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Background

Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of HOMECs by tumour -secreted factors in their microenvironment. We have previously shown that ovarian cancer cells secrete a range of factors with possible roles in metastatic angiogenesis including the lysosomal proteases cathepsin D (CD) and cathepsin L (CL). However, the role of these proteases in ovarian cancer metastasis to the omentum is not fully understood.

Aim

To investigate whether proliferative effects of CD and CL are dependent on their catalytic activity in HOMECs.

To investigate the intracellular signalling kinases activated by CD and CL.

Method

HOMEC proliferation was assessed by using a colorimetric (WST1) assay. Potential signalling pathways were examined by phosphokinase array and ELISAs. pH experiments were carried out examine whether the observed effects were due to the catalytic activity of CD and CL.

Result

CD and CL (50 ng/ml) significantly increased HOMEC proliferation to 141 ± 27% (p = 0.001, n = 50) and 151% ± 34% (p = 0.001, n = 45) respectively vs. control (100%) 72 h post-treatment. Inhibitors of CD and CL enzyme activity had no effect on HOMEC proliferation and subsequent pH data suggest a nonproteolytic mitogenic activity of these cathepsins. Both proteins induced phosphorylation of ERK1/2, AKT and p38α to ∼2, ∼1.5 and ∼1.5 folds respectively relative to total levels (compared to control).

Conclusion

CD and CL induced proliferation in HOMECs. CD and CL may non- proteolytically contribute to pro-angiogenic responses of the omental microvasculature. Induction of phosphorylation of proliferative kinases ERK1/2, AKT and p38α suggest possible downstream signalling cascades of these proteins.

Background

Glioblastomas (GBL) are most aggressive brain tumors in adults. They are often radio- and chemotherapy resistant. The standard therapy of GBL includes surgery followed by radiotherapy with concomitant chemotherapy with temozolomide named temodal (imidazotetrazine derivative). Methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene promoter is considered as predictor of better outcome of temodal therapy in comparison with the cases without MGMT promoter methylation (Stupp et al., 2009). The aim of this study is finding of additional predictors of temozolomide therapy effectiveness.

Materials and methods

We obtained primary cultures of GBL. The rate of GBL sensitivity to temozolomide was revealed by MTT test. Expression of multidrug resistance genes and genes which mediate temozolomide sensitivity (YB1, MDR1, MRP1, LRP, BCRP and MGMT) was determined by real time PCR. Protein localization and expression was revealed by immunohistochemical technique.

Results

We developed 14 primary GBL cultures. Staining for nestin was used for demonstration of neuronal origin of cell cultures. Analysis of gene expression and the rate of cell proliferation of the cultures showed that the rate of YB1 gene expression in slow proliferating cultures is significantly lower than in quickly proliferating cell populations. Cell cultures differed in temodal sensitivity 2–2.5-fold. We analyzed whether GBL cell sensitivity to temodal and the rate of several genes’ expression are correlated. We did not find the correlation between cell temodal sensitivity and the rate of MGMT gene expression. These data are in accordance with some results of another authors (Mullins et al., 2013; Brennan et al., 2013). However, we revealed positive correlation of the temodal sensitivity of GBL cultures and MVP/LRP expression (r = 0.5592, p = 0.04). We found also the trend to negative correlation between GBL temodal sensitivity and YB1 gene expression (r = −0.43602, p = 0.02) as well as MDR1 expression (r = −0.4195, p = 0.02).

Conclusion

The rate of temodal sensitivity of GBL primary cultures is connected with the rate of the expression of MVP/LRP, YB1 and MDR1genes. It is likely that YB-1 protein affects temodal sensitivity by influence on DNA reparation. YB1 could also have an effect on MDR1 expression. MVP/LRP expression is independent factor of GBL prognosis.

This paper was completed with partial support from the Russian Foundation for Basic Research – Russia (Grant No. 23-04-40204).

Melanoma is one of the aggressive cancer types. Mutations that lock the BRAF protein in an active state may cause excessive signaling in the pathway, leading to uncontrolled cell growth and survival. Primarily among the BRAF mutations observed in melanoma, over 90% are supposed to be at codon 600, resulting in substitution of glutamic acid for valine, V600E (T > A transversion) located in exon 15, BRAFV600E. Of particular interest is BRAF negative melanoma. This type of melanoma is not sensitive to BRAF inhibitors and approaches to therapy require further study. We aimed to investigate the frequency of BRAF V600 mutations in 80 patients with primary melanoma and determine the relationship between mutations and clinical/pathologic features. Genomic DNA was extracted from biopsy specimens with prevalent percentage of tumor cells by DNA-sorb B isolation kit (Amplisense, Russia). BRAF V600E mutation was estimated by real-time PCR-based assay for the BRAF V600E mutation allele-specific DNA test (BioLink, Russia). Breslow thickness was assessed by applying commercial Infinity Capture, Infinity Analyze Software. The lymphocytic infiltration was determined in all tumors and classified as “brisk”, “nonbrisk”, and “absent” according to criteria established by Clark et al. Tumor infiltrating lymphocytes (TIL) were identified as lymphocytes within tumor nodes. “Brisk” infiltrate was determined in case of a diffuse presence of lymphocytes within tumor, “non-brisk” infiltrate was in focal location of lymphocytes and “absent” if no lymphocytes were present in a tumor. Mitotic activity was determined as mitotic count on 10 high power fields. For all patients, clinical and pathologic features were tested for significant association with BRAF V600E mutation status using simple cross tabulations, Fisher’s exact test, Pearson’s χ² test, and/or non-parametric Mann–Whitney U test. The P values lower 0.05 were considered as significant. BRAF V600E mutation was detected in 41.25% of tested tumours. Patients with BRAF-mutant and non-BRAF mutant melanoma were matched by age and gender. Superficial spreading melanomawas observed in 66.2% of patients with wild-type BRAF, nodular melanoma in 21.2%, both lentigo-melanoma and acral-lentiginous melanomain 6.3% and mucosal melanoma in 1 3.0% of patients. In wild-type BRAF melanoma patients, 59.7% tumors had “brisk” infiltrate, 14.8% – “non-brisk”, and 12 25.5% had no infiltrate. There was no found correlation between BRAF status and tumor localization, clinico-pathological type of tumor, TIL status, Breslow thickness and mitotic rate. However, when cases were stratified by age, it was revealed that melanoma patients aged above 80 years were preferentially BRAF-negative (p < 0.05). BRAF-negative melanomas occurred significantly more frequent in superficial spreading type of the tumor. The localization of melanomas was differe