Autophagy In Colon Cancer Research Articles

An approach for an enhanced anticancer activity of ferulic acid-loaded polymeric micelles via MicroRNA-221 mediated activation of TP53INP1 in caco-2 cell line

Ferulic acid (FA) has powerful antioxidant and antitumor activities, but it has low bioavailability owing to its poor water solubility. Our aim is to formulate polymeric mixed micelles loaded with FA to overcome its poor solubility and investigate its potential anticancer activity via miRNA-221/TP53INP1 axis-mediated autophagy in colon cancer. A D-optimal design with three factors was used for the optimization of polymeric mixed micelles by studying the effects of each of total Pluronics mixture (mg), Pluronic P123 percentage (%w/w), and drug amount (mg) on both entrapment efficiency (EE%) and particle size. The anticancer activity of FA and Tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles formula (O2) was assessed by MTT and flow cytometry. O2 showed an EE% of 99.89%, a particle size of 13.86 nm, and a zeta potential of − 6.02 mv. In-vitro drug release studies showed a notable increase in the release rate of FA from O2, as compared to the free FA. The (IC50) values for FA from O2 and free FA were calculated against different cell lines showing a prominent IC50 against Caco-2 (17.1 µg/ml, 191 µg/ml respectively). Flow cytometry showed that FA caused cell cycle arrest at the G2/M phase in Caco-2. RT-PCR showed that O2 significantly increased the mRNA expression level of Bax and CASP-3 (4.72 ± 0.17, 3.67 ± 0.14), respectively when compared to free FA (2.59 ± 0.13, 2.14 ± 0.15), while miRNA 221 levels were decreased by the treatment with O2 (0.58 ± 0.02) when compared to free FA treatment (0.79 ± 0.03). The gene expression of TP53INP1 was increased by the treatment with O2 compared to FA at P < 0.0001. FA-loaded TPGS mixed micelles showed promising results for enhancing the anticancer effect of FA against colorectal cancer, probably due to its enhanced solubility. Thus, FA-loaded TPGS mixed micelles could be a potential therapeutic agent for colorectal cancer by targeting miRNA-221/TP53INP1 axis-mediated autophagy.

Novel GSH-responsive prodrugs derived from indole-chalcone and camptothecin trigger apoptosis and autophagy in colon cancer

Antineoplastic agents that target tubulin have shown efficacy as chemotherapeutic drugs, yet they are often constrained by multidrug resistance (MDR) and unwanted side effects. A multi-targeted strategy demonstrates great potency in reducing toxicity and enhancing efficacy and provides an alternative way for attenuating MDR. In this study, a series of dual-targeted anti-cancer agents based on indole-chalcone derivatives and the camptothecin (CPT) scaffold were synthesized. Among them, 14-1 demonstrated superior anti-proliferative activity than its precursor 13-1, CPT or their physical mixtures against tested cancer cells, including multidrug-resistant variants, while exhibited moderate cytotoxicity toward human normal cells. Mechanistic studies revealed that 14-1 acted as a glutathione-responsive prodrug, inducing apoptosis by substantially enhancing intracellular uptake of CPT, inhibiting tubulin polymerization, increasing the accumulation of intracellular reactive oxygen species, and initiating a mitochondrion-dependent apoptotic pathway.Moreover, 14-1 notably induced autophagy and suppressed topoisomerase I activity to further promote apoptosis. Importantly, 14-1 displayed potent inhibitory effect on tumor growth in paclitaxel (PTX)-resistant colorectal cancer (HCT-116/PTX) xenograft models without inducing obvious toxicity compared with CPT- or combo-treated group. These results suggest that 14-1 holds promise as a novel candidate for anti-cancer therapy, particularly in PTX-resistant cancers.

KLF4 targets RAB26 and decreases 5-FU resistance through inhibiting autophagy in colon cancer

ABSTRACT Background Accumulating studies demonstrated that resistance of colon cancer (CC) to 5-fluorouracil (5-FU) contributes to adverse prognosis. We investigated how Kruppel-like factor 4 (KLF4) affected 5-FU resistance and autophagy in CC cells. Methods KLF4 expression and its downstream target gene RAB26 in CC tissues was analyzed by bioinformatics analysis, and the effect of abnormal KLF4 expression on prognoses of CC patients was predicted. Luciferase reporter assay detected the targeted relationship between KLF4 and RAB26. The viability and apoptosis of CC cells were analyzed by CCK-8 and flow cytometry. The formation of intracellular autophagosomes was detected by confocal laser scanning microscopy and immunofluorescence staining. The mRNA and protein levels were assayed by qRT-PCR and western blot. A xenograft animal model was constructed to verify the function of KLF4. Rescue assay was employed to verify whether KLF4/RAB26 could affect 5-FU resistance in CC cells through autophagy. Results KLF4 and RAB26 were lowly expressed in CC. KLF4 correlated with patients’ survival. KLF4 was down-regulated in 5-FU resistant CC cells. KLF4 overexpression suppressed the proliferation and 5-FU resistance of CC cells, and inhibited LC3 II/I expression and autophagosome formation. Autophagy activator Rapamycin or sh-RAB26 treatment reversed the impact of KLF4 overexpression on 5-FU resistance. In vivo assay verified that KLF4 inhibited 5-FU resistance in CC cells. Rescue experiments revealed that KLF4 targeted RAB26 to inhibit CC cell autophagy, resulting in decreasing the resistance to 5-FU. Conclusion KLF4 strengthened the sensitivity of CC cells to 5-FU by targeting RAB26 to restrain autophagy pathway.

Ellagic acid induces apoptosis and autophagy in colon cancer through the AMPK/mTOR pathway

Ellagic acid (EA), found in fruits and foods, has been shown to be effective in the treatment of breast, colon and bladder cancer. However, due to the complexity of colon cancer, the therapeutic mechanism of EA for colon cancer is still unclear. Cell Counting Kit-8 (CCK-8) assay were employed to investigate the cell proliferation. Western blotting and flow cytometry assays were utilized to investigate apoptosis and autophagy in CRC cells (HCT116), respectively. Moreover, western blotting and luciferase reporter assays were evaluated the effect of EA on AMPK/mTOR pathway. Through flow cytometry analysis, EA could promote the apoptosis of HCT116 cells. In addition, EA can reduce the phosphorylation of mTOR, promoted phosphorylation of AMPK, and induced autophagy in HCT116 cells. Also, Dorsomorphin pretreatment can reduce the expression of autophagy protein, which indicates that EA induces autophagy through AMPK/mTOR pathway. These results suggest that EA inhibits the growth of colon cancer through AMPK/mTOR pathway and induces apoptosis and protective autophagy.

Methylation Drives SLC2A1 Transcription and Ferroptosis Process Decreasing Autophagy Pressure in Colon Cancer.

Colon cancer is a common malignant tumor in the digestive tract, with relatively high rates of morbidity and mortality. It is the third most common type of tumor in the world. The effective treatment of advanced colon cancer is limited, so it is particularly important to study the new pathogenesis of colon cancer. Ferroptosis is a nonapoptotic regulated cell death mode driven by iron-dependent lipid peroxidation, a process which has been discovered in recent years. Autophagy involves lysosomal degradation pathways that promote or prevent cell death. High levels of autophagy are associated with ferroptosis, but a clear association has not yet been made between ferroptosis and autophagy in colon cancer. Through the analysis of transcriptome expression profiling data in colon cancer, we obtained the common upregulated genes and downregulated genes by recording the intersection of the differentially expressed genes in each dataset. Solute Carrier Family 2 Member 1 (SLC2A1) was identified by combining autophagy genes obtained from GeneCards and ferroptosis genes obtained from FerrDb. In order to explore the clinical significance and prognostic value of SLC2A1, we utilized massive databases to conduct an in-depth exploration of the methylation of SLC2A1, and we also investigated the differences in immune infiltration between tumor and normal tissues. We found that there are abundant methylation sites in SLC2A1 and that the methylation of SLC2A1 is correlated with the immunosuppression of tumor tissue. We discovered that during the induction of environmental factors, the transcription and methylation levels of SLC2A1 were greatly increased, autophagy and ferroptosis were inhibited, and the immune system was defective, resulting in a poor prognosis for patients. These results suggest that the autophagy and ferroptosis-related gene SLC2A1 is involved in the tumor immune regulation of colon cancer, and SLC2A1 may become a new therapeutic target for colon cancer.

Inhibition of autophagy triggers melatonin-induced apoptosis in glioblastoma cells

BackgroundAutophagy is considered to be another restorative focus for the treatment of brain tumors. Although several research have demonstrated that melatonin induces autophagy in colon cancer and hepatoma cells, there has not been any direct evidence of whether melatonin is capable of inducing autophagy in human glioma cells.ResultsIn the present research, we report that melatonin or its agonist, agomelatine, induced autophagy in A172 and U87-MG glioblastoma cells for a concentration-and time-dependent way, which was significantly attenuated by treatment with luzindole, a melatonin receptor antagonist. Furthermore, by suppressing autophagy at the late-stage with bafilomycin A1 and early stage with 3-MA, we found that the melatonin-induced autophagy was activated early, and the autophagic flux was complete. Melatonin treatment alone did not induce any apoptotic changes in the glioblastoma cells, as measured by flow cytometry. Western blot studies confirmed that melatonin alone prominently upregulated the levels of Beclin 1 and LC3 II, which was accompanied by an increase in the expression of Bcl-2, whereas it had no effect on the expression of Bax in the glioblastoma cells. Remarkably, co-treatment with 3-MA and melatonin significantly enhanced the apoptotic cell population in the glioblastoma cells, along with a prominent decrease in the expression of bcl-2 and increase in the Bax expression levels, which collectively indicated that the disruption of autophagy triggers the melatonin-induced apoptosis in glioblastoma cells.ConclusionsThese results provide information indicating that melatonin may act as a common upstream signal between autophagy and apoptosis, which may lead to the development of new therapeutic strategies for glioma.

Functions and Implications of Autophagy in Colon Cancer.

Autophagy is an essential function to breakdown cellular proteins and organelles to recycle for new nutrient building blocks. In colorectal cancer, the importance of autophagy is becoming widely recognized as it demonstrates both pro- and anti-tumorigenic functions. In colon cancer, cell autonomous and non-autonomous roles for autophagy are essential in growth and progression. However, the mechanisms downstream of autophagy (to reduce or enhance tumor growth) are not well known. Additionally, the signals that activate and coordinate autophagy for tumor cell growth and survival are not clear. Here, we highlight the context- and cargo-dependent role of autophagy in proliferation, cell death, and cargo breakdown.

FuFangChangTai Decoction Activates Macrophages via Inducing Autophagy.

The traditional Chinese medicine decoction FuFangChangTai (FFCT) has been used in the therapy of colon cancer clinically, yielding alleviated toxicity and enhanced immunity. In our previous study, FFCT exerted its antitumor activity not only by inducing apoptosis but also by activating autophagy to eliminate tumor cells. However, its mechanism is not well understood. The purpose of this study was to investigate the relationship between macrophages activation and FFCT-induced autophagy. Results showed that FFCT could induce autophagy in colon cancer, as demonstrated by increased level of intracellular autophagy marker LC3 II in CT26.WT cells by fluorescence microscope and western blot assay. FFCT also facilitated numbers of vesicular bodies with bilayer membrane in CT26.WT cells, which were indicative of autophagosomes formation. Autophagosomes secreted by FFCT-treated CT26.WT cells can activate M1 type macrophages, accompanied with increased expression of costimulatory molecules CD86 and CD40 on the surface of RAW264.7 cells, and more inflammatory cytokines secretion, such as TNF-α, IL-6, MCP-1, and IL-1β. mRNA expressions of M2 macrophages markers, such as IL-10, CD206, Arg-1, and FIZZ-1, were downregulated. And this process helps regulate the polarization of macrophages and promote the immune response. These findings support a mechanism of FFCT-induced autophagy and provide novel evidence demonstrating that macrophages are involved in FFCT-induced autophagy progression.

Activation of the SphK1/ERK/p-ERK pathway promotes autophagy in colon cancer cells.

Sphingosine kinase 1 (SphK1) is a master kinase that catalyzes the synthesis of sphingosine 1 phosphate and participates in the regulation of cell proliferation and autophagy. The present study aimed to assess the effects of the activation of the SphK1/extracellular signal-regulated kinase (ERK)/phosphorylated (p-)ERK pathway in the regulation of autophagy in colon cancer (HT-29) cells. Inverted fluorescence microscopy was used to detect the expression of green fluorescent protein (GFP) in the SphK1-overexpressing HT-29 cells [SphK1(+)-HT-29] and the negative control HT-29 cells (NC-HT-29). Western blotting was used to detect the protein expression levels of SphK1, ERK1/2, p-ERK1/2, as well as those of the autophagy-associated markers LC3A, ATG5, and ULK1. Protein localization and expression of the LC3A antibody were detected by immunofluorescence. The results demonstrated that GFP was similarly expressed in SphK1(+)-HT-29 and NC-HT-29 cells. However, significantly increased SphK1 mRNA and protein expression levels were detected in SphK1(+)-HT-29 cells compared with in NC-HT-29 cells, which resulted in upregulated ERK/p-ERK. Furthermore, the protein expression levels of the three autophagy-associated markers increased. LC3A protein was localized in the cytoplasm of SphK1(+)-HT-29 cells, indicating autophagy. In summary, the findings of the present study suggested that activation of the SphK1/ERK/p-ERK pathway promotes autophagy in colon cancer HT-29 cells.

Expression analysis of LC3B and p62 indicates intact activated autophagy is associated with an unfavorable prognosis in colon cancer.

Autophagy is a lysosomal degradation and recycling process implicated in cancer progression and therapy resistance. We assessed the impact of basal autophagy in colon cancer (CC) in vitro and ex vivo. Functional autophagy was demonstrated in CC cell lines (LoVo; HT-29) showing a dose-dependent increase of the autophagy markers LC3B, p62 and autophagic vesciles upon increasing concentrations of the autophagy inhibitor chloroquine, which was demonstrated by immunoblotting, immunofluorescence and electron microscopy. Next, tissue microarrays with 292 primary resected CC, with cores from different tumor regions, and normal mucosa were analyzed by immunohistochemistry for LC3B and p62. CC tissue showed LC3B dot-like, p62 dot-like, cytoplasmic and nuclear staining in various levels without significant intratumoral heterogeneity. Tumoral LC3B and p62 expression was significantly higher than in normal tissue (p<0.001). No associations between staining patterns and pathological features (e.g. TNM categories; grading) were observed. Both low LC3B dot-like and low p62 dot-like-cytoplasmic staining were associated with worse overall survival (p=0.005 and p=0.002). The best prognostic discrimination, however, was seen for a combination of LC3B dot-like/p62 dot-like-cytoplasmic staining: high expression of both markers, indicative of impaired activated autophagy, was associated with the best overall survival. In contrast, high LC3B dot-like/low p62 dot-like-cytoplasmic expression, indicative of intact activated autophagy, was associated with the worst outcome (p<0.001 in univariate and HR=0.751; CI=0.607-0.928; p=0.008 in multivariate analysis). These specific expression patterns of LC3B and p62 pointing to different states of autophagy associated with diverging clinical outcomes highlighte the potential significance of basal autophagy in CC biology.

Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer.

Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed.

P-199 Degradation of CONNEXIN 43 in the plasma membrane by autophagy in colon cancer

P-199 Degradation of CONNEXIN 43 in the plasma membrane by autophagy in colon cancer

Effect of panitumumab on autophagy in colon cancer

e22151 Background: Panitumumab, a human monoclonal antibody raised against EGFR, has been approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) for the treatment of patients with EGFR-expressing mCRC and wild type kras. The ratio of reduced/oxidised form of glutathione (GSH/GSSG) is an indicator of the redox status in cells. The aim of the current study was to investigate the effect of panitumumab on the redox status of colon cancer cell lines Caco-2, DLD-1 and HT-29 regarding proliferation, apoptosis, necrosis, cell cycle arrest and autophagy. Methods: Cell proliferation was measured by MTT assay. Apoptosis and necrosis were detected by annexin v/propidium iodide assay. Cell cycle arrest was estimated by propidium iodide assay. Autophagy was detected by immunobloting and GSH levels were measured by spectrophotometrical analysis. kras mutations were detected by sequencing analysis. Results: Caco-2, DLD-1 and HT-29 cell lines differ in the expression levels of EGFR and HER-2. Kras mutation analysis in previous studies and in the current study showed that DLD-1 cells express mutated kras while Caco-2 and HT-29 cells express wild type of kras. Panitumumab decreased proliferation only in DLD-1 cells 48 h after its application besides the mutated kras. However, panitumumab did not affect DLD-1 cell apoptosis, necrosis or cell cycle progression 24 and 48 h after cells treatment. Interestingly, panitumumab increased protein levels of beclin 1, an indicator of autophagy, 24 h after its addition in cells. Moreover, an increase in GSH levels was noted 48 h after cells treatment with panitumumab. Conclusions: This is the first study to show that panitumumab, an EGFR inhibitor, affects colon cancer cell proliferation independently of kras mutations and EGFR protein levels through the induction of autophagy. The inhibition in cell proliferation was followed by an increase in GSH levels reflecting an imbalance on the redox status of cells. No significant financial relationships to disclose.

New anti-proliferative agent, MK615, from Japanese apricot “Prunus mume ” induces striking autophagy in colon cancer cells in vitro

瞄准：调查 MK615 的反肿瘤的效果，从日本李属的一篇摘录(李属亩我) ，对结肠癌房间。方法：三根结肠癌房间线， SW480，科罗拉多，和 WiDr，与 MK615 是有教养的。生长抑制被细胞增长试金评估，杀死活动由 lactate 脱氢酶试金是坚定的。apoptosis 的正式就职被附属建筑在 V 流动血细胞计数评估。词法变化被光和电子学习显微镜学，并且免疫荧光与 Atg8 染色。结果：MK615 禁止了生长和受溶解的 SW480，以一种剂量依赖者方式的科罗拉多和 WiDr 房间。Annexin V 流动血细胞计数证明 MK615 在 6 h 孵化，在点， apoptotic 房间的出现分别地为 SW480，科罗拉多，和 WiDr 房间是 68.0% ， 65.7% 和 64.7% 以后导致了 apoptosis。光和电子显微镜学，和与 Atg8 染色的免疫荧光表明 MK615 在所有三根房间线导致了巨大的细胞质的液泡(autophagosomes ) 。结论：MK615 对结肠癌房间有反肿瘤的效果。效果可以被 apoptosis 和 autophagy 的正式就职施加。