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Published in final edited form as: Nutr Cancer. 2022 May 23;74(10):3670–3678. doi: 10.1080/01635581.2022.2077385

Fermented Soy Drink (Q-CAN® PLUS) Induces Apoptosis and Reduces Viability of Cancer Cells

Xinshou Ouyang a, Yonglin Chen a, Boodapati S Tejaswi a, Suyavaran Arumugam a, Eric Secor b, Theresa R Weiss c, Michael Leapman d, Ather Ali c
PMCID: PMC10986312  NIHMSID: NIHMS1967591  PMID: 35603899

Abstract

This study tested the ability of a fermented soy product to induce tumor cell toxicity and to assess if this was due to fermentation of soy, and to the genistein content. Four cancer cell lines were cultured without additive, with fermented soy (Q-CAN® PLUS), nonfermented soy, or genistein, and cell viability was examined at 24 h, 48 h, and 72 h. The sensitivity of the cell lines to apoptosis by Q-CAN PLUS was tested with the Annexin V assay. All cell lines demonstrated a dose and time response reduction in tumor cell viability with exposure to Q-CAN PLUS (IC50 at 24 h 3.8 mg/mL to 9 mg/mL). Unfermented soy did not show reduction in viability of any cell line within the same concentration range. The IC50 of genistein for each of the cell lines was significantly greater than for Q-CAN PLUS. All four tumor cell lines demonstrated apoptosis in response to Q-CAN PLUS. Q-CAN PLUS reduces viability and increases apoptosis of cancer cells in a concentration- and fermentation-dependent manner. Taking into consideration the IC50 of genistein and the concentration of genistein in Q-CAN PLUS, the genistein content of Q-CAN PLUS is not responsible for the majority reduction in tumor cell viability. This suggests that fermentation of soy results in the production of metabolites that reduce cancer cell viability and induce cellular apoptosis, and play a major role in addition to any effects produced by their genistein content.

Introduction

Fermentation is the process of the conversion of food items by select microorganisms to obtain beneficial storage and nutritional effects (1). Fermentation, along with salting and drying, is one of the main ways to allow for food storage, and does so by removing substrate for spoilage organism and by the production of lactate, alcohols, and carbon dioxide making for an environment that would not support growth of most bacteria. In addition, fermentation shares features with cooking by releasing nutrients from hard to digest foods and increasing their available nutritional value. Fermentation, and consumption of fermented foods, covers a wide range of practices and there is a large literature on positive and negative associations with a range of diseases including cancers (2).

Soybeans have long been recognized as sources of high-quality protein and beneficial lipids with several health benefits (3). Unfermented soybeans are difficult to digest due to the high amount of protein enzyme inhibitors and indigestible sugar structures. The benefits of fermented soybeans have been recognized for many years and have recently been examined (4). Consumption of fermented soybean foods is associated with many health benefits including reduced risks of type 2 diabetes (T2D) and blood pressure (5-7), improved fasting blood glucose and other metabolic syndrome symptoms (8), improved plasma triglyceride levels (9), and protection against the development of insulin resistance and nonalcoholic fatty liver disease (NAFLD) (10). There is a large amount of data regarding the association of soy and cancer, particularly in relation to the isoflavone content of soy (11, 12). The epidemiological data are mostly supportive with a reduced risk of breast cancer in Chinese populations with high dietary soy, and this has been confirmed in populations in the United States with relatively lower soy consumption (13, 14). A wide range of biological pathways have been proposed to be responsible for this antitumor effect and these range from the soy isoflavones having selective estrogen receptor modulation ability, to their ability to alter a wide range of gene expression programs (15, 16).

There are several challenges in extending beyond associative studies of fermented foods and cancer, or other health effects of fermented foods. The development of food fermentation processes has mostly been empiric and iterative with little understanding of the biochemical processes involved. Also, the biochemistry is very complicated and for most fermented foods there is not even an accurate quantification of their biochemical constituents, let alone the biochemical pathways that resulted in their production. Finally, many studies use experimental or locally obtained fermented products making confirmation of their findings, and further testing by other groups, almost impossible. We chose to use a widely available fermented soy product (Q-CAN® PLUS), as this would allow for verification and further studies by the wider scientific community.

Materials and Methods

Cell Lines

Four cancer cell lines A498 (renal cell), DU145 (prostate), MCF-7 (breast), and FaDu (squamous) were obtained from American Type Culture Collection (ATCC), Manassas, VA.

Soy Products

Fermented soy product was provided by BESO Biological Research Inc. (Diamond Bar, CA), and its stability has been previously tested (17). The fermented soy product was added into Roswell Park Memorial Institute Medium (RPMI) 1640 or Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at concentration of 10 mg/ml. The suspension was mixed thoroughly and passed through 0.22-μm pore size membrane. The effluent was collected and used for cancer cells stimulation.

Total soy isoflavone content was measured utilizing the Isoflavone (ASOF_S:11) assay: Official Methods of Analysis of AOAC INTERNATIONAL, (2005) 18th ED., AOAC INTERNATIONAL Gaithersburg, MD, USA, Official Methods 2001.10.

Reagents

Genistein, Sulforhodamine B (SRB), trichloroacetic acid, acetic acid, and Tris were purchased from Sigma Aldrich (St Louis, MO). Fetal bovine serum, penicillin, streptomycin, RPMI 1640 medium, and DMEM medium were purchased from Gibco (Gaithersburg, MD). FITC-Annexin V was purchased from eBioscience (San Diego, CA).

Cell Viability Assay

The SRB assay relies on the stoichiometric binding of SRB dye to proteins (18). The amount of dye is a proxy for cell mass and thus the number of cells in a sample. In the assay, cultured cells are fixed on plates, stained with SRB, washed and dried, then the bound dye is solubilized and the absorbance of the dye in solution is measured at OD 515 nm. Cells that have lost their integrity are not retained on the plate and are thus not present for staining. The SRB assay therefore provides information on the cumulative loss of cellular viability, and not the percentage of cells with loss of viability at the time of testing.

Cancer cells were plated in 96-well plates and cultured overnight. Culture medium was removed the next day. Fresh culture medium containing gradient concentrations of soy product or genistein was added to cancer cells. Cell viability was examined by SRB assay separately after 24 h, 48 h, and 72 h stimulation. In brief, cancer cells were fixed by 10% trichloroacetic acid at 4 °C for 1 h, and washed with ddH2O. Plates were dried in air. Dried plates were incubated with 0.4% SRB (dissolved with 1% acetic acid solution) at room temperature for 30 min, and washed with 1% acetic acid solution. Plates were dried in air. About 10 mM Tris solution (pH = 10.5) was added to dissolve SRB. Absorbance at 515 nm was read. The percentage of viability was normalized to untreated control group.

Apoptosis Assays

Cancer cells were plated in 6-well plates and cultured overnight. Culture medium was removed the next day. Fresh culture medium containing gradient concentrations of soy product was added to cancer cells. Cell apoptosis was examined by FITC-Annexin V staining separately after 24 h, 48 h, and 72 h stimulation. Adherent as well as cells in the culture medium were collected and stained with Annexin V. Flow cytometry was performed on a LSRII (BD Biosciences, San Jose, CA), and data were analyzed with Flowjo software (Tree Star Inc., Ashland, USA). The Annexin assay therefore provides information on the percentage of cells undergoing apoptosis at the time of the assay, and the effects of Q-CAN PLUS are reported as EC50 values. It does not provide information on the percentage of cells that have previously undergone apoptosis, lost cellular integrity, and are no-longer available to be examined.

For western blot analysis, cancer cells were seeded in complete DMEM (10% FBS and PenStrep) in 6-well plates at 0.3 × 106cells/ml count. Primary hepatocytes were isolated from collagenase digested mouse liver and were seeded in collagen-coated 6-well plates at 0.65 × 106 cells/ml. The cells were treated for 48 h with increasing concentrations of Q-CAN PLUS fermented soy extract under standard conditions. After 48 h, the cells were washed with phosphate buffered saline and protein was extracted with ice cold RIPA buffer. The protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a 4%–12% Bis–Tris NUPAGE gel with 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. The proteins samples mixed with NUPAGE LDS sample buffer 4× (NP008) and sample reducing agent (NP004) and head denatured at 95 °C for 5 min and then loaded in equal amounts into separate lanes. Bolt antioxidant (BT0005) was added to the inner tank buffer to prevent sample oxidation during run. The gel was run at 120 V until dye front disappeared and then was transferred into a polyvinylidene difluoride (PVDF) membrane using semi-dry transfer (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were blocked using 5% Bovine serum albumin (BSA) in 1× Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) for 1 h at room temperature followed by incubation with poly adenosyl ribose polymerase (PARP) primary rabbit IgG antibody (Catalog no. 9542T, Cell Signaling Technology; 1:1000 dilution, Danvers, MA) and β-actin primary mouse IgG antibody (Catalog no. sc-47778, Santa Cruz Biotechnology, Inc.; 1:2000 dilution) for overnight at 4 °C. The membranes were then washed and incubated with Goat anti-rabbit antibody (Catalog no. 7074, Cell Signaling Technology; 1:10,000 dilution) and Goat anti-mouse antibody (Catalog no. 43593, Cell Signaling Technology; 1:10,000 dilution) for 1 h at room temperature. The membranes were washed and subjected to ECL reaction and images were captured using Hyblot CL film (Denville Scientific, USA).

Statistical Analysis

The IC50 values of cell viability assay was automatically calculated with GraphPad software (GraphPad Software, San Diego, CA) using the inhibitor vs. normalized response-variable slope equation. No extra curve fitting restraints were included. The EC50 values of Annexin V assay was automatically calculated with GraphPad software using the agonist vs. response-variable slope equation. No extra curve fitting restraints were included. All data are from three independent experiments and expressed as mean ± SEM.

Results and Discussion

Fermented Soy Results in Decreased Viability of Cancer Cell Lines

Fermented soy (Q-CAN PLUS) was selected because it is well characterized and widely available. It was tested in the concentration range of 0–10 mg/ml because tumor cells in the gastrointestinal (GI) tract could be expected to be exposed to such concentrations. This was a 72 h experiment, and although tumor cells inside the body would not be in direct contact with these concentrations for an extended period of time, they may build up components over extended periods of time.

The data from the exposure of the four cell lines to Q-CAN PLUS gave very stable dose–response curves validating the choice of the dose range and assay (Figure 1). The IC50 was approximately in the middle of the dose–response curve, and gradually reduced with increased exposure time. For the four cell lines, the IC50 was between from 3.8 mg/ml to 9 mg/ml, with the toxicity at 24 h being greatest for breast with the following sequence from highest to lowest toxicity: breast > prostate > renal > squamous. This hierarchy of toxicity was fairly stable and at 72 h was breast > prostate > squamous > renal.

Figure 1.

Figure 1.

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Q-CAN PLUS reduces the viability of cancer cells in a dose- and time-dependent manner. The cancer cells were subjected to increasing concentrations of Q-CAN PLUS extract (0, 2, 4, 6, 8, and 10 mg/ml) for 24, 48, and 72 h. The graphs represent the viability of A498 cells (a–c), DU145 cells (d–f), MCF7 cells (g–i), and FaDu cells (j–l) subjected to Q-CAN PLUS for 24, 48, and 72 h, respectively. The viability of cell lines A498 (renal cell), DU145 (prostate), MCF-7 (Breast), and FaDu (squamous cell) were all reduced in a concentration- and time-dependent manner.

As discussed in Introduction, fermentation was likely initially a process to enhance preservation of food, but the changes induced by fermentation have been reported to have a wide range of beneficial effects. To address the question if the tumor cell toxicity demonstrated by Q-CAN PLUS was a generic feature of soy, or developed after fermentation, we also tested unfermented soy powder on the same cell lines in the same concentration range (0–10 mg/ml) and did not see a decrease in SRB signal (data not shown). In fact, there was a slight increase at all time points, which is consistent with increase in cell mass as would be expected in culture in the absence of cytotoxicity.

Genistein Results in Decreased Viability of Cancer Cell Lines

Genistein is an isoflavone that is found in a number of plants with fava beans and soybeans being major food sources. Genistein has been reported to have a very wide range of biological properties including being an antioxidant, stimulating autophagy, activation of Nrf2, and inhibition of a number of receptors including glycine and nicotinic receptors (19-21). Genistein has been shown to reduce tumor cell proliferation and induce tumor cell apoptosis, making it relevant to ask if the effects of Q-CAN PLUS in Figure 1 were due to its genistein content (22). The genistein composition of Q-CAN PLUS was quantified as detailed in Materials and Methods and was 0.055%. An IC50 of Q-CAN PLUS in the range of 3.8–9 mg/ml therefore corresponds to a genistein concentration of 0.038 and 0.09 mg/ml. We therefore performed a dose–response curve of genistein with the same cell lines and under the same conditions as for Q-CAN PLUS. As Figure 2 shows, this resulted in a dose–response curve demonstrating the ability of genistein to inhibit viability of the same four cancer cell lines. The IC50 of genistein for each of the cell lines was significantly greater than for Q-CAN PLUS (Figure 1). Taking into consideration the IC50 of genistein and the concentration of genistein in Q-CAN PLUS, it is clear that the genistein content of Q-CAN PLUS is not responsible for the majority reduction in tumor cell viability seen in Figure 1. Genistein may however be a contributing component to it.

Figure 2.

Figure 2.

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Genistein shows reduced IC50 values for corresponding dosage and time as compared to Q-CAN PLUS in cancer cell cytotoxicity. The cancer cells were subjected to increasing concentrations of genistein (0, 5, 10, 15, 20, and 25 mg/ml) for 24, 48, and 72 h. The graphs represent the viability of A498 cells (a–c), DU145 cells (d–f), MCF7 cells (g–i), and FaDu cells (j–l) subjected to genistein for 24, 48, and 72 h, respectively. The corresponding IC50 values of genistein were higher for all four cells lines when compared to Q-CAN PLUS. These results portray that Q-CAN PLUS fermented soy extract has higher anticancer potential when compared to genistein.

Fermented Soy Results in Increased Apoptosis of a Cancer Cell Line

Complex food products like soy contain many hundreds of molecules and this complexity increases after fermentation, with an associated change in functional properties such as radical scavenging (23). We wished to assess if the loss of viability of tumor cells with Q-CAN PLUS was due to nonspecific effects, or due to the specific process of apoptosis. A key feature of apoptosis is the loss of plasma membrane polarity resulting in molecules such as phosphatidylserine being exposed to the extracellular side of the plasma membrane (24). This phenomenon is used to identify apoptotic cells by staining with fluorescent-labeled Annexin V that binds phosphatidylserine (25). Detection of Annexin V is by flow cytometry. Initially, the flowcytometry is gated on cell-sized particles to ensure that signal is not being recorded from subparticular components, and then the percentage of Annexin V positive cells are quantified.

Supplemental Figure 1 shows representative Annexin V plots for DU145 cells treated with Q-CAN PLUS for 24 h, demonstrating a dose response curve of increasing apoptotic state as demonstrated by Annexin V positivity. Figure 3 shows that Q-CAN PLUS induces apoptosis in all four cell lines, but with much greater variability than for the viability data. For DU145 (prostate) and MCF-7 (breast), there is a high degree of apoptosis even at the earliest time point, but for A498 (renal) and FaDU (squamous) the percentage of apoptosis does not increase until 72 h,. The main point from these data is that Q-CAN PLUS does induce Annexin positivity and apoptosis. There can be a number of reasons for the lack of exact match of IC50 and EC50between the viability and the Annexin data for A498 and FaDU. Annexin V positivity is a transient phase in the process of apoptosis (25). After cells become Annexin V positive they form blebs and lose an intact cell structure. The Annexin V staining therefore only detects the number of cells that are going through that Annexin V positive phase at the time of the assay, and not cells that have undergone apoptosis in the past. It is possible that A498 and FaDU cells spend much less time in the Annexin V positive stage before they lose their cellular structure and are therefore not present to give an Annexin V signal.

Figure 3.

Figure 3.

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Q-CAN PLUS increased the apoptosis of cancer cells in a dose- and time-dependent manner. The cancer cells were subjected to increasing concentrations of Q-CAN PLUS extract (0, 2, 4, 6, 8, and 10 mg/ml) for 24, 48, and 72 h. The graphs represent the viability of A498 cells (a–c), DU145 cells (d–f), MCF7 cells (g–i), and FaDu cells (j–l) subjected to Q-CAN PLUS for 24, 48, and 72 h, respectively. The apoptosis of all four cell lines was increased in a concentration- and time-dependent manner as demonstrated by increased Annexin V positivity.

To further confirm that the nature of the cell death was via apoptosis we performed quantification of cleaved PARP in all four cell lines (MCF-7, DU145, FaDu, and A498) at the full dose range of Q-CAN PLUS at 48 h. As can be seen from Figure 4a-h there was a dose-dependent increase in cleaved PARP for three of the cell lines (MCF-7, DU145, and FaDu) but not for A498. For primary cells (hepatocytes), there was no decrease in viability at any of the concentrations (2, 4, 6, 8, and 10 mg/ml) or time points (24, 48, and 72 h) (Figure 4i-m). This is in significant contrast to the data on cancer cell lines in (Figure 1).

Figure 4.

Figure 4.

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Q-CAN PLUS exhibits apoptotic effects on cancer cells and nontoxicity in primary cells. Cancer cell lines (MCF-7, DU145, Fadu, and A498) and primary hepatocytes from C57bl/6J mouse were treated with varying concentrations of Q-CAN PLUS aqueous suspension (2, 4, 6, 8, and 10 mg/ml) for 48 h, and their whole cell protein was extracted and subjected to immunoblotting for cleaved and whole forms of PARP. a, c, e, g, i: represent western blots for total and cleaved forms of PARP for the aforementioned cell lines with β-actin as loading control. The blots were quantified using Image J software and were statistically evaluated (b, d, f, h, m) represented as ratio of β-actin density (mean ± SEM; N = 3; mean significant at P ≤ 0.05). Cytotoxicity changes for primary hepatocytes were explored using SRB assay and are represented as % viability in (i–k) for 24-, 48-, and 72-h treatment of Q-CAN PLUS respectively (mean ± SD for N = 3).

Loss of mitochondrial potential is a mechanism of apoptotic cell death, and to obtain mechanistic information on how Q-CAN PLUS induces apoptotic cell death we examined the ability of Q-CAN PLUS to inhibit the normal mitochondrial potential. DU145 cells were subjected to increasing concentrations of Q-CAN PLUS (2–10 mg/ml) aqueous extract for 24 h, and mitochondrial membrane potential was quantified by staining with Mitotracker red. Results show that Q-CAN PLUS-fermented soy extract treatment significantly reduced the mitochondrial membrane potential in cancer cells (Figure 5a,c), and this was quantified (Figure 5b,d). One mechanism of mitochondrial damage and loss of mitochondrial potential is the production of excessive mitochondrial reactive oxygen species (ROS). MCF and DU145 cells treated with increasing concentrations of Q-CAN PLUS extract (2–10 mg/ml) for 24 h and mitochondrial ROS quantified by staining with mitoSOX red. Figure 5a,c shows representative confocal images of MCF and DU145 cells stained for mitoSOX red. Figure 5b,d shows quantifications of Figure 5a,c, respectively (values are represented as mean ± S.D.; values are significant at P ≤ 0.05). These results show a very significant, Q-CAN PLUS dose-mediated increase in mitochondrial ROS. To confirm the mechanistic importance of this increase in mitochondrial ROS in Q-CAN PLUS-mediated increase in cancer cell death, mitochondrial ROS was titrated with the mitochondrial targeted antioxidant Mitotempo. Viability bar graphs of MCF and DU145 cells respectively are shown comparing cells treated with Q-CAN PLUS alone for 24 h versus pretreatment with (10 μM) Mitotempo followed by Q-CAN PLUS for 24 h (Figure 5i,j). These data demonstrate that pretreatment with Mitotempo, a mitochondrial specific antioxidant, which scavenges mitochondrial superoxide, significantly protected cancer cells against Q-CAN PLUS-induced cytotoxicity, confirming that increase in mitochondrial ROS is a mechanism for Q-CAN PLUS-induced cell death.

Figure 5.

Figure 5.

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QCAN plus fermented soy extract induces cancer cell toxicity by inducing mitochondrial ROS-induced cell death. a and c: representative confocal images of MCF and DU145 cells subjected to increasing concentrations of Q-CAN PLUS (2–10 mg/ml) aqueous extract for 24 h and stained for Mitotracker red to quantify membrane potential. Q-CAN PLUS fermented soy extract treatment significantly reduces the mitochondrial membrane potential in cancer cells (a and c). b and d: quantifications of (a) and (c). MCF and DU145 cells treated with increasing concentrations of Q-CAN PLUS extract (2–10 mg/ml) for 24 h were stained with mitoSOX red for detection and quantification of mitochondrial ROS. e and g: representative confocal images of MCF and DU145 cells stained for mitoSOX red. f and h: quantifications of (e) and (g) respectively. Viability bar graphs with and without an antioxidant (Mitotempo) of MCF and DU145 cells respectively are shown in (i) and (j) comparing cells treated with Q-CAN PLUS alone for 24 h vs. pretreatment with (10 μM) Mitotempo followed by Q-CAN PLUS for 24 h. Observations reveal that pretreatment with Mitotempo a mitochondria-specific antioxidant which scavenges mitochondrial superoxide, significantly protected the cancer cells against Q-CAN PLUS-induced cytotoxicity (values are represented as mean ± S.D.; values are significant at P ≤ 0.05).

Collectively, these results demonstrate that Q-CAN PLUS can reduce tumor cell viability and induce apoptosis of four tumor cell lines that are representative of major cancer groups (renal cell, prostate, breast, and squamous). Interestingly, unfermented soy protein does not have the antitumor effect of Q-CAN Plus, and the genistein content of Q-CAN PLUS does not explain the entire antitumor effect. This strongly argues that fermentation of soy results in the production of metabolites with potent antitumor effects and these are distinct from genistein, which however may be contributing to them. The identification of antitumor compounds in Q-CAN Plus is now of great interest. In addition to isolfavones such as genistein, these compounds may include fermentation metabolites such as ketones and medium chain fatty acids, as both have been shown to possess antitumor effects (26). The presence of multiple antitumor compounds also brings into play the concept of synergy, which is well established in cancer chemotherapy (27, 28). It is very possible that the antitumor effects of fermentation metabolites also have synergy and testing this experimentally is a high priority. Q-CAN Plus is used at a concentration (6–9 g/ml) which is a thousandfold higher than the demonstrated IC50 for cell viability at 24 h, suggesting that Q-CAN PLUS may be having these effects in vivo, at least for cells of the lining of the gastrointestinal tract which would be in direct contact with Q-CAN PLUS. Finally, the ready availability of Q-CAN PLUS is of practical value as it will allow for the confirmation and expansion of these findings.

Supplementary Material

Supplementary Figure

Funding

This work was supported by a research grant from BESO Biological Research, Inc. to Yale School of Medicine.

Footnotes

Disclosure Statement

The authors declare no conflict of interest.

Supplemental data for this article is available online at at https://doi.org/10.1080/01635581.2022.2077385

Data Availability Statement

Data will be available from Dr. Xinshou Ouyang on request at xinshou.ouyang@yale.edu

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure
NIHMS1967591-supplement-Supplementary_Figure.pptx (114.2KB, pptx)

Data Availability Statement

Data will be available from Dr. Xinshou Ouyang on request at xinshou.ouyang@yale.edu

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