Colorectal Cancer Cell LoVo Research Articles

Isobavachalcone induces apoptosis of colorectal cancer cells mediated by interaction with survivin: Experimental and theoretical analyses

Isobavachalcone (IBC) as a bioactive chalcone isolated from the seeds of Psoralea corylifolia Linn has shown promising anticancer activities. However, its main anticancer mechanism in colorectal cancer cells has not been well-explored. Recently, the interaction of therapeutic compounds with survivin, an antiapoptotic marker, has been indicated to be a potential approach for advancing anticancer compounds. Therefore, in this study, IBC was evaluated for its anticancer activities against metastatic colorectal adenocarcinoma, LoVo, followed by exploring its interaction with survivin. It was observed that incubation of the human LoVo colorectal cancer cells with the IBC at 24 h resulted in a significant reduction in cell viability with an IC50 of about 35 µM as well as a significant membrane damage. Also, it was deduced that IBC could result in the formation of excessive ROS, lipid peroxidation, reduction of SOD/CAT activity, as well as downregulation of Nrf2 and HO-1 mRNA levels. Further studies showed that IBC could induce MMP reduction, cytochrome c (Cyt c) release, overexpression of Bax/Bcl-2 mRNA ratio as well as caspase-9 and −3. Moreover, it was deduced that IBC mitigated the expression of survivin at mRNA and protein levels. Then, binding parameters indicated that IBC strongly binds to one molecule of survivin at physiological temperature. Also, it was revealed that IBC substantially induced the conformational changes of survivin. Ultimately, through hydrogen bonding and hydrophobic forces, IBC has a substantial binding affinity with survivin (–9.80 kcal/mol) at a region that is critical to the dimerization process, according to molecular docking studies. Overall, this study showed that IBC may be used in future research to assess its clinical and in vivo behaviors in the modulation of colorectal cancer mediated by deactivation and downregulation of antiapoptotic survivin.

SGLT2 inhibitor promotes mitochondrial dysfunction and ER-phagy in colorectal cancer cells

BackgroundSodium-glucose transporter 2 (SGLT2) inhibitors (iSGLT2) are approved medications for type 2 diabetes. Recent studies indicate that iSGLT2 inhibit the growth of some cancer cells. However, the mechanism(s) remains to be fully elucidated.MethodsThe SGLT2 levels were determined in normal colon CCD 841 CoN and, HCT 116, HT-29, SW480 and LoVo colorectal cancer (CRC) cell lines by quantitative real-time PCR and western blot. The effect of iSGLT2 canagliflozin on cell proliferation was examined using CCK-8, as its role on CRC cells metabolism and tumorigenesis has been evaluated by XF HS Seahorse Bioanalyzer and flow cytometric analyses. Transient gene silencing experiments and analysis of protein–protein interaction network were conducted to evaluate the SGLT2 molecular targets in CRC cells.ResultsData showed that the treatment with iSGLT2 (50 µM) for 72 h induced cell cycle arrest (p < 0.001), impaired glucose and energetic metabolism (p < 0.001), promoted apoptotic cell death and ER stress flowing into autophagy (p < 0.001) in HCT 116 and HT-29 cells. These cellular events were accompanied by sirtuin 3 (SIRT3) upregulation (p < 0.01), as also supported by SIRT3 transient silencing experiments resulting in the attenuation of the effects of iSGLT2 on the cellular metabolic/energetic alterations and the induction of programmed cell death. The identification and validation of dipeptidyl peptidase 4 (DPP4) as potential common target of SGLT2 and SIRT3 were also assessed.ConclusionsThese results deepened knowledge on the iSGLT2 contribution in limiting CRC tumorigenesis unveiling the SGLT2/SIRT3 axis in the cytotoxic mechanisms.Graphical

Neutrophil-to-lymphocyte ratio in colorectal tissue affects prognosis in patients with colorectal cancer

The objective of this investigation was to analyse the correlation between the neutrophil-to-lymphocyte ratio (NLR) status in the immune microenvironment (IME) and the prognostic outcomes of patients who have undergone radical surgery for colorectal cancer (CRC). In light of the continued prevalence of CRC in China, this study utilised Kaplan-Meier and Cox regression analyses to assess the prognostic relevance of NLR status in IME among patients with CRC. Furthermore, cellular experiments, such as cell scratching, were conducted to elucidate the underlying mechanisms of NLR's impact on CRC. The NLR status in IME has been found to have a significant impact on the prognosis of patients with CRC. Patients who exhibit elevated intratumoural and extratumoural NLR are associated with a poor prognosis. Experimental evidence indicates that tumour-associated neutrophil (TAN) augments the migratory, invasive, and proliferative potential of HT-29, HCT-116 and LOVO colorectal cancer cells, while concurrently reducing their sensitivity to oxaliplatin. Conversely, lymphocytes have demonstrated cytotoxic effects on HT-29 cells. The NLR status in IME may serve as a prognostic biomarker for resectable CRC.

Activation of M2 macrophage autophagy by rapamycin increases the radiosensitivity of colorectal cancer xenografts.

Tumor-associated macrophages (TAMs) are intimately involved in cancer radiochemotherapy resistance. However, the mechanism by which macrophages affect radiosensitivity through autophagy remains unclear. The purpose of our study was to investigate how activating autophagy in type-II macrophages (M2) by using rapamycin (RAP) would affect the radiosensitivity of colorectal cancer (CRC) xenografts. A nude mouse CRC model was established by injecting LoVo CRC cells. After tumor formation, supernatant from M2 cells (autophagy-unactivated), autophagy-activated M2 cells, or autophagy-downregulated M2 cells was injected peritumorally. All tumor-bearing mice were irradiated with 8-Gy X-rays twice, and the radiosensitivity of CRC xenografts was analyzed in each group. The mass, volume, and microvessel density (MVD) of tumors in the autophagy-unactivated M2 group significantly increased; however, supernatant from M2 cells that were autophagy-activated by rapamycin significantly decreased tumor weight, volume, and MVD compared with negative control. Combining bafilomycin A1 (BAF-A1) with RAP treatment restored the ability of the M2 supernatant to increase tumor mass, volume, and MVD. Immunohistochemical and Western blot results showed that compared with the negative control group, supernatant from M2 cells that were not activated by autophagy downregulated the expression of Livin and Survivin in tumor tissues; activation of M2 autophagy further downregulated the protein levels. Therefore, autophagy-activated M2 supernatant can downregulate the expression of the antiapoptotic genes Livin and Survivin in CRC xenografts, improving the radiosensitivity of CRC by inducing apoptosis in combination with radiotherapy and inhibiting the growth of transplanted tumors.

Novel Stable Protease Inhibitor from Phoenix dactylifera(L.) Flowers with Antimicrobial and Antitumoral Activities.

A novel protease inhibitor isolated from date palm Phoenix dactylifera(L.) flowers (PIDF) was purified and characterized. A heat and acidic treatment step followed by ethanol precipitation and reverse-phase high-performance chromatography was applied to purify this natural protease inhibitor to homogeneity with a single band of about 19 kDa. The stability study depicted that PIDF was fully stable at 40 °C and retained 65% of its initial activity after heating at 50 °C for 24 h. Its thermal stability at 70 °C was markedly enhanced by adding calcium, bovine serum albumin, and sorbitol as well as by metal divalent cations, especially Mg2+ and Hg2+. This protease inhibitor showed high inhibitory activity against therapeutic proteases, including pepsin, trypsin, chymotrypsin, and collagenase, and acted as a potent inhibitor of some commercial microbial proteases from Aspergillus oryzae, Bacillus. sp, and Bacillus licheniformis. Moreover, a potent antibacterial spectrum against Gram (+) and Gram (-) bacterial strains and an efficient antifungal effect were observed. Its cytotoxicity toward human colorectal cancer cell LoVo and HCT-116 lines suggested that PIDF could serve as a new therapeutic target inhibiting human colorectal cancer.

A Study on Thymoquinone: Antioxidant Capacity and Anticancer Activities in LoVo Colorectal Cancer Cells.

Thymoquinone has antioxidant and anticancer effects. This study investigates the cytotoxic, genotoxic, and apoptotic effects of black seed and its active ingredient, thymoquinone on colorectal cancer cells. The antioxidant content of Black seed methanolic extracts (BSME) with different concentrations (50, 500 and 1000 μg/mL) were determined by the photometric methods. The reactive oxygen production (iROS) of BSME and thymoquinone on colorectal cancer cells (LoVo) and normal epithelial cells (CCD18Co) were analyzed by the fluorometric methods. A luminometric glutathione kit was employed to observe the changes in intracellular glutathione (GSH) levels. Cytotoxicity was determined by the ATP method, genotoxicity was determined by Comet Assay, and the apoptosis was identified by the Acridine Orange/Ethidium Bromide (AO/EB) double dye method. The cytotoxicity was increased by BSME and thymoquinone in LoVo cells in a dose-dependent manner (p<0.001). BSME and thymoquinone also increased iROS, and induced apoptosis and DNA damage (p<0.001). High doses of BSME and thymoquinone on cancer and healthy cells have cytotoxic, genotoxic and apoptotic effects with pro-oxidant effects. Colorectal cancer cells are more sensitive than healthy cells.

The radiation- and chemo-sensitizing capacity of diclofenac can be predicted by a decreased lactate metabolism and stress response

BackgroundAn enhanced aerobic glycolysis (“Warburg effect”) associated with an increase in lactic acid in the tumor microenvironment contributes to tumor aggressiveness and resistance to radiation and chemotherapy. We investigated the radiation- and chemo-sensitizing effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac in different cancer cell types.MethodsThe effects of a non-lethal concentration of diclofenac was investigated on c-MYC and Lactate Dehydrogenase (LDH) protein expression/activity and the Heat shock Protein (HSP)/stress response in human colorectal (LS174T, LoVo), lung (A549), breast (MDA-MB-231) and pancreatic (COLO357) carcinoma cells. Radiation- and chemo-sensitization of diclofenac was determined using clonogenic cell survival assays and a murine xenograft tumor model.ResultsA non-lethal concentration of diclofenac decreases c-MYC protein expression and LDH activity, reduces cytosolic Heat Shock Factor 1 (HSF1), Hsp70 and Hsp27 levels and membrane Hsp70 positivity in LS174T and LoVo colorectal cancer cells, but not in A549 lung carcinoma cells, MDA-MB-231 breast cancer cells and COLO357 pancreatic adenocarcinoma cells. The impaired lactate metabolism and stress response in diclofenac-sensitive colorectal cancer cells was associated with a significantly increased sensitivity to radiation and 5Fluorouracil in vitro, and in a human colorectal cancer xenograft mouse model diclofenac causes radiosensitization.ConclusionThese findings suggest that a decrease in the LDH activity and/or stress response upon diclofenac treatment predicts its radiation/chemo-sensitizing capacity.

CircMSH3 is a potential biomarker for the diagnosis of colorectal cancer and affects the distant metastasis of colorectal cancer

Objectives To identify the most significantly differentially expressed circular RNAs (circRNAs) in colorectal cancer (CRC) tissues in terms of their expression levels and circularity, and to analyze the relationship between their expression levels and the clinical characteristics of patients. Methods circRNA RNA-seq technology was used to screen differentially expressed circRNAs in CRC. Sanger sequencing was used to identify circRNA back-splice junction sites. The relative expression levels of hsa_circ_0003761 (circMSH3) in CRC tissues and cell lines were detected by quantitative real-time fluorescence PCR technology. An RNA-protein pull-down assay was used to detect protein binding to circRNAs. Dual-luciferase reporter gene vectors were constructed to verify that circRNAs bind to microRNAs. Results Four hundred twenty circRNAs were found to be upregulated, and 616 circRNAs were downregulated. circMSH3 was derived from the MutS homolog 3 (MSH3) gene and was formed by a loop of exons 9, 10, 11, and 12. In 110 pairs of CRC and adjacent tissues, circMSH3 expression was 4.487-fold higher in CRC tissues. circMSH3 was also highly expressed in the HT-29 and LOVO CRC cell lines. The expression level of circMSH3 was associated with distant metastasis in CRC patients (P = 0.043); the area under the curve (AUC) of circMSH3 for CRC diagnosis was 0.75, with a sensitivity and specificity of 70.9% and 66.4%, respectively. circMSH3 could bind to a variety of proteins, mainly those involved in RNA transcription, splicing, cell cycle, and cell junctions. Furthermore, circMSH3 could bind to miR-1276, miR-942-5p, and miR-409-3p. Conclusion circMSH3 is a potential biomarker for the diagnosis of CRC and affects the distant metastasis of CRC. Multiple RNA-binding protein binds to circMSH3, and circMSH3 binds to miR-1276, miR-942-5p, and miR-409-3p, thereby affecting the expression of circMSH3.

Investigation of ATR Inhibitor VX970 as a Radiosensitizer in Colorectal Cancer Cells

Colorectal cancer (CRC) is the second leading cause of combined cancer-related mortality in males and females in the U.S. Traditional treatment of locally advanced rectal cancer consists of neoadjuvant chemoradiation followed by surgery and adjuvant chemotherapy. Emerging data suggests that higher response rates can be achieved with total neoadjuvant therapy (TNT) where delivery of all chemotherapy and radiation therapy (RT) occurs prior to surgery. In addition, for patients with a complete response to TNT, non-operative management (NOM) can be considered. However, despite the use of TNT, pathologic complete response rates remain below 40% and NOM is only achieved in approximately 50% of rectal cancer patients. A strong need remains for more active anti-cancer therapies in rectal cancer to both reduce pelvic recurrence and facilitate NOM. Here, we tested the hypothesis that inhibition of the ataxia telangiectasia and Rad3-related protein kinase (ATR), a critical regulator of cellular DNA damage response, could increase the sensitivity of CRC to RT. VX970, a highly potent and selective ATR inhibitor, was investigated as a radiosensitizer in SW48 and LoVo CRC cell models. In vitro, IC50 of VX970 was assessed by alamarBlue cytotoxicity assay, while radiosensitivity was revealed by radiation clonogenic assays (0, 2, 4, 6, 8 Gy). ATR activity was determined by p-Chk1 using immunoblotting, and cell cycle distribution was analyzed by propidium iodide flow cytometry. CRC xenografts were generated using both LoVo and SW48 cells injected in the left flanks of athymic nude mice to explore the radiosensitizing effects of VX970 in vivo. Tumors were allowed to grow to 100-150 mm3, and the mice were randomized into multiple groups [vehicle alone, RT alone (10 Gy/5 fractions), VX970 alone, and VX970+RT]. Mouse weights and tumor size were measured three times weekly. Comparison of treatment groups was performed using the log-rank test with P<0.05 considered significant. The IC50 concentrations of VX970 on SW48 and LoVo cells were about 500 and 100 nM, respectively. VX970 at doses of 3 nM did not alter the viability of CRC cells, but significantly sensitized CRC cells to radiotherapy (P<0.05), with DER of 1.43 and 1.59, respectively, in SW48 and LoVo cells. VX970 efficiently attenuated p-Chk1 expression and significantly abrogated radiation induced G2/M cell cycle arrest (P<0.05). In addition, VX970 in combination with radiotherapy significantly prolonged tumor growth delay of CRC xenografts compared to radiation alone (P<0.05), with minimal toxicity observed. Inhibition of the ATR-Chk1 pathway by targeting ATR kinase with VX970 sensitizes CRC to radiotherapy in vitro and in vivo. Our findings support that ATR inhibition by VX970 is a promising new approach to improve the therapeutic ratio of radiotherapy for patients with CRC and warrants further clinical testing.

Phytochemical analysis and anticancer activity of the Pithecellobium dulce seed extract in colorectal cancer cells

Abstract Colorectal cancer remains a challenging medical issue worldwide, and utilizing natural products and plants to produce novel, effective and safe therapies against this disease is continuously a sought-after strategy. Fruit and leaf extracts of Pithecellobium dulce (P. dulce) showed potential anticancer properties as they induced apoptosis of breast cancer and Dalton’s lymphoma ascites cells. Thus, the main objective of the current study is to determine whether the seed extract of P. dulce will affect apoptosis, cell cycle, migration, and inflammation of LoVo colorectal cancer cells. The high-resolution liquid chromatography–mass spectrometry was used to determine the chemical composition of the P. dulce seed extract, which revealed the presence of 35 phytochemicals. The findings of this study indicated a significant cytotoxic effect of seeds of this plant in colorectal cancer characterized by induction of apoptosis, cell cycle arrest, and reduction of migration. In addition, the seed extract suppressed several genes that are essential for cancer progression such as MMP2, MMP9, and IL-8, and, on the other hand, upregulated pro-apoptotic genes such as BAX and P53. This study has established P. dulce as a potential and valuable source for providing future therapies against colorectal cancer and other cancers.

Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

DYRK2 downregulation in colorectal cancer leads to epithelial–mesenchymal transition induction and chemoresistance

Colorectal cancer (CRC) is among the most prominent causes of cancer-associated mortality in the world, with chemoresistance representing one of the leading causes of treatment failure. However, the mechanisms governing such chemoresistance remain incompletely understood. In this study, the role of DYRK2 as a mediator of CRC cell drug resistance and the associated molecular mechanisms were assessed by evaluating human tumor tissue samples, CRC cell lines, and animal model systems. Initial analyses of The Cancer Genome Atlas database and clinical tissue microarrays revealed significant DYRK2 downregulation in CRC in a manner correlated with poor prognosis. We further generated LoVo CRC cells that were resistant to the chemotherapeutic drug 5-FU, and found that such chemoresistance was associated with the downregulation of DYRK2 and a more aggressive mesenchymal phenotype. When DYRK2 was overexpressed in these cells, their proliferative, migratory, and invasive activities were reduced and they were more prone to apoptotic death. DYRK2 overexpression was also associated with enhanced chemosensitivity and the inhibition of epithelial–mesenchymal transition (EMT) induction in these LoVo 5-FUR cells. Co-immunoprecipitation assays revealed that DYRK2 bound to Twist and promoted its proteasomal degradation. In vivo studies further confirmed that the overexpression of DYRK2 inhibited human CRC xenograft tumor growth with concomitant Twist downregulation. Overall, these results thus highlight DYRK2 as a promising therapeutic target in CRC worthy of further investigation.

Association Mining Identifies MAL2 as a Novel Tumor Suppressor in Colorectal Cancer.

IntroductionColorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. However, the driver genes that promote CRC metastasis remain poorly understood. Association mining mines and extracts the repeated correlations and relevance in a dataset to predict the appearance of other data items according to the appearance of one item.MethodsHere, the Apriori algorithm was used to find the frequent mutational gene sets (FMGSs) and hidden association rules (ARs) within these FMGSs from 383 CRCs with whole exome sequencing datasets. The weighted correlation network analysis (WGCNA) was used to identify the hub genes in CRC. CCK8, colony formation, cell migration and invasion assays were adopted to detect the roles of hub genes in CRC.ResultsIntriguingly, we found that MAL2 (myelin and lymphocyte protein 2) was associated with TP53 and APC in stage IV of CRC, and further subnetwork exploration based on WGCNA identified MAL2 as a potent hub gene. To validate the metastasis-related role of MAL2 in CRC, a lentivirus-based overexpression system was utilized to construct MAL2-overexpressing human CRC LOVO cells. Overexpression of MAL2 remarkably inhibited CRC cell proliferation and invasion.ConclusionOur results highlighted that MAL2 acts as a tumor suppressor in CRC and could serve as a potential therapeutic target.

PTPN6-EGFR Protein Complex: A Novel Target for Colon Cancer Metastasis.

This study investigates the expression of nonreceptor protein tyrosine phosphatase 6 (PTPN6) gene in different colon cancer cells and its effect on malignant biological behavior. The expression level of PTPN6 mRNA in different colon cancer cell lines was detected by qPCR. CCK-8, clone formation assay, scratch assay, and transwell assay were used to detect the effect of knockdown or overexpression of the PTPN6 gene on the malignant biological behavior of colon cancer cells. CO-IP assay was used to detect the interaction protein of PTPN6. PTPN6 was highly expressed in colorectal cancer tissues. High expression of PTPN6 is associated with poor prognosis in patients with colon cancer. PtPN6 knockdown inhibited the proliferation, invasion, migration, and clonogenesis of colorectal cancer LOVO and SW480 cells. At the same time, the knockdown of PTPN6 inhibited the EMT process in colorectal cancer. CO-IP results showed that PTPN6 had a protein-protein interaction with EGFR. Overexpression of EGFR increased the carcinogenic effect of PTPN6. The high expression of the PTPN6 gene can promote the proliferation, migration, and invasion of colon cancer cells. PTPN6 can interact with EGFR. PTPN6-EGFR complex may be an important factor affecting the biological characteristics of colon cancer cells and a potential therapeutic target.

The effect of glycyrrhetinic acid on the proliferation and inva-sion of colorectal cancer LoVo cells

目的:研究不同浓度的甘草次酸对大肠癌LoVo细胞增殖和侵袭的影响。方法:将大肠癌LoVo细胞分为对照组,甘草次酸低、中、高剂量组(甘草次酸浓度分别为50, 100, 200 μmol/L)和5氟尿嘧啶组(5氟尿嘧啶浓度为100 μmol/L ),各组细胞经过药物孵育24和48 h后进行检测。通过四氮唑蓝试验检测甘草次酸对各组细胞增殖率的影响;通过Annexin V/PI双标流式细胞术检测各组细胞的凋亡率;Transwell 小室法检测各组细胞的侵袭能力;通过蛋白质印迹法检测检各组细胞的NF-κB蛋白表达。结果:与对照组相比,甘草次酸中、高剂量组和5氟尿嘧啶组中细胞抑制率均显著降低(P＜0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中细胞凋亡率均显著升高(P＜0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中大肠癌LoVo 细胞侵袭能力显著降低(P＜ 0.05);甘草次酸中、高剂量组和5氟尿嘧啶组中NF-κB相对表达量均显著降低(P＜0.05)。结论:浓度为100 μmol/L和200 μmol/L甘草次酸可以抑制大肠癌LoVo细胞的增殖,降低侵袭能力,这些作用的机制与抑制NF-κB蛋白的表达有关。.

S-Adenosylmethionine Increases the Sensitivity of Human Colorectal Cancer Cells to 5-Fluorouracil by Inhibiting P-Glycoprotein Expression and NF-κB Activation.

Colorectal cancer (CRC) is the second deadliest cancer worldwide despite significant advances in both diagnosis and therapy. The high incidence of CRC and its poor prognosis, partially attributed to multi-drug resistance and antiapoptotic activity of cancer cells, arouse strong interest in the identification and development of new treatments. S-Adenosylmethionine (AdoMet), a natural compound and a nutritional supplement, is well known for its antiproliferative and proapoptotic effects as well as for its potential in overcoming drug resistance in many kinds of human tumors. Here, we report that AdoMet enhanced the antitumor activity of 5-Fluorouracil (5-FU) in HCT 116p53+/+ and in LoVo CRC cells through the inhibition of autophagy, induced by 5-FU as a cell defense mechanism to escape the drug cytotoxicity. Multiple drug resistance is mainly due to the overexpression of drug efflux pumps, such as P-glycoprotein (P-gp). We demonstrate here that AdoMet was able to revert the 5-FU-induced upregulation of P-gp expression and to decrease levels of acetylated NF-κB, the activated form of NF-κB, the major antiapoptotic factor involved in P-gp-related chemoresistance. Overall, our data show that AdoMet, was able to overcome 5-FU chemoresistance in CRC cells by targeting multiple pathways such as autophagy, P-gp expression, and NF-κB signaling activation and provided important implications for the development of new adjuvant therapies to improve CRC treatment and patient outcomes.

Hydroxyphenyl Butanone Induces Cell Cycle Arrest through Inhibition of GSK3β in Colorectal Cancer.

Background Colorectal cancer (CRC) is among the top three gastrointestinal malignancy in morbidity and mortality. The abnormal activation of Wnt/β-catenin pathway is considered to be a key factor in the occurrence and development of CRC. Novel inhibitor discovery against key factor in WNT pathway is important for CRC treatment and prevention. Methods Cell proliferation was detected after hydroxyphenyl butanone treatment in human colorectal cancer HCT116, LOVO, and normal colonic epithelial NCM460 cells. Colony formation, cell invasion ability, and cell cycle were detected with and without GSK-3β knockdown. Results Hydroxyphenyl butanone induces cycle arresting on G1-S phase of colorectal cancer cell line through GSK3β in Wnt/β-catenin pathway and inhibits malignant biological manifestations of cell proliferation, colony formation, and invasion. The inhibition in the high concentration group is stronger than that in the low concentration group, and the antitumor effect is different for different tumor cells. Under the same concentration of natural hydroxyphenyl butanone, the inhibition on normal colonic epithelial cells is significantly lower than that on tumor cells. The natural hydroxyphenyl butanone with medium and low concentration could promote the proliferation of normal colonic epithelial cells. Conclusion This study illustrated natural hydroxyphenyl butanone as new inhibitor of GSK3β and revealed the mechanisms underlying the inhibitory effects in colorectal cancer.

Abstract 1983: Investigation of the effects of nutrient restriction on endolysosomal signaling in colorectal cancer (CRC) cells

Abstract Cancer cells grown under the stress of nutrient restriction can rewire endo-lysosomal activation and initiate autophagy as a survival mechanism. Colorectal cancer (CRC) cells are known to be particularly susceptible to the restriction of specific nutrients such as glucose and glutamine; nonetheless this susceptibility can vary considerably between cell lines. Caco-2, T84 and LoVo CRC cell lines were cultured for 2h - 48h in a pre-optimized starvation medium containing low serum, glucose and L-glutamine and pathways associated with autophagy, as well as lysosomal trafficking, biogenesis and degradation were examined in these cells. With nutrient restriction, all three cell lines showed induction of autophagy via an increase in LC3-II and p-AMPK. However, autophagic flux was inhibited in starved T84 and LoVo cells reflected by an increase in the cargo protein p62; starved Caco-2 cells showed normal autophagic flux. We therefore hypothesized a recalibration of nutrient sensing signals via the endo-lysosomal pathway in T84 and LoVo cells under nutrient restricted conditions. Nutrient restricted LoVo cells showed a surprising increase in mTORC1 activity and a decrease in the mRNA expression of MCOLN1, a cation-permeable channel on lysosomes and a transcriptional target of the master transcription regulator of lysosomal biogenesis Transcription factor EB (TFEB). Nutrient restricted T84 and Caco-2 cells showed a decrease in mTORC1 activity and a modest increase in MCOLN1 expression. A remarkable increase in the protein expression of early (Rab5) and late (Rab7) endosomal markers was observed in LoVo and T84 but not in Caco-2 cells. Additionally, treatment of both unstarved and starved T84 and LoVo cells with the v-ATPase inhibitor Bafilomycin A1 (Baf) led to an accumulation of Rab5 and Rab7; this accumulation was reversed in unstarved but not in starved cells upon withdrawal of Baf. The mRNA expression of Rab5 and Rab7 either decreased or remained unchanged in the starved T84 cells, implicating either post-transcriptional regulation via miRNAs or enhanced protein synthesis. Small RNA sequencing and subsequent confirmation with RT-qPCR of nutrient restricted cells indicated the differential expression of a network of miRNAs targeting protein involved in endo-lysosomal signalling. Additionally, the activation of the mTORC1 pathway is strongly suggestive of enhanced protein synthesis in response to the stress of starvation. Overall, our data suggests that nutrient restriction can lead to a highly rapid deregulation in endo-lysosomal signalling via post-transcriptional mechanisms. Future studies will indicate whether such deregulation provides a survival advantage to cancer cells and whether perturbation of nutrient sensing pathways can be used as a therapeutic option. Citation Format: Aliye E. Gulec, Hepsen H. Husnugil, Goksu Oral, Ilir Sheraj, Sreeparna Banerjee. Investigation of the effects of nutrient restriction on endolysosomal signaling in colorectal cancer (CRC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1983.

Gene Expression Profile of the Human Colorectal Carcinoma LoVo Cells Treated With Sporamin and Thapsigargin.

Sporamin, a proteinase inhibitor isolated from the sweet potato (Ipomoea batatas), has shown promising anticancer effect against colorectal cancer (CRC) in vitro and in vivo but its mechanisms of action are poorly understood. In the present study, high throughput RNA sequencing (RNA-seq) technology was applied to explore the transcriptomic changes induced by sporamin in the presence of thapsigargin (TG), a non-12-O-tetradecanolphorbol-13-acetate type cancer promoter, in the LoVo human CRC cells. Cellular total RNA was extracted from the cells after they were treated with vehicle (CTL), 1 μM of thapsigargin (TG), or 1 μM of TG plus 30 μM of sporamin (TGSP) for 24 h. The migratory capacity of the cells was determined by wound healing assay. The gene expression profiles of the cells were determined by RNA-seq on an Illumina platform. GO enrichment analysis, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and transcription factors (TF) prediction were all performed based on the differentially expressed genes (DEGs) across groups with a series of bioinformatics tools. Finally, the effect and potential molecular targets of the sporamin at the transcriptome level were evaluated. Sporamin significantly inhibited the migration of cells induced by TG. Among the 17915 genes detected in RNA-seq, 46 DEGs were attributable to the effect of sporamin. RT-PCR experiment validated that the expression of RGPD2, SULT1A3, and BIVM-ERCC5 were up-regulated while NYP4R, FOXN1, PAK6, and CEACAM20 were down-regulated. Sporamin enhanced the mineral absorption pathway, worm longevity regulating pathway, and pyrimidine metabolism pathway. Two TFs (SMIM11A and ATOH8) were down-regulated by sporamin. HMOX1 (up-regulated) and NME1-NME2 (down-regulated) were the main nodes in a PPI network consisting of 16 DEGs that were modulated by sporamin in the presence of TG. Sporamin could favorably alter the gene expression profile of CRC cells, up-regulating the genes that contribute to the homeostasis of intracellular metal ions and the activities of essential enzymes and DNA damage repairment. More studies are warranted to verify its effect on specific genes and delineate the mechanism of action implicated in the process.

α-Difluoromethylornithine-Induced Cytostasis is Reversed by Exogenous Polyamines, Not by Thymidine Supplementation.

Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072–81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.