Effect of a novel piperazine compound on cancer cells

Many drugs have been developed for anticancer chemotherapy. However, more anti-cancer drugs should be developed from potential chemicals to circumvent the disadvantages of existing drugs. Most anti-cancer chemicals induce apoptosis in cancer cells. This study tested the efficiency of a new chemical, the piperazine derivative 1-[2-(Allylthio) benzoyl]-4-(4-methoxyphenyl) piperazine (CB01), on glioblastoma (U87) and cervix cancer (HeLa) cells. CB01 was highly cytotoxic to these cells (IC50S < 50 nM) and induced the traditional apoptotic symptoms of DNA fragmentation and nuclear condensation at 40 nM. Western-blot analysis of the cell lysates revealed that the intracellular apoptotic marker proteins, such as cleaved caspase-3, cytochrome c, and Bax, were highly upregulated in the CB01-treated cells. Furthermore, increased activities of caspase-3 and -9, but not caspase-8, were observed. Therefore, these results suggest that CB01 can act as an anticancer chemotherapeutic by stimulating the intrinsic mitochondrial signaling pathway to induce cytotoxicity and apoptosis in cancer cells.

Antifungal and Anti-Biofilm Effects of Caffeic Acid Phenethyl Ester on Different Candida Species

This study investigated the effect of CAPE on planktonic growth, biofilm-forming abilities, mature biofilms, and cell death of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis strains. Our results showed a strain- and dose-dependent effect of CAPE on Candida, and the MIC values were between 12.5 and 100 µg/mL. Similarly, the MBIC values of CAPE ranging between 50 and 100 µg/mL highlighted the inhibition of the biofilm-forming abilities in a dose-dependent manner, as well. However, CAPE showed a weak to moderate biofilm eradication ability (19-49%) on different Candida strains mature biofilms. Both caspase-dependent and caspase-independent apoptosis after CAPE treatment were observed in certain tested Candida strains. Our study has displayed typical apoptotic hallmarks of CAPE-induced chromatin margination, nuclear blebs, nuclear condensation, plasma membrane detachment, enlarged lysosomes, cytoplasm fragmentation, cell wall distortion, whole-cell shrinkage, and necrosis. In conclusion, CAPE has a concentration and strain-dependent inhibitory activity on viability, biofilm formation ability, and cell death response in the different Candida species.

Nuclear Opening and Histone Release Are Essential for Nuclear but Not Chromatin Condensation during Terminal Erythropoiesis

Nuclear condensation and enucleation are characteristic processes in mammalian terminal erythropoiesis. These processes are associated with the transient nuclear opening formation that mediates partial histone release to the cytoplasm. Our previous report showed that caspases are involved in the cleavage of nuclear lamina to enable histone release. However, it remains unclear whether nuclear opening formation and histone release regulate the genomic three-dimensional organization during nuclear condensation. To answer this question, we cultured E13.5 mouse fetal liver Ter119 negative erythroid progenitor cells in erythropoietin (EPO) containing medium for 48 h with or without the presence of caspase inhibitor. As expected, caspase inhibitor blocked nuclear opening formation and histone release, and significantly reduced nuclear condensation and enucleation. We next performed a Hi-C sequencing to investigate chromatin structural change during terminal differentiation and nuclear condensation. To this end, the cultured fetal liver erythroid cells with or without caspase inhibitor were harvested at 30 h right before enucleation for Hi-C sequencing. The sequencing results showed that cells at 30 h contain significantly more interactions than freshly isolated erythroid progenitors, which is consistent with chromatin condensation during terminal erythropoiesis. Further analysis showed that increased interactions mainly accumulate as inter-chromosomal interactions, suggesting inter-chromosome interaction is the dominant structural force driving erythrocyte chromatin condensation. Surprisingly, there were no significant chromatin structural changes between caspase inhibitor treated and mock-treated cells when compared at 30 h. We also performed ATAC-seq and RNA-seq with the same experiment settings, both corresponded to Hi-C sequencing and showed little difference under caspase inhibitor treatment. These results indicate that although histone release and nuclear condensation are compromised with the inhibition of caspases, chromatin stays condensed with well-organized three-dimensional structure and appropriate gene expression regulations. To further confirm this phenomenon, we generated caspase-3 and -7 double knock out (cas3cas7-/-) mice. Cas3cas7-/- mice are embryonically lethal due to defective cardiac development. The hematopoietic tissues in these mice have not been well studied. We harvested fetal liver Ter119 negative erythroid progenitor cells from E13.5 cas3cas7-/- mice and the cells from the littermate (cas7-/-, cas3+/-cas7-/-) mice were used as controls. We first cultured Ter119 negative fetal liver erythroid progenitors in EPO containing medium for 48 h. Immunofluorescence analysis showed that the nuclear opening was significantly inhibited, and the nuclear size significantly increased in the erythroid cells from cas3cas7-/- mice due to failure of histone release into cytoplasm. Flow cytometry analysis showed that enucleation was significantly impaired in cas3cas7-/- cells, but the cells could still differentiate although with lower efficiency. We further performed an in vivo assay in which E13.5 cas3cas7-/- fetal liver cells were transplanted into wild type lethally irradiated recipient mice. EPO medium cultured bone marrow lineage negative cells from these transplanted mice showed significant reduction in nuclear opening and histone release, and enlargement of nuclear size. However, these mice survived well despite anemia. These results indicate a portion of orthochromatic erythroblasts managed to enucleate even with the less condensed nuclei. Overall, our study demonstrates that nuclear opening and histone release are essential for nuclear condensation but have minimal effects on chromatin condensation or the regulation of gene expression in terminal erythropoiesis. Appropriate nuclear condensation is important for efficient enucleation. However, orthochromatic erythroblasts could still manage to enucleate although with low efficacies. Disclosures No relevant conflicts of interest to declare.

The Natural Pigment Violacein Potentially Suppresses the Proliferation and Stemness of Hepatocellular Carcinoma Cells In Vitro

Hepatocellular carcinoma (HCC) is a malignant type of primary liver cancer with high incidence and mortality, worldwide. A major challenge in the treatment of HCC is chemotherapeutic resistance. It is therefore necessary to develop novel anticancer drugs for suppressing the growth of HCC cells and overcoming drug resistance for improving the treatment of HCC. Violacein is a deep violet-colored indole derivative that is produced by several bacterial strains, including Chromobacterium violaceum, and it possesses numerous pharmacological properties, including antitumor activity. However, the therapeutic effects of violacein and the mechanism underlying its antitumor effect against HCC remain to be elucidated. This study is the first to demonstrate that violacein inhibits the proliferation and stemness of Huh7 and Hep3B HCC cells. The antiproliferative effect of violacein was attributed to cell cycle arrest at the sub-G1 phase and the induction of apoptotic cell death. Violacein induced nuclear condensation, dissipated mitochondrial membrane potential (MMP), increased generation of reactive oxygen species (ROS), activated the caspase cascade, and upregulated p53 and p21. The anticancer effect of violacein on HCC cells was also associated with the downregulation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2 signaling. Violacein not only suppressed the proliferation and formation of tumorspheres of Huh7 and Hep3B cancer stem-like cells but also reduced the expression of key markers of cancer stemness, including CD133, Sox2, Oct4, and Nanog, by inhibiting the signal transducer and activator of transcription 3 (STAT3)/AKT/ERK pathways. These results suggest the therapeutic potential of violacein in effectively suppressing HCC by targeting the proliferation and stemness of HCC cells.

A histone variant condenses flowering plant sperm via chromatin phase separation

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state. Flowering plant sperm cells lack protamines, yet have small, transcriptionally active nuclei with chromatin condensed by an unknown mechanism. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin, demonstrating that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, thus achieving nuclear compaction without reducing transcription. H2B.8 also intermixes inactive AT-rich chromatin and GC-rich pericentromeric heterochromatin, altering higher-order chromatin architecture. Altogether, our results reveal a novel mechanism of nuclear compaction via global aggregation of unexpressed chromatin. We propose that H2B.8 is a flowering plant evolutionary innovation that achieves nuclear condensation compatible with active transcription.

Carnosic Acid Alleviates Levodopa-Induced Dyskinesia and Cell Death in 6-Hydroxydopamine-lesioned Rats and in SH-SY5Y Cells

The present study investigated the impact of carnosic acid (CA) from rosemary on the levodopa (L-dopa)-induced dyskinesia (LID) in rats treated with 6-hydroxydopamine (6-OHDA). To establish the model of LID, 6-OHDA-lesioned rats were injected intraperitoneally with 30 mg/kg L-dopa once a day for 36 days. Rats were daily administrated with 3 or 15 mg/kg CA by oral intubation prior to L-dopa injection for 4 days. Rats pretreated with CA had reduced L-dopa-induced abnormal involuntary movements (AIMs) and ALO scores (a sum of axial, limb, and orofacial scores). Moreover, the increases of dopamine D1-receptor, p-DARPP-32, ΔFosB, p-ERK1/2, and p-c-Jun ser63, along with the decrease in p-c-Jun ser73, induced by L-dopa in 6-OHDA-treated rats were significantly reversed by pretreatment with CA. In addition, we used the model of SH-SY5Y cells to further examine the neuroprotective mechanisms of CA on L-dopa-induced cytotoxicity. SH-SY5Y cells were treated with CA for 18 h, and then co-treated with 400 μM L-dopa for the indicated time points. The results showed that pretreatment of CA attenuated the cell death and nuclear condensation induced by L-dopa. By the immunoblots, the reduction of Bcl-2, p-c-Jun ser73, and parkin and the induction of cleaved caspase 3, cleaved Poly (ADP-ribose) polymerase, p-ERK1/2, p-c-Jun ser63, and ubiquitinated protein by L-dopa were improved in cells pretreated with CA. In conclusion, CA ameliorates the development of LID via regulating the D1R signaling and prevents L-dopa-induced apoptotic cell death through modulating the ERK1/2-c-Jun and inducing the parkin. This study suggested that CA can be used to alleviate the adverse effects of LID for PD patients.

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis

Background Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. Results We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X. Conclusions We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.

Ferroptosis: a cell death connecting oxidative stress, inflammation and cardiovascular diseases

Ferroptosis, a recently identified and iron-dependent cell death, differs from other cell death such as apoptosis, necroptosis, pyroptosis, and autophagy-dependent cell death. This form of cell death does not exhibit typical morphological and biochemical characteristics, including cell shrinkage, mitochondrial fragmentation, nuclear condensation. The dysfunction of lipid peroxide clearance, the presence of redox-active iron as well as oxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids are three essential features of ferroptosis. Iron metabolism and lipid peroxidation signaling are increasingly recognized as central mediators of ferroptosis. Ferroptosis plays an important role in the regulation of oxidative stress and inflammatory responses. Accumulating evidence suggests that ferroptosis is implicated in a variety of cardiovascular diseases such as atherosclerosis, stroke, ischemia-reperfusion injury, and heart failure, indicating that targeting ferroptosis will present a novel therapeutic approach against cardiovascular diseases. Here, we provide an overview of the features, process, function, and mechanisms of ferroptosis, and its increasingly connected relevance to oxidative stress, inflammation, and cardiovascular diseases.

Deacetylation Induced Nuclear Condensation of HP1γ Promotes Multiple Myeloma Drug Resistance

Acquired chemoresistance to proteasome inhibitors (PIs) is a major obstacle that results in failure to manage patients with multiple myeloma (MM) in the clinic; however, the key regulators and underlying mechanisms are still unclear. In this study, we found that high levels of a chromosomal modifier, heterochromatin protein 1 gamma (HP1γ), are accompanied by a low acetylation level in bortezomib-resistant (BR) MM cells, and aberrant DNA repair capacity is correlated with HP1γ overexpression. Mechanistically, the deacetylation of HP1γ at lysine 5 by histone deacetylase 1 (HDAC1) alleviates HP1γ ubiquitination, and the stabilized HP1γ recruits the mediator of DNA damage checkpoint 1 (MDC1) to induce DNA damage repair. Simultaneously, deacetylation modification and MDC1 recruitment enhance the nuclear condensate of HP1γ, which facilitates the chromatin accessibility of genes governing sensitivity to PIs, such as FOS, JUN and CD40. Thus, targeting HP1γ stability using the HDAC1/2 inhibitor, romidepsin, sensitizes PIs treatment and overcomes drug resistance both in vitro and in vivo. Our findings elucidate a previously unrecognized role of HP1γ in the acquired drug resistance of MM and suggest that targeting HP1γ may be efficacious for overcoming drug resistance in MM patients.