Migration Of SW480 Cells Research Articles

Antitumor Effects of Tryptanthrin on Colorectal Cancer by Regulating the Mitogen-Activated Protein Kinase Signaling Pathway and Targeting Topo I and IDO1.

Tryptanthrin (TRYP) is an indole quinazoline alkaloid with a range of pharmaceutical activities, but the specific mechanism of TRYP against colorectal cancer (CRC) remains obscure. The purpose of this study was to evaluate the antitumor effects of TRYP on CRC models both in vitro and in vivo and further analyze its concrete mechanisms. The results of the in vitro experiment show that TRYP effectively inhibited the proliferation and migration of SW620 cells, arrested the cell cycle at the S phase, and induced cell apoptosis. Deeply, TRYP dramatically increased the expression of Bax and cleaved caspase 3 while decreasing the expression of Bcl-2. The results of transcriptome sequencing implied that the inhibitory effects of TRYP were closely related to the mitogen-activated protein kinase (MAPK) signaling pathway, and the results of western blotting verified that TRYP could decrease the expression of p-Erk and increase the expression of p-p38 and p-Jnk. Besides, our results identified that topoisomerase I (Topo I) and indole amine 2,3-dioxygenase 1 (IDO1) were the targets of TRYP. In vivo, the results showed that different TRYP doses significantly inhibited tumor growth in mice, induced different degrees of necrosis in tumor tissues, decreased the expression level of Ki67 protein, and increased the apoptotic signal in tumor tissues. The findings demonstrated the inhibitory effects of TRYP on CRC, and the mechanisms were tightly connected to inhibiting the activity of Topo I and IDO1 and regulating the expression of the MAPK signaling pathway. Especially, it was first identified that TRYP could directly inhibit Topo I to arrest SW620 at the S phase. Therefore, this work established a scientific basis for the development of TRYP.

Multi-omics analysis of the biological function of the VEGF family in colon adenocarcinoma

The vascular endothelial growth factor (VEGF) family plays a crucial role in cancer progression, but the prognostic significance and biological functions of VEGF family members in colon adenocarcinoma (COAD) remain unclear. Using data from The Cancer Genome Atlas, Gene Expression Omnibus, Gene Set Cancer Analysis, cBioPortal, GeneMANIA, String, MethSurv and starBase database, we identified vascular endothelial growth factor B (VEGFB) as a key gene associated with COAD prognosis, with its abnormal expression linked to methylation dysregulation. In vitro experiments confirmed VEGFB expression was significantly higher in colon cancer tissues compared to normal tissues, as shown by Real-time quantitative PCR and immunohistochemistry. Cell Counting Kit-8 and colony formation assay showed that decreased VEGFB expression in SW480 cells resulted in decreased cell viability and proliferation ability. Scratch assay showed that VEGFB downregulation impaired SW480 cell migration. In addition, our research suggests that VEGFB not only promotes angiogenesis but is also involved in the tumor microenvironment and immune regulation. The SHNG17-miR-375-VEGFB regulatory axis provides a potential therapeutic target for COAD, highlighting VEGFB’s role in immune activation during anti-angiogenic therapy and potential reversal of drug resistance.

Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

Resveratrol can Reduce the Aggressiveness of Hypoxic Colon Cancer Cells

Colorectal cancer is a significant global health problem characterized by the development of metastasis due to fast cell growth, tolerance to low oxygen levels, and the formation of new blood vessels. Notable advancements have been seen in the management of these instances; however, uncertainties persist about drug resistance and its accompanying adverse effects. Resveratrol, a natural polyphenol derived from several plants, has diverse pharmacological characteristics. The anticancer impact of this has piqued the curiosity of several researchers. The objective of our research was to investigate the impact of resveratrol on the proliferation, migration, and expression of angiogenic factors in hypoxic colorectal cancer cells by the use of resveratrol. Hypoxia was chemically induced using cobalt chloride. Serially diluted concentrations of resveratrol (200, 100, 50, 25, 12.5, and 6.25 \(\mu\)g/ml) were employed to assess the cytotoxic effect through the MTT assay. Smaller concentrations (below IC50) were utilized to investigate the impact of resveratrol on the migration of SW480 cells using the wound healing assay (50 \(\mu\)g/ml). The impact of resveratrol on the expression of vascular endothelial growth factor (VEGF) was assessed using the enzyme-linked immunosorbent assay (ELISA). The findings shown that resveratrol has the ability to effectively decrease cell proliferation in a dose-dependent way, inhibit cell migration and angiogenesis, via suppression of VEGF and HIF-1\(\alpha\). The significance of this etude lies in the enduring inability to effectively manage metastatic colorectal cancer. Suggesting that resveratrol might function as an adjunctive therapy and a useful supplement for those suffering from very metastatic colorectal cancer. The specific method by which resveratrol works is yet unknown, hence more study is needed to conduct experiments in living organisms and clinical trials.

Anti-Cancer Study of Carvacrol in Hypoxic-Induced Colorectal Cancer Cell

Introduction: Colorectal cancer presents a complex global health challenge, characterized by complications arising from metastasis development due to rapid proliferation, adaptation to hypoxia, and angiogenesis. Carvacrol, a natural monoterpenoid phenol derived from various plants, has interest for its diverse pharmacological properties, particularly its antitumor effects. Methods: We aimed to assess the inhibitory effect of carvacrol on cell migration and proliferation in the hypoxic colorectal cancer cell line (SW480). Chemical induction of hypoxia using cobalt chloride and serially diluted concentrations of carvacrol (400, 200, 100, 50, 25, and 12.5 µg/ml) were employed to evaluate the cytotoxic effect via the MTT assay. Smaller concentrations (low IC50) were used to examine the impact of carvacrol on SW480 cell migration through a cell migration assay (50, 25, 12.5, and 6.25 µg/ml). Results: The results demonstrated a significant, dose-dependent reduction in both cell proliferation and migration with carvacrol doses. Conclusion: The findings suggest that carvacrol could be useful as an adjuvant therapy and a promising addition for patients with highly metastatic colorectal cancer.

Hedyotis diffusa–Sculellaria barbata (HD–SB) suppresses the progression of colorectal cancer cells via the hsa_circ_0039933/hsa-miR-204-5p/wnt11 axis

Our previous study confirmed that the combination of Hedyotis diffusa (HD) and Scutellaria barbata (SB) significantly inhibited colorectal cancer cell proliferation and the WNT signaling pathway. However, the exact molecular modulation remains unclear. In this study, colorectal cancer cells (SW620) were treated with 1 mg/mL HD–SB for 24 h, and high-throughput sequencing of circRNAs was performed. The level of hsa_circ_0039933 in three colorectal cancer cell lines (HT-29, SW620, and HCT116) was verified by qPCR. After transfection of hsa_circ_0039933 overexpression plasmids or small interfering RNAs, CCK8, apoptosis, cell migration, and cell invasion were utilized to evaluate the function of hsa_circ_0039933 in the progression of colorectal cancer cells. We identified hsa_circ_0039933, which was downregulated in HD–SB-induced colorectal cancer cells and positively related to colorectal cancer progression. In SW620 cells with relatively high expression of hsa_circ_0039933, interfering with the expression of hsa_circ_0039933 inhibited the proliferation, invasion, and migration of SW620 cells. In HCT116 cells with relatively low expression of hsa_circ_0039933, overexpression of hsa_circ_0039933 promoted the proliferation and invasion and migration ability of HCT116. Mechanistically, hsa_circ_0039933 targeted hsa-miR-204-5p to increase the expression of wnt11, leading to the activation of the Wnt pathway, thereby promoting the proliferation of colorectal cancer cells. This work revealed the potential molecular mechanism of HD–SB for the treatment of colorectal cancer, which was to inhibit the Wnt signaling pathway through the hsa_circ_0039933/hsa-miR-204-5p/wnt11 axis, then suppressing proliferation, migration, and invasion in the colorectal cancer cell.

Hypoxia Increases ATX Expression by Histone Crotonylation in a HIF-2α-Dependent Manner.

Autotaxin (ATX), the key enzyme that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC), is involved in tumorigenesis through the ATX-LPA axis and is regarded as a valuable target in tumor therapy. Hypoxia is a major feature of solid tumors and contributes to tumor development with striking alterations in the gene expression profile. Here, we show that hypoxia induces ATX expression in a hypoxia-inducible factor (HIF) 2α-dependent fashion in human colon cancer SW480 cells. HIF-2α is directly bound to specific hypoxia response elements (HREs) in the ATX promoter. Under hypoxic conditions, knockout or inhibition of ATX suppressed the migration of SW480 cells, which could be rescued by the addition of LPA, suggesting that the induction of ATX during hypoxia promotes cancer cell migration through the ATX-LPA axis. Further studies showed that ATX expression was induced by HIF-2α through recruiting p300/CBP, which led to crotonylation but not acetylation of histone H3 in the ATX promoter region during hypoxia. Moreover, elevation of cellular histone crotonylation levels could induce ATX expression under normoxic conditions. In conclusion, our findings reveal that ATX is induced in SW480 cells during hypoxia by histone crotonylation in a HIF-2α-dependent manner, while as a novel mechanism of ATX expression regulation, the upregulation of ATX expression by histone crotonylation is not confined to hypoxia.

Phosphorylation-mediated interaction between human E26 transcription factor 1 and specific protein 1 is required for tumor cell migration.

Transcription factors, human E26 transcription factor 1 (Ets1) and specific protein 1 (Sp1), are known to induce gene expression in tumorigenicity. High Ets1 expression is often associated with colorectal tumorigenesis. In this study, we discover that metastasis and clone formation in SW480 cells mainly depend on the direct interaction between Ets1 and Sp1 instead of high Ets1 expression. The interaction domains are further addressed to be the segment at Sp1(626-708) and the segment at Ets1(244-331). In addition, the phosphorylation inhibition of Ets1 at Tyr283 by either downregulation of Src kinase or Src family inhibitor treatment decreases the interaction between Sp1 and Ets1 and suppresses SW480 migration. Either administration or overexpression of the peptides harboring the interaction segment strongly inhibits the colony formation and migration of SW480 cells. Our findings suggest that the interaction between Ets1 and Sp1 rather than Ets1 alone promotes transformation in SW480 cells and provide new insight into the Ets1 and Sp1 interaction as an antitumour target in SW480 cells.

ABCA12 Promotes Proliferation and Migration and Inhibits Apoptosis of Pancreatic Cancer Cells Through the AKT Signaling Pathway

Background: As a malignant tumor, pancreatic cancer is difficult to detect in its early stage. Pancreatic cancer progresses rapidly and has a short survival time. Most cases have metastasized to distant organs before diagnosis. The mechanism of induction of pancreatic cancer is not fully understood. Methods: In this study, bioinformatics predicted ATP binding cassette subfamily A member 12 (ABCA12) expression in pancreatic tissues and performed survival analysis, risk assessment, and enrichment analysis. The expression of ABCA12 in 30 pairs of clinical samples was detected by immunohistochemistry and we analyzed its correlation with clinical information. Both reverse transcription polymerase chain reaction (RT–PCR) and western blot analysis were used to detect mRNA and protein expression in cell lines. Two different siRNAs and SW1990 cell line were used to construct pancreatic cancer cell models with ABCA12 knockdown. Cell viability was evaluated by cell counting kit-8 (CCK-8) and EdU proliferation assays. Wound healing assays and Transwell assays were used to measure the ability of cell migration and invasion. Flow cytometry was used to investigate the effect of ABCA12 on the proliferation cycle and apoptosis of pancreatic cancer. Western blot analysis detected changes in apoptosis, migration, and other pathway proteins in SW1990 cells after transfection. Results: ABCA12 is highly expressed in pancreatic cancer tissues and cells. After ABCA12 was knocked down, the proliferation, invasion, and migration of SW1990 cells were significantly reduced, and apoptosis was increased. The changes in pathway proteins suggested that ABCA12 may regulate the progression of pancreatic cancer through the AKT pathway. Conclusion: We found that ABCA12 is differentially expressed in pancreatic tissues and cells. ABCA12 can also affect the biological behavior of pancreatic cancer cells effectively, which may serve as a new target for pancreatic cancer diagnosis and treatment.

Gnetum montanum extract induces apoptosis by inhibiting the activation of AKT in SW480 human colon cancer cells

Context Gnetum montanum Markgr. (Gnetaceae) is used to treat rheumatic arthralgia and bruises in the clinic. Objective To exam the activity and mechanism of G. montanum extract (GME) against colon cancer cells SW480. Materials and methods The anti-proliferative activity of GME (0–120 μg/mL) on SW480 cells was determined using MTS assay at 24, 48, and 72 h. The in vitro activity of GME (0–120 μg/mL) on SW480 cells was investigated using flow cytometry and western blotting analysis. The in vivo activity of GME was evaluated using xenograft tumour model of zebrafish and nude mice. The chemical composition of GME was detected by using HPLC–MS/MS. Results The IC50 value SW480 cells viability by GME were 126.50, 78.25, and 50.77 μg/mL, respectively, for 24, 48, and 72 h. The experiments showed that apoptotic cells and G2/M phase cells increased from 20.81 to 61.53% (p < 0.01) and 25.76 to 34.93% with 120 μg/mL GME, respectively. GME also down-regulated the protein expression of P-AKT, P-GSK-3β, P-PDK1, P-c-Raf, caspase-3, and Bcl-2, and up-regulated the expression cleaved caspase-3, cleaved PARP, and Bax. In vivo study found that GME can significantly inhibit the growth and migration of SW480 cells in xenograft zebrafish. GME reduced the nude mice tumour weight to approximately 32.19% at 28 mg/kg/day and to 53.17% (p < 0.01) at 56 mg/kg/day. Forty-two compounds were identified from the GME. Discussion and conclusions GME has a significant antitumor effect on colon cancer cells SW480, and it has the potential to be developed as an anticancer agent.

Danshensu Attenuated Epithelial-Mesenchymal Transformation and Chemoresistance of Colon Cancer Cells Induced by Platelets.

The interactions between platelets and tumor cells are well-known to play important roles in the progression of malignant tumors. Danshensu, a main water-soluble component of Salvia miltiorrhiza, can resist platelet aggregation and exert significant anti-tumor effects on various types of tumors. However, whether Danshensu could inhibit the progression of malignant tumors by suppressing the activities of platelets had not been reported. The effects of Danshensu on the platelet activity and epithelial-mesenchymal transformation (EMT)-like invasive phenotype of SW620 colon cancer cells were assessed by stimulating with the supernatants from co-cultured platelets and SW620 cells with direct contact (SCP). The expression and secretion of proteins were determined by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Hematoxylin and eosin (H&E) staining was performed to analyzed the histopathology of tumor tissues and immunohistochemical staining was conducted to examine the protein expression in tumors. Co-incubation of SW620 cells with platelets directly or SCP both generated long spindle-shaped invasive phenotype. Pretreatment of platelets with Danshensu (25 μM) inhibited the morphological changes of SW620 cells induced by SCP, which was associated with the inhibitory effects of Danshensu on platelet secretion. Danshensu diminished the secretion of a list of biological factors in SCP, including interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-1β and vascular endothelial growth factor (VEGF) that are all involved in tumor cell EMT and chemoresistance. Moreover, Danshensu up-regulated the expression of E-cadherin but down-regulated the levels of N-cadherin and Vimentin, resulting in the repression of SW620 cell migration. It was also shown that Danshensu enhanced the sensitivity of SW620 cells to oxaliplatin by suppressing the expression of MDR1. Furthermore, Danshensu could not only reduced the growth of subcutaneous tumors and liver metastasis that induced by SCP, but also down-regulated the expression of MDR1 in vivo. Mechanistic studies revealed that Danshensu suppressed the activation of the TGF-β/Smad signaling pathway. Danshensu attenuated EMT-like characteristics and chemoresistance by inhibiting secretion capability of platelets and activation of the TGF-β/Smad signaling pathway, suggesting that it may be optimized to be a therapeutic agent for fighting against colon cancer.

The long non-coding RNA T cell leukemia homeobox 1 neighbor enhances signal transducer and activator of transcription 5A phosphorylation to promote colon cancer cell invasion, migration, and metastasis

ABSTRACT Colon cancer is among the most prevalent gastrointestinal tumor types. The long noncoding RNA (lncRNA) T cell leukemia homeobox 1 neighbor (TLX1NB) is up-regulated in colorectal cancer (CRC). However, the functional role of this lncRNA in colon cancer remains unknown. In our study, we investigated the clinical significance of TLX1NB in colon cancer through bioinformatics analysis and explored its role in migration, invasion and metastasis of colon cancer cell with a series of experiments. Firstly, TLX1NB was up-regulated in colon cancer tissues and increased TLX1NB expression was significantly associated with advanced N stages. In wound healing assays and transwell assays, TLX1NB overexpression promoted HCT116 cell migration and invasion while TLX1NB knockdown inhibited SW620 cell migration and invasion. In vivo, TLX1NB knockdown suppressed pulmonary metastasis of SW620 cell and vimentin expression but increased E-cadherin expression. Then, TLX1NB overexpression enhanced signal transducer and activator of transcription 5A (STAT5A) phosphorylation and TLX1NB knockdown suppressed STAT5A phosphorylation. Moreover, the inhibition of STAT5A phosphorylation reversed TLX1NB overexpression-associated increase in HCT116 cell migratory and invasive activity. In conclusion, TLX1NB enhances STAT5A phosphorylation to promote colon cancer cell invasion, migration, and metastasis.

Open Reading Frame-3a gene of the 2019 novel coronavirus inhibits the occurrence and development of colorectal cancer

Intestinal microecology is composed of bacteria, fungi and viruses. As a part of intestinal microecology, viruses participate in the occurrence and development of colorectal cancer. The 2019-nCoV was detected in stool samples from patients during COVID-19, suggesting that the 2019-nCoV may be associated with intestinal microecology. However, the relationship of the 2019-nCoV and CRC is unclear. The aim of this study is to explore the role of Open Reading Frame-3a (ORF3a) of the 2019-nCoV in CRC. After the pCDH-CMV-MCS-EF1-Puro vector that provides high expression of ORF3a was transfected into the SW480 CRC cell line, immunofluorescence was used to determine the localization of ORF3a in SW480 cells. The proliferation, migration, invasion, apoptosis, and cell cycle progression of SW480 cells were measured using the Cell Counting Kit-8 (CCK-8), wound healing, Transwell assay, flow cytometry, the TUNEL assay, and propidium iodide single staining. The results showed that ORF3a inhibited the proliferation, invasion, and migration of SW480 cells and induced their apoptosis after 24, 48, 72 h. Meanwhile, ORF3a inhibited the cell cycle and blocked SW480 CRC cells in the G1 phase. In in vivo experiments, high ORF3a expression was associated with decreased tumor volume, tumor weight, relative tumor volume, and tumor activity. ORF3a inhibited the growth and induced apoptosis and necrosis of tumor tissues. Based on this, we demonstrated that ORF3a might play a role in CRC, providing a new direction for the prevention and treatment of CRC.

Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) decoction suppresses colorectal cancer via downregulation of Wnt5/\u03b2-Catenin signal

BackgroundThe decoction of Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) has been reported as a potential antitumor agent for colorectal cancer (CRC) in experimental and clinical studies, but its underlying mechanism is still unclear.MethodsThe current research aims to explore the potential of Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) decoction (AR decoction) in the treatment of CRC and explore the underlying mechanism. SW620 cells were transient transfection to overexpress or knock down wnt 5 or β-Catenin. Astragalus membranaceus (Huangqi) and Rhizoma curcumae (Ezhu) -containing serum (AR-CS) was used to interfere with SW620 cells. Additional AR-CS, Wnt5 inhibitor (IWP-4), and β-Catenin inhibitor (JW55) were used to intervene in SW620 cells. Furthermore, subcutaneously injection of SW620 cells into the right flank of nude mice replicated xenograft mice, which were treated with AR decoction for 21 days.ResultsAR-CS significantly reduced the mRNA and protein expression levels of Wnt5, β-Catenin, ARF6, and N-Cadherin in SW620 cells, while inhibiting the proliferation and migration of SW620 cells. In cells overexpressing Wnt5 or β-Catenin, these effects of AR-CS were significantly suppressed. On the contrary, the inhibitory effect of AR-CS on the mRNA and protein levels of ARF6 and N-Cadherin and cell proliferation and migration of SW620 was enhanced, when Wnt5 or β-Catenin were knocked down or suppressed by the inhibitors. Moreover, in the mouse model of xenograft tumors, AR decoction not only reduced the tumor volume and inhibited the mRNA levels and protein levels of Wnt5, β-Catenin, ARF6, and N-Cadherin in the tumor, but also inhibit the protein levels of LRP5, LRP6, TCF-4, and LEF1.The histopathology of mice also showed increased apoptosis in tumor tissues, and AR decoction treatment did not cause pathological damage to the kidney and liver.ConclusionsOur results provide evidence that AR decoction inhibits Wnt5/β-catenin signaling and inhibits the development of CRC, which is a promising traditional medicine in the clinical treatment of CRC.

Lysophosphatidylcholine acyltransferase 1 is involved in the regulation of exosome secretion and uptake in colorectal cancer cells

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a phospholipid acyltransferase that promotes phospholipid synthesis and plasma membrane reconstruction. Exosomes play an important role in tumor metastasis. The release and uptake of exosomes are key steps of their functions and depend on plasma membrane fusion and plasma membrane receptors, respectively. The purpose of this study was to explore whether LPCAT1-induced plasma membrane remodeling would change the secretion and uptake behavior of exosomes in tumor cells. We first confirmed the abnormally high expression of LPCAT1 in colorectal cancer cells by quantitative real-time PCR (qPCR) and Western blot analysis. Then, SW620 cells were used as exosome source cells, and SW480 cells were used as exosome receiver cells. Exosomes from SW620 cells could effectively promote the migration of SW480 cells. When LPCAT1 expression was reduced via siRNA knockdown in the source cells, the secretion of exosomes was downregulated, thus weakening the pro-migratory effects of exosomes on target cells. Conversely, when LPCAT1 was knocked down in target cells, the uptake of exosomes in target cells also decreased sharply. These results undoubtedly revealed that LPCAT1 is functionally associated with the release and internalization of exosomes in colorectal cancer cells and could affect the paracrine effects of exosomes, preliminarily extending the classical metabolic function of LPCAT1 to exosome-related pathways.

Lysophosphatidylcholine acyltransferase 1 is involved in the regulation of exosome secretion and uptake in colorectal cancer cells

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a phospholipid acyltransferase that promotes phospholipid synthesis and plasma membrane reconstruction. Exosomes play an important role in tumor metastasis. The release and uptake of exosomes are key steps of their functions and depend on plasma membrane fusion and plasma membrane receptors, respectively. The purpose of this study was to explore whether LPCAT1-induced plasma membrane remodeling would change the secretion and uptake behavior of exosomes in tumor cells. We first confirmed the abnormally high expression of LPCAT1 in colorectal cancer cells by quantitative real-time PCR (qPCR) and Western blot analysis. Then, SW620 cells were used as exosome source cells, and SW480 cells were used as exosome receiver cells. Exosomes from SW620 cells could effectively promote the migration of SW480 cells. When LPCAT1 expression was reduced via siRNA knockdown in the source cells, the secretion of exosomes was downregulated, thus weakening the pro-migratory effects of exosomes on target cells. Conversely, when LPCAT1 was knocked down in target cells, the uptake of exosomes in target cells also decreased sharply. These results undoubtedly revealed that LPCAT1 is functionally associated with the release and internalization of exosomes in colorectal cancer cells and could affect the paracrine effects of exosomes, preliminarily extending the classical metabolic function of LPCAT1 to exosome-related pathways.

Abstract PO-057: Targeting ErbB2 degradation via the ubiquitin–proteasome pathway to inhibit the metastasis of pancreatic cancer

Abstract Objective: Pancreatic cancer is currently one of the most lethal human cancers and continues to be a major unsolved health problem in the 21st century. Since pancreatic cancer has a high metastatic potential, chemotherapy is inevitable for most pancreatic cancer patients, whereas the clinical responses to anticancer agents are not satisfied. In this work, we designed and synthesized a novel compound, termed as 6-MPA, and found that 6-MPA could improve the proteasomal degradation of ErbB2 pancreatic cancer cells. Methods: The inhibitory effects of 6-MPA on migration and invasion was measured by scratch experiment, wound healing assay and transwell experiment. Clone formation and CCK-8 assay was used to determine the cytotoxicity of 6-MPA. Real-time PCR and western blotting was used to determine the expression of indicated genes or proteins respectively. Immunoprecipitation was used to characterize protein–protein interactions. Results: 4, 8, and 16 μM of 6-MPA exhibited an inhibitory effects on the migration of SW1990 cells and the inhibition rates reached 40.2±10.1%, 58.2±11.2%, and 65.2±9.9% in vitro, respectively. RNA-seq showed that 6-MPA treatment manipulated ErbB2 level and its downstream PI3K/AKT/mTOR signaling pathways, which was verified by western blotting assay. Treatment with 6-MPA could enhance the interaction between ErbB2 and ubiquitin, and thus promote the proteasomal degradation of ErbB2. Conclusion: In this study, we obtained a novel small molecule 6-MPA, which could specifically degrade ErbB2 through the activation of protein ubiquitinination. And we found that 6-MPA possessed potent inhibitory effects on migration and invasion of pancreatic cancer cells. Therefore, our work illustrated a novel structure skeleton that could be used to target ErbB2, and potentially suppressed the metastasis of pancreatic cancer. Citation Format: Bo Zhang, Fei Teng, Nengming Lin. Targeting ErbB2 degradation via the ubiquitin–proteasome pathway to inhibit the metastasis of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-057.

MiR-146a suppresses the expression of CXCR4 and alters survival, proliferation and migration rate in colorectal cancer cells

CXCR4 plays an important role in colorectal cancer (CRC) development and metastasis. Some previous studies have indicated CXCR4 as a therapeutic target in cancer. CXCR4 is known as a direct target of miR-146a. The present study aimed to investigate how exogenous induction of miR-146a affects CXCR4 gene and protein expression and also proliferation, apoptosis and migration of CRC cells. Transfection of Caco-2 and SW480 cells by a synthetic miR-146a mimic led to downregulation of CXCR4 expression at both gene and protein levels. It also downregulated expression of several miR-146a targets, including GSK3B, IRAK1, TRAF6, AKT2, SMAD4, EGFR and NFKB1, mostly in SW480 cells. Overexpression of miR-146a resulted in a partial cell cycle arrest in the both cell lines, while the apoptotic rate was also decreased. In regards to epithelial-mesenchymal transition factors, VIM was downregulated in the both cell lines, but SNAI1 was upregulated in Caco-2 cells. The wound closure assay showed a reduction in cell migration in SW480 cells, but an opposite effect was detected in Caco-2 cells following transfection with miR-146a mimic. Therefore, our results are indicating that overexpression of miR-146a, despite downregulation of oncogenic CXCR4, may not lead to a universal tumor suppressive effect in all CRC cells, and this is possibly due to differences in miR-146a effects on signaling pathways in each cell type. Selection of miR-146a for tumor suppression requires enough details regarding the signaling profile of cancer cells otherwise it may produce unexpected outcome.

Tumor suppressive effect of scavenger receptor class A member 5 overexpression in colorectal cancer by regulating PI3K/AKT/mTOR pathway

BackgroundColorectal cancer (CRC) exhibits high risks of morbidity and mortality.ObjectiveTo investigate the effect of scavenger receptor class A member 5 (SCRAR5) on CRC and its mechanism on modulation of cancer development.MethodsThe SCRAR5 expression in four kinds of CRC cell lines (SW620, SW480, HT29, and HCT116) was measured by quantitative PCR and western blotting, respectively. The effects of SCRAR5 abnormal expression on cell proliferation, apoptosis, and migration were analyzed by CCK-8 assay, EdU assay, colony-forming assay, flow cytometry assay, Transwell assay and wound healing assay, respectively. Meanwhile, the involvements of PI3K/AKT/mTOR pathway with the role of SCRAR5 were investigated by western blotting. Afterwards, the in vivo effects of SCRAR5 abnormal expression on CRC xenograft mice were finally investigated by evaluating tumor volume, apoptosis and Ki67 expression.ResultsSCRAR5 was lowly expressed in CRC cell lines, especially SW480 cells. Up-regulation of SCRAR5 significantly promoted cell apoptosis, reduced cell proliferation and migration in SW480 cells. Notably, SCRAR5 overexpression obviously inhibited the phosphorylation levels of PI3K, AKT, and mTOR. Reversely, SCRAR5 silence exhibited promoting effects on HT29 cells. Consistently, in vivo experiments also revealed that SCRAR5 overexpression remarkably suppressed tumor volume and Ki67 expression, as well as promoted cell apoptosis.ConclusionsOverall, up-regulating of SCRAR5 obviously inhibited CRC tumor growth in vitro and in vivo, which might be related to PI3K/AKT/mTOR pathway.

Exosomes derived from colon cancer cells and plasma of colon cancer patients promote migration of SW480 cells through Akt/mTOR pathway

ObjectiveTo investigate the effect of exosomes derived from colon cancer (CC) cells and plasma of CC patients on migration of SW480 cells. MethodsThe exosomes derived from culture medium of human colon epithelial cell line NCM460 and CC cell line SW620 were isolated by ultracentrifugation. The exosomes derived from plasma of CC patients and healthy controls were isolated by size exclusion chromatography (SEC). The particle size and morphology of exosomes were identified by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) respectively, and exosomal markers were detected by Western blotting. The uptake of fluorescent DiI labeled exosomes by SW480 cells was observed by confocal microscopy. Transwell assay was used to detect the effect of exosomes on the migration of SW480 cells. The expression level of associated proteins in signaling pathway were analyzed by Western blotting. Rapamycin, an inhibitor of mTOR, was used to study the role of mTOR signaling pathway on exosomes mediated migration of SW480 cells. ResultsThe results of NTA and TEM showed that the particle size of the isolated exosomes was about 120 nm, which were small vesicles with membrane structure. The expressions of exosomal markers Alix, TSG101 and CD63 could be detected. The exosomes were evidenced by a red fluorescent signal inside the cytoplasm of SW480 recipient cells, and could promote the migration of SW480 cells, which is associated with Akt/mTOR signaling pathway. Compared with the control group, plasma exosomes derived from CC patients could significantly promote the migration of SW480 cells. Inhibition the activity of mTOR signaling could attenuate the migration of SW480 cells. ConclusionsExosomes derived from CC cells and plasma of CC patients could promote the migration of SW480 cells, which is associated with Akt/mTOR signaling pathway.