Resveratrol induces apoptosis and alters gene expression in human fibrosarcoma cells.

BACKGROUND/AIM: Metastatic fibrosarcomas still represent a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin have been proven effective in fewer than 30% of all cases disseminated of fibrosarcoma. Elderly patients with cardiac disease are not suitable for systemic chemotherapy with doxorubicin. We therefore tested the apoptotic effects of the natural and well-tolerated compound resveratrol on human fibrosarcoma cells (HT1080). MATERIALS AND METHODS: Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Gene expression was analyzed by RNA microarrays. RESULTS: Application of resveratrol induced apoptotic cell death and significantly reduced proliferation of HT1080 cells. Correspondingly, expression of apoptosis-associated genes was altered in microarray analysis. CONCLUSION: This in vitro study demonstrates the anticancer activity of resveratrol against human fibrosarcoma cells. These results provide experimental support for in vivo trials assessing the effect of the natural polyphenol resveratrol.

Pro­apoptotic effects of pycnogenol on HT1080 human fibrosarcoma cells.

Complete surgical resection with clear margins remains the mainstay of therapy for localised fibrosarcomas. Nevertheless, metastatic fibrosarcomas still represent a therapeutic dilemma. Commonly used chemotherapeutic agents like doxorubicin have proven to be effective in <30% of all cases of disseminated fibrosarcoma. Especially elderly patients with cardiac subdisease are not suitable for systemic chemotherapy with doxorubicin. Therefore we tested the apoptotic effects of the well-tolerated pine bark extract pycnogenol and its constituents on human fibrosarcoma cells (HT1080). Ten healthy subjects (six females, four males, mean age 24.8 ± 6 years) received a single dose of 300 mg pycnogenol orally. Blood plasma samples were obtained before and 6 h after intake of pycnogenol. HT1080 cells were treated with these plasma samples. Additionally, HT1080 were incubated separately with catechin, epicatechin and taxifolin that are known as the main constituents of pycnogenol. Vital, apoptotic and necrotic cells were quantified using flow cytometric analysis. Gene expression was analyzed by RNA microarray. The results showed that single application of taxifolin, catechin and epicatechin reduced cell viability of HT1080 cells only moderately. A single dose of 300 mg pycnogenol given to 10 healthy adults produced plasma samples that led to significant apoptotic cell death ex vivo whereas pycnogenol-negative serum displayed no apoptotic activity. Microarray analysis revealed remarkable expression changes induced by pycnogenol in a variety of genes, which are involved in different apoptotic pathways of cancer cells [Janus kinase 1 (JAK1), DUSP1, RHOA, laminin γ1 (LAMC1), fibronectin 1 (FN1), catenin α1 (CTNNA1), ITGB1]. In conclusion, metabolised pycnogenol induces apoptosis in human fibrosarcoma cells. Pycnogenol exhibits its pro-apoptotic activity as a mixture and is more effective than its main constituents catechin, epicatechin and taxifolin indicating that the metabolised components interact synergistically. These results provide experimental support for in vivo trials assessing the effect of the pine bark extract pycnogenol.

Expression profiling identifies genes that predict recurrence of breast cancer after adjuvant CMF-based chemotherapy.

Cyclophosphamide, methotrexate and 5-fluorouracile (CMF)-based chemotherapy for adjuvant treatment of breast cancer reduces the risk of relapse. In this exploratory study, we tested the feasibility of identifying molecular markers of recurrence in CMF-treated patients. Using Affymetrix U133A GeneChips, RNA samples from 19 patients with primary breast cancer who had been uniformly treated with adjuvant CMF chemotherapy were analyzed. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. An additional independent validation set of 51 patients and 21 genes were analyzed by quantitative RT-PCR. Applying different algorithms to evaluate our microarray data, we identified two gene expression signatures of 21 and 12 genes containing eight overlapping genes, that predict recurrence in 19 cases with high accuracy (94%). Quantitative RT-PCR demonstrated that six genes from the combined signatures (CXCL9, ITSN2, GNAI2, H2AFX, INDO, and MGC10986) were significantly differentially expressed in the recurrence versus the non-recurrence group of the 19 cases and the independent breast cancer patient cohort (n = 51) treated with CMF. High expression levels of CXCL9, ITSN2, and GNAI2 were associated with prolonged disease-free survival (DFS) (P = 0.029, 0.018 and 0.032, respectively). When patients were stratified by combined CXCL9/ITSN2 or CXCL9/FLJ22028 tumor levels, they exhibited significantly different disease-free survival curves (P = 0.0073 and P = 0.005, respectively). Finally, the CXCL9/ITSN2 and CXCL9/FLJ22028 ratio was an independent prognostic factor (P = 0.034 and P = 0.003, respectively) for DFS by multivariate Cox analysis in the 70-patient cohort. Our data highlight the feasibility of a prognostic assay that is applicable to therapeutic decision-making for breast cancer. Whether the biomarker profile is chemotherapy-specific or whether it is a more general indicator of bad prognosis of breast cancer patients remains to be explored.

Synergistic apoptotic effects of taurolidine and TRAIL on squamous carcinoma cells of the esophagus.

The treatment of choice for esophageal cancer is considered surgical resection, but a median survival of around 20 months after treatment is still discouraging. The value of adjuvant or neoadjuvant radiation or chemotherapy is limited and to date, benefits have only been described for certain tumor stages. Therefore, new therapeutic options are required. As alternative chemotherapeutics, we tested the antibiotic taurolidine (TRD) on KYSE 270 human esophageal carcinoma cells alone and in combination with rhTRAIL (TNF related apoptosis-inducing ligand). Viability, apoptosis and necrosis were visualized by TUNEL assay and quantitated by FACS analysis. Gene expression was analysed by RNA microarray. The most effective concentration of TRD as single substance (250 micromol/l) induced apoptosis to a maximum of 40% after 12-h dose dependently, leaving 4% viable cells after 48 h; by comparison, rhTRAIL did not have a significant effect. The combination of both substances doubled the effect of TRD alone. Gene expression profiling revealed that TRD downregulated endogenous TRAIL, TNFRSF1A, TRADD, TNFRSF1B, TNFRSF21, FADD, as well as MAP2K4, JAK2 and Bcl2, Bcl2l1, APAF1 and caspase-3. TNFRSF25, cytochrome-c, caspase-1, -8, -9, JUN, GADD45A and NFKBIA were upregulated. TRAIL reduced endogenous TRAIL, Bcl2l1 and caspase-1 expression. BIRC2, BIRC3, TNFAIP3, and NFKBIA were upregulated. The combined substances upregulated endogenous TRAIL, NFKBIA and JUN, whereas DFFA and TRAF3 were downregulated compared to TRD as single substance. We conclude that TRD overcomes TRAIL resistance in KYSE 270 cells. Synergistic effects are dependent on the same and on distinct apoptotic pathways which, jointly triggered, result in an amplified response. Several apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway, were differentially regulated by the substances on gene expression level. Additionally transcription factors seem to be influenced, NFKB in particular. Endogenous TRAIL expression is increased by the combination of substances, whereas it is reduced by each single substance. Taking into consideration that the non-toxic TRD was able to reduce rhTRAIL toxicity and dose, combined therapy with TRD and rhTRAIL may offer new options for treatment in esophageal cancer.