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In the present study we examined a hypothesis that salinomycin, an antibiotic ionophore, might efficiently potentiate the cytotoxic effect of dichloroacetate (DCA), a metabolic inhibitor, on cancer cells. We used two human colorectal cancer derived cell lines - DLD-1 and HCT116. First, we performed series of dose response experiments in 2D cell culture in mono- and combination therapy and evaluated effects, based on Chou-Talalay method. This analysis revealed that salinomycin in combination with DCA acted synergistically in both cell lines. Secondly, in order to recapitulate the in vivo tumour architecture, we tested doses, selected from 2D experiments, in 3D multicellular spheroid culture in mono- and in combination therapy. In such conditions, the effect of salinomycin and DCA was additive in DLD-1 and synergistic in HCT116 cell lines. Further, we demonstrate that synergistic effect of compounds might be related with inhibitory effect of DCA on multidrug resistance proteins (MRPs) and with reduction of intracellular pH that in cancer cells is elevated. The activity of MRPs as well as potency of certain drugs is pH-dependent. Finally in order to disclose the rationale for sensitivity variations to combination therapy between two cell lines, we have investigated gene expression profiling of DLD-1 and HCT116 cell lines in 2D and 3D cell cultures and presume that decreased sensitivity of DLD1 cells to combination therapy could be attributed to increased stemness of DLD-1 cells in 3D cultures compared to HCT116 cells.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor with the highest incidence rates in Lithuania. CcRCC is characterized by the highest mortality rate of genitourinary cancers, therefore, new diagnostic and/or prognostic biomarkers are urgently needed. Considering this, the current study aimed to develop a gene-specific DNA methylation tool for non-invasive and early kidney cancer diagnosis and follow-up. Genome-wide DNA methylation profiling utilizing microarrays allowed the identification of a set of novel presumable ccRCC-specific DNA methylation biomarkers, having moderate to high diagnostic and/or prognostic potential. Moreover, identified DNA methylation alterations at the regulatory regions of selected genes appeared to be amenable for non-invasive detection in the urine samples of ccRCC patients, where they outperformed the diagnostic and prognostic value of previously described biomarkers and even some other parameters currently used in the clinical practice. Thus, the results showed the promising potential of the chosen genes as candidates for further development of non-invasive tools for kidney cancer patients testing. Although further comprehensive verification is mandatory to prove the clinical significance of the provided DNA methylation biomarkers for ccRCC, the obtained results seem to be rather promising and will definitely encourage these validation studies.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor with the highest incidence rates in Lithuania. CcRCC is characterized by the highest mortality rate of genitourinary cancers, therefore, new diagnostic and/or prognostic biomarkers are urgently needed. Considering this, the current study aimed to develop a gene-specific DNA methylation tool for non-invasive and early kidney cancer diagnosis and follow-up. Genome-wide DNA methylation profiling utilizing microarrays allowed the identification of a set of novel presumable ccRCC-specific DNA methylation biomarkers, having moderate to high diagnostic and/or prognostic potential. Moreover, identified DNA methylation alterations at the regulatory regions of selected genes appeared to be amenable for non-invasive detection in the urine samples of ccRCC patients, where they outperformed the diagnostic and prognostic value of previously described biomarkers and even some other parameters currently used in the clinical practice. Thus, the results showed the promising potential of the chosen genes as candidates for further development of non-invasive tools for kidney cancer patients testing. Although further comprehensive verification is mandatory to prove the clinical significance of the provided DNA methylation biomarkers for ccRCC, the obtained results seem to be rather promising and will definitely encourage these validation studies.

Although local PCa treatment is quite effective and aggressive disease develops for a minority of patients, such PCa cases are associated with worse prognosis and shorter survival; also there is a lack of reliable biomarkers for the evaluation of response to treatment. Blood- and urine-circulating RNA can be used as lowly-invasive biomarkers for diagnostic and prognostic purposes in PCa. We found that miR-148a, -365, -375, and -429 are PCa-specific and have significant associations with clinical-pathological parameters of PCa. Also we demonstrated that the combination of urinary miR-148a and miR-375 is a sensitive molecular tool for noninvasive diagnosis of PCa, while the combination of miR-19b and miR-19a/b specifically predicts biochemical recurrence. Castration resistant prostate cancer (CRPC) remains incurable. Due to the heterogeneous nature of CRPC, approximately 30% of patients are already resistant to these new drugs. In patients treated with abiraterone acetate (AA), urinary miR-148 predicts progression-free survival, while the combination of miR-148a and miR-375 – overall survival. One of the resistance mechanisms could be AR splicing variants (AR-Vs) which lack a ligand binding domain and are constitutively active. We demonstrated that higher level of AR-FL and -V1 transcripts in AA-treated CRPC patients’ blood predicts progression-free survival and overall survival.

Although local PCa treatment is quite effective and aggressive disease develops for a minority of patients, such PCa cases are associated with worse prognosis and shorter survival; also there is a lack of reliable biomarkers for the evaluation of response to treatment. Blood- and urine-circulating RNA can be used as lowly-invasive biomarkers for diagnostic and prognostic purposes in PCa. We found that miR-148a, -365, -375, and -429 are PCa-specific and have significant associations with clinical-pathological parameters of PCa. Also we demonstrated that the combination of urinary miR-148a and miR-375 is a sensitive molecular tool for noninvasive diagnosis of PCa, while the combination of miR-19b and miR-19a/b specifically predicts biochemical recurrence. Castration resistant prostate cancer (CRPC) remains incurable. Due to the heterogeneous nature of CRPC, approximately 30% of patients are already resistant to these new drugs. In patients treated with abiraterone acetate (AA), urinary miR-148 predicts progression-free survival, while the combination of miR-148a and miR-375 – overall survival. One of the resistance mechanisms could be AR splicing variants (AR-Vs) which lack a ligand binding domain and are constitutively active. We demonstrated that higher level of AR-FL and -V1 transcripts in AA-treated CRPC patients’ blood predicts progression-free survival and overall survival.

Combination therapy is the cornerstone of cancer treatment. The simultaneous application of cytotoxic drugs potentiates their efficacy compared with monotherapy because it targets the key pathways in a synergistic or an additive manner. Such therapy is likely to diminish drug resistance, while simultaneously providing cytotoxic benefits, such as inhibition of tumor growth, decrease of cancer stem cell population, reduction of metastatic potential, and induction of apoptosis. In the present study, we investigated the effects of two drugs – dichloroacetate and salinomycin and the combination of both agents on the colorectal as well as lung cancer cell lines in vitro in 2D and 3D cell culture models. We also have tested therapeutic potential of this combination therapy in vivo in an allograft C57BL/6 mice model. In vitro studies were dedicated to investigate potential mechanism of salinomycin+dichloroacetate action and with in vivo studies we analyzed effects of this therapy on tumor growth, metastatic site formation, and the expression of tumor progression markers.

Early diagnosis of prostate cancer (PCa) and patients’ stratification into different risk groups are critical for successful management of the disease. DNA methylation of tumor suppressor genes (TSGs) is a potential source of diagnostic and prognostic biomarkers. The present study focused on identification and validation of novel DNA methylation biomarkers of PCa. During the first stage of experimental work, several TSGs were selected for methylation analysis in prostate tumors followed by evaluation of the most potential biomarkers for noninvasive screening in urine. Although the obtained results were promising, the relatively low specificity for PCa encouraged to search for biomarkers with excelling performance. The second stage of the study began with genome-wide DNA methylation profiling, which led to identification of a set of novel putative biomarkers with diagnostic and/ or prognostic value that might even surpass currently available tools. The prostate cancer dataset of The Cancer Genome Atlas project was utilized as an independent validation cohort. Promoter methylation of the newly identified genes was also demonstrated to be detectable in urine of PCa patients, which shows its potential application for noninvasive testing. Although additional validation is required, the obtained results seem to be rather promising. Protein-coding and miRNA host genes with newly identified methylation differences in PCa might be attractive targets for epigenetic therapeutics.

Early diagnosis of prostate cancer (PCa) and patients’ stratification into different risk groups are critical for successful management of the disease. DNA methylation of tumor suppressor genes (TSGs) is a potential source of diagnostic and prognostic biomarkers. The present study focused on identification and validation of novel DNA methylation biomarkers of PCa. During the first stage of experimental work, several TSGs were selected for methylation analysis in prostate tumors followed by evaluation of the most potential biomarkers for noninvasive screening in urine. Although the obtained results were promising, the relatively low specificity for PCa encouraged to search for biomarkers with excelling performance. The second stage of the study began with genome-wide DNA methylation profiling, which led to identification of a set of novel putative biomarkers with diagnostic and/ or prognostic value that might even surpass currently available tools. The prostate cancer dataset of The Cancer Genome Atlas project was utilized as an independent validation cohort. Promoter methylation of the newly identified genes was also demonstrated to be detectable in urine of PCa patients, which shows its potential application for noninvasive testing. Although additional validation is required, the obtained results seem to be rather promising. Protein-coding and miRNA host genes with newly identified methylation differences in PCa might be attractive targets for epigenetic therapeutics.