Methylthioacetic acid, a derivative of aroma compounds from Cucumis melo var. conomon dose-dependently triggers differentiation and apoptosis of RCM-1 human colorectal cancer cells

Abstract

Methylthioacetic acid (MTA) is an acid-hydrolyzed derivative of a natural aroma compound, methylthioacetic acid ethyl ester isolated from Cucumis melo var. conomon (Katsura-uri, Japanese Picking Melon), and induces a villiform-like structure dome in RCM-1 human colorectal cancer cell culture. Thus far, the physiological and molecular properties of MTA-mediated dome formation remain unknown. Herein, MTA (not more than 2 mM) was demonstrated to differentiate the unorganized cell mass into the dome in RCM-1 cell culture by disclosing the correlation between dome formation and several intestinal differentiation markers such as alkaline phosphatase activity and the protein levels of dipeptidyl peptidase 4, villin, and Krüppel-like factor 4. Dome formation in RCM-1 cell culture was additively enhanced by the simultaneous administration of MTA and butyric acid (BA), suggesting that MTA directs the differentiation of RCM-1 cells, potentially through the same or similar pathway(s) shared with BA. Notably, a high dose of MTA (2 mM or more) elevated several apoptosis markers, such as DNA fragmentation, caspase-3/7 activity, and cleavage of poly(ADP-ribose) polymerase. Altogether, in addition to RCM-1 cell differentiation, MTA triggers apoptosis. These results indicate that MTA is a potential anticarcinogenic agent applicable in differentiation therapy and traditional chemotherapy against colorectal cancers.

INTRODUCTION

Cancer is the second leading cause of human death globally, accounting for an estimated 9.6 million deaths, or one in six deaths, in 2018 (World Health Organization (WHO)). Among over 100 types of cancers, colorectal and lung cancers are very common regardless of gender. To date, different medical treatments, including surgery, chemotherapy, and radiation therapy, have been employed as cancer therapies (Miller et al., 2019). These treatments are used alone or in combination, and are administered according to the type, location, and grade of the cancer. These standard therapeutic approaches are effective for curing various cancers; however, patients often suffer from detrimental side effects due to the non-specific activities of cytotoxic agents and radiation toward vigorously dividing normal cells (Dickens and Ahmed, 2021). Moreover, these traditional treatments often render surviving tumor cells more aggressive and resistant to treatment, resulting in cancer recurrence (Johnson et al., 2014; Vasan et al., 2019).

Differentiation therapy is an alternative to traditional therapies, in which chemical agents are used to reprogram aberrant machinery in cancer cells to restore their normal developmental pathway toward terminal differentiation, senescence, and ultimately cell death, such as apoptosis, thereby alleviating cancer symptoms (Sugihara and Saya, 2013; Lin et al., 2018; Sell and Ilic, 2019). To date, the most successful example of differentiation therapy involves the use of all-trans retinoic acid (ATRA) for the treatment of a hematologic cancer, acute promyelocytic leukemia (Huang et al., 1988; Nowak et al., 2009). In contrast, the use of differentiation therapy for solid cancers has been lagging owing to limited knowledge on these neoplastic cells, which may be due to their complicated nature (Yu and Ding, 2020). Nonetheless, many differentiation approaches have been reported for solid tumors with reasonable but limited success. For example, several chemical substances, including cAMP, butyric acid (BA), retinoids such as ATRA, and peroxisome proliferator-activated receptor γ (PPARγ) agonists such as 15-deoxy-∆12,14- prostaglandin J2 (15d-PGJ2: natural) and rosiglitazone (ROSI: synthetic), have been proven to induce the differentiation of various solid tumor cells (Cox et al., 1999; Orchel et al., 2005; Cerbone et al., 2007; Yang et al., 2012). Although these findings are not satisfactory, they suggest the feasibility of differentiation therapy for solid malignancies. Therefore, to develop differentiation therapies for solid cancers, novel chemical compounds with sufficient pharmacological efficacy and fewer side effects must be discovered and the underlying mechanisms in the cellular differentiation pathways triggered by these compounds must be elucidated.

Plants are a rich source of natural compounds with different biological activities including cancer chemotherapeutic agents (Seca and Pinto, 2018). An heirloom vegetable Katsura-uri (Cucumis melo var. conomon) is a Japanese pickling melon and is mainly cultivated in the Kyoto area of Japan (Sasaki et al., 2017). To date, we have identified six fragrant compounds from the fully ripened Katsura-uri, including four compounds containing sulfur atom, i.e., 3-methylthiopropionic acid ethyl ester (MTPE), methylthioacetic acid ethyl ester (MTAE), acetic acid 3-methylthio propyl ester (AMTP) and acetic acid 2-methylthio ethyl ester (AMTE). We also reported that each of these four compounds possessed at least one of the following biological activities including an inhibitory effect on carcinogenesis: an inductive and potential forming of the villiform-like structure (dome) (MTPE and MTAE), an antimutagenic activity (MTAE and AMTP), and antioxidative activity (all four sulfur-containing compounds) (Nakamura et al., 2010). Therefore, Katsura-uri melon is expected to be a potent natural source for anticarcinogenic and antioxidative substances.

RCM-1, a human colorectal cancer cell line, is established from colorectal cancer tissue diagnosed as a well-differentiated rectal adenocarcinoma (Kataoka et al., 1989). We used the number of domes formed in the RCM-1 cell culture as a guide for screening to isolate the above-mentioned MTPE and MTAE from Katsura-uri and piperitenone oxide (PO) from spearmint (Mentha spicata) as well (Nakamura et al., 2008, 2010, 2014). Furthermore, we identified methylthioacetic acid (MTA), an acid-hydrolyzed derivative of MTAE (Aoi et al., 2015) to have the most potent dome-inducing activity among the 37 MTPE and MTAE analogs and it is involved in the cell cycle arrest of RCM-1 (Kamimura et al., 2020). On the other hand, the physiological and molecular properties of MTA remain unknown.

In this study, we demonstrate that MTA can trigger the differentiation and apoptosis of RCM-1 cells in a dose-dependent manner. The MTA pathway leading to cell differentiation perhaps is similar to the pathway for BA. Furthermore, actinomycin D (AD) and 5-fluorouracil (5-FU), known apoptosis inducers, enhance dome formation in RCM-1 cell culture. Accordingly, it is implied that a close relationship can exist between differentiation and apoptosis.

MATERIALS AND METHODS

Chemicals

In this study, 15d-PGJ2 (Calbiochem-Novabiochem Corp., CA, USA), 5-FU (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), AD (AdipoGen Life Sciences Inc., CA, USA), MTA (Matrix Scientific, SC, USA), and ROSI (Tokyo Chemical Industry) dissolved in 100% dimethyl sulfoxide (DMSO), and BA (Tokyo Chemical Industry) dissolved in water, were employed. At 24 hr after cell seeding, the chemicals were added to the culture at the specified concentrations with 0.1% DMSO. The cells were then incubated for a defined period for each assay.

Cell culture

RCM-1 cells were cultured as previously described (Kamimura et al., 2020), with minor modifications. Briefly, culture volumes were changed according to the experimental purpose; 1 × 10⁵ cells were seeded in a 96-well plate as a small-scale culture for the dome formation, cell viability, DNA fragmentation, and caspase activity assays, while 5.8 × 10⁵ cells were seeded in a 24-well plate for the trypan blue exclusion assay and immunoblotting.

Dome formation assay

The dome formation assay was performed according to Kamimura et al. (2020). The domes were observed and counted under an inverted microscope (IX73, Olympus Corp., Tokyo, Japan). Dome area was measured and analyzed using ImageJ software (https://imagej.nih.gov/ij/, National Institutes of Health, MD, USA).

ALP activity assay

Alkaline phosphatase (ALP) activity was measured using a LabAssay ALP kit, according to the manufacturer’s instructions (Fujifilm Wako Pure Chemical Corp., Osaka, Japan).

CCK-8 assay

The Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to biochemically evaluate cell viability. Briefly, 10 μL of CCK-8 solution was added to each well containing 100–200 μL of cell culture and incubated at 37°C for 2 hr. Subsequently, the absorbance at 450 nm was measured using the microplate reader, Mithras LB940 (Berthold Technologies GmbH & Co., KG, Bad Wildbad, Germany).

Trypan blue exclusion assay

A trypan blue exclusion assay was performed to microscopically evaluate cell viability. Briefly, the cells were harvested by trypsin (0.25%) digestion and mixed with 0.4% trypan blue (Sigma-Aldrich Co., MO, USA) at a ratio of 1:4. After 3 min of incubation, viable (colorless) and dead (blue) cells were counted using a Bürker–Türk hemocytometer.

Immunoblot analysis

The extract from RCM-1 cells was prepared and subjected to immunoblotting. The cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with a protease inhibitor cocktail (1:50, Nacalai Tesque, Kyoto, Japan), and the protein concentration in the lysate was determined using a BCA protein assay (Cat. no. T9300A, Takara Bio Inc., Shiga, Japan). Immunoblotting was performed as described by Kamimura et al. (2020). The primary antibody reactions were performed using the following antibodies appropriately diluted to each target protein: anti-cleaved poly(ADP-ribose) polymerase (PARP; 1:1,000, #5625, Cell Signaling Technology, Inc., MA, USA), anti-dipeptidyl peptidase 4 (DPP4; 1:1,000, #67138, Cell Signaling Technology), anti-Krüppel-like factor 4 (KLF4; 1:500, #12173, Cell Signaling Technology), anti-villin (1:500, 66096-1-Ig, Proteintech Group, Inc., IL, USA), and anti-β-actin (1:10,000, 66009-1-Ig, Proteintech). The secondary antibody reactions were carried out using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:3,000, #7074, Cell Signaling Technology; sheep anti-mouse IgG, 1:5,000, NA931, GE Healthcare, IL, USA). Protein bands were detected using WSE-7120 EzWestLumi plus solution (ATTO Co., Tokyo, Japan) and a chemiluminescence CCD imaging system (Ez-Capture MG; ATTO).

DNA fragmentation assay

Cytoplasmic histone-associated DNA fragmentation was quantified using Cell Death Detection ELISA^PLUS (Cat. no. 11774425001, Roche Diagnostics GmbH, Mannheim, Germany). Following the reaction, the absorbances at the test (405 nm) and reference (490 nm) wavelengths were measured using the Mithras LB940 microplate reader. The subtracted values were then normalized to the total protein content.

Caspase-3/7 fluorescence assay

Caspase-3/7 activity was measured using a Caspase-3/7 Fluorescence Assay Kit (Cat. no. 10009135, Cayman Chemical Co., MI, USA), according to the manufacturer’s instructions. Fluorescence intensity (excitation light: 485 nm; emission light: 535 nm) was measured using the microplate reader, Infinite 200 PRO (Tecan Trading AG, Männedorf, Switzerland). The fluorescence values were normalized to the total protein content.

Statistical analysis

Data are presented as mean with standard deviation (S.D.) for each experiment. Statistical significance was determined using one-way ANOVA, followed by the Tukey–Kramer honestly significant difference test for multiple comparisons (p < 0.05).

RESULTS

MTA affects dome formation in the RCM-1 cell culture both dose- and time-dependently

Whereas we reported that MTA enhanced dome formation in RCM-1 cell culture (Kamimura et al., 2020), the physiology of such a dome formation is so far not well understood. Therefore, we further conducted the study as to the dose- and time-dependency of MTA-triggered dome development and the effect of MTA on several intestinal differentiation markers. As shown in Fig. 1A, the number of domes gradually increases along with increasing the MTA dose and provides a peak at 1 mM MTA, followed by decreasing at concentrations above 1 mM. No dome is formed at 4 mM when counting at 48 hr after MTA addition. Dome formation by MTA was evaluated at later time points (Fig. 1B). The number of domes in the culture treated with 1 and 2 mM MTA increases from 48 to 96 hr while the number per se declines slightly at 96 hr from at 72 hr. Additionally, 1 mM MTA constantly shows a higher effect on increasing the number of domes than 2 mM MTA.

Fig. 1

Domes generated in the RCM-1 cell culture as markers of morphological differentiation. RCM-1 cells were cultured with different doses of MTA for specified durations. (A and B) The number of domes formed during the early (A; 48 hr) and late (B; 48, 72, and 96 hr) stages of MTA treatment. (C) ALP activity fluctuated with MTA treatment. (D) Surface area of the dome affected by MTA. The horizontal bars show the mean area of the individual domes formed in the same treatment. (E) Three differentiation marker proteins, DPP4, villin, and KLF4, accumulated in the presence of 1 mM MTA. Immunoblotting was performed as described in Materials and Methods, and β-actin was used as a control for equal loading of the proteins. The data are presented as mean (S.D.) (n ≥ 3). Alphabetical letters indicate statistically significant differences among the MTA treatments.

The activity of ALP, a known biochemical differentiation marker, was measured at the early time points to clarify whether the activity is correlated with MTA-mediated dome formation, although increased ALP activity after 96 hr of incubation with MTA has already been reported (Kamimura et al., 2020). As shown in Fig. 1C, although ALP activity gradually increases over time of culture with or without of MTA (1 and 2 mM), the activity is significantly higher in MTA-treated cells than in non-treated cells at 48, 72, and 96 hr after MTA addition. Furthermore, dome formation is constant in MTA-treated cells from 48 to 96 hr of culture. ALP activity is higher in the cell culture treated with 1 and 2 mM MTA than that treated with 4 mM MTA, which entirely inhibits dome formation (Fig. 1A and 1B). The result suggests that the increase in ALP activity is closely associated with dome development enhanced by MTA. Interestingly, 2 mM MTA induces greater ALP activity than 1 mM, while 1 mM MTA increases the number of domes more effectively than 2 mM thereof. On the other hand, a morphometric analysis reveals that the average area of individual domes triggered by 2 mM MTA is larger than that triggered by 1 mM (Fig. 1D).

We further evaluated whether MTA affects the levels of the known molecular differentiation markers DPP4, villin, and KLF4 (Dudouet et al., 1987; Coudrier et al., 1988; Darmoul et al., 1992; Imai et al., 1992; Katz et al., 2002; Flandez et al., 2008; Hu et al., 2011), via immunoblotting. As shown in Fig. 1E, all three proteins are accumulated more in the MTA (1 mM)-treated cells than untreated cells at 48 and 72 hr of incubation. Therefore, these results indicate that MTA induces the differentiation of the unorganized cell mass into the villiform-like structure dome in the RCM-1 cell culture.

MTA and BA cooperatively enhance dome formation in the RCM-1 cell culture

It has been reported that BA and PPARγ agonists enhance dome formation and apoptosis in a variety of cell lines (Gum et al., 1987; Heerdt et al., 1994; Litvak et al., 1998; Shimada et al., 2002; Buda et al., 2003; Orchel et al., 2005; Cerbone et al., 2007; Shin et al., 2009; Tsuda et al., 2010). On the other hand, in our previous study, there was no increase of the number of domes when RCM-1 cell culture was treated with BA (Kamimura et al., 2020). In contrast, at this time, BA enhances the dome formation even though in a very narrow range of concentrations. As shown in Fig. 2A, the number of domes is mostly unchanged or even decreases from that of the mock control between 0 and 8 mM BA, where 0.1 mM BA results in the slight increase. Administration of 1 mM of BA or higher amounts completely inhibits dome formation, which is consistent with the previous result. RCM-1 cells are morphologically different from the normal cells, and the growth of RCM-1 cells does not reach confluence when 2 mM or more BA is added (Supplementary Fig. S1A), which indicates the occurrence of BA-mediated apoptosis. The above speculation is also supported by the remarkable increase of caspase-3/7 activity (Supplementary Fig. S2). Because 0.1 mM BA provides a slight increase, the effect of BA on dome formation using a narrow range of concentrations between 0 and 250 µM (Fig. 2B) was examined. At 50 and 100 µM, BA slightly increases the number of domes, but not statistically significant relative to the mock control (0 µM BA). It is shown that 250 µM BA enhances dome formation at 4.6-fold of the control in the RCM-1 cell culture. Meanwhile, 15d-PGJ2 and ROSI, PPARγ agonists do not totally increase the number of domes in a broad range of concentrations (Fig. 2C and 2D) and rather than increase, the number of domes gradually decreases by 1 μM of 15d-PGJ2 and there is no dome formation over 1 µM, and also by 0.1 µM of ROSI and the dome formation is strongly inhibited beyond 0.1 µM. Supplementary Fig. S1B shows that the growth of RCM-1 cells is delayed at 50 μM of 15d-PGJ2 and Supplementary Fig. S1C shows that RCM-1 cells is not grown to confluence in the presence of ROSI at 200 and 300 µM.

Fig. 2

Effect of BA and PPARγ agonists on dome formation in the RCM-1 cell culture and the relationship between BA and MTA. RCM-1 cells were incubated for 48 hr with one of the three differentiation inducers: BA and PPARγ agonists (15d-PGJ2 and ROSI) at the concentrations indicated in the graphs. (A and B) The number of domes formed in the BA-treated culture (0–8 mM in A; 0–250 μM in B). (C and D) The number of domes formed in the PPARγ agonist-treated culture (0–20 μM of 15d-PGJ2 in C; 0–300 μM of ROSI in D). (E) The number of domes increased by the simultaneous administration of BA (either 0.1 or 0.25 mM) with MTA (0.1, 0.25, or 1 mM). Data are presented as mean (S.D.) (n = 4). Different alphabetical letters indicate statistically significant differences among the treatments.

Followingly, we examined how a co-administration of MTA (0.1, 0.25, or 1 mM) and BA (either 0.1 or 0.25 mM) affects dome formation to evaluate an association between both compounds. As shown in Fig. 2E, the number of domes additively increases when 0.1 mM BA is applied with either 0.1 or 0.25 mM MTA compared to the sole administration of 0.1 mM BA and 0.1 or 0.25 mM MTA. Meanwhile, no additive effect is observed when 0.25 mM BA and either one of 0.1 and 0.25 mM MTA are co-administered. Furthermore, 0.25 mM BA decreases the number of domes, which is enhanced by 1 mM MTA; the number of domes is in a lower level than the level that a single compound administration provides, even though such level is still higher than that of the mock control.

High-dose MTA triggers the apoptosis of RCM-1 cells

It is unclear whether MTA affects the viability and death of RCM-1 cells and the extent thereto. The CCK-8 assay was conducted to estimate the proportion of living cells, which is almost synonymous with cell viability. As shown in Fig. 3A, the living cell proportion gradually declines in accordance with an increase of MTA dose. The proportion thereof is almost the same level when less than 0.5 mM MTA is administered to the cells and obviously decreases when 1 and 2 mM MTA are administered. When 4 mM MTA is administered, the proportion of living cells is nearly 60% of that of the untreated control, and dome formation never takes place (Fig. 1A and 1B). A trypan blue exclusion assay was subsequently performed to estimate the number of dead cells (Fig. 3B). Following cultivation in the absence of MTA, since approximately 30% of RCM-1 cells are stained blue, it is suggested that cells are naturally died during the culture. When MTA is administered, the death rate of cells is getting higher along with increasing MTA dose. The death rate is 50% when the cells were treated with 4 mM MTA. These results indicate that MTA is not only enhancing dome formation, but also lowering viability of cells and eventually leading cells to death. In addition, RCM-1 cells are no longer morphologically normal when 4 mM or more MTA is administered (Supplementary Fig. S1D).

Fig. 3

Effect of MTA on the viability and death of RCM-1 cells. RCM-1 cells were cultured with different doses of MTA for 48 hr. (A) Cell viability determined using the CCK-8 assay. Data are presented as mean (S.D.) (n = 4). (B) Cell death determined by the trypan blue exclusion assay. Data are presented as mean (S.D.) (n = 8). Different alphabetical letters in the graphs (A and B) indicate statistically significant differences among the samples.

We investigated whether the apoptotic pathway is involved in cell death caused by MTA. First, DNA fragmentation in MTA-treated cells was examined (Fig. 4A). When 1 and 2 mM MTA are applied, dome formation is constantly enhanced (Fig. 1) and DNA fragmentation gradually increases compared with that of the mock control. In fact, the value is three-fold higher than that of the control when 4 mM or more of MTA is administered. Caspase activity was then measured in MTA-treated cells using a caspase-3/7 fluorescence assay (Fig. 4B). The effector caspases’ activity is dose-dependently increased by MTA, which is similar to the results for DNA fragmentation. The activity is slightly induced when 1 mM MTA is added, and continues to increase as the MTA dose is increased up to 6 mM. To further validate the relevance of apoptosis in this process, we conducted immunoblot analysis as to the accumulation of cleaved PARP protein that is an indicator of the progression of apoptosis. As shown in Fig. 4C, cleaved PARP is detected very faintly when 1 mM MTA is administered, the protein level thereof increases dose-dependently, and the amount of protein upon the administration of 4–6 mM MTA is maximum. Taken together, these results indicate that MTA-triggered cell death is mediated by apoptosis.

Fig. 4

Effect of MTA on the apoptotic markers in RCM-1 cells. RCM-1 cells were treated with different doses of MTA for 48 hr. (A) DNA fragmentation determined using Cell Death Detection ELISA^PLUS. (B) Caspase-3/7 activity determined using a Caspase-3/7 Fluorescence Assay Kit. This assay was concomitantly carried out with or without a caspase-3/7 inhibitor to determine the specificity of enzyme activity. (C) Accumulation of the cleaved PARP protein as revealed by immunoblotting. Each value in the graphs (A and B) is expressed as a fold-increase relative to that of the mock control. The data are presented as mean (S.D.) (n = 3). Different alphabetical letters indicate significant differences among the samples. β-Actin was used as a control for equal protein loading.

The apoptosis inducers AD and 5-FU enhance dome formation in the RCM-1 cell culture

We were also interested in the relationship between apoptosis induction and dome formation. First, we tested whether AD and 5-FU, known apoptosis inducers, can enhance dome formation in RCM-1 cell culture because MTA dose-dependently enhances dome formation at a low dose (Fig. 1) but causes cell death at a high dose (Figs. 3 and 4). As shown in Fig. 5A, AD increases the number of domes at extremely low doses in the range of 0.001 to 0.01 µM to be higher than the mock control (DMSO), but no dome formation is observed at 0.1 µM or higher doses. Based on microscopic observations, RCM-1 cells are not grown to confluence in the presence of a high dose of AD, which is suggested due to the cytotoxicity of AD (Supplementary Fig. S1E). This result is consistent with AD-mediated increase in caspase-3/7 activity (Supplementary Fig. S2). 5-FU also increases the number of domes in a narrow range of concentrations (1–50 µM) and there is a peak of the number thereof at 10 µM (Fig. 5B). The enhancement of dome formation by 5-FU, on the other hand, is much weaker than by AD. 5-FU also has a smaller effect on the growth of RCM-1 cells (Supplementary Fig. S1F) and capase-3/7 activity (Supplementary Fig. S2).

Fig. 5

Effect of two apoptosis inducers, AD and 5-FU, on dome formation. RCM-1 cells were cultured with different doses of two apoptosis inducers, AD and 5-FU for 48 hr. The number of domes formed in the chemical-treated culture (0–50 µM for AD in A; 0–200 µM for 5-FU in B). The presentation styles align with those in Fig. 2.

DISCUSSION

To date, we have isolated dome-inducing compounds such as MTPE and MTAE from Katsura-uri (C. melo var. conomon), and PO from spearmint (M. spicata) using the number of domes formed in the RCM-1 cell culture as a guide for screening (Nakamura et al., 2008, 2010, 2014). Furthermore, we found that among the 37 MTPE and MTAE analogs MTA had the most potent dome-inducing activity against the RCM-1 cell culture (Kamimura et al., 2020). It is reported that MTPE and MTA enhanced dome formation and also increased the activity of ALP, a biochemical differentiation marker at the later time points of administration of these compounds (Nakamura et al., 2008; Kamimura et al., 2020). And, the domes developed in RCM-1 cell culture have not been validated as a reliable marker yet, although those formed in various cell lines are recognized as a morphological differentiation marker (Lever, 1979; Fantini et al., 1986; Gum et al., 1987; Zucchi et al., 2002). Therefore, we are interested in whether and how domes are related to the differentiation of RCM-1 cells. The result shows that MTA (0.125–1 mM) dose- and time-dependently increases the number of domes in the RCM-1 cell culture and concomitantly increases the activity of ALP in the same manner (Fig. 1A–1C). Meanwhile, a high dose of MTA (4 mM) has no effect on dome formation at all and lowers ALP activity. The correlation between the number of domes and ALP activity supports that domes formed in the RCM-1 cell culture should be regarded as a morphological differentiation marker. Interestingly, 2 mM MTA more intensely induces ALP activity than 1 mM, although 2 mM MTA is less effective on increasing the number of domes than 1 mM MTA (Fig. 1A–1C). In contrast, the average area of the domes in the 2 mM MTA-treated RCM-1 cell culture is larger than that of domes in the 1 mM MTA-treated culture (Fig. 1D). It is suggested that MTA-induced ALP activity is correlated with the increased number and average area of domes.

Immunoblot analyses revealed that three molecular differentiation markers, DPP4, villin, and KLF4, are increased by 1 mM MTA and accumulated at 48 and 72 hr after MTA addition (Fig. 1E). Of note, the number of domes greatly increases at the same time points (Fig. 1B). DPP4 and villin are a serine protease and a cytoskeletal protein interlining the microvilli, respectively, and are localized to the intestinal brush borders in colon cancer cell lines, such as HT-29 (Coudrier et al., 1988; Darmoul et al., 1992). Therefore, it is indicated that MTA directs the differentiation of RCM-1 cancer cells into enterocytes with microvilli because of the increased level of both marker proteins together with the increased activity of ALP that reportedly accumulates in the microvilli of differentiated enterocytes at the brush border domain in normal intestinal tissues (Domar et al., 1992). In contrast, KLF4 is a zinc-finger transcription factor that is reportedly required for the terminal differentiation of goblet cells in the colon (Katz et al., 2002). Accordingly, MTA might generate more than one type of differentiated cells in the RCM-1 cell culture (e.g., enterocytes and goblet cells). Taken together, these results strongly suggest that the domes formed in the RCM-1 cell culture are available as a reliable differentiation indicator; it is similar to those used in two well-known models of colon cancer cell lines, HT-29 and Caco-2 (Fantini et al., 1986; Mariadason et al., 2000). The results also confirm that MTA is certainly an inducer of RCM-1 cell differentiation and that MTPE, MTAE, and PO are the differentiation inducers because these compounds enhance dome formation in the RCM-1 cell culture (Nakamura et al., 2008, 2010, 2014).

BA is a short-chain fatty acid with four carbon atoms that is derived from bacterial fermentation of dietary fibers (Mortensen et al., 1988) and habitually exists in millimolar concentrations in the intestinal lumen (Cummings, 1981). It is reported that 2 mM BA enhanced dome formation in colon cancer cell lines, such as LS174T and Caco-2 (Gum et al., 1987; Mariadason et al., 2000). But RCM-1 cell culture treated with the relevant millimolar concentrations of BA provided a different outcome in our previous study (Kamimura et al., 2020). Herein, BA enhances dome formation in RCM-1 cell culture in the low and narrow concentration range of approximately 100–250 µM (Fig. 2A and 2B). Such findings suggest that RCM-1 cell line is more sensitive to BA in terms of dome formation than the two lines described above. MTA is structurally similar to BA because only the sulfur atom in MTA is replaced by a carbon atom in BA. Like BA, MTA increases the number of domes in RCM-1 cell culture at a range from 0.125 to 2 mM (Fig. 1A). Furthermore, co-application of MTA and BA shows an additive effect on dome formation in a low concentration range (0.1 mM BA and 0.1 or 0.25 mM MTA) (Fig. 2E). These results indicate that MTA and BA enhance dome formation in the RCM-1 cell culture, at least partly through the same or similar pathway(s). Therefore, MTA might be more suitable for inducing the differentiation of some types of colon cancer cells because MTA is less toxic than BA and exerts differentiation-inducing ability over a broad concentration range against RCM-1 cell line (Fig. 1 and Fig. 2). In addition, PPARγ agonists 15d-PGJ2 and ROSI, which reportedly enhanced dome formation in Caco-2 (Cerbone et al., 2007), do not increase the number of domes in the RCM-1 cell culture (Fig. 2C and 2D). The result infers that the PPARγ-mediated differentiation pathway has been hampered or lost in RCM-1 cell line. Collectively, these findings suggest the importance of combination of differentiation inducers and cancer cell types.

Some differentiation agents, such as BA, 15d-PGJ2, and ROSI, induce the apoptosis of colon cancer cell lines (Orchel et al., 2005; Cerbone et al., 2007; Shin et al., 2009). In this study, 4 mM MTA has no effect on dome formation at all and decreases ALP activity (Fig. 1A–1C), and also more than 2.5 mM of MTA induces an abnormal cell morphology (Supplementary Fig. S1D). Both the cell viability assay (Fig. 3) and the apoptosis marker analysis (Fig. 4) strongly suggest that MTA at 2 mM or more triggers the apoptosis of RCM-1 cells. Thus, MTA has the dual effects of inducing the differentiation and apoptosis of RCM-1 cells, as with the effect of BA on HT-29 and SW620 (Heerdt et al., 1994; Orchel et al., 2005), and that of 15d-PGJ2 on HT-29 and Caco-2 (Shimada et al., 2002; Cerbone et al., 2007). Interestingly, the growth of RCM-1 cells is retarded by 2 mM or more BA, 50 μM or more 15d-PGJ2, or 200 μM or more ROSI (Supplementary Fig. S1). Furthermore, BA and 15d-PGJ2 increase caspase-3/7 activity in the RCM-1 cells (Supplementary Fig. S2). These results indicate that BA and 15d-PGJ2 in addition to MTA trigger the apoptosis of RCM-1 cells. Of note, AD (1 and 10 nM) and 5-FU (10 μM), known apoptosis inducers, enhance dome formation in the RCM-1 cell culture (Fig. 5). Together, these findings suggest a close relationship between differentiation and apoptosis. Although the mechanism linking the two biological events remains unknown, the differentiation and apoptosis triggered by each agent may operate through the similar pathway(s) as previously reported for BA (Heerdt et al., 1994; Litvak et al., 1998).

Molecular mechanisms underlying MTA-mediated differentiation and apoptosis remain unclear. As mentioned above, MTA and BA, which are structurally related compounds dose-dependently induce cell differentiation and apoptosis in the RCM-1 cell culture, so that both compounds enable RCM-1 cells to trigger these biological events through the same or similar pathway(s). Since MTA per se should exist in the human gut only when ingested, it is reasonable to speculate that MTA uses pre-existing BA pathway(s) toward cell differentiation and apoptosis, which has been evolved in the colon epithelial cells exposed to BA produced in the colon intestinal lumen (Hamer et al., 2008). As BA has so far been intensively studied compared with MTA, and as a result, an extensive knowledge on BA-mediated differentiation and apoptosis should be accumulated. In fact, various literatures have reported the involvement of BA in different cellular processes such as cell cycle control (Heerdt et al., 1997; Litvak et al., 1998), epigenetic regulation, e.g., inhibition of histone deacetylase (Davie, 2003), signal transduction (Bordonaro et al., 2002; Orchel et al., 2005), and oxidative control (Domokos et al., 2010). Therefore, one of the feasible and rational approaches to clear MTA functions is to analyze whether and how MTA affects the BA-related pathway. In any case, understanding the mechanisms underlying MTA pathway is necessary and critical to develop a new therapeutic application using MTA.

In conclusion, we demonstrated that MTA, a derived ingredient of an aroma compound (MTAE) of Katsura-uri melon (C. melo var. conomon) had a dual role as an inducer of differentiation and apoptosis to RCM-1 human colorectal cancer cells. Furthermore, our results, together with those of others studies imply a close relationship between differentiation and apoptosis. Collectively, the present study indicates that MTA can be a novel and potential candidate for anticancer agent based on the cell differentiation therapy and the traditional cytotoxic chemotherapy for colorectal cancers.

ACKNOWLEDGMENTS

We thank Drs. Shoji Ookutsu and Kozue Sakao of Kagoshima University for technical support, helpful advice, and discussion to properly proceed our study. This work was partly supported by Japan Society for the Promotion of Science [grant numbers 19H01610, 21H03696].

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Aoi,  W.,  Takeda,  K.,  Sasaki,  A.,  Hasegawa,  Y.,  Nakamura,  Y.,  Park,  E.Y.,  Sato,  K.,  Iwasa,  M.,  Nakayama,  A.,  Minamikawa,  M.,  Kobayashi,  Y.,  Shirota,  K. and  Suetome,  N. (2015): The effect of Katsura-uri (Japanese pickling melon, Cucumis melo var. conomon) and its derived ingredient methylthioacetic acid on energy metabolism during aerobic exercise. Springerplus, 4, 377.
•  Bordonaro,  M.,  Lazarova,  D.L.,  Augenlicht,  L.H. and  Sartorelli,  A.C. (2002): Cell type- and promoter-dependent modulation of the Wnt signaling pathway by sodium butyrate. Int. J. Cancer, 97, 42-51.
•  Buda,  A.,  Qualtrough,  D.,  Jepson,  M.A.,  Martines,  D.,  Paraskeva,  C. and  Pignatelli,  M. (2003): Butyrate downregulates alpha2beta1 integrin: a possible role in the induction of apoptosis in colorectal cancer cell lines. Gut, 52, 729-734.
•  Cerbone,  A.,  Toaldo,  C.,  Laurora,  S.,  Briatore,  F.,  Pizzimenti,  S.,  Dianzani,  M.U.,  Ferretti,  C. and  Barrera,  G. (2007): 4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells. Free Radic. Biol. Med., 42, 1661-1670.
•  Coudrier,  E.,  Arpin,  M.,  Dudouet,  B.,  Finidori,  J.,  Garcia,  A.,  Huet,  C.,  Pringault,  E.,  Robine,  S.,  Sahuquillo-Merino,  C. and  Louvard,  D. (1988): Villin as a structural marker to study the assembly of the brush border. Protoplasma, 145, 99-105.
•  Cox,  M.E.,  Deeble,  P.D.,  Lakhani,  S. and  Parsons,  S.J. (1999): Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression. Cancer Res., 59, 3821-3830.
•  Cummings,  J.H. (1981): Short chain fatty acids in the human colon. Gut, 22, 763-779.
•  Darmoul,  D.,  Lacasa,  M.,  Baricault,  L.,  Marguet,  D.,  Sapin,  C.,  Trotot,  P.,  Barbat,  A. and  Trugnan,  G. (1992): Dipeptidyl peptidase IV (CD 26) gene expression in enterocyte-like colon cancer cell lines HT-29 and Caco-2. Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA levels during cell differentiation. J. Biol. Chem., 267, 4824-4833.
•  Davie,  J.R. (2003): Inhibition of histone deacetylase activity by butyrate. J. Nutr., 133 (Suppl), 2485S-2493S.
•  Dickens,  E. and  Ahmed,  S. (2021): Principles of cancer treatment by chemotherapy. Surgery, 39, 215-220.
•  Domar,  U.,  Nilsson,  B.,  Baranov,  V.,  Gerdes,  U. and  Stigbrand,  T. (1992): Expression of intestinal alkaline phosphatase in human organs. Histochemistry, 98, 359-364.
•  Domokos,  M.,  Jakus,  J.,  Szeker,  K.,  Csizinszky,  R.,  Csiko,  G.,  Neogrady,  Z.,  Csordas,  A. and  Galfi,  P. (2010): Butyrate-induced cell death and differentiation are associated with distinct patterns of ROS in HT29-derived human colon cancer cells. Dig. Dis. Sci., 55, 920-930.
•  Dudouet,  B.,  Robine,  S.,  Huet,  C.,  Sahuquillo-Merino,  C.,  Blair,  L.,  Coudrier,  E. and  Louvard,  D. (1987): Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones. J. Cell Biol., 105, 359-369.
•  Fantini,  J.,  Abadie,  B.,  Tirard,  A.,  Remy,  L.,  Ripert,  J.P.,  el Battari,  A. and  Marvaldi,  J. (1986): Spontaneous and induced dome formation by two clonal cell populations derived from a human adenocarcinoma cell line, HT29. J. Cell Sci., 83, 235-249.
•  Flandez,  M.,  Guilmeau,  S.,  Blache,  P. and  Augenlicht,  L.H. (2008): KLF4 regulation in intestinal epithelial cell maturation. Exp. Cell Res., 314, 3712-3723.
•  Gum,  J.R.,  Kam,  W.K.,  Byrd,  J.C.,  Hicks,  J.W.,  Sleisenger,  M.H. and  Kim,  Y.S. (1987): Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase. J. Biol. Chem., 262, 1092-1097.
•  Hamer,  H.M.,  Jonkers,  D.,  Venema,  K.,  Vanhoutvin,  S.,  Troost,  F.J. and  Brummer,  R.J. (2008): Review article: the role of butyrate on colonic function. Aliment. Pharmacol. Ther., 27, 104-119.
•  Heerdt,  B.G.,  Houston,  M.A. and  Augenlicht,  L.H. (1994): Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res., 54, 3288-3293.
•  Heerdt,  B.G.,  Houston,  M.A. and  Augenlicht,  L.H. (1997): Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth Differ., 8, 523-532.
•  Hu,  R.,  Zuo,  Y.,  Zuo,  L.,  Liu,  C.,  Zhang,  S.,  Wu,  Q.,  Zhou,  Q.,  Gui,  S.,  Wei,  W. and  Wang,  Y. (2011): KLF4 Expression Correlates with the Degree of Differentiation in Colorectal Cancer. Gut Liver, 5, 154-159.
•  Huang,  M.E.,  Ye,  Y.C.,  Chen,  S.R.,  Chai,  J.R.,  Lu,  J.X.,  Zhoa,  L.,  Gu,  L.J. and  Wang,  Z.Y. (1988): Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood, 72, 567-572.
•  Imai,  K.,  Maeda,  M.,  Fujiwara,  H.,  Kariya,  M.,  Takakura,  K.,  Kanzaki,  H. and  Mori,  T. (1992): Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells. Hum. Reprod., 7, 1189-1194.
•  Johnson,  B.E.,  Mazor,  T.,  Hong,  C.,  Barnes,  M.,  Aihara,  K.,  McLean,  C.Y.,  Fouse,  S.D.,  Yamamoto,  S.,  Ueda,  H.,  Tatsuno,  K.,  Asthana,  S.,  Jalbert,  L.E.,  Nelson,  S.J.,  Bollen,  A.W.,  Gustafson,  W.C.,  Charron,  E.,  Weiss,  W.A.,  Smirnov,  I.V.,  Song,  J.S.,  Olshen,  A.B.,  Cha,  S.,  Zhao,  Y.,  Moore,  R.A.,  Mungall,  A.J.,  Jones,  S.J.,  Hirst,  M.,  Marra,  M.A.,  Saito,  N.,  Aburatani,  H.,  Mukasa,  A.,  Berger,  M.S.,  Chang,  S.M.,  Taylor,  B.S. and  Costello,  J.F. (2014): Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science, 343, 189-193.
•  Kamimura,  M.,  Sasaki,  A.,  Watanabe,  S.,  Tanaka,  S.,  Fukukawa,  A.,  Takeda,  K.,  Nakamura,  Y.,  Nakamura,  T.,  Kuramochi,  K.,  Otani,  Y.,  Hashimoto,  F.,  Ishimaru,  K.,  Matsuo,  T. and  Okamoto,  S. (2020): Chemical and molecular bases of dome formation in human colorectal cancer cells mediated by sulphur compounds from Cucumis melo var. conomon. FEBS Open Bio, 10, 2640-2655.
•  Kataoka,  H.,  Nabeshima,  K.,  Komada,  N. and  Koono,  M. (1989): New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol., 57, 157-165.
•  Katz,  J.P.,  Perreault,  N.,  Goldstein,  B.G.,  Lee,  C.S.,  Labosky,  P.A.,  Yang,  V.W. and  Kaestner,  K.H. (2002): The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development, 129, 2619-2628.
•  Lever,  J.E. (1979): Inducers of mammalian cell differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK). Proc. Natl. Acad. Sci. USA, 76, 1323-1327.
•  Lin,  B.,  Srikanth,  P.,  Castle,  A.C.,  Nigwekar,  S.,  Malhotra,  R.,  Galloway,  J.L.,  Sykes,  D.B. and  Rajagopal,  J. (2018): Modulating Cell Fate as a Therapeutic Strategy. Cell Stem Cell, 23, 329-341.
•  Litvak,  D.A.,  Evers,  B.M.,  Hwang,  K.O.,  Hellmich,  M.R.,  Ko,  T.C. and  Townsend,  C.M. Jr. (1998): Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27Kip1. Surgery, 124, 161-170.
•  Mariadason,  J.M.,  Rickard,  K.L.,  Barkla,  D.H.,  Augenlicht,  L.H. and  Gibson,  P.R. (2000): Divergent phenotypic patterns and commitment to apoptosis of Caco-2 cells during spontaneous and butyrate-induced differentiation. J. Cell. Physiol., 183, 347-354.
•  Miller,  K.D.,  Nogueira,  L.,  Mariotto,  A.B.,  Rowland,  J.H.,  Yabroff,  K.R.,  Alfano,  C.M.,  Jemal,  A.,  Kramer,  J.L. and  Siegel,  R.L. (2019): Cancer treatment and survivorship statistics, 2019. CA Cancer J. Clin., 69, 363-385.
•  Mortensen,  P.B.,  Holtug,  K. and  Rasmussen,  H.S. (1988): Short-chain fatty acid production from mono- and disaccharides in a fecal incubation system: implications for colonic fermentation of dietary fiber in humans. J. Nutr., 118, 321-325.
•  Nakamura,  Y.,  Hasegawa,  Y.,  Shirota,  K.,  Suetome,  N.,  Nakamura,  T.,  Chomnawang,  M.T.,  Thirapanmethee,  K.,  Khuntayaporn,  P.,  Boonyaritthongchai,  P.,  Wongs-Aree,  C.,  Okamoto,  S.,  Shigeta,  T.,  Matsuo,  M.,  Park,  E.Y. and  Sato,  K. (2014): Differentiation-inducing effect of piperitenone oxide, a fragrant ingredient of spearmint (Mentha spicata), but not carvone and menthol, against human colon cancer cells. J. Funct. Foods, 8, 62-67.
•  Nakamura,  Y.,  Nakayama,  Y.,  Ando,  H.,  Tanaka,  A.,  Matsuo,  T.,  Okamoto,  S.,  Upham,  B.L.,  Chang,  C.C.,  Trosko,  J.E.,  Park,  E.Y. and  Sato,  K. (2008): 3-Methylthiopropionic acid ethyl ester, isolated from Katsura-uri (Japanese pickling melon, Cucumis melo var. conomon), enhanced differentiation in human colon cancer cells. J. Agric. Food Chem., 56, 2977-2984.
•  Nakamura,  Y.,  Watanabe,  S.,  Kageyama,  M.,  Shirota,  K.,  Shirota,  K.,  Amano,  H.,  Kashimoto,  T.,  Matsuo,  T.,  Okamoto,  S.,  Park,  E.Y. and  Sato,  K. (2010): Antimutagenic; differentiation-inducing; and antioxidative effects of fragrant ingredients in Katsura-uri (Japanese pickling melon; Cucumis melo var. conomon). Mutat. Res., 703, 163-168.
•  Nowak,  D.,  Stewart,  D. and  Koeffler,  H.P. (2009): Differentiation therapy of leukemia: 3 decades of development. Blood, 113, 3655-3665.
•  Orchel,  A.,  Dzierzewicz,  Z.,  Parfiniewicz,  B.,  Weglarz,  L. and  Wilczok,  T. (2005): Butyrate-induced differentiation of colon cancer cells is PKC and JNK dependent. Dig. Dis. Sci., 50, 490-498.
•  Sasaki,  A.,  Nakamura,  Y.,  Kobayashi,  Y.,  Aoi,  W.,  Nakamura,  T.,  Shirota,  K.,  Suetome,  N.,  Fukui,  M.,  Shigeta,  T.,  Matsuo,  T.,  Okamoto,  S.,  Park,  E.Y. and  Sato,  K. (2017): A new strategy to protect Katsura-uri (Japan’s heirloom pickling melon, Cucumis melo var. conomon) from extinction. Journal of Ethnic Foods, 4, 44-50.
•  Seca,  A.M. and  Pinto,  D.C. (2018): Plant Secondary Metabolites as Anticancer Agents: Successes in Clinical Trials and Therapeutic Application. Int. J. Mol. Sci., 19, 263.
•  Sell,  S. and  Ilic,  Z. (2019): Comparison of survivor scores for differentiation therapy of cancer to those for checkpoint inhibition: half full or half empty. Tumour Biol., 41, 1010428319873749.
•  Shimada,  T.,  Kojima,  K.,  Yoshiura,  K.,  Hiraishi,  H. and  Terano,  A. (2002): Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells. Gut, 50, 658-664.
•  Shin,  S.W.,  Seo,  C.Y.,  Han,  H.,  Han,  J.Y.,  Jeong,  J.S.,  Kwak,  J.Y. and  Park,  J.I. (2009): 15d-PGJ2 induces apoptosis by reactive oxygen species-mediated inactivation of Akt in leukemia and colorectal cancer cells and shows in vivo antitumor activity. Clin. Cancer Res., 15, 5414-5425.
•  Sugihara,  E. and  Saya,  H. (2013): Complexity of cancer stem cells. Int. J. Cancer, 132, 1249-1259.
•  Tsuda,  H.,  Ochiai,  K.,  Suzuki,  N. and  Otsuka,  K. (2010): Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J. Periodontal Res., 45, 626-634.
•  Vasan,  N.,  Baselga,  J. and  Hyman,  D.M. (2019): A view on drug resistance in cancer. Nature, 575, 299-309.
• World Health Organization (WHO). Cancer. Retrieved from https://www. who.int/health-topics/cancer#tab=tab_1. Accessed July 18, 2022.
•  Yang,  Q.J.,  Zhou,  L.Y.,  Mu,  Y.Q.,  Zhou,  Q.X.,  Luo,  J.Y.,  Cheng,  L.,  Deng,  Z.L.,  He,  T.C.,  Haydon,  R.C. and  He,  B.C. (2012): All-trans retinoic acid inhibits tumor growth of human osteosarcoma by activating Smad signaling-induced osteogenic differentiation. Int. J. Oncol., 41, 153-160.
•  Yu,  C. and  Ding,  S. (2020): Therapeutic strategies targeting somatic stem cells: chemical approaches. Bioorg. Med. Chem., 28, 115824.
•  Zucchi,  I.,  Bini,  L.,  Albani,  D.,  Valaperta,  R.,  Liberatori,  S.,  Raggiaschi,  R.,  Montagna,  C.,  Susani,  L.,  Barbieri,  O.,  Pallini,  V.,  Vezzoni,  P. and  Dulbecco,  R. (2002): Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc. Natl. Acad. Sci. USA, 99, 8660-8665.