Proliferation Of Colon Cancer Cells Research Articles

A Quassinoid Diterpenoid Eurycomanone from Eurycoma longifolia Jack Exerts Anti-Cancer Effect through Autophagy Inhibition

Eurycomanone (EN) is one of the representative quassinoid diterpenoids from roots of Eurycoma longifolia Jack, a natural medicine that is widely distributed in Southeast Asia. Previous studies showed that EN induces cancer cell apoptosis and exhibits anti-cancer activity, but the molecular mechanism of EN against cancer has still not been elucidated. In this study, we examined the regulatory effect of EN on autophagy to reveal the mechanism of EN-mediated colon cancer growth inhibition. First, we found that EN is able to inhibit colon cancer cell proliferation and colony formation. The angiogenesis level in cancer cells was inhibited as well. Next, the treatment of EN led to the suppression of autophagy, which was characterized by the downregulation of the LC3-II level and the formation of GFP-LC3 puncta under EN treatment in colon cancer. Moreover, we revealed that the mTOR signaling pathway was activated by EN in a time- and concentration-dependent manner. Finally, autophagy induction protected colon cancer cells from EN treatment, suggesting that autophagy improves cell survival. Taken together, our findings revealed the mechanism of EN against colon cancer through inhibiting autophagy and angiogenesis in colon cancer, supporting that the autophagy inhibitor EN could be developed to be a novel anti-cancer agent.

Expression of SAMHD1 and its mutation on prognosis of colon cancer.

The expression of sterile α motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) and its mutation play a key role in the prognosis of colon cancer. The aim of the present study was to investigate the mechanism and the role of SAMHD1 in colon cancer. Microarray data from 187 patients with colon cancer and 45 adjacent normal tissue obtained from the Gene Expression Omnibus (GEO) were analyzed. A protein-protein interaction (PPI) network was constructed to identify key genes associated with colon cancer prognosis. Cox proportional hazard regression and survival analyses were performed to identify the potential for SAMHD1 to serve as a prognostic biomarker. Immunohistochemistry (IHC) and immunofluorescence (IF) were performed to assess the expression levels and distribution of SAMHD1 in tissues and cells. Western blotting (WB) and Cell Counting Kit-8 (CCK-8) assays were used to identify the proliferation and apoptotic effects of SAMHD1 on HT-29 (Cas9-SAMHD1) cell lines. A total of 6,905 consistently differentially expressed genes were identified in the GEO database. Through the PPI network, SAMHD1 was found to be associated with Kirsten rat sarcoma virus (KRAS). SAMHD1 expression was negatively associated with KRAS. Proportional hazards regression and survival analyses demonstrated that low expression of SAMHD1 was associated with increased patient mortality. IHC and IF results demonstrated that SAMHD1 expression in patients with colon cancer was decreased compared with controls (both P<0.05). CCK-8 and WB results showed that proliferation was significantly promoted, and the expression levels of apoptosis-related proteins were significantly inhibited in the D137N and D311A groups as a result of a mutation in the deoxynucleoside triphosphohydrolase (dNTPase) site (both P<0.05 vs. wild-type). Proliferation was inhibited and apoptosis-related protein expression levels were promoted in the wild-type (WT) and D137N groups following 20 µg/ml 5-fluorouracil (5-FU) treatment (both P<0.05). WB and CCK-8 results showed cell proliferation was promoted and cell apoptosis-related protein expression was inhibited in the D137N group following treatment with 20 µg/ml 5-FU (all P<0.05) compared with the WT group. In conclusion, SAMHD1 expression was low in colon cancer. The dNTPase function of SAMHD1 may inhibit colon cancer cell proliferation and may enhance apoptosis. In addition, first-line chemotherapy with 5-FU has a time-dependent effect, which may provide novel options for clinical treatment of colon cancer.

Targeting the lncRNA FGD5-AS1/miR-497-5p/PD-L1 Axis Inhibits Malignant Phenotypes in Colon Cancer (CC).

Long noncoding RNAs (lncRNAs) regulate cancer progression and drug resistance. However, the role of lncRNA FGD5-AS1 in regulating colon cancer (CC) progression is still largely unknown. Hence, this study investigated the role of lncRNA FGD5-AS1 in regulating colon cancer (CC) progression and found that lncRNA FGD5-AS1 regulated miR-497-5p/PD-L1 axis to promote cancer progression in CC cells in vitro and in vivo. Specifically, we found that lncRNA FGD5-AS1 and PD-L1 tended to be high-expressed, while miR-497-5p was low-expressed in CC tissues and cell lines compared to the normal adjacent tissues and cells. Next, we found that lncRNA FGD5-AS1 positively regulated PD-L1 in CC cells by sponging miR-497-5p. Finally, our gain- and loss-of-function experiments evidenced that the lncRNA FGD5-AS1/miR-497-5p/PD-L1 axis regulates CC progression. Functionally, the data suggested that lncRNA FGD5-AS1 positively regulated while miR-497-5p negatively modulated malignant phenotypes, including cell proliferation, viability, invasion, migration, epithelial-mesenchymal transition (EMT), and tumorigenesis in CC cells. Interestingly, the inhibiting effects of lncRNA FGD5-AS1 ablation on CC development were abrogated by both silencing miR-497-5p and upregulating PD-L1. This study found that lncRNA FGD5-AS1 sponged miR-497-5p to upregulate PD-L1, resulting in CC progression, and provided novel agents for CC diagnosis and prognosis.

AMP-activated protein kinase β1 or β2 deletion enhances colon cancer cell growth and tumorigenesis.

Abnormal metabolism is a major hallmark of cancer and has been validated as a therapeutic target. Adenine monophosphate-activated protein kinase (AMPK), an αβγ heterotrimer, performs essential functions in cancer progression due to its central role in maintaining the homeostasis of cellular energy. While the contributions of AMPKα and AMPKγ subunits to cancer development have been established, specific roles of AMPKβ1 and AMPKβ2 isoforms in cancer development are poorly understood. Here, we show the functions of AMPKβ1 and AMPKβ2 in colon cancer. Specifically, deletion of AMPKβ1 or AMPKβ2 leads to increased cell proliferation, colony formation, migration, and tumorigenesis in HCT116 and HT29 colon cancer cells. Interestingly, the AMPKβ1 and AMPKβ2 isoforms have slightly different effects on regulating cancer metabolism, as colon cancer cells with AMPKβ1 knockout showed decreased rates of glycolysis-related oxygen consumption, while AMPKβ2 deletion led to enhanced rates of oxygen consumption due to oxidative phosphorylation. These results demonstrate that functional AMPKβ1 and AMPKβ2 inhibit growth and tumorigenesis in colon cancer cells, suggesting their potential as effective targets for colon cancer therapy.

Anticancer Properties of N,N-dibenzylasparagine as an Asparagine (Asp) analog, Using Colon Cancer Caco-2 Cell Line.

This study was conducted to investigate the potential anticancer properties of N, N-dibenzyl asparagine (NNDAsp), as an Asparagine (Asp) analog, using colon cancer Caco-2 cell and the normal NCM-460 cell line. Cell viability rate and levels of produced lactate dehydrogenase (LDH) were achieved upon treatment with NNDAsp compared to Asp treatment using MTT assay and LDH production kit. The protein expression profile of asparagine synthetase (ASNS) was achieved by using ELISA and flow cytometry assay. The levels of released inflammatory cytokines, including interleukin-1 alpha (IL-1α) and IL-1 beta (IL-β), were monitored using an ELISA assay. Our findings showed significant inhibition of colon cancer cell proliferation accompanied by a high level of produced LDH in a dose-dependent of an NNDAsp treatment without detectable toxic effect in normal cells. Interestingly, NNDAsp showed competitive inhibition of ASNS protein expression, in almost 3% of stained cancer cells, compared to 18% and 35% of untreated cells and cells pre-treated with Asp, respectively. Likewise, the concentration of ASNS protein was dramatically depleted in a dose and time-dependent of NNDAsp treatment in comparison with Asp treatment indicated by ELISA assay. Furthermore, as an apoptotic indicator, the expression of P53 and Caspase 3 (Caps3) was significantly increased in Caco-2 cells treated with NNDAsp at both RNA and protein levels. In contrast, their expression was markedly depleted in Asp-treated cells. In addition, the expression of both IL-1α and IL-1 β was markedly increased in Caco-2 cells in a dose and time-dependent of NNDAsp exogenous treatment. Moreover, targeting of ASNS by the Asp analog, NNDAsp, was further confirmed by the docking analysis of inhibitors ligands and crystal structure of ASNS protein. These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells.

2,3,5,6-Tetramethylpyrazine Targets Epithelial-Mesenchymal Transition by Abrogating Manganese Superoxide Dismutase Expression and TGFβ-Driven Signaling Cascades in Colon Cancer Cells.

Epithelial-mesenchymal transition (EMT) is a crucial process in which the polarized epithelial cells acquire the properties of mesenchymal cells and gain invasive properties. We have previously demonstrated that manganese superoxide dismutase (MnSOD) can regulate the EMT phenotype by modulating the intracellular reactive oxygen species. In this report, we have demonstrated the EMT-suppressive effects of 2,3,5,6-Tetramethylpyrazine (TMP, an alkaloid isolated from Chuanxiong) in colon cancer cells. TMP suppressed the expression of MnSOD, fibronectin, vimentin, MMP-9, and N-cadherin with a parallel elevation of occludin and E-cadherin in unstimulated and TGFβ-stimulated cells. Functionally, TMP treatment reduced the proliferation, migration, and invasion of colon cancer cells. TMP treatment also modulated constitutive activated as well as TGFβ-stimulated PI3K/Akt/mTOR, Wnt/GSK3/β-catenin, and MAPK signaling pathways. TMP also inhibited the EMT program in the colon cancer cells-transfected with pcDNA3-MnSOD through modulation of MnSOD, EMT-related proteins, and oncogenic pathways. Overall, these data indicated that TMP may inhibit the EMT program through MnSOD-mediated abrogation of multiple signaling events in colon cancer cells.

The Potential Mechanisms of Cinobufotalin Treating Colon Adenocarcinoma by Network Pharmacology.

Network pharmacology, as a novel way using bioinformatics to explore drug targets and interactions in cancer, broadens our understanding of drug action, thereby facilitating drug discovery. Here, we utilized network pharmacology to explore the role and mechanism by which cinobufotalin functions in colon adenocarcinoma (COAD). We found that cinobufotalin represses the growth and proliferation of colon cancer cells, and integrated public databases for targets reported to be associated with COAD, together with those predicted to be targets of cinobufotalin. Targets overlapped between COAD-associated proteins and cinobufotalin target proteins were used to filter candidate targets of cinobufotalin in COAD. The following proteins were thought to occupy a key position in COAD-cinobufotalin target networks: SRC, PIK3R1, MAPK1, PIK3CA, HSP90AA1, CTNNB1, GRB2, RHO1, PTPN11, and EGFR. The networks regulated by cinobufotalin were involved mainly in extracellular signal stimulation and transduction, including MAPK signaling pathway, PI3K-AKT signaling pathway, and JAK-STAT signaling pathway. Besides, transcriptome sequencing results also indicated that cinobufotalin inhibits the response of colon cancer cells to extracellular stimulation and promotes cell apoptosis. Molecular docking results showed that cinobufotalin matches in the pocket of the top candidate cinobufotalin target proteins (SRC, PIK3R1, MAPK1 and PIK3CA). These findings demonstrate cinobufotalin can be developed as potential anti-cancer therapeutics.

Identification and verification of the role of crucial genes through which methionine restriction inhibits the progression of colon cancer cells.

Studies have shown that methionine restriction (MR) can inhibit tumor progression, but its mechanism in colon cancer is unknown. Through DESeq2 and Edge analysis of the GSE72131 and GSE103602 datasets, 649 co-upregulated and 532 co-downregulated genes affected by MR were identified, respectively. Enrichment analysis showed that these genes were closely associated with tumor progression. Combined with the differentially expressed genes of colon cancer in The Cancer Genome Atlas database, MR affected 330 dysregulated genes in colon cancer. On this basis, a transcriptional regulatory and competing endogenous RNA network was established and F transcription factor 1 and microRNA 17-92a-1 Cluster Host Gene were identified as a key transcription factor and long non-coding RNA, respectively. In addition, four genes (FA Complementation Group I, Holliday Junction Recognition Protein, Karyopherin Subunit Alpha 2 and Kinesin Family Member 15) were identified by analyzing the relationship between dysregulated genes and overall survival. Finally, western blotting, reverse transcription-quantitative PCR, Transwell and other in vitro experiments verified that MR inhibits HCT116 colon cancer cell proliferation, metastasis and invasion, induces apoptosis and downregulates 6 hub genes. Collectively, the present study identified potential targets for MR to inhibit colon cancer progression and contributed to the clinical application of MR.

The novel FGFR inhibitor F1-7 induces DNA damage and cell death in colon cells.

Fibroblast growth factor receptor (FGFR) signaling influenced tumour occurrence and development. Overexpression of FGFR had been observed in many types of cancers, including colon cancer. FGFR inhibitor is considered to be effective in treating colon cancer patients. First, the kinase inhibition rate was determined. MTT, western blotting, colony formation, EdU and comet assays were performed to evaluate the anti-tumour effects of F1-7 in vitro. RNA-seq and bioinformatics analysis were used for further verification. Additionally, a xenograft model was generated to investigate the anti-tumour effect of F1-7. F1-7 can inhibit the proliferation of colon cancer cells in vitro. It could significantly inhibit FGFR phosphorylation and its downstream signaling pathway. Whole-genome RNA-seq analysis found that the changed genes were not only functionally focused on MAPK signaling pathway but also related to cell apoptosis and ferroptosis. Experimental evidence demonstrated that F1-7 can directly increase the level of cellular DNA damage. The occurrence of DNA damage led to cell cycle arrest and inhibition of cell metastasis and cell apoptosis. Mouse model experiments also confirmed that F1-7 could inhibit tumour growth by inhibiting the FGFR pathway. F1-7 exhibits anti-tumour activity by inhibiting the FGFR pathway. It could be a novel therapeutic agent for targeting colon cancer cells.

Abstract 864: Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2

Abstract Extracted from the root tuber of Ophiopogon japonicas, ophiopogon is well known to have an anti-cancer effect. However, the underlying mechanisms are still largely unknown. Here, we report that Ophiopogon D (OP-D) can inhibit colon cancer cell proliferation and induce apoptosis by inhibiting c-Myc expression through activation of p53 and CNOT2 regulation. Our results showed that OP-D induced p53 expression via ribosomal protein L5 or L11 and inhibited c-Myc expression through CNOT2 in a dose-dependent manner. Additionally, OP-D regulated cyclin D1 and CDK4 which are well known as cell cycle regulatory proteins. Consistently, OP-D inhibited the phosphorylation of AKT expression in a dose-dependent manner. Furthermore, OP-D shortened c-Myc’s half-life in a time-dependent manner. Furthermore, CNOT2 knockdown enhanced the inhibitory effect of OP-D on c-Myc in colon cancer cells. Interestingly, OP-D has increased the apoptotic effect of colon cancer cells when combined with doxorubicin or 5-FU, a treatment already used clinically. Altogether, our results suggested that OP-D regulates colon cancer cell survival and induces apoptosis by inhibiting c-Myc expression via activation of p53 and CNOT2 regulation. Citation Format: Hyun Min Ko, Wona Jee, Do-il Park, Somi Park, Ye-Rin Park, Hyeung-Jin Jang, Ji Hoon Jung. Ophiopogonin D increase apoptosis by activating p53 via ribosomal protein L5 and L11 and inhibiting the expression of c-Myc via CNOT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 864.

Abstract 3250: Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers

Abstract Colon cancer is the third leading cause in cancer related deaths in United States of America. Chemoresistance (drug resistance) of tumors is the primary reason for the failure of chemotherapy and a major cause of mortality in colon cancer. The molecular mechanisms involved in chemoresistance or methods to chemosensitization of cancer cells to chemotherapy remain elusive. Recent studies suggest that gut microbiota and microbial metabolites play a crucial role in the development and progression of colon cancer. Urolithin A (UroA) is a gut microbial metabolite derived from ellagitannin/ellagic acid rich-diets such as pomegranate and berries. Here, we identified that UroA and its novel structural analogue (UAS03) chemosensitize 5FU resistant (5FUR) colon cancer cells to 5FU treatment. Our data suggested that combination of 5FU with UroA or UAS03 significantly reduced cell viability of 5FUR colon cancer cell lines with combination index (CI) less than 1 suggesting their synergism. Moreover, UroA or UAS03 in combination with 5FU significantly inhibited 5FUR colon cancer cell proliferation, migration as well as induced apoptosis. Mechanistically, UroA or UAS03 treatment down regulated drug transporters (MDR1, BCRP and MRP2) leading to decreased efflux of drug (e.g., 5FU) and potentially responsible for chemosensitization. Furthermore, treatment with these compounds also modulated EMT makers (e.g., downregulated Snail and β-catenin; upregulated ZO-1 and E-cadherin) to reduce EMT transition. Treatment of UroA or UAS03 in combination with 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Taken together, utilization of UroA or UAS03 in combination with 5FU treatment may provide better therapeutic options for 5FUR colon cancer alleviation. Citation Format: Sweta Ghosh, Rajbir Singh, Zachary M. Vanwinkle, Haixun Guo, Praveen K. Vemula, Ajay Goel, Bodduluri Haribabu, Venkatakrishna Rao Jala. Microbial metabolite, Urolithin A acts as chemo-sensitizing adjuvant in conventional drug therapies via modulation of drug transporters and EMT markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3250.

Upregulation of INHBA mediated by the transcription factor BHLHE40 promotes colon cancer cell proliferation and migration

BackgroundColon cancer is highly prevalent, and cell proliferation and migration are major reasons for its progression to malignancy. The upregulation of INHBA, a glycoprotein hormone that regulates the secretion of pituitary hormones, is documented to be oncogenic in numerous cancers, consisting of breast, gastric, and ovarian cancer. Herein, we assessed the role of INHBA in the proliferation along with the migration of colon cancer cells.MethodsTCGA datasets were used to assess INHBA expression and its correlation with prognosis in colon cancer patients. Analyses on JASPAR, PROMO, and ENCODE databases, uncovered high correlation between INHBA and BHLHE40. Western blot and RT‐qPCR analysis were used to determine protein and mRNA levels. Cell transfection inhibited the expression of INHBA and BHLHE40. Cell proliferation rates were determined using CCK8 analysis. Wound healing assays were adopted to explore cell migration.ResultsINHBA is markedly elevated in colon cancer tissues along with cells and is a predictive factor for patient's prognosis with colon cancer. INHBA silencing suppressed colon cancer cell proliferation and migration. Furthermore, we confirmed the association of INHBA with BHLHE40 in colon cancer cells. BHLHE40 could directly modulates INHBA expression. Here, we show that BHLHE40 modulates the expression of INHBA, which influences the proliferation, and migration of colon cancer cells.ConclusionINHBA acts as an oncogene in colon cancer and it can be regulated by the transcription factor BHLHE40.

M7G-Related DNA Damage Repair Genes are Potential Biomarkers for Predicting Prognosis and Immunotherapy Effectiveness in Colon Cancer Patients.

Objective: m7G is a post-transcriptional modification modality, however, limited research has been conducted on its role in colon cancer. DNA damage repair (DDR) is an important factor that contributes to colon cancer development, growth and chemoresistance. This study aimed to explore whether m7G-related DNA damage repair genes may be used as biomarkers to predict the prognosis of colon cancer patients. Methods: We use non-negative matrix factorization (NMF) to type CRC patients into. Risk models were constructed using different expression genes in two clusters. We assessed the reliability of risk models with DCA curves, and a Nomogram. Meanwhile, The receiver operating characteristic and C-index curves were used to compare the predictive significance of the constructed risk models with other studies. In additional, we examined the significance of risk models on patients’ immunity microenvironment and response to immune therapy. Finally, we used a series of cellular experiments to validate the effect of model genes on the malignant progression of CRC cells. Results: Twenty-eight m7G genes were obtained from the GSEA database. Multivariate Cox and LASSO Cox regression analysis was performed and eleven m7G-related DDR genes were identified for constructing the risk model. Survival and stage of CRC patients were worser in the high-risk group than in the low-risk group for both the training and test sets. Additionally, the different immune microenvironment status of patients in the high- and low-risk groups, suggesting that patients in the low-risk group may be more sensitive to immunotherapy, particularly immune checkpoint inhibitors. Finally, we found that depletion of ATP2A1, one of the risk genes in our model, influence the biologic behaviour of CRC cells significantly. Conclusion: The m7G-related DDR genes can be used as important markers for predicting patient prognosis and immunotherapy response. Our data suggest that ATP2A1 may promote the proliferation of colon cancer cells. These findings may provide new therapeutic targets for the treatment of colon cancer.

Study on the Mechanism of Diosgenin Targeting STAT3 to Inhibit Colon Cancer Proliferation and Migration.

To elucidate regulatory effects and molecular mechanisms of diosgenin on colon cancer, this study administered diosgenin at concentrations of 10 (low), 50 (medium), and 100 μmol/L (high concentration group) at the cell level, respectively. EdU, colony formation, and Transwell assays were implemented to determine SW480 cellular proliferation and migration. Assays of flow cytometry and TUNEL were employed to estimate cell apoptosis. Additionally, nude mouse tumorigenesis assay was used to further verify the regulatory function of diosgenin on colon cancer. The target protein of diosgenin was predicted via molecular docking. The results showed that all three concentrations of diosgenin could reduce colon cancer cellular proliferation and migration, and after diosgenin treatment, colon cancer cellular apoptosis was markedly increased, and the 100 μmol/L diosgenin group produced the most satisfactory inhibition on colon cancer cell proliferation. Ki67 expression was markedly reduced whereas those of Bax and caspase3 were greatly increased after diosgenin treatment. The nude mouse tumorigenesis assay indicated that the parameters of tumorous volume and mass of diosgenin treatment group were greatly decreased as compared to control, and as the concentration of diosgenin increased, the inhibitory effect was more significant. Molecular docking indicated that STAT3 served as a target protein of diosgenin. Moreover, after diosgenin treatment on colon cancer cells, the STAT3 expression was markedly reduced. The STAT3 overexpression would counteract the inhibitory effect of 50 μmol/L diosgenin in both suppressing colon cancer cellular proliferation and migration and promoting apoptosis. Taken together, all our outcomes demonstrated the diosgenin effects in not only inhibiting colon cancer cellular proliferation and migration but also promoting cancerous cellular apoptosis. Diosgenin is a regulatory player in targeting and regulating STAT3.

Muscarinic receptor activation in colon cancer selectively augments pro-proliferative microRNA-21, microRNA-221 and microRNA-222 expression.

Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.

Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth.

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3’-[(1,1’-Biphenyl)-4’,4’-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers’ therapy.

Aspirin and celecoxib revealed anticancer and immunomodulatory effects on colon cancer cells

Colorectal cancer is one of the most common malignancies in the world. Clinical evidence suggests that non-steroidal anti-inflammatory drugs (NSAIDs ) were found to reduce the risk of colonic adenoma and colorectal cancer occurrence or recurrence and are more beneficial in acute inflammatory disorders than in chronic inflammatory diseases, implying that NSAIDs target the early stages of the inflammatory response. Celecoxib inhibits tumor initiation and tumor cell proliferation. Aspirin inhibits the constitutive isoform of the platelet enzyme cyclooxygenase- 1 (COX-1) and the inducible isoform cyclooxygenase-2 (COX-2) which is expressed by cytokines, and some growth factors. In this study, different concentrations of both Celecoxib and Aspirin in various sets were applied to investigate their effects on colon cancer cells (SW480) proliferation and cytokines production. Our result reported a significant decrease in cells viability after the treatment with aspirin and celecoxib alone or in combination. Celecoxib significantly decreased IL-6 levels, while Aspirin treatment showed no significant change in IL-6, IL-12, and TNF- α levels. In conclusion, non-steroidal anti-inflammatory drugs (NSAIDs) have pronounced anti-proliferative effects on colorectal cancer cell lines.

The Effects of Ovalbumin on Proliferation, Migration, and Stemness Properties of Chemoresistant SW480 Colon Cancer Cells

Egg peptides are factors in the embryonic environment with many significant biological activities, such as anticancer activity. Therefore, the current study investigates the effect of egg ovalbumin (OVA) on survival, cell cycle, self-renewal ability, stemness properties, and migration in SW480 colon cancer cells and 5-fluorouracil (5FU) resistant subgroup. MTT test was performed to assess cell viability. Flow cytometry was employed to analyze the cell cycle. Clonogenic assay and spheroid formation were used to assess the effect of OVA on self-renewal and stemness properties. Wound healing assay and RT-PCR were performed to analyze migration and gene mRNA expression. We demonstrated that OVA (8 and 12 mg/ml) attenuated cell viability, induced cell-cycle arrest, inhibited colony formation, and non-significantly reduced spheroid formation and migration in both cell lines. Furthermore, OVA downregulated the expression of Nanog, c-Myc, and NDRG1 in both cells, suggesting a stemness and self-renewal attenuation by OVA. In conclusion, OVA exposure inhibited the 5FU-SW480 chemo-resistant subpopulation growth by inducing cell cycle arrest and diminishing self-renewal and partially stemness properties of colon cancer cells.

Cytotoxic Activity, Cell Cycle Inhibition, and Apoptosis-Inducing Potential of Athyrium hohenackerianum (Lady Fern) with Its Phytochemical Profiling.

In the present study, we investigated the cytotoxic effects of Athyrium hohenackerianum ethanolic extract (AHEE) on the proliferation of breast, lung, and colon cancer cells. The AHEE was tested for its effect on the progression of the cell cycle, followed by induction of apoptosis determination by flow cytometry. Real-time qRT-PCR was also utilized to observe the initiation of apoptosis. In addition, GC-MS was performed in order to identify the active phytochemicals present in the AHEE. A cytotoxic activity with an IC50 value of 123.90 μg/mL against HCT-116 colon cancer cells was exhibited by AHEE. Following propidium iodide staining, annexin-V/PI, and clonogenic assays, AHEE treatment results in cell arrest in the S phase, causing an increase in the early and late phases of apoptosis and displaying antiproliferative potential, respectively. The morphological alterations were further monitored using acridine orange/ethidium bromide (AO/EB) staining. When compared with the control cells, features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed. The apoptosis was also confirmed by detecting the increased expression of p53 and caspase-3 along with the downregulation of Bcl-2. GC-MS analysis revealed that trans-linalool oxide, loliolide, phytol, 4,8,12,16-tetramethylheptadecan-4-olide, and gamma-sitosterol were the major phytochemical constituents. Based on these findings, it can be suggested that AHEE causes cellular death via apoptosis, which should be further explored for the identification of active compounds responsible for these observed effects. Therefore, AHEE can be used in the pharmaceutical development of anticancer agents for cancer therapeutics.

Tripartite motif 52 (TRIM52) promotes proliferation, migration, and regulation of colon cancer cells associated with the NF-κB signaling pathway.

With the advancement of early detection and treatment, the incidence of colon cancer (CC) has declined steadily worldwide; however, the mortality remains unacceptably high. Tripartite motif 52 (TRIM52) is a member of the family of highly conserved RBCC (a RING-finger, two B-boxes, and a predicted alpha-helical Coiled-Coil domain were linked to the N-terminal region in sequence) proteins with more than 70 isoforms, which plays an important role in tumorigenesis through different signaling pathways. How it regulates the development of CC remains unknown. Western blot was used to reveal that TRIM52 protein expression is up-regulated in CC cells. The Analysis of The Cancer Genome Atlas (TCGA) database was used to find the different expressions of TRIM52 between colon cancer tissues and normal colonic epithelial tissues. Cell proliferation assays, migration and invasion assays, and apoptosis were used to verify the changes in cell function after knockdown or overexpression of TRIM52 in CC cells. After that, the key proteins of the nuclear factor (NF)-κB signaling pathway were validated by western blot to explore the role of TRIM52 in the NF-κB signaling pathway. Finally, in order to explore the potential sites of TRIM52, LPS and PDTC were employed to activate and block the NF-κB signaling pathway, and the key proteins of the NF-κB signaling pathway were validated by western blot. TGCA database revealed that TRIM52 expression was elevated in CC tissues and correlated with prognosis. It was verified that TRIM52 promoted the proliferation, migration, and invasion of CC cells, and inhibited cell apoptosis. Most of the tripartite motif proteins (TRIMs) have ubiquitin ligase activity related to their highly conserved RING structure. Detection of the key proteins of the NF-κB signaling pathway in CC cells revealed that TRIM52 activated the NF-κB signaling pathway. We confirmed that TRIM52 promotes proliferation, migration, and invasion while inhibiting apoptosis of CC cells. The regulatory effect of TRIM52 on CC cells is related to the activation of the NF-κB signaling pathway. As TRIM52 acted as an upstream stimulator, stimulating the transfer of P65 into the nucleus to activate the NF-κB signaling pathway, it may provide a potential target for prognosis prediction and treatment of CC.