Inhibition of long non-coding RNA ROR reverses resistance to Tamoxifen by inducing autophagy in breast cancer.

This study explored the mechanism underlying long non-coding RNA ROR regulating autophagy on Tamoxifen resistance in breast cancer. Cancer tissues and adjacent normal tissues were collected from 74 breast cancer patients. Human breast cancer BT474 cells were assigned into blank, phosphate buffered saline, Tamoxifen, negative control + Tamoxifen, siROR + Tamoxifen, 3-methyladenine + Tamoxifen, and siROR + 3-methyladenine + TA groups. The expression of long non-coding RNA ROR and expressions of multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA were detected using quantitative real-time polymerase chain reaction. The expressions of light chain 3, Beclin 1, multi-drug resistance-associated P-glycoprotein, and glutathione S-transferase-π protein were determined using western blotting. Cell proliferation, invasion, and migration abilities were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay, and scratch test, respectively. The long non-coding RNA ROR expression was higher in the breast cancer tissues than that in the adjacent normal tissues. Compared with the blank group, light chain 3 and Beclin 1 expressions were increased in the siROR + Tamoxifen group but decreased in the 3-methyladenine + Tamoxifen group; these data indicated that downregulated long non-coding RNA ROR promoted autophagy. In comparison with the blank group, multi-drug resistance-associated P-glycoprotein and glutathione S-transferase-π messenger RNA and protein expressions were reduced in the siROR + Tamoxifen group but elevated in the 3-methyladenine + Tamoxifen group, suggesting that downregulated long non-coding RNA ROR suppressed the drug resistance to Tamoxifen and the inhibition of autophagy reversed the effect of long non-coding RNA ROR on drug resistance. Compared with the Tamoxifen, negative control, and siROR + 3-methyladenine + Tamoxifen groups, the cell proliferation, invasion, and migration in the siROR + Tamoxifen group were much decreased; these results implied that downregulated long non-coding RNA ROR suppressed BT474 cell proliferation, invasion, and migration and reversed the effect of Tamoxifen on the BT474 cells. These results indicate that inhibition of long non-coding RNA ROR reverses resistance to Tamoxifen by inducing autophagy in breast cancer.

Increased CDCA2 Level Was Related to Poor Prognosis in Hepatocellular Carcinoma and Associated With Up-Regulation of Immune Checkpoints.

Background: Cell division cycle-associated protein 2 (CDCA2) is a member of cell cycle-related proteins. CDCA2 plays a role in the regulation of protein phosphatase 1(PP1) γ-dependent DNA damage response (DDR) and H3 phosphorylation. CDCA2 promotes the tumorigenesis and development of several types of cancers by promoting the proliferation of tumor cells. However, the relationship between CDCA2 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is unknown. Methods: Gene expression information and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. The expression of CDCA2 and its correlation to clinical characteristics in HCC were analyzed. The expression level of CDCA2 was validated in HCC cell lines. The relationship between CDCA2 expression and the survival of patients with HCC was analyzed by using Kaplan-Meier method. The prognostic value of CDCA2 in HCC was estimated by Cox regression analysis. The expression difference of CDCA2 between HCC and normal tissues and its correlation to survival were verified in independent datasets. Gene set enrichment analysis (GSEA) was used to screen the CDCA2-related signaling pathways. Results: Cell division cycle-associated protein 2 expression was upregulated in HCC tissues (p < 0.001) and increased CDCA2 was correlated to increased T stage, pathologic stage, histologic grade, and alpha-fetoprotein (AFP) level (p < 0.001). In addition, CDCA2 was overexpressed in HCC cell lines HepG2 and LM3. High CDCA2 expression level was associated with poor overall survival [hazard ratio (HR) = 1.69; 95% CI, 1.20-1.40, p = 0.003], disease specific survival (HR = 1.73; 95% CI, 1.11-2.71, p = 0.016), and progress free interval (HR = 1.74; 95% CI, 1.30-2.34, p < 0.001). Overexpression of CDCA2 and its correlation to poor survival in HCC were verified in Gene Expression Omnibus (GEO) datasets and Kaplan-Meier plotter database. Increased CDCA2 expression was associated with upregulation of PD-L1 (Spearman's coefficient = 0.207, p < 0.001), PD-L2 (Spearman coefficient's = 0.118, p < 0.05), and CTLA4 (Spearman's coefficient = 0.355, p < 0.001). GSEA showed that homologous recombination pathway, insulin signaling pathway, mitogen-activated protein kinase (MAPK) pathway, mismatch repair pathway, mechanistic target of rapamycin (mTOR) pathway, Notch pathway, T cell receptor pathway, toll like receptor pathway, and WNT pathway were enriched in CDCA2 high expression phenotype. Conclusion: Cell division cycle-associated protein 2 may serve as an independent biomarker for poor prognosis in HCC and increased CDCA2 expression was associated with upregulation of immune checkpoints.

Chemoradiotherapy and Increased Prescription Dose in Esophageal Squamous Cell Cancer: A Retrospective Study.

To analyze the outcomes and adverse events of patients with esophageal squamous cell carcinoma (ESCC) treated with definitive chemoradiation with modified radiotherapy volume and increased radiation dose. This was a retrospective analysis of patients with ESCC treated with definitive chemoradiotherapy at the Sun Yat-sen University Cancer Center (02/2015 to 02/2017). The dose to the planning gross tumor volume (PGTV) and planning clinical tumor volume (PTV1) was 66-68 Gy (2.0-2.2 Gy/fraction). The dose to the planning regional lymph node drainage area volume (PTV2) was 46 Gy (2.0 Gy/fraction). Treatment response, adverse events, progression-free survival (PFS), overall survival (OS), and locoregional failure-free survival (LRFFS) were analyzed. Twenty-six patients were included. The median follow-up was 31 (range, 4.3-51.3) months. Sixteen (61.5%) patients had a complete response, and four (15.4%) achieved a partial response. The objective response rate was 76.9%, and the disease control rate was 80.8%. The median PFS and OS were not achieved. The 4-year PFS was 63.9%, and the 4-year OS was 71.0%. Grade 1-2 and 3-4 radiation-related esophagitis was observed in 15 (57.7%) and one (4.5%) patients, respectively. Grade 1-2 and 3-4 radiation-related pneumonitis was observed in 12 (46.2%) and one (4.5%) patients, respectively. No patients developed radiation-related heart or skin damage. The modified target volume definition and increased dose of definitive radiotherapy combined with chemotherapy in patients with ESCC had low toxicity and might improve survival, but additional trials are necessary to prove the superiority of this strategy.

Radio-Susceptibility of Nasopharyngeal Carcinoma: Focus on Epstein- Barr Virus, MicroRNAs, Long Non-Coding RNAs and Circular RNAs.

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer. As a neoplastic disorder, NPC is a highly malignant squamous cell carcinoma that is derived from the nasopharyngeal epithelium. NPC is radiosensitive; radiotherapy or radiotherapy combining with chemotherapy are the main treatment strategies. However, both modalities are usually accompanied by complications and acquired resistance to radiotherapy is a significant impediment to effective NPC therapy. Therefore, there is an urgent need to discover effective radio-sensitization and radio-resistance biomarkers for NPC. Recent studies have shown that Epstein-Barr virus (EBV)-encoded products, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), which share several common signaling pathways, can function in radio-related NPC cells or tissues. Understanding these interconnected regulatory networks will reveal the details of NPC radiation sensitivity and resistance. In this review, we discuss and summarize the specific molecular mechanisms of NPC radio-sensitization and radio-resistance, focusing on EBV-encoded products, miRNAs, lncRNAs and circRNAs. This will provide a foundation for the discovery of more accurate, effective and specific markers related to NPC radiotherapy. EBVencoded products, miRNAs, lncRNAs and circRNAs have emerged as crucial molecules mediating the radio-susceptibility of NPC. This understanding will improve the clinical application of markers and inform the development of novel therapeutics for NPC.

Long noncoding RNA cancer susceptibility candidate 9 promotes doxorubicin­resistant breast cancer by binding to enhancer of zeste homolog 2.

The present study aimed to investigate the effect of the long noncoding RNA cancer susceptibility candidate 9 (CASC9) on doxorubicin (DOX)­resistant breast cancer and to reveal the potential underlying mechanisms. The expression of CASC9 in breast cancer tissues and cell lines, in addition to drug­resistant breast cancer cells (MCF­7/DOX), was detected by reverse transcription­quantitative polymerase chain reaction. Subsequently, MCF­7/DOX cells were transfected with the silencing vector pS­CASC9, containing enhancer of zeste homolog 2 (EZH2), multidrug resistance protein 1 (MDR1) or control small interfering (si)RNAs. The viability, apoptosis, migration and invasion of the transfected cells were assessed via an MTT assay, flow cytometry and a Transwell assay, respectively. The expression levels of apoptosis­associated proteins (apoptosis regulator Bcl­2, apoptosis regulator BAX, caspase­3 and caspase­9) were determined by western blotting. An RNA pull­down assay was performed to identify CASC9­binding candidates. In addition, the expression levels of the MDR1 gene and its encoded protein, P­glycoprotein, were detected. CASC9 expression was upregulated in breast cancer tissues and cell lines, and drug­resistant breast cancer cells. CASC9 knockdown significantly inhibited the growth and metastasis of drug­resistant breast cancer cells, and decreased the half­maximal inhibitory concentration DOX in MCF­7/DOX cells. The RNA pull­down assay revealed that CASC9 engaged EZH2; EZH2 siRNA significantly inhibited the cell growth, metastasis and chemoresistance of MCF­7/DOX cells. Additionally, EZH2 may regulate the MDR1 gene. The present study demonstrated the oncogenic role of CASC9 in drug­resistant breast cancer by binding to EZH2 and regulating the MDR1 gene. Modulation of CASC9 expression may be a promising target in the therapy of breast cancer and drug­resistant breast cancer.

MiR-99a suppresses cell invasion and metastasis in nasopharyngeal carcinoma through targeting HOXA1.

BACKGROUND: Recent studies reported that miRNAs play important roles in the carcinogenesis and progression of nasopharyngeal carcinoma (NPC). Therefore, further studies are warranted to better elucidate the function and mechanism of miRNAs in NPC. METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the miR-99a expression in NPC cell lines and tissue samples. Wound healing, transwell migration and invasion, and lung metastatic colonization assays were performed to determine NPC cell migratory, invasive and metastatic abilities of NPC cells. Luciferase reporter assays, quantitative RT-PCR and Western blotting were used to validate the target of miR-99a. RESULTS: We found that miR-99a was significantly downregulated in NPC cell lines and tissue samples. Ectopic overexpression of miR-99a significantly inhibited NPC cell migration and invasion in vitro, and suppressed lung macroscopic and microscopic metastatic colonization in vivo. Conversely, silencing of miR-99a significantly promoted the migratory and invasive abilities of NPC cells. Furthermore, HOXA1 was validated as a direct target of miR-99a, and ectopic expression of HOXA1 could rescue the suppressive effect of miR-99a overexpression on NPC cell migration and invasion. CONCLUSION: Together, these results indicated that miR-99a could inhibit NPC invasion and metastasis by targeting HOXA1, thus providing a novel potential target for miRNA-based treatment for NPC patients in the future.

Anti-proliferative and pro-apoptotic effect of carvacrol on human hepatocellular carcinoma cell line HepG-2.

Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.

Parathion degradation and its intermediate formation by Fenton process in neutral environment.

The purpose of this study is to investigate parathion degradation by Fenton process in neutral environment. The initial parathion concentration for all the degradation experiments was 20 ppm. For hydrogen ion effect on Fenton degradation, the pH varied from 2 to 8 at the [H2O2] to [Fe²(+)] ratio of 2-2 mM, and the result showed pH 3 as the most effective environment for parathion degradation by Fenton process. Apparent degradation was also observed at pH 7. The subsequent analysis for parathion degradation was conducted at pH 7 because most environmental parathion exists in the neutral environment. Comparing the parathion degradation results at various Fenton dosages revealed that at Fe²(+) concentrations of 0.5, 1.0 and 1.5 mM, the Fenton reagent ratio ([H2O2]/[Fe²(+)]) for best-removing performance were found as 4, 3, and 2, resulting in the removal efficiencies of 19%, 48% and 36%, respectively. Further increase in Fe²(+) concentration did not cause any increase of the optimum Fenton reagent ratio for the best parathion removal. The result from LC-MS also indicated that hydroxyl radicals might attack the PS double bond, the single bonds connecting nitro-group, nitrophenol, or the single bond within ethyl groups of parathion molecules forming paraoxons, nitrophenols, nitrate/nitrite, thiophosphates, and other smaller molecules. Lastly, the parathion degradation by Fenton process at the presence of humic acids was investigated, and the results showed that the presence of 10 mg L⁻¹ of humic acids in the aqueous solution enhanced the parathion removal by Fenton process twice as much as that without the presence of humic acids.