Pharmaceutical Approach to Develop Novel Photosensitizer Nanoformulation: An Example of Design and Characterization Rationale of Chlorophyll α Derivative.

In this study, we described physico-chemical properties of novel nanoformulation of photosensitizer-pyropheophorbide α 17-diethylene glycol ester (XL) (chlorophyll α derivative), revealing insights into antitumor activity and maintaining quality, meeting the pharmaceutical approach of new nanoformulation design. Our formulation, based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles, increased XL solubility and selective tumor-targeted accumulation. In our research, we revealed, for the first time, that XL binding to polyvinyl alcohol (PVA) enhances XL photophysical activity, providing the rationale for PVA application as a stabilizer for nanoformulations. Results of FTIR, DSC, and XRD revealed the physical interactions between XL and excipients, including PVA, indicating that the encapsulation maintained XL binding to PVA. The encapsulated XL exhibited higher photophysical activity compared to non-encapsulated substance, which can be attributed to the influence of residual PVA. Gamma-irradiation led to degradation of XL; however, successful sterilization of the samples was achieved through the filtration. Importantly, the encapsulated and sterilized XL retained cytotoxicity against both 2D and 3D tumor cell models, demonstrating the potential of the formulated NP-XL for photodynamic therapy applications, but lacked the ability to reactivate epigenetically silenced genes. These findings provide valuable insights into the design and characterization of PLGA-based nanoparticles for the encapsulation of photosensitizers.

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.

Histone Methyltransferases as a New Target for Epigenetic Action of Vorinostat.

Epigenetic genome regulation during malignant cell transformation is characterized by the aberrant methylation and acetylation of histones. Vorinostat (SAHA) is an epigenetic modulator actively used in clinical oncology. The antitumor activity of vorinostat is commonly believed to be associated with the inhibition of histone deacetylases, while the impact of this drug on histone methylation has been poorly studied. Using HeLa TI cells as a test system allowing evaluation of the effect of epigenetically active compounds from the expression of the GFP reporter gene and gene knockdown by small interfering RNAs, we showed that vorinostat not only suppressed HDAC1, but also reduced the activity of EZH2, SUV39H1, SUV39H2, and SUV420H1. The ability of vorinostat to suppress expression of EZH2, SUV39H1/2, SUV420H1 was confirmed by Western blotting. Vorinostat also downregulated expression of SUV420H2 and DOT1L enzymes. The data obtained expand our understanding of the epigenetic effects of vorinostat and demonstrate the need for a large-scale analysis of its activity toward other enzymes involved in the epigenetic genome regulation. Elucidation of the mechanism underlying the epigenetic action of vorinostat will contribute to its more proper use in the treatment of tumors with an aberrant epigenetic profile.

Insights into the Mechanism of Curaxin CBL0137 Epigenetic Activity: The Induction of DNA Demethylation and the Suppression of BET Family Proteins.

The development of malignant tumors is caused by a complex combination of genetic mutations and epigenetic alterations, the latter of which are induced by either external environmental factors or signaling disruption following genetic mutations. Some types of cancer demonstrate a significant increase in epigenetic enzymes, and targeting these epigenetic alterations represents a compelling strategy to reverse cell transcriptome to the normal state, improving chemotherapy response. Curaxin CBL0137 is a new potent anticancer drug that has been shown to activate epigenetically silenced genes. However, its detailed effects on the enzymes of the epigenetic system of transcription regulation have not been studied. Here, we report that CBL0137 inhibits the expression of DNA methyltransferase DNMT3a in HeLa TI cells, both at the level of mRNA and protein, and it decreases the level of integral DNA methylation in Ca Ski cells. For the first time, it is shown that CBL0137 decreases the level of BET family proteins, BRD2, BRD3, and BRD4, the key participants in transcription elongation, followed by the corresponding gene expression enhancement. Furthermore, we demonstrate that CBL0137 does not affect the mechanisms of histone acetylation and methylation. The ability of CBL0137 to suppress DNMT3A and BET family proteins should be taken into consideration when combined chemotherapy is applied. Our data demonstrate the potential of CBL0137 to be used in the therapy of tumors with corresponding aberrant epigenetic profiles.

Synthesis of Novel Lipophilic Polyamines via Ugi Reaction and Evaluation of Their Anticancer Activity.

Natural polyamines (PAs) are involved in the processes of proliferation and differentiation of cancer cells. Lipophilic synthetic polyamines (LPAs) induce the cell death of various cancer cell lines. In the current paper, we have demonstrated a new method for synthesis of LPAs via the multicomponent Ugi reaction and subsequent reduction of amide groups by PhSiH3. The anticancer activity of the obtained compounds was evaluated in the A-549, MCF7, and HCT116 cancer cell lines. For the first time, it was shown that the anticancer activity of LPAs with piperazine fragments is comparable with that of aliphatic LPAs. The presence of a diglyceride fragment in the structure of LPAs appears to be a key factor for the manifestation of high anticancer activity. The findings of the study strongly support further research in the field of LPAs and their derivatives.

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.

HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes.

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors-vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors-decitabine, 5-azacytidine, RG108; HMT inhibitors-UNC0638, BIX01294, DZNep; a chromatin remodeler-curaxin CBL0137; and BET inhibitors-JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation.

Prevention of Colorectal Carcinogenesis by DNA-Binding Small-Molecule Curaxin CBL0137 Involves Suppression of Wnt Signaling.

Chemoprevention is considered a valid approach to reduce the incidence of colorectal cancer, one of the most common malignancies worldwide. Here, we investigated the tumor-preventive activity of curaxin CBL0137. This compound represents a new class of nonmutagenic DNA-binding small molecules that alter chromatin stability and inhibit the function of the histone chaperone FACT. Among downstream effects of CBL0137 treatment are activation of p53 and type I interferons and inhibition of NFκB, HSF1, and MYC. In addition, our data show that in both human and mouse colorectal cancer cells in vitro, CBL0137 inhibits the APC/WNT/ß-catenin signaling pathway, which plays a key role in colon carcinogenesis. Using quantitative RT-PCR and microarray hybridization, we have demonstrated decreased expression of multiple components and downstream targets of the WNT pathway in colon cancer cells treated with CBL0137. At the same time, CBL0137 induced expression of WNT antagonists. Inhibition of WNT signaling activity by CBL0137 was also confirmed by luciferase reporter assay. Tumor-preventive activity of CBL0137 in vivo was tested in a murine model of colorectal carcinogenesis induced by 1,2-dimethylhydrazine (DMH), which is known to involve WNT pathway dysregulation. After DMH subcutaneous treatment, mice were administered CBL0137 in drinking water. Efficacy of CBL0137 in suppressing development of colorectal cancer in this model was evidenced by reduced incidence of adenocarcinomas and adenomas in both males and females and decrease in tumor multiplicity. These data support the prospective use of CBL0137 in chemoprevention of colorectal cancer as well as of other malignances associated with activated WNT signaling.