Autophagy induced by a sulphamoylated estrone analogue contributes to its cytotoxic effect on breast cancer cells

Background

Autophagy can either be protective and confer survival to stressed cells, or it can contribute to cell death. The antimitotic drug 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) is an in silico-designed 17-β-estradiol analogue that induces both autophagy and apoptosis in cancer cells. The aim of the study was to determine the role of autophagy in ESE-15-ol-exposed human adenocarcinoma breast cancer cells; knowledge that will contribute to future clinical applications of this novel antimitotic compound. By inhibiting autophagy and determining the cytotoxic effects of ESE-15-ol-exposure, deductions could be made as to whether the process may confer resistance to the drug, or alternatively, contribute to the cell death process.

Methods and results

Spectophometrical analysis via crystal violet staining was used to perform cytotoxicity studies. Morphology studies were done using microscopic techniques namely polarization-optical transmitted light differential interference light microscopy, fluorescent microscopy using monodansylcadaverine staining and transmission electron microscopy. Flow cytometry was used to quantify the autophagy inhibition and assess cell viability. Results obtained indicated that 3-methyladenine inhibited autophagy and increased cell survival in both MCF-7 and MDA-MB-231 cell lines.

Conclusion

This in vitro study inferred that autophagy inhibition with 3-methyladenine does not confer increased effectiveness of ESE-15-ol in inducing cell death. Thus it may be concluded that the autophagic process induced by ESE-15-ol exposure in MCF-7 and MDA-MB-231 cells plays a more significant role in cell death than conferring survival.

     

Effect of ATP and Bax on the apoptosis of <Emphasis Type="Italic">Eimeria tenella</Emphasis> host cells

Background

Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells.

Results

The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P?<?0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24–120 h (P?<?0.05 or P?<?0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those in the untreated group.

Conclusions

ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.

     

Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis

Background

Oxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that oxidative stress-induced apoptosis could be a potential mechanism mediating chromosome breakages in nasopharyngeal epithelial cells. Additionally, caspase-activated DNase (CAD) may be the vital player in mediating the chromosomal breakages during oxidative stress-induced apoptosis. Chromosomal breakage occurs during apoptosis and chromosome rearrangement. Chromosomal breakages tend to cluster in certain regions, such as matrix association region/scaffold attachment region (MAR/SAR). We hypothesised that oxidative stress-induced apoptosis may result in chromosome breaks preferentially at the MAR/SAR sites. The AF9 gene at 9p22 was targeted in this study because 9p22 is a deletion site commonly found in NPC.

Results

By using MAR/SAR recognition signature (MRS), potential MAR/SAR sites were predicted in the AF9 gene. The predicted MAR/SAR sites precisely match to the experimentally determined MAR/SARs. Hydrogen peroxide (H₂O₂) was used to induce apoptosis in normal nasopharyngeal epithelial cells (NP69) and NPC cells (HK1). Nested inverse polymerase chain reaction was employed to identify the AF9 gene cleavages. In the SAR region, the gene cleavage frequency of H₂O₂-treated cells was significantly higher than that of the non-treated cells. A few chromosomal breakages were detected within the AF9 region which was previously found to be involved in the mixed lineage leukaemia (MLL)-AF9 translocation in an acute lymphoblastic leukaemia patient. As for the non-SAR region, no significant difference in the gene cleavage frequency was found between the untreated control and H₂O₂-treated cells. Furthermore, H₂O₂-induced cleavages within the SAR region were reduced by caspase-3 inhibitor, which indirectly inhibits CAD.

Conclusions

These results reaffirm our previous findings that oxidative stress-induced apoptosis could be one of the potential mechanisms underlying chromosome breakages in nasopharyngeal epithelial cells. MAR/SAR may play a vital role in defining the location of chromosomal breakages mediated by oxidative stress-induced apoptosis, where CAD is the major nuclease.

     

Synthesis and inhibitory effect of 10-chlorocanthin-6-one on ovarian cancer HO8910PM cells

Objective

To synthesize and determine the antitumor activity of 10-chlorocanthin-6-one in ovarian cancer HO8910PM cells.

Results

Among the synthesized canthin-6-one analogs, 10-chlorocanthin-6-one was the most cytotoxic (IC₅₀ = 4.9 μM), as demonstrated by a dose-dependent cytotoxicity assay. Moreover, 10-chlorocanthin-6-one induced apoptosis through the activation of poly(ADP-ribose) polymerase and caspase-3 cleavage, upregulation of Bcl-2, and downregulation of Bim, x-linked inhibitor of apoptosis protein (XIAP), and survivin in HO8910PM cells. Furthermore, Bim RNA, upregulated in a concentration-dependent manner, and knockdown of Bim via short-hairpin RNAs attenuated the inhibitory effects of 10-chlorocanthin-6-one on HO8910PM cell growth.

Conclusions

10-Chlorocanthin-6-one inhibits cell proliferation and induces apoptosis in H08910PM cells. The underlying molecular mechanisms of 10-chlorocanthin-6-one include activation of the Bim-mediated mitochondrial apoptotic pathway via upregulation of Bim and downregulation of Bcl-2, XIAP, and survivin. These data suggest that Bim is a potential target of 10-chlorocanthin-6-one, further demonstrating its potential use in the prevention and treatment of ovarian cancer.

     

Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study

Background

Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample.The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female’s erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C.

Results

No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress.

Conclusions

From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.     

A dominant negative mutant of TLK1 causes chromosome missegregation and aneuploidy in normal breast epithelial cells

Background

In Arabidopsis thaliana, the gene Tousled encodes a protein kinase of unknown function, but mutations in the gene lead to flowering and leaf morphology defects. We have recently cloned a mammalian Tousled-L ike K inase (TLK1B) and found that it phosphorylates specifically histone H3, in vitro and in vivo. We now report the effects that overexpression of a kinase-dead mutant of TLK1B mediates in a normal diploid cell line.

Results

Expression of a kinase-dead mutant resulted in reduction of phosphorylated histone H3, which could have consequences in mitotic segregation of chromosomes. When analyzed by FACS and microscopy, these cells displayed high chromosome number instability and aneuploidy. This phenomenon was accompanied by less condensed chromosomes at mitosis; failure of a number of chromosomes to align properly on the metaphase plate; failure of some chromosomes to attach to microtubules; and the occasional presentation of two bipolar spindles. We also used a different method (siRNA) to reduce the level of endogenous TLK1, but in this case, the main result was a strong block of cell cycle progression suggesting that TLK1 may also play a role in progression from G1. This block in S phase progression could also offer a different explanation of some of the later mitotic defects.

Conclusions

TLK1 has a function important for proper chromosome segregation and maintenance of diploid cells at mitosis in mammalian cells that could be mediated by reduced phosphorylation of histone H3 and condensation of chromosomes, although other explanations to the phenotype are possible.

     

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.

Abstract

Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

     

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 μM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.     

Knockout of <Emphasis Type="Italic">CTNNB1</Emphasis> by CRISPR-Cas9 technology inhibits cell proliferation through the Wnt/β-catenin signaling pathway

Objective

To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway.

Results

CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of β-catenin significantly weakened adhesion ability and inhibited proliferation rate (P < 0.01) of HEK 293T cells. Nevertheless, deletion of β-catenin did not affect apoptosis of HEK 293T cells, which was analyzed by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. In addition, expression level of GSK-3β, CCND1, and CCNE1 detected by qPCR and expression level of N-Cadherin and cyclin D1 detected by western blotting were significantly decreased (P < 0.01) while expression of γ-catenin detected by western blotting was significantly increased (P < 0.001).

Conclusions

Knockout of CTNNB1 disturbed Wnt/β-catenin signaling pathway and significantly inhibited adhesion and proliferation of HEK 293T cells.

     

RNA binding protein QKI contributes to WT1 mRNA and suppresses apoptosis in ST cells

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.     

Classification of a new phytoplasmas subgroup 16SrII-W associated with <Emphasis Type="Italic">Crotalaria</Emphasis> witches’ broom diseases in Oman based on multigene sequence analysis

Background

Crotalaria aegyptiaca, a low shrub is commonly observed in the sandy soils of wadis desert and is found throughout all regions in Oman. A survey for phytoplasma diseases was conducted. During a survey in a wild area in the northern regions of Oman in 2015, typical symptoms of phytoplasma infection were observed on C. aegyptiaca plants. The infected plants showed an excessive proliferation of their shoots and small leaves.

Results

The presence of phytoplasma in the phloem tissue of symptomatic C. aegyptiaca leaf samples was confirmed by using Transmission Electron Microscopy (TEM). In addition the extracted DNA from symptomatic C. aegyptiaca leaf samples and Orosius sp. leafhoppers were tested by PCR using phytoplasma specific primers for the 16S rDNA, secA, tuf and imp, and SAP11 genes. The PCR amplifications from all samples yielded the expected products, but not from asymptomatic plant samples. Sequence similarity and phylogenetic tree analyses of four genes (16S rDNA, secA, tuf and imp) showed that Crotalaria witches’ broom phytoplasmas from Oman is placed with the clade of Peanut WB (16SrII) close to Fava bean phyllody (16SrII-C), Cotton phyllody and phytoplasmas (16SrII-F), and Candidatus Phytoplasma aurantifolia’ (16SrII-B). However, the Crotalaria’s phytoplasma was in a separate sub-clade from all the other phytoplasmas belonging to Peanut WB group. The combination of specific primers for the SAP11 gene of 16SrII-A, ?B, and -D subgroup pytoplasmas were tested against Crotalaria witches’ broom phytoplasmas and no PCR product was amplified, which suggests that the SAP11 of Crotalaria phytoplasma is different from the SAP11 of the other phytoplasmas.

Conclusion

We propose to assign the Crotalaria witches’ broom from Oman in a new lineage 16SrII-W subgroup depending on the sequences analysis of 16S rRNA, secA, imp, tuf, and SAP11 genes. To our knowledge, this is the first report of phytoplasmas of the 16SrII group infecting C. aegyptiaca worldwide.

     

SfP53 and filamentous actin (F-actin) are the targets of viral pesticide AcMNPV-<Emphasis Type="Italic">Bm</Emphasis>K IT (P10/PH) in host <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> 9 cells

Objectives

To analyze the anti-insect mechanism of viral pesticide AcMNPV-BmK IT(P10/PH) in the host Spodoptera frugiperda 9 (Sf9) cells.

Results

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV)- mediated expression of BmK IT, regulated by P10 protein promoter (P10) and polyhedrosis promoter (PH), promoted the replication of progeny virus in host Sf9 cells. AcMNPV-BmK IT(P10) could accelerate the budding process (or speed) of budded virus (BV) in Sf9 cells. The impact of AcMNPV-BmK IT(P10) on the nuclear polymerization of filamentous actin (F-actin) participated in regulating the accelerated budding process. Unexpectedly, both AcMNPV-BmK IT(P10) and AcMNPV-BmK IT(PH) delayed the nuclear polymerization of F-actin and promoted the clearance of F-actin in the nucleus. SfP53, an important apoptosis factor, was involved in the regulation of AcMNPV-BmK IT(P10/PH) in Sf9 cells. AcMNPV-BmK IT(P10/PH) could also delay and promote the nuclear recruitment of SfP53 after 27 h post infection (h p.i.).

Conclusion

SfP53 and F-actin are the targets of viral pesticide AcMNPV-BmK IT (P10/PH) in host Sf9 cells, which provides the experimental basis for the development of recombinant baculovirus biopesticides.

     

Inhibitory effects of YCW and MOS from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella pullorum</Emphasis> adhesion to Caco-2 cells

Background

For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.

Methods

YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.

Results

The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.

Conclusions

These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.

     

Use of the yeast-like cells of <Emphasis Type="Italic">Tremella fuciformis</Emphasis> as a cell factory to produce a <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> hydrophobin

Objectives

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens.

Results

The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate.

Conclusion

Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.