Abstract
Background/Aim: Fucoxanthinol (FxOH), a marine carotenoid, induces apoptosis and anoikis in human colorectal cancer (CRC) DLD-1 cells via the down-regulation of chloride intracellular channel 4 (CLIC4) expression, a key molecule for apoptosis. However, whether FxOH is susceptible to CLIC4 expression and its regulatory mechanisms in human CRC cells remains unknown. We investigated the inhibitory effects of FxOH on six types of human CRC cells with CLIC4 regulation. Materials and Methods: The association between FxOH and CLIC4 was investigated using gene knockdown, overexpression, and transcriptome analyses. Results: CLIC4 expression in CRC cells was a significant factor associated with sensitivity to FxOH. CLIC4 regulates many cancer-related signals and participates in growth inhibition in FxOH-treated DLD-1 cells. Both CLIC4 knockdown and overexpression attenuated the inhibitory effects of FxOH on DLD-1 cells. Conclusion: Our findings suggest that the protein expression of CLIC4 and its regulating mechanisms play significant roles regarding cell death in human CRC cells by FxOH treatment. Further investigation by in vitro and in vivo models is needed to determine the effect of CLIC4.
Colorectal cancer (CRC) is one of most prevalent cancers, with the third highest number of new cases (1.9 million per a total of 19.3 million) and second highest number of new deaths (0.9 million per a total of 10.0 million) worldwide, as estimated using the Global Cancer Observatory: CANCER TODAY 2020 database (1). The global incidence and mortality rates of CRC are expected to increase to more than 2.2 million new cases and 1.1 million deaths, respectively, by 2030 (2).
Point mutations in certain driver genes, such as adenomatous polyposis coli, Kirsten-ras (KRAS) and tumor protein p53 (Tp53), and aberrant regulations of many other genes and processes, such as signal transduction, tumor microenvironment formation, immune suppression, and gut microbiota alteration, are considered to contribute to carcinogenesis and disease progression (3-14).
The chloride intracellular channel (CLIC) protein family is a group of small globular proteins (28 kDa) comprising seven members: CLIC1, CLIC2, CLIC3, CLIC4, CLIC5A, CLIC5B, and CLIC6. They are ubiquitously expressed in various tissues and involved in intracellular trafficking, membrane remodeling, and other homeostatic mechanisms (15). Among these, CLIC4 has been well-studied for its association with cancer development. CLIC4 is a key molecule for apoptosis, angiogenesis, cell adhesion, wound healing, and tumorigenesis (14-20). In addition, CLIC4 expression is regulated by KRAS and Tp53 (21,22). We recently revealed that the number of CLIC4 high-expressing cells tended to decrease with the progression of pathological grades in colorectal malignant tissues of patients with CRC (23). These findings were also consistent with those of a previous study (24). Moreover, high expression levels of CLIC4 protein are suggested to be correlated with poor prognosis in patients with CRC and the aggressive potential of CRC stem-like cells (25). However, the role of CLIC4 in CRC development remains unknown.
A dominant marine pigment of fucoxanthin (Fx), a nonprovitamin A carotenoid, is an essential photosynthetic lipid for brown algae and microalgae (26). Humans can also obtain Fx via edible brown algae, such as Undaria pinnatifida (wakame) and Himanthalia elongata (sea spaghetti). Toxicological studies have revealed that Fx is a safe compound with little side effects in humans and rodents (27-29). Dietary Fx is metabolically converted to cis-Fx and fucoxanthinol (FxOH; Figure 1A) in the intestine, and subsequently to amarouciaxanthin A (Amx A) and cis-Amx A in the liver in both humans and mice. FxOH and cis-FxOH are the main plasma metabolites in humans when ingested with brown algae or its extract (30-32). No clinical and epidemiological data are available regarding the anticancer effects of Fx. However, anti-cancer potentials for Fx and FxOH have been well elucidated in vivo and in vitro (33-37).
Effects of FxOH on cell growth and CLIC4 expression in six of human colorectal cancer (CRC) cells, and on cell growth in DLD-1 cells with CLIC4 knockdown. (A) Fucoxanthinol (FxOH) structure. Molecular weight, 616.87 g/mol (C40H56O5). (B) Six of human CRC cells, DLD-1, HT-29, HCT116, Colo205, LoVo, and KM12C, were treated with FxOH (1.0 and 5.0 μM) for 2 days. Cell viability was determined using a tetrazolium salt [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) assay. Mean±SE (n=6). *p<0.05. (C) CLIC4 protein levels in six human colorectal cancer cells treated with FxOH (5.0 μM) for 1 day were measured by western blotting. (D) The correlation plots between growth inhibition and CLIC4 expression in human CRC cells treated with FxOH. X-axis, CLIC4 expression in each type of control cell that was set as 100% on the CLIC4 expression of control DLD-1 cells; Y-axis, the ratio of growth inhibitions in six of CRC cells treated with FxOH (1.0 and 5.0 μM) as shown in Figure 1A. (E) DLD-1 cells were treated with Clic4 dsiRNA for 2 days. The CLIC4 protein level was measured by western blotting. NC, negative control. (F) The CLIC4 knockdown DLD-1 cells were treated with FxOH (5.0 μM) for 1 day. The cell viability was assessed by a WST-1 assay. Mean±SE (n=6). a, *p<0.05 vs. negative control cells with vehicle alone; b, *p<0.05 vs. negative control cells with FxOH; c, *p<0.05 vs. CLIC4 knockdown cells with vehicle alone. (G) CLIC4 protein levels in negative control and CLIC4 knockdown in DLD-1 cells with and without FxOH treatment were measured by western blotting, respectively.
Recently, we showed that FxOH treatment can induce apoptosis in human CRC DLD-1 cells via the suppression of CLIC4 signals comprised of CLIC4, integrin β1, Na+/H+ exchanger regulatory factor 2 (NHERF2), and phosphorylated Smad2, which are all significant regulators of CRC development (38-42). Interestingly, CLIC4 knockdown attenuated the apoptotic potential of FxOH in cells (38). Moreover, alterations in cancer-related signals, such as the cell cycle, adhesion, and phosphoinositide 3-kinase (PI3K)/serine-threonine kinase protein B (AKT) expression, and the induction of anchor-dependent cell detachment (anoikis) were observed in FxOH-treated DLD-1 cells (38, 43-45). These findings led us to assume that sensitivity to apoptosis and anoikis induction by FxOH in human CRC cells may depend on the protein expression levels of CLIC4 and its regulatory mechanisms. However, the detailed mechanism remains unclear.
In the present study, we investigated the growth inhibitory effect of FxOH via CLIC4 regulation in human CRC cells and elucidated its underlying molecular mechanisms.
Materials and Methods
Chemicals. All-trans-FxOH (purity ≥ 98%) was kindly donated by Dr. Hayato Maeda (Hirosaki University, Japan) (Figure 1). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Human CRC cells (DLD-1, HT-29, HCT116, Colo205, LoVo, and KM12C) were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine (4 mM), penicillin (40,000 U/l), and streptomycin (40 mg/L). Lipofectamine RNAiMAX, Lipofectamine 3000, Opti-MEM I, RNAlater, penicillin/streptomycin, and GlutaMAX were obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). DynaMarker RNA high for easy electrophoresis was obtained from the BioDynamics Laboratory (Tokyo, Japan). Anti-β-actin, anti-CLIC4, and anti-interferon induced protein with tetratricopeptide repeats 1 (IFIT1) antibodies were obtained from GeneTex (Irvine, CA). Anti-claudin-2 (CLDN2) and anti-c-KIT antibodies were purchased from Novus Biologicals (Littleton, CO, USA) and R&D Systems (Minneapolis, MN, USA), respectively. The premixed water-soluble tetrozolium (WST)-1 cell proliferation assay system was obtained from Takara Bio (Shiga, Japan). All other reagents and solvents used in this study were of high grade.
Cell viability. DLD-1, HT-29, HCT116, Colo205, LoVo, and KM12C cells were seeded at a density of 5×104 cells/ml in a 24-well plate in 10% FBS/DMEM. After adherence for 3.5 h, the medium was replaced with fresh 1% FBS/DMEM medium containing FxOH (final concentration, 1.0 and 5.0 μmol/l) or vehicle (dimethyl sulfoxide, DMSO) and incubated for 1 day. Cell viability was evaluated using a WST-1 assay and measured at 450 nm wavelength using an enzyme-linked immunosorbent assay reader (TECAN Japan, Tokyo, Japan).
CLIC4 knockdown and overexpression. Two 27-mer dicer-substrate short interfering RNAs (dsiRNAs) encoding CLIC4 mRNA in Homo sapiens were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The CLIC4 dsiRNAs used were as follows: dsiRNA-1, 5’-GCUCUCUAUUCUAGUUGAUAAAACT-3’ and dsiRNA-2, 5’-CAAAUUCUCUGCAUAUAUCAAGAAT-3’. A dsiRNA sequence of the negative control (NC), 5’-GUGUUCU ACACCAUUACUCAAUUCUUA-3’, was prepared for CLIC4 knockdown. A CLIC4 ORF plasmid cDNA (pcDNA) 3.1(+)-CMV mammalian expression vector (Clone, OHu22609D) was constructed for CLIC4 overexpression using GenScript (Piscataway, NJ, USA). Lipofectamine RNAiMAX and Opti-MEM I reduced serum medium were used to prepare dsiRNA complexes, according to the manufacturer’s instructions. For CLIC4 knockdown, 5.5 μl of dsiRNA or NC was added to 30 μl of Lipofectamine RNAiMAX and mixed with 1,000 μl of Opti-MEM I medium. DLD-1 cells were seeded in 100-mm dishes at a density of 8×104 cells/ml and allowed to adhere for 1 d for both CLIC4 knockdown and overexpression. The dsiRNA -1, -2, or NC complex was then added to 10 ml of 10% FBS/DMEM (final concentration of dsiRNA, 10 nmol/l) for 1 day. The medium was then changed to 10% FBS/DMEM containing the new dsiRNA complex for 1 day.
Lipofectamine 3000 and Opti-MEM I reduced-serum medium were used to prepare pcDNA complexes, according to the manufacturer’s instructions. For CLIC4 overexpression, 4 μg of pcDNA solution or vehicle (Tris-ethylenediaminetetraacetic acid, TE buffer) was mixed with 200 μl of Opti-MEM I reduced-serum medium (mixture-A). Subsequently, Lipofectamine 3000 was mixed with 200 μl of Opti-MEM I reduced-serum medium at room temperature for 5 min (mixture-B). Mixtures A and B were mixed at room temperature for 20 min, added to 10 ml of cell culture medium (final concentration of pcDNA, 0.4 μg/ml), and incubated at 37°C for 1 day. The medium was then changed to 10% FBS/DMEM containing the new pcDNA complex for 1 day.
Cells with or without CLIC4 knockdown and overexpression were seeded at a density of 5×104 cells/ml in a 24-well plate in 10% FBS/DMEM. After adherence for 3.5 h, the cells were reintroduced into the medium containing FxOH (final concentration, 5 μmol/l) or vehicle (DMSO) in 1% FBS/DMEM and incubated for 1 day. Cell growth was measured using a tetrazolium salt [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) cell viability assay.
Total RNA extraction and purification. Total RNA was extracted from each whole cell and purified using the RNeasy Mini Kit and RNase-Free DNase Set (QIAGEN, Valencia, CA, USA), according to the manufacturer’s instructions. The concentration of total RNA was determined using Nanodrop ND-1000 (NanoDrop, Wilmington, DE, USA). Total RNA was quantified using 1.3% agarose gel electrophoresis based on 18S and 28S rRNAs and DynaMarker RNA High for Easy Electrophoresis.
Transcriptome analysis. Transcriptome analysis of each whole cell was performed using a Clariom™ S human assay (GeneChip) with the standard enzymes and reagents kit (Thermo Fisher Scientific, Carlsbad, CA, USA). First, 50 ng of total RNA was mixed with the poly(A)-RNA controls. First-strand cDNA was enzymatically generated from RNA templates and second-strand cDNA was subsequently synthesized. In vitro transcription using T7 RNA polymerase was conducted for the massive preparation of singlestranded cRNA from double-stranded cDNA. The resulting singlestranded cRNA (15 μg) was purified using a magnetic purification bead and converted into second-cycle single-stranded cDNA. The cDNA template was then purified using a magnetic purification bead, fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1, and biotin-labeled with terminal deoxynucleotidyl transferase. The labeled cDNA (2.3 μg) was then hybridized to GeneChip, washed, and stained using Affymetrix Fluidics Station 450 with stain solutions (Affymetrix, Santa Clara, CA, USA). GeneChip was scanned using the Affymetrix GeneChip Scanner 3000 system (Affymetrix). Data were analyzed using Transcriptome Analysis Console (TAC) software version 4.0.2 (Applied Biosystems, Foster City, CA, USA). The genes with significant differences between the two groups were evaluated using ≥2.0 and ≤-2.0-fold with cutoff p-value [one-way analysis of variance (ANOVA), p<0.05]. Principal coordinate analysis (PCoA), volcano plots, hierarchy clustering, and wikipathways were performed using the TAC software.
Western blotting analysis. Cells were collected and lysed using the lysis buffer. Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Ten micrograms of whole cell lysate were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and then electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Amersham Bioscience, Little Chalfont, UK). The PVDF membrane was washed with Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and blocked with 1% w/v bovine serum albumin (BSA) in TBS-T (1% BSA/TBS-T) at room temperature for 1 h. Subsequently, the membrane was incubated with each primary antibody (diluted 1:1,000) in 1% BSA/TBS-T at 4°C overnight. The membrane was washed with TBS-T and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies in 1% BSA/TBS-T at room temperature for 1 h. Protein bands in the membrane were observed using a chemiluminescence assay (Millipore, Billerica, MA, USA).
Statistical analysis. All values are represented as the mean±standard error. Statistical analysis was performed using one-way ANOVA with Tukey–Kramer post hoc tests for multiple comparisons. Differences were considered to be statistically significant at *p<0.05 and exact p-Values were calculated by an exact test on edge R in the TAC software.
Results
Effect of FxOH on the growth of human CRC cells. The effect of FxOH on the growth of six human CRC cells was determined. The growth of DLD-1, HT-29, HCT116, Colo205, and KM12C cells was significantly inhibited by FxOH treatment in a dose-dependent manner. However, little significant difference in LoVo cell growth was observed (Figure 1B). In addition, FxOH treatment clearly decreased CLIC4 protein expression levels in DLD-1 and HCT116 cells, but not in HT-29, Colo205, LoVo, and KM12C cells (Figure 1C). An inverse correlation was found between the growth inhibitory level of each cell with FxOH treatment and CLIC4 expression level in each control cell (100% in that of DLD-1 control cell): R2 values in FxOH 1.0 and 5.0 μmol/l were 0.5131 and 0.3608, respectively (Figure 1D). DLD-1 cells treated with DsiRNA for two days showed decreased CLIC4 protein expression compared to NC cells (Figure 1E). CLIC4 knockdown and NC cells were collected, seeded onto a culture plate, and treated with FxOH (5.0 μmol/l). Cell growth in CLIC4 knockdown control cells was significantly decreased compared with that in NC control cells. The inhibitory effect of FxOH on cell growth in CLIC4 knockdown cells with FxOH was significantly attenuated compared to that of the cell growth in NC cells with FxOH (Figure 1F). The protein expression levels of CLIC4 were decreased in NC cells with FxOH treatment, whereas those in CLIC4 knockdown DLD-1 cells were not detected with or without FxOH treatment (Figure 1G).
Transcriptome alterations in human CRC DLD-1 cells following CLIC4 knockdown. First, the transcriptome changes in CLIC4 knockdown DLD-1 cells were determined. The PCoA plot showed major genetic differences between CLIC4 knockdown and NC cells (Figure 2A). Hierarchical clustering of 956 genes revealed large differences between CLIC4 knockdown and NC DLD-1 cells (Figure 2B). The volcano plot revealed that the levels of both gene fold differences and p-values on the down-regulated genes in CLIC4 knockdown control cells were higher than those in the NC cells (Figure 2C). Overall, 454 up-regulated and 502 down-regulated genes (a total of 956 genes) were obtained in CLIC4 knockdown control cells compared to NC cells (Figure 2D). Pathway analysis showed that 14 of the top 20 gene sets, namely nuclear receptor (NR), vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), endothelin, nuclear factor-erythroid 2-related factor 2 (NRF2), PI3K/AKT, adhesion, interleukin (IL)-18, epithelial–mesenchymal transition (EMT), transforming growth factor (TGF)-β, and insulin signaling, were involved in gene groups for cancer development, including three types of microRNA (miRNA) regulation (Figure 2E, black circle).
Gene expression profiles in DLD-1 cells with or without CLIC4 knockdown. Gene expression levels between the CLIC4 knockdown (KD) DLD-1 cells (group 1) and the negative control cells (group 2) were subjected to Clariom™ S human assays and TAC software (n=3). (A) PCoA plot indicating genetic differences among different samples. (B) Hierarchical cluster analysis for 956 genes between groups 1 and 2. (C) Volcano plots between groups 1 and 2. (D) Number of up- (≥2.0-fold) and down-regulated (≤-2.0-fold) genes between groups 1 and 2. (E) The distribution of the top 20 gene sets that changed between group 1 and 2. Black circles indicate the gene set involving cancer development. The p-Value was determined using two-sided Fisher’s exact test. The value implies an index measuring bias of the gene set in DLD-1 cells with CLIC4 knockdown.
Effect of FxOH on the growth of CLIC4-overexpressing human CRC DLD-1 cells. The effect of FxOH on the growth of CLIC4-overexpressing DLD-1 cells was also determined. DLD-1 cells treated with CLIC4 pDNA for two days showed increased CLIC4 protein expression levels compared to NC cells (Figure 3A). The CLIC4 overexpression and NC cells were collected, seeded onto a culture plate, and treated with FxOH (5.0 μmol/l). Cell growth in CLIC4 overexpression control cells was significantly increased compared with that in NC control cells. The inhibitory effect of FxOH on cell growth in CLIC4 overexpression cells was significantly attenuated compared to that of the cell growth in NC cells (Figure 3B). The protein expression levels of CLIC4 decreased in NC cells with FxOH treatment, whereas those in CLIC4 overexpression cells with FxOH treatment did not change either with or without FxOH treatment (Figure 3C).
Effects of FxOH on cell growth and CLIC4 expression in DLD-1 cells with CLIC4 overexpression. DLD-1 cells were treated with Clic4 plasmid DNA [(p)DNA] for 2 days. (A) The CLIC4 protein level was measured by western blotting. NC, negative control. (B, C) The CLIC4 overexpression DLD-1 cells were treated with FxOH (5.0 μM) for 1 day. (B) Cell viability was assessed by a tetrazolium salt [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) assay. Mean±SE (n=6-10). a, *p<0.05 vs. negative control cells with vehicle alone; b, *p<0.05 vs. negative control cells with FxOH; c, *p<0.05 vs. CLIC4 overexpression cells with vehicle alone. (C) CLIC4 protein levels in negative control and CLIC4 overexpression DLD-1 cells with and without FxOH treatment were measured by western blotting, respectively.
Transcriptome alterations in CLIC4-overexpressing human CRC DLD-1 cells. Transcriptomic changes in the CLIC4-overexpressing DLD-1 cells were determined. The PCoA plot showed major genetic differences between CLIC4 overexpression and NC cells (Figure 4A). Hierarchical clustering of 69 genes showed large differences between CLIC4 overexpression and NC cells (Figure 4B). The volcano plot revealed that the levels of both gene fold differences and p-values on the up-regulated genes in CLIC4 overexpression control cells were higher than those in the NC cells (Figure 4C). Overall, 38 up-regulated and 31 down-regulated genes (a total of 69 genes) were obtained in CLIC4-overexpressing control cells compared to NC cells (Figure 4D). Pathway analysis showed that 12 of the top 20 gene sets, namely NR, interferon-α, p53 transcription, insulin, immune response, PI3K/AKT, adhesion, VEGF, MAPK, microRNA regulation, and type I- and II- interferon signals, were involved in gene groups for cancer development (Figure 4E, black circle).
Gene expression profiles in DLD-1 cells with or without CLIC4 overexpression. Gene expression levels between the CLIC4 overexpression (OE) DLD-1 cells (group 1) and the negative control cells (group 2) were subjected to Clariom™ S human assays and TAC software (n=3). (A) PCoA plot indicating genetic differences among different samples. (B) Hierarchical cluster analysis for 69 genes between groups 1 and 2. (C) Volcano plots between groups 1 and 2. (D) Number of up- (≥2.0-fold) and down-regulated (≤-2.0-fold) genes between groups 1 and 2. (E) The distribution of the top 20 gene sets that changed between group 1 and 2. Black circles indicate the gene set involving cancer development. The p-Value was determined using two-sided Fisher’s exact test. The value implies an index measuring bias of the gene set in DLD-1 cells with CLIC4 knockdown.
Expression levels of two proteins up-regulated in both CLIC4 knockdown and -overexpressing DLD-1 cells. Table I, Table II, Table III, and Table IV show the significantly altered genes in DLD-1 cells with CLIC4 knockdown and overexpression in comparison with each negative control. Six genes (≥2.0-fold), CLDN2, endoplasmic reticulum protein 27 (ERP27), IFIT1, fibroblast growth factor binding protein 1 (FGFBP1), solute carrier family 1 member 3 (SLC1A3), and lipase G (LIPG), were identified as genes up-regulated in cells with both CLIC4 knockdown and overexpression (Table I and Table III). Three genes, FERM domain containing kindlin 2 (FERMT2), G protein-coupled receptor 1 (GPR1), and very low-density lipoprotein receptor (VLDLR), were identified as the genes up-regulated in CLIC4 knockdown and down-regulated in CLIC4-overexpressing cells (Table I and Table IV). Four genes, TSC22 domain family member 3 (TSC22D3), chromosome 22 putative open reading frame 46 (C22orf46), transmembrane protein 154 (TMEM154), and leucine rich adaptor protein 1 like (LURAP1L), were identified as the genes down-regulated in cells with both CLIC4 knockdown and overexpression (Table II and Table IV). Nine genes, keratin associated protein 3-1 (KRTAP3-1), IFIT2, 2’-5’-oligoadenylate synthetase 1 (OAS1), TNF receptor superfamily member 19 (TNFRSF19), ankyrin repeat domain 13C (ANKRD13C), Purkinje cell protein 4 (PCP4), JUN, calcium voltage-gated channel auxiliary subunit beta 3 (CACNB3), and semaphorin 3C (SEMA3C), were identified as the genes down-regulated in CLIC4 knockdown and up-regulated in CLIC4-overexpressing cells (Table II and Table III). Based on these transcriptome alterations, the protein expression levels of IFIT1 and CLDN2 were determined in DLD-1 cells with CLIC4 knockdown and overexpression. The protein expression levels of CLDN2 increased in both CLIC4 knockdown and overexpressing cells. CLIC4 overexpression in cells increased IFIT1 protein expression but was not detected in CLIC4 knockdown and NC cells (Figure 5).
Up-regulated gene profile in DLD-1 cells with CLIC4 knockdowna.
Down-regulated gene profile in DLD-1 cells with CLIC4 knockdowna.
Up-regulated gene profile in DLD-1 cells with CLIC4 overexpressiona.
Down-regulated gene profile in DLD-1 cells with CLIC4 overexpressiona.
Protein expression levels in DLD-1 cells with CLIC4 knockdown or overexpression. DLD-1 cells were treated with Clic4 dsiRNA or plasmid DNA [(p)DNA] for 2 days. The CLIC4 protein level was measured by western blotting. NC, negative control.
Discussion
The present study demonstrated that FxOH most strongly suppressed the growth of DLD-1 cells with the highest expression of CLIC4 compared to the other five types of human CRC cells. Both CLIC4 knockdown and overexpression modulated the growth of DLD-1 cells by altering many signal transduction pathways. Unexpectedly, both CLIC4 knockdown and overexpression reduced the sensitivity of DLD-1 cells to FxOH. This is the first report suggesting the significance of the protein expression of CLIC4 and its regulating mechanisms in human CRC cells for the sensitivity to FxOH.
First, we confirmed the effect of FxOH on cell growth in six types of human CRC cells and on CLIC4 expression. We found an inverse association between sensitivity to FxOH in each cell and CLIC4 expression in each control cell. Notably, the sensitivities of DLD-1 and HCT116 cells with higher CLIC4 expression to FxOH were higher than those of LoVo and HT-29 cells with lower CLIC4 expression (Figure 1B-D). However, a minor difference was observed in the microsatellite instability, CpG island methylation phenotype, chromosomal instability pathway, and mutational characteristics among these four types of human CRC cells (46). We examined the molecular mechanisms triggered by CLIC4 knockdown and overexpression in DLD-1 cells.
Transcriptome changes based on CLIC4 knockdown and overexpression demonstrated that 15 cancer-related signals, namely, NR, insulin, PI3K/AKT, adhesion, VEGF, MAPK, NRF2, endothelin, IL-18, EMT, TGF-β, interferon, p53, immune response, and miRNA regulation (CLIC4-related signals), were significantly altered in CRC cells compared to the negative control cells (Figure 2A-E, Figure 4A-E and Figure 6A). FxOH treatment (5.0 μmol/l) has been reported to induce apoptosis and anoikis, anchor-dependent cell death, in DLD-1 cells via attenuation of integrin signaling (44, 45). In the two previous reports, we displayed 11 cancer-related signals among the top 20 signals altered on the FxOH-treated DLD-1cells (Figure 6B): NR, VEGF, adhesion (containing integrin signals), NRF2, MAPK, PI3K/AKT, insulin, IL-18, microRNA regulation, EGFR, and cell cycle (43). Among these 11 cancer-related signals, 9 signals (82%), i.e., NR, VEGF, adhesion, NRF2, MAPK, PI3K/AKT, insulin, IL-18, and miRNA regulation, were overlapped with the CLIC4-related signals. Thus, CLIC4 is suggested to be a central regulator of apoptosis and anoikis induction by FxOH in DLD-1 cells (Figure 6B).
Possible mechanisms underlying DLD-1 cells with CLIC4 alteration by FxOH treatment. (A) Venn diagram of signals changed in DLD-1 cells with CLIC4 knockdown and overexpression. Blue and red correspond to signals altered in CLIC4 knockdown and overexpressing DLD-1 cells, respectively. Black, signals altered in both CLIC4 knockdown and overexpressing DLD-1 cells. These 15 signals were represented as 15 cancer-related signals altered by both CLIC4 knockdown and overexpression (CLIC4-related signals). (B) Nine of CLIC4-related signals, NR, VEGF, adhesion, NRF2, MAPK, PI3K/AKT, insulin, IL-18, and miRNA regulation among the 15 of CLIC4-related signals, were overlapped with the top 11 cancer-related signals, NR, VEGF, adhesion, NRF2, MAPK, PI3K/AKT, insulin, IL-18, miRNA regulation, EGFR, and cell cycle, altered in FxOH-treated DLD-1 cells, which were represented based on previous reports (38, 43, 44). EGFR and cell cycle among 11 cancer-related signals were represented as other signals without overlap to CLIC4-related signals. The overlay of each signal group suggests that the CLIC4-related signals play significant role for apoptosis and anoikis inductions in FxOH-treated DLD-1 cells.
CLIC4 regulates or is regulated by molecules, such as the G-protein-coupled receptor, Rho A, F-actin, integrin, NHERF2, KRAS, p53, and TNF-α, and signals, such as apoptosis, angiogenesis, cell adhesion, and TGF-β (16-18, 21, 22, 39-42, 47). In the present study, we discovered many novel CLIC4-related molecules and signals affecting the development of cancer cells, other than those described in previous studies (Figure 2A-E, Figure 4A-E, Table I, Table II, Table III, and Table IV). Genes exhibited the opposite expression pattern in DLD-1 cells between CLIC4 knockdown and overexpression; FERMT2, GPR1, VLDLR, KRTAP3-1, IFIT2, OAS1, TNFRSF19, ANKRD13C, PCP4, JUN, CACNB3 and SEMA3C were expected to be involved in growth inhibition by CLIC4 knockdown and in CRC development by CLIC4 overexpression (Table I, Table II, Table III, and Table IV). On the other hand, common cell conditions acquired in DLD-1 cells by both CLIC4 knockdown and overexpression were the decreased sensitivity to FxOH exposure (Figure 1F and Figure 3B). Therefore, common up-regulated genes, like CLDN2, ERP27, IFTĨ1, FGFBP1, SLC1A3 and LIPG, or common down-regulated genes, including TSC22D3, C22orf46, TMEM154 and LURAP1L, in the cells by both CLIC4 knockdown and overexpression may be involved in the decreased sensitivity to FxOH (Table I, Table II, Table III, and Table IV). These results were another novel finding. In addition, CLDN2 and IFIT1 protein expression levels increased in DLD-1 cells following CLIC4 knockdown and/or overexpression (Figure 5). CLDN2 is a tight junction modulator that is involved in cancer proliferation, migration, and metastasis. CLDN2 expression contributes to chemoresistance in CRC cells and is associated with poor outcomes in patients with CRC (48, 49). ERP27 is an endoplasmic reticulum protein, and the gene is highly expressed in patients with CRC (50). IFIT1 is an interferon-stimulated inflammation-related protein that promotes cell invasion, metastasis, and tumor development (51). FGFBP1 releases fibroblast growth factors-interacting fibroblast growth factor receptors from extracellular matrix and enhances the migration and invasion of cancer cells (52). SLC1A3 is a glutamate/aspartate transporter that promotes tumor metabolism and progression via activation of the PI3K/AKT signal (53). LIPG, a protein exhibiting lipase activity, plays a significant role in plasma high-density lipoprotein metabolism and augments tumor formation and metastasis (54). However, the functions of TSC22D3, C22orf46, TMEM154, and LURAP1L in cancer cells remain unclear. Little is known about the associations among CLIC4 and CLDN2, ERP27, IFIT1, FGFBP1, SLC1A3, LIPG, TSC22D3, C22orf46, TMEM154, or LURAP1L.
Several studies have demonstrated the effects of CLIC4 knockdown or overexpression in various cancer cells. CLIC4 knockdown can attenuate cell-matrix adhesion, cellular spreading, and integrin signals in human cervical cancer (HeLa) and breast cancer (MDA-MB-231) cells, while enhancing their cell motility (16). Thus, there is a case in which CLIC4 knockdown not only unilaterally weakens the development of cancer cells but also leads to the induction of an alternative signal related to development. In addition, photodynamic therapy (PDT) suppresses the invasion ability with the decrease in CLIC4 and metalloproteinase 9 (MMP9) expression levels in human A375 melanoma and MDA-MB-231 cells, but CLIC4 overexpression enhances the invasion ability and attenuates PDT-induced suppressive effect on the invasion abilities of both cells (55). These two prior findings led us to speculate that induction of complete cell death in cancer cells targeting CLIC4 expression using a combination of FxOH and a CLIC4 expression-altering agents may be difficult.
In summary, an inverse association between the FxOH sensitivities in six types of human CRC cells and the protein expression level of CLIC4 in each control cell was found. In addition, CLIC4 knockdown and overexpression suggested that CLIC4 was involved in 11 signals among the top 20 cancer-associated signals altered by apoptosis and anoikis induction in DLD-1 cells by FxOH treatment. Interestingly, both CLIC4 knockdown and overexpression attenuated the FxOH-induced growth inhibition in DLD-1 cells by FxOH treatment. CLDN2, ERP27, IFIT1, FGFBP1, SLC1A3, LIPG, TSC22D3, C22orf46, TMEM154, and LURAP1L induced by CLIC4 alteration may be key molecules responsible for the decreased sensitivity of these cells. These results suggest that CLIC4 must be specifically traced for cell death in human CRC cells by FxOH treatment. Further studies are needed to elucidate the contribution of CLIC4 to the effects of Fx and FxOH in CRC.
Acknowledgements
This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (no. 20K05879).
Footnotes
Authors’ Contributions
R. Y. and M. T. conceived, designed the study, and wrote the paper. R.Y., A. K., S. Y., T. O., and M. T performed the experiments. A. K., H. K., J. H., and H. M. reviewed and edited the manuscript.
Conflicts of Interest
None declared.
- Received March 30, 2022.
- Revision received April 24, 2022.
- Accepted April 26, 2022.
- Copyright © 2022 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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