Suppressed Colorectal Cancer Growth Research Articles

Methylation of NRIP3 Is a Synthetic Lethal Marker for Combined PI3K and ATR/ATM Inhibitors in Colorectal Cancer.

The aim of this study was to investigate the epigenetic regulation and underlying mechanism of NRIP3 in colorectal cancer (CRC). Eight cell lines (SW480, SW620, DKO, LOVO, HT29, HCT116, DLD1, and RKO), 187 resected margin samples from colorectal cancer tissue, 146 cases with colorectal adenomatous polyps, and 308 colorectal cancer samples were used. Methylation-specific PCR, Western blotting, RNA interference assay, and a xenograft mouse model were used. NRIP3 exhibited methylation in 2.7% (5/187) of resected margin samples from colorectal cancer tissue, 32.2% (47/146) of colorectal adenomatous polyps, and 50.6% (156/308) of CRC samples, and the expression of NRIP3 was regulated by promoter region methylation. The methylation of NRIP3 was found to be significantly associated with late onset (at age 50 years or older), poor tumor differentiation, lymph node metastasis, and poor 5-year overall survival in CRC (all P < 0.05). In addition, NRIP3 methylation was an independent poor prognostic marker ( P < 0.05). NRIP3 inhibited cell proliferation, colony formation, invasion, and migration, while induced G1/S arrest. NRIP3 suppressed CRC growth by inhibiting PI3K-AKT signaling both in vitro and in vivo . Methylation of NRIP3 sensitized CRC cells to combined PI3K and ATR/ATM inhibitors. NRIP3 was frequently methylated in both colorectal adenomatous polyps and CRC. The methylation of NRIP3 may potentially serve as an early detection, late-onset, and poor prognostic marker in CRC. NRIP3 is a potential tumor suppressor. NRIP3 methylation is a potential synthetic lethal marker for combined PI3K and ATR/ATM inhibitors.

The Mechanistic Target of Rapamycin Complex 1 Pathway Contributes to the Anti-Tumor Effect of Granulocyte-Macrophage-Colony-Stimulating Factor-Producing T Helper Cells in Mouse Colorectal Cancer

ABSTRACT Introduction The role of granulocyte-macrophage-colony-stimulating factor-producing T helper (ThGM) cells in colorectal cancer (CRC) development remains unclear. This study characterizes the function of ThGM cells in mouse CRC. Methods Mouse CRC was induced by administrating azoxymethane and dextran sulfate sodium. The presence of ThGM cells in CRC tissues and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in ThGM cells was detected by flow cytometry. The impact of mTORC1 signaling on ThGM cell function was determined by in vitro culture. The effect of ThGM cells on CRC development was evaluated by adoptive transfer assays. Results ThGM cells, which expressed granulocyte-macrophage-colony-stimulating factor (GM-CSF), accumulated in CRC tissues. mTORC1 signaling is activated in CRC ThGM cells. mTORC1 inhibition by rapamycin suppressed ThGM cell differentiation and proliferation and resulted in the death of differentiating ThGM cells. mTORC1 inhibition in already differentiated ThGM cells did not induce significant cell death but decreased the expression of GM-CSF, interleukin-2, and tumor necrosis factor-alpha while impeding cell proliferation. Furthermore, mTORC1 inhibition diminished the effect of ThGM cells on driving macrophage polarization toward the M1 type, as evidenced by lower expression of pro-inflammatory cytokines, major histocompatibility complex class II molecule, and CD80 in macrophages after co-culture with rapamycin-treated ThGM cells. Lentivirus-mediated knockdown/overexpression of regulatory-associated protein of mTOR (Raptor) confirmed the essential role of mTORC1 in ThGM cell differentiation and function. Adoptively transferred ThGM cells suppressed CRC growth whereas mTORC1 inhibition abolished this effect. Conclusion mTORC1 is essential for the anti-CRC activity of ThGM cells.

P53-responsive CMBL reprograms glucose metabolism and suppresses cancer development by destabilizing phosphofructokinase PFKP

Aerobic glycolysis is critical for cancer progression and can be exploited in cancer therapy. Here, we report that the human carboxymethylenebutenolidase homolog (carboxymethylenebutenolidase-like [CMBL]) acts as a tumor suppressor by reprogramming glycolysis in colorectal cancer (CRC). The anti-cancer action of CMBL is mediated through its interactions with the E3 ubiquitin ligase TRIM25 and the glycolytic enzyme phosphofructokinase-1 platelet type (PFKP). Ectopic CMBL enhances TRIM25 binding to PFKP, leading to the ubiquitination and proteasomal degradation of PFKP. Interestingly, CMBL is transcriptionally activated by p53 in response to genotoxic stress, and p53 activation represses glycolysis by promoting PFKP degradation. Remarkably, CMBL deficiency, which impairs p53's ability to inhibit glycolysis, makes tumors more sensitive to a combination therapy involving the glycolysis inhibitor 2-deoxyglucose. Taken together, our study demonstrates that CMBL suppresses CRC growth by inhibiting glycolysis and suggests a potential combination strategy for the treatment of CMBL-deficient CRC.

Hybrid membrane camouflaged Prussian blue nanoparticles with cinobufagin loading for chemo/photothermal therapy of colorectal cancer

The natural compound of cinobufagin (CS-1) has showed potential application for colorectal cancer therapy. However, the limitation of hydrophobicity and cardiotoxicity appealed for the improvement of compound formulation. Meanwhile, sole chemotherapy for colorectal cancer (CRC) is facing the challenges of finite accumulation of drugs in tumor sites and nonspecific toxicity. In order to overcome these limitations, cinobufagin loaded in Prussian Blue nanoparticles (PC NPs) were prepared for CRC therapy by combining with photothermal therapy (PTT). The hybrid cell membranes coating was adopted to endow immune escape and tumor homing capacity of PC@M NPs. Both in vitro and in vivo assay demonstrated that the chemo/photothermal therapy not only suppressed CRC growth, but also inhibited the metastasis of primary by downregulating the expression of Vimentin, matrix metalloproteinase-9 (MMP-9) and hypoxia-inducible factor1-alpha (HIF-1α). Moreover, this strategy modulated the intestinal microbiota of CRC-bearing mice to a normal level in some extent. Taken together, this proposed nanoplatform presents a potential strategy for CRC therapy.

CircRNA ITGA7 restrains growth and enhances radiosensitivity by up-regulating SMAD4 in colorectal carcinoma.

Circular RNAs have been reported to be widely involved in cancer cell tumorigenesis and drug resistance; here, the aim of this study was to investigate whether circRNA Integrin Subunit Alpha 7 (ITGA7) (circ_ITGA7) was associated with the tumor growth and radiosensitivity of colorectal cancer (CRC). We found that circ_ITGA7 expression was lower in CRC tissues and cells than those in the normal tissues and cell lines according to quantitative real-time polymerase chain reaction. As shown by cell counting kit-8 assay, flow cytometry, colony formation assay, and xenograft experiment, ectopic overexpression of circ_ITGA7 remarkably restrained CRC tumor growth and enhanced radiosensitivity in vitro and in vivo. Mechanistically, circ_ITGA7 could target microRNA (miR)-766 to prevent the degradation of its target gene mothers against decapentaplegic homolog 4 (SMAD4), the binding between miR-766 and circ_ITGA7 or SMAD4 was first verified by dual-luciferase activity assay. Additionally, miR-766 up-regulation reversed the inhibitory effects of circ_ITGA7 on CRC growth and radiosensitivity. Besides that, inhibition of miR-766 reduced CRC cell growth and sensitized cells to radiotherapy, and these effects mediated by miR-766 inhibitor were rescued by the silencing of SMAD4. In all, circ_ITGA7 suppressed CRC growth and enhanced radiosensitivity by up-regulating SMAD4 through sequestering miR-766, providing an insight for the further development of CRC treatment.

Degradation of HIF-1α induced by curcumol blocks glutaminolysis and inhibits epithelial-mesenchymal transition and invasion in colorectal cancer cells.

Colorectal cancer (CRC) has high morbidity and mortality. Epithelial-mesenchymal transition (EMT) is associated with CRC progression and metastasis. Glutaminolysis is essential for malignancy of cancer cells. Here, we examined the effects of curcumol on CRC EMT. We observed that curcumol suppressed invasion and migration in human CRC cells associated with upregulation of epithelial markers E-cadherin and Zonula occludens 1 and downregulation of mesenchymal markers N-cadherin and Vimentin as well as EMT-related transcription factors Snail and Twist. Curcumol increased intracellular levels of glutamine but decreased intracellular levels of glutamate, α-ketoglutarate, ATP, glutathione, and tricarboxylic acid cycle metabolites, suggesting interruption of glutaminolysis. Next, curcumol repressed glutaminase 1 (Gls1) mRNA and protein expression, and overexpression of Gls1 promoted EMT and abolished curcumol effects on CRC cell EMT. Molecular examinations showed that curcumol stimulated protein degradation of hypoxia-inducible factor-1α (HIF-1α) and prevented its nuclear accumulation in CRC cells. HIF-1α agonist deferoxamine (DFO) promoted HIF-1α binding to Gls1 promoter and increased Gls1 expression but abolished curcumol's inhibitory effects on Gls1 expression. DFO also enhanced EMT and invasion and migration in CRC cells and eliminated curcumol effects. Furthermore, mouse CRC models were established with in vivo overexpression of HIF-1α and Gls1. Curcumol effectively inhibited CRC growth, metastasis, and EMT in mice, which was abrogated by overexpression of HIF-1α or Gls1. Altogether, stimulation of HIF-1α degradation was required for curcumol to disrupt EMT and repress invasion and migration in CRC cells through inhibiting Gls1-mediated glutaminolysis. Curcumol could be a promising candidate for intervention of CRC metastasis. • Curcumol inhibits EMT and blocks glutaminolysis in CRC cells. • Inhibition of Gls1 is required for curcumol blockade of glutaminolysis and EMT. • Curcumol induces HIF-1α degradation leading to inhibition of Gls1 and blockade of glutaminolysis and EMT. • Curcumol suppresses CRC growth and metastasis via inhibiting HIF-1α, glutaminolysis and EMT in mice.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Hsa_circ_0062682 Promotes Serine Metabolism and Tumor Growth in Colorectal Cancer by Regulating the miR-940/PHGDH Axis

Colorectal cancer (CRC) is one of the most common malignancies globally. Increasing evidence indicates that circular RNAs (circRNAs) play a pivotal role in various cancers. The present study focused on exploring the role of a functionally unknown circRNA, hsa_circ_0062682 (circ_0062682), in CRC. By online analyses and experimental validations, we showed that circ_0062682 expression was aberrantly increased in CRC tissues compared with paired normal tissues. Increased expression of circ_0062682 in CRC notably correlated with a poor prognosis and advanced tumor stage. Functional experiments showed that circ_0062682 knockdown reduced CRC growth both in vitro and in vivo. Mechanistically, we revealed that circ_0062682 could sponge miR-940 and identified D-3-phosphoglycerate dehydrogenase (PHGDH), a key oxidoreductase involved in serine biosynthesis, as a novel target of miR-940. Silencing miR-940 expression could mimic the inhibitory effect of circ_0062682 knockdown on CRC proliferation. The expression of PHGDH was downregulated in circ_0062682-depleted or miR-940 overexpressing CRC cells at both the mRNA and protein levels. Circ_0062682 knockdown suppressed CRC growth by decreasing PHGDH expression and serine production via miR-940. Taken together, these data demonstrate, for the first time, that circ_0062682 promotes serine metabolism and tumor growth in CRC by regulating the miR-940/PHGDH axis, suggesting circ_0062682 as a potential novel therapeutic target for CRC.

CDKN2B antisense RNA 1 suppresses tumor growth in human colorectal cancer by targeting MAPK inactivator dual-specificity phosphatase 1.

Aberrant expression of long noncoding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) has been detected in human colorectal cancer (CRC). This study aimed to investigate the role of CDKN2B-AS1 and the underlying mechanism in human CRC. Gain- and loss-of-function assays were performed to explore the role of CDKN2B-AS1 in the malignant behavior of HCT116 and SW480 CRC cells in vitro and in vivo. RNA pull-down assay was conducted to identify the target of CDKN2B-AS1 in CRC cells. The physical and functional interactions between CDKN2B-AS1 and the target were examined. CDKN2B-AS1 inhibited CRC cell proliferation and migration while promoting apoptosis in vitro via activation of mitogen-activated protein kinase kinases (MEK)/extracellular signal-regulated kinase (ERK)/p38 signaling. CDKN2B-AS1 bound to mitogen-activated protein kinase (MAPK) inactivator dual-specificity phosphatase 1 (DUSP1) in CRC cells. In contrast to CDKN2B-AS1, DUSP1 promoted CRC cell proliferation, suppressed apoptosis and inactivated MEK/ERK/p38 signaling in CRC cells. Furthermore, CDKN2B-AS1 overexpression attenuated DUSP1 expression in normal colonic myofibroblasts and CRC cells. Overexpression of DUSP1 effectively countered the activation of MEK/ERK/p38 signaling induced by CDKN2B-AS1 overexpression or further blocked MEK/ERK/p38 signaling suppressed by CDKN2B-AS1 silencing. In the mouse xenograft model, CDKN2B-AS1 suppressed CRC growth, whereas DUSP1 promoted CRC growth. CDKN2B-AS1 induced cell apoptosis while suppressing EMT (epithelial-mesenchymal transition), whereas DUSP1 suppressed cell apoptosis while inducing EMT in CRC, as evidenced by the alterations in the protein levels of apoptosis and EMT markers in tumor tissue samples. CDKN2B-AS1 regulates CRC cell growth and survival by targeting MAPK inactivator DUSP1.

Circular RNA circFAT1(e2) Promotes Colorectal Cancer Tumorigenesis via the miR-30e-5p/ITGA6 Axis.

circRNAs (circular RNAs) are a family of noncoding RNAs and have diverse physiological and pathological functions. However, the functions and mechanisms of circRNAs in the development and progression of colorectal cancer (CRC) remain largely unknown. Here, we aimed to explore the functions and roles of circFAT1(e2) in CRC. qRT-PCR revealed that circFAT1(e2) in CRC tumor tissues was upregulated compared with that in adjacent normal tissues and was also upregulated in CRC cell lines. Small interfering RNAs (siRNAs) against circFAT1(e2) were used to decrease the expression of circFAT1(e2) in HCT116 and RKO cells in vitro. The roles of circFAT1(e2) in CRC cell metastasis and proliferation were then determined by transwell and CCK-8 assays. The results showed that circFAT1(e2) silencing markedly suppressed CRC growth. Moreover, we identified circFAT1(e2) as a promoter of CRC metastasis. Knockdown of circFAT1(e2) evidently reduced HCT116 and RKO cell migration and invasion. Furthermore, the regulatory relationship between circFAT1(e2) and its target miRNAs was verified by a luciferase reporter assay. We demonstrated that circFAT1(e2) could sponge miR-30e-5p, which regulated the expression level of integrin α6 (ITGA6), the downstream target gene of miR-30e-5p. Rescue assays demonstrated that knockdown of miR-30e-5p enhanced CRC proliferation and migration via ITGA6. Taken together, our results reveal the novel oncogenic roles of circFAT1(e2) in CRC through the miR-30e-5p/ITGA6 axis.

Vacuolar Membrane ATPase Activity 21 Predicts a Favorable Outcome and Acts as a Suppressor in Colorectal Cancer.

Extracellular and/or intracellular manipulation of pH in tumor may have noticeable potential in cancer treatment. Although the assembly factor genes of V0 domain of the V-ATPase complex are required for intracellular pH homeostasis, their significance in colorectal cancer (CRC) remains largely unknown. Here, we used bioinformatics to identify the candidates from known assembly factor genes of the V0 domain, which were further evaluated by immunohistochemistry (IHC) in CRC and adjacent normal specimens from 661 patients. Univariate and multivariate Cox analyses were used to evaluate factors contributing to prognosis. The effects of variations in the expression of VMA21 on tumor growth were assessed in vitro and in vivo. Of five known assembly factors, only VMA21 showed differential expression between CRC and adjacent normal tissues at both mRNA and protein levels. Patients with high VMA21 expression had higher differentiation grade and longer disease-specific survival (DSS) at stages I–III disease. High VMA21 expression in tumors was also an independent predictor of DSS (hazard ratio, 0.345; 95% confidence interval, 0.123–0.976), with covariates included TNM stage and differentiation grade. VMA21 overexpression decreased CRC growth, whereas VMA21 knockdown increased CRC growth in vitro and in vivo. VMA21 expression suppresses CRC growth and predicts a favorable DSS in patients with stage I-III disease.

ATF3 inhibits the growth and stem cells-like features of SW620 colorectal cancer cells in vitro

Background and objective: Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumor development depending on the cellular context or cancer cell type. However, the effect of ATF3 on stem cells-like features in colorectal cancer (CRC) has yet to be elaborated. Methods: In this study, we overexpressed ATF3 in SW620 CRC cells to investigate its effects on stem cells-like features. Results: Our results indicated that overexpressing ATF3 inhibited the proliferation, invasion, migration, and sphere formation capacity of SW620 CRC cells. ATF3 overexpression also decreased the size of tumorspheres and reduced expression of the cancer stem cell markers CD44 and CD133 in SW620 cells. Conclusion: In summary, our study revealed that ATF3 suppresses CRC growth and stem cells-like features. ATF3 is considered a potential target in CRC therapy.

Circular RNA hsa_circRNA_102958 promotes tumorigenesis of colorectal cancer via miR-585/CDC25B axis.

Purpose: Circular RNAs (circRNAs) are recently identified new noncoding RNAs and play an important role in tumorigenesis. Previous studies have indicated that hsa_circRNA_102958 is a potential diagnostic indicator in gastric cancer. However, its role in colorectal cancer (CRC) is poorly understood.Methods: qRT-PCR and ISH were used to test gene expression in tissues. Survival rate was analzyed by Kaplan-Meier curve. Luciferase reporter assay was used to determine the interaction between circRNA and miRNA or between miRNA and mRNA. Western blotting was used to test protein expression. CCK8 and colony formation assay was used to analyze proliferation. Transwell assay was used for migration and invasion determination.Results: In our research, we found that hsa_circRNA_102958 expression was significantly increased in CRC tissues, compared to adjacent normal controls. Increased hsa_circRNA_102958 levels in CRC patients indicated a poor prognosis. The effects of hsa_circRNA_102958 on CRC cell proliferation, migration and invasion were then determined by CCK8, colony formation and Transwell assays. We showed that hsa_circRNA_102958 silencing markedly suppressed CRC growth, migration and invasion. Furthermore, hsa_circRNA_102958 was identified as a sponge for miR-585. We demonstrated that hsa_circRNA_102958 promoted CDC25B expression through inhibiting miR-585 in CRC. Rescue assays illustrated that CDC25B overexpression reversed the suppressive effects of hsa_circRNA_102958 silencing on CRC.Conclusion: Taken together, our findings revealed the novel oncogenic roles of hsa_circRNA_102958 in CRC through miR-585/CDC25B axis.

MiR-125a-5p suppresses colorectal cancer progression by targeting VEGFA.

BackgroundMiR-125a-5p has been reported to be involved in the development and progression of various cancers. However, the biological function and underlying mechanisms in colorectal cancer(CRC) still remain unclear. Here, we explored the potential biological roles of miR-125a-5p in CRC.MethodsThe expression of miR-125a-5p was detected using quantitative real-time PCR (qRT-PCR), biological functions of miR-125a-5p were assessed by cell counting kit-8, wound-healing, transwell invasion, and human umbilical vein endothelial cell (HUVEC) tube formation assays in vitro and animal experiments in vivo.ResultsWe found that miR-125a-5p was downregulated in CRC tissues and cell lines, it inhibited CRC cell proliferation, migration, and invasion and reduced the ability of HUVECs to form tubes. Moreover, we verifed that miR-125a-5p suppressed CRC growth and metastasis in vivo. Additionally, we showed that VEGFA, a direct target gene of miR-125a-5p, could reverse the inhibitory effect caused by miR-125a-5p overexpression.ConclusionmiR-125a-5p might serve as a tumor suppressor in CRC and could be regarded as a potential therapeutic candidate for CRC.

RETRACTED ARTICLE: CircHIPK3 promotes colorectal cancer growth and metastasis by sponging miR-7

Mounting evidences indicate that circular RNAs (circRNAs) have a vital role in human diseases, especially cancers. More recently, circHIPK3, a particularly abundant circRNA, was proposed to be involved in tumorigenesis. However, its role in colorectal cancer (CRC) has not been explored. In this study, we found circHIPK3 was significantly upregulated in CRC tissues and cell lines, at least in part, due to c-Myb overexpression and positively correlated with metastasis and advanced clinical stage. Moreover, Cox multivariate survival analysis showed that high-level expression of circHIPK3 was an independent prognostic factor of poor overall survival (OS) in CRC (hazard ratio [HR] = 2.75, 95% confidence interval [CI] 1.74–6.51, p = 0.009). Functionally, knockdown of circHIPK3 markedly inhibited CRC cells proliferation, migration, invasion, and induced apoptosis in vitro and suppressed CRC growth and metastasis in vivo. Mechanistically, by using biotinylated-circHIPK3 probe to perform RNA pull-down assay in CRC cells, we identified miR-7 was the only one microRNA that was abundantly pulled down by circHIPK3 in both HCT116 and HT29 cells and these interactions were also confirmed by biotinylated miR-7 pull-down and dual-luciferase reporter assays. Overexpression of miR-7 mimicked the effect of circHIPK3 knockdown on CRC cells proliferation, migration, invasion, and apoptosis. Furthermore, ectopic expression of circHIPK3 effectively reversed miR-7-induced attenuation of malignant phenotypes of CRC cells by increasing the expression levels of miR-7 targeting proto-oncogenes (FAK, IGF1R, EGFR, YY1). Remarkably, the combination of circHIPK3 silencing and miR-7 overexpression gave a better effect on tumor suppression both in vitro and in vivo than did circHIPK3 knockdown or miR-7 overexpression alone. Taken together, our data indicate that circHIPK3 may have considerable potential as a prognostic biomarker in CRC, and support the notion that therapeutic targeting of the c-Myb/circHIPK3/miR-7 axis may be a promising treatment approach for CRC patients.

Matrine Ameliorates Colorectal Cancer in Rats via Inhibition of HMGB1 Signaling and Downregulation of IL-6, TNF-α, and HMGB1.

Matrine may be protective against colorectal cancer (CRC), but how it may work is unclear. Thus, we explored the underlying mechanisms of matrine in CRC. Matrine-related proteins and CRC-related genes and therapeutic targets of matrine in CRC were predicted using a network pharmacology approach. Five targets, including interleukin 6 (IL-6), the 26S proteasome, tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-β1) and p53, and corresponding high-mobility group box 1 (HMGB1) signaling and T helper cell differentiation were thought to be associated with matrine's mechanism. Expression of predicted serum targets were verified in a 1,2-dimethylhydrazine dihydrochloride-induced CRC model rats that were treated with matrine (ip) for 18 weeks. Data show that matrine suppressed CRC growth and decreased previously elevated expression of IL-6, TNF-α, p53, and HMGB1. Matrine may have had a therapeutic effect on CRC via inhibition of HMGB1 signaling, and this occurred through downregulation of IL-6, TNF-α, and HMGB1.

CLCA1 suppresses colorectal cancer aggressiveness via inhibition of the Wnt/beta-catenin signaling pathway

BackgroundChloride channel accessory 1 (CLCA1) belongs to the calcium-sensitive chloride conductance protein family, which is mainly expressed in the colon, small intestine and appendix. This study was conducted to investigate the functions and mechanisms of CLCA1 in colorectal cancer (CRC).MethodsThe CLCA1 protein expression level in CRC patients was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and western blotting analysis. Using CRISPR/Cas9 technology, CLCA1-upregulated (CLCA1-ACT) and CLCA1-knockout cells (CLCA1-KO), as well as their respective negative controls (CLCA1-ACT-NC and CLCA1-KO-NC), were constructed from the SW620 cell line. Cell growth and metastatic ability were assessed both in vitro and in vivo. The association of CLCA1 with epithelial-mesenchymal transition (EMT) and other signaling pathways was determined by western blotting assays.ResultsThe expression level of CLCA1 in CRC tissues was significantly decreased compared with that in adjacent normal tissue (P< 0.05). Meanwhile, the serum concentration of CLCA1 in CRC patients was also significantly lower when compared with that of healthy controls (1.48 ± 1.06 ng/mL vs 1.06 ± 0.73 ng/mL, P = 0.0018). In addition, CLCA1 serum concentration and mRNA expression level in CRC tissues were inversely correlated with CRC metastasis and tumor stage. Upregulated CLCA1 suppressed CRC growth and metastasis in vitro and in vivo, whereas inhibition of CLCA1 led to the opposite results. Increased expression levels of CLCA1 could repress Wnt signaling and the EMT process in CRC cells.ConclusionsOur findings suggest that increased expression levels of CLCA1 can suppress CRC aggressiveness. CLCA1 functions as a tumor suppressor possibly via inhibition of the Wnt/beta-catenin signaling pathway and the EMT process.

Self-assembling HA/PEI/dsRNA-p21 ternary complexes for CD44 mediated small active RNA delivery to colorectal cancer

Our previous work proved that sequence specific double strand RNA (dsRNA-p21) effectively activated p21 gene expression of colorectal cancer (CRC) cells and consequently suppressed CRC growth. However, efficient delivery system is a significant challenge to achieve sufficient therapy. In this study, a self-assembled HA/PEI/dsRNA-p21 ternary complex (TC-dsRNA-p21) was developed for the tumor-target delivery of dsRNA-p21 into CRC cells. Hyaluronic acid (HA) was introduced to shield the PEI/dsRNA-p21 binary complexes (BC-dsRNA-p21) for reducing the cytotoxicity of PEI and for increasing the tumor-targeted intracellular uptake by cancer cells through HA-CD44 mediated endocytosis. Comparing to the BC-dsRNA-p21, the TC-dsRNA-p21 showed increase in size, decrease in zeta potential, low cytotoxicity as well as high stability in physiological conditions due to the anionic shielding. Confocal microscopy analysis and flow cytometry confirmed that TC-dsRNA-p21 had high transfection efficiency in the CD44-abundant Lovo cells, as compared with binary complex. In vitro physiological experiment showed that, comparing to the control group, the TC-dsRNA-p21 effectively activated the expression of p21 mRNA and P21 protein, causing blockage of cell cycle at G0/G1 phase and suppression of cancer cell proliferation as well as colony formation. Furthermore, in vivo distribution experiment demonstrated that the TC-dsRNA-p21 could effectively accumulate at rectal wall for up to 10 h, following in situ application. These findings indicated that TC-dsRNA-p21 might hold great potential for delivering dsRNA-p21 to treat CRC.

Metformin suppresses CRC growth by inducing apoptosis via ADORA1.

Accumulating evidence suggests that the anti-diabetic drug, metformin, exerts anti-proliferative effects in many types of cancers. However, the function and mechanisms of metformin in human colorectal cancer (CRC) remain unknown. Here, we show that metformin induces growth inhibition and apoptosis through activating AMPK-mTOR pathway in human colorectal cancer cells. Notably, metformin treatment significantly up-regulated adenosine A1 receptor (ADORA1) expression in human colorectal cancer cells, while suppression of ADORA1 activity by its specific inhibitor rescued the growth inhibition induced by metformin. Moreover, ADORA1-mediated growth inhibition and apoptosis induced by metformin is AMPK-mTOR pathway dependent in human colorectal cancer cells. Taken together, these results indicate that metformin suppresses human colorectal cancer growth by inducing apoptosis via ADORA1, which provide evidence the anti-neoplastic effects of metformin in the treatment of human colorectal cancer.

Inhibitory effects of Hedyotis diffusa Willd. on colorectal cancer stem cells.

Cancer stem cells (CSCs) are proposed to be closely correlated with the development and progression of tumors, as well as with chemo- and radioresistance. Targeting CSCs may therefore be a promising potential strategy for the treatment of cancer. Currently, natural products have received great interest due to their therapeutic efficacy and reduced adverse effects compared with modern chemotherapeutics. As a significant component of a number of traditional Chinese medicine formulas, the medicinal herb Hedyotis diffusa Willd. (HDW) has long been utilized in China to clinically treat a variety of malignancies, including colorectal cancer (CRC). Previously, the authors of the present study reported that HDW suppressed CRC growth through multiple mechanisms, including promoting apoptosis, and inhibiting cell proliferation and tumor angiogenesis. To additionally investigate its mode of action, the present study isolated a stem-like side population (SP) from colorectal cancer HT-29 cells to investigate the effect of ethanol extract of HDW on CSCs. It was observed that HDW was able to markedly downregulate the expression of CSC marker leucine-rich repeat-containing G-protein coupled receptor 5 and also significantly decrease the proportion of SP in HT-29 cells, in a dose-dependent manner. Furthermore, HDW treatment significantly and dose-dependently inhibited the viability and sphere formation, and induced cell morphological changes of isolated HT-29 SP cells. In addition, HDW greatly suppressed the messenger RNA expression of several critical genes that mediate CSC features, including ATP-binding cassette, sub-family B, member 1, β-catenin, c-Myc, proliferating cell nuclear antigen and survivin. In conclusion, the present study indicates that HDW may exert inhibitory effects on cancer stem cells.