrointestinal tract conditions [33]. However, there remains the absence of a standard protocol of PDNV isolation, as there is presently no consensus among researchers. In addition, specific protein markers for PDNVs are still controversial due to the robust diversity between different species of plants. Hence, the precise understanding of the composition and biological functions of PDNVs may help establish a standard isolation method and improve therapeutic applications.

Conclusion

In conclusion, this study demonstrated the anticancer effect of FDNVs against CRC cells with low toxicity to normal colon epithelial cells, indicating its selective anticancer property. Furthermore, the anticancer effect of FDNVs was mediated through disruption of intracellular redox homeostasis and induction of apoptosis pathway. Thus, FDNVs may be a promising intervention for CRC patients.

Supporting information

S1 Fig. Total protein concentration of samples from qEV column.

Protein concentrations of 30 fractions from qEV were determined by BCA Protein Assay.

(TIF)

Click here for additional data file.^{(811.5KB, tif)}

S1 Table. The discriminative putatively identified metabolites of FDNVs.

The metabolites were identified based on ChemSpider online databases, with rigorous statistical validation.

(DOCX)

Click here for additional data file.^{(27KB, docx)}

S2 Table. The minimal data set underlying the results.

(DOCX)

Click here for additional data file.^{(63.1KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This research project is supported by Mahidol University (Basic Research Fund: fiscal year 2021), Faculty of Science, Mahidol University, the Central Instrument Facility (CIF) Grant, Faculty of Science, Mahidol University and partially supported by Postdoctoral fellowship award from Mahidol University (grant number MD-PD_2021_12). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0266044.r001

Decision Letter 0

Lay-Hong Chuah

1 Nov 2021

PONE-D-21-31088Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)PLOS ONE

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Reviewer #4: Partly

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Reviewer #4: Yes

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5. Review Comments to the Author

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Reviewer #1: In this manuscript, the authors report the nanovesicles derived from fingerroot possess the anticancer activity against CRC cell lines (HT-29 and HCT116) and are inert to the normal colon epithelial cells. But this manuscript is too preliminary in science. I suggest the authors to analyze the fingerroot derived nanovesicle components by metabolomics, proteomics, and transcriptomics, and investigate the cellular internalization mechanism of fingerroot derived nanovesicles. A better understanding of fingerroot derived nanovesicle component and cellular uptake is essential to potentiate the capacity of nanovesicles to induce phenotypic changes in recipient cells. The introduction and discussion sections in this manuscript are also poorly constructed and written. Although their approach itself is of interest, the presentation does not suffice to show the novelty and significance that meet the standards of rigor required by the journal to be considered for publication.

Reviewer #2: The present work is interesting as authors isolated EVs from fingerroot for the treatment of CRC. I have carefully reviewed the articles and my comments are as follows:

Introduction:

1) Line 47-48. The unsatisfactory response rate is referring to all the drugs used for colon cancer? Early or late stage or patient? This has to be clear otherwise it shows confusing statement. Do the drugs used for chemotherapy are all very low response rate?

2) Authors should check the grammars in the article. For instance, Line 44, “Surgical resection of tumour and metastasis….”. The metastasis is not needed and it is confusing. It means “metastasis” is another treatment. Line 49, “cancellous” should be corrected to “cancerous”.

Methods:

1) Any reason why the concentration of EVs were used as the parameter for treatment instead of number of particles?

Results and Discussion:

1) Authors have indicated the total volume of 500 uL of EVs was isolated. However, what was the EVs protein concentration for fraction 7-9? If the concentration was diluted, more EVs in the form of PBS were added into the wells, resulting in the differential response if the volume of PBS was not standardised throughout the plate. Could authors provide more details on this? I concerned the effect was due to lack of media at high concentration in comparison to the control. For instance, 100 ug/mL FDNV, how many uL of PBS were actually added into the well? Was the same volume of PBS was added to the control? If the concentration of FDNV was diluted, end up the well has more PBS than the media. Fig 4A apoptosis, all the cells died, was it due to the PBS or FDNVs? The dose-dependent effect was then due to increasing volume of PBS or FDNVs? This has to be clarified.

2) Authors claimed that the EVs were successfully isolated from fingerroot. Although these were supported by size, however, the internal marker (E.g.: HSP70), external markers and the markers that are just present in plant but not in EVs should be shown. Although these have been well defined for mammals cells, authors should performed the best efforts to show the plant’s EVs markers.

3) Fig 2: Statistical analysis should be indicated in the figure.

4) Authors should discuss why there is a differential uptake in cancer cells in comparison to non-cancerous cells. What are the possible underlying mechanisms? This is interesting but no further detailed mechanisms were reported.

5) Although apoptosis was confirmed in the studied, however, the underlying mechanisms were not defined. Authors should at least investigated on certain pathway.

Reviewer #3: This work describes a method to isolate extracellular vesicles from Boesenbergia rotunda. The nanovesicles were further investigated for their anti-cancer properties on colon cancer cells. Based on in vitro results, the study concluded selective anti-cancer property of Boesenbergia rotunda derived nanovesicles.

Some comments as below:

1. The active constituent from Boesenbergia rotunda nanovesicles was not characterized / described

2. In lines 48-52, the authors specified that development of new anticancer agents for colorectal cancer is needed due to side effects in existing chemotherapy. However, the authors did not show the efficacy of Boesenbergia rotunda nanovesicles in comparison with existing chemotherapy.

3. In lines 67-69, the authors specified that limitation of crude Boesenbergia rotunda extract was non-specific cytotoxicity on non-cancerous cells. However, the authors did not compare the efficacy of crude Boesenbergia rotunda extract vs Boesenbergia rotunda nanovesicles in the context of selective cytotoxicity.

4. Thus, the purpose of developing Boesenbergia rotunda nanovesicles warrants further elaboration.

5. Unable to read Figure 3 due to low resolution.

6. The authors described Boesenbergia rotunda nanovesicles being similar to extracellular vesicles obtained from other edible plants. It will be informative to elaborate the similarities for the benefit of readers not familiar in this space.

7. ROS-induced apoptosis was demonstrated in colorectal cancer cells after treatment of Boesenbergia rotunda nanovesicles. It would be interesting to know, if similar mechanisms are observed in non-cancerous cells, i.e. the underlying mechanisms leading to the selective anti-cancer properties of Boesenbergia rotunda nanovesicles.

Reviewer #4: In this manuscript, authors isolated nanovesicles from fingerroot (FDNV) using differential centrifugation and size exclusion chromatography. Then, they used isolated FDNVs to treat two human colorectal cancer cell lines (HT-29 and HCT116) and one normal human colon cells line (CCD 841 CoN) and observed cytotoxicity in cancer cell lines but not in the normal cell line. Next, they investigated the uptake of FDNVs in all three cells lines. Following the confirmation of uptake of FDNVs in cancer cell lines, they examined the apoptosis percentage and the possible underlying mechanism that leads to apoptosis in both cancer cells line. Overall, I believe that the authors are off to a good start, however, several control experiments are missing (will explain in detail in the major comments section). This manuscript is within the scope of the journal and delivers a great scientific story. Hence, recommendation with major revision is advised.

Major comments:

1. In the introduction (and briefly in the discussion section, Line 413), authors introduced the concept of extracellular vesicles (EV). However, the whole manuscript is about FDNVs that are derived and isolated from the homogenate of fingerroot, which are not EVs. EVs are vesicles that are excreted (e.g., exosomes), hence, “extracellular” in the name. It is very misleading to have EV introduced in the introduction section and mentioned briefly in the discussion section, and it is wrong to equivalize FDNVs with EVs in the method section. Authors should make extinct differentiation in the manuscript between these two concepts.

2. For FDNV internalization, it seems the uptake was very localized for both cancer cell lines. Would that be the case for the normal cell line? It seems that there were way fewer number of cells in the frame for CCD 841 CoN comparing to the other two (based on DAPI staining) so that no uptake was captured under the microscope?

3. The apoptosis assay lacks the normal cell line control. If no apoptosis observed in normal cell line, then it will strengthen the conclusion of differential cytotoxicity and uptake in normal cell line.

4. Authors concluded that “FDNVs increased ROS generation and decreased GSH levels” in both CRC cell lines. Here, I think that this conclusion is a little premature and lacks two control experiments:

1) How did authors exclude the possibility that the increased intracellular ROS is not because/contaminated with the abundant ROS from peroxisome isolated from fingerroot that released into the cell through FDNV uptake? The differential centrifugation final pellets (after 100,000 *g) would contain all small membrane vesicles derived from fingerroot (lysosomes, endosomes, peroxisomes, microsomes, etc.) and all these vesicles are very similar in terms of size, so the SEC might not be sufficient to separate these organelles. Therefore, FDNVs are very likely to contain peroxisomes. A control experiment with just FDNVs (no cells) should be done to assess the extent of ROS contamination from FDNVs.

2) Both ROS and GSH measurement lacked a normal cell line control. Does the ROS and GSH amount stay the same in normal colon cell line? I understand that no cytotoxicity was observed in the normal colon cell line, but believes this control is necessary to strengthen the conclusions.

3) Additional question: why FDNV at 50 µg/mL generates more intracellular ROS but also have more cellular GSH than positive control hydrogen peroxide?

5. It is an interesting choice that the authors used µg/mL as their unit to describe the amount of FDNVs they treated the cells with. I assumed that this unit came from BCA assay, which is for total protein concentration. Is there a reason that the authors normalize all FDNVs treatment to total protein concentration? Is the potential active ingredient from fingerroot a protein? Does the total protein concentration of FDNV correlates with the # of FDNVs? The authors have NTA assay data, why not use the # FDNVs/mL?

6. All data should be reported in ± standard deviation (S.D.) instead of S.E.M. because you are reporting variabilities among your experiment replicates.

Minor comments:

1. Authors should state what medium (e.g., water, PBS, or etc.) they blended fingerroot in or no other liquid was added for homogenization in the method section.

2. Fig 1 C lacks statistical analysis.

3. Page 16, Line 353, please define PDNV in the discussion section (i.e., plant derived nano-vesicles (PDNV)).

4. Page 17 Line 367, differential centrifugation was used in this manuscript. Mentioning density gradient might confuse the reader. The authors should clarify to avoid confusion.

5. Page 17 Line 387-388, don’t need to capitalize pinostrobin, linoleic acid and phospholipase D.

6. Page 19 Line 435, add “chromatography” after “size exclusion”.

7. While the study appears to be sound, there are many typos, especially in introduction and discussion, making it difficult to follow. I advise the authors to re-read and revise the manuscript to improve the flow and readability of the text in introduction and discussion.

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Reviewer #1: No

Reviewer #2: Yes: Jhi Biau Foo

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Reviewer #4: No

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PLoS One. 2022 Apr 4;17(4):e0266044. doi: 10.1371/journal.pone.0266044.r002

Author response to Decision Letter 0

27 Feb 2022

Authors’ response to the editor and reviewers’ comments

Manuscript: PONE-D-21-31088

Title: Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

Dear Editor,

Thank you for considering our manuscript for publication in PLOS ONE. We carefully read all the comments and suggestions from the editors and reviewers and revised the manuscript accordingly. Overall, we agree with the majority of the comments and revised the manuscript following the suggestions.

What follows are our point-by-point responses to the comments from the editors and reviewers. Changes to the manuscript are also mentioned (track changes).

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Response: Thank you for your valuable suggestion. We have checked the templates and made the adjustments to meet the journal requirements.

Response: No permits were required for the described study, which complied with all relevant

regulations.

3. Thank you for stating the following financial disclosure:

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: We have added "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript" in the cover letter.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: We have already uploaded study’s minimal underlying data set as Supporting Information files (S2 Table).

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Fig share or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Response: The data is not a core part of the research being presented in this study. We, therefore, removed the phrase “data not shown” that refers to these data from this revised manuscript.

Response to Reviewers’ Comments:

Reviewer #1:

1) I suggest the authors to analyze the fingerroot derived nanovesicle components by metabolomics, proteomics, and transcriptomics.

Response: We appreciate for reviewer’s perspective. We have now performed the metabolomic analysis of FDNVs by LC-MS/MS. The method for LC-MS/MS has been incorporated in the Materials and Methods section (pages 6-7, lines 126-151). The distribution of the identified metabolites in FDNVs is shown in new Fig 1E. List of the discriminative putatively identified metabolites (58 named metabolites) of FDNVs is also presented in new S1 Table. Alkaloids were the most common FDNVs metabolite subtype (53%; 31/58), followed by phenolics (21%; 12/58), lipids (14%; 8/58), and organic compounds (12%; 7/58). Importantly, the phenolic compounds naringenin chalcone, pinostrobin, and pinocembrin were found in FDNVs. These phenolic compounds have been found as promising bioactive compounds in fingerroot juice [1]. Thus, these secondary metabolites may serve as medicinal agents that underlie the therapeutic action of FDNVs, which can improve our understanding of how FDNVs exhibit biological activities This detail information has been incorporated in the Result (page 13, lines 278-292). The discussion regarding the possible biological activity of the identified metabolites in FDNVs is now stated in the Discussion section (page 22, lines 498-506).

2) I suggest the authors to investigate the cellular internalization mechanism of fingerroot derived nanovesicles. A better understanding of fingerroot derived nanovesicle component and cellular uptake is essential to potentiate the capacity of nanovesicles to induce phenotypic changes in recipient cells.

Response: We thank the reviewer for the suggestion and we agree with the reviewer. In this revised manuscript, we investigated the uptake mechanism of FDNVs in colorectal cells. The uptake of FDNVs was examined in the presence of pinocytosis and phagocytosis inhibitors. As shown in new Fig 4, the uptake of FDNVs in HT29 and HCT116 were markedly inhibited by filipin (Fig 4A) and cytochalasin D (Fig 4B), which are the inhibitors of caveolae-mediated endocytosis and phagocytosis, respectively (P > 0.001). Conversely, treatment with amiloride, an inhibitor of micropinocytosis, and chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not affect the uptake of FDNVs in both cancer cell lines (Fig 4A). These data suggest that the internalization of FDNVs in colorectal cancer cells is partly via caveolae-mediated endocytosis and phagocytosis pathways. This related information has been incorporated in the Material and Methods (page 10, lines 218-228), Results (pages 16-17, Line 341-353 and 363-372) and Discussion (pages 23-24, lines 522-542) sections.

3) The introduction and discussion sections in this manuscript are also poorly constructed and written. Although their approach itself is of interest, the presentation does not suffice to show the novelty and significance that meet the standards of rigor required by the journal to be considered for publication.

Response: Thank you for your valuable comments. We have edited and revised the introduction and discussion of the manuscript.

Reviewer #2

Introduction:

Response: Thank you for your valuable suggestion. Chemotherapeutic drugs were applied to the high-risk stage II-IV CRC patients [2, 3]. Unfortunately, the overall response rate of advanced colorectal cancer to 5-fluorouracil (5-FU), the first-line drug, is only 10-15% [4]. Therefore, combining 5-FU, leucovorin, and capecitabine with oxaliplatin was recommended. However, a significant improvement in overall survival (OS) was observed only for stage III colon cancer [3, 5]. These data indicate an unsatisfactory response rate for colon cancer treatments. However, we revised the introduction in this manuscript according to reviewers #1 and #4 comments, emphasizing systemic toxicity to normal cells. Therefore, we design not to include the explanation of the unsatisfactory response rate mentioned in the previous version of the manuscript in this revised manuscript.

Response: Thank you for your comments. The revised manuscript had been carefully checked to eliminate grammatical errors. In addition, we revised the introduction in this manuscript according to reviewers #1 and #4 comments; therefore, the sentence “Surgical resection of tumor and metastasis….” was removed.

Methods:

1) Any reason why the concentration of EVs were used as the parameter for treatment instead of number of particles?

Response: We thank the reviewer for pointing this out. The particles/ml may represent the amount of EVs better than the total protein concentration (µg/ml) due to the possibility of other protein contamination in the sample. However, specific equipment like Nanoparticle tracking analysis (NTA) is required to measure the number of particles in the sample. Therefore, several laboratories, including us, measured the protein concentration using the BCA method to determine the concentration of EVs instead of the number of particles. Indeed, the total protein representing the concentration of EVs have been widely used to study the biological functions of plant-derived EVs; for example, EVs derived from citrus limon [6], ginger [7], and corn [8]. In addition, we found that the number of particles from isolated FDNVs correlated with the protein concentration (Fig 1B and S1 Fig); therefore, we used protein concentration as a parameter for treatment instead of particle number.

Results and Discussion:

1) Authors have indicated the total volume of 500 uL of EVs was isolated. However, what was the EVs protein concentration for fraction 7-9? If the concentration was diluted, more EVs in the form of PBS were added into the wells, resulting in the plate. Could authors provide more details on this? I concerned the effect was due to lack of media at high concentration in comparison to the control. For instance, 100 ug/mL FDNV, how many uL differential response if the volume of PBS was not standardised throughout the of PBS were actually added into the well? Was the same volume of PBS was added to the control? If the concentration of FDNV was diluted, end up the well has more PBS than the media. Fig 4A apoptosis, all the cells died, was it due to the PBS or FDNVs? The dose-dependent effect was then due to increasing volume of PBS or FDNVs? This has to be clarified.

Response: Thank you for your valuable comments and suggestions. After ultracentrifugation, the vesicle pellet was resuspended with 1 ml PBS. Then, the FDNVs were purified using size exclusion chromatography (iZON). Fraction 8 (500 µl in PBS) was selected for all experiments based on the data of NTA and TEM, as mentioned in the result section (page12, lines 260-271). The protein concentration of this fraction was approximately 0.4 µg/ul (BCA protein assay), and total protein was 200 µg (500 µl x 0.4 µg/µl).

To increase the concentration of FDNVs, we combined four vesicle pellets from ultracentrifugation before performing size exclusion chromatography. Thus, the total protein concentration was increased to approximately 1.5 µg/µl, and the total protein was 750 µg (500 µl x 1.5 µg/µl). These samples were used to study the activity of FDNVs. To investigate the effect of FDNVs on apoptosis induction, we prepared 100 µg/ml FDNVs by adding 67 µl of FDNVs (67 µl x 1.5 µg/µl = 100 µg) to 933 µl culture media and incubated with cells for 48 h. In the vehicle control group, we mixed 67 µl PBS with 933 µl culture media. We found no significant effect of PBS in the untreated condition (Fig 5). Therefore, the effect of 100 µg/ml FDNVs on apoptosis induction is not due to the dilution of the medium with PBS. This information has been incorporated in the Materials and Methods (page 8, line 178) section.

2) Authors claimed that the EVs were successfully isolated from fingerroot. Although these were supported by size, however, the internal marker (E.g.: HSP70), external markers and the markers that are just present in plant but not in EVs should be shown. Although these have been well defined for mammal cells, authors should perform the best efforts to show the plant’s EVs markers.

Response: We thank the reviewer for pointing this out, and we agree with the reviewer. Although the guideline on the nomenclature and minimal information for studies of EVs have been recommended [9]. However, the markers guideline for plant-derived nanovesicles is currently unavailable due to insufficient information in the field of plant EVs [10]. In addition, the antibody specifically to plat proteins is also limited. However, as per your suggestion, we attempted to examine the expression of protein markers that were previously reported in mammalian cells-derived EVs, including TSG101, flotillin, CD81, by western blotting. In addition, calnexin was included as a negative marker for EVs. Unfortunately, we failed to detect the expression of all protein markers of mammalian cells-derived EVs in FDNVs (Figure 1 below). Therefore, these markers may not be specific for plant-derived EVs, or the antibodies failed to detect these plat proteins.

Figure 1: The expression of protein markers of mammalian cells-derived EVS in FDNVs by western blotting.

3) Fig 2: Statistical analysis should be indicated in the figure.

Response: We thank the reviewer for the comment, we analyzed and provided the statistics in the revised Figure 2 and the result (pages 14-15, lines 297-322).

Response: We agree with the reviewer and thank you for your suggestion. We found that CRC cells were more susceptible to FDNV uptake than normal colon cells (Fig 3). Moreover, the uptake of FDNVs in CRC cells was markedly inhibited by filipin (new Fig 4A) and cytochalasin D (new Fig 4B), which are the inhibitors of caveolae-mediated endocytosis and phagocytosis, respectively (P > 0.001). Conversely, treatment with amiloride, an inhibitor of micropinocytosis, and chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not affect the uptake of FDNVs in both CRC cell lines (new Fig 4A). These data indicate that the internalization of FDNVs in colorectal cancer cells is partly via caveolae-mediated endocytosis and phagocytosis pathways. However, these inhibitors did not affect the FDNVs internalization in normal colon epithelial cells (CCD 841 CoN) (Figure 2 below). This result indicates cell-dependent FDNVs uptake.

Figure 2: The inhibition of FDNVs internalization in normal colon epithelial cells (CCD 841 CoN). ns: not significant.

Indeed, the internalization of plant nanovesicles in cancer cells via phagocytosis and caveolae-mediated endocytosis mechanisms has been reported. For example, ginger-derived nano-lipids loaded with doxorubicin were mainly internalized via the phagocytosis pathway into CRC cancer cells that were significantly inhibited by cytochalasin D [7]. Moreover, the internalization of plant sap-derived extracellular vesicles breast cancer cells was mediated by phagocytosis and caveolae-mediated endocytosis [11]. Caveolin-1, the principal structural component of caveolae, is involved in caveolae-mediated endocytosis [12]. Although caveolin-1 function in cancer is controversial, overexpression of caveolin-1 has been reported in colon cancer [13]. Therefore, the caveolae-mediated endocytosis may be more effective in CRC, resulting in larger amounts of FDNVs internalization than in normal colon epithelial cells. In addition, the internalization of garlic-derived nanovesicles is mediated by interaction with the CD98 heavy chain (CD98hc) in liver cancer cells (HepG2) [14]. Expression levels of CD98hc protein were higher in CRC tissues than in matched normal tissues [15]. Therefore, upregulation of CD98hc might support the uptake of FDNVs in CRC cells. Taken together, these specific properties may help cancer to gain nanovesicles uptake inside the cells and explain the greater toxicity of FDNVs toward CRC cells. However, additional experiments are required to understand the FDNVs uptake mechanism in cancer cells. This information has been incorporated in the discussion (pages 23-24, lines 522-542).

5) Although apoptosis was confirmed in the studied, however, the underlying mechanisms were not defined. Authors should at least investigate on certain pathway.

Response: We completely agree with the reviewer. Intrinsic apoptosis pathway is characterized by mitochondria dysfunction-mediated cytochrome C release and subsequent activation of caspases-9 and caspases-3 [16]. In this revised manuscript, we determined the expression of the apoptosis-associated genes in HT-29 and HCT 116 cells treated with FDNVs by quantitative RT-PCR (new Fig 6). Treatment with FDNVs at 12.5 and 25 µg/ml markedly increased the expression of caspase-3 and caspase-9 in HT-29 and HCT116 cells (new Fig 6A and B). Aa increase in the expression of Bax, a pro-apoptotic gene, was also observed after treatment with 25 µg/ml FDNVs in both CRC cell lines. In contrast, the expression of Bcl-2, an anti-apoptotic gene, was decreased in both CRC cell lines after treatment with FDNVs at 25 µg/ml. These results indicate the anticancer effect of FDNVs was partially mediated through intrinsic apoptosis pathway. This information has been incorporated in the Materials and Methods (pages 8-9, lines 185-202), Results (pages 18-19, lines 403-412 and 421-426) and Discussion (page 24, lines 543-551) sections.

Reviewer #3:

1) The active constituent from Boesenbergia rotunda nanovesicles was not characterized / described

Response: We appreciate for reviewer’s perspective. We have now performed the metabolomic analysis of FDNVs by LC-MS/MS. The method for LC-MS/MS has been incorporated in the Materials and Methods (pages 6-7, lines 126-151) section. The distribution of the identified metabolites in FDNVs is shown in new Fig 1E. List of the discriminative putatively identified metabolites (58 named metabolites) of FDNVs is also presented in new S1 Table. Alkaloids were the most common FDNVs metabolite subtype (52%; 31/59), followed by phenolics (22%; 13/59), lipids (13%; 8/59), and organic compounds (12%; 7/59). Importantly, the phenolic compounds naringenin chalcone, pinostrobin, and pinocembrin were found in FDNVs. These phenolic compounds have been found as promising bioactive compounds in fingerroot juice [1]. Thus, these secondary metabolites may serve as medicinal agents that underlie the therapeutic action of FDNVs, which can improve our understanding of how FDNVs exhibit biological activities. This detail information has been incorporated in the Result (page 13, lines 278-284). The discussion regarding the possible biological activity of the identified metabolites in FDNVs is now stated in the Discussion (page 22, lines 498-506) section.

2) In lines 48-52, the authors specified that development of new anticancer agents for colorectal cancer is needed due to side effects in existing chemotherapy. However, the authors did not show the efficacy of Boesenbergia rotunda nanovesicles in comparison with existing chemotherapy.

Response: We appreciate the reviewer’s perspective. Currently, 5-fluorouracil (5-FU), a chemotherapeutic drug, was the first-line therapy for most CRC patients worldwide [17]. However, non-specific toxicity toward normal colon epithelial cells (CCD 841 CoN) of 5-FU has been reported [18]. Moreover, doxorubicin has been used as an adjuvant chemotherapy drug for CRC at advanced stages [19]. However, cytotoxicity of doxorubicin has also been reported against CCD 841 CoN [20]. This information indicates the non-selective cytotoxic effect of the conventional chemotherapeutic agents. Therefore, in this manuscript, we emphasize the selective activity of FDNVs toward CRC cells. Interestingly, we demonstrated that FDNVs exhibited cytotoxicity against CRC cells but not normal colon epithelial cells. Therefore, we did not compare the cytotoxicity of FDNVs with the conventional chemotherapeutic drugs.

3) In lines 67-69, the authors specified that limitation of crude Boesenbergia rotunda extract was non-specific cytotoxicity on non-cancerous cells. However, the authors did not compare the efficacy of crude Boesenbergia rotunda extract vs Boesenbergia rotunda nanovesicles in the context of selective cytotoxicity. Thus, the purpose of developing Boesenbergia rotunda nanovesicles warrants further elaboration.

Response: We thank the reviewer for this suggestion. We compared the cytotoxic selectivity between fingerroot extract and its nanovesicles. In contrast to the selective cytotoxic effect of FDNVs, the fingerroot extract exhibited dose- and time-dependent effects against both cancer cells and normal human colon epithelial cells (Fig 2 and Table 2). Cytotoxicity of the fingerroot extract was significantly observed at 25 µg/ml after 24 h of treatment toward all tested cells (P < 0.001). Additionally, there was no difference between the IC50 values of the fingerroot extract against all tested cells. These results indicate the selective cytotoxic effect of FDNVs on colorectal cancer cells with relatively low cytotoxicity toward normal colon cells. This information has been incorporated in the Materials and Methods (page 8, line 167) and Results (pages 14-15, lines 297-322) sections.

4) Unable to read Figure 3 due to low resolution.

Response: With all due respect to the reviewer. We have improved the resolution of the figures.

6) The authors described Boesenbergia rotunda nanovesicles being similar to extracellular vesicles obtained from other edible plants. It will be informative to elaborate the similarities for the benefit of readers not familiar in this space.

Response: We thank the reviewer for this valuable suggestion. We have added the result and discussion on the similarities of FDNVs to other nanovesicles from edible plants. This information has been incorporated in the Results (pages 12-13, lines 275-276) and Discussion (pages 21-23, lines 510-514, lines 528-530)

7) ROS-induced apoptosis was demonstrated in colorectal cancer cells after treatment of Boesenbergia rotunda nanovesicles. It would be interesting to know, if similar mechanisms are observed in non-cancerous cells, i.e. the underlying mechanisms leading to the selective anti-cancer properties of Boesenbergia rotunda nanovesicles.

Response: We thank the reviewer for this valuable suggestion. We have determined the effect of FDNVs on apoptosis induction in normal colon epithelial cells (CCD 841 CoN). As shown in new Fig 5C, there was no significant induction of early apoptosis in all tested concentrations of FDNVs compared to untreated control. Statistically significant induction of apoptosis was only found in the presence of 5% DMSO in human colon epithelial cells (32.2 ± 5.1 %, P < 0.001). More than 90% of cells remained viable even at a high concentration of FDNVs, indicating that FDNVs exhibited low cytotoxicity against normal colon cells. In contrast, treatment with 5% DMSO resulted in a significant reduction of cell viability (P < 0.001) relative to control. Late apoptosis and necrosis were not significantly different in all tested conditions. These results demonstrated that FDNVs displayed selective induction of apoptosis-mediated cell death in cancerous but not normal, cells. This information has been incorporated in the Results (pages 18-19, lines 391-402 and 414-419) sections.

Moreover, we determined the effect of FDNVs on the intracellular ROS level in normal colon epithelial cells (CCD 841 CoN). In contrast to CRC cells, only H2O2 causes significantly increased ROS (P < 0.05) and reduced GHS (P < 0.001) levels (new Fig. 7C). These data suggest that FDNVs showed selective cytotoxicity towards cancer cells through increased ROS production. This information has been incorporated in the Results (pages 19-21, lines 428-465) sections.

Reviewer #4

Major comments:

1) In the introduction (and briefly in the discussion section, Line 413), authors introduced the concept of extracellular vesicles (EV). However, the whole manuscript is about FDNVs that are derived and isolated from the homogenate of fingerroot, which are not EVs. EVs are vesicles that are excreted (e.g., exosomes), hence, “extracellular” in the name. It is very misleading to have EV introduced in the introduction section and mentioned briefly in the discussion section, and it is wrong to equivalize FDNVs with EVs in the method section. Authors should make extinct differentiation in the manuscript between these two concepts.

Response: Thank you for your valuable suggestion. We agree that EVs and nanovesicles (NVs) have different concepts. According to the recent report [10], the term “plant-derived nanovesicles (PDNV)” is suggested for vesicular fractions obtained from plant tissues when destructive processes are used and when natural release into the extracellular space cannot be established. Therefore, based on the method used to isolate vesicles, “EVs” is now changed to “nanovesicles (NVs)” throughout the manuscript.

2) For FDNV internalization, it seems the uptake was very localized for both cancer cell lines. Would that be the case for the normal cell line? It seems that there were way fewer number of cells in the frame for CCD 841 CoN comparing to the other two (based on DAPI staining) so that no uptake was captured under the microscope?

Response: We thank the reviewer for pointing this out, and we agree with the reviewer. The size of CCD 841 CoN cells is bigger than CRC cells; therefore, a low number of cells was observed under the same magnification. However, we quantified the fluorescence intensity using the same number of cells as shown in Fig 3D.

3) The apoptosis assay lacks the normal cell line control. If no apoptosis observed in normal cell line, then it will strengthen the conclusion of differential cytotoxicity and uptake in normal cell line.

4) Authors concluded that “FDNVs increased ROS generation and decreased GSH levels” in both CRC cell lines. Here, I think that this conclusion is a little premature and lacks two control experiments:

4.1) How did authors exclude the possibility that the increased intracellular ROS is not because/contaminated with the abundant ROS from peroxisome isolated from fingerroot that released into the cell through FDNV uptake? The differential centrifugation final pellets (after 100,000 *g) would contain all small membrane vesicles derived from fingerroot (lysosomes, endosomes, peroxisomes, microsomes, etc.) and all these vesicles are very similar in terms of size, so the SEC might not be sufficient to separate these organelles. Therefore, FDNVs are very likely to contain peroxisomes. A control experiment with just FDNVs (no cells) should be done to assess the extent of ROS contamination from FDNVs.

Response: We thank the reviewer for this constructive suggestion. We have additionally determined the ROS level in FDNVs (Figure 3 below). Briefly, 12.5-50 mg/ml FDNVs were stained with CM-H2DCFDA for 30 min. HT-29 cells treated with 50 mg/ml FDNVs and 200 mM H2O2 were positive controls. Then the fluorescence signal was determined using EnVision® multimode plate reader (Ex/Em: ∼492–495/517–527 nm). We found that treatments with 50 mg/ml FDNVs (P < 0.001) and 200 mM H2O2 (P < 0.001) significantly induced intracellular ROS levels in HT29 when compared with untreated cells. However, no fluorescent signal was detected in FDNVs. These results indicate no ROS contamination from FDNVs.

Figure 3: Induction of intracellular ROS level in FDNVs-treated normal colon epithelial cells (CCD 841 CoN). **P < 0.01, ***P < 0.001 (one-way ANOVA).

4.2) Both ROS and GSH measurement lacked a normal cell line control. Does the ROS and GSH amount stay the same in normal colon cell line? I understand that no cytotoxicity was observed in the normal colon cell line, but believes this control is necessary to strengthen the conclusions.

Response: We thank the reviewer for pointing this out, and we agree with the reviewer. We determined the effect of FDNVs on the intracellular ROS and GSH levels in normal colon epithelial cells (CCD 841 CoN). In contrast to CRC cells, only H2O2 causes significantly increased ROS (P < 0.05) and reduced GHS (P < 0.001) levels (new Fig 7C). These data further support the selective cytotoxicity of FDNVs towards cancer cells through increased ROS production. This information has been incorporated in the Results (pages 19-21, lines 428-465) sections.

4.3) Additional question: why FDNV at 50 µg/mL generates more intracellular ROS but also have more cellular GSH than positive control hydrogen peroxide?

Response: We thank the reviewer for pointing this out. The cellular antioxidant mechanisms play critical roles in protecting the cells and organisms from oxidative damage. Several antioxidant mechanisms, both enzymatic and non-enzymatic systems, have been reported to counteract oxidative stress in human cells and organisms [21]. Moreover, several antioxidant mechanisms of Boesenbergia rotunda have been reported [22]. However, other antioxidant mechanisms may involve oxidative stress induced by FDNVs, which is different from the oxidative stress induced by H2O2. Therefore, further experiments may be required to study the effects of FDNVs on oxidative stress defense mechanisms.

5) It is an interesting choice that the authors used µg/mL as their unit to describe the amount of FDNVs they treated the cells with. I assumed that this unit came from BCA assay, which is for total protein concentration. Is there a reason that the authors normalize all FDNVs treatment to total protein concentration? Is the potential active ingredient from fingerroot a protein? Does the total protein concentration of FDNV correlates with the # of FDNVs? The authors have NTA assay data, why not use the # FDNVs/mL?

6) All data should be reported in ± standard deviation (S.D.) instead of S.E.M. because you are reporting variabilities among your experiment replicates.

Response: Thank you for your suggestion. All data are now reported as means ± standard deviation (SD).

Minor comments:

1. Authors should state what medium (e.g., water, PBS, or etc.) they blended fingerroot in or no other liquid was added for homogenization in the method section.

Response: There was no liquid during the blending process. We have incorporated this information in the section of Materials and Methods (page 4, lines 86-88).

2. Fig 1 C lacks statistical analysis.

Response: Fig 1C (zeta potential) was changed to Fig 1D in the revised manuscript. In addition, we have performed the statistical analysis of Fig 1D.

3. Page 16, Line 353, please define PDNV in the discussion section (i.e., plant derived nano-vesicles (PDNV)).

Response: We have revised according to your suggestion (page 21, lines 479).

4. Page 17 Line 367, differential centrifugation was used in this manuscript. Mentioning density gradient might confuse the reader. The authors should clarify to avoid confusion.

Response: We thank the reviewer for your suggestion. In this revised manuscript, “density gradient centrifugation-based methods” is now replaced with “sucrose density-gradient separation methods”. (page 22, lines 494-495).

5. Page 17 Line 387-388, don’t need to capitalize pinostrobin, linoleic acid and phospholipase D.

Response: We have corrected it according to your suggestion (page 24, lines 557).

6. Page 19 Line 435, add “chromatography” after “size exclusion”.

Response: We have deleted this phrase according to revision.

Response: We thank the reviewer for this constructive comment. We have edited and revised the introduction and discussion of the manuscript.

End of Response to the Reviewers

We appreciate the reviewers for not only their time but also their constructive and helpful comments, which helped us improve our manuscript. In addition to the changes described above, we noticed and fixed minor errors from our originally submitted version. We also carefully re-read the manuscript and made some further minor changes to improve clarity and readability. We think the manuscript is now ready for publication, yet please do not hesitate to contact us should any further questions arise or if you feel additional corrections are necessary.

Sincerely,

Nittaya Boonmuen on behalf of all authors

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PLoS One. doi: 10.1371/journal.pone.0266044.r003

Decision Letter 1

Lay-Hong Chuah

14 Mar 2022

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

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Acceptance letter

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24 Mar 2022

PONE-D-21-31088R1

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

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Supplementary Materials

S1 Fig. Total protein concentration of samples from qEV column.

Protein concentrations of 30 fractions from qEV were determined by BCA Protein Assay.

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S1 Table. The discriminative putatively identified metabolites of FDNVs.

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Data Availability Statement

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PERMALINK

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.)

Saharut Wongkaewkhiaw

Amaraporn Wongrakpanich

Sucheewin Krobthong

Witchuda Saengsawang

Arthit Chairoungdua

Nittaya Boonmuen

Roles

Abstract

Introduction

Materials and methods

Isolation and purification of fingerroot-derived nanovesicles (FDNVs) and fingerroot extract

Determinations of protein concentration and size distribution of FDNVs

Zeta potential measurements

Transmission Electron Microscopy (TEM)

Metabolomic profiling of FDNVs

Cell culture

Cytotoxicity assay

Apoptosis assay

Quantitative real-time PCR

Cellular uptake of FDNVs

Inhibition of the cellular uptake of FDNVs

Measurements of intracellular reactive oxygen species (ROS) levels

Measurements of intracellular glutathione (GSH) levels

Statistical analysis

Results

Isolation, characterization, and metabolite profiling of FDNVs

Fig 1. Characterization of FDNVs.

Table 1. Size of FDNVs in fractions 7, 8 and 9 using nanoparticle tracking analysis (n = 3).

Cytotoxicity of FDNVs and fingerroot extract on colorectal cancer cells

Fig 2. Cytotoxic effects of FDNVs and fingerroot extract on colorectal cancer and normal colon epithelial cell lines.

Table 2. IC50 values of FDNVs and fingerroot extract against colorectal cancer cells.

Cellular uptake of FDNVs

Fig 3. Internalization of FDNVs in colorectal cancer and normal colon epithelial cells.

Fig 4. The inhibition of FDNVs internalization in colorectal cells.

FDNVs induce colorectal cancer cells apoptosis

Fig 5. Representative FACS quantitative analyses showing FDNVs-induced apoptosis.

Fig 6. Effect of FDNVs on apoptosis-related genes expression.

FDNVs increased ROS generation but decreased glutathione levels in colorectal cancer cells

Fig 7. Induction of intracellular ROS and GSH in FDNVs-treated cells.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Lay-Hong Chuah

Roles

Author response to Decision Letter 0

Decision Letter 1

Lay-Hong Chuah

Roles

Acceptance letter

Lay-Hong Chuah

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 2. IC₅₀ values of FDNVs and fingerroot extract against colorectal cancer cells.