The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer.

Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance.

Establishment and characterization of GCSR1, a multi-drug resistant signet ring cell gastric cancer cell line.

Signet ring cell gastric cancer (SRCGC) has very poor prognosis worldwide, and studying its molecular characteristics is urgent for improving the outcome. However, few well-characterized SRCGC cell lines are available for research. Therefore, we established a novel cell line GCSR1, from a Chinese male SRCGC patient. Cell morphology of GCSR1 in culture, maintained in vitro for over 90 passages, is similar to the cells from the patient. GCSR1 cells proliferated in vitro with a doubling time of 67.65 h. Karyotyping showed they were aneuploid. Missense mutation occurred in codon 193 of P53 and deletion occurred in exons 1 and 3 of P16. Results of CCK8 assay revealed that GCSR1 was more resistant to 5-fluorouracil (5-FU) and mitomycin (MMC) than other gastric cancer cell lines. Stem cell marker assay by flow cytometry showed that GCSR1 had high proportion of CD44+ and/or CD133+ cells. It formed colonies easily in soft agar and generated xenograft tumors in nude mice. In conclusion, GCSR1 is a well-established, well-characterized multi-drug resistant cell line with abundant cancer stem cells.

Heterogeneity of chemosensitivity in esophageal cancer using ATP-tumor chemosensitivity assay.

AIM: Current chemotherapy for esophageal cancer is conducted on the basis of empirical information from clinical trials, which fails to take into account the known heterogeneity of chemosensitivity between patients. This study was aimed to demonstrate the degree of heterogeneity of chemosensitivity in esophageal cancers. METHODS: A total of 42 esophageal cancer specimens were collected. The heterogeneity of chemosensitivity in esophageal cancer specimens was examined using an ex vivo ATP-tumor chemosensitivity assay (ATP-TCA). RESULTS: Thirty eight specimens produced evaluable results (90.5%). The most active single agent tested was nedaplatin, to which 28.9% of samples were sensitive. Combinations of chemotherapy agents exhibited much higher sensitivity: cisplatin + paclitaxel was sensitive in 16 of 38 (42.1%) of samples, while nedaplatin+paclitaxel was more effective, which was sensitive in 20 of 38 cases (52.6%). CONCLUSION: There was a marked heterogeneity of chemosensitivity in esophageal cancer. Chemosensitivity testing may provide a practical method for testing new regimens before clinical trials in esophageal cancer patients.

[Study of the metastasis-associated genes and its copy numbers variation in highly metastatic epithelial ovarian cancer].

OBJECTIVE: To investigate the relationship of the metastasis-associated genes and its copy numbers variation in the highly metastatic human epithelial ovarian cancer cell line HO-8910PM. METHODS: The differentially expressed genes and its copy number variation between HO-8910PM cell line and normal ovarian tissues was detected by human genome U133A 2.0 gene chip and human mapping 10K array 2.0 gene chip, and the data was analyzed by bioinformatics. Some of metastasis-associated genes were validated the results of single nucleotide polymorphism (SNP) and cDNA chips by fluorescence in situ hybridization (FISH) and real-time quantitative PCR. RESULTS: Integrate analysis of two gene chips data showed that there were 385 differentially expressed genes in the same and 379 SNP positional point (6 of them, included 2 genes) between HO-8910PM cell line and normal ovarian tissues, these copy number amplification of 379 SNP positional point of chromosome were > or = 3, which had 240, deletion < or = 1 had 139. Chromosome location analysis showed that there were 385 differentially expressed genes located at all chromosomes, and 261 of them (67.8%, 261/385) located at 10 chromosomes, included that 34 (8.8%), 33 (8.6%), 28 (7.3%), 27 (7.0%), 25 (6.5%), 24 (6.2%) of them located at chromosome 3, 2, 9, 10, 1 and 11 respectively, and 23 (6.0%) of them at chromosome 6 and 12 each, 22 (5.7%) of them at chromosome 4 and 5 each. For the function of differentially expressed genes, the results showed that 99 (25.7%) genes belonged to the family of enzymes and their regulators, 54 (14.0%) genes associated with signal transduction, 50 (13.0%) genes associated with nucleic acid binding, and 36 (9.4%) genes associated with protein binding. CONCLUSION: We have demonstrated that there are 4 kinds of differentially expressed genes related to metastasis of ovarian cancer, which belonged to the families enzyme and its regulator, nucleic acid binding, signal transduction and protein binding, and located at chromosome 1, 2, 3, 4, 5, 6, 9, 10, 11 and 12.

Identification of differentially expressed genes in the high and low metastatic human ovarian cancer cell lines and analyses of their chromosomal localizations and functions.

Oligonucleotide microarrays were used to study the differences of gene expressions in high (H) and low (L) metastatic ovarian cancer cell lines and in normal ovarian tissues (C). Bioinformatics was used to identify novel genes and their functions as well as chromosomal localizations. A total of 409 genes were differentially expressed between the high and low metastatic ovarian cancer cell lines. Of them, 271 genes were up regulated (Signal Log Ratio[SLR] > or = 1), and 138 genes were down regulated (SLR < or = -1). Except one gene whose location was unknown, all these genes were localized randomly on all the chromosomes, with a majority of them localized to Chromosomes 1, 6, 2, 17, 3, 5 and 11. Chromosome 1 contained, 43 of them (10.7%), the most for a single chromosome. A total of 264 genes (64.7%) were localized on the short arm of the chromosome (q). Functional classification showed that the 104 (25.4%) genes coding for enzymes and enzyme regulators made up the largest functional group, followed by signal transduction activity genes (43, 10.5%), nucleic acid binding activity genes (42, 10.3%), and proteins binding activity genes (34, 8.3%). These four groups accounted for 54.5% of all the differentially expressed genes. In addition, the functions of 76 genes (18.6%) were unknown. Tumor metastasis is the result of a number of genes acting in concert. The four functional groups of genes classified among these genes and their abnormalities would be the focus of further studies on ovarian cancer metastasis.

[Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer cell lines].

OBJECTIVE: To find the key proteins associated with metastasis of ovarian cancer, and find potential diagnostic markers and therapeutic targets of this malignancy. METHODS: A comparative proteomic strategy, in a combination of two-dimensional electrophoresis separation and mass spectrometry identification, was adopted to search for proteome alternations in an ovarian cancer mother cell line HO-8910 and its highly metastatic cell subline HO-8910PM. RESULTS: Twenty-one significantly different spots (two-fold increase or decrease) were detected between the two cell lines, of which 17 candidate proteins were successfully identified and characterized. Compared with those in HO-8910 mother cell line, 16 proteins were significantly up-regulated, while 5 proteins down-regulated in the highly metastatic cell subline HO-8910PM. The seventeen identified proteins could be functionally classified into 7 groups as following: zinc finger protein, calcium-binding protein, DNA repair and synthesis protein, cell regulatory protein, metabolism-related protein, cell surface antigen, cell signals and transducing protein. CONCLUSIONS: The results suggest that an obviously differential proteomic expression exists between the human ovarian cancer mother cell line HO-8910 and highly metastatic cell subline HO-8910PM. It provides a clue for further identification of metastasis-related proteins, novel diagnostic markers as well as therapeutic targets of this malignancy.

[Establishment of human multidrug-resistant lung carcinoma cell line (D6/MVP)].

OBJECTIVE: To establish human multidrug-resistant lung carcinoma cell line (D6/MVP) with its characteristics studied. METHODS: Intermittent administration of high-dose MMC, VDS and DDP (MVP) was used to induce human lung carcinoma cell line (D6) to a multidrug-resistant variety (D6/MVP). MTT assay was used to study the multidrug resistance of D6/MVP to multianticarcinogen. Flow cytometry was used to study the cell cycle distribution and the expression of P-gp, multidrug resistance-associated protein (MRP) and GSH/GST. RESULTS: 1. D6/MVP was resistant to many anti-tumor agents, with the IC(50) 13.3 times higher and the drug resistance 2 - 6 times higher than D6, 2. The multiplication time of D6/MVP was prolonged and the cell number of S-phase decreased while that of G1- and G(2)-phase increased and 3. The expression of P-gp and MRP was enhanced significantly (96.2% vs 51.7%), but the expression of GSH/GST kept stable. CONCLUSION: D6/MVP is a multidrug-resistant cell line possessing the basic characteristics of drug-resistance.