Activation of a positive feedback loop involving IL-6 and aromatase promotes intratumoral 17ß-estradiol biosynthesis in endometrial carcinoma microenvironment.

Tumor-stroma interactions contribute greatly to intratumoral estrogen biosynthesis in endometrial carcinoma, but the mechanisms involved remain largely unknown. Previous study demonstrated that intratumoral aromatase upregulation in stromal cells participated in this process, but the specific aromatase-regulators have not been reported. In the present study, we found that aromatase expression in intratumoral stroma, but not in tumor epithelium, correlated positively with interleukin 6 (IL-6) expression in cancer epithelial cells by immunohistochemistry, which was confirmed using laser capture microdissection/real-time reverse transcription-PCR. With stimulation by exogenous IL-6, aromarase expression was increased in stromal cells not but not in cancer cells. Aromatase mRNA levels in endometrial cancer cells were not influenced by cocultivation with intratumoral stromal cells. When cocultured with 17ß-estradiol (E2 )-treated cancer cells, aromatase mRNA in stromal cells was significantly elevated and increased IL-6 protein levels were detected in E2 -treated culture medium. Next, we demonstrated that E2 -induced IL-6 production was through cooperation between estrogen receptor α and nuclear factor-kappa B. Furthermore, an IL-6 receptor blocking antibody could attenuate the upregulation of aromatase expression in stromal cells and the E2 concentration in coculture systems of cancer and stromal cells. The results were confirmed by an orthotopic nude endometrial carcinoma model in vivo. These studies elucidated the activation of a positive feedback loop, that is, IL-6 stimulated by E2 in endometrial cancer cells induced aromatase expression in stromal cells, promoting enhanced intratumoral E2 synthesis. Blocking of this tumor-stroma interaction may be a therapeutic strategy to overcome in situ estrogen biosynthesis in endometrial carcinoma.

Interleukin 6 promotes endometrial cancer growth through an autocrine feedback loop involving ERK-NF-κB signaling pathway.

Interleukin (IL)-6 as an inflammation factor, has been proved to promote cancer proliferation in several human cancers. However, its role in endometrial cancer has not been studied clearly. Previously, we demonstrated that IL-6 promoted endometrial cancer progression through local estrogen biosynthesis. In this study, we proved that IL-6 could directly stimulate endometrial cancer cells proliferation and an autocrine feedback loop increased its production even after the withdrawal of IL-6 from the medium. Next, we analyzed the mechanism underlying IL-6 production in the feedback loop and found that its production and IL-6-stimulated cell proliferation were effectively blocked by pharmacologic inhibitors of nuclear factor-kappa B (NF-κB) and extra-cellular signal-regulated kinase (ERK). Importantly, activation of ERK was upstream of the NF-κB pathways, revealing the hierarchy of this event. Finally, we used an orthotopic nude endometrial carcinoma model to confirm the effects of IL-6 on the tumor progression. Taken together, these data indicate that IL-6 promotes endometrial carcinoma growth through an expanded autocrine regulatory loop and implicate the ERK-NF-κB pathway as a critical mediator of IL-6 production, implying IL-6 to be an important therapeutic target in endometrial carcinoma.

[Analysis of gene expression profiles in gastric cancer cell cycle].

OBJECTIVE: To detect the gene expression profile in gastric cancer cell cycle and explain the mechanism of gastric cancer cell proliferation by a genomic study. METHODS: Gastric cancer cells MKN45 were synchronized at G2/M and G1/S point by nocodazole-thymidine and double thymidine methods. The synchronizing degree of cells was monitored by flow cytometry. The gene expression profiles at G2/M point, M/G1 transition, G1 early phase, G1 late phase, G1/S point, S early phase, S late phase, G2 early phase and G2 late phase in MKN45 cell cycling were examined using cDNA microarray chips. Hierarchy analysis was conducted with a professional software package and the up-regulated genes at G1 late and G2 phase were analyzed according to gene database. Furthermore, the mRNA level of cyclin E, cyclin B, plk1 and STK15 in above mentioned nine points were measured by quatitative PCR. RESULTS: 2001 genes were detected to be available at all 9 points via software processing, out of which 959 appeared up-regulated or down-regulated. 379 genes showed to be up-regulated at late G1 (147) or G2 phases (232), 40 at S and M phases (also up-regulated at G1 late and G2 phases). The 147 up-regulated genes at G1 late phase are involved in DNA metabolism, transcription and translation, protein transportation, ubiquitination and signal transduction, etc. The 232 up-regulated genes in G2 phase are involved in RNA synthesis and processing, intracellular protein transportation, cytoskeleton synthesis, signal transduction, apoptosis and anti-apoptosis, transcription regulation, ubiquitination, mitosis regulation and oncogene expression, etc. The mRNA level of 4 genes detected by quantitative PCR during cell cycle was in agreement with that detected by microarray. CONCLUSION: During MKN45 cell cycling, the preparation for DNA synthesis and chromosome separation are conducted in G1 and G2, which are implicated in multiple genes, may be the main impetus of driving MKN45 cell cycle. Some of these genes may be related to tumor over-proliferation. The cDNA microarray technique has characteristic features such as reliability and can provide a great deal for future research on cell cycle related genes in gastric cancer.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest.

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

[Polo like kinase 1 expression and prognostic value in gastric carcinomas].

OBJECTIVE: To identify the expression of polo like kinase 1 (plk1) and to discuss its relationship with the clinicopathological parameters and prognosis in gastric carcinoma. METHODS: Plk1 protein expression levels in 89 cases of resected gastric carcinomas were detected by immunohistochemistry method, the relations between plk1 expression levels and the survival periods were estimated by Kaplan-Meier curve. RESULTS: The positive rate of plk1 expression in gastric cancer tissues was 42.7% (38/89), significantly higher than that (13.5%) in the adjacent noncancerous tissues (12/89) (P<0.01). The expression levels of plk1 were closely related to tumor differentiation, invasion and TNM stage (P<0.05). Patients with plk1-positive expression had worse prognosis than those with plk1-negative expression in gastric cancer patients (P<0.05). CONCLUSIONS: Plk1 may promote carcinogenesis and gastric cancer development, its overexpression can be a novel marker for diagnosing certain biological behaviours and predicting prognosis in gastric cancer.

[Inhibition of polo like kinase gene expression induces apoptosis in gastric cancer cells].

OBJECTIVE: To observe the effect of inhibition of polo like kinase1 (plk1) gene expression on apoptosis induction in gastric cancer cell line-MKN45 and discuss the vital role of plk1 proliferation and viability of gastric cancer cells. METHODS: The plk1 expression was inhibited by chemically synthesized siRNA. The plk1 mRNA and protein level were respectively measured by real-time quantitative PCR and Western blotting. The spindle morphological change was observed by immunofluorescence staining and confocal microscopy. The change of cell cycle distribution and apoptosis rate was detected by flow-cytometry. Pro caspase3 level was also detected by western blotting. RESULTS: After treatment by siRNA targeting plk1, plk1 mRNA and protein level decreased obviously, the cell mitotic spindle became obscure and lost cohesiveness, more MKN45 cells accumulated at G(2)/M phase (P< 0.05), apoptosis rate of plk1 siRNA treated MKN45 cells was higher than that of control cells at 48 h and 72 h (P< 0.05) with pro-caspase3 level decreasing at 72 h. CONCLUSIONS: Inhibition of plk1 gene expression induces apoptosis in MKN45 cells through the pathway of caspase3. Plk1 gene play a key role in viability of MKN45 cells.