Upregulation of programmed death ligand-1 in tumor-associated macrophages affects chemotherapeutic response in ovarian cancer cells.

To better understand the mechanism of chemoresistance in ovarian cancer cells, we aimed to investigate the influence of macrophages on the tumor cell response to carboplatin and identify the genes associated with chemoresistance. We mimicked the tumor microenvironment (TME) using a co-culture technique and compared the proliferation of ovarian cells with and without macrophages. We also examined M1 and M2 marker expression and the expression of key TME genes. Post the co-culture, we treated ovarian cancer cells with carboplatin and elucidated the function of programmed death-ligand 1 (PD-L1) in carboplatin chemoresistance. We investigated CD68 and PD-L1 expression in normal and cancerous ovarian tissues using immunohistochemistry (IHC). Finally, we analyzed the association between CD68 or PD-L1 expression and survival outcomes. Inducible nitric oxide synthase (iNOS) was downregulated, while the gene expression of M2 macrophage markers was increased in ovarian cancer cells. PD-L1, vascular endothelial growth factor (VEGF), Interleukin (IL)-6, IL-10, IL-12, signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma 2 (BCL2), multidrug resistance 1 (MDR1), and colony stimulating factor 1 (CSF-1) were upregulated. Notably, PD-L1 was upregulated in both the ovarian cancer cells and macrophages. Ovarian cancer cells co-cultured with macrophages exhibited statistically significant carboplatin resistance compared to single-cultured ovarian cancer cells. PD-L1 silencing induced chemosensitivity in both types of co-cultured ovarian cancer cells. However, IHC results revealed no correlation between PD-L1 expression and patient survival or cancer stage. CD68 expression was significantly increased in cancer cells compared to normal or benign ovarian tumor cells, but it was not associated with the survival outcomes of ovarian cancer patients. Our study demonstrated that ovarian cancer cells interact with macrophages to induce the M2 phenotype. We also established that PD-L1 upregulation in both ovarian cancer cells and macrophages is a key factor for carboplatin chemoresistance.

Loss of RUNX3 is significantly associated with advanced tumor grade and stage in endometrial cancers.

Loss of runt­related transcription factor 3 (RUNX3) has been reported in various cancers, and one of the mechanisms mediating loss of RUNX3 expression is DNA methylation. However, the role of RUNX3 expression and its DNA methylation status as prognostic factors in endometrial cancer remain unclear. In the present study, the expression and promoter methylation of RUNX3 was examined in endometrial cancer tissues and cell lines, as well as their association with endometrial cancer prognosis. Fifty­five endometrial cancer tissues and two endometrial cancer cell lines (HEC1­α and Ishikawa) were studied. RUNX3 expression and promoter methylation were examined using reverse transcription­polymerase chain reaction (RT­PCR), methylation specific PCR (MS­PCR), and immunohistochemical staining. The demethylating agent 5­aza­2'­deoxycytidine (ADC) was used to reverse the methylation of the RUNX3 promoter. Loss of RUNX3 expression was observed in 50.9% (27/53) of endometrial cancer tissues and in the HEC1­α cell line by immunohistochemistry and RT­PCR, respectively. Methylation of the RUNX3 promoter was observed in 62.2% (33/53) of endometrial cancer tissues, 12.5% (1/8) of normal endometrial tissues, and the HEC1­α cell line by MS­PCR. Tumor grade and stage were significantly correlated with loss of RUNX3 expression. The expression of RUNX3 was restored by treatment with ADC and resulted in growth inhibition in HEC1­α cells. The present results suggested that methylation may serve a critical role in the silencing of RUNX3 and loss of RUNX3 expression may serve as a prognostic marker in endometrial cancer.

Polymerase chain reaction-based fluorescent Luminex assay to detect the presence of human papillomavirus types.

Becuase 40% of human papillomavirus (HPV) infections are mixed infections, the accurate identification of high-risk HPV genotypes in mixed infections is important for defining a woman's risk for progression to cervical cancer. Thus, advanced Luminex-based HPV genotyping has been developed to simultaneously detect the presence of multiple HPV types. Here, we describe the development of a Luminex-based HPV genotyping that combines polymerase chain reaction amplification with hybridization to fluorescence-labeled polystyrene bead microarrays (Luminex suspension array technology). New HPV type-specific oligonucleotide probes and YBT L1/GP6-1 primers were used to detect the HPV types in 132 clinical samples. We simultaneously evaluated the usefulness of this technique on clinical samples. We detected 15 specific HPV types (6, 16, 18, 31, 35, 42, 51, 52, 55, 56, 58, 59, 66, 67 and 68) examined with specificity without known cross-reaction to other HPV types. The detection limit for the different HPV types was above 500 plasmids. We compared the performance of the Luminex-based assay to the established HPV DNA microarray chip for polymerase chain reaction products derived from 53 clinical samples. The evaluation showed excellent agreement. The Luminex-based HPV genotyping was a sensitive, reproducible technique for the simultaneous genotyping of all clinically relevant genital HPV types. This assay system may be used to provide critical clinical information for early detection of HPV, especially in cases where the HPV copy numbers are low and the latency period of HPV infection is prolonged.

Searching for pathogenic gene functions to cervical cancer.

OBJECTIVES: Molecular pathology of cervical cancers associated with human papillomavirus (HPV) infection is presently unclear. In an effort to clarify this issue, we investigated gene expression profiles and pathogenic cellular processes of cervical cancer lesions. METHODS: Tissues of 11 patients (invasive cancer stages Ib-IIIa) were investigated by a cDNA microarray of 4700 genes, hierarchical clustering and the Gene Ontology (GO). RESULTS: We identified 74 genes showing a more than 2-fold difference in their expression in at least 8 out of 11 patients. Among these, 33 genes were up-regulated, in contrast, 41 genes were down-regulated. The gene expression profiles were classified into mutually dependent 345 function sets, resulting in 611 cellular processes according to the GO. The GO analysis showed that cervical carcinogenesis underwent complete down-regulation of cell death, protein biosynthesis, and nucleic acid metabolism. Also, genes belonging to nucleic acid binding and structural molecule activity were significantly down-regulated. In contrast, significant up-regulation was shown in skeletal development, immune response, and extracellular activity. CONCLUSIONS: These data suggest that the regulated genes and cellular processes could be further used for predicting prognosis and diagnosis of cervical cancer patients, and further investigation and functional characterization of the identified genes is warranted.

cDNA Microarray Analysis of Gene Expression Profiles Associated with Cervical Cancer.

PURPOSE: The molecular pathology of cervical cancers associated with human papillomavirus infection is presently unclear. In an effort to clarify this issue, the gene expression profiles and pathogenic cellular processes of cervical cancer lesions were investigated. MATERIALS AND METHODS: Cervical cancer biopsies were obtained from patients at the Department of Obstetrics and Gynecology, The Catholic University of Korea. The disease status was assigned according to the International Federation of Gynecology and Obstetrics. The tissue samples of 11 patients (invasive cancer stage Ib- IIIa) were investigated by a cDNA microarray of 4, 700 genes, hierarchical clustering and the Gene Ontology (GO). Total RNA from cervical cancer and non-lesional tissues were labeled with Cy5 and Cy3. The HaCaT human epithelial keratinocyte cell line was used as a negative control cell. The stages of invasive cancer were Ib to IIIb. All specimens were obtained by punch-biopsies and frozen in liquid nitrogen until required. RESULTS: 74 genes, showing more than a 2 fold difference in their expressions, were identified in at least 8 of the 11 patients. Of these genes, 33 were up-regulated and 41 were down-regulated. The gene expression profiles were classified into 345 mutually dependent function sets, resulting in 611 cellular processes according to their GO. The GO analysis showed that cervical carcinogenesis underwent complete down-regulation of cell death, protein biosynthesis and nucleic acid metabolism. The genes related to nucleic acid binding and structural molecule activity were also significantly down-regulated. In contrast, significant up-regulation was shown in the skeletal development, immune response and extracellular activity. CONCLUSION: These data are suggestive of potentially significant pathogenetic cellular processes, and showed that the down-regulated functional profiling has an important impact on the discovery of pathogenic pathways in cervical carcinogenesis. GO analysis can also overcome the complexity of the expression profiles of the cDNA microarray via a cellular process level approach. Thereby, a valuable prognostic candidate gene, with real relevance to disease-specific pathogenesis, can be found at the cellular process levels.