Human Colorectal Cancer Growth Research Articles

Linderapyrone analogue LPD-01 as a cancer treatment agent by targeting importin7.

The Wnt/β-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/β-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/β-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/β-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (β-catenin) was not decreased, suggesting that 1 inhibited the Wnt/β-catenin signaling pathway without affecting β-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-β-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/β-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/β-catenin signaling pathway.

IFI6 predicts prognosis and promotes cell growth of human colorectal cancer

Purpose: To analyze the effect of IFI6 in colorectal cancer (CRC).
 Methods: Differentially-expressed genes were analyzed using a volcano plot. Metascape and STRING were used for functional enrichment and protein-protein interaction network analyses, respectively. Expression of IFI6 was analyzed using the online platforms TIMER, UALCAN, and GEPIA. The correlation between IFI6 and poor prognosis was analyzed using prognoscan, while Western blotting was used to determine the expressions of related proteins. Cell proliferation was performed by CCK8 and foci formation assays. Cell apoptosis was assessed using flow cytometry.
 Results: Differentially-expressed genes between CRC cells and alisertib-resistant CRC cells comprised 84 upregulated genes and 358 downregulated genes (p < 0.05). Enrichment analysis showed that differential genes were mainly involved in interferon α/β signaling, antiviral mechanism of interferon-stimulated genes, and positive regulation of the immune response. IFI6, a hubgene in the protein-protein interaction network, was highly expressed in CRC (p < 0.001) and correlated with poor prognoses (p = 0.028). IFI6 knockdown inhibited cell viability and colony formation and induced cell apoptosis by upregulation of p53, p21, and Bax.
 Conclusion: These results suggest that IFI6 may be a biomarker for the diagnosis of human CRC.

FIT links c-Myc and P53 acetylation by recruiting RBBP7 during colorectal carcinogenesis.

Colorectal cancer (CRC) poses one of the most serious threats to human health worldwide, and abnormally expressed c-Myc and p53 are deemed the pivotal driving forces of CRC progression. In this study, we discovered that the lncRNA FIT, which was downregulated in CRC clinical samples, was transcriptionally suppressed by c-Myc in vitro and promoted CRC cell apoptosis by inducing FAS expression. FAS is a p53 target gene, and we found that FIT formed a trimer with RBBP7 and p53 that facilitated p53 acetylation and p53-mediated FAS gene transcription. Moreover, FIT was capable of retarding CRC growth in a mouse xenograft model, and FIT expression was positively correlated with FAS expression in clinical samples. Thus, our study elucidates the role of the lncRNA FIT in human colorectal cancer growth and provides a potential target for anti-CRC drugs.

Targeting fat mass and obesity-associated protein mitigates human colorectal cancer growth in vitro and in a murine model.

Colorectal cancer (CRC) remains a significant cause of cancer related mortality. Fat mass and obesity-associated protein (FTO) is a m6A mRNA demethylase that plays an oncogenic role in various malignancies. In this study we evaluated the role of FTO in CRC tumorigenesis. Cell proliferation assays were conducted in 6 CRC cell lines with the FTO inhibitor CS1 (50-3200 nM) (± 5-FU 5-80 mM) and after lentivirus mediated FTO knockdown. Cell cycle and apoptosis assays were conducted in HCT116 cells (24 h and 48 h, 290 nM CS1). Western blot and m6A dot plot assays were performed to assess CS1 inhibition of cell cycle proteins and FTO demethylase activity. Migration and invasion assays of shFTO cells and CS1 treated cells were performed. An in vivo heterotopic model of HCT116 cells treated with CS1 or with FTO knockdown cells was performed. RNA-seq was performed on shFTO cells to assess which molecular and metabolic pathways were impacted. RT-PCR was conducted on select genes down-regulated by FTO knockdown. We found that the FTO inhibitor, CS1 suppressed CRC cell proliferation in 6 colorectal cancer cell lines and in the 5-Fluorouracil resistant cell line (HCT116-5FUR). CS1 induced cell cycle arrest in the G2/M phase by down regulation of CDC25C and promoted apoptosis of HCT116 cells. CS1 suppressed in vivo tumor growth in the HCT116 heterotopic model (p< 0.05). Lentivirus knockdown of FTO in HCT116 cells (shFTO) mitigated in vivo tumor proliferation and in vitro demethylase activity, cell growth, migration and invasion compared to shScr controls (p< 0.01). RNA-seq of shFTO cells compared to shScr demonstrated down-regulation of pathways related to oxidative phosphorylation, MYC and Akt/ mTOR signaling pathways. Further work exploring the targeted pathways will elucidate precise downstream mechanisms that can potentially translate these findings to clinical trials.

A recombinant Chinese measles virus vaccine strain rMV-Hu191 inhibits human colorectal cancer growth through inducing autophagy and apoptosis regulating by PI3K/AKT pathway

The potential therapeutic effects of oncolytic measles virotherapy have been verified against plenty of malignancies. However, the oncolytic effects and underlying mechanisms of the recombinant Chinese measles virus vaccine strain Hu191 (rMV-Hu191) against human colorectal cancer (CRC) remain elusive. In this study, the antitumor effects of rMV-Hu191 were evaluated in CRC both in vitro and in vivo. From our data, rMV-Hu191 induced remarkably caspase-dependent apoptosis and complete autophagy in vitro. In mice bearing CRC xenografts, tumor volume was remarkably suppressed and median survival was prolonged significantly with intratumoral treatment of rMV-Hu191. To gain further insight into the relationship of rMV-Hu191-induced apoptosis and autophagy, we utilized Rapa and shATG7 to regulate autophagy. Our data suggested that autophagy was served as a protective role in rMV-Hu191-induced apoptosis in CRC. PI3K/AKT signaling pathway as one of the common upstream pathways of apoptosis and autophagy was activated in CRC after treatment with rMV-Hu191. And inhibition of PI3K/AKT pathway using LY294002 was accompanied by enhanced apoptosis and decreased autophagy which suggested that PI3K/AKT pathway promoted rMV-Hu191-induced autophagy and inhibited rMV-Hu191-induced apoptosis. This is the first study to demonstrate that rMV-Hu191 could be used as a potentially effective therapeutic agent in CRC treatment. As part of the underlying cellular mechanisms, apoptosis and autophagy were involved in the oncolytic effects generated by rMV-Hu191. And the cross-talk between these two processes and the PI3K/AKT signaling pathway was well identified.

Morusin inhibits the growth of human colorectal cancer HCT116‑derived sphere‑forming cells via the inactivation of Akt pathway.

The existence of colorectal cancer stem-like cells (CSC) is responsible for the failure of current treatments against colorectal cancer. Therefore, novel therapies need be developed to target CSCs. Some natural agents, including morusin have been proposed as possible candidates for this purpose. Morusin has been shown to exert antitumor effects. In the present study, it is demonstrated that morusin exerts antitumor effects on colorectal CSCs (CCSCs). The viability of human CCSCs was enhanced when the CCSCs formed spheroids in a serum-free and non-adhesive floating culture system. HCT116 sphere cells exhibited an increased proliferative capacity and a higher expression of stemness markers [octamer-binding transcription factor 4 (Oct4), Sox2 and Nanog]. Morusin inhibited the development of cancer spheroids and suppressed the growth of sphere cells via the induction of cell cycle arrest. Similarly, morusin decreased the expression levels of the stemness markers, Nanog and Oct4. The data partially revealed the molecular mechanisms involved: β-catenin signaling maintains the growth of CSCs and directly modulates the expression of Nanog and Oct4. Morusin suppressed the activity of β-catenin signaling via the inactivation of Akt; the executive β-catenin/TCF4 complex and the downstream targets, c-Myc, survivin and cyclin D1, were also downregulated. Moreover, the morusin-induced inactivation of Akt also increased the expression of p21Cip1/WAF1 and p27Kip, which can block the cell cycle by interacting with cyclin-dependent kinase (CDK) complexes. On the whole, the present study demonstrates that morusin inhibited the growth of colorectal cancer sphere cells, which were enriched with CCSCs via the inactivation of the Akt pathway.

Ursolic acid induces apoptosis and anoikis in colorectal carcinoma RKO cells

BackgroundUrsolic acid (UA) is an anti-cancer herbal compound. In the present study, we observed the effects of UA on anchorage-dependent and -independent growth of human colorectal cancer (CRC) RKO cells.MethodsRKO cells were cultured in conventional and detached condition and treated with UA. Cell viability was evaluated by CCK-8 assay. Cell cycle was analyzed by flow cytometry. Apoptosis was identified by Hoechst 33258 staining and flow cytometry analysis. Activities of caspases were measured by commercial kits. Reactive oxygen species (ROS) was recognized by DCFH-DA fluorescent staining. Anoikis was identified by EthD-1 fluorescent staining and flow cytometry analysis. Expression and phosphorylation of proteins were analyzed by western blot.ResultsUA inhibited RKO cell viability in both a dose- and time-dependent manner. UA arrested the cell cycle at the G0/G1 phase, and induced caspase-dependent apoptosis. UA inhibited Bcl-2 expression and increased Bax expression. In addition, UA up-regulated the level of ROS that contributed to UA activated caspase-3, − 8 and − 9, and induced apoptosis. Furthermore, UA inhibited cell growth in a detached condition and induced anoikis in RKO cells that was accompanied by dampened phosphorylation of FAK, PI3K and AKT. UA also inhibited epithelial-mesenchymal transition (EMT) as indicated by the down-regulation of N-Cad expression and up-regulation of E-Cad expression.ConclusionsUA induced caspase-dependent apoptosis, and FAK/PI3K/AKT singling and EMT related anoikis in RKO cells. UA was an effective anti-cancer compound against both anchorage-dependent and -independent growth of RKO cells.

Extraction, characterization of xylan from Azadirachta indica (neem) sawdust and production of antiproliferative xylooligosaccharides

Xylan extracted from neem sawdust gave 22.5%, (w/w) yield. The extracted xylan was composed of xylose and glucuronic acid at a molar ratio of 8:1 and with a molecular mass, ~66 kDa. FTIR and NMR analyses indicated a backbone of xylan substituted with 4-O-methyl glucuronic acid at the O-2 position. FESEM analysis showed a highly porous and granular surface structure of xylan. A thermogravimetric study of xylan showed thermal denaturation at 271 °C. The hydrolysis of xylan by recombinant endo-β-1,4-xylanase produced a mixture of xylooligosaccharides ranging from degree of polymerization 2–7. Xylooligosaccharides inhibited cell growth of human colorectal cancer (HT-29) cells but did not affect the mouse fibroblast cells confirming its biocompatibility. Western blotting, DNA laddering and flow cytometric analysis displayed inhibition of HT-29 cells by xylooligosaccharides. FLICA staining and mitochondrial membrane potential analyses confirmed the activation of the intrinsic pathway of apoptosis. The study amply indicated that the xylooligosaccharides produced from neem xylan could be potentially used as an antiproliferative agent.

Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K

We have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K+ channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.

Combination of Cetuximab and Oncolytic Virus Canerpaturev Synergistically Inhibits Human Colorectal Cancer Growth

The naturally occurring oncolytic herpes simplex virus canerpaturev (C-REV), formerly HF10, proved its therapeutic efficacy and safety in multiple clinical trials against melanoma, pancreatic, breast, and head and neck cancers. Meanwhile, patients with colorectal cancer, which has increased in prevalence in recent decades, continue to have poor prognosis and morbidity. Combination therapy has better response rates than monotherapy. Hence, we investigated the antitumor efficacy of cetuximab, a widely used anti-epidermal growth factor receptor (EGFR) monoclonal antibody, and C-REV, either alone or in combination, in vitro and in an in vivo human colorectal xenograft model. In human colorectal cancer cell lines with different levels of EGFR expression (HT-29, WiDr, and CW2), C-REV exhibited cytotoxic effects in a time- and dose-dependent manner, irrespective of EGFR expression. Moreover, cetuximab had no effect on viral replication in vitro. Combining cetuximab and C-REV induced a synergistic antitumor effect in HT-29 tumor xenograft models by promoting the distribution of C-REV throughout the tumor and suppressing angiogenesis. Application of cetuximab prior to C-REV yielded better tumor regression than administration of the drug after the virus. Thus, cetuximab represents an ideal virus-associated agent for antitumor therapy, and combination therapy represents a promising antitumor strategy for human colorectal cancer.

Growth Suppression of Human Colorectal Cancer Cells with Mutated KRAS by 3-Deaza-cytarabine in 3D Floating Culture.

During screening for compounds that selectively suppress growth of human colorectal cancer (CRC) spheroids with mutant (mt) KRAS, the uridine analogue, 5-bromouridine (BrUrd) was identified and its derivatives were explored. DNA incorporation in two-dimensional (2D) and three-dimensional floating (3DF) cultures was examined with the uridine analogue, 5-ethynyl-2'-deoxyuridine (EdU). The area of HKe3 CRC spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in 3DF culture with 11 BrUrd derivatives. EdU was strongly incorporated into newly-synthesized DNA from HKe3-mtKRAS cells compared to HKe3-wtKRAS in 2D and 3DF culture. 3-Deaza-cytarabine, which has properties of BrUrd and cytidine, was the most effective inhibitor of HKe3-mtKRAS spheroids with the least toxicity to HKe3-wtKRAS. Growth suppression of 3-deaza-cytarabine was stronger than cytarabine in 2D culture, and toxicity was lower than gemcitabine in long-term 3DF culture. 3-Deaza-cytarabine exhibits properties useful for the treatment of CRC patients with mtKRAS.

Effect of CEACAM-1 knockdown in human colorectal cancer cells.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) is the major antigen of the CD66 cluster of granulocyte differentiation antigens. The present study aimed to assess the biological function of CEACAM-1 on the growth of human colorectal cancer (CRC) cells in vitro. Treatment of cultured CRC HCT-8 cells with CEACAM-1-specific siRNA successfully downregulated CEACAM-1 expression by 61% compared with control cells. The effects of CEACAM-1 downregulation on HCT-8 cell proliferation and apoptosis were then assessed via Cell Counting kit-8 assay and flow cytometry, respectively. The results demonstrated that siRNA-induced CEACAM-1 downregulation significantly inhibited proliferation and increased apoptosis, but had no significant effect on cell cycle progression in HCT-8 cells. Together, these results suggest that CEACAM-1 activity is critical to CRC growth, and thus, CEACAM-1 may be a promising therapeutic target for the treatment of CRC.

MiR-760 suppresses human colorectal cancer growth by targeting BATF3/AP-1/cyclinD1 signaling

BackgroundRecent studies have reported that microRNAs (miRNAs) often function as negative post-transcriptional regulators with altered expression levels found in colorectal cancer (CRC). There have been few studies on miRNAs that regulate the oncogenic alterations in CRC. Here, we aim to explore the anti-cancer miRNA and the potential mechanisms by which miRNAs modulate CRC progression.MethodsWe performed an integrated analysis of CRC miRNA expression datasets in The Cancer Genome Atlas (TCGA). The miRNA with the lowest expression, miR-760, was validated in an independent validation sample cohort of 76 CRC tissues. Functional assays, such as CCK-8 assay, colony formation assay, and CFSE staining, were used to determine the oncogenic role of miR-760 in human CRC progression. Furthermore, western blotting and dual-luciferase reporter assay were used to determine the mechanism by which miR-760 promotes proliferation of CRC cells. Xenograft nude mouse models were used to determine the role of miR-760 in CRC tumorigenicity in vivo. Immunohistochemical assays were conducted to study the relationship between miR-760 expression and basic leucine zipper transcriptional factor ATF-like 3 (BATF3) expression in human CRC samples.ResultsmiR-760 was markedly downregulated in CRC tissues, and low miR-760 expression was associated with poor prognosis among CRC patients. Upregulation of miR-760 suppressed CRC cell proliferation, whereas downregulation of miR-760 promoted CRC proliferation in vitro. Additionally, we identified BATF3 as a direct target of miR-760, and that the essential biological function of miR-760 during CRC progression both in vitro and in vivo is to suppress the expression of BATF3 and downstream cyclinD1 via AP-1 transcription factor. Finally, we showed a significant correlation between miR-760 and BATF3 expression in CRC tissues.ConclusionsmiR-760 inhibited CRC growth by downregulating BATF3/AP-1/ cyclinD1 signaling.

Pim1 supports human colorectal cancer growth during glucose deprivation by enhancing the Warburg effect

Cancer cells metabolize glucose mainly by glycolysis and are well adapted to metabolic stress. Pim1 is an oncogene that promotes colorectal cancer (CRC) growth and metastasis, and its expression is positively correlated with CRC progression. However, the mechanism underlying Pim1 overexpression during CRC progression and the role of Pim1 in CRC metabolism remains unclear. In the present study, we discovered that Pim1 expression was significantly upregulated in response to glucose deprivation‐induced metabolic stress by AMP‐activated protein kinase signaling. Pim1 promoted CRC cell proliferation in vitro and tumorigenicity in vivo. Clinical observations showed that Pim1 expression was higher in CRC tissues than in adjacent normal tissues. Pim1 overexpression in CRC tissues not only predicted CRC prognosis in patients but also showed a positive relationship with 18F‐fluorodeoxyglucose uptake. Further in vitro experiments showed that Pim1 promoted the Warburg effect and that Pim1 expression was positively correlated with hexokinase 2 and lactate dehydrogenase A expression. Pim1‐silenced cells were more vulnerable to glucose starvation, and Pim1‐induced tumor proliferation or tolerance to glucose starvation was attenuated by blocking the Warburg effect. In conclusion, glucose deprivation is one of the mechanisms that leads to elevated Pim1 expression in CRC, and Pim1 upregulation ensures CRC growth in response to glucose deprivation by facilitating the Warburg effect in a compensatory way.

Effects of PHA-665752 and Cetuximab Combination Treatment on In Vitro and Murine Xenograft Growth of Human Colorectal Cancer Cells with KRAS or BRAF Mutations.

It remains unknown whether blockade of c-Met signaling and epidermal growth factor receptor signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. In this study, we investigated the effects of the c-Met inhibitor PHA-665752 alone and in combination with cetuximab on the growth of human CRC cells in vitro and in mouse xenografts. Human CRC cell lines (Caco2, HCT-116, and HT-29) and mice bearing HCT-116 xenografts were treated with cetuximab in the absence or presence of PHA-665752. Cell viability and apoptosis were examined using the MTT and TUNEL assays, respectively. Vimentin was measured by immunohistochemistry as a marker for epithelial-to-mesenchymal transition. Western blotting was used to determine signaling protein expression levels. The MTT assay showed that the growth of Caco2, HCT-116, and HT-29 cells was inhibited by PHA-665752 in a dose-dependent manner, but only Caco2 cell growth was suppressed by cetuximab. Combination treatment with PHA-665752 and cetuximab inhibited the proliferation of Caco2 cells and RAS mutant CRC cell lines. However, relative to the PHA-665752-alone treatment group, HT-29 cells with a BRAF mutation showed no noticeable effect. The mean tumor volume in mice treated with cetuximab in combination with PHA-665752 was significantly smaller than that in the mice treated with only cetuximab (P = 0.033) or PHA-665752 (P < 0.01). Similarly, the expression of vimentin in the mice treated with PHA-665752 in combination with cetuximab was significantly lower than that in the mice treated with cetuximab or PHA-665752 alone (P < 0.05 in each case). TUNEL assays revealed that treatment with PHA-665752 in combination with cetuximab markedly increased CRC cell apoptosis. Western blotting analysis of signaling protein expression showed that PHA- 665752 inhibited Met phosphorylation (P < 0.05). In addition, treatment with cetuximab alone or in combination with PHA-665752 effectively inhibited EGFR phosphorylation (P < 0.05). Combination treatment with PHA-665752 and cetuximab suppressed in vitro and in vivo CRC cell growth more than treatment with either agent alone did.

Effects of PHA-665752 and vemurafenib combination treatment on in vitro and murine xenograft growth of human colorectal cancer cells with BRAFV600E mutations.

It remains unknown whether blockade of B-Raf proto-oncogene, serine/threonine kinase (BRAF)V600E signaling and MET proto-oncogene, receptor tyrosine kinase (c-Met) signaling is effective in suppressing the growth of human colorectal cancer (CRC) cells. The present study investigated the effects of the vemurafenib alone and in combination with c-Met inhibitor PHA-665752 on the growth of human CRC cells in vitro and in mouse xenografts. HT-29 and RKO CRC cell lines with BRAFV600E mutations and mice bearing HT-29 xenografts were treated with vemurafenib in the absence or presence of PHA-665752. Cell viability and cycle phase were respectively examined by using the MTT and flow cytometry assay. Immunohistochemistry was conducted to detect the protein expression levels of hepatocyte growth factor (HGF), phosphorylated (p)-c-Met, p-AKT serine/threonine kinase (AKT) and p-extracellular signal-regulated kinase (p-ERK). The MTT assay demonstrated that the growth of RKO and HT-29 cells was inhibited by PHA-665752 in a time- and dose-dependent manner (P<0.05), however no significant suppressive effects were observed with vemurafenib. Relative to the PHA-665752 or vemurafenib stand-alone treatment groups, the combination of PHA-665752 and vemurafenib had a significant inhibitory effect on the proliferation of CRC cell lines (P<0.05). The mean tumor volume in mice treated with vemurafenib in combination with PHA-665752 was significantly smaller compared with those treated with only vemurafenib or PHA-665752 (P<0.05). Flow cytometry assay revealed that the G0/G1 phase frequency was significantly increased in the combination group compared with any other treatment groups (P<0.05). Immunohistochemistry demonstrated that vemurafenib in combination with PHA-665752 effectively induced the expression of p-c-Met, p-AKT and p-ERK, however had no effect on HGF.

Specific up-regulation of p21 by a small active RNA sequence suppresses human colorectal cancer growth.

The double stranded small active RNA (saRNA)- p21-saRNA-322 inhibits tumor growth by stimulating the p21 gene expression. We focused our research of p21-saRNA-322 on colorectal cancer because 1) p21 down-regulation is a signature abnormality of the cancer, and 2) colorectal cancer might be a suitable target for in situ p21-saRNA-322 delivery. The goal of the present study is to learn the activity of p21-saRNA-322 in colorectal cancer. Three human colorectal cancer cell lines, HCT-116, HCT-116 (p53–/−) and HT-29 were transfected with the p21-saRNA-322. The expression of P21 protein and p21 mRNA were measured using the Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The effect of p21-saRNA-322 on cancer cells was evaluated in vitro; and furthermore, a xenograft colorectal tumor mode in mice was established to estimate the tumor suppressing ability of p21-saRNA-322 in vivo. The results showed that in all three colorectal cancer cell lines, the expression of p21 mRNA and P21 protein were dramatically elevated after p21-saRNA-322 transfection. Transfection of p21-saRNA-322 caused apoptosis and cell cycle arrest at the G0/G1. Furthermore, anti-proliferation effect, reduction of colonies formation and cell senescence were observed in p21-saRNA-322 treated cells. Animal studies showed that p21-saRNA-322 treatment significantly inhibited the HT-29 tumor growth and facilitated p21 activation in vivo. These results indicated that, p21-saRNA-322-induceded up-regulation of p21 might be a promising therapeutic option for the treatment of colorectal cancer.

Inhibitory effect and mechanism of exogenous mammalian sterile 20-like kinase 1 on the growth of human colorectal cancer.

The present study aimed to observe the inhibitory effect and preliminary mechanism of exogenous mammalian sterile 20-like kinase 1 (MST1) on the growth of colorectal cancer SW480 cells. The SW480 cells were randomly divided into the following groups: Control, empty enhanced green fluorescent protein (EGFP) plasmid (pEGFP-N1), MST1 EGFP plasmid (pEGFP-MST1), 20 µmol/l fluorouracil (5-FU) and pEGFP-MST1 + 5-FU. An MTS colorimetric assay was used to detect cell viability, Hoechst 33342 staining was used to observe cell apoptosis, and western blotting and immunohistochemistry were used to detect the levels of the proteins MST1, yes-associated protein (YAP), phospho-YAP1 (Ser127), p53 and p53 upregulated modulator of apoptosis (PUMA). In addition, nude mice were injected with SW480 cells to assess the tumor inhibition rates. Compared with the control group, the growth inhibition and apoptosis rates, the levels of MST1, p53 and PUMA, and the ratios of phospho-YAP1/YAP in the pEGFP-MST1 and pEGFP-MST1 + 5-FU groups were increased significantly (P<0.01). Additionally, relative to the control group, the tumor inhibition rates in the nude mice transplanted with SW480 cells of the pEGFP-MST1 and pEGFP-MST1 + 5-FU groups were 48.52±1.63 and 87.28±2.58%, respectively, and the positive rates of phospho-YAP1 (Ser127) protein in nuclei increased significantly (P<0.01). Overall, exogenous MST1 effectively inhibited the proliferation and growth of transplanted human colorectal cancer cells and promoted cancer cell apoptosis. The mechanism involved may be associated with the increase of intracellular phospho-YAP1 (Ser127) protein.

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.

Oncogenic retinoic acid receptor α promotes human colorectal cancer growth through simultaneously regulating p21 transcription and GSK3β/β-catenin signaling

Retinoic acid receptor α (RARα) plays important roles in the progression of several cancers such as leukemia, breast cancer, and lung cancer. In this study, we demonstrated that RARα protein was frequently overexpressed in human CRC specimens and CRC cell lines. RARα knockdown decreased cell survival, proliferation, and colony formation in vitro and tumorigenic potential in nude mice. Specifically, RARα knockdown inhibited cell cycle progression at G1 phase through upregulation of cell cycle inhibitor p21, and downregulation of cyclinD1. Furthermore, RARα was directly recruited to the p21 promoter to inhibit the expression of p21. Simultaneously, RARα contributed to the progression of CRC cells in part due to upregulation of cyclinD1 via activation of GSK3β/β-catenin pathway. Molecular mechanism studies revealed RARα interacted with GSK3β and led to decreased expression of GSK3β at ser9, followed by increased β-catenin expression. Taken together, our results signified the importance of RARα in CRC and demonstrated that RARα promotes CRC progression through suppressing p21 transcription and enhancing GSK3β/β-catenin signaling. RARα might become a potential molecular target for the treatment of CRC.