miR-495 sensitizes MDR cancer cells to the combination of doxorubicin and taxol by inhibiting MDR1 expression.

MDR1 is highly expressed in MDR A2780DX5 ovarian cancer cells, MDR SGC7901R gastric cancer cells and recurrent tumours. It pumps cytoplasmic agents out of cells, leading to decreased drug accumulation in cells and making cancer cells susceptible to multidrug resistance. Here, we identified that miR-495 was predicted to target ABCB1, which encodes protein MDR1. To reduce the drug efflux and reverse MDR in cancer cells, we overexpressed a miR-495 mimic in SGC7901R and A2780DX cells and in transplanted MDR ovarian tumours in vivo. The results indicated that the expression of MDR1 in the above cells or tumours was suppressed and that subsequently the drug accumulation in the MDR cells was decreased, cell death was increased, and tumour growth was inhibited after treatment with taxol-doxorubicin, demonstrating increased drug sensitivity. This study suggests that pre-treatment with miR-495 before chemotherapy could improve the curative effect on MDR1-based MDR cancer.

Erratum: Thioredoxin 1 supports colorectal cancer cell survival and promotes migration and invasion under glucose deprivation through interaction with G6PD: Erratum.

[This corrects the article DOI: 10.7150/ijbs.71809.].

Inhibition of thioredoxin-1 enhances the toxicity of glycolysis inhibitor 2-deoxyglucose by downregulating SLC1A5 expression in colorectal cancer cells.

BACKGROUND: Targeting glycolysis in cancer is an attractive approach for therapeutic intervention. 2-Deoxyglucose (2DG) is a synthetic glucose analog that inhibits glycolysis. However, its efficacy is limited by the systemic toxicity at high doses. Understanding the mechanism of 2DG resistance is important for further use of this drug in cancer treatment. METHODS: The expression of thioredoxin-1 (Trx-1) in colorectal cancer (CRC) cells treated with 2DG was detected by Western blotting. The effect of Trx-1 on the cytotoxicity of 2DG in CRC cells was examined in vitro and in vivo. The molecular mechanism involved in Trx-1-mediated activation of the SLC1A5 gene promoter activity was elucidated using in vitro models. RESULTS: Inhibition glycolysis with 2DG increased the expression of Trx-1 in CRC cells. Overexpression of Trx-1 decreased the cytotoxicity of 2DG, whereas knockdown of Trx-1 by shRNA significantly increased the cytotoxicity of 2DG in CRC cells. The Trx-1 inhibitor PX-12 increased the cytotoxicity of 2DG on CRC cells both in vitro and in vivo. In addition, Trx-1 promoted SLC1A5 expression by increasing the promoter activity of the SLC1A5 gene by binding to SP1. We also found that the SLC1A5 expression was upregulated in CRC tissues, and inhibition of SLC1A5 significantly enhanced the inhibitory effect of 2DG on the growth of CRC cells in vitro and in vivo. Overexpression of SLC1A5 reduced the cytotoxicity of 2DG in combination with PX-12 treatment in CRC cells. CONCLUSION: Our results demonstrate a novel adaptive mechanism of glycolytic inhibition in which Trx-1 increases GSH levels by regulating SLC1A5 to rescue cytotoxicity induced by 2DG in CRC cells. Inhibition of glycolysis in combination with inhibition of Trx-1 or SLC1A5 may be a promising strategy for the treatment of CRC.

Nuclear translocation of thioredoxin-1 promotes colorectal cancer development via modulation of the IL-6/STAT3 signaling axis through interaction with STAT3.

Background: Thioredoxin 1 (Trx-1) is a small redox protein predominantly localized in the cytoplasm. Its expression is increased in several cancers, including colorectal cancer (CRC). However, the function of Trx-1 translocation to the nucleus in cancer is not clear. In this study, we investigated the role of Trx-1 nuclear translocation in development of CRC. Methods: Expression of Trx-1 and STAT3 was analyzed by Western blot and immunofluorescence. Endogenous interaction of Trx-1, STAT3, and karyopherin α1 in CRC cells was analyzed by co-immunoprecipitation. Trx-1 and pSTAT3 nuclear staining in human CRC tissues was analyzed by immunohistochemistry. A mouse model of AOM/DSS induced colitis-associated cancer (CAC) was utilized to investigate the antitumor effect of PX-12, a Trx-1 inhibitor. A knockin mouse with the Txn1(KK81-82EE) mutation was generated via CRISPR/Cas9, and CAC was induced in knockin and wild-type mice. Results: Nuclear translocation of Trx-1 was induced by IL-6, and inhibition of this translocation reversed IL-6-induced epithelial-to-mesenchymal transition, invasion and metastasis. Karyopherin α1 was found to specifically mediate IL-6-induced translocation of the Trx-1-pSTAT3 complex into the nucleus. Nuclear Trx-1 expression was closely correlated with lymph node metastasis and distant metastasis in human CRC. In addition, nuclear staining of Trx-1 showed significant positive correlation with nuclear staining of pSTAT3 in human CRC tissues. PX-12, an inhibitor of Trx-1, significantly impaired the activation of STAT3 and suppressed the development of AOM/DSS-induced CAC in mice. Moreover, AOM/DSS-induced nuclear Trx-1 expression was suppressed in Txn1(KK81-82EE) mice, which inhibited STAT3 activation and cancer progression. Conclusions: These results provide new insights into the mechanisms of STAT3 activation triggered by IL-6 and identify nuclear translocation of Trx-1 as a potential therapeutic target for the treatment of CRC and CAC.

The Curcumin Analog Da0324 Inhibits the Proliferation of Gastric Cancer Cells via HOTAIRM1/miR-29b-1-5p/PHLPP1 Axis.

Background: Our previous study has shown that Da0324, a curcumin analog, exhibited significantly improved stability and antitumor activity. However, the molecular mechanisms of action of Da0324 remain poorly understood. Long non-coding RNA (lncRNA) has been shown to play a key role in tumor progression. Here, we aim to investigate the molecular mechanisms underlying the anti-cancer activity of Da0324 by regulating the lncRNA HOTAIRM1. Methods: Gastric cancer cell lines were treated with Da0324 and/or transfected with lentiviral vector expressing HOTAIRM1 shRNA, and/or miR-29b-1-5p mimics and/or small interference RNA (siRNA) against PHLPP1, or HOTAIRM1 siRNA or lentiviral vector expressing HOTAIRM1, as needed. The expression of HOTAIRM1, miR-29b-1-5p and PHLPP1 in GC cells was determined by Real-Time PCR. Cell growth was examined by CCK-8 assay and colony formation assay in vitro. The targeted relationship between HOTAIRM1 and miR-29b-1-5p was verified by luciferase reporter gene assay. PHLPP1 protein expression was examined by Western blotting. Results: Da0324 increased the expression of HOTAIRM1 in GC cells. HOTAIRM1 expression was significantly down-regulated in GC tissues, and the low expression of HOTAIRM1 was associated with the shorter survival rate of GC patients based on the TCGA database. Knockdown of HOTAIRM1 promoted GC cell proliferation whereas overexpression of HOTAIRM1 inhibited GC cell proliferation as demonstrated by CCK-8 and colony formation assays. Moreover, knockdown of HOTAIRM1 reversed the Da0324-mediated growth inhibition of GC cells. Furthermore, HOTAIRM1 acted as a sponge for miR-29b-1-5p and PHLPP1 is regulated by the HOTAIRM1/miR-29b-1-5p axis in GC cells. Overexpression of miR-29b-1-5p or knockdown of PHLPP1 reversed the ability of Da0324 to inhibit the growth of GC cells. Conclusions: Our data suggest that Da0324 exerts antitumor activity by regulating HOTAIRM1/miR-29b-1-5p/PHLPP1 axis in GC cells, and provide new insights into the anti-cancer mechanism of Da0324.

Thioredoxin 1 supports colorectal cancer cell survival and promotes migration and invasion under glucose deprivation through interaction with G6PD.

Overcoming energy stress is a critical step for cells in solid tumors. Under this stress microenvironment, cancer cells significantly alter their energy metabolism to maintain cell survival and even metastasis. Our previous studies have shown that thioredoxin-1 (Trx-1) expression is increased in colorectal cancer (CRC) and promotes cell proliferation. However, the exact role and mechanism of how Trx-1 is involved in energy stress are still unknown. Here, we observed that glucose deprivation of CRC cells led to cell death and promoted the migration and invasion, accompanied by upregulation of Trx-1. Increased Trx-1 supported CRC cell survival under glucose deprivation. Whereas knockdown of Trx-1 sensitized CRC cells to glucose deprivation-induced cell death and reversed glucose deprivation-induced migration, invasion, and epithelial-mesenchymal transition (EMT). Furthermore, we identified glucose-6-phosphate dehydrogenase (G6PD) interacting with Trx-1 by HuPortTM human protein chip, co-IP and co-localization. Trx-1 promoted G6PD protein expression and activity under glucose deprivation, thereby increasing nicotinamide adenine dinucleotide phosphate (NADPH) generation. Moreover, G6PD knockdown sensitized CRC cells to glucose deprivation-induced cell death and suppressed glucose deprivation-induced migration, invasion, and EMT. Inhibition of Trx-1 and G6PD, together with inhibition of glycolysis using 2-deoxy-D-glucose (2DG), resulted in significant anti-tumor effects in CRC xenografts in vivo. These findings demonstrate a novel mechanism and may represent a new effective therapeutic regimen for CRC.

Construction and Validation of a Novel Ferroptosis-Related lncRNA Signature to Predict Prognosis in Colorectal Cancer Patients.

Background: Colorectal cancer (CRC) ranks as the third most common malignancy worldwide but a reliable prognostic biomarker of CRC is still lack. Thus, the purpose of our study was to explore whether ferroptosis - related lncRNAs could predict the prognosis of CRC. Methods: The mRNA expression profiling of colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) patients in The Cancer Genome Atlas (TCGA) database were downloaded. Univariate Cox and multivariate Cox regression analyses was used to obtain prognostic differently expressed ferroptosis-related lncRNAs (DE-FLs) and a risk signature was developed. Quantitative polymerase chain reaction (q-PCR) was used to validated the different expressions of DE-FLs. The calibration curves, C-index and the receiver operating characteristic (ROC) curves were applied to evaluate the accuracy of nomogram. Gene set enrichment analyses (GSEA) were carried out to explore the biological mechanism between high- and low-risk group and the potential regulated pathway of prognostic DE-FLs in CRC. Results: Forty-nine DE-FLs were identified between CRC and normal tissue. Then, a 4-DE-FLs (AC016027.1, AC099850.3, ELFN1-AS1, and VPS9D1-AS1) prognostic signature model was generated. AC016027.1 was downregulated in CRC tissue; VPS9D1-AS1 and ELFN1-AS1 were upregulated by q-PCR. The model had a better accuracy presenting by 1-, 3-, and 5-years ROC curve (AUC ≥0.6), and identified survival probability (p < 0.05) well. Moreover, the risk signature could play as an independent factor of CRC (p < 0.05). Further, a nomogram including age, pathologic stage, T stage, and risk score with good prognostic capability (C-index = 0.789) was constructed. In addition, we found biological pathways mainly related to metabolism and apoptosis were down-regulated in high-risk group who with poor outcome. Finally, the functional enrichment showed prognostic DE-FLs may significantly impact bile secretion in CRC. Conclusion: A risk model and nomogram based on ferroptosis-related lncRNAs were created to predict 1-, 3-, and 5-years survival probability of CRC patients. Our data suggested that the prognostic lncRNAs could serve as valuable prognostic marker.

Exosome-transmitted miRNA-335-5p promotes colorectal cancer invasion and metastasis by facilitating EMT via targeting RASA1.

Exosomal microRNA (miRNA) secretion has been characterized as a vital factor in intercellular communication among cancer cells. However, little is known about cancer-secreted miRNAs specifically involved in metastasis of colorectal cancer (CRC). Here, we found that exosomes derived from metastatic CRC cell line SW620 promoted migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells. The profiling of exosome miRNAs revealed that microRNA (miR)-335-5p was highly expressed in exosomes from metastatic SW620 cells compared to those derived from primary SW480 cells. miR-335-5p was transmitted from metastatic SW620 cells to CRC cells via exosomes and promoted migration, invasion, and EMT of CRC cells. Moreover, exosome-transmitted miRNA-335-5p promotes CRC cell invasion and metastasis by facilitating EMT via targeting RAS p21 protein activator 1 (RASA1). Overexpression of RASA1 abolished the promotive effects of exosomal miR-335-5p on CRC cell migration, invasion, and EMT. Collectively, our data revealed that exosomal miR-335-5p derived from metastatic CRC cells promotes CRC cell invasion and metastasis by facilitating EMT via targeting RASA1, which may serve as a potential therapeutic target for CRC metastasis.

BCAT1 decreases the sensitivity of cancer cells to cisplatin by regulating mTOR-mediated autophagy via branched-chain amino acid metabolism.

Cisplatin is one of the most effective chemotherapy drugs and is widely used in the treatment of cancer, including hepatocellular carcinoma (HCC) and cervical cancer, but its therapeutic benefit is limited by the development of resistance. Our previous studies demonstrated that BCAT1 promoted cell proliferation and decreased cisplatin sensitivity in HCC cells. However, the exact role and mechanism of how BCAT1 is involved in cisplatin cytotoxicity remain undefined. In this study, we revealed that cisplatin triggered autophagy in cancer cells, with an increase in BCAT1 expression. The cisplatin-induced up-regulation of BCAT1 decreased the cisplatin sensitivity by regulating autophagy through the mTOR signaling pathway. In addition, branched-chain amino acids or leucine treatment inhibited cisplatin- or BCAT1-mediated autophagy and increased cisplatin sensitivity by activating mTOR signaling in cancer cells. Moreover, inhibition of autophagy by chloroquine increased cisplatin sensitivity in vivo. Also, the knockdown of BCAT1 or the administration of leucine activated mTOR signaling, inhibited autophagy, and increased cisplatin sensitivity in cancer cells in vivo. These findings demonstrate a new mechanism, revealing that BCAT1 decreases cisplatin sensitivity in cancer cells by inducing mTOR-mediated autophagy via branched-chain amino acid leucine metabolism, providing an attractive pharmacological target to improve the effectiveness of chemotherapy.

Sodium Butyrate Selectively Kills Cancer Cells and Inhibits Migration in Colorectal Cancer by Targeting Thioredoxin-1.

BACKGROUND: Sodium butyrate (NaB) is a short-chain fatty acid which is produced by bacterial fermentation of nondigestible dietary fiber and has been reported to exert anti-tumor effects in many tumors including colorectal cancer (CRC). However, the role of thioredoxin-1 (Trx-1) in NaB-induced anti-tumor effect has not been completely clarified. MATERIALS AND METHODS: Effects of NaB on the growth of CRC cell lines HT29 and SW480 were detected by the Cell Counting Kit-8 (CCK-8) and colony formation assays. The apoptotic cells were determined by flow cytometry, and cell migration was assessed by a Transwell assay. Western blot analysis was used to test the Trx-1 and epithelial-to-mesenchymal transition (EMT)-related proteins level. Reactive oxygen species (ROS) level was determined and N-acetylcysteine (NAC) recovery experiment was performed in CRC cells. In addition, mice xenograft model was established to test the effect of NaB on CRC growth in vivo. Further, the effects of NaB on CRC cells with overexpression or knockdown were tested by the CCK-8 and Transwell assays. RESULTS: NaB treatment significantly inhibited cell growth and decreased Trx-1 protein expression in CRC cells but not in normal colon epithelial cells. NaB also induced apoptosis, inhibited colony formation, migration and EMT in CRC cells. Besides, NaB increased ROS level in CRC cells and NAC reversed NaB-induced inhibition of cell proliferation. Moreover, downregulation of Trx-1 significantly enhanced NaB-induced inhibitory effects on cell growth and migration, whereas overexpression of Trx-1 attenuated NaB-induced inhibitory effects on growth and migration in CRC cells. CONCLUSION: These findings indicate that the NaB-mediated anti-tumor effects on CRC cells are related to downregulation of Trx-1.

A Novel DNA Aptamer Targeting S100P Induces Antitumor Effects in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a prevalent malignancy with poor prognosis and survival. As a Ca2+ binding protein, S100P plays a role in calcium-dependent signal transduction pathways that involve in diverse biological processes. Our previous studies have shown that S100P is overexpressed in CRC tissues and regulates cell growth, invasion, and metastasis in CRC. Therefore, S100P is expected to be an effective target for CRC therapy. Aptamers are short single-stranded oligonucleotides that could serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purposes. In this study, we generated a novel DNA aptamer against S100P (AptS100P-1) by way of the SELEX process and high-throughput sequencing. The binding assay showed that AptS100P-1 had a high affinity for S100P protein. Further experiments indicated that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, AptS100P-1 was capable of binding to cells and had an inhibitory effect on CRC cell growth in vitro and in vivo. Also, AptS100P-1 inhibited the migration and epithelial-mesenchymal transition of CRC cells expressing S100P. These results indicate a novel DNA aptamer targeting S100P, which might be a potential therapeutic strategy for targeting S100P against S100P-expressing CRC.

Increased Thioredoxin-1 Expression Promotes Cancer Progression and Predicts Poor Prognosis in Patients with Gastric Cancer.

BACKGROUND: Thioredoxin-1 (Trx-1) is a small redox protein, which plays an important role in many biological processes. Although increased expression of Trx-1 in various solid tumors has been reported, the prognostic significance and function of Trx-1 in human gastric cancer (GC) are still unclear. Here, we investigated the clinical and prognostic significance of Trx-1 expression and the function and mechanism of Trx-1 in human GC. METHODS: We analyzed Trx-1 mRNA expression from the GEO database and Trx-1 protein expression in 144 GC tissues using immunohistochemistry. Effects of Trx-1 on GC cell were assessed in vitro and in vivo through Trx-1 knockdown or overexpression. The antitumor effects of the Trx-1 inhibitor, PX-12, on GC cells were investigated. PTEN and p-AKT expressions were evaluated by Western blotting. RESULTS: Increased Trx-1 expression was found in GC tissues and associated with poor prognosis and aggressive clinicopathological characteristics in patients with GC. High Trx-1 expression predicted poor prognosis, and its expression was an independent prognostic factor for overall survival of GC patients. Knockdown of Trx-1 expression inhibited GC cell growth, migration, and invasion in vitro and tumor growth and lung metastasis in vivo. Conversely, overexpression of Trx-1 promoted GC cell growth, migration, and invasion. We also found that PX-12 inhibited GC cell growth, migration, and invasion. Overexpression of Trx-1 caused a decrease in PTEN and increase in p-AKT levels whereas silencing Trx-1 caused an increase in PTEN and decrease in p-AKT levels in GC cells. Inhibition of AKT signaling pathway by MK2206 also inhibited GC cell growth, migration, and invasion. CONCLUSION: Our results indicate that Trx-1 may be a promising prognostic indicator and therapeutic target for GC patients.