Colorectal Cancer Cell Lines SW480 Research Articles

PTPN11 is a potential biomarker for type 2 diabetes mellitus complicated with colorectal cancer

Epidemiological surveys have shown that the incidence of type 2 diabetes mellitus (T2DM) and malignancies is rapidly increasing worldwide and has become a major disease that threatens human life. In this study, we quantitatively analyzed the proteome of tumor tissues and adjacent normal tissues from six patients withT2DM combined with colorectal cancer (CRC) and eight non-diabetic CRC, focusing on the effect of T2DM on tumor tissues. We analyzed the functional enrichment of differentially expressed proteins (DEPs) using clusterProfiler in R and the expression level of protein tyrosine phosphatase non-receptor type 11 (PTPN11) and other key proteins in the TIMER and GEPIA2 databases. The HPA database was used to validate PTPN11 protein expression. The correlation between PTPN11 expression and clinicopathological features was analyzed by UALCAN database. The impact of PTPN11 on clinical prognosis was evaluated utilizing Kaplan-Meier Plotter. The correlation between PTPN11 expression and tumor-infiltrated immune cells was investigated via TIMER and TISIDB databases. Gene set enrichment analysis (GSEA) was performed to examined the pathway of PTPN11 enrichment in CRC using data from The Cancer Genome Atlas (TCGA) database. Furthermore, small interfering (si) RNA was used to knock down PTPN11 in CRC cell line SW480. Western blot analysis was used to detect PTPN11 expression in tissue samples or cells and the effect of PTPN11 knockdown on key proteins related to PI3K/AKT and cell cycle pathway in SW480 cells. Cell proliferation and wound healing assays were used to detect the effects of cell proliferation and migration after knockdown of PTPN11 or treatment with high glucose. We found that metabolic pathways such as oxidative phosphorylation, glycolysis/gluconeogenesis, and insulin secretion were significantly enriched in tumor tissues from diabetic patients compared to non-diabetic patients. In addition, PTPN11, a marker gene associated with T2DM and CRC, were mined in diabetic tumor tissues. PTPN11 showed high expression in diabetic tumor tissues compared to normal tissues. High PTPN11 expression predicted poor prognosis in CRC. PTPN11 expression was strongly associated with immune infiltrating cells in CRC. GSEA analysis revealed that PTPN11 was enriched in cancer-related pathways. Western blotting analysis indicated that PTPN11 knockdown reduced the protein levels of p-PI3K, p-AKT, CDK1 and CYCLIN D, without altering PI3K and AKT protein levels. Cell proliferation and wound healing data showed that PTPN11 and high glucose could increase the proliferation and migration ability. These findings showed that PTPN11 may be a potential key biomarker for CRC in patients with diabetes, which will provide new potential targets for future intervention of T2DM complicated with CRC.

Blockade of CaV3 calcium channels and induction of G0/G1 cell cycle arrest in colon cancer cells by gossypol.

Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.

JianPiTongLuo (JPTL) Recipe regulates anti-apoptosis and cell proliferation in colorectal cancer through the PI3K/AKT signaling pathway

BackgroundJianPiTongLuo Recipe (JPTL Recipe) is a traditional Chinese medicine formula commonly used in the clinical treatment of colorectal cancer. Clinical studies have found that it can significantly improve the prognosis of patients with colorectal cancer. However, its mechanisms of action are not well understood, which has limited its further clinical application. MethodsWe investigated the potential mechanisms of action of the JianPiTongLuo (JPTL) Recipe on colorectal cancer (CRC) using a multi-step approach. Initially, network pharmacology and bioinformatics analyses were conducted using databases such as TCMSP, HERB, BATMAN-TCM, and STRING to identify active components of JPTL Recipe and predict their therapeutic targets. Interaction networks and functional enrichment analyses were constructed to hypothesize relevant biological processes and pathways. In vitro studies involved treating human CRC cell lines HCT116, LoVo and SW480 with varying concentrations of JPTL Recipe extract, measuring cell viability with the CCK-8 assay, assessing apoptosis via flow cytometry, and analyzing signaling pathways through Western blotting. To corroborate these findings, in vivo experiments were performed on BALB/c nude mice implanted with HCT116 cells, divided into control, JPTL Recipe-treated, 5-fluorouracil (5-FU)-treated, and JPTL Recipe combined with 5-FU groups, with tumor growth and histological changes monitored. Mechanistic studies focused on the PI3K/AKT signaling pathway, examining the phosphorylation status of key pathway proteins using immunofluorescence and Western blot analyses to elucidate JPTL Recipe 's interaction with pathway activity. ResultsWe demonstrated that JPTL Recipe effectively inhibits colorectal cancer cell proliferation, anti-apoptotic ability, and exerts synergistic therapeutic effects with fluorouracil. Further analysis revealed that JPTL Recipe affects the activity of colorectal cancer cells by inhibiting the phosphorylation of the PI3K/AKT signaling pathway. ConclusionIn summary, we have discovered and confirmed that the traditional Chinese medicine compound JPTL Recipe can serve as a novel adjuvant therapy for colorectal cancer, offering a new treatment approach for the integration of traditional Chinese and Western medicine in the treatment of colorectal cancer.

Fibroblasts Promote Resistance to KRAS Silencing in Colorectal Cancer Cells.

Colorectal cancer (CRC) responses to KRAS-targeted inhibition have been limited due to low response rates, the mechanisms of which remain unknown. Herein, we explored the cancer-associated fibroblasts (CAFs) secretome as a mediator of resistance to KRAS silencing. CRC cell lines HCT15, HCT116, and SW480 were cultured either in recommended media or in conditioned media from a normal colon fibroblast cell line (CCD-18Co) activated with rhTGF-β1 to induce a CAF-like phenotype. The expression of membrane stem cell markers was analyzed by flow cytometry. Stem cell potential was evaluated by a sphere formation assay. RNAseq was performed in KRAS-silenced HCT116 colonospheres treated with either control media or conditioned media from CAFs. Our results demonstrated that KRAS-silencing up-regulated CD24 and down-regulated CD49f and CD104 in the three cell lines, leading to a reduction in sphere-forming efficiency. However, CAF-secreted factors restored stem cell marker expression and increased stemness. RNA sequencing showed that CAF-secreted factors up-regulated genes associated with pro-tumorigenic pathways in KRAS-silenced cells, including KRAS, TGFβ, NOTCH, WNT, MYC, cell cycle progression and exit from quiescence, epithelial-mesenchymal transition, and immune regulation. Overall, our results suggest that resistance to KRAS-targeted inhibition might derive not only from cell-intrinsic causes but also from external elements, such as fibroblast-secreted factors.

The Role of Matrix Metalloproteinase-2 (MMP2) in Colorectal Cancer Progression: Correlation With Clinicopathological Features and Impact on Cellular Processes.

Background Colorectal cancer (CRC) is a prevalent and deadly disease characterized by significant molecular complexity. Matrix metalloproteinase-2 (MMP2) has been implicated in cancer progression due to its role in extracellular matrix degradation, yet comprehensive studies linking MMP2 expression to CRC progression and its molecular mechanisms remain needed. Methodology This study involved 90 CRC patients, with tumor and adjacent normal tissues analyzed via immunohistochemistry (IHC) to assess MMP2 expression. The human CRC cell line SW480 was treated with an MMP2 inhibitor, ARP100, and evaluated for changes in cell migration, invasion, proliferation, and apoptosis using various assays, including MTT, wound-healing, transwell, caspase activity, and western blot analysis. Results High MMP2 expression was significantly associated with advanced tumor stages, lymph node involvement, and metastasis in CRC patients. Compared to normal tissues, MMP2 expression was markedly higher in cancerous tissues. Inhibition of MMP2 in SW480 cells resulted in reduced migration, invasion, and proliferation, and induced apoptosis, evidenced by increased caspase 3 and 9 activities and higher levels of cleaved caspase proteins. Conclusion Elevated MMP2 expression is correlated with advanced CRC and aggressive tumor characteristics. MMP2 inhibition can suppress CRC cell invasiveness, migration, and proliferation while promoting apoptosis, suggesting its potential as a therapeutic target in CRC treatment.

Mechanical shear flow regulates the malignancy of colorectal cancer cells.

Colorectal cancer (CRC) is notable for its high mortality and high metastatic characteristics. The shear force generated by bloodstream provides mechanical signals regulating multiple responses of cells, including metastatic cancer cells, dispersing in blood vessels. We, therefore, studied the effect of shear flow on circulating CRC cells in the present study. The CRC cell line SW620 was subjected to shear flow of 12.5 dynes/cm2 for 1 and 2 h separately. Resulting elevated caspase-9 and -3 indicated that shear flow initiated the apoptosis of SW620. Enlarged cell size associated with a higher level of cyclin D1 was coincident with the flow cytometric results indicating that the cell cycle was arrested at the G1 phase. An elevated phosphor-eNOSS1177 increased the production of nitric oxide and led to reactive oxygen species-mediated oxidative stress. Shear flow also regulated epithelial-mesenchymal transition (EMT) by increasing E-cadherin and ZO-1 while decreasing Snail and Twist1. The migration and invasion of sheared SW620 were also substantially decreased. Further investigations showed that mitochondrial membrane potential was significantly decreased, whereas mitochondrial mass and ATP production were not changed. In addition to the shear flow of 12.5 dynes/cm2, the expressions of EMT were compared at lower (6.25 dynes/cm2) and at higher (25 dynes/cm2) shear flow. The results showed that lower shear flow increased mesenchymal characteristics and higher shear flow increased epithelial characteristics. Shear flow reduces the malignancy of CRC in their metastatic dispersal that opens up new ways to improve cancer therapies by applying a mechanical shear flow device.

Expression and Prognosis of Differential Gene Troponin T1 Between Right and Left Colon Cancers.

Colorectal cancer (CRC) is one of the most common digestive tract tumors in humans. At present, many scholars believe that the primary site of the tumor has a direct and profound impact on its curative effect. There are significant differences in the expression of many genes, tumor microenvironment, and prognosis between the left and right colon. However, there is a lack of detailed studies on whether the differentially expressed genes in the left and right colon significantly impact the prognosis of patients with CRC. Troponin T1 ( TNNT1 ) is an important gene that affects the prognosis difference between left and right colon cancer screening from "The Cancer Genome Atlas" database. By analyzing the differential gene expression data and clinical data of the left and right hemicolons in the database, the online prognostic database was used to screen the key molecules that significantly affect the tumor immune microenvironment and patient prognosis and to predict their functions and pathways. Quantitative reverse transcription-polymerase chain reaction was used to verify the expression difference of TNNT1 in CRC cell lines SW480 and HCT116, and normal human colorectal epithelial cell line FHC. The relationship between TNNT1 expression in 88 pairs of CRC samples and clinical information and pathologic parameters of patients with CRC was analyzed to judge the impact of TNNT1 expression on patient survival. Database analysis showed that TNNT1 was significantly overexpressed in CRC, and TNNT1 was one of the main differential genes between left colon cancer (LCC) and right colon cancer (RCC). The expression of TNNT1 was significantly increased in RCC, which could lead to poor prognosis of patients. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of TNNT1 was significantly up-regulated in CRC cell lines SW480 and HCT116. Eighty-eight immunohistochemistry (IHC) of CRC tissues and adjacent tissues suggested that the expression of TNNT1 in CRC was significantly higher than that in normal adjacent tissues. By analyzing the clinical information and pathologic indicators matched with these clinical samples, we found that high TNNT1 expression in the primary tumor location (right colon) and high N stage (N2, N3) were unfavorable factors affecting the prognosis of patients with CRC. Multivariate Cox regression analysis suggested that high expression of TNNT1 may be an independent risk factor for the prognosis of patients with CRC. As one of the main differential genes between LCC and RCC, TNNT1 is representative to some extent. Its high expression may be one of the reasons why the prognosis of patients with RCC is worse than that of patients with LCC.

Research on the Effect and Mechanism of Selenium on Colorectal Cancer Through TRIM32.

The intake of selenium (Se) in the human body is negatively correlated with the risk of colorectal cancer (CRC), but its mechanism in the occurrence and development of CRC is not clear. This study aimed to evaluate the therapeutic effect of Se on CRC, and explore the anti-tumor effect of Se supplementation on CRC and its molecular mechanism. In this study, we utilized colony formation assay, cell scratch test, Transwell migration, and flow cytometry to assess cell proliferation, migration, and apoptosis. Our findings demonstrate that Se effectively suppresses the growth and proliferation of CRC cell lines HCT116 and SW480 and promoting cellular apoptosis. In vivo experiments demonstrated a significant inhibitory effect of Se on tumor growth. CRC-related datasets were extracted from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases for differential expression analysis of TRIM32 and survival analysis. We found that TRIM32 was highly expressed in tumor tissues of CRC patients and correlated with a poor prognosis. Furthermore, through RNA sequencing analysis, we identified TRIM32 as a gene that was significantly decreased after Se treatment in HCT116 cells. This finding was subsequently validated by Western blot results. Moreover, TRIM32 knockdown combined with Se treatment significantly inhibited cell growth proliferation and migration and further induced apoptosis of colorectal cancer cells. In conclusion, our findings provided evidence that Se inhibited the growth of colorectal cancer cells by down-regulating TRIM32.

Synthesis and SAR investigation of biphenylaminoquinoline derivatives with benzyloxy substituents as promising anticancer agents.

The biphenyl scaffold represents a prominent privileged structure within the realms of organic chemistry and drug development. Biphenyl derivatives have demonstrated notable biological activities, including antimicrobial, anti-inflammatory, anti-HIV, and the treatment of neuropathic pain. Importantly, their anticancer abilities should not be underestimated. In this context, the present study involves the design and synthesis of a series of biphenyl derivatives featuring an additional privileged structure, namely the quinoline core. We have also diversified the substituents attached to the benzyloxy group at either the meta or para position of the biphenyl ring categorized into two distinct groups: [4,3']biphenylaminoquinoline-substituted and [3,3']biphenylaminoquinoline-substituted compounds. We embarked on an assessment of the cytotoxic activities of these derivatives in colorectal cancer cell line SW480 and prostate cancer cell line DU145 for exploring the structure-activity relationship. Furthermore, we determined the IC50 values of selected compounds that exhibited superior inhibitory effects on cell viability against SW480, DU145 cells, as well as MDA-MB-231 and MiaPaCa-2 cells. Notably, [3,3']biphenylaminoquinoline derivative 7j displayed the most potent cytotoxicity against these four cancer cell lines, SW480, DU145, MDA-MB-231, and MiaPaCa-2, with IC50 values of 1.05 μM, 0.98 μM, 0.38 μM, and 0.17 μM, respectively. This highly promising outcome underscores the potential of [3,3']biphenylaminoquinoline 7j for further investigation as a prospective anticancer agent in future research endeavors.

MRI-traceable multifunctional magnetic mesoporous silica nanoparticles (MMSN) capped with graphene quantum dots (GQD) as a theragnostic system in colorectal cancer treatment

In the study, a multifunctional nanosystem composed of magnetic core-mesoporous silica nanoparticles (MMSN) capped with graphene quantum dots (GQD) was proposed as a theragnostic platform against colorectal cancer cells. The theragnostic carrier was well characterized and had an average particle size of 140 nm, with a specific surface of 475.82 m2 g−1. The superparamagnetic nanoparticles showed a hysteresis loop, with a maximum magnetization of 14.93 emu.g−1 and a remanence of 2.64 emu.g−1. The functionalization of MMSN was confirmed using the ninhydrin test. Regorafenib (REGO), an anticancer drug, was used with a loading capacity of 77.5 μg mg−1. The drug release profiles from the carrier and the sensitivity of the interaction between MMSN and GQD to pH were examined, revealing that more drug release was observed in acidic pH. The cytotoxicity of the MMSN-REGO/GQD system was evaluated on colorectal cancer cell line SW-480 using MTT assay, and the results indicated that no cytotoxicity was observed for the MMSN/GQD carrier. Furthermore, the IC50 of REGO was significantly reduced from 85 μg ml−1 to 6 μg ml−1 in the loaded form. The response of MMSN/GQD nanoparticles to the external magnetic field exhibited the capability of the theragnostic system as an MRI contrast agent. Generally, biocompatible, theragnostic, pH-responsive, multifunctional MMSN/GQD can be considered promising candidates for treatment, MRI imaging, and hyperthermia in colorectal cancer treatment.

Knockdown of SETD5 Inhibits Colorectal Cancer Cell Growth and Stemness by Regulating PI3K/AKT/mTOR Pathway.

SET domain-containing 5 (SETD5), a member of protein lysine methyltransferase family, is expressed in multiple cancers, making it potential therapeutic targets. However, the role of SETD5 in colorectal cancer remains largely unknown. The expression of SETD5 in the 30 pairs colorectal cancer tissues samples and cell lines were determined by qRT-PCR. The functions of SETD5 was detected by knocked-down or overexpression in colorectal cancer cell lines SW480 and HCT116 cells. Cell proliferative activity, cell death, and stemness characteristics were assessed. BEZ235, a PI3K/AKT/mTOR pathway inhibitor, was used to perform rescue experiment to analyze whether SETD5 exerted its effects through activating PI3K/AKT/mTOR pathway. SETD5 was substantially upregulated in colorectal cancer, and correlated to metastasis and clinical stage of patients. Knockdown of SETD5 inhibited SW480 and HCT116 cell growth, as evidenced by the inhibition of cell viability and clone-forming. Moreover, Knockdown of SETD5 suppressed the capability of tumor sphere formation of SW480 and HCT116 cells, and reduced the expression of stemness-related proteins Nanog and Sox2. Further western blot analysis revealed that SETD5 knockdown inhibited the phosphorylation of proteins associated with the PI3K/AKT/mTOR pathway. In contrast, overexpression of SETD5 exerted the opposite effects. Mechanistically, by blocking PI3K/AKT/mTOR pathway with BEZ235, the effects of SETD5 overexpression on cell viability and Nanog and Sox2 protein expression were reversed. Our results substantiated that SETD5 functioned as an oncogene by promoting cell growth and stemness in colorectal cancer cells through activating the PI3K/AKT/mTOR signaling pathway.

Abstract P52: Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway

Abstract Colorectal cancer is the third most common type of cancer and the second most in terms of mortality rate worldwide. 5-fluorouracil (5-FU), the fluoropyrimidine drug, that interferes with DNA replication and repair processes, is the first-line-of-treatment against colorectal cancer. However, cells gain resistance over time and relapses occur upon continuous 5-FU therapy. Interestingly, 5-FU promotes stemness in colorectal cancer via Wnt signaling pathway which is critical for its progression. Studies have pointed out the possibility of using a Wnt signaling inhibitor along with 5-FU as a better strategy to overcome the poor outcomes of current 5-FU based therapies. We have previously shown that menadione (vitamin K3) suppresses Wnt signaling pathway in colorectal cancer cells. In the present study, we attempted to evaluate if the combination of menadione and 5-FU can exert a synergistic effect in the human colorectal cancer cell lines HCT116 and SW620. Analysis of the cell viability assay results using CompuSyn software confirmed that the combination indeed acts synergistically. The combination efficiently downregulated the expression of the Wnt signal transducer protein, β-catenin, and the expression of other proteins in the pathway such as phospho-GSK3β in the cell lines HCT116 and SW620. Docking of the individual drugs and their combination on β-catenin using Autodock Vina showed that 5-FU and menadione in combination bind to key protein-protein interaction (PPI) sites of β-catenin whereby they may inhibit β-catenin-TCF interaction or activate the β-catenin destruction complex. Taken together, our data possibly explains that the synergistic action of 5-FU and menadione in colorectal cancer cells may be due to the simultaneous binding of the two drugs to multiple PPI sites thereby blocking the activity of β-catenin. Citation Format: Vidya P Warrier, Sankaran Venkatachalam, Sakthivel Ramasamy, M Michael Gromiha, Devarajan Karunagaran. Combination of 5-fluorouracil and Menadione Exerts Synergistic Effect on Colorectal Cancer Cells by Targeting Wnt/β-catenin Signaling Pathway [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P52.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

Abstract 3334: Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response

Abstract Integrated stress response (ISR) activation has been linked to many human diseases, including cancer and neurological diseases. The ISR controls protein synthesis and proteostasis. ISR main effector ATF4 transcriptional factor regulates the balance between survival and cell death. Under acute ISR, ATF4 promotes cell adaptation and survival. Sustained ISR leads to ATF4-mediated apoptosis. In many cancers, mutations or overexpression of oncogenes, results in enhanced cell proliferation and increased protein synthesis, which activate the ISR. Therefore, protein translation is reduced to maintain proteostasis. In other words, many cancers are primed by the ISR. Therefore, additional push by small molecule ISR-inducers will disrupt the balance and force cancer cells to enter apoptosis. Nelfinavir activates ATF4 through inhibition of eIF2α specific phosphatases CReP and GADD34. PG3 upregulates ATF4 through the HRI/eIF2α/ATF4 pathway. We hypothesize that combined treatment of PG3 and Nelfinavir will sustain upregulation of ATF4, and lead to ATF4-mediated cell apoptosis. Cell viability assays indicated that the combined treatment potently and synergistically inhibited cell viability in colorectal cancer cell lines HT29, SW480 and HCT116 p53−/−, osteosarcoma cell line U2OS and breast cancer cell lines MCF7 and T47D. The combined treatment sustained the induction of ATF4 and enhanced the expression of its proapoptotic target genes, CHOP and PUMA, and cell apoptosis. We confirmed that the enhanced induction of ATF4, CHOP and PUMA is through the ISR because ISRIB (ISR inhibitor) blocked the combined treatment-induced upregulation of ATF4, CHOP and PUMA. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Combined treatment with PG3 and Nelfinavir induced synergistic apoptosis though sustained activation of integrated stress response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3334.

Effect and potential mechanism of oncometabolite succinate promotes distant metastasis of colorectal cancer by activating STAT3

To investigate the effect of Oncometabolite succinate on colorectal cancer migration and invasion and to initially explore the underlying mechanism.Succinate acid detection kit detected the succinate content in tissues. The growth of colorectal cancer cells was measured by cck-8 assay, wound-healing migration assay and transwell migration and invasion assays, and then explored the level of epithelial-mesenchymal transition (EMT) and STAT3/ p-STAT3 expression by western blot analysis and quantitative real-time PCR for mRNA expression. We found that succinate levels were significantly higher in carcinoma tissues than paracancerous tissues. After succinate treatment, the colorectal cancer cell lines SW480 and HCT116 had enhanced migration and invasion, the expression of biomarkers of EMT was promoted, and significantly increased phosphorylation of STAT3. In vivo experiments also showed that succinate can increase p-STAT3 expression, promote the EMT process, and promote the distant metastasis of colorectal cancer in mice.Succinate promotes EMT through the activation of the transcription factor STAT3, thus promoting the migration and invasion of colorectal cancer.

Effect of some aspirin analogues on SW480 Colorectal cancer cell line: Tip of the iceberg

Abstract Colorectal cancer (CRC) is the third most common cancer and second leading cause of death with approximately 1.9 million cases and almost a million deaths worldwide in 2020. It is predicted that the global incidences of CRC will be over 3 million in 2040. Aspirin has been shown to have cytotoxic and immunomodulatory effects on CRC cell lines. However, due to side effects such as gastrointestinal bleeding in older patients, there has always a quest for safer and more effective analogues, leading to the synthesis of aspirin analogues. One of the primary regulators of the mitochondria-mediated pathway to apoptosis is the family known as BCL-2 proteins, which are broadly grouped into pro-apoptotic proteins such as BAX, BAK, BIK, BAD and anti-apoptotic proteins such as BCL-2 and BCL-XL. Phase contrast images of the SW480 CRC cells treated with aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin at 0.5 mM for 24 h, 48 h and 72 h were taken at 100X magnification. Western blot was used to detect BAX and BCL-2 proteins by treating CRC cells with aspirin and its analogues, and analysed by SDS-PAGE and immunoblotting. The blots were then probed with primary antibodies BCL-2 and BAX, after which the HRP bound protein-labelled antibody was visualised using ECL and exposed to CL-XPosure™ Film. It was observed that the isomers of aspirin (O-ASP), meta-aspirin (M-ASP) and para-aspirin (P-ASP) significantly increased the density of BAX Ab. O-ASP, M-ASP, P-ASP and ortho-thioaspirin (O-TASP) significantly reduced the density of BCL-2 Ab, thus proposing that the anti-apoptotic effect of this cell line is reversed/reduced by these aspirin analogues. The results of this study suggest that M-ASP and P-ASP have its antiproliferative effect on the SW480 CRC cell line via driving the pro-apoptotic effect of BAX protein and decreasing the expression of anti-apoptotic of BCL-2 protein, thus, encouraging apoptosis in this CRC cell line.

Sample Preparation of Adherent Cell Lines for Flow Cytometry: Protocol Optimization—Our Experience with SW-480 colorectal cancer cell line

Sample Preparation of Adherent Cell Lines for Flow Cytometry: Protocol Optimization—Our Experience with SW-480 colorectal cancer cell line

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480.

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells.

The power of mumps virus: Matrix protein activates apoptotic pathways in human colorectal cell lines

New therapeutic approaches can significantly impact the control of colorectal cancer (CRC), which is increasing worldwide. In this study, we investigated the potential of targeting viral proteins to combat cancer cells. Specifically, we examined the anticancer potential of the matrix (M) protein of the mumps virus Hoshino strain in SW480 CRC cell lines. To begin, we individually transfected SW480 cells with pcDNA3 plasmids containing the mumps virus M gene. We then investigated the percentage of cell death, caspase activity, and the expression levels of genes involved in apoptosis pathways. Following this, we performed bioinformatics analysis on the M protein to identify any similarities with Bcl-2 family members and their viral homologs. Our diagnostic methods showed that treatment with the mumps M protein induced apoptosis and upregulated the expression and activity of pro-apoptotic proteins in SW480 CRC cells compared to the control and vector groups. Based on our bioinformatics studies, we proposed that the BH3 motif in the M protein may trigger apoptosis in CRC cells by interacting with cellular Bax. Overall, our study showed for the first time that the mumps virus M protein could be considered as a targeted treatment for CRC by inducing apoptotic pathways.

Targeting the survival kinase DYRK1B: A novel approach to overcome radiotherapy-related treatment resistance

BackgroundCancer cell survival under stress conditions is a prerequisite for the development of treatment resistance. The survival kinase DYRK1B is a key regulator of stress survival pathways and might thereby also contribute to radiation resistance. Here we investigate the strategy of targeting DYRK1B in combination with ionizing radiation (IR) to enhance tumor cell killing under stress conditions. MethodsDYRK1B expression, ROS formation and DNA damage were investigated under serum-starvation (0.1% FBS), hypoxia (0.2%, 1% O2) and IR. The combined treatment modality of IR and DYRK1B inhibition was investigated in 2D and in spheroids derived from the colorectal cancer cell line SW620, and in primary patient-derived colorectal carcinoma (CRC) organoids. ResultsExpression of DYRK1B was upregulated under starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in CRC-organoids but not in healthy tissue-derived organoids. ConclusionCombined treatment with the DYRK1B inhibitor AZ191 and IR resulted in (supra-) additive tumor cell killing in colorectal tumor cell systems and in primary CRC organoids. Mechanistic investigations support the rational to target the stress-enhanced survival kinase DYRK1B in combination with irradiation to overcome hypoxia- and starvation-induced treatment resistances.