Irreversible inhibition of estrogen receptor α signaling and the emergence of hormonal resistance in MCF7 breast cancer cells induced by DNA damage agents.

Combining chemotherapy and hormone therapy is a prevalent approach in breast cancer treatment. While the cytotoxic impact of numerous chemotherapy drugs stems from DNA damage, the exact role of these DNA alterations in modulating estrogen receptor α (ERα) machinery remains elusive. The present study aimed to analyze the impact of DNA damage agents on ERα signaling in breast cancer cells and assess the signaling pathways mediating the influence of DNA damage drugs on the ERα machinery. Cell viability was assessed using the MTT method, while the expression of signaling proteins was analyzed by immunoblotting. ERα activity in the cells treated with various drugs (17ß-estradiol, tamoxifen, 5-fluorouracil) was assessed through reporter gene assays. In vitro experiments were conducted on MCF7 breast cancer cells subjected to varying durations of 5-fluorouracil (5-FU) treatment. Two distinct cell responses to 5-FU were identified based on the duration of the treatment. A singular dose of 5-FU induces pronounced DNA fragmentation, temporally suppressing ERα signaling while concurrently activating AKT phosphorylation. This suppression reverses upon 5-FU withdrawal, restoring normalcy within ten days. However, chronic 5-FU treatment led to the emergence of 5-FU-resistant cells with irreversible alterations in ERα signaling, resulting in partial hormonal resistance. These changes mirror those observed in cells subjected to UV-induced DNA damage, underscoring the pivotal role of DNA damage in shaping estrogen signaling alterations in breast cancer cells. In summary, the results of the present study suggested that the administration of DNA damage agents to cancer cells can trigger irreversible suppression of estrogen signaling, fostering the development of partial hormonal resistance. This outcome may ultimately impede the efficacy of combined or subsequent chemo- and hormone therapy strategies.

Genetic, Epigenetic and Transcriptome Alterations in Liposarcoma for Target Therapy Selection.

Liposarcoma (LPS) is one of the most common adult soft-tissue sarcomas (STS), characterized by a high diversity of histopathological features as well as to a lesser extent by a spectrum of molecular abnormalities. Current targeted therapies for STS do not include a wide range of drugs and surgical resection is the mainstay of treatment for localized disease in all subtypes, while many LPS patients initially present with or ultimately progress to advanced disease that is either unresectable, metastatic or both. The understanding of the molecular characteristics of liposarcoma subtypes is becoming an important option for the detection of new potential targets and development novel, biology-driven therapies for this disease. Innovative therapies have been introduced and they are currently part of preclinical and clinical studies. In this review, we provide an analysis of the molecular genetics of liposarcoma followed by a discussion of the specific epigenetic changes in these malignancies. Then, we summarize the peculiarities of the key signaling cascades involved in the pathogenesis of the disease and possible novel therapeutic approaches based on a better understanding of subtype-specific disease biology. Although heterogeneity in liposarcoma genetics and phenotype as well as the associated development of resistance to therapy make difficult the introduction of novel therapeutic targets into the clinic, recently a number of targeted therapy drugs were proposed for LPS treatment. The most promising results were shown for CDK4/6 and MDM2 inhibitors as well as for the multi-kinase inhibitors anlotinib and sunitinib.

Perspectives of Cell Sensitivity/Resistance Assay in Soft Tissue Sarcomas Chemotherapy.

Treatment of highly malignant soft tissue sarcomas (STSs) requires multicomponent therapy including surgery, radiotherapy, and chemotherapy. Despite the advancements in targeted cancer therapies, cytostatic drug combinations remain the gold standard for STS chemotherapy. The lack of algorithms for personalized selection of STS chemotherapy leads to unhelpful treatment of chemoresistant tumors, causing severe side effects in patients. The goal of our study is to assess the applicability of in vitro chemosensitivity/resistance assays (CSRAs) in predicting STS chemoresistance. Primary cell cultures were obtained from 148 surgery samples using enzymatic and mechanical disaggregation. CSRA was performed using resazurin-based metabolic activity measurement in cells cultured with doxorubicin, ifosfamide, their combination and docetaxel, gemcitabine, and also their combination for 7 days. Both the clinical data of patients and the CSRA results demonstrated a higher resistance of some cancer histotypes to specific drugs and their combinations. The correlation between the CSRA results for doxorubicin and ifosfamide and clinical responses to the combination chemotherapy with these drugs was demonstrated via Spearman rank order correlation. Statistically significant differences in recurrence-free survival were also shown for the groups of patients formed, according to the CSRA results. Thus, CSRAs may help both practicing physicians to avoid harmful and useless treatment, and researchers to study new resistance markers and to develop new STS drugs.

Targeting Features of Curaxin CBL0137 on Hematological Malignancies In Vitro and In Vivo.

The anticancer activity of Curaxin CBL0137, a DNA-binding small molecule with chromatin remodulating effect, has been demonstrated in different cancers. Herein, a comparative evaluation of CBL0137 activity was performed in respect to acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia and multiple myeloma (MM) cultured in vitro. MTT assay showed AML and MM higher sensitivity to CBL0137's cytostatic effect comparatively to other hematological malignancy cells. Flow cytometry cell cycle analysis revealed an increase in subG1 and G2/M populations after CBL0137 cell treatment, but the prevalent type of arrest varied. Apoptosis activation by CBL0137 measured by Annexin-V/PI dual staining was more active in AML and MM cells. RT2 PCR array showed that changes caused by CBL0137 in signaling pathways involved in cancer pathogenesis were more intensive in AML and MM cells. On the murine model of AML WEHI-3, CBL0137 showed significant anticancer effects in vivo, which were evaluated by corresponding changes in spleen and liver. Thus, more pronounced anticancer effects of CBL0137 in vitro were observed in respect to AML and MM. Experiments in vivo also indicated the perspective of CBL0137 use for AML treatment. This in accordance with the frontline treatment approach in AML using epigenetic drugs.

Effects of G-Quadruplex-Binding Plant Secondary Metabolites on c-MYC Expression.

Guanine-rich DNA sequences tending to adopt noncanonical G-quadruplex (G4) structures are over-represented in promoter regions of oncogenes. Ligands recognizing G4 were shown to stabilize these DNA structures and drive their formation regulating expression of corresponding genes. We studied the interaction of several plant secondary metabolites (PSMs) with G4s and their effects on gene expression in a cellular context. The binding of PSMs with G4s formed by the sequences of well-studied oncogene promoters and telomeric repeats was evaluated using a fluorescent indicator displacement assay. c-MYC G4 folding topology and thermal stability, as well as the PMS influence on these parameters, were demonstrated by UV-spectroscopy and circular dichroism. The effects of promising PSMs on c-MYC expression were assessed using luciferase reporter assay and qPR-PCR in cancer and immortalized cultured cells. The ability of PMS to multi-targeting cell signaling pathways was analyzed by the pathway-focused gene expression profiling with qRT-PCR. The multi-target activity of a number of PSMs was demonstrated by their interaction with a set of G4s mimicking those formed in the human genome. We have shown a direct G4-mediated down regulation of c-MYC expression by sanguinarine, quercetin, kaempferol, and thymoquinone; these effects being modulated by PSM's indirect influence via cell signaling pathways.

Soft Tissue Sarcoma Study: Association of Genetic Alterations in the Apoptosis Pathways with Chemoresistance to Doxorubicin.

Soft tissue sarcomas (STS) are heterogeneous cancers with more than 100 histological subtypes, different in molecular alterations, which make its personalized therapy very complex. Gold standard of chemotherapy for advanced STS includes combinations of Doxorubicin and Ifosfamide or Gemcitabine and Docetaxel. Chemotherapy is efficient for less than 50% of patients and it is followed by a fast development of drug resistance. Our study was directed to the search of genetic alterations in cancer cells associated with chemoresistance of undifferentiated pleomorphic and synovial sarcomas to the abovementioned genotoxic drugs. We analyzed chemoresistance of cancer cells in vitro using primary STS cultures and performed genetic analysis for the components of apoptotic signaling. In 27% of tumors, we revealed alterations in TP53, ATM, PIK3CB, PIK3R1, NTRK1, and CSF2RB. Cells from STS specimens with found genetic alterations were resistant to Dox, excluding the only one case when TP53 mutation resulted in the substitution Leu344Arg associated with partial oligomerization loss and did not cause total loss of TP53 function. Significant association between alterations in the components of apoptosis signaling and chemoresistance to Dox was found. Our data are important to elaborate further the therapeutic strategy for STS patients with alterations in apoptotic signaling.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells.

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Molecular Mechanisms of Anticancer Activity of N-Glycosides of Indolocarbazoles LCS-1208 and LCS-1269.

Novel indolocarbazole derivatives named LCS were synthesized by our research group. Two of them were selected as the most active anticancer agents in vivo. We studied the mechanisms of anticancer activity in accordance with the previously described effects of indolocarbazoles. Cytotoxicity was estimated by MTT assay. We analyzed LCS-DNA interactions by circular dichroism in cholesteric liquid crystals and fluorescent indicator displacement assay. The effect on the activity of topoisomerases I and II was studied by DNA relaxation assay. Expression of interferon signaling target genes was estimated by RT-PCR. Chromatin remodeling was analyzed-the effect on histone H1 localization and reactivation of epigenetically silenced genes. LCS-induced change in the expression of a wide gene set was counted by means of PCR array. Our study revealed the cytotoxic activity of the compounds against 11 cancer cell lines and it was higher than in immortalized cells. Both compounds bind DNA; binding constants were estimated-LCS-1208 demonstrated higher affinity than LCS-1269; it was shown that LCS-1208 intercalates into DNA that is typical for rebeccamycin derivatives. LCS-1208 also inhibits topoisomerases I and IIα. Being a strong intercalator and topoisomerase inhibitor, LCS-1208 upregulates the expression of interferon-induced genes. In view of LCSs binding to DNA we analyzed their influence on chromatin stability and revealed that LCS-1269 displaces histone H1. Our analysis of chromatin remodeling also included a wide set of epigenetic experiments in which LCS-1269 demonstrated complex epigenetic activity. Finally, we revealed that the antitumor effect of the compounds is based not only on binding to DNA and chromatin remodeling but also on alternative mechanisms. Both compounds induce expression changes in genes involved in neoplastic transformation and target genes of the signaling pathways in cancer cells. Despite of being structurally similar, each compound has unique biological activities. The effects of LCS-1208 are associated with intercalation. The mechanisms of LCS-1269 include influence on higher levels such as chromatin remodeling and epigenetic effects.

Current Approaches for Personalized Therapy of Soft Tissue Sarcomas.

Soft tissue sarcomas (STS) are a highly heterogeneous group of cancers of mesenchymal origin with diverse morphologies and clinical behaviors. While surgical resection is the standard treatment for primary STS, advanced and metastatic STS patients are not eligible for surgery. Systemic treatments, including standard chemotherapy and newer chemical agents, still play the most relevant role in the management of the disease. Discovery of specific genetic alterations in distinct STS subtypes allowed better understanding of mechanisms driving their pathogenesis and treatment optimization. This review focuses on the available targeted drugs or drug combinations based on genetic aberration involved in STS development including chromosomal translocations, oncogenic mutations, gene amplifications, and their perspectives in STS treatment. Furthermore, in this review, we discuss the possible use of chemotherapy sensitivity and resistance assays (CSRA) for the adjustment of treatment for individual patients. In summary, current trends in personalized management of advanced and metastatic STS are based on combination of both genetic testing and CSRA.