The Combination of Inositol Hexaphosphate and Inositol Inhibits Metastasis of Colorectal Cancer Cells by Upregulating Claudin 7

Abstract

Inositol hexaphosphate (IP6), a widely found natural bioactive substance in grains, effectively inhibits the progression of colorectal cancer (CRC) when used in combination with inositol (INS). We previously showed that supplementation of IP6 and INS upregulated the claudin 7 gene in orthotropic CRC xenografts in mice. The aim of this study was to elucidate the role of claudin 7 in the inhibition of CRC metastasis by IP6 and INS, and explore the underlying mechanisms. We found that IP6, INS and their combination inhibited the epithelial–mesenchymal transition (EMT) of colon cancer cell lines (SW480 and SW620), as indicated by upregulation of claudin 7 and E-cadherin, and downregulation of N-cadherin. The effect of IP6 and INS was stronger compared to either agent alone (combination index < 1). Furthermore, the silencing of the claudin 7 gene diminished the anti-metastatic effects of IP6 and INS on SW480 and SW620 cells. Consistent with in vitro findings, the combination of IP6 and INS suppressed CRC xenograft growth in a mouse model, which was neutralized by claudin 7. Taken together, the combination of IP6 and INS can inhibit CRC metastasis by blocking EMT of tumor cells through upregulation of claudin 7.

INTRODUCTION

Colorectal cancer (CRC) is the 4th most common malignancy and the second leading cause of cancer-related deaths worldwide.^1–3) Despite advances in treatment, the prognosis of the patient remains poor.⁴⁾ Metastasis is the main cause of the high mortality rates associated with CRC.^5,6)

In recent years, research focus on the development of anticancer drugs has increasingly shifted to natural products. Inositol hexaphosphate (IP6), also known as phytic acid, is a natural bioactive substance present in whole grains.⁷⁾ Studies have reported inhibitory effects of IP6 against leukemia, human rhabdomyosarcoma, prostate cancer, breast cancer and CRC.^8–13) Furthermore, epidemiological studies have shown that regular intake of foods rich in phytochemicals, such as IP6, is associated with a reduced risk of CRC.¹⁴⁾ IP6 also reduced the appearance of abnormal crypt lesions and inhibited the formation of azoxymethane (AOM)-induced colon tumors in a rat model.¹⁵⁾ Furthermore, IP6 is known to decrease HCT-29 cell viability and induce apoptosis through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway,¹⁶⁾ whereas IP6 hydrolysates can competitively bind to AKT to inhibit colorectal tumor growth.¹⁷⁾ Inositol (INS), the molecular skeleton of IP6 and a type of B vitamin, is ubiquitous in animal and plant species and is used mainly to treat diabetes, hepatitis, and polycystic ovary syndrome.^18,19) Furthermore, INS can also reduce the viability of cells from lung, breast, and colon cancer cells.^20–22) Several in vitro and in vivo studies have demonstrated stronger therapeutic effects of IP6 and INS compared to either agent alone.^21,22) Some studies have also shown that IP6 and INS have very low toxicity in humans.^23,24) In a previous study, we found that the combination of IP6 and INS inhibited the growth of orthotropic CRC xenografts in a mouse model. At the molecular level, IP6 and INS significantly increased the claudin 7 (Supplementary Table 1) gene in the tumors.^25,26)

Tight junctions (TJs) are multiprotein complexes that form a barrier between adjacent cells, and their loss of function and a shift in cell polarity are linked to tumor development.^27,28) The claudin family of proteins consists of 27 members and is a key component of TJs. Claudin 7 is mainly expressed in the colon and is downregulated in various cancers.²⁹⁾ Epithelial–mesenchymal transition (EMT) is a key process that facilitates tumor metastasis³⁰⁾ through loss of cell polarity and intercellular adhesion, allowing tumor cells to invade adjacent and distant tissues.³¹⁾ Downregulation of claudin 7 in CRC cells promotes EMT, invasion, and migration, and ectopic expression of claudin 7 can inhibit EMT progression and metastasis.^32,33) Therefore, claudin 7 is a promising target of IP6 and INS in the treatment of CRC.

In this study, we established stable claudin 7-silenced SW480 and SW620 colon cancer cell lines to explore its role in the therapeutic effects of IP6 and INS against CRC. Our findings provide new information on the clinical use of the combination of IP6 and INS for the treatment of metastatic CRC.

MATERIALS AND METHODS

Cell Culture

The SW480 and SW620 human colon cancer cell lines were purchased from Wuhan Punosai Life Technology Ltd. (Wuhan, China). Cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10 mg/mL) at 37 °C and 95% humidity under 5% CO₂.

Lentiviral Transfection

Lentiviruses and related reagents were purchased from Shanghai Jikai Medical Technology Ltd. Both SW480 and SW620 cells were transfected with LV-CLDN7-Rnai and negative control lentiviruses. The stably transfected SW480/CLDN7, SW480/NC, SW620/CLDN7 and SW620/NC cell lines were screened with 4 µg/mL puromycin. The silence of claudin 7 was verified by real-time quantitative PCR (qRT-PCR).

RNA Extraction and RT-PCR

Total RNA was isolated from the culture cells using the RNAprep FastPure Cell Total RNA Extraction Kit and reverse transcribed using the Goldenstar™ RT6 cDNA synthesis mix. RT-qPCR was performed using the 2 × T5 Fast qPCR mix on the RocheLight Cycler 96 (Basel, Switzerland). The expression of the target gene was calculated relative to that of the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

In Vitro Intervention

The SW480 and SW620 cells were divided into the untreated control and IP6, INS, and IP6 + INS (1 : 1 v/v) treatment groups. Likewise, SW480/NC and SW620/NC cell lines were divided into the control and IP6 + INS intervention groups. The SW480/CLDN7 and SW620/CLDN7 cell lines were treated with IP6 and INS. The dose of drug for all experiments except the Cell Counting Kit-8 (CCK8) assay and the establishment of tumor xenograft was 200 mg/L.

Cell Viability Assay

The viability of the differentially treated cells was evaluated by the CCK-8 assay. Briefly, the cells were seeded in 96-well plates at the density of 5 × 10³ cells per well, and then treated with various concentrations (0, 50, 100, 150, and 200 mg/L) of IP6, INS and IP6 + INS (1 : 1). After 72 h, 10% CCK-8 reagent in 100 µL medium was added to each well, and the cells were incubated for 2 h. The absorbance of each well was measured at 450 nm, and viability rate was calculated based on standard curves.

Cooperative Determination

Data obtained from cell viability measurements were standardized for control groups. CompuSyn 2.0 software was used to calculate the combination index (CI). The CI value represents the interaction pattern between the two drugs. CI = 1 indicates an additive effect, CI < 1 indicates a synergistic effect, and CI > 1 indicates an antagonistic effect.

Wound Healing Assay

Cells were seeded in 24-well plates at the density of 2 × 10⁶ cells/well and cultured till 90% confluent. The monolayer was then scratched longitudinally with a 200 µL sterile pipette tip, and washed three times with PBS to remove the dislodged cells. Serum-free medium was then added and the cells were incubated for 48 h. The wound area was photographed at 0 and 48 h using an inverted optical microscope, and the migration rate was calculated on the basis of the wound coverage.

Cell Invasion Assay

Cells were seeded in the upper compartment of Transwell chambers in 100 µL serum-free medium at the density of 5 × 10⁵ cells/mL along with the different drugs, and the lower compartments were filled with 600 µL DMEM medium containing 10% FBS. After 48 h of culture, unmigrated cells were gently removed with a cotton swab, and cells that had migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde (1 mL) and stained with 0.1% crystal violet at room temperature. The number of migrated cells was counted in five random fields under a microscope and the average was calculated for each group.

Western Blot

The total protein was extracted from the cells using the radio immunoprecipitation assay (RIPA) lysis solution and quantified using the bicinchoninic acid (BCA) kit. Samples were diluted in loading buffer and denatured in boiling water for 5 min, and 15 µg protein per sample were separated on 10 and 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels. The protein bands were transferred to a polyvinylidene difluoride membrane, which was then blocked with 5% skim milk powder in Tris-buffered saline (TBS) buffer for two hours at room temperature. After washing three times with TBS buffer, the membrane was incubated overnight with anti-claudin 7, anti-E-cadherin, anti-N-cadherin (anti-N-cadherin antibodies are from Immunoway and ZEN BIO, in Fig. 2a, SW480 uses ZENBIO’s N-cadherin antibody (383341), Immunoway-branded antibodies were used in SW620 cells in Fig. 2b and SW480 and SW620 cells in Fig. 4 (YT2988)) and anti-GAPDH antibodies at 4 °C. The blots were then incubated for 2 h with secondary antibodies on a shaker at room temperature. Positive bands were developed using an enhanced chemiluminescence (ECL) solution and their density was measured using ImageJ software.

Establishment of in Vivo Xenograft Model

In this study 18 male BALB/C nude mice aged 6 weeks were used. Mice were raised under pathogen-free conditions (12/12 h light/dark cycle, 25 °C, 50% humidity), with food and water provided ad libitum. After one week of adaptive feeding, mice were injected subcutaneously with 2 × 10⁶ SW620/NC or SW620/CLDN7 cells into their right axilla. The animals were randomly divided into the following 3 groups (n = 6 each): 1) control (SW620/NC cells and normal saline), 2) IP6 + INS (SW620/NC and 130 mg/kg drugs) and 3) IP6 + INS KD (SW620/CLDN7 and 130 mg/kg drugs). The respective drugs were administered once a day by gavage for 24 d. The mice were sacrificed after the treatment regimen and the tumors were harvested, weighed and measured to calculate the volume.

Statistical Analysis

SPSS23.0 (IBM SPSS Statistics) was used for all statistical analyzes. The different groups were compared using ANOVA and the least significant difference (LSD) post hoc test. p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Metastasis is a complex, multistage process that mainly involves the loss of tumor cell polarity and contact inhibition. In this study, we analyzed the potential antimetastatic effects of IP6 and INS, and the underlying mechanisms, in CRC cells. We used the primary CRC cell line SW480 and the SW620 cell line derived from lymph node metastasis from the same patient. These two cell lines represent the biological characteristics of CRC cells at different stages of metastasis, which can more comprehensively verify the inhibitory effect of antimetastatic drugs. As shown in Fig. 1, IP6 and/or INS decreased viability (Fig. 1b, CI < 1 indicating synergistic effect) and invasiveness (Figs. 1c, d) of both cell lines, and the inhibitory effect of their combination was stronger compared to that of either drug. Therefore, IP6 and INS can inhibit the initiation and metastasis of colorectal tumors, which is consistent with our previous findings.

Fig. 1. IP6 and INS Inhibited the Malugnant Potential of CRC Cells

(a) CCK-8 assays were performed to determine viability activity. SW480 and SW620 cells were treated with different concentrations (0, 50, 100, 150, and 200 mg/L) of IP6, INS, and IP6 + INS. Cell viability was detected by CCK-8 for 72 h. (b) Combination index (CI) of SW480 and SW620 cells treated with IP6 and INS. CI < 1, synergistic activity; CI = 1, additive effect; CI > 1, antagonism. (c) Transwells were used to detect invasion ability. SW480 and SW620 cells were treated with control, IP6, INS and IP6 + INS for 48 h. (d) Cell migration ability was evaluated by scratch test. SW480 and SW620 cells were treated with control, IP6, INS and IP6 + INS for 48 h. All experiments were carried out three times. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the IP6 + INS group.

The occurrence and development of CRC involve a complex network of multiple signaling pathways.³⁴⁾ Prior transcriptomics studies by our group showed that IP6 + INS upregulated claudin 7 in tumor tissues from a mouse model of CRC liver metastasis.²⁶⁾ Claudin 7 is a key member of the claudin family of TJ proteins, which play a key role in maintaining cell polarity and integrity.³⁵⁾ It is downregulated in several malignancies, including CRC,³⁶⁾ nasopharyngeal carcinoma,³⁷⁾ ovarian carcinoma³⁸⁾ and esophageal squamous cell carcinoma,³⁹⁾ and correlates significantly with poor differentiation and metastasis in CRC.³⁶⁾ In addition, claudin 7 knockout mice spontaneously develop intestinal adenoma.⁴⁰⁾ In this study, we found that IP6 and INS significantly increased the expression level of claudin 7 (Fig. 2) in primary and metastatic CRC cells, indicating that the drug combination may prevent CRC metastasis by upregulating claudin 7.

Fig. 2. IP6 or INS Alone or in Combination Modulated Claudin 7 and EMT in CRC Cells

(a, b) Western blot was used to detect the expression of claudin 7 and EMT markers (E-cadherin and N-cadherin) in SW480 (a) and SW620 (b) cells treated with IP6, INS and IP6 + INS. GAPDH was used as a reference gene. All experiments were carried out three times. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the IP6 + INS group.

EMT results in the loss of polarity and adhesion capacity of tumor cells and allows then to invade the extracellular matrix, eventually promoting their migration and distant metastasis.^30,31,41,42) At the molecular level, EMT is characterized by the downregulation of E-cadherin and upregulation of N-cadherin.⁴³⁾ E-cadherin is a Ca-dependent cell-cell adhesion factor that is essential for tissue development.⁴⁴⁾ The main function of N-cadherin, which is expressed primarily in the nervous system, is to control the adhesion of neuronal cells.⁴⁵⁾ It is aberrantly elevated in several human malignancies, including pancreatic cancer, prostate cancer, and breast cancer.⁴⁶⁾ Previous studies have shown that IP6 + INS can inhibit the expression of EMT-related proteins, such as N-cadherin, in tumor-bearing mice.^47,48) Furthermore, there is evidence that loss of claudin 7 can promote EMT and induce invasion and metastasis of CRC cells,³²⁾ whereas its ectopic expression can reverse the EMT process and inhibit metastasis.³³⁾ In this study, we found that IP6 + INS upregulated E-cadherin and downregulated N-cadherin in SW480 and SW620 cells (Fig. 2), indicating that these drugs can inhibit EMT in primary and metastatic CRC cells.

To determine whether these changes involve claudin 7, we silenced the gene in both CRC cell lines and treated them with IP6 and INS (Fig. 3). Silencing claudin 7 mitigated the inhibitory effect of IP6 + INS on metastasis (Figs. 3c–e) and EMT (Fig. 4) of SW480 and SW620 cells, clearly indicating that upregulation of claudin 7 is critical for the therapeutic effects of IP6 and INS against CRC. Consistent with in vitro findings, IP6 + INS effectively inhibited CRC xenograft growth in vivo (Fig. 5), and antitumor effects were considerably diminished with claudin 7 silencing (Fig. 5).

Fig. 3. Claudin 7 Is Successfully Silenced in CRC Cells, and Silence of Claudin 7 Decreases the Inhibitory Effects of IP6 + INS on CRC Cell Viability, Invasion, and Migration

(a) SW480 and SW620 cells were cultured 48 h after transfection and then observed under the fluorescence microscope. (b) The mRNA expression of claudin 7 after transfection. GAPDH was used as an internal reference. (c) SW480 and SW620 cells transfected with lentivirus were treated with IP6 + INS for 72 h and cell viability activity was detected by CCK-8. (d) The invasiveness of SW480 and SW620 cells treated with IP6 + INS for 48 h after transfer of lentivirus was detected by the Transwell invasion assay. (e) SW480 and SW620 cells transfected with lentivirus were treated with IP6 + INS for 48 h, and changes in cell migration ability were detected by scratch. All experiments were carried out three times. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the IP6 + INS group.

Fig. 4. Claudin 7 Silence Reverses the Inhibitory Effects of IP6 + INS on EMT in CRC Cells

(a, b) The expressions of claudin 7, E-cadherin, and N-cadherin in SW480 and SW620 cells were detected by Western blot. All experiments were carried out three times. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the IP6 + INS group.

Fig. 5. Effect of Claudin 7 on IP6 + INS Inhibition of Tumor Growth in a CRC Xenograft Model

The model was established by injecting SW620/NC cells and SW620/CLDN7 cells into the right axilla of mice. Mice were treated with saline (Control group) and IP6 + INS. (a) Weight of tumor tissues in different groups. (b) Progression of tumor volume as a function of time. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the IP6 + INS group.

In conclusion, our findings suggest that IP6 + INS can inhibit the metastatic potential of CRC cells by blocking the EMT process upregulation by upregulation of claudin 7, thus, providing novel insights into the treatment of CRC using plant-derived compounds.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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