Curcumin Suppresses Colon Cancer In Vitro and In Vivo

Objective: To discuss In Vitro and In Vivo the effects of curcumin on colon cancer. Material and Methods: SW620 cell and nude mice with tumor were respectively divided into 3 groups: NC, low, middle, high and 5-Fu groups. Measuring the cell activity by MTT, the cell cycle and cell apoptosis using flow cytometry and relative proteins by WB assay in cell experiment. Evaluating tumor volume and weight, cell apoptosis rate by TUNEL assay and relative proteins by Immunohistochemistry (IHC). Results: Compared with NC group, the SW620 cell activity was significantly depressed with cell apoptosis and G1 phase rates increasing and PI3K, AKT and P53 proteins expression were significantly differences in curcumin treated groups with dose-dependent by WB assay; In Vivo study, the tumor volume and size were significantly suppressed and positive cell number were significantly up-regulation in curcumin treated groups with dose-dependent, and PI3K, AKT and P53 proteins expression were significantly differences in curcumin treated groups with dose-dependent by IHC. Conclusions: Curcumin had anti-tumor effects to colon cancer via regulation PI3K/AKT/P53 pathway In Vivo and vitro study.

Metformin and Calcitriol Enhance 5-Fluorouracil Tumoricidal Effects in Colon Cancer By Modulating The PI3K/Akt/PTEN/mTOR Network

Purpose: Chemoresistance to 5-Fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, calcitriol, and/or metformin single/dual/triple regimens as complementary/alternative therapies. Methods: Ninety male mice were divided into: negative and positive (PC) controls, 5-FU, Cal, Met, 5-FU/Cal, 5-FU/Met, Cal/Met, and 5-FU/Cal/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. The therapeutic regimens were also applied in the SW480 and SW620 CRC cell lines. Results: The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR alongside reduced p21/PTEN/Cytochrome-C/Caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/Cal/Met most prominent regimen. All protocols also decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, increased pro-apoptotic molecules with apoptosis index, and 5-FU/Cal/Met revealed the strongest anti-cancer effects. In vitro, all therapies equally induced G1-phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in G0/G1-phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/Cal/Met disclosed the lowest percentage (81%) of viable SW620 cells. Moreover, 5-FU/Cal/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2) and the PI3K/Akt/mTOR molecules alongside highest expression of cell cycle inhibitors (p21/p27), pro-apoptotic markers (BAX/Cytochrome-C/Caspase-3), and PTEN in both cell lines. Conclusions: Metformin monotherapy was superior to calcitriol, whereas the 5-FU/metformin protocol showed better anti-cancer effects relative to the other dual therapies. However, the 5-FU/Cal/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells

Background A new strategy, particularly a novel combination, for immunotherapy in microsatellite stable metastatic colorectal cancer (mCRC) treatment needs to be formulated. Studies on the interferon-γ (IFN-γ)/ Janus kinase (JAK)/ signal transducer and activator of transcription (STAT)1 pathway provide new directions in this regard. Methods Our study applies three colon cancer cell lines, including microsatellite stable (MSS) cell lines, which are SW480 and SW620, and microsatellite instability-high (MSI-H) cell line, which is DLD-1. We compared the expressions of immune surface markers on colon cancer cells in response to IFN-γ. We elucidated these mechanisms, which involved the upregulation of immune surface markers. Furthermore, we examined real-world clinical samples using the PerkinElmer Opal multiplex system and NanoString analysis. Results We established that the baseline expression of major histocompatibility complex (MHC) class I alleles and programmed death-ligand 1 (PD-L1) were generally low in cell line models. The immune surface markers were significantly increased after IFN-γ stimulation on SW480 but were notably unresponsive on the SW620 cell line. We discovered that STAT1 and phosphorylated STAT1 (pSTAT1) were downregulated in the SW620 cell line. We verified that the STAT1/pSTAT1 could be restored through the application of proteasome inhibitors, especially bortezomib. The expression of MHC class I as downstream signals of STAT1 was also up-regulated by proteasome inhibitors. The similar results were reproduced in DLD-1 cell line, which was also initially unresponsive to IFN-γ. In real-world samples of patients with mCRC, we found that higher STAT1 expression in tumor cells was strongly indicative of a highly immunogenic microenvironment, with significantly higher expression levels of MHC class I and PD-L1, not only on tumor cells but also on non-tumor cells. Furthermore, tumor infiltrating lymphocytes (TILs) were increased in the positive-STAT1 group. Through NanoString analysis, we confirmed that the mRNA expressions of IFN-γ, human leukocyte antigen (HLA)-A, HLA-E, and HLA-G were also significantly higher in the positive-STAT1 group than those in the negative-STAT1 group. Conclusion Our study provides a novel rationale for the addition of bortezomib, a proteasome inhibitor, into new immunotherapy combinations.

Qualitative and quantitative top-down proteomics of human colorectal cancer cell lines identified 23000 proteoforms and revealed drastic proteoform-level differences between metastatic and non-metastatic cancer cells

Understanding cancer metastasis at the proteoform level is crucial for discovering new protein biomarkers for cancer diagnosis and drug development. Proteins are the primary effectors of function in biology and proteoforms from the same gene can have drastically different biological functions. Here, we present the first qualitative and quantitative top-down proteomics (TDP) study of a pair of isogenic human metastatic and non-metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). This study pursues a global view of human CRC proteome before and after metastasis in a proteoform-specific manner. We identified 23,319 proteoforms of 2,297 genes from the CRC cell lines using capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS), representing nearly one order of magnitude improvement in the number of proteoform identifications from human cell lines compared to literature data. We identified 111 proteoforms containing single amino acid variants (SAAVs) using a proteogenomic approach and revealed drastic differences between the metastatic and non-metastatic cell lines regarding SAAVs profiles. Quantitative TDP analysis unveiled statistically significant differences in proteoform abundance between the SW480 and SW620 cell lines on a proteome scale for the first time. Ingenuity Pathway Analysis (IPA) disclosed that many differentially expressed genes at the proteoform level had diversified functions and were closely related to cancer. Our study represents a milestone in TDP towards the definition of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.

Polyphenolic QTOF-ESI MS Characterization and the Antioxidant and Cytotoxic Activities of Prunus domestica Commercial Cultivars from Costa Rica

There is an increased interest in plum research because of their metabolites’ potential bioactivities. In this study, the phenolic profiles of Prunus domestica commercial cultivars (Methley, Pisardii and Satsuma) in Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry using a quadrupole-time-of-flight analyzer (UPLC-ESI-QTOF MS) on enriched phenolic extracts obtained through Pressurized Liquid Extraction (PLE) under acidic and neutral extraction conditions. In total, 41 different phenolic compounds were identified in the skin and flesh extracts, comprising 11 flavan-3-ols, 14 flavonoids and 16 hydroxycinnamic acids and derivatives. Neutral extractions for the skins and flesh from all of the cultivars yielded a larger number of compounds, and were particularly rich in the number of procyanidin trimers and tetramers when compared to the acid extractions. The total phenolic content (TPC) and antioxidant potential using the DPPH and ORAC methods exhibited better results for neutral extracts with Satsuma skins and Methley flesh, which showed the best values (685.0 and 801.6 mg GAE/g extract; IC50 = 4.85 and 4.39 µg/mL; and 12.55 and 12.22 mmol TE/g extract, respectively). A Two-Way ANOVA for cytotoxicity towards AGS gastric adenocarcinoma and SW620 colon adenocarcinoma indicated a significant difference (p < 0.05) for PLE conditions, with better results for neutral extractions, with Satsuma skin delivering the best results (IC50 = 60.7 and 46.7 µg/mL respectively) along with Methley flesh (IC50 = 76.3 and 60.9 µg/mL, respectively). In addition, a significant positive correlation was found between TPC and ORAC (r = 0.929, p < 0.05), as well as a significant negative correlation (p < 0.05) between TPC and cytotoxicity towards AGS and SW620 cell lines (r = −0.776, and −0.751, respectively). A particularly high, significant, negative correlation (p < 0.05) was found between the number of procyanidins and cytotoxicity against the AGS (r = −0.868) and SW620 (r = −0.855) cell lines. Finally, the PCA clearly corroborated that neutral extracts are a more homogenous group exhibiting higher antioxidant and cytotoxic results regardless of the part or cultivar; therefore, our findings suggest that PLE extracts under neutral conditions would be of interest for further studies on their potential health benefits.

LncRNA CRNDE Promoted Colorectal Cancer Cells Reisitance To Paclitaxel Through Wnt/β-Catenin Signaling Pathway

Background The lncRNA colorectal neoplasia differentially expressed (lncRNA CRNDE) is commonly over-expressed in different human cancers and involved in different biological functions. Paclitaxel(PTX) is a tricyclic diterpenoid compound which often used as a natural anticancer drugs in cancer treatments. Although there have many research reports about the mechanisms of LncRNA involved in PTX treatment, there are no any research about lncRNA CRNDE and PTX resistance in colorectal cancer. The purpose of this study is to investigate the mechanisims of LncRNA CRNDE involving PTX resistance in colorectal cancer. Results We constructed lncRNA CRNDE over-expression vector and transfected it into SW620 cell. CCK8, Transwell experiments proved that over-expression of lnc CRNDE increased SW620 cells proliferation and invasion, while the si-CRNDE group was significantly decreased. over-expression CRNDE can significantly up-regulate β-catenin, c-myc, APC and Axin2 expression and affect the expression of cyclinD1 and CDK4 after treated with PTX. Conclusion lncRNA CRNDE promotes CRCs proliferation, invasion and migration. Over-expression of LncRNA CRNDE enhanced the reisitance of CRC to PTX through inhibition of Wnt/ β-catenin signaling pathway.

Exosomal miR-224-5p from colorectal cancer cells promotes malignant transformation of human colon normal cells by promoting cell proliferation through downregulation of CMTM4

Background Interactions between malignant cells and neighboring normal cells is important for carcinogenesis. In addition, cancer cell-derived exosomes have been shown to promote the malignant transformation of recipient cells, but the mechanisms remain unclear. Methods The level of miR-224-5p in colorectal cancer (CRC) cell-derived exosomes was determined by RT-qPCR assay. In addition, PKH26 dye-labeled exosomes were used to assess the efficacy of the transfer of exosomes between SW620 and CCD 841 CoN cells. Results In this study, we found that overexpression of miR-224-5p significantly promoted the proliferation, migration and invasion of SW620 cells. In addition, miR-224-5p can be transferred from SW620 cells to CCD 841 CoN cells via exosomes. SW620 cell-derived exosomes overexpressing miR-224-5p markedly promoted proliferation, migration and invasion of CCD 841 CoN cells. Meanwhile, SW620 cell-derived exosomal miR-224-5p notably decreased the expression of CMTM4 in CCD 841 CoN cells. Furthermore, SW620 cell-derived exosomal miR-224-5p significantly inhibited tumor growth in a xenograft model in vivo. Conclusion These findings suggested that SW620 cell-derived exosomal miR-224-5p could promote malignant transformation and tumorigenesis in vitro and in vivo via downregulation of CMTM4, suggesting that miR-224-5p might be a potential target for therapies in CRC.

Effect of Targeting Leucine-Zipper-Like Transcription Regulator 1 Gene on Colon Cancer Cells

LZTR1 is associated with several diseases, including liver cancer, childhood cancer, and schwannomas. However, LZTR1’s role in colon cancer and its mechanism of action have not been reported. The colon cancer tissues and adjacent tissues were collected to measure the expression of LZTR1 by Real time PCR. Colon cancer SW620 cell lines were cultured and randomly divided into control group and LZTR1 group followed by analysis of LZTR1 expression by real time PCR, cell proliferation by MTT assay, Caspase3 activity, Bcl-2 and Bax level by Real time PCR, cell invasion by Transwell chamber; NF-κB/VEGF expression by Western blot. LZTR1 expression was significantly reduced in colon cancer tissues compared to adjacent tissues (P <0.05) and negatively correlated with colon cancer TNM stage, tumor size, and lymph node metastasis, and positively associated with tumor differentiation (P <0.05). LZTR1 plasmid transfection into SW620 cells can significantly up-regulate LZTR1, inhibit tumor cell proliferation and invasion, increase Caspase 3 activity and Bax level, downregulate Bcl-2, NF-κB and VEGF (P <0.05). LZTR1 expression is reduced in colon cancer tissues, which is related to its clinicopathological characteristics. Up-regulation of LZTR1 can regulate apoptosis and inhibit tumor proliferation and invasion by regulating NF-κB/VEGF signaling pathway.

Death Receptor 5 (TNFRSF10B) Is Upregulated and TRAIL Resistance Is Reversed in Hypoxia and Normoxia in Colorectal Cancer Cell Lines after Treatment with Skyrin, the Active Metabolite of Hypericum spp

Skyrin (SKR) is a plant bisanthraquinone secondary metabolite from the Hypericum genus with potential use in anticancer therapy. However, its effect and mechanism of action are still unknown. The negative effect of SKR on HCT 116 and HT-29 cancer cell lines in hypoxic and normoxic conditions was observed. HCT 116 cells were more responsive to SKR treatment as demonstrated by decreased metabolic activity, cellularity and accumulation of cells in the G1 phase. Moreover, an increasing number of apoptotic cells was observed after treatment with SKR. Based on the LC-MS comparative proteomic data from hypoxia and normoxia (data are available via ProteomeXchange with the identifier PXD019995), SKR significantly upregulated Death receptor 5 (DR5), which was confirmed by real-time qualitative PCR (RT-qPCR). Furthermore, multiple changes in the Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-activated cascade were observed. Moreover, the reversion of TRAIL resistance was observed in HCT 116, HT-29 and SW620 cell lines, even in hypoxia, which was linked to the upregulation of DR5. In conclusion, our results propose the use of SKR as a prospective anticancer drug, particularly as an adjuvant to TRAIL-targeting treatment to reverse TRAIL resistance in hypoxia.

Silencing Circ_0005075 Inhibits the Invasion and Migration of Colon Cancer Cells and Induces Apoptosis

Colon cancer is a common malignancy of the digestive tract, mostly occurring at the junction of the rectum and colon. It easily invades multiple internal organs and tissues, causing systemic injury and mortality to patients. The occurrence and development of colon cancer involve multiple genes and multiple factors. It is greatly significant to identify oncogenes and tumor suppressor genes that influence the development of colon cancer and to study their mechanism of action for colon cancer diagnosis and treatment. Furthermore, circRNA are involved in the development of several types of tumors by affecting miRNA regulation on target gene expression. In the present study, circ_0005075 siRNA and circ_0005075 siRNA + mir-335 inhibitor were transfected into colon cancer SW620 cells, and circ_0005075 gene expression was significantly decreased after transfecting circ_0005075 siRNA into SW620 cells. Silencing circ_0005075 expression significantly inhibited SW620 cell proliferation, invasion, and migration, and promoted apoptosis, downregulated PCNA and vimentin expression, and upregulated E-cadherin and cleaved caspase3 expression. After circ_0005075 siRNA + mir-335 was transfected, it inhibited circ_0005075 and mir-335 expression, which significantly affected the vigor, invasion and migration ability, apoptosis rate of SW620 cells, as well as PCNA, E-cadherin, vimentin, and cleaved caspase3 expression. Therefore, the present study demonstrated that circ_0005075 silencing inhibited the proliferation, invasion, and migration of SW620 cells and promoted apoptosis by upregulating mir-335 expression.