Project description:Two isogenic human colorectal cancer cell lines (primary SW480 cell line and its lymph node metastatic variant SW620 cell line),as an in vitro metastatic model. We have demonstrated that SW620 cell line possesses high metastasis potential and SW480 cell linepossesses low metastatic potential. We want to compare the whole cell microRNAs profiles of two isogenic colorectal cancer cell lines (SW480 and SW620 cell line), to gain an insight into the molecular events of colon cancer metastasis.
Project description:Two isogenic human colorectal cancer cell lines (primary SW480 cell line and its lymph node metastatic variant SW620 cell line),as an in vitro metastatic model. We have demonstrated that SW620 cell line possesses high metastasis potential and SW480 cell linepossesses low metastatic potential.
Project description:Metastasis is a complex process involving multiple steps. We were interested in the role of microRNAs (miRNAs) in the process of liver colonization by colorectal cancer cells. We hypothesized that the comparison between non-metastatic versus metastatic isogenic cell line should thus offer valuable insight to the molecular mechanisms involved in developing metastatic behavior. KM12C/KM12SM and SW480/SW620 are probably the best available models of isogenic cell lines differing in metastatic properties for colorectal cancer. Our first goal was to identify miRNAs that contribute to the metastatic traits of the isogenic colorectal cancer cell lines, KM12C/KM12SM and SW480/SW620. Total RNA was extracted from cells using the mirVana kit (Ambion). Total RNA (1 µg) from KM12C and SW480 (poorly metastatic) and KM12SM and SW620 (highly metastatic) cells was used to analyze the global miRNA expression profiling with TaqMan Megaplex human array A (v2.0) and B (v3.0) (Applied Biosystems).
Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.
Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.
Project description:Colorectal cancer is recognized as one of the leading causes of cancer death amongst both sexes in the U.S. Despite rigorous screening, those diagnosed with colon cancer often face poor prognosis, and approximately 70% of affected patients will develop metastatic relapse. The investigation of colon carcinoma cell lines’ genetic variability and response to chemotherapy panels may aid in targeting therapies to improve outcomes. This study aims to find correlations between metastasis status, gene variability, and drug response. We used two cell lines that were isolated from the same 51-year-old male with colorectal adenocarcinoma: a primary tumor-derived line (SW480) and a secondary metastasized-derived line (SW620). Live cell imaging using time-lapse microscopy over three days exhibited differential cell death responses following treatment with multiple chemotherapeutic agents, particularly against Cediranib and Nu-7441, with SW620 demonstrating greater sensitivity. Western blots revealed changes in DNA repair machinery expression (particularly NHEJ proteins) between SW480 and SW620. RNA sequencing via Gene Ontology corroborated our findings, demonstrating upregulated DNA repair and metabolic survival genes including TGM2 in SW620 and PROM1 in SW480. Both cell lines overcame higher concentrations of Nu-7441, but an SW620 line grown in non-attachment plates and then reattached (SW620F) exhibited greater resistance against Nu-7441, expanding upon gene adaptive behavior previously seen. Correlation between drug response and gene expression crucial to cell growth and successful metastasis may reveal new biomarkers to target potential treatments.
Project description:In this study, acyl-biotinyl exchange (ABE) and magnetic bead enrichment techniques were used to treat protein extracts from human colorectal cancer tissues, primary SW480 cell line and lymph node metastasis SW620 cell line. The proteomic results of endogenous and potential S-palmitoylated proteins were analyzed by mass spectrometry.
Project description:Four cancer cell line, ie SW480-Vector, SW480-TET2, SW620-Vector and SW620-TET2 were treated with tgfb1, repsox and control. Then the total RNA of these samples were extracted and sequenced with Illumina Nextseq 500.