Copper deprivation enhances the chemosensitivity of pancreatic cancer to rapamycin by mTORC1/2 inhibition.

Cuproplasia, or copper-dependent cell proliferation, has been observed in varieties of solid tumors along with aberrant copper homeostasis. Several studies reported good response of patients to copper chelator assisted neoadjuvant chemotherapy, however, the internal target molecules are still undetermined. Unravel copper-associated tumor signaling would be valuable to forge new links to translate biology of copper into clinical cancer therapies. We evaluated the significance of high-affinity copper transporter-1 (CTR1) by bioinformatic analysis, and in 19 pairs of clinical specimens. Then, with the help of gene interference and chelating agent, enriched signaling pathways were identified by KEGG analysis and immunoblotting. Accompanying biological capability of pancreatic carcinoma-associated proliferation, cell cycle, apoptosis, and angiogenesis were investigated. Furthermore, a combination of mTOR inhibitor and CTR1 suppressor has been assessed in xenografted tumor mouse models. Hyperactive CTR1 was investigated in pancreatic cancer tissues and proven to as the key point of cancer copper homeostasis. Intracellular copper deprivation induced by CTR1 gene knock-down or systematic copper chelation by tetrathiomolybdate suppressed proliferation and angiogenesis of pancreatic cancer cell. PI3K/AKT/mTOR signaling pathway was suppressed by inhibiting the activation of p70(S6)K and p-AKT, and finally inhibited mTORC1 and mTORC2 after copper deprivation. Additionally, CTR1 gene silencing successfully improved the anti-cancer effect of mTOR inhibitor rapamycin. Our study reveals that CTR1 contributes to pancreatic tumorigenesis and progression, by up-regulating the phosphorylation of AKT/mTOR signaling molecules. Recovering copper balance by copper deprivation addresses as promising strategy for improved cancer chemotherapy.

EGCG Enhances the Chemosensitivity of Colorectal Cancer to Irinotecan through GRP78-MediatedEndoplasmic Reticulum Stress.

This study aimed to explore the role of GRP78-mediated endoplasmic reticulum stress (ERS) in the synergistic inhibition of colorectal cancer by epigallocatechin-3-gallate (EGCG) and irinotecan (IRI). Findings showed that EGCG alone or in combination with irinotecan can significantly promote intracellular GRP78 protein expression, reduce mitochondrial membrane potential and intracellular ROS in RKO and HCT 116 cells, and induce cell apoptosis. In addition, glucose regulatory protein 78 kDa (GRP78) is significantly over-expressed in both colorectal cancer (CRC) tumor specimens and mouse xenografts. The inhibition of GRP78 by small interfering RNA led to the decrease of the sensitivity of CRC cells to the drug combination, while the overexpression of it by plasmid significantly increased the apoptosis of cells after the drug combination. The experimental results in the mouse xenografts model showed that the combination of EGCG and irinotecan could inhibit the growth of subcutaneous tumors of HCT116 cells better than the two drugs alone. EGCG can induce GRP78-mediated endoplasmic reticulum stress and enhance the chemo-sensitivity of colorectal cancer cells when coadministered with irinotecan.

Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution.

The analysis of single living cells, including intracellular delivery and extraction, is essential for monitoring their dynamic biochemical processes and exploring intracellular heterogeneity. However, owing to the 2D view in bright-field microscopy and optical distortions caused by the cell shape and the variation in the refractive index both inside and around the cells, achieving spatially undistorted imaging for high-precision manipulation within a cell is challenging. Here, an accurate and visual system is developed for single-cell spatial manipulation by correcting the aberration for simultaneous bright-field triple-view imaging. Stereo information from the triple view enables higher spatial resolution that facilitates the precise manipulation of single cells. In the bright field, we resolved the spatial locations of subcellular structures of a single cell suspended in a medium and measured the random spatial rotation angle of the cell with a precision of ±5°. Furthermore, we demonstrated the visual manipulation of a probe to an arbitrary spatial point of a cell with an accuracy of <1 pixel. This novel system is more accurate and less destructive for subcellular content extraction and drug delivery.

EGCG synergizes the therapeutic effect of irinotecan through enhanced DNA damage in human colorectal cancer cells.

Irinotecan is a kind of alkaloid with antitumour activity, but its low solubility and high toxicity limit its application. Epigallocatechin-3-gallate (EGCG) is one of the main bioactive components in tea. The epidemiological investigation and animal and cell experiments show that EGCG has a preventive and therapeutic effect on many kinds of tumours. Here, colorectal cancer cells RKO and HCT116 were employed, and the CCK8 proliferation test was used to screen the appropriate concentration of EGCG and irinotecan, and the effects of single and/or combined drugs on migration, invasion, DNA damage, cell cycle and autophagy of tumour cells were investigated. The results showed that EGCG combined with irinotecan (0.5 µmol L- ) not only had a stronger inhibitory effect on tumour cells than EGCG or irinotecan alone but also prevented tumour cell migration and invasion. EGCG alone did not cause DNA damage in colorectal cancer cells, but its combination with irinotecan could induce S or G2 phase arrest by inhibiting topoisomerase I to cause more extensive DNA damage. EGCG also induced apoptosis by promoting autophagy with irinotecan synergistically. These results indicated that EGCG in combination with irinotecan could be a promising strategy for colorectal cancer.

Usnic Acid Inhibits Proliferation and Migration through ATM Mediated DNA Damage Response in RKO Colorectal Cancer Cell.

BACKGROUND: Usnic Acid (UA), also known as lichenol, has been reported to have inhibitory effects on a variety of cancer cells, but its specific mechanism remained to be elucidated. Tumor chemotherapy drugs, especially DNA damage chemotherapeutic drugs, target Chromosomal DNA, but their spontaneous and acquired drug resistance are also an urgent problem to be solved. Therefore, drug combination research has become the focus of researchers. METHODS: Here, we evaluated the tumor-suppressing molecular mechanism of UA in colorectal cancer cells RKO from the perspective of the ATM-mediated DNA damage signaling pathway through H2O2 simulating DNA damage chemotherapeutic drugs. CCK8 cell proliferation assay was used to determine the inhibition of RKO cells by hydrogen peroxide and UA alone or in combination, and wound healing assay was applied to determine the effect of the drug on cell migration. RESULTS: Transfected cells with miRNA18a-5p mimics and inhibitors, MDC and DCFH-DA staining for the measurement of autophagy and ROS, cell cycle and apoptosis were detected by flow cytometry, expressions of microRNA and mRNA were determined by fluorescence quantitative PCR, and protein by Western blot. DISCUSSION: We found that UA can upregulate ATM via miR-18a to activate the DNA damage signaling pathway and inhibit the proliferation and migration of RKO cells in a concentration-dependent manner. CONCLUSION: At the same time, DNA damage responses, including cell cycle, autophagy, apoptosis and ROS levels, are also regulated by UA. Therefore, UA combined with DNA damage chemotherapeutic drugs may be an effective treatment for cancer.

[Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer].

OBJECTIVE: To examine copper transporter 1 (CTR1) expression in pancreatic carcinoma cells, orthotopic xenograft pancreatic tumor model and clinical samples, and verify the effect of copper chelating agent ammonium tetrathiomolybdate (TM) regulate the expression of CTR1 in pancreatic carcinoma cells and the inhibition of pancreatic carcinoma. METHODS: The expressions of copper transporter CTR1 and antioxidant protein 1 (ATOX1) in 22 clinical pancreatic ductal carcinoma and paracancer tissues 0.5-1 cm away from the tumor were measured by immunohistochemistry (IHC). PANC-1 cells were used to construct 5 orthotopic xenograft pancreatic tumor of nude mice models. Pancreatic cancer tissues and corresponding normal pancreatic tissues were collected, and the expressions of CTR1 and ATOX1 were detected by IHC and compared with clinical tissues. The proliferation of pancreatic carcinoma cells PANC-1 treated with 10, 30, 50, 100 µmol/L TM for 24 h, 48 h, 72 h was measured by CCK8 assay. The migration abilities of PANC-1 cells treated with 50 µmol/L TM for 24 h, 48 h were detected by scratch test. The expressions of CTR1, vascular endothelial growth factor (VEGF) and CyclinD1 proteins in PANC-1 cells treated with 10, 30, 50, 100 µmol/L TM for 48 h were measured by Western blot. Then the subcutaneous tumor-bearing model of nude mice were established with PANC-1 cells, and the growth of tumor was observed after oral administration of 0.3 mg/d and 1.0 mg/d of TM, respectively. RESULTS: The immunohistochemical results indicated that 19 of the 22 clinical pancreatic ductal cancer tissues of carcinoma patients had high expression of CTR1, and the same high expression of CTR1 was found in the orthotopic transplanted tumor tissues of PANC-1 nude mice. The proliferation inhibition of PANC-1 cells increased with the concentration of TM increased and the treatment time prolonged. The expressions of intracellular CTR1, VEGF and CyclinD1 all decreased with the concentration of TM increased. The cell migration ability decreased after the PANC-1 cells treated with TM. The tumor growth of PANC-1 tumor-bearing nude mice was inhibited after different doses of TM were delivered. The reduction in tumor volume and weight was more pronounced in the high-dose TM group (P<0.05). CONCLUSION: The expression of CTR1 is abnormally elevated in pancreatic carcinoma, and treatment with copper chelating agent for this target may help to inhibit pancreatic carcinoma.

Metformin mediated microRNA-7 upregulation inhibits growth, migration, and invasion of non-small cell lung cancer A549 cells.

Metformin, a medication widely used in the treatment of type 2 diabetes mellitus, has a possible antitumor effect in type 2 diabetes mellitus patients. MicroRNA-7 is a significant microRNA in non-small cell lung cancer. Metformin has an inhibitory effect on lung cancer and regulates the expression of certain microRNAs, but there is no report connecting metformin with microRNA-7 in lung cancer. Thus, we used qPCR to measure microRNA-7 expression in A549 non-small cell lung cancer cells treated with metformin. We used CCK8, cell scratch, and Transwell assays to test the growth, migration, and invasion of A549 cells. Western blotting was used to measure the expression level of relevant proteins in A549 cells. We found that microRNA-7 was dramatically upregulated by metformin via AMPK in a dose- and time-dependent manner. Both metformin and microRNA-7 mimic reduced A549 cell growth, migration, and invasion. Metformin downregulated the levels of p-NF-κB p65, p-Erk1/2, p-AKT, and p-mTOR proteins. The treatment with the microRNA-7 mimic had the same result. The decrease of these proteins caused the inhibition of A549 cell growth, migration, and invasion. Our discovery revealed that metformin, via increasing the expression of microRNA-7 mediated by AMPK, regulates the AKT/mTOR, MAPK/Erk, and NF-κB signaling pathways, thereby suppressing A549 cell growth, migration, and invasion.

[The Effect of Methylation Level of microRNA Promoter on the Expression of microRNAs and on the Proliferation, Migration and Invasion of Lung Cancer Cells].

OBJECTIVE: To study the effect of methylation level of microRNA promoter on the expression of microRNAs (miRNA34a, miRNA34b, miRNA148a, miRNA203a) and on the proliferation, migration and invasion of lung cancer A549 cells. METHODS: The proliferation of A549 cells treated with different concentrations of demethylated drug 5-aza-2'-deoxycytidine (5-Aza-CdR) was measured by CCK8 assay and calculated the inhibitory rate in 24 h, 48 h and 72 h, respectively. After 72 h of treatment with 20 µmol/L 5-Aza-CdR, methylation-specific PCR (MSP) was used to detect the methylation level of A549 cells in miRNAs gene promoter regions, and real-time quantitative PCR (real-time PCR) was used to test the expression of miRNAs. The migration abilities of A549 cells treated with 20 µmol/L 5-Aza-CdR in 24 h and 48 h were performed with wound healing assay, while the invasion abilities in 48 h were evaluated by Transwell assay, respectively. RESULTS: The proliferation inhibition rate of A549 cells gradually increased with the treatment concentration of 5-Aza-CdR increased and the treatment time prolonged. Compared with the control group, the methylated band of the experimental group was weaker and the unmethylated band was stronger, and the miRNAs gene promoter regions methylation level of the experimental group was lower than that of the control group. The expression level of miRNAs was significantly increased in the experimental group (P<0.05) . The migration and invasion of the experimental group of A549 cells were inhibited compared with the control group (P<0.05) . CONCLUSION: 5-Aza-CdR can reverse methylation levels of miRNAs promoter regions and upregulate the expression level of miRNA34a, miRNA34b, miRNA148a, miRNA203a, resulting in significantly inhibiting the proliferation, migration and invasion of lung cancer cells.

[The Study of Methylated Regulation MicroRNA in Pancreatic Carcinoma Cell and Its Effect on Cell Proliferation,Migration and Invasion].

OBJECTIVE: To study the epigenetic regulation of pancreatic carcinoma related microRNA (miR34a,miR34b,miR148a and miR203a) expression by gene promoter methylation,and its effect on the proliferation,migration and invasion of pancreatic carcinoma cells. METHODS: The pancreatic carcinoma cells were divided into two groups:control group and treatment group.Control group was treated with 0 µmol/L DNA methyltransferase inhibitor 5-Aza-CdR and treatment group was treated with 60 µmol/L 5-Aza-CdR. The methylation status of microRNA gene promoter regions was detected by MSP (methylation-specific PCR). The microRNAs' expression levels were evaluated by real-time PCR. The CCK-8 assay,wound healing assay and Transwell assay were employed to study the proliferation,migration and invasion of pancreatic carcinoma cells,respectively. RESULTS: The results of MSP showed that the methylated band of the treated group was weaker than that of the untreated group and the unmethylated band of the treated group was stronger than that of the untreated group. Real-time PCR results showed that the relative expression levels of microRNAs in the treatment group were higher than those in the control group ( P<0.05). The CCK-8 assay showed that inhibition rate of the treatment group showed dose-dependent effect with the increase of drug concentration. Wound healing assay showed that the wound healing rate of Treatment group was lower than that of untreated group ( P<0.01). The results of transwell assay showed that the number of migrated cells in the treated group was less than that in the untreated group ( P<0.01). CONCLUSION: Decreased methylation levels in microRNA promoter region caused by 5-Aza-CdR treatment increased the expression of miR34a,miR34b ,miR148a and miR203a,leading to inhibition of the proliferation,migration and invasion of pancreatic carcinoma cells.

[Study on the Effect of Overexpression of miR-18a on Cellular Proliferation and Migration by Targeting ATM in Human Colorectal Cancer Cells].

OBJECTIVES: To study the regulation to colon cancer cellular biological properties through miR-18a targeting ataxia-telangiectasia mutated gene (ATM). METHODS: A target of miR-18a was predicted by using bioinformatics tools. The miR-18a mimics and inhibitors were designed and synthesized. The expression of endogenous miR-18a in colon cancer cell line HCT116 was up-regulated or down-regulated by transfection. The effect of overexpression of miR-18a on cellular proliferation, invasion and migration via regulation of ATM gene expression was confirmed in vitro by using qRT-PCR, Western blot, MTT assay, clone forming assay and Transwell method, respectively. RESULTS: ATM was identified as a potential target gene of miR-18a in the bioinformatics analysis. In addition, through transient transfection leading to the overexpression of miR-18a in HCT116 cell, the expression level of ATM was decreased. Down-regulation of HCT116 cell proliferation activity while significantly reducing HCT116 cell clone forming ability, lateral migration ability and longitudinal invasion ability were observed after transfected with miR-18a mimics. All of the changes were related to the overexpression of miR-18a. CONCLUSIONS: miR-18a inhibited the proliferation and migration of colon cance cell HCT116 through negative regulation of ATM expression.