Colorectal Cancer Cell Lines SW480 Research Articles

Increasing miR-150 and lowering HMGA2 inhibit proliferation and cycle progression of colon cancer in SW480 cells.

High mobility group protein A2 (HMGA2) is a kind of oncogene that regulates cell proliferation and cycle. HMGA2 up-regulation is related to the occurrence of multiple tumors including colorectal cancer. MiR-150 is found down-regulated in colorectal cancer tissue. Bioinformatics analysis shows the complementary targeted relationship between miR-150 and the 3'-UTR of HMGA2. This study explores the role of microRNA-150 (miR-150) in regulating HMGA2 expression, colorectal cancer cell proliferation, and cycle. Colorectal cancer patients were enrolled to collect cancer and para-carcinoma tissues. MiR-150 and HMGA2 expressions were tested in tissue. MiR-150, HMGA2, and Cyclin A levels in colorectal cancer cell line SW480, and normal colorectal epithelial cell line FHC were compared. The targeted relationship between miR-150 and the 3'-UTR of HMGA2 was evaluated by dual luciferase reporter gene assay. SW480 cells were divided into five groups, including miR-control, miR-150 mimic, small interfere normal control (si-NC), si-HMGA2, and miR-150 mimic + si-HMGA2. Cell cycle was determined by using flow cytometry. The cell proliferation was detected by using the cell counting kit 8 (CCK-8) test. HMGA2 expression was significantly increased, while miR-150 levels were significantly declined in colorectal cancer tissue compared with that in para-carcinoma tissue (p<0.05). HMGA2 and Cyclin A levels were higher significantly, whereas miR-150 expression was lower significantly in SW480 cells compared to that in FHC cells (p<0.05). MiR-150 targeted band to the 3'-UTR of HMGA. MiR-150 mimic and/or si-HMGA2 significantly reduced HMGA2 and Cyclin A expressions, blocked cell cycle in the G0/G1 phase, and attenuated cell proliferation. We observed that miR-150 down-regulated Cyclin A expression to block colorectal cancer cell cycle and inhibit proliferation through targeted inhibiting HMGA2.

Knockdown of PRL-3 increases mitochondrial superoxide anion production through transcriptional regulation of RAP1.

BackgroundPhosphatase of regenerating liver-3 (PRL-3) has been shown to be highly expressed in various types of cancers and is related to poor prognosis. Our previous study showed that silencing of PRL-3 leads to increased reactive oxygen species (ROS). However, the mechanism of PRL-3 regulating ROS is not clear.Materials and methodsPRL-3 or Repressor activator protein 1 (RAP1) was knockdown in human colorectal cancer cell lines HCT116 and SW480. The mRNA level was measured by quantitative real-time (qRT)-PCR and the protein level was measured by western blot. ROS was detected by specific oxidationsensitive fluorescent probes. Cell cycle was analyzed through flow cytometry. Luciferase assay and chromatin immunoprecipitation (ChIP) were performed to investigate the regulation of RAP1 by PRL-3. Gene expression correlation was analyzed through an interactive web server. Statistical analysis was performed with SPSS software.ResultsKnockdown of PRL-3 significantly increases mitochondrial superoxide anion, mitochondria membrane potential, and induces cell cycle arrest. Decreased PRL-3-induced mitochondrial superoxide anion accumulation is related to the downregulation of RAP1, which could also affect the level of mitochondria superoxide anion. PRL-3 regulates the expression of RAP1 through binding to the promoter of rap1 gene. PRL-3 could regulate the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) through the mediation of RAP1. Both PRL-3 and RAP1 could regulate the expression of manganese superoxide dismutase 2 (SOD2) and the uncoupling protein 2 (UCP2), which may be related to PRL-3 suppression induced mitochondria superoxide anion.ConclusionOur study presents the first evidence that PRL-3 is involved in the regulation of mitochondria superoxide anion as a transcriptional factor.

The anti-metastatic effect of ginsenoside Rb2 in colorectal cancer in an EGFR/SOX2-dependent manner.

Ginsenoside Rb2, a saponin from Panax ginseng, has been shown to have many functions. However, the effect of ginsenoside Rb2 on the metastasis of colorectal cancer (CRC) remains unknown. CRC cell lines HT29 and SW620 were used to determine the effects of ginsenoside Rb2 on the colony‐forming, migration, invasion, and wound‐healing abilities of CRC cells in vitro. Further, ginsenoside Rb2 was given intraperitoneally at 5 mg/kg of mouse body weight to check its effect on the metastasis of CRC cells in vivo. Ginsenoside Rb2 decreased colony‐forming ability, migration, invasion, and wound healing of CRC cells in vitro, although it did not affect cell proliferation. As a possible mechanism, we found that ginsenoside Rb2 down‐regulated the expression of stemness and Epithelial–mesenchymal transition (EMT)‐related genes via the EGFR/SOX2 signaling axis; these were partially rescued by either exogenous EGF treatment or ectopic expression of SOX2. More importantly, ginsenoside Rb2 significantly reduced the number of metastatic nodules in the livers, lungs, and kidneys in a mouse model of metastasis. These results suggest that ginsenoside Rb2 could be used to treat the metastasis of CRC therapeutically or as a supplement.

CCL19 suppresses angiogenesis through promoting miR-206 and inhibiting Met/ERK/Elk-1/HIF-1\u03b1/VEGF-A pathway in colorectal cancer

The mechanisms underlying the role of chemokines in tumor angiogenesis is still not fully understood. In this study, we detected the influence of CCL19 on colorectal cancer (CRC) angiogenesis. The expression of CCL19 and CD31 in CRC tissues were detected by immunohistochemistry. Human CRC cell lines SW1116 and SW620 stably transfected with CCL19 lentivirus and CCL19 shRNA, and HUVEC stably transfected with CCR7 shRNA were used in our study. Our study showed that CCL19 was significantly low-expressed in CRC tissues and positively related to highly tumor microvessel density. In vitro, we observed that CCL19 high-expressed SW1116 supernatant was able to inhibit proliferation, migration, and sprouting responses of HUVEC, whereas CCL19 low-expressed SW620 supernatant can promote HUVEC angiogenesis. Additionally, we further demonstrated that these functions maybe achieved through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in a CCR7-dependent manner. Mice angiogenesis model also confirmed that elevated expression of CCL19 inhibit the angiogenesis of CRC in vivo. In summary, our results supported that CCL19 can inhibit CRC angiogenesis through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway. This may be a novel therapeutic option for anti-vascular treatment in CRC.

4′-hydroxywogonin inhibits colorectal cancer angiogenesis by disrupting PI3K/AKT signaling

Angiogenesis is fundamental for solid tumor growth and metastasis, and anti-angiogenic therapy has been an important therapeutic option for cancer treatment. Colorectal cancer (CRC) represents the fourth leading cause of cancer-related death worldwide. The current studies were aimed at investigating the anti-angiogenic effects of the natural compound 4′-hydroxywogonin (4′-HW) on CRC-related angiogenesis. Human CRC cell line SW620 cells and normal human intestinal epithelial HIEC cells were cultured and treated with interleukin-6 to mimic the tumor inflammatory microenvironment. Our data showed that 4′-HW reduced the viability of SW620 cells in a concentration- and time-dependent manner. 4′-HW also suppressed the proliferation of SW620 cells, but had little effect on the viability of HIEC cells. Moreover, 4′-HW concentration-dependently decreased the mRNA and protein expression of vascular endothelial growth factor-A (VEGF-A), the predominant pro-angiogenic cytokine in tumor angiogenesis. Subsequently, 4′-HW concentration-dependently inhibited the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT. PI3K inhibitor wortmannin, similar to 4′-HW, significantly downregulated the VEGF-A expression in SW620 cells, and combination of wortmannin and 4′-HW produced more significant effects. Finally, human umbilical vein endothelial cells (HUVECs) incubated with the conditioned medium of 4′-HW-treated SW620 cells exhibited impaired angiogenic capacity at Matrigel. Incubation with the neutralizing antibody against VEGF-Aalone also suppressed the angiogenic properties of HUVECs in vitro. Collectively, 4′-HW decreased the viability and reduced angiogenesis in CRC, which was associated with downregulation of VEGF-A expression by disrupting the PI3K/AKT pathway. Our discoveries suggested 4′-HW as a promising anticancer agent against CRC targeting angiogenesis.

Extracellular Vesicles Derived from Hypoxic Colorectal Cancer Cells Confer Metastatic Phenotype to Non-metastatic Cancer Cells.

Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and stemness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naïve cancer cells.

Blue light emitting diodes irradiation causes cell death in colorectal cancer by inducing ROS production and DNA damage.

The light emitting diodes (LEDs) irradiation has been demonstrated to be potential therapeutic strategies for several diseases. However, the blue LED effects remain largely unknown in colorectal cancer (CRC), which is a major cause of morbidity and mortality throughout the world. In this study, we determined the effects of blue LED irradiation, the maximal light emission at 470 nm in wavelength, in human CRC cell lines SW620 and HT29. The cells were irradiated with blue LED light for 0 J/cm2, 72 J/cm2, 144 J/cm2, 216 J/cm2 and 288 J/cm2 respectively. We found that irradiation with blue LED light induced a marked decrease of live cells and an increase of dead cells. Additionally, lower cell proliferation and a remarkably increase of cell apoptosis were observed in blue LED-irradiated cells as compared with non-irradiated control group. The cell migration was significantly inhibited by blue LED irradiation 24, 48 and 72 h later compared with non-treated group. Blue LED-treated CRC cells further displayed a remarkably inhibition of EMT process in CRC cells. Finally, we found the accumulation of ROS production and DNA damage were induced by blue LED irradiation. These results indicated that blue LED irradiation inhibits CRC cell proliferation, migration and EMT process as well as induces cell apoptosis, which may result from increased ROS accumulation and induction of DNA damage.

Effects of saponins of patrinia villosa against invasion and metastasis in colorectal cancer cell through NF-κB signaling pathway and EMT

BackgroundResearch has indicated that Herba Patriniae can suppress the growth of Several kinds of tumor cells in vitro and in vivo, thus displaying favorable antitumor activity. However, research regarding the effect of saponins of Patrinia villosa against CRC cell has not been reported. In the current study, We have revealed that the effects of saponins of patrinia villosa on colorectal cancer (CRC) cell invasion and epithelial-mesenchymal transition (EMT) as well as its underlying mechanism. MethodsThe CRC EMT model was induced through repeated TGF-β1 stimulations on human CRC cell line SW480. Effects of saponins of patrinia villosa at various concentrations on CRC SW480 cell and EMT model cell proliferation were detected using MTT method, so as to select the optimal action concentration. Meanwhile, effects on SW480 cell and EMT model cell invasion were determined through Scratch assay and Transwell assay. Moreover, changes in expression of EMT-related proteins E-cadherin, N-cadherin and NF-ΚBp65 in each group were detected through Western blotting. ResultsSaponins of patrinia villosa at various concentrations could markedly inhibit the proliferation rate of CRC cell in an obvious concentration-dependent manner. Meanwhile, saponins of patrinia villosa at various concentrations could also remarkably suppress migration of cell developing EMT. In addition, the protein expression of E-cadherin and N-cadherin was down-regulated with the increase in saponins of patrinia villosa concentration, while that of NF-KBp65 was notably down-regulated. ConclusionSaponins of patrinia villosa can act against tumor invasion and metastasis through inhibiting EMT in human CRC cell line, which may be achieved through down-regulating the NF-κB signaling pathway.

Nicotine downregulates microRNA-200c to promote metastasis and the epithelial-mesenchymal transition in human colorectal cancer cells.

Cigarette smoking is the most well-established risk factor for colorectal cancer (CRC). However, the mechanisms of smoking-associated colorectal carcinogenesis are poorly understood. The effects of prediagnosis tobacco use on clinical characteristics, overall survival (OS), and recurrence-free survival (RFS) were analyzed in 396 patients with CRC. Associations between smoking status and OS and RFS were evaluated using Cox's proportional hazards regression. Furthermore, the effects of nicotine on the CRC cell lines SW620 and HT-29 were evaluated using in vitro assays. "Ever smoking" was associated with elevated serum carcinoembryonic antigen, American Joint Committee on Cancer T category, metastasis, and poorer OS and RFS in patients with CRC (OS: hazard ratio [HR] = 1.74, 95% confidence interval [CI], 1.07-2.81, p = 0.025; RFS: HR = 1.66, 95% CI: 1.18-2.34, p = 0.004). MicroRNA (miR)-200c was downregulated in CRC and tumor-adjacent tissues from ever smokers compared with the corresponding tissues from never smokers with CRC. Nicotine inhibited miR-200c expression in a dose- and time-dependent manner in SW620 and HT-29 CRC cell lines. Nicotine induced cell proliferation, migration, and invasion and promoted the epithelial-mesenchymal transition in SW620 and HT-29 cells, and these effects were attenuated by overexpression of miR-200c. Our findings support the adverse effects of prediagnosis cigarette smoking on prognosis and clinical behavior in CRC. We demonstrate a novel oncogenic mechanism by which nicotine promotes growth and metastasis in CRC by downregulating miR-200c.

The regulatory effects of metformin on the [SNAIL/miR-34]:[ZEB/miR-200] system in the epithelial-mesenchymal transition(EMT) for colorectal cancer(CRC)

The epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression, metastasis and drug resistance. The transcription factor(TF) and microRNA (miR) chimeric [SNAIL/miR-34]:[ZEB/miR-200] unit is the core regulatory system for the EMT process. Here, we proposed to assess the anti-EMT abilities and explore the inherent pharmacological mechanisms of the classic hypoglycaemic agent metformin for colorectal cancer(CRC).For the EMT model, the TGF-β-induced CRC cell lines SW480 and HCT116 were treated with metformin. The viability, migration and invasion abilities of the cells were evaluated with the Cell Counting Kit-8, wound-healing and trans-well assay. The alterations of the [SNAIL/miR-34]:[ZEB/miR-200] system and the EMT markers E-cadherin and vimentin were detected by western blot, qPCR and immunofluorescent staining.Metformin exhibited inhibitory effects on the proliferation, migration and invasion of the CRC SW480 cells. The up-regulation of E-cadherin and the down-regulation of vimentin for both SW480 and HCT116 cells revealed the anti-EMT abilities of metformin. For the [SNAIL/miR-34]:[ZEB/miR-200] system, metformin increased miR-200a, miR-200c and miR-429 levels and decreased miR-34a, SNAIL1 and ZEB1 levels in the TGF-β-induced EMT. From immunofluorescence, we observed increased E-cadherin and ZEB1 co-expression in metformin-treated cells.Metformin may perform bidirectional regulations of the [SNAIL/miR-34]:[ZEB/miR-200] system in the EMT process for colorectal cancer. Such regulation is expressed as the inhibition of EMT in general as well as an increased higher proportion of E/M hybrid cells in the total population.

Abstract 1867: Combined and sequential chemotherapy efficacy of trifluridine/tipiracil and regorafenib in colorectal cancer cell lines

Abstract Background: Trifluridine/tipiracil (FTD/TPI or TFTD, also known as TAS-102) is recommended for treating metastatic colorectal cancers refractory to standard chemotherapies. Regorafenib is an oral multikinase inhibitor that blocks the activity of several protein kinases associated with angiogenesis, oncogenesis, and the tumor microenvironment. We evaluated the antitumor effects of FTD/TPI combined with regorafenib on a colorectal tumor xenograft mouse model. We also investigated whether cytotoxicity was enhanced when FTD was used sequentially with regorafenib in vitro. Method: The colorectal cancer cell lines SW620 and COLO 205 were implanted into nude mice subcutaneously. FTD/TPI (150 mg/kg/day) was orally administered twice daily from days 1 to 14 and regorafenib (10 mg/kg/day) was administered once a day at approximately 2-4 h after FTD/TPI. For the in vitro cytotoxic assay, SW620 cells were treated with FTD and regorafenib as follows: (1) exposure to 0.1-4.0 µM FTD alone, (2) sequential exposure to 0.1-4.0 µM FTD for 24 h followed by 10 µM regorafenib for 24 h, or (3) sequential exposure to 10 µM regorafenib for 24 h followed by 0.1-4.0 µM FTD for 24 h. All treatments were evaluated by a colony formation assay. Molecules involved in mechanism of action of FTD and regorafenib and in apoptosis were evaluated by western blot. Results: Tumor growth inhibition by combination therapy was 86.4% and 92.3% for SW620 and COLO 205 xenografts, respectively; this was significantly superior to the growth inhibition observed after either monotherapy for the two evaluated cancer xenografts (P &amp;lt; 0.01). Thus, FTD/TPI combined with regorafenib is significantly more effective than either monotherapy in colorectal cancer xenografts. This combination therapy was tolerated without 20% body-weight reduction and drug-related death. FTD followed by regorafenib treatment in SW620 cells showed stronger cell killing effect than FTD alone. In contrast, regorafenib followed by FTD treatment attenuated its cell killing effect rather than that to FTD alone. In comparison with FTD alone, FTD followed by regorafenib reduced thymidine synthase (TS) and inhibited phosphorylation of ERK1/2 (pERK1/2) with the induction of proapoptotic marker, cleavage of PARP. In contrast, regorafenib followed by FTD induced TS and pERK1/2. Thus, FTD followed by regorafenib treatment is more effective than regorafenib followed by FTD treatment in vitro. Conclusion: FTD/TPI combined with regorafenib is significantly more effective than either monotherapy in preclinical models. Moreover, FTD followed by regorafenib is more effective than vice versa in vitro, suggesting that in patients sequential therapy with FTD/TPI prior to regorafenib may be helpful in treating colorectal cancer. Citation Format: Kazuaki Matsuoka, Fumio Nakagawa, Junji Uchida, Teiji Takechi. Combined and sequential chemotherapy efficacy of trifluridine/tipiracil and regorafenib in colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1867.

Pea lectin inhibits cell growth by inducing apoptosis in SW480 and SW48 cell lines

Globally, colorectal cancer is the third most common type of malignant tumor, after lung and breast. Here anticancer property of pea lectin was evaluated against colorectal cancer cell lines SW480 and SW48. The cells were treated with different doses of lectin for 3 days in vitro and the inhibitory effects were found in a dose dependent manner. At the high dose(1.0 mg/ml) 62% and 63% cell growth inhibitions were observed for SW48 and SW480 cell lines, respectively. Cell growth inhibition was further studied by colony formation of the cell lines and the numbers of colonies in SW480+pea lectin and SW48+pea lectin cells were noted significantly lower in comparison to that of control cells. Cell morphological study revealed that pea lectin induced apoptosis both in SW48 and SW480 cell lines, which was further confirmed by caspase inhibitors. Involvement of intrinsic mitochondrial pathway in the apoptosis progression was confirmed by caspase inhibitors and increased of caspase-3 & -9 proteins expressions. Expression levels of p53 and p21 protein were also increased significantly in both cell lines with the decreased of PARP1 protein expression. G2/M and G0/G1 cell cycle arrested was noted in SW48 and SW480 cell lines, respectively, after treatment with pea lectin.

Manganese-Enhanced Magnetic Resonance Imaging for Detection and Characterization of Colorectal Cancers.

Here, we investigated the diagnostic performance of manganese (Mn)-enhanced magnetic resonance imaging (MEMRI) in colorectal cancer (CRC). The ability of CRC cell lines SW620 and SW480 to uptake Mn was evaluated and compared with a normal colon cell using MEMRI. Subcutaneous xenografts in nude mice underwent MRI examination at tumor sizes of 5, 10, and 15 mm. Contrast enhancement was compared between gadolinium (Gd)- and Mn-enhanced MRI. SW620 and SW480 cell lines took up more Mn2+ than normal cells, resulting in 4.5 and 2 times greater T1 value shortening than normal cell using in vitro MEMRI (P < .001). Most xenografts (17/23) enhanced markedly on MEMRI. A heterogeneous enhancement pattern invariably noted whether Mn or Gd agents were administered, but tumors imaged using MEMRI showed a greater degree of enhancement with a larger extent of enhanced area than those imaged using Gd-enhanced MRI. The numbers of markedly Mn-enhanced cases were more in the 5-mm-size tumor group than in 10- or 15-mm-size tumor groups. Overall, MEMRI could enhance CRCs and it showed potential in detecting early small lesions and markedly enhancing tumors that had minimal Gd enhancement.

A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities via Inhibition of NF-κB Signaling in KRAS-Mutant Colon Cancer Cells.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence "MIRTX." MIRTX directly targeted the 3'-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977-87. ©2018 AACR.

Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture.

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air–liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.

The kallikrein-Kinin system modulates the progression of colorectal liver metastases in a mouse model

BackgroundThe Kallikrein-Kinin System (KKS) has been found to play a role in tumor progression in several cancers. The KKS metabolic cascade depends on signalling through two cross talking receptors; bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R). Activation of the Kinin receptor is responsible for multiple pathophysiologic functions including increase of vascular permeability and induction of host inflammatory responses that exert diverse effects on tumor growth.MethodsB1R and B2R expression on mouse and human CRC cell lines was investigated. Changes in tumor growth and progression was assessed in male CBA mice bearing colorectal liver metastases (CRLM) following treatment with B1R or B2R blockers. In vitro cultures of human SW-480 and mouse colorectal cancer (MoCR) cell lines were examined for changes in their proliferation and migration properties following treatment with B1R or B2R blockers.ResultsBoth colorectal cancer cell lines tested strongly positive for B1R and B2R expression. Inhibition of both receptors retarded tumor growth but only B1R blockade significantly reduced tumor load and increased tumor apoptosis. Blockade of either receptor reduced tumor vascularization in vivo and significantly inhibited proliferation and migration of colorectal cancer cells in vitro.ConclusionTaken together, the present study demonstrated that kinin receptor blockade inhibited tumor growth and reduced its invading properties suggesting that KKS manipulation could be a novel target in colorectal cancer therapy.

Effects of secreted frizzled-related protein 1 on proliferation, migration, invasion, and apoptosis of colorectal cancer cells

BackgroundSecreted frizzled-related protein 1 (SFRP1) is a member of the SFRPs family that modulates the Wnt signal transduction pathway. Recent studies have showed down-regulation of SFRP1 expression in colorectal cancer (CRC). We aimed to evaluate the effect of SFRP1 on the proliferation, migration, invasion and apoptosis of CRC cells in vitro.Materials and methodsWe used real-time fluorescence quantification (RT-PCR) and Western blotting to detect SFRP1 expression in CRC, pericarcinomatous tissues and CRC cell lines. We assessed the influence of overexpression and knockdown of SFRP1 on CRC cell proliferation, migration, invasion, and apoptosis, Western blotting was used to evaluate protein levels of Wnt, β-catenin, and apoptosis-related proteins.ResultsThe expression of SFRP1 was significantly decreased in CRC tissues. Among the six CRC cell lines (sw-480, sw1116, caco-2, ht-29, colo-205, and hct-116), RT-PCR revealed that sw1116 cells had the lowest expression of SFRP1, while caco-2 cells had the highest SFRP1 expression. SFRP1 overexpression in sw1116 cells significantly suppressed cell proliferation while SFRP1 knockdown in caco-2 cells significantly increase the cell proliferation. In addition, overexpression of SFRP1 in sw1116 cells remarkedly suppressed cell migration and invasion, whereas knockdown of SFRP1 in caco-2 cells resulted in significant enhancement of migration and invasion. Furthermore, SFRP1 overexpression in sw1116 cells promoted cell apoptosis. Western blotting showed that SFRP1 overexpression significantly decreased the protein levels of Wnt, β-catenin and apoptosis-related proteins, including MMP2, MMP9, Twist, CDK1, TGF, and Bcl2.ConclusionOur results demonstrate that SFRP1 suppresses cell proliferation, migration and invasion, and promotes apoptosis in CRC cells.

Long noncoding RNA RP4 functions as a competing endogenous RNA through miR-7-5p sponge activity in colorectal cancer

AIMTo investigate the role of long noncoding RNA (lncRNA) RP4 in colorectal cancer.METHODSLentivirus-mediated lncRNA RP4 overexpression and knockdown were performed in the colorectal cancer cell line SW480. Cell proliferation, tumor growth, and early apoptosis were evaluated by a cell counting kit-8 assay, an in vivo xenograft tumor model, and annexin V/propidium iodide staining, respectively. Analysis of the lncRNA RP4 mechanism involved assessment of the association of its expression with miR-7-5p and the SH3GLB1 gene. Western blot analysis was also performed to assess the effect of lncRNA RP4 on the autophagy-mediated cell death pathway and phosphatidylinositol-3-kinase (PI3K)/Akt signaling.RESULTSCell proliferation, tumor growth, and early apoptosis in SW480 cells were negatively regulated by lncRNA RP4. Functional experiments indicated that lncRNA RP4 directly upregulated SH3GLB1 expression by acting as a competing endogenous RNA (ceRNA) for miR-7-5p. This interaction led to activation of the autophagy-mediated cell death pathway and de-repression of PI3K and Akt phosphorylation in colorectal cancer cells in vivo.CONCLUSIONOur results demonstrated that lncRNA RP4 is a ceRNA that plays an important role in the pathogenesis of colorectal cancer, and could be a potential therapeutic target for colorectal cancer treatment.

MiR-29b reverses oxaliplatin-resistance in colorectal cancer by targeting SIRT1.

Oxaliplatin is a commonly used chemotherapeutic drug for the treatment of advanced colorectal cancer. However, acquired drug resistance against oxaliplatin remains a major obstacle for efficient use of it, and mechanisms underlying oxaliplatin resistance are still required to be explored. In the present study, we exposed colorectal cancer cell line SW480 to oxaliplatin for a long time to obtain oxaliplatin-resistant colorectal cancer cell model (OR-SW480). We found that intracellular expression of miR-29b was decreased when the SW480 cells became oxaliplatin-resistant. More importantly, overexpression of miR-29b resensitized OR-SW480 cells to oxaliplatin treatment. Mechanically, gene of SIRT1 was identified to be the target of miR-29b. Overexpression of miR-29b in oxaliplatin-treated OR-SW480 decreased the expression of SIRT1 to enhance the ROS production and JNK phosphorylation, and thus promoting apoptosis via activation of caspase 9, 7 and 3. On the other hand, expression plasmid of SIRT1, N-acetyl cysteine or SP600125 (JNK specific inhibitor) abolished the effect of miR-29b on oxaliplatin-treated OR-SW480. We therefore demonstrated that miR-29b reverses oxaliplatin-resistance in colorectal cancer by targeting SIRT1/ROS/JNK pathway.

Proteome Analysis of Colorectal Cancer Cell Line SW480 Released Extracellular Vesicles

The detection and profiling of disease-specific extracellular vesicles (EVs) from body fluids has been challenging research area during recent years. However, the question – can EVs surface proteins be exploited as a credible tool for early cancer diagnosis – is still not answered. Objective of the current study was to find out whether hypoxia induces differences in protein profiles of EVs released from hypoxic human colorectal cancer cells SW480 (EVHyp) and EVs released from these cells grown in normoxic conditions – EVNorm. Obtained results show differences in EVs surface protein samples. Some protein fragments were found only or mostly in EVHyp surface protein samples. Finding of one or two such EVHyp protein fragments allows us to suggest that deciphered EVHyp surface proteins might be indices of hypoxia-induced proteome changes and might serve as a hint to find a cancer specific protein.