Sodium Butyrate Enhances the Cytotoxic Effect of Etoposide in HDACi-Sensitive and HDACi-Resistant Transformed Cells.

To overcome the problem of antitumor agent toxicity for normal cells, a combined therapy using drugs with synergistic effects seems to be more effective. We investigated the molecular mechanisms of the sensitization of tumor cells resistant and sensitive to histone deacetylase inhibitors (HDACis) upon etoposide treatment together with the HDACi sodium butyrate (NaBut). We showed that NaBut enhances the cytotoxic effect of etoposide in both HDACi-sensitive and HDACi-resistant cells due to the accumulation of the Bax protein and the dissociation of Ku70-Bax inhibitory complexes. In HDACi-resistant cells, NaBut causes the cytoplasmic accumulation of Bax dissociated from mitochondria in complexes with Ku70 proteins. The increased phosphorylation of the pro-apoptotic Bad protein due to the NaBut-induced activation of Erk and Akt kinases is one of the possible reasons for the accumulation of Bax in the cytoplasm. Despite the inactivation of Bax in HDACi-resistant cells, its accumulation in the cytoplasm upon NaBut treatment makes it possible to enhance the apoptotic response against agents activating the intrinsic pathway of apoptosis. Thus, HDACis involved in combined therapy mediate the sensitization of tumor cells to genotoxic drugs, regardless of the cells' resistance to HDACis.

Autocrine regulation of tumor cell repopulation by Hsp70-HMGB1 alarmin complex.

BACKGROUND: Cancer recurrence is regulated by a variety of factors, among which is the material of dying tumor cells; it is suggested that remaining after anti-cancer therapy tumor cells receive a signal from proteins called damage-associated molecular patterns (DAMPs), one of which is heat shock protein 70 (Hsp70). METHODS: Two models of tumor repopulation were employed, based on minimal population of cancer cells and application of conditioned medium (CM). To deplete the CMs of Hsp70 affinity chromatography on ATP-agarose and immunoprecipitation were used. Cell proliferation and the dynamics of cell growth were measured using MTT assay and xCELLigence technology; cell growth markers were estimated using qPCR and with the aid of ELISA for prostaglandin E detection. Immunoprecipitation followed by mass-spectrometry was employed to identify Hsp70-binding proteins and protein-protein interaction assays were developed to reveal the above protein complexes. RESULTS: It was found that CM of dying tumor cells contains tumor regrowth-initiating factors and the removal of one of them, Hsp70, caused a reduction in the relapse-activating capacity. The pull out of Hsp70 alone using ATP-agarose had no effect on repopulation, while the immunodepletion of Hsp70 dramatically reduced its repopulation activity. Using proteomic and immunochemical approaches, we showed that Hsp70 in conditioned medium binds and binds another abundant alarmin, the High Mobility Group B1 (HMGB1) protein; the complex is formed in tumor cells treated with anti-cancer drugs, persists in the cytosol and is further released from dying tumor cells. Recurrence-activating power of Hsp70-HMGB1 complex was proved by the enhanced expression of proliferation markers, Ki67, Aurka and MCM-10 as well as by increase of prostaglandin E production and autophagy activation. Accordingly, dissociating the complex with Hsp70 chaperone inhibitors significantly inhibited the pro-growth effects of the above complex, in both in vitro and in vivo tumor relapse models. CONCLUSIONS: These data led us to suggest that the abundance of the Hsp70-HMGB1 complex in the extracellular matrix may serve as a novel marker of relapse state in cancer patients, while specific targeting of the complex may be promising in the treatment of cancers with a high risk of recurrence.

HDAC Inhibitor Sodium Butyrate Attenuates the DNA Repair in Transformed but Not in Normal Fibroblasts.

Many cancer therapy strategies cause DNA damage leading to the death of tumor cells. The DNA damage response (DDR) modulators are considered as promising candidates for use in combination therapy to enhance the efficacy of DNA-damage-mediated cancer treatment. The inhibitors of histone deacetylases (HDACis) exhibit selective antiproliferative effects against transformed and tumor cells and could enhance tumor cell sensitivity to genotoxic agents, which is partly attributed to their ability to interfere with DDR. Using the comet assay and host-cell reactivation of transcription, as well as γH2AX staining, we have shown that sodium butyrate inhibited DNA double-strand break (DSB) repair of both endo- and exogenous DNA in transformed but not in normal cells. According to our data, the dysregulation of the key repair proteins, especially the phosphorylated Mre11 pool decrease, is the cause of DNA repair impairment in transformed cells. The inability of HDACis to obstruct DSB repair in normal cells shown in this work demonstrates the advantages of HDACis in combination therapy with genotoxic agents to selectively enhance their cytotoxic activity in cancer cells.

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination.

Clinical tests based on whole-genome sequencing are generally focused on a single task approach, testing one or several parameters, although whole-genome sequencing (WGS) provides us with large data sets that can be used for many supportive analyses. In spite of low genome coverage, data of WGS-based non-invasive prenatal testing (NIPT) contain fully sequenced mitochondrial DNA (mtDNA). This mtDNA can be used for variant calling, ancestry analysis, population studies and other approaches that extend NIPT functionality. In this study, we analyse mtDNA pool from 645 cell-free DNA (cfDNA) samples of pregnant women from different regions of Russia, explore the effects of transportation and storing conditions on mtDNA content, analyse effects, frequency and location of mitochondrial variants called from samples and perform haplogroup analysis, revealing the most common mitochondrial superclades. We have shown that, despite the relatively low sequencing depth of unamplified mtDNA from cfDNA samples, the mtDNA analysis in these samples is still an informative instrument suitable for research and screening purposes.

Expression of Adenoviral E1A in Transformed Cells as an Additional Factor of HDACi-Dependent FoxO Regulation.

The adenoviral early region 1A (E1A) protein has proapoptotic and angiogenic activity, along with its chemosensitizing effect, making it the focus of increased interest in the context of cancer therapy. It was previously shown that E1A-induced chemosensitization to different drugs, including histone deacetylases inhibitors (HDACi), appears to be mediated by Forkhead box O (FoxO) transcription factors. In this study, we explore the relationship between E1A expression and the modulation of FoxO activity with HDACi sodium butyrate (NaBut). We show here that the basal FoxO level is elevated in E1A-expressing cells. Prolonged NaBut treatment leads to the inhibition of the FoxO expression and activity in E1A-expressing cells. However, in E1A-negative cells, NaBut promotes the transactivation ability of FoxO over time. A more detailed investigation revealed that the NaBut-induced decrease of FoxO activity in E1A-expressing cells is due to the NaBut-dependent decrease in E1A expression. Therefore, NaBut-induced inhibition of FoxO in E1A-positive cells can be overcome under unregulated overexpression of E1A. Remarkably, the CBP/p300-binding domain of E1Aad5 is responsible for stabilization of the FoxO protein. Collectively, these data show that the expression of E1A increases the FoxO stability but makes the FoxO level more sensitive to HDACi treatment.

p21Waf1 deficiency does not decrease DNA repair in E1A+cHa-Ras transformed cells by HDI sodium butyrate.

This study aimed to explore a role of p21Waf1 in γH2AX foci formation and DNA repair as assessed by a Host-Cell Reactivation Assay in wild-type (p21Waf+/+) and p21Waf1-deficient E1A+Ras-transformed cells. p21Waf1+/+ cells have low γH2AX background compared to p21Waf1-/- cells. The treatment with histone deacetylase inhibitor (HDI) sodium butyrate (NaBut) causes to accumulation of γH2AX in p21Waf+/+ cells with little effect in p21Waf-/- cells. Moreover, NaBut inhibits DNA repair in wt cells but not in p21Waf1-/- cells. This could be explained by the weakening of GADD45 and PCNA proteins binding in NaBut-treated p21Waf1-expressing cells but not in p21Waf1-/- cells. We suggest that in wt-ERas cells NaBut activates both p21Waf1 expression and a release of p21Waf1 from the complexes with E1A that leads to suppression of DNA repair and promotes γH2AX persistency. The absence of p21Waf1 is by itself considered by the cell as stressful factor with formation of γH2AX. But the lack of p21Waf1 interferes with an inhibitory effect of NaBut to inhibit DNA repair and thereby to stop concomitant accumulation of harmful mutations. We conclude that p21Waf1 is directly involved in control of genome integrity and DNA repair acting through modulation of the components of the DNA repair machinery.

Time-dependent modulation of FoxO activity by HDAC inhibitor in oncogene-transformed E1A+Ras cells.

HDAC inhibitors (HDACIs) induce irreversible cell cycle arrest and senescence in mouse embryonic fibroblasts transformed with E1A and c-Ha-Ras oncogenes (E1A+Ras cell line). The aging rate has been associated with the production of high levels of Reactive Oxygen Species (ROS). Specific increases of ROS level have been demonstrated as potentially critical for induction and maintenance of cell senescence process. It's known that HDACs regulate the ROS-dependent FoxO factors, which are responsible for cell growth, proliferation, and longevity. The characteristic ROS increase during aging may be responsible for the decreased HDAC activity, which facilitates the senescent-like phenotype. The objective of this study was to investigate the impact of FoxO transcription factors on HDACIs-induced senescence of E1A+Ras oncogenes transformed cells. This study shows the specific time-dependent effect of HDACI sodium butyrate treatment on FoxO proteins in E1A+Ras cells. Indeed, short-term treatment with NaB results in FoxO activation, which takes place through nuclear translocation, and accompanied by accumulation of such ROS scavengers as MnSOD and SOD2. However, prolonged treatment leads to extensive FoxO degradation and increased intracellular levels of ROS. This degradation is connected with NaB-induced activation of Akt kinase. All of these findings establish that one of the possible mechanism involved in NaB-induced senescence of transformed cells is mediated through down-regulation of FoxO transcription factors and ROS accumulation.

Correction: Time-dependent modulation of FoxO activity by HDAC inhibitor in oncogene-transformed E1A+Ras cells.

[This corrects the article DOI: 10.3934/genet.2018.1.41.].