Effect of aging on the formation and growth of colonic epithelial organoids by changes in cell cycle arrest through TGF-ß-Smad3 signaling.

BACKGROUND: This study aimed to investigate how aging alters the homeostasis of the colonic intestinal epithelium and regeneration after tissue injury using organoid models and to identify its underlying molecular mechanism. METHODS: To investigate aging-related changes in the colonic intestinal epithelium, we conducted organoid cultures from old (older than 80 weeks) and young (6-10 weeks) mice and compared the number and size of organoids at day 5 of passage 0 and the growth rate of organoids between the two groups. RESULTS: The number and size of organoids from old mice was significantly lower than that from young mice (p < 0.0001) at day 5 of passage 0. The growth rate of old-mouse organoids from day 4 to 5 of passage 0 was significantly slower than that of young-mouse organoids (2.21 times vs. 1.16 times, p < 0.001). RNA sequencing showed that TGF-ß- and cell cycle-associated genes were associated with the aging effect. With regard to mRNA and protein levels, Smad3 and p-Smad3 in the old-mouse organoids were markedly increased compared with those in the young-mouse organoids. Decreased expression of ID1, increased expression of p16INK4a, and increased cell cycle arrest were observed in the old mouse-organoids. Treatment with SB431542, a type I TGF-ß receptor inhibitor, significantly increased the formation and growth of old-mouse organoids, and TGF-ß1 treatment markedly suppressed the formation of young-mouse organoids. In the acute dextran sulfate sodium-colitis model and its organoid experiments, the colonic epithelial regeneration after tissue injury in old mice was significantly decreased compared with young mice. CONCLUSIONS: Aging reduced the formation ability and growth rate of colonic epithelial organoids by increasing cell cycle arrest through TGF-ß-Smad3-p16INK4a signaling.

Tumor-Suppressive Effect of Metformin via the Regulation of M2 Macrophages and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment of Colorectal Cancer.

Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells. Metformin, one of the most common diabetes drugs, has shown anti-inflammatory and anti-tumor effects. However, the effects of metformin on inflammatory cells of the tumor microenvironment and its underlying mechanisms remain unclarified. In this study, we investigated the effect of metformin on M2 macrophages and MDSCs using monocyte THP-1 cells and a dextran sodium sulfate (DSS)-treated ApcMin/+ mouse model of colon cancer. Metformin decreased the fractions of MDSCs expressing CD33 and arginase, as well as M2 macrophages expressing CD206 and CD163. The inhibitory effect of metformin and rapamycin on MDSCs and M2 macrophages was reversed by the co-treatment of Compound C (an AMP-activated protein kinase (AMPK) inhibitor) or mevalonate. To examine the effect of protein prenylation and cholesterol synthesis (the final steps of the mevalonate pathway) on the MDSC and M2 macrophage populations, we used respective inhibitors (YM53601; SQLE inhibitor, FTI-277; farnesyl transferase inhibitor, GGTI-298; geranylgeranyl transferase inhibitor) and found that the MDSC and M2 populations were suppressed by the protein prenylation inhibitors. In the DSS-treated ApcMin/+ mouse colon cancer model, metformin reduced the number and volume of colorectal tumors with decreased populations of MDSCs and M2 macrophages in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK activation and subsequent mTOR inhibition, leading to the downregulation of the mevalonate pathway.

Suppressive effect of α-mangostin for cancer stem cells in colorectal cancer via the Notch pathway.

BACKGROUND: Since colon cancer stem cells (CSCs) play an important role in chemoresistance and in tumor recurrence and metastasis, targeting of CSCs has emerged as a sophisticated strategy for cancer therapy. α-mangostin (αM) has been confirmed to have antiproliferative and apoptotic effects on cancer cells. This study aimed to evaluate the selective inhibition of αM on CSCs in colorectal cancer (CRC) and the suppressive effect on 5-fluorouracil (5-FU)-induced CSCs. METHODS: The cell viability assay was performed to determine the optimal concentration of αM. A sphere forming assay and flow cytometry with CSC markers were carried out to evaluate the αM-mediated inhibition of CSCs. Western blot analysis and quantitative real-time PCR were performed to investigate the effects of αM on the Notch signaling pathway and colon CSCs. The in vivo anticancer efficacy of αM in combination with 5-FU was investigated using a xenograft mouse model. RESULTS: αM inhibited the cell viability and reduced the number of spheres in HT29 and SW620 cells. αM treatment decreased CSCs and suppressed the 5-FU-induced an increase in CSCs on flow cytometry. αM markedly suppressed Notch1, NICD1, and Hes1 in the Notch signaling pathway in a time- and dose-dependent manner. Moreover, αM attenuated CSC markers CD44 and CD133, in a manner similar to that upon DAPT treatment, in HT29 cells. In xenograft mice, the tumor and CSC makers were suppressed in the αM group and in the αM group with 5-FU treatment. CONCLUSION: This study shows that low-dose αM inhibits CSCs in CRC and suppresses 5-FU-induced augmentation of CSCs via the Notch signaling pathway.

IL-6 and IL-8, secreted by myofibroblasts in the tumor microenvironment, activate HES1 to expand the cancer stem cell population in early colorectal tumor.

Interaction between a tumor and its microenvironment is important for tumor initiation and progression. Cancer stem cells (CSCs) within the tumor interact with a microenvironmental niche that controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts into the microenvironment of early colorectal cancer tumors and its molecular mechanism. By messenger RNA microarray analysis, expression of HES1, a Notch signaling target, significantly increased in Caco-2 cells cocultured with 18Co cells (pericryptal myofibroblasts), compared to its expression in Caco-2 cells cultured alone. Caco-2 cells cultured in 18Co-conditioned media (CM) showed a significant increase in CD133+CD44+ cells and HES1 expression compared to that in Caco-2 cells cultured in regular media. Significant amounts of interleukin-6 (IL-6) and IL-8 were detected in 18Co-CM compared to levels in regular media. The 18Co-CM-induced increase in CD133+CD44+ cells was attenuated by IL-6- and IL-8-neutralizing antibodies. Furthermore, these neutralizing antibodies and inhibitors of STAT3 and gamma-secretase reduced the expression of HES1 induced in Caco-2 cells cultured in 18Co-CM. Immunohistochemical analysis of human tissues revealed that IL-6, IL-8, and HES1 expression increased from normal to adenoma, and from adenoma to cancer tissues. In addition, IL-6 and HES1 expression was positively correlated in early colorectal cancer tissues. In conclusion, the increase of CSCs by myofibroblasts could be mediated by IL-6/IL-8-induced HES1 activation in the tumor microenvironment. Based on these data, the IL-6/IL-8-mediated Notch/HES1 and STAT3 pathway, through which CSCs interact with their microenvironment, might be a potential target for the prevention and treatment of colorectal tumors.

Publisher Correction: Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism.

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Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism.

Metformin has been known to suppress cancer stem cells (CSCs) in some cancers. However, the differential effects of metformin on CSCs and their mechanisms have not been reported. Herein, metformin induced pAMPK activation and pS6 suppression in metformin-sensitive (HT29) cells, but not in metformin-resistant (SW620) cells. The oxygen consumption rate was higher in HT29 cells than in SW620 cells and showed a prominent decrease after metformin treatment in HT29 cells. In glutamine-depleted medium, but not in low-glucose medium, SW620 cells became sensitive to the CSC-suppressing effect of metformin. A combination of metformin and glutaminase C inhibitor (compound 968) suppressed CSCs in SW620 cells and enhanced that effect in HT29 cells. SW620 cells showed higher expression of glutaminase 1 and glutamine transporter (ASCT2) than HT29 cells, especially ASCT2 in CSCs. Knockdown of glutaminase 1, ASCT2, and c-Myc induced significant CSC-suppression and enhanced CSC-suppressing effect of metformin and compound 968. In xenografts and human cancer organoids, combined treatment with metformin and compound 968 showed the same results as those shown in vitro. In conclusion, the effect of metformin on CSCs varies depending on the AMPK-mTOR and glutamine metabolism. The inhibition of glutamine pathway could enhance the CSC-suppressing effect of metformin, overcoming metformin resistance.

Long-Term Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Dextran Sulfate Sodium-Induced Murine Chronic Colitis.

BACKGROUND/AIMS: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown beneficial effects in experimental colitis models, but the underlying mechanisms are not fully understood. We investigated the long-term effects of BM-MSCs, particularly in mice with chronic colitis. METHODS: Chronic colitis was induced by administering 3% dextran sulfate sodium (DSS) in a series of three cycles. BMMSCs were injected intravenously into DSS-treated mice three times during the first cycle. On day 33, the therapeutic effects were evaluated with clinicopathologic profiles and histological scoring. Inflammatory mediators were measured with real-time polymerase chain reaction. RESULTS: Systemic infusion of BM-MSCs ameliorated the severity of colitis, and body weight restoration was significantly promoted in the BMMSC- treated mice. In addition, BM-MSC treatment showed a sustained beneficial effect throughout the three cycles. Microscopic examination revealed that the mice treated with BM-MSCs had fewer inflammatory infiltrates, a lesser extent of inflammation, and less crypt structure damage compared with mice with DSS-induced colitis. Anti-inflammatory cytokine levels of interleukin-10 were significantly increased in the inflamed colons of BM-MSC-treated mice compared with DSS-induced colitis mice. CONCLUSIONS: Systemic infusion of BM-MSCs at the onset of disease exerted preventive and rapid recovery effects, with long-term immunosuppressive action in mice with repeated DSS-induced chronic colitis.

Nonsteroidal anti-inflammatory drugs suppress cancer stem cells via inhibiting PTGS2 (cyclooxygenase 2) and NOTCH/HES1 and activating PPARG in colorectal cancer.

Cancer stem cells (CSCs) play a pivotal role in cancer relapse or metastasis. We investigated the CSC-suppressing effect of nonsteroidal anti-inflammatory drugs (NSAIDs) and the relevant mechanisms in colorectal cancer. We measured the effect of NSAIDs on CSC populations in Caco-2 or SW620 cells using colosphere formation and flow cytometric analysis of PROM1 (CD133)(+) CD44(+) cells after indomethacin treatment with/without prostaglandin E2 (PGE2) or peroxisome proliferator-activated receptor γ (PPARG) antagonist, and examined the effect of indomethacin on transcriptional activity and protein expression of NOTCH/HES1 and PPARG. These effects of indomethacin were also evaluated in a xenograft mouse model. NSAIDs (indomethacin, sulindac and aspirin), celecoxib, γ-secretase inhibitor and PPARG agonist significantly decreased the number of colospheres formation compared to controls. In Caco-2 and SW620 cells, compared to controls, PROM1 (CD133)(+) CD44(+) cells were significantly decreased by indomethacin treatment, and increased by 5-fluorouracil (5-FU) treatment. This 5-FU-induced increase of PROM1 (CD133)(+) CD44(+) cells was significantly attenuated by combination with indomethacin. This CSC-inhibitory effect of indomethacin was reversed by addition of PGE2 and PPARG antagonist. Indomethacin significantly decreased CBFRE and increased PPRE transcriptional activity and their relative protein expressions. In xenograft mouse experiments using 5-FU-resistant SW620 cells, the 5-FU treatment combined with indomethacin significantly reduced tumor growth, compared to 5-FU alone. In addition, treatment of indomethacin alone or combination of 5-FU and indomethacin decreased the expressions of PROM1 (CD133), CD44, PTGS2 (cyclooxygenase 2) and HES1, and increased PPARG expression. NSAIDs could selectively reduce the colon CSCs and suppress 5-FU-induced increase of CSCs via inhibiting PTGS2 (cyclooxygenase 2) and NOTCH/HES1, and activating PPARG.

Differential expression of CD133 based on microsatellite instability status in human colorectal cancer.

The association between the types of genomic instability and cancer stem cell (CSC) has not been elucidated. We aimed to investigate the expressions of CSC markers with respect to microsatellite instability (MSI) status in human colorectal cancer (CRC). Immunostainings for CD133, CD44, and CD166, and K-ras mutation analysis were performed on 50 MSI-high (MSI-H), and 50 microsatellite stable (MSS) CRC tissues. In 11 MSS and MSI-H CRC cell lines, CD133 expression and DNA methylation statuses of the CD133 promoter were determined. The proportion of CD133 positive cells and the ability of colosphere formation were compared between HCT116 cells and HCT116 + Chr3 cells (hMLH1-restored HCT116 cells). Immunohistochemistry for CSC markers revealed that high CD133 expression was more frequent in MSS cancers than in MSI-H (P < 0.001, 74.0% vs. 28.0%, respectively), and related with short disease-free survival. Neither CD44 nor CD166 expression differed significantly with respect to MSI status. K-ras mutation showed no association with expressions of CD133, CD44, or CD166. CD133 expression was relatively high in the MSS cell lines compared to those in MSI-H, and showed a reverse correlation with DNA methylation of the CD133 promoter. hMLH1-restored HCT116 cells increased proportions of CD133 positive cells and colosphere forming ability, compared to those in HCT116 cells. In conclusion, high levels of CD133 expression were observed more frequently in MSS CRC than in MSI-H, suggesting that differential expression of colon CSC markers may be linked to tumor characteristics dependent on MSI status.

Mesenchymal stem cell transfer suppresses airway remodeling in a toluene diisocyanate-induced murine asthma model.

PURPOSE: Severe asthma is characterized by high medication requirements to maintain good disease control or by persistent symptoms despite high medication use. The transfer of bone marrow-derived mesenchymal stem cells (BMDMSCs) to the injured lungs is a possible treatment for severe asthma. This study investigated the therapeutic effects of BMDMSCs in airway remodeling and inflammation in an experimental toluene diisocyanate (TDI)-induced asthma animal model of severe asthma. METHODS: BMDMSCs were transferred into rats after TDI inhalation. Bronchoalveolar lavage (BAL) cell profiles, histological changes including an inflammatory index and goblet cell hyperplasia, and the airway response to methacholine using plethysmography were analyzed. Smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA) protein expression were observed in lung tissue using immunohistochemical staining. The collagen content was measured in lung tissue sections and lung extracts using Masson's trichrome staining and an immunoassay kit. RESULTS: The numbers of inflammatory cells in BAL fluid, histological inflammatory index, airway response to methacholine, number of goblet cells, and amount of collagen were increased in TDI-treated rats compared with sham rats (P=0.05-0.002). BMDMSC transfer significantly reduced the TDI-induced increase in the inflammatory index and numbers of eosinophils and neutrophils in BAL fluid to levels seen in sham-treated rats (P<0.05). BMDMSC transfer significantly reduced the number of goblet cells, collagen deposition, and immune staining for SMA and PCNA with concomitant normalization of the airway response to methacholine. CONCLUSIONS: The systemic transfer of BMDMSCs effectively reduced experimental TDI-induced airway inflammation and remodeling and airway hyperreactivity.

Ym1 and Ym2 expression in a mouse model exposed to diesel exhaust particles.

BACKGROUND: Chitinase may play a role in regulating allergic diseases. OBJECTIVE: We studied the role of chitinase in a mouse model exposed to diesel exhaust particles (DEP). Mice were exposed to intranasal DEP (0.6 mg/mL) for 5 days and challenged with aerosolized DEP (6 mg/m(3)) on days 6-8. Enhanced pause (Penh), as an airway obstruction marker, was measured on day 9, and bronchoalveolar lavage (BAL) fluid and lung tissues were collected on day 10. The expression of Ym1 and Ym2 mRNA was assessed in lung tissue extracts by reverse transcription-polymerase chain reaction. RESULTS: DEP induced significant increases in methacholine-induced Penh and IL-4 levels in BAL fluid relative to the control group. Peribronchial and perivascular inflammatory cell infiltrates were prominent in the DEP group. DEP induced Ym1 and Ym2 mRNA expression in lung tissue extracts relative to the control group. CONCLUSION: These results demonstrate that DEP induced airway hyperresponsiveness and Ym mRNA expression via a Th2 cell-biased response, suggesting that chitinase may play an important role in airway inflammation and responsiveness upon exposure to DEP in a mouse model, and may therefore be involved in regulating allergic diseases.